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Sample records for programming enzyme specificity

  1. Programming of enzyme specificity by substrate mimetics: investigations on the Glu-specific V8 protease reveals a novel general principle of biocatalysis.

    PubMed

    Wehofsky, N; Bordusa, F

    1999-01-25

    In this paper the universal validity of the substrate mimetic concept in enzymatic C-N ligations was expanded to anionic leaving groups based on the specificity determinants of Glu-specific endopeptidase from Staphylococcus aureus (V8 protease). In an empirical way a specific mimetic moiety was designed from simple structure-function relationship studies. The general function of the newly developed substrate mimetics to serve as an artificial recognition site for V8 protease have been examined by hydrolysis kinetic studies. Enzymatic peptide syntheses qualify the strategy of substrate mimetics as a powerful concept for programming the enzyme specificity in the direction of a more universal application of enzymes in the general area of biocatalysis. PMID:9989609

  2. [The synthesis of specific enzyme inhibitors].

    PubMed

    Iakovleva, G M

    1987-04-01

    The review deals with directed synthesis of specific enzyme inhibitors. They are classified within the framework of the mechanistic approach, namely, stable analogues of substrates, which form enzyme complexes mimicking the Michaelis complex or those which influence the chemical stages of enzyme catalysis; conformational inhibitors; substrate analogues participating in enzyme reactions and producing modified products; suicide inhibitors; stage inhibitors (inhibitors influencing certain stages of enzyme reaction); transition state analogues; multisubstrate analogues and collected substrates. Types of chemical modification used in synthesis of the specific inhibitors are discussed. Some possibilities of the quantity structure-activity relationship methods, computer modelling and molecular graphics in designing the optimal structure of inhibitors are mentioned. PMID:3300658

  3. Main factors providing specificity of repair enzymes.

    PubMed

    Nevinsky, G A

    2011-01-01

    Specific and nonspecific DNA complex formation with human uracil-DNA glycosylase, 8-oxoguanine-DNA glycosylase, and apurine/apyrimidine endonuclease, as well as with E. coli 8-oxoguanine-DNA glycosylase and RecA protein was analyzed using the method of stepwise increase in DNA-ligand complexity. It is shown that high affinity of these enzymes to any DNA (10(-4)-10(-8) M) is provided by a large number of weak additive contacts mainly with DNA internucleoside phosphate groups and in a less degree with bases of nucleotide links "covered" by protein globules. Enzyme interactions with specific DNA links are comparable in efficiency with weak unspecific contacts and provide only for one-two orders of affinity (10(-1)-10(-2) M), but these contacts are extremely important at stages of DNA and enzyme structural adaptation and catalysis proper. Only in the case of specific DNA individual for each enzyme alterations in DNA structure provide for efficient adjustment of reacting enzyme atoms and DNA orbitals with accuracy up to 10-15° and, as a result, for high reaction rate. Upon transition from nonspecific to specific DNA, reaction rate (k(cat)) increases by 4-8 orders of magnitude. Thus, stages of DNA and enzyme structural adaptation as well as catalysis proper are the basis of specificity of repair enzymes. PMID:21568843

  4. Enzyme Specific Activity in Functionalized Nanoporous Supports

    SciTech Connect

    Lei, Chenghong; Soares, Thereza A.; Shin, Yongsoon; Liu, Jun; Ackerman, Eric J.

    2008-03-26

    Enzyme specific activity can be increased or decreased to a large extent by changing protein loading density in functionalized nanoporous support, where organophosphorus hydrolase can display a constructive orientation and thus leave a completely open entrance for substrate even at higher protein loading density, but glucose oxidase can not.

  5. 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. PMID:26191550

  6. [Enzyme labeling methods and it's specificities].

    PubMed

    Kambegawa, A

    1995-09-01

    Enzyme labeled antigen for use in ELISA of Hapten (steroids, prostanoid, carbohydrate, nucleic acid, peptide, herbicide, insecticide and antibiotic) have usually been prepared by condensation of carboxy group of hapten with amino groups of lysine residue in enzyme. The horseradish peroxidase (HRP) is best suitable as labeling enzyme, therefore it is small molecular, and substrate turnover is much higher compared to the other enzymes. The mixed anhydride and carbodiimide methods have mainly been used the preparation of hapten conjugate BSA, but not satisfactory for enzyme labeling. The N-hydroxysuccinimide ester (NHS: active ester) method is satisfactory with respect to reproducibility and sensitivity. The sensitivity is related to the bridging phenomenon: One of the disadvantages of the homologous labels is that the antibody shows an affinity not only for the Hapten but also for the bridge which connects the Hapten to carrier protein. We have developed a sensitive bridge heterologous EIA for progesterone (P) using geometrical isomers of P-3 (E/Z) (O-carboxymethyl) oxime-N-hydroxysuccinimide esters [ef Ab of P-3 (E) CMO-BSA/P-3 (Z) CMO-HRP]3). The sensitivity of heterologous proved to be higher than a homologous EIA or a conventional RIA. It seem like that a 1:1 steroid-enzyme conjugate is suitable for obtaining a high sensitivity. The avidin-biotin (AB) system provides great versatility, since by conjugation with an appropriate label, the AB assay can be used with any chosen detector. Furthermore, IgG can be labels with biotin without significantly influencing their immunological activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7474375

  7. 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 ...

  8. 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…

  9. 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.

  10. Maternal Undernutrition Programs Offspring Adrenal Expression of Steroidogenic Enzymes

    PubMed Central

    Khorram, Naseem M.; Magee, Thomas R.; Wang, Chen; Desai, Mina; Ross, Michael; Khorram, Omid

    2011-01-01

    The aim of this study was to determine the influence of maternal undernutrition (MUN) on maternal and offspring adrenal steoridogenic enzymes. Pregnant Sprague-Dawley rats were 50% food-restricted from day 10 of gestation until delivery. Control animals received ad libitum food. Offspring were killed on day 1 of life (P1) and at 9 months. We determined the messenger RNA (mRNA) expression of steroidogneic enzymes by real-time reverse transcriptase polymerized chain reaction (RT-PCR). Maternal undernutrition inhibited maternal adrenal expression of P450 cholesterol side-chain cleavage enzyme (CYP11A1), 11 beta-hydroxylase (CYP11B1), aldosterone synthase (CYP11B2), and adrenocorticotropic hormone (ACTH) receptor (ACTH-R; MC2 gene) compared with control offspring. There was a marked downregulation in the expression of CYP11B1, CYP11B2, 11 β-hydroxysteroid dehydrogenase type 1 and 2 (HSD1 and HSD2), CYP11A1, ACTH receptor, steroidogenic acute regulatory protein (STAR), and mineralocorticoid receptor (MCR; NR3C2 gene) mRNA in P1 MUN offspring (both genders), with no changes in glucocorticoid receptor (GCR). Quantitative immunohistochemical analysis confirmed the PCR data for GCR and MCR in P1 offspring and demonstrated lower expression of leptin receptor protein (Ob-Ra/Ob-Rb) and mRNA in P1 MUN offspring. In 9-month adult male MUN offspring, the expression of HSD1, CYP11A1, CYP11B2, Ob-Ra/Ob-Rb, and GCR mRNA were significantly upregulated with a trend toward an increase in ACTH-R and a decrease in 17 alpha-hydroxylase (CYP17A1) expression. In adult female MUN offspring, similar to males, the expression of CYP11A1, ACTH-R, and Ob-Rb mRNA were increased, whereas GCR and CYP17A1 mRNA were decreased. These results indicate that the adrenal gland is a target of nutritional programming. In utero undernutrition has a global suppressive effect on maternal and P1 offspring adrenal steroidogenic enzymes in association with reduced circulating corticosterone levels in P1 offspring

  11. Nonprocedural-dataflow specification of concurrent programs

    SciTech Connect

    Prywes, N.; Szymanski, B.; Shi, Y.

    1983-01-01

    A previously developed nonprocedural specification language model and its program generator are extended to concurrent programming. The specification of concurrent programs in model and the generation of the corresponding object programs is illustrated through a resource allocation example which consists of a number of parallel processes that request and release resources and a monitor process that allocates requested resources. The discussion is particularized by the example of the dining philosophers problem. To provide background, the author reviews the methodology of the program generator for (1) checking the global and local consistency and completeness of the specification, and (2) generating a schedule of program events to attain efficiency in program execution. 20 references.

  12. Pairwise specificity and sequential binding in enzyme catalysis: thymidylate synthase.

    PubMed

    Finer-Moore, J S; Montfort, W R; Stroud, R M

    1990-07-31

    The structures of thymidylate synthase (TS) from Escherichia coli, in ternary complexes with substrate and an analogue of the cofactor, are the basis of a stereochemical model for a key reaction intermediate in the catalyzed reaction. This model is used to compare the reaction chemistry and chirality of the transferred methyl group with structures of the components, to identify those residues that participate, and to propose a stereochemical mechanism for catalysis by TS. Effects of chemical modification of specific amino acid residues and site-directed mutations of residues are correlated with structure and effects on enzyme mechanism. The ordered binding sequence of substrate deoxyuridine monophosphate and methylenetetrahydrofolate can be understood from the structure, where each forms a large part of the binding site for the other. The catalytic site serves to orient the reactants, which are sequestered along with many water molecules within a cavernous active center. Conformational changes during the reaction could involve nearby residues in ways that are not obvious in this complex. PMID:2223755

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

    PubMed

    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

  14. 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

  15. 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.

  16. Tissue Specificity of Human Angiotensin I-Converting Enzyme

    PubMed Central

    Kryukova, Olga V.; Tikhomirova, Victoria E.; Golukhova, Elena Z.; Evdokimov, Valery V.; Kalantarov, Gavreel F.; Trakht, Ilya N.; Schwartz, David E.; Dull, Randal O.; Gusakov, Alexander V.; Uporov, Igor V.; Kost, Olga A.; Danilov, Sergei M.

    2015-01-01

    Background Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, as well as in reproductive functions, is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells. ACE also presents as a soluble form in biological fluids, among which seminal fluid being the richest in ACE content - 50-fold more than that in blood. Methods/Principal Findings We performed conformational fingerprinting of lung and seminal fluid ACEs using a set of monoclonal antibodies (mAbs) to 17 epitopes of human ACE and determined the effects of potential ACE-binding partners on mAbs binding to these two different ACEs. Patterns of mAbs binding to ACEs from lung and from seminal fluid dramatically differed, which reflects difference in the local conformations of these ACEs, likely due to different patterns of ACE glycosylation in the lung endothelial cells and epithelial cells of epididymis/prostate (source of seminal fluid ACE), confirmed by mass-spectrometry of ACEs tryptic digests. Conclusions Dramatic differences in the local conformations of seminal fluid and lung ACEs, as well as the effects of ACE-binding partners on mAbs binding to these ACEs, suggest different regulation of ACE functions and shedding from epithelial cells in epididymis and prostate and endothelial cells of lung capillaries. The differences in local conformation of ACE could be the base for the generation of mAbs distingushing tissue-specific ACEs. PMID:26600189

  17. Enzyme family-specific and activity-based screening of chemical libraries using enzyme microarrays.

    PubMed

    Funeriu, Daniel P; Eppinger, Jörg; Denizot, Lucile; Miyake, Masato; Miyake, Jun

    2005-05-01

    The potential of protein microarrays in high-throughput screening (HTS) still remains largely unfulfilled, essentially because of the difficulty of extracting meaningful, quantitative data from such experiments. In the particular case of enzyme microarrays, low-molecular-weight fluorescent affinity labels (FALs) can function as ideally suited activity probes of the microarrayed enzymes. FALs form covalent bonds with enzymes in an activity-dependent manner and therefore can be used to characterize enzyme activity at each enzyme's address, as predetermined by the microarraying process. Relying on this principle, we introduce herein thematic enzyme microarrays (TEMA). In a kinetic setup we used TEMAs to determine the full set of kinetic constants and the reaction mechanism between the microarrayed enzymes (the theme of the microarray) and a family-wide FAL. Based on this kinetic understanding, in an HTS setup we established the practical and theoretical methodology for quantitative, multiplexed determination of the inhibition profile of compounds from a chemical library against each microarrayed enzyme. Finally, in a validation setup, K(i)(app) values and inhibitor profiles were confirmed and refined. PMID:15821728

  18. Cell-specific activity of neprilysin 2 isoforms and enzymic specificity compared with neprilysin.

    PubMed Central

    Rose, Christiane; Voisin, Stéphanie; Gros, Claude; Schwartz, Jean-Charles; Ouimet, Tanja

    2002-01-01

    Neprilysin (NEP) 2 is a recently cloned glycoprotein displaying a high degree of sequence identity with neprilysin (EC 3.4.24.11), the prototypical member of the M13 subfamily of metalloproteases. Whereas NEP is involved in the metabolism of several bioactive peptides by plasma membranes of various cells, the enzymic properties and physiological functions of NEP2 are unknown. Here we characterize the cell-expression modalities and enzymic specificity of two alternatively spliced isoforms of NEP2 in Chinese hamster ovary and AtT20 cells. In the two cell lines, both isoforms are type II glycoproteins inserted in the endoplasmic reticulum as inactive precursors. Maturation detected by Western-blot analysis of glycosidase digests was cell-specific and more efficient in the endocrine cell line. The enzymic activity of both isoforms semi-purified from AtT20 cells reveals comparable specificities in terms of model substrates, pH optima and inhibitory patterns. NEP2 activity was compared with that of NEP regarding potencies of transition-state inhibitors, modes of hydrolysis, maximal hydrolysis rates and apparent affinities of bioactive peptides. Although all transition-state inhibitors of NEP inhibited NEP2 activity, albeit with different potencies, and many peptides were cleaved at the same amide bond by both peptidases, differences could be observed, i.e. in the hydrolysis of gonadotropin-releasing hormone and cholecystokinin, which occurred at different sites and more efficiently in the case of NEP2. Differences in cleavage of bioactive peptides, in cell-trafficking patterns and in tissue distribution indicate that NEP and NEP2 play distinct physiological roles in spite of their high degree of sequence identity. PMID:11964170

  19. 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.

  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. Automatic program generation from specifications using PROLOG

    NASA Technical Reports Server (NTRS)

    Pelin, Alex; Morrow, Paul

    1988-01-01

    An automatic program generator which creates PROLOG programs from input/output specifications is described. The generator takes as input descriptions of the input and output data types, a set of transformations and the input/output relation. Abstract data types are used as models for data. They are defined as sets of terms satisfying a system of equations. The tests, the transformations and the input/output relation are also specified by equations.

  2. An inhibitory C-terminal region dictates the specificity of A-adding enzymes

    PubMed Central

    Tretbar, Sandy; Neuenfeldt, Anne; Betat, Heike; Mörl, Mario

    2011-01-01

    For efficient aminoacylation, tRNAs carry the conserved 3′-terminal sequence C-C-A, which is synthesized by highly specific tRNA nucleotidyltransferases (CCA-adding enzymes). In several prokaryotes, this function is accomplished by separate enzymes for CC- and A-addition. As A-adding enzymes carry an N-terminal catalytic core identical to that of CCA-adding enzymes, it is unclear why their activity is restricted. Here, it is shown that C-terminal deletion variants of A-adding enzymes acquire full and precise CCA-incorporating activity. The deleted region seems to be responsible for tRNA primer selection, restricting the enzyme’s specificity to tRNAs ending with CC. The data suggest that A-adding enzymes carry an intrinsic CCA-adding activity that can be reactivated by the introduction of deletions in the C-terminal domain. Furthermore, a unique subtype of CCA-adding enzymes could be identified that evolved out of A-adding enzymes, suggesting that mutations and deletions in nucleotidyltransferases can lead to altered and even more complex activities, as a simple A-incorporation is converted into sequence-specific addition of C and A residues. Such activity-modifying events may have had an important role in the evolution of tRNA nucleotidyltransferases. PMID:22167803

  3. Specification and control of behavioral robot programs

    NASA Astrophysics Data System (ADS)

    Kahn, Philip

    1992-04-01

    Behavioral robotics is the specification and control of robots as a composition, coordination, and adaptation of more primitive sub-behaviors. The past decade has led to the development of many useful algorithms that can support valuable behavioral function in robots (e.g., perception, robotics, planning, domain rules). Increased availability of these component algorithms increases the need for robot control methods that can select between gross behaviors in addition to providing control within a given behavior. For example, gross behavioral changes may consider when and whether to attend to new stimuli, and if and how that stimuli can lead to new or different behaviors. This paper describes an approach being developed for the specification and control of these types of behavioral programs. The first section introduces a task oriented approach to behavioral robot program specification and control. The second section then describes a Behavioral Architecture for Robot Tasks (BART) being developed. A BART language is being built to provide a portable tool to support various robot programming and execution strategies, evidence accrual methods, and domain representations. This language is being used to develop behavioral programs that control tanks and tank platoons.

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

    PubMed

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

    2015-08-01

    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. PMID:26080680

  5. 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.

  6. Interconversion of the specificities of human lysosomal enzymes associated with Fabry and Schindler diseases.

    PubMed

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

    2010-07-01

    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-GAL(SA)) 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(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(SA) to the wild-type enzymes shows that active sites of alpha-GAL(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. PMID:20444686

  7. DNA-Linked Enzyme-Coupled Assay for Probing Glucosyltransferase Specificity.

    PubMed

    Sukovich, David J; Modavi, Cyrus; de Raad, Markus; Prince, Robin N; Anderson, J Christopher

    2015-07-17

    Traditional enzyme characterization methods are low-throughput and therefore limit engineering efforts in synthetic biology and biotechnology. Here, we propose a DNA-linked enzyme-coupled assay (DLEnCA) to monitor enzyme reactions in a high-throughput manner. Throughput is improved by removing the need for protein purification and by limiting the need for liquid chromatography mass spectrometry (LCMS) product detection by linking enzymatic function to DNA modification. We demonstrate the DLEnCA methodology using glucosyltransferases as an illustration. The assay utilizes cell free transcription/translation systems to produce enzymes of interest, while UDP-glucose and T4-β-glucosyltransferase are used to modify DNA, which is detected postreaction using qPCR or a similar means of DNA analysis. OleD and two glucosyltransferases from Arabidopsis were used to verify the assay's generality toward glucosyltransferases. We further show DLEnCA's utility by mapping out the substrate specificity for these enzymes. PMID:25621860

  8. 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

  9. Prediction of Detailed Enzyme Functions and Identification of Specificity Determining Residues by Random Forests

    PubMed Central

    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. PMID:24416252

  10. Combining Structure and Sequence Information Allows Automated Prediction of Substrate Specificities within Enzyme Families

    PubMed Central

    Röttig, Marc; Rausch, Christian; Kohlbacher, Oliver

    2010-01-01

    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/. PMID:20072606

  11. Highly specific detection of genetic modification events using an enzyme-linked probe hybridization chip.

    PubMed

    Zhang, M Z; Zhang, X F; Chen, X M; Chen, X; Wu, S; Xu, L L

    2015-01-01

    The enzyme-linked probe hybridization chip utilizes a method based on ligase-hybridizing probe chip technology, with the principle of using thio-primers for protection against enzyme digestion, and using lambda DNA exonuclease to cut multiple PCR products obtained from the sample being tested into single-strand chains for hybridization. The 5'-end amino-labeled probe was fixed onto the aldehyde chip, and hybridized with the single-stranded PCR product, followed by addition of a fluorescent-modified probe that was then enzymatically linked with the adjacent, substrate-bound probe in order to achieve highly specific, parallel, and high-throughput detection. Specificity and sensitivity testing demonstrated that enzyme-linked probe hybridization technology could be applied to the specific detection of eight genetic modification events at the same time, with a sensitivity reaching 0.1% and the achievement of accurate, efficient, and stable results. PMID:26345863

  12. Hybrid reuteransucrase enzymes reveal regions important for glucosidic linkage specificity and the transglucosylation/hydrolysis ratio.

    PubMed

    Kralj, Slavko; van Leeuwen, Sander S; Valk, Vincent; Eeuwema, Wieger; Kamerling, Johannis P; Dijkhuizen, Lubbert

    2008-12-01

    The reuteransucrase enzymes of Lactobacillus reuteri strain 121 (GTFA) and L. reuteri strain ATCC 55730 (GTFO) convert sucrose into alpha-d-glucans (labelled reuterans) with mainly alpha-(1-->4) glucosidic linkages (50% and 70%, respectively), plus alpha-(1-->6) linkages. In the present study, we report a detailed analysis of various hybrid GTFA/O enzymes, resulting in the identification of specific regions in the N-termini of the catalytic domains of these proteins as the main determinants of glucosidic linkage specificity. These regions were divided into three equal parts (A1-3; O1-3), and used to construct six additional GTFA/O hybrids. All hybrid enzymes were able to synthesize alpha-glucans from sucrose, and oligosaccharides from sucrose plus maltose or isomaltose as acceptor substrates. Interestingly, not only the A2/O2 regions, with the three catalytic residues, affect glucosidic linkage specificity, but also the upstream A1/O1 regions make a strong contribution. Some GTFO derived hybrid/mutant enzymes displayed strongly increased transglucosylation/hydrolysis activity ratios. The reduced sucrose hydrolysis allowed the much improved conversion of sucrose into oligo- and polysaccharide products. Thus, the glucosidic linkage specificity and transglucosylation/hydrolysis ratios of reuteransucrase enzymes can be manipulated in a relatively simple manner. This engineering approach has yielded clear changes in oligosaccharide product profiles, as well as a range of novel reuteran products differing in alpha-(1-->4) and alpha-(1-->6) linkage ratios. PMID:19016850

  13. 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.

  14. 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.

  15. How well do the substrates KISS the enzyme? Molecular docking program selection for feruloyl esterases

    PubMed Central

    Udatha, D. B. R. K. Gupta; Sugaya, Nobuyoshi; Olsson, Lisbeth; Panagiotou, Gianni

    2012-01-01

    Molecular docking is the most commonly used technique in the modern drug discovery process where computational approaches involving docking algorithms are used to dock small molecules into macromolecular target structures. Over the recent years several evaluation studies have been reported by independent scientists comparing the performance of the docking programs by using default ‘black box’ protocols supplied by the software companies. Such studies have to be considered carefully as the docking programs can be tweaked towards optimum performance by selecting the parameters suitable for the target of interest. In this study we address the problem of selecting an appropriate docking and scoring function combination (88 docking algorithm-scoring functions) for substrate specificity predictions for feruloyl esterases, an industrially relevant enzyme family. We also propose the ‘Key Interaction Score System’ (KISS), a more biochemically meaningful measure for evaluation of docking programs based on pose prediction accuracy. PMID:22435086

  16. Sequence specific inhibition of human type II phospholipase A2 enzyme activity by phosphorothioate oligonucleotides.

    PubMed Central

    Bennett, C F; Chiang, M Y; Wilson-Lingardo, L; Wyatt, J R

    1994-01-01

    Phosphorothioate oligonucleotides were identified which directly inhibited human type II phospholipase A2 (PLA2) enzyme activity in a sequence specific manner. The minimum pharmacophore common to all oligonucleotides which inhibited PLA2 enzyme activity consisted of two sets of three or more consecutive guanosine residues in a row. These oligonucleotides appear to form G quartets resulting in the formation of oligonucleotide aggregates. Additionally, a phosphorothioate backbone was required to be effective inhibitors of type II PLA2. The activity of one oligodeoxynucleotide, IP 3196 (5'-GGGTGGGTATAGAAGGGCTCC-3') has been characterized in more detail. IP 3196 inhibited PLA2 enzyme activity when the substrate was presented in the form of a phospholipid bilayer but not when presented in the form of a mixed micelle with anionic detergents. Human type II PLA2 was 50-fold more sensitive to inhibition by IP 3196 than venom and pancreatic type I enzymes. These data demonstrate that phosphorothioate oligonucleotides can specifically inhibit human type II PLA2 enzyme activity in a sequence specific manner. PMID:8065936

  17. Cell Type–Specific Localization of Transcripts Encoding Nine Consecutive Enzymes Involved in Protoberberine Alkaloid Biosynthesis

    PubMed Central

    Samanani, Nailish; Park, Sang-Un; Facchini, Peter J.

    2005-01-01

    Molecular clones encoding nine consecutive biosynthetic enzymes that catalyze the conversion of l-dopa to the protoberberine alkaloid (S)-canadine were isolated from meadow rue (Thalictrum flavum ssp glaucum). The predicted proteins showed extensive sequence identity with corresponding enzymes involved in the biosynthesis of related benzylisoquinoline alkaloids in other species, such as opium poppy (Papaver somniferum). RNA gel blot hybridization analysis showed that gene transcripts for each enzyme were most abundant in rhizomes but were also detected at lower levels in roots and other organs. In situ RNA hybridization analysis revealed the cell type–specific expression of protoberberine alkaloid biosynthetic genes in roots and rhizomes. In roots, gene transcripts for all nine enzymes were localized to immature endodermis, pericycle, and, in some cases, adjacent cortical cells. In rhizomes, gene transcripts encoding all nine enzymes were restricted to the protoderm of leaf primordia. The localization of biosynthetic gene transcripts was in contrast with the tissue-specific accumulation of protoberberine alkaloids. In roots, protoberberine alkaloids were restricted to mature endodermal cells upon the initiation of secondary growth and were distributed throughout the pith and cortex in rhizomes. Thus, the cell type–specific localization of protoberberine alkaloid biosynthesis and accumulation are temporally and spatially separated in T. flavum roots and rhizomes, respectively. Despite the close phylogeny between corresponding biosynthetic enzymes, distinct and different cell types are involved in the biosynthesis and accumulation of benzylisoquinoline alkaloids in T. flavum and P. somniferum. Our results suggest that the evolution of alkaloid metabolism involves not only the recruitment of new biosynthetic enzymes, but also the migration of established pathways between cell types. PMID:15722473

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

    SciTech Connect

    Jeyakanthan, Jeyaraman; Drevland, Randy; Gayathri, Dasara; Velmurugan, Devadasan; Shinkai, Akeo; Graham, David E

    2010-01-01

    The aconitase family of hydro-lyase enzymes includes three classes of proteins that catalyze the isomerization of -hydroxyacids to -hydroxyacids. Besides aconitase, isopropylmalate isomerase (IPMI) proteins specifically catalyze the isomerization of , -dicarboxylates with hydrophobic -chain groups, and homoaconitase (HACN) proteins catalyze the isomerization of tricarboxylates with variable chain length -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 leads 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 2 and 3 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 structural and kinetic results will

  19. 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...

  20. Engineered catalytic biofilms: Site-specific enzyme immobilization onto E. coli curli nanofibers.

    PubMed

    Botyanszki, Zsofia; Tay, Pei Kun R; Nguyen, Peter Q; Nussbaumer, Martin G; Joshi, Neel S

    2015-10-01

    Biocatalytic transformations generally rely on purified enzymes or whole cells to perform complex transformations that are used on industrial scale for chemical, drug, and biofuel synthesis, pesticide decontamination, and water purification. However, both of these systems have inherent disadvantages related to the costs associated with enzyme purification, the long-term stability of immobilized enzymes, catalyst recovery, and compatibility with harsh reaction conditions. We developed a novel strategy for producing rationally designed biocatalytic surfaces based on Biofilm Integrated Nanofiber Display (BIND), which exploits the curli system of E. coli to create a functional nanofiber network capable of covalent immobilization of enzymes. This approach is attractive because it is scalable, represents a modular strategy for site-specific enzyme immobilization, and has the potential to stabilize enzymes under denaturing environmental conditions. We site-specifically immobilized a recombinant α-amylase, fused to the SpyCatcher attachment domain, onto E. coli curli fibers displaying complementary SpyTag capture domains. We characterized the effectiveness of this immobilization technique on the biofilms and tested the stability of immobilized α-amylase in unfavorable conditions. This enzyme-modified biofilm maintained its activity when exposed to a wide range of pH and organic solvent conditions. In contrast to other biofilm-based catalysts, which rely on high cellular metabolism, the modified curli-based biofilm remained active even after cell death due to organic solvent exposure. This work lays the foundation for a new and versatile method of using the extracellular polymeric matrix of E. coli for creating novel biocatalytic surfaces. PMID:25950512

  1. Enzyme-gold cytochemistry of seed xyloglucans using two xyloglucan-specific hydrolases. Importance of prior heat-deactivation of the enzymes.

    PubMed

    Vian, B; Nairn, J; Reid, J S

    1991-03-01

    Two pure, homogeneous xyloglucan-hydrolyzing enzymes from germinated nasturtium seeds have been used to localize xyloglucans specifically in seed cell walls. The enzymes, a novel endo (1----4)-beta-D-glucanase which shows absolute specificity towards xyloglucans and a beta-D-galactosidase which is capable of removing galactosyl residues from polymeric xyloglucans, were used to stabilize gold sols. The complexes were applied to ultrathin sections of nasturtium (Tropaeolum majus L) and tamarind (Tamarindus indica L) seeds. The gold complexes prepared from the active enzyme proteins retained enzyme activity, and such complexes gave extremely weak section-labelling or no labelling at all. When the enzymes were subjected to heat-deactivation before being used to stabilize the gold sols, gold complexes were obtained which lacked enzyme activity, but which gave strong, specific labelling of xyloglucans in ultrathin sections. The specificity of the labelling was checked by substrate-competition, by pretreatment of sections with the active and heat-denaturated enzymes and by comparing the labelling of xyloglucan-containing storage cells with other cell types in the same section. The labelling was maximal at the pH which was optimal for the active enzyme. We conclude that the enzyme-gold complexes which retain high activity against the substrate to be localized are likely to be unsuitable as cytochemical probes because they may cause in situ substrate modification. In the case of the enzyme complexes described here the specific localization obtained with the gold complexes prepared from heat deactivated enzymes may be attributable to the retention by the heat-treated enzymatically-inactive proteins of substrate recognition.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1783560

  2. 7 CFR 3052.235 - Program-specific audits.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Program-specific audits. (a) Program-specific audit guide available. In many cases, a program-specific audit guide will be available to provide specific guidance to the auditor with respect to internal control, compliance requirements, suggested audit procedures, and audit reporting requirements....

  3. 29 CFR 99.235 - Program-specific audits.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... § 99.235 Program-specific audits. (a) Program-specific audit guide available. In many cases, a program-specific audit guide will be available to provide specific guidance to the auditor with respect to internal control, compliance requirements, suggested audit procedures, and audit reporting requirements....

  4. 7 CFR 3052.235 - Program-specific audits.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Program-specific audits. (a) Program-specific audit guide available. In many cases, a program-specific audit guide will be available to provide specific guidance to the auditor with respect to internal control, compliance requirements, suggested audit procedures, and audit reporting requirements....

  5. 29 CFR 99.235 - Program-specific audits.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... § 99.235 Program-specific audits. (a) Program-specific audit guide available. In many cases, a program-specific audit guide will be available to provide specific guidance to the auditor with respect to internal control, compliance requirements, suggested audit procedures, and audit reporting requirements....

  6. 29 CFR 99.235 - Program-specific audits.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... § 99.235 Program-specific audits. (a) Program-specific audit guide available. In many cases, a program-specific audit guide will be available to provide specific guidance to the auditor with respect to internal control, compliance requirements, suggested audit procedures, and audit reporting requirements....

  7. 29 CFR 99.235 - Program-specific audits.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... § 99.235 Program-specific audits. (a) Program-specific audit guide available. In many cases, a program-specific audit guide will be available to provide specific guidance to the auditor with respect to internal control, compliance requirements, suggested audit procedures, and audit reporting requirements....

  8. 29 CFR 99.235 - Program-specific audits.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... § 99.235 Program-specific audits. (a) Program-specific audit guide available. In many cases, a program-specific audit guide will be available to provide specific guidance to the auditor with respect to internal control, compliance requirements, suggested audit procedures, and audit reporting requirements....

  9. 7 CFR 3052.235 - Program-specific audits.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Program-specific audits. (a) Program-specific audit guide available. In many cases, a program-specific audit guide will be available to provide specific guidance to the auditor with respect to internal control, compliance requirements, suggested audit procedures, and audit reporting requirements....

  10. 7 CFR 3052.235 - Program-specific audits.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Program-specific audits. (a) Program-specific audit guide available. In many cases, a program-specific audit guide will be available to provide specific guidance to the auditor with respect to internal control, compliance requirements, suggested audit procedures, and audit reporting requirements....

  11. 7 CFR 3052.235 - Program-specific audits.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Program-specific audits. (a) Program-specific audit guide available. In many cases, a program-specific audit guide will be available to provide specific guidance to the auditor with respect to internal control, compliance requirements, suggested audit procedures, and audit reporting requirements....

  12. Specificity and catalysis hardwired at the RNA-protein interface in a translational proofreading enzyme

    NASA Astrophysics Data System (ADS)

    Ahmad, Sadeem; Muthukumar, Sowndarya; Kuncha, Santosh Kumar; Routh, Satya Brata; Yerabham, Antony S. K.; Hussain, Tanweer; Kamarthapu, Venu; Kruparani, Shobha P.; Sankaranarayanan, Rajan

    2015-06-01

    Proofreading modules of aminoacyl-tRNA synthetases are responsible for enforcing a high fidelity during translation of the genetic code. They use strategically positioned side chains for specifically targeting incorrect aminoacyl-tRNAs. Here, we show that a unique proofreading module possessing a D-aminoacyl-tRNA deacylase fold does not use side chains for imparting specificity or for catalysis, the two hallmark activities of enzymes. We show, using three distinct archaea, that a side-chain-stripped recognition site is fully capable of solving a subtle discrimination problem. While biochemical probing establishes that RNA plays the catalytic role, mechanistic insights from multiple high-resolution snapshots reveal that differential remodelling of the catalytic core at the RNA-peptide interface provides the determinants for correct proofreading activity. The functional crosstalk between RNA and protein elucidated here suggests how primordial enzyme functions could have emerged on RNA-peptide scaffolds before recruitment of specific side chains.

  13. A fluorescence assay for elucidating the substrate specificities of deubiquitinating enzymes

    SciTech Connect

    Yin, Si-Tao; Huang, Hao; Zhang, Yu-Hang; Zhou, Zi-Ren; Song, Ai-Xin; Hong, Fa-Shui; Hu, Hong-Yu

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer A deubiquitinating enzyme has its unique substrate specificity for deubiquitination. Black-Right-Pointing-Pointer We have established an activity assay for ubiquitin C-terminal hydrolases. Black-Right-Pointing-Pointer This assay can be applicable to other deubiquitinating enzymes. -- Abstract: Ubiquitin C-terminal hydrolases (UCHs) are a representative family of deubiquitinating enzymes (DUBs), which specifically cleave ubiquitin (Ub) chains or extensions. Here we present a convenient method for characterizing the substrate specificities of various UCHs by fluorescently mutated Ub-fusion proteins (Ub{sup F45W}-Xaa) and di-ubiquitin chains (Ub{sup F45W}-diUb). After removal of the intact substrate by Ni{sup 2+}-NTA affinity, the enzymatic activities of UCHs were quantitatively determined by recording fluorescence of the Ub{sup F45W} product. The results show that three UCHs, i.e. UCH-L1, UCH-L3 and UCH37/UCH-L5, are distinct in their substrate specificities for the Ub-fusions and diUb chains. This assay method may also be applied to study the enzymatic activities and substrate specificities of other DUBs.

  14. 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.

  15. 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.

  16. 2 CFR 200.507 - Program-specific audits.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... MANAGEMENT AND BUDGET GUIDANCE Reserved UNIFORM ADMINISTRATIVE REQUIREMENTS, COST PRINCIPLES, AND AUDIT REQUIREMENTS FOR FEDERAL AWARDS Audit Requirements Audits § 200.507 Program-specific audits. (a) Program-specific audit guide available. In many cases, a program-specific audit guide will be available to...

  17. 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

  18. 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. PMID:23800623

  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

    2013-11-01

    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

  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. 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…

  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. 38 CFR 41.235 - Program-specific audits.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...-specific audits. (a) Program-specific audit guide available. In many cases, a program-specific audit guide will be available to provide specific guidance to the auditor with respect to internal control, compliance requirements, suggested audit procedures, and audit reporting requirements. The auditor...

  4. 38 CFR 41.235 - Program-specific audits.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...-specific audits. (a) Program-specific audit guide available. In many cases, a program-specific audit guide will be available to provide specific guidance to the auditor with respect to internal control, compliance requirements, suggested audit procedures, and audit reporting requirements. The auditor...

  5. 38 CFR 41.235 - Program-specific audits.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...-specific audits. (a) Program-specific audit guide available. In many cases, a program-specific audit guide will be available to provide specific guidance to the auditor with respect to internal control, compliance requirements, suggested audit procedures, and audit reporting requirements. The auditor...

  6. 38 CFR 41.235 - Program-specific audits.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...-specific audits. (a) Program-specific audit guide available. In many cases, a program-specific audit guide will be available to provide specific guidance to the auditor with respect to internal control, compliance requirements, suggested audit procedures, and audit reporting requirements. The auditor...

  7. Functional characterization and substrate specificity of spinosyn rhamnosyltransferase by in vitro reconstitution of spinosyn biosynthetic enzymes.

    PubMed

    Chen, Yi-Lin; Chen, Yi-Hsine; Lin, Yu-Chin; Tsai, Kuo-Chung; Chiu, Hsien-Tai

    2009-03-13

    Spinosyn, a potent insecticide, is a novel tetracyclic polyketide decorated with d-forosamine and tri-O-methyl-L-rhamnose. Spinosyn rhamnosyltransferase (SpnG) is a key biocatalyst with unique sequence identity and controls the biosynthetic maturation of spinosyn. The rhamnose is critical for the spinosyn insecticidal activity and cell wall biosynthesis of the spinosyn producer, Saccharopolyspora spinosa. In this study, we have functionally expressed and characterized SpnG and the three enzymes, Gdh, Epi, and Kre, responsible for dTDP-L-rhamnose biosynthesis in S. spinosa by purified enzymes from Escherichia coli. Most notably, the substrate specificity of SpnG was thoroughly characterized by kinetic and inhibition experiments using various NDP sugar analogs made by an in situ combination of NDP-sugar-modifying enzymes. SpnG was found to exhibit striking substrate promiscuity, yielding corresponding glycosylated variants. Moreover, the critical residues presumably involved in catalytic mechanism of Gdh and SpnG were functionally evaluated by site-directed mutagenesis. The information gained from this study has provided important insight into molecular recognition and mechanism of the enzymes, especially SpnG. The results have made possible the structure-activity characterization of SpnG, as well as the use of SpnG or its engineered form to serve as a combinatorial tool to make spinosyn analogs with altered biological activities and potency. PMID:19126547

  8. N-terminal modifications of cellular proteins: The enzymes involved, their substrate specificities and biological effects

    PubMed Central

    Varland, Sylvia; Osberg, Camilla; Arnesen, Thomas

    2015-01-01

    The vast majority of eukaryotic proteins are N-terminally modified by one or more processing enzymes. Enzymes acting on the very first amino acid of a polypeptide include different peptidases, transferases, and ligases. Methionine aminopeptidases excise the initiator methionine leaving the nascent polypeptide with a newly exposed amino acid that may be further modified. N-terminal acetyl-, methyl-, myristoyl-, and palmitoyltransferases may attach an acetyl, methyl, myristoyl, or palmitoyl group, respectively, to the α-amino group of the target protein N-terminus. With the action of ubiquitin ligases, one or several ubiquitin molecules are transferred, and hence, constitute the N-terminal modification. Modifications at protein N-termini represent an important contribution to proteomic diversity and complexity, and are essential for protein regulation and cellular signaling. Consequently, dysregulation of the N-terminal modifying enzymes is implicated in human diseases. We here review the different protein N-terminal modifications occurring co- or post-translationally with emphasis on the responsible enzymes and their substrate specificities. PMID:25914051

  9. 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

  10. 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

    2016-02-01

    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. PMID:26689635

  11. 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.

  12. Switching of Self-Assembly in a Peptide Nanostructure with a Specific Enzyme

    PubMed Central

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

    2012-01-01

    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. PMID:22408645

  13. Threonine aldolases: perspectives in engineering and screening the enzymes with enhanced substrate and stereo specificities.

    PubMed

    Fesko, Kateryna

    2016-03-01

    Threonine aldolases have emerged as a powerful tool for asymmetric carbon-carbon bond formation. These enzymes catalyse the unnatural aldol condensation of different aldehydes and glycine to produce highly valuable β-hydroxy-α-amino acids with complete stereocontrol at the α-carbon and moderate specificity at the β-carbon. A range of microbial threonine aldolases has been recently recombinantly produced by several groups and their biochemical properties were characterized. Numerous studies have been conducted to improve the reaction protocols to enable higher conversions and investigate the substrate scope of enzymes. However, the application of threonine aldolases in organic synthesis is still limited due to often moderate yields and low diastereoselectivities obtained in the aldol reaction. This review briefly summarizes the screening techniques recently applied to discover novel threonine aldolases as well as enzyme engineering and mutagenesis studies which were accomplished to improve the catalytic activity and substrate specificity. Additionally, the results from new investigations on threonine aldolases including crystal structure determinations and structural-functional characterization are reviewed. PMID:26810201

  14. Human fibroblast collagenase: glycosylation and tissue-specific levels of enzyme synthesis.

    PubMed Central

    Wilhelm, S M; Eisen, A Z; Teter, M; Clark, S D; Kronberger, A; Goldberg, G

    1986-01-01

    Human skin fibroblasts secrete collagenase as two proenzyme forms (57 and 52 kDa). The minor (57-kDa) proenzyme form is the result of a partial posttranslational modification of the major (52-kDa) proenzyme through the addition of N-linked complex oligosaccharides. Human endothelial cells as well as fibroblasts from human colon, cornea, gingiva, and lung also secrete collagenase in two forms indistinguishable from those of the skin fibroblast enzyme. In vitro tissue culture studies have shown that the level of constitutive synthesis of this fibroblast-type interstitial collagenase is tissue specific, varies widely, and correlates with the steady-state level of a single collagenase-specific mRNA of 2.5 kilobases. The tumor promoter, phorbol 12-myristate 13-acetate, apparently blocks the control of collagenase synthesis resulting in a similarly high level of collagenase expression (approximately equal to 3-7 micrograms of collagenase per 10(6) cells per 24 hr) in all examined cells. The constitutive level of synthesis of a 28-kDa collagenase inhibitor does not correlate with that of the enzyme. Phorbol 12-myristate 13-acetate stimulates the production of this inhibitor that in turn modulates the activity of collagenase in the conditioned media. As a result, the apparent activity of the enzyme present in the medium does not accurately reflect the rate of its synthesis and secretion. Images PMID:3012533

  15. 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

  16. Adapting Scheduling Programs for Educational Specifications.

    ERIC Educational Resources Information Center

    Glass, Thomas E.

    1984-01-01

    Planners of a new Arizona school used the district's existing computerized master scheduling program to identify instructional spaces needed. Total classroom needs by subject were translated into total square footage requirements. (MLF)

  17. 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

  18. 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... DEVELOPMENT CENTERS § 130.350 Specific program responsibilities. (a) Policy development. SBA will establish Program policies and procedures to improve the delivery of services by SBDCs to the small...

  19. Development of Diubiquitin-Based FRET Probes To Quantify Ubiquitin Linkage Specificity of Deubiquitinating Enzymes.

    PubMed

    Geurink, Paul P; 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; Ovaa, Huib

    2016-05-01

    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

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

    PubMed Central

    Geurink, Paul P.; 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; Ovaa, Huib

    2016-01-01

    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. Frequencies and Specificities of “Enzyme-Only” Detected Erythrocyte Alloantibodies in Patients Hospitalized in Austria: Is an Enzyme Test Required for Routine Red Blood Cell Antibody Screening?

    PubMed Central

    Habres, Claudia; Wallner, Franz; Mayr, Barbara; Halwachs-Baumann, Gabriele

    2014-01-01

    The aim of this study was to determine the frequencies and specificities of “enzyme-only” detected red blood cell (RBC) alloantibodies in the routine antibody screening and antibody identification in patients hospitalized in Austria. Routine blood samples of 2420 patients were investigated. The antibody screening was performed with a 3-cell panel in the low-ionic strength saline- (LISS-) indirect antiglobulin test (IAT) and with an enzyme-pretreated (papain) 3-cell panel fully automated on the ORTHO AutoVue Innova System. The antibody identification was carried out manually with an 11-cell panel in the LISS-IAT and with an enzyme-pretreated (papain) 11-cell panel. In total 4.05% (n = 98) of all patients (n = 2420) had a positive RBC antibody screening result. Of them 25.51% (25/98) showed “enzyme-only” detected specific or nonspecific RBC alloantibodies. Rhesus and Lewis system antibodies were found the only specificities of “enzyme-only” RBC alloantibodies: all in all 4.8% (4/98) were detected with anti-E, 3.06% (3/98) with anti-Lea, 3.06% (3/98) with anti-D after anti-D prophylaxis and 1.02% (1/98) with anti-e. In total, 14.29% (14/98) showed a nonspecific RBC alloantibody result with the enzyme test. The results of the present study demonstrate that a high number of unwanted positive reactions with the enzyme technique overshadows the detection of “enzyme-only” RBC alloantibodies. (Trial Registration: K-37-13). PMID:24790773

  2. [Detection of IgE specific for egg yolk by enzyme immunoassay. Description of a case].

    PubMed

    Carrillo Díaz, T; Cuevas Agustín, M; Moneo Goiri, I; Ibáñez Sandín, M D; Ureña Vilardell, V

    1986-01-01

    Food allergy is a common disease in our country, especially affecting atopic children. Egg-white hypersensitivity is frequently found in these patients. However, egg-yolk hypersensitivity is not usually reported in patients with egg allergy. This article describes a young patient with egg-yolk hypersensitivity, a 12 year old female patient with a medical history of contact urticaria, angioedema and severe acute bronchospasm shortly after the intake of small amounts of egg-yolk. All these episodes required treatment in emergency care units because of the severity of the symptoms. The patient did not describe any other food hypersensitivity and remained symptom-free after the intake of boiled or fried egg-white. She had clinical symptoms of grass pollen hypersensitivity and was therefore on specific immunotherapy at the time of the study. The skin prick-tests were positive to grass pollen and egg-yolk and were negative to mites, moulds, animal dander and to the common food tested (milk, fish, peanut, almond and hazel-nut). Total serum IgE was 1.160 UL/ml. The patient had a positive RAST to egg-white (0.0 PRU/ml) as well as to egg-yolk (8.6 PRU/ml). Furthermore, an indirect enzyme immunoassay as well as a reverse enzyme immunoassay also revealed the presence of specific IgE antibodies. The reverse enzyme immunoassay uses microtiter plates as a solid surface. These plates are coated with a monospecific antihuman IgE antibody. Thereafter, the serum samples are incubated overnight in the wells. After several washings, the presence of specific antibodies is revealed by means of a peroxidase conjugated allergen.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3515886

  3. An enzyme combination assay for serum sphingomyelin: Improved specificity through avoiding the interference with lysophosphatidylcholine.

    PubMed

    Kimura, Takehide; Kuwata, Hideyuki; Miyauchi, Kazuhito; Katayama, Yuki; Kayahara, Norihiko; Sugiuchi, Hiroyuki; Matsushima, Kazumi; Kondo, Yuki; Ishitsuka, Yoichi; Irikura, Mitsuru; Irie, Tetsumi

    2016-04-01

    Serum sphingomyelin (SM) has predictive value in the development of atherosclerosis. Furthermore, SM plays important roles in cell membrane structure, signal transduction pathways, and lipid raft formation. A convenient enzymatic method for SM is available for routine laboratory practice, but the enzyme specificity is not sufficient because of nonspecific reactions with lysophosphatidylcholine (LPC). Based on the differential specificity of selected enzymes toward choline-containing phospholipids, a two-step assay for measuring SM was constructed and its performance was evaluated using sera from healthy individuals on a Hitachi 7170 autoanalyzer. Results from this assay were highly correlated with theoretical serum SM concentrations estimated by subtracting phosphatidylcholine (PC) and LPC concentrations from that of total phospholipids determined using previously established methods. There was a good correlation between the results of SM assayed by the proposed method and the existing enzymatic method in sera from healthy individuals. Moreover, the proposed method was superior to the existing method in preventing nonspecific reactions with LPC present in sera. The proposed method does not require any pretreatment, uses 2.5 μl of serum samples, and requires only 10 min on an autoanalyzer. This high-throughput method can measure serum SM with sufficient specificity for clinical purposes and is applicable in routine laboratory practice. PMID:26792376

  4. 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.

  5. 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

    2016-06-01

    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. PMID:27174700

  6. 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.

  7. Rethinking Assessment in an Indigenous Specific Program

    ERIC Educational Resources Information Center

    Fleet, Alma; Kitson, Rosalind

    2009-01-01

    Nonstandard entry programs into higher education include worthy goals and problematic processes. Although effective practices in teacher education would seem to be well established, complications arise when good intentions intersect with university protocols, issues of power, history, rights, and cultural complexities. This article reports on an…

  8. A novel hatching enzyme from starfish Asterias amurensis: purification, characterization, and cleavage specificity.

    PubMed

    Li, Zhi Jiang; Kim, Sang Moo

    2013-02-01

    Hatching enzyme (HE) is of importance to degrade egg membrane to let the larvae be free. HE was purified and characterized from starfish blastula. The specific activity and the purification ratio of the purified HE with 110.9 kDa of molecular weight were 449.62 U/mg and 7.42-fold, respectively. Its optimal pH and temperature for activity were pH 8.0 and 30 °C, respectively. This enzyme was relatively stable in the range of pH 4.0-6.0 and 30-40 °C. This enzyme was inhibited by ethylene diamine tetraacetic acid (EDTA) and ethylene glycol tetraacetic acid, and also done moderately by Leupeptin, tosyl-lysine chloromethyl ketone, tosyl-phenylalanine chloromethyl ketone, and phenyl-methanesulfonyl fluoride. Zn(2+) ion activated HE activity strongly and recovered the EDTA-pretreated activity more than did Ca(2+), Mg(2+), and Cu(2+). Based on the results above, the starfish HE was classified as a zinc metallo- and trypsin-like serine protease. The values of Km, Vmax, and Kcat of the starfish HE on dimethyl casein were 0.31 mg/ml, 0.17 U/ml, and 122.70 s(-1), respectively, whereas 1.09 mg/ml, 0.12 U/ml, and 771.98 s(-1) on type I collagen. Therefore, the starfish HE could be a potential cosmeceutical because of its strong cleavage specificity on type I collagen. PMID:23306897

  9. Sinapis phylogeny and evolution of glucosinolates and specific nitrile degrading enzymes.

    PubMed

    Agerbirk, Niels; Warwick, Suzanne I; Hansen, Paul R; Olsen, Carl E

    2008-12-01

    Levels of sinalbin (4-hydroxybenzylglucosinolate) and 28 other glucosinolates were determined in leaves and roots of 20 species that were either phylogenetically close to Sinapis alba, Sinapis arvensis, or Sinapis pubescens (tribe Brassiceae, Brassicaceae), or were expected to contain arylalkyl nitrilase activity. Comparison with a molecular phylogenetic tree based on ITS DNA sequences identified two separate occurrences of sinalbin. The first in a group of species related to S. alba (including members of the genera Coincya and Kremeriella); and the second in S. arvensis, nested among sinalbin deficient species. Significant 4-hydroxyphenylacetonitrile degrading enzyme activity was found in both S. alba and S. arvensis, but in S. alba the major product was the corresponding carboxylic acid, while in S. arvensis the major product was the amide. Both investigated enzyme activities, nitrilase and nitrile hydratase, were specific, accepting only certain arylacetonitriles such as 4-hydroxy and 4-methoxyphenylacetonitrile. Only the S. alba enzyme required an oxygen in para position of the substrate, as found in sinalbin. Indole-3-acetonitrile, arylcyanides, and arylpropionitriles were poor substrates. The nitrilase activity of S. alba was quantitatively comparable to that reported in the monocot Sorghum bicolor (believed to be involved in cyanogenic glycoside metabolism). Glucosinolates derived from methionine were found in all Sinapis clades. Glucosinolate patterns suggested a complex evolution of glucosinolates in the investigated species, with several apparent examples of abrupt changes in glucosinolate profiles including chain length variation and appearance of glucosinolates derived from branched-chain amino acids. NMR data for desulfated homosinalbin, 9-methylsulphonylnonylglucosinolate, 3-methylpentylglucosinolate and related glucosinolates are reported, and a facultative connection between sinalbin and specific nitrilases is suggested. PMID:18995873

  10. 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. PMID:25844554

  11. Substrate and Enzyme Specificity of the Kinetic Isotope Effects Associated with the Dioxygenation of Nitroaromatic Contaminants.

    PubMed

    Pati, Sarah G; Kohler, Hans-Peter E; Pabis, Anna; Paneth, Piotr; Parales, Rebecca E; Hofstetter, Thomas B

    2016-07-01

    Compound-specific isotope analysis (CSIA) is a promising approach for tracking biotransformation of organic pollutants, but isotope fractionation associated with aromatic oxygenations is only poorly understood. We investigated the dioxygenation of a series of nitroaromatic compounds to the corresponding catechols by two enzymes, namely, nitrobenzene and 2-nitrotoluene dioxygenase (NBDO and 2NTDO) to elucidate the enzyme- and substrate-specificity of C and H isotope fractionation. While the apparent (13)C- and (2)H-kinetic isotope effects of nitrobenzene, nitrotoluene isomers, 2,6-dinitrotoluene, and naphthalene dioxygenation by NBDO varied considerably, the correlation of C and H isotope fractionation revealed a common mechanism for nitrobenzene and nitrotoluenes. Similar observations were made for the dioxygenation of these substrates by 2NTDO. Evaluation of reaction kinetics, isotope effects, and commitment-to-catalysis based on experiment and theory showed that rates of dioxygenation are determined by the enzymatic O2 activation and aromatic C oxygenation. The contribution of enzymatic O2 activation to the reaction rate varies for different nitroaromatic substrates of NBDO and 2NTDO. Because aromatic dioxygenation by nonheme iron dioxygenases is frequently the initial step of biodegradation, O2 activation kinetics may also have been responsible for the minor isotope fractionation reported for the oxygenation of other aromatic contaminants. PMID:26895026

  12. 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.

  13. INAKT--an interactive non-linear regression program for enzyme inactivation and affinity labelling studies.

    PubMed

    Christophersen, A; McKinley-McKee, J S

    1984-01-01

    An interactive program for analysing enzyme activity-time data using non-linear regression analysis is described. Protection studies can also be dealt with. The program computes inactivation rates, dissociation constants and promotion or inhibition parameters with their standard errors. It can also be used to distinguish different inactivation models. The program is written in SIMULA and is menu-oriented for refining or correcting data at the different levels of computing. PMID:6546558

  14. Nicotinamide Adenine Dinucleotide-specific "Malic" Enzyme in Kalanchoë daigremontiana and Other Plants Exhibiting Crassulacean Acid Metabolism.

    PubMed

    Dittrich, P

    1976-02-01

    NAD-specific "malic" enzyme (EC 1.1.1.39) has been isolated and purified 1200-fold from leaves of Kalanchoë daigremontiana. Kinetic studies of this enzyme, which is activated 14-fold by CoA, acetyl-CoA, and SO(4) (2-), suggest allosteric properties. Cofactor requirements show an absolute specificity for NAD and for Mn(2+), which cannot be replaced by NADP or Mg(2+). For maintaining enzyme activity in crude leaf extracts a thiol reagent, Mn(2+), and PVP-40 were required. The latter could be omitted from purified preparations. By sucrose density gradient centrifugation NAD-malic enzyme could be localized in mitochondria. A survey of plants with crassulacean acid metabolism revealed the presence of NAD-malic enzyme in all 31 plants tested. Substantial levels of this enzyme (121-186 mumole/hr.mg of Chl) were detected in all members tested of the family Crassulaceae. It is proposed that NAD-malic enzyme in general supplements activity of NADP-malic enzyme present in these plants and may be specifically employed to increase internal concentrations of CO(2) for recycling during cessation of gas exchange in periods of severe drought. PMID:16659473

  15. 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…

  16. 47 CFR 76.75 - Specific EEO program requirements.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 4 2011-10-01 2011-10-01 false Specific EEO program requirements. 76.75 Section 76.75 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) BROADCAST RADIO SERVICES MULTICHANNEL VIDEO AND CABLE TELEVISION SERVICE Equal Employment Opportunity Requirements § 76.75 Specific EEO program requirements. Under the terms...

  17. A Condensing Enzyme from the Seeds of Lesquerella fendleri That Specifically Elongates Hydroxy Fatty Acids1

    PubMed Central

    Moon, Hangsik; Smith, Mark A.; Kunst, Ljerka

    2001-01-01

    Lesquerella fendleri seed oil contains up to 60% hydroxy fatty acids, nearly all of which is the 20-carbon hydroxy fatty acid lesquerolic acid (d-14-hydroxyeicos-cis-11-enoic acid). Previous work suggested that lesquerolic acid in L. fendleri was formed by the elongation of the 18-carbon hydroxy fatty acid, ricinoleic acid. To identify a gene encoding the enzyme involved in hydroxy fatty acid elongation, an L. fendleri genomic DNA library was screened using the coding region of the Arabidopsis Fatty Acid Elongation1 gene as a probe. A gene, LfKCS3, with a high sequence similarity to known very long-chain fatty acid condensing enzymes, was isolated. LfKCS3 has a 2,062-bp open reading frame interrupted by two introns, which encodes a polypeptide of 496 amino acids. LfKCS3 transcripts accumulated only in the embryos of L. fendleri and first appeared in the early stages of development. Fusion of the LfKCS3 promoter to the uidA reporter gene and expression in transgenic Arabidopsis resulted in a high level of β-glucuronidase activity exclusively in developing embryos. Seeds of Arabidopsis plants transformed with LfKCS3 showed no change in their very long-chain fatty acid content. However, when these Arabidopsis plants were crossed with the transgenic plants expressing the castor oleate 12-hydroxylase, significant amounts of 20-carbon hydroxy fatty acids accumulated in the seed, indicating that the LfKCS3 condensing enzyme specifically catalyzes elongation of 18-carbon hydroxy fatty acids. PMID:11743108

  18. 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. PMID:24911589

  19. Differences in the Glucuronidation of Resveratrol and Pterostilbene: Altered Enzyme Specificity and Potential Gender Differences

    PubMed Central

    Dellinger, Ryan W.; Gomez Garcia, Angela M.; Meyskens, Frank L.

    2015-01-01

    Summary Resveratrol, a natural polyphenol found in grapes, berries and other plants, has been proposed as an ideal chemopreventative agent due to its plethora of health promoting activities. However, despite its lofty promise as a cancer prevention agent its success in human clinical trials has been limited due to its poor bioavailability. Thus, interest in other natural polyphenols is intensifying including the naturally occurring dimethylated analog of resveratrol, pterostilbene. The UDP-glucuronosyltransferase (UGT) family of enzymes plays a vital role in the metabolism of both resveratrol and pterostilbene. The current study sought to elucidate the UGT family members responsible for the metabolism of pterostilbene and to examine gender differences in the glucuronidation of resveratrol and pterostilbene. We demonstrate that UGT1A1 and UGT1A3 are mainly responsible for pterostilbene glucuronidation although UGT1A8, UGT1A9 and UGT1A10 also had detectable activity. Intriguingly, UGT1A1 exhibits the highest activity against both resveratrol and pterostilbene despite altered hydroxyl group specificity. Using pooled human liver microsomes, enzyme kinetics were determined for pterostilbene and resveratrol glucuronides. In all cases females were more efficient than males, indicating potential gender differences in stilbene metabolism. Importantly, the glucuronidation of pterostilbene is much less efficient than that of resveratrol, indicating that pterostilbene will have dramatically decreased metabolism in humans. PMID:23965644

  20. An essential ubiquitin-conjugating enzyme with tissue and developmental specificity in th nematode Caenorhabditis elegans.

    PubMed Central

    Zhen, M; Schein, J E; Baillie, D L; Candido, E P

    1996-01-01

    The ubc-2 gene in Caenorhabditis elegans encodes a ubiquitin-conjugating enzyme (E2) homologous to yeast UBC4 and UBC5. UBC4 and UBC5 are individually dispensable class I E2 enzymes involved in the degradation of short-lived and abnormal proteins. Transgenic analysis using ubc-2-lacZ fusions and in situ immunofluorescence indicate that ubc-2 is abundantly expressed in most tissues of embryos and early larvae, but becomes specific to the nervous system in L4 larvae and adults. This suggests that the functions of this type of E2 are developmentally regulated in C.elegans. This hypothesis is supported by antisense analysis, which shows that blocking the expression of ubc-2 has a more severe effect in early developmental stages than in later stages. Through complementation of previously identified essential genes in the vicinity of ubc-2, we demonstrate that ubc-2 corresponds to let-70, a gene essential for C.elegans larval development. One let-70(ubc-2) allele contains a His75-->Tyr substitution, while another has an altered splice donor site. Images PMID:8670823

  1. A sensitive and specific two-site enzyme-immunoassay for human calcitonin using monoclonal antibodies.

    PubMed

    Seth, R; Motté, P; Kehely, A; Wimalawansa, S J; Self, C H; Bellet, D; Bohuon, C; MacIntyre, I

    1988-11-01

    A highly sensitive, specific and rapid two-site enzyme-immunometric assay (EIA) for the measurement of immunoreactive (ir) human calcitonin (hCT) in human plasma was developed using high-affinity monoclonal antibodies. The assay was validated in terms of sensitivity, specificity and reproducibility and its performance compared with that of a radioimmunoassay (RIA) employing a polyclonal antiserum. The sensitivity of the overnight EIA (2 pmol/l) was comparable with the long-incubation (7 days) RIA. The overnight RIA had a sensitivity of 10 pmol/l. The inter- and intra-assay variations of the EIA were less than 12%. Some related and non-related peptides were compared with synthetic hCT for cross-reactivity in the assay and were found to be negative. The mean recovery of added synthetic hCT from plasma of normal volunteers was 96%. Both RIA and EIA have been applied to the measurement of ir-hCT in normal volunteers and in patients with medullary carcinoma of the thyroid. In both groups, the level of ir-hCT measured by EIA was found to be lower than that measured by RIA, presumably due to the ability of the more specific EIA to detect only the 'mature' form of the hormone. EIA offers an attractive alternative to the more cumbersome and lengthy RIA in current usage, with the added advantage of employing a non-isotopic label. PMID:3058855

  2. 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…

  3. 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.

  4. [Value of prostate-specific antigen measurements with newly developed enzyme immunoassay (MARKIT-M PA)].

    PubMed

    Arai, Y; Onishi, H; Oishi, K; Takeuchi, H; Yoshida, O

    1992-10-01

    Serum prostate-specific antigen (PSA) levels in patients with prostate cancer and benign prostate hypertrophy (BPH) were investigated with a newly developed enzyme immunoassay (MARKIT-M PA, Dainippon Pharmaceutical Co. Ltd., Osaka, Japan). Sensitivity of the assay system is 0.5 ng/ml and the detection range is 0.5-100 ng/ml. There was a high linear correlation (r = 0.987) between the assay and MARKIT-F PA, and values obtained with the assay were almost equal to those yielded by MARKIT-F PA assay. Using the BPH group as a negative control, the upper cut-off value in BPH patients was determined to be 3.6 ng/ml. Of the 48 patients with untreated prostate cancer, 77% was detectable by means of MARKIT-M PA assay. Using the BPH group as a negative control, specificity and efficiency were 93% and 86%, respectively. In another group of 27 BPH patients whose blood samples were taken immediately after digital prostatic examination, PSA was elevated in 15%. During follow-up of prostate cancer patients, PSA was elevated in 82% at the time of clinically detectable progression. In 15 patients whose disease was clinically well controlled, all levels of PSA were observed to be negative. These findings suggests that detection of serum PSA with this assay is of great use both in the diagnosis and monitoring of prostate cancer patients. PMID:1282772

  5. Role of enzyme-peptide substrate backbone hydrogen bonding in determining protein kinase substrate specificities.

    PubMed

    Thomas, N E; Bramson, H N; Miller, W T; Kaiser, E T

    1987-07-14

    As part of a search for peptides that have specificity for selected protein kinases, the possibility that adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) recognizes the hydrogen-bonding potential of its peptide substrates was investigated. A-Kinase catalyzes the phosphorylation of five N alpha-methylated and four depsipeptide derivatives of Leu-Arg-Arg-Ala-Ser-Leu-Gly (peptide 1) at rates that differ by at least 7 orders of magnitude. These peptide 1 analogues each lack the ability to donate a hydrogen bond at selected positions in the peptide chain. If a particular amide hydrogen of a peptide amide is involved in hydrogen bonding, which is important for enzyme recognition, the prediction is that peptides which contain an ester or a N-methylated bond at that position in peptide 1 will be comparatively poor substrates. In contrast, if a depsipeptide has a reactivity comparable to that of peptide 1 but the analogous N-methylated peptide has a poor reactivity with A-kinase, the result might indicate that the N-methyl group causes unfavorable steric effects. The depsipeptide that lacks a Leu6 amide proton is a good substrate for A-kinase, but the corresponding N-methylated peptide is phosphorylated far less efficiently. This result and others presented in this paper suggest that although enzyme-substrate hydrogen bonding may play some role in A-kinase catalysis of phosphoryl group transfer, other explanations are necessary to account for the relative reactivities of N alpha-methylated and depsi-containing peptide 1 analogues.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3663600

  6. Message based event specification for debugging nondeterministic parallel programs

    SciTech Connect

    Damohdaran-Kamal, S.K.; Francioni, J.M.

    1995-02-01

    Portability and reliability of parallel programs can be severely impaired by their nondeterministic behavior. Therefore, an effective means to precisely and accurately specify unacceptable nondeterministic behavior is necessary for testing and debugging parallel programs. In this paper we describe a class of expressions, called Message Expressions that can be used to specify nondeterministic behavior of message passing parallel programs. Specification of program behavior with Message Expressions is easier than pattern based specification techniques in that the former does not require knowledge of run-time event order, whereas that later depends on the user`s knowledge of the run-time event order for correct specification. We also discuss our adaptation of Message Expressions for use in a dynamic distributed testing and debugging tool, called mdb, for programs written for PVM (Parallel Virtual Machine).

  7. 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... management specifications, the following procedure applies: (1) The Flight Standards District Office that... proposed amendment. (2) The Flight Standards District Office that issued the program manager's...

  8. 47 CFR 76.75 - Specific EEO program requirements.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... MULTICHANNEL VIDEO AND CABLE TELEVISION SERVICE Equal Employment Opportunity Requirements § 76.75 Specific EEO... necessary. Nothing in this section shall be interpreted to require a multichannel video programming...) In addition to using such recruitment sources, a multichannel video programming...

  9. 47 CFR 76.75 - Specific EEO program requirements.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... MULTICHANNEL VIDEO AND CABLE TELEVISION SERVICE Equal Employment Opportunity Requirements § 76.75 Specific EEO... necessary. Nothing in this section shall be interpreted to require a multichannel video programming...) In addition to using such recruitment sources, a multichannel video programming...

  10. 47 CFR 76.75 - Specific EEO program requirements.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... MULTICHANNEL VIDEO AND CABLE TELEVISION SERVICE Equal Employment Opportunity Requirements § 76.75 Specific EEO... necessary. Nothing in this section shall be interpreted to require a multichannel video programming...) In addition to using such recruitment sources, a multichannel video programming...

  11. 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.

  12. 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.

  13. Glucose-Specific Enzyme IIA Has Unique Binding Partners in The Vibrio cholerae Biofilm

    PubMed Central

    Pickering, Bradley S.; Smith, Daniel R.; Watnick, Paula I.

    2012-01-01

    ABSTRACT Glucose-specific enzyme IIA (EIIAGlc) is a central regulator of bacterial metabolism and an intermediate in the phosphoenolpyruvate phosphotransferase system (PTS), a conserved phosphotransfer cascade that controls carbohydrate transport. We previously reported that EIIAGlc activates transcription of the genes required for Vibrio cholerae biofilm formation. While EIIAGlc modulates the function of many proteins through a direct interaction, none of the known regulatory binding partners of EIIAGlc activates biofilm formation. Therefore, we used tandem affinity purification (TAP) to compare binding partners of EIIAGlc in both planktonic and biofilm cells. A surprising number of novel EIIAGlc binding partners were identified predominantly under one condition or the other. Studies of planktonic cells revealed established partners of EIIAGlc, such as adenylate cyclase and glycerol kinase. In biofilms, MshH, a homolog of Escherichia coli CsrD, was found to be a dominant binding partner of EIIAGlc. Further studies revealed that MshH inhibits biofilm formation. This function was independent of the Carbon storage regulator (Csr) pathway and dependent on EIIAGlc. To explore the existence of multiprotein complexes centered on EIIAGlc, we also affinity purified the binding partners of adenylate cyclase from biofilm cells. In addition to EIIAGlc, this analysis yielded many of the same proteins that copurified with EIIAGlc. We hypothesize that EIIAGlc serves as a hub for multiprotein complexes and furthermore that these complexes may provide a mechanism for competitive and cooperative interactions between binding partners. PMID:23131828

  14. Divergence in Ubiquitin Interaction and Catalysis among the Ubiquitin-Specific Protease Family Deubiquitinating Enzymes.

    PubMed

    Tencer, Adam H; Liang, Qin; Zhuang, Zhihao

    2016-08-23

    Deubiquitinating enzymes (DUBs) are responsible for reversing mono- and polyubiquitination of proteins and play essential roles in numerous cellular processes. Close to 100 human DUBs have been identified and are classified into five families, with the ubiquitin-specific protease (USP) family being the largest (>50 members). The binding of ubiquitin (Ub) to USP is strikingly different from that observed for the DUBs in the ubiquitin C-terminal hydrolase (UCH) and ovarian tumor domain protease (OTU) families. We generated a panel of mutant ubiquitins and used them to probe the ubiquitin's interaction with a number of USPs. Our results revealed a remarkable divergence of USP-Ub interactions among the USP catalytic domains. Our double-mutant cycle analysis targeting the ubiquitin residues located in the tip, the central body, and the tail of ubiquitin also demonstrated different crosstalk among the USP-Ub interactions. This work uncovered intriguing divergence in the ubiquitin-binding mode in the USP family DUBs and raised the possibility of targeting the ubiquitin-binding hot spots on USPs for selective inhibition of USPs by small molecule antagonists. PMID:27501351

  15. Polyol specificity of recombinant Arabidopsis thaliana sorbitol dehydrogenase studied by enzyme kinetics and in silico modeling

    PubMed Central

    Aguayo, M. Francisca; Cáceres, Juan Carlos; Fuentealba, Matías; Muñoz, Rodrigo; Stange, Claudia; Cabrera, Ricardo; Handford, Michael

    2015-01-01

    Polyols are enzymatically-produced plant compounds which can act as compatible solutes during periods of abiotic stress. Nicotinamide adenine dinucleotide+-dependent SORBITOL DEHYDROGENASE (SDH, E. C. 1.1.1.14) from Arabidopsis thaliana L. sorbitol dehydrogenase (AtSDH) is capable of oxidizing several polyols including sorbitol, ribitol, and xylitol. In the present study, enzymatic assays using recombinant AtSDH demonstrated a higher specificity constant for xylitol compared to sorbitol and ribitol, all of which are C2 (S) and C4 (R) polyols. Enzyme activity was reduced by preincubation with ethylenediaminetetraacetic acid, indicating a requirement for zinc ions. In humans, it has been proposed that sorbitol becomes part of a pentahedric coordination sphere of the catalytic zinc during the reaction mechanism. In order to determine the validity of this pentahedric coordination model in a plant SDH, homology modeling, and Molecular Dynamics simulations of AtSDH ternary complexes with the three polyols were performed using crystal structures of human and Bemisia argentifolii (Genn.) (Hemiptera: Aleyrodidae) SDHs as scaffolds. The results indicate that the differences in interaction with structural water molecules correlate very well with the observed enzymatic parameters, validate the proposed pentahedric coordination of the catalytic zinc ion in a plant SDH, and provide an explanation for why AtSDH shows a preference for polyols with a chirality of C2 (S) and C4 (R). PMID:25755662

  16. Development of an enzyme-linked immunosorbent assay specific to Sudan red I.

    PubMed

    Xu, Ting; Wei, Ke Yi; Wang, Jia; Eremin, Sergei A; Liu, Shang Zhong; Li, Qing X; Li, Ji

    2010-10-01

    To obtain antibodies to develop an enzyme-linked immunosorbent assay (ELISA) for the analysis of Sudan red I, haptens were designed and synthesized via four different strategies: (i) attachment of a spacer at the para position of the benzene ring, (ii) attachment of a spacer at the naphthol part, (iii) attachment of a spacer at the hydroxyl group of the Sudan red I molecule, and (iv) use of a fragment of the target molecule. A total of 10 haptens were used to generate immunogens, coating antigens, and polyclonal antibodies. One of the heterologous ELISAs developed exhibited an IC(50) of 1.6 ng/ml, a limit of detection (LOD) of 0.03 ng/ml, and a dynamic range between 0.1 and 14 ng/ml. The assay had 13% cross-reactivity with Para red and negligible cross-reactivity with other structure-related compounds. This ELISA was much more specific than those published previously. This assay was used to determine Sudan red I residues in tomato sauce and chili powder samples after simple pretreatment. The results were validated by comparison with high-performance liquid chromatography (HPLC). The average recoveries of Sudan red I by ELISA and HPLC were in ranges of 70-97% and 82-114%, respectively, indicating suitability of the developed ELISA for screening of Sudan red I in foods. PMID:20522332

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

    PubMed

    Alcorn, S W; Pascho, R J

    2000-05-01

    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 Renibacterium 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. PMID:10826838

  18. A novel pH–enzyme-dependent mesalamine colon-specific delivery system

    PubMed Central

    Jin, Lei; Ding, Yi-cun; Zhang, Yu; Xu, Xiao-qing; Cao, Qin

    2016-01-01

    The aim of the present study was to design a new pH–enzyme double-dependent mesalamine colon-specific delivery system. The drug release behaviors in vitro and pharmacokinetics and biodistribution in vivo were further evaluated. The mean particle diameters of mesalamine-coated microparticles were 312.2 µm. In vitro, a small amount of mesalamine was released in HCl at a pH of 1.2 and PBS medium at a pH of 7.4 for 5 hours, and 71% of the entrapped mesalamine was further released during the subsequent 20 hours of incubation. A greater area under the plasma concentration–time curve (AUC)0–t was obtained for the coated microparticles (1.9-fold) compared to the suspensions group, which indicated that the encapsulated mesalamine had mostly been absorbed in rats over the period of 12 hours. The AUC0–t of the coated microparticles in colon was 2.63-fold higher compared to the suspensions (P<0.05). Hence, mesalamine-coated microparticles are considered to maintain the drug concentration within target ranges for a long period of time. PMID:27382255

  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. 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

  1. Specific starch digestion of maize alpha-limit dextrins by recombinant mucosal glucosidase enzymes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Starch digestion requires two luminal enzymes, salivary and pancreatic alpha-amylase (AMY), and four small intestinal mucosal enzyme activities from the N- and C-terminals of maltase-glucoamylase (MGAM) and sucrose-isomaltase (SI) complexes. AMY is not a requirement for starch digestion to glucose b...

  2. 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.

  3. Compartment-specific expression of collagens and their processing enzymes in intrapulmonary arteries of IPAH patients

    PubMed Central

    Hoffmann, Julia; Marsh, Leigh M.; Pieper, Mario; Stacher, Elvira; Ghanim, Bahil; Kovacs, Gabor; König, Peter; Wilkens, Heinrike; Haitchi, Hans Michael; Hoefler, Gerald; Klepetko, Walter; Olschewski, Horst; Olschewski, Andrea

    2015-01-01

    Alterations in extracellular matrix (ECM) have been implicated in the pathophysiology of pulmonary hypertension. Here, we have undertaken a compartment-specific study to elucidate the expression profile of collagens and their processing enzymes in donor and idiopathic pulmonary arterial hypertension (IPAH) pulmonary arteries. Predominant intimal, but also medial and perivascular, remodeling and reduced lumen diameter were detected in IPAH pulmonary arteries. Two-photon microscopy demonstrated accumulation of collagen fibers. Quantification of collagen in pulmonary arteries revealed collagen accumulation mainly in the intima of IPAH pulmonary arteries compared with donors. Laser capture-microdissected pulmonary artery profiles (intima+media and perivascular tissue) were analyzed by real-time PCR for ECM gene expression. In the intima+media of IPAH vessels, collagens (COL4A5, COL14A1, and COL18A1), matrix metalloproteinase (MMP) 19, and a disintegrin and metalloprotease (ADAM) 33 were higher expressed, whereas MMP10, ADAM17, TIMP1, and TIMP3 were less abundant. Localization of COLXVIII, its cleavage product endostatin, and MMP10, ADAM33, and TIMP1 was confirmed in pulmonary arteries by immunohistochemistry. ELISA for collagen XVIII/endostatin demonstrated significantly elevated plasma levels in IPAH patients compared with donors, whereas circulating MMP10, ADAM33, and TIMP1 levels were similar between the two groups. Endostatin levels were correlated with pulmonary arterial wedge pressure, and established prognostic markers of IPAH, right atrial pressure, cardiac index, 6-min walking distance, NH2-terminal pro-brain natriuretic peptide, and uric acid. Expression of unstudied collagens, MMPs, ADAMs, and TIMPs were found to be significantly altered in IPAH intima+media. Elevated levels of circulating collagen XVIII/endostatin are associated with markers of a poor prognosis. PMID:25840998

  4. Restricted Substrate Specificity for the Geranylgeranyltransferase-I Enzyme in Cryptococcus neoformans: Implications for Virulence

    PubMed Central

    Selvig, Kyla; Ballou, Elizabeth R.; Nichols, Connie B.

    2013-01-01

    Proper cellular localization is required for the function of many proteins. The CaaX prenyltransferases (where CaaX indicates a cysteine followed by two aliphatic amino acids and a variable amino acid) direct the subcellular localization of a large group of proteins by catalyzing the attachment of hydrophobic isoprenoid moieties onto C-terminal CaaX motifs, thus facilitating membrane association. This group of enzymes includes farnesyltransferase (Ftase) and geranylgeranyltransferase-I (Ggtase-1). Classically, the variable (X) amino acid determines whether a protein will be an Ftase or Ggtase-I substrate, with Ggtase-I substrates often containing CaaL motifs. In this study, we identify the gene encoding the β subunit of Ggtase-I (CDC43) and demonstrate that Ggtase-mediated activity is not essential. However, Cryptococcus neoformans CDC43 is important for thermotolerance, morphogenesis, and virulence. We find that Ggtase-I function is required for full membrane localization of Rho10 and the two Cdc42 paralogs (Cdc42 and Cdc420). Interestingly, the related Rac and Ras proteins are not mislocalized in the cdc43Δ mutant even though they contain similar CaaL motifs. Additionally, the membrane localization of each of these GTPases is dependent on the prenylation of the CaaX cysteine. These results indicate that C. neoformans CaaX prenyltransferases may recognize their substrates in a unique manner from existing models of prenyltransferase specificity. It also suggests that the C. neoformans Ftase, which has been shown to be more important for C. neoformans proliferation and viability, may be the primary prenyltransferase for proteins that are typically geranylgeranylated in other species. PMID:24014765

  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. Compartment-specific expression of collagens and their processing enzymes in intrapulmonary arteries of IPAH patients.

    PubMed

    Hoffmann, Julia; Marsh, Leigh M; Pieper, Mario; Stacher, Elvira; Ghanim, Bahil; Kovacs, Gabor; König, Peter; Wilkens, Heinrike; Haitchi, Hans Michael; Hoefler, Gerald; Klepetko, Walter; Olschewski, Horst; Olschewski, Andrea; Kwapiszewska, Grazyna

    2015-05-15

    Alterations in extracellular matrix (ECM) have been implicated in the pathophysiology of pulmonary hypertension. Here, we have undertaken a compartment-specific study to elucidate the expression profile of collagens and their processing enzymes in donor and idiopathic pulmonary arterial hypertension (IPAH) pulmonary arteries. Predominant intimal, but also medial and perivascular, remodeling and reduced lumen diameter were detected in IPAH pulmonary arteries. Two-photon microscopy demonstrated accumulation of collagen fibers. Quantification of collagen in pulmonary arteries revealed collagen accumulation mainly in the intima of IPAH pulmonary arteries compared with donors. Laser capture-microdissected pulmonary artery profiles (intima+media and perivascular tissue) were analyzed by real-time PCR for ECM gene expression. In the intima+media of IPAH vessels, collagens (COL4A5, COL14A1, and COL18A1), matrix metalloproteinase (MMP) 19, and a disintegrin and metalloprotease (ADAM) 33 were higher expressed, whereas MMP10, ADAM17, TIMP1, and TIMP3 were less abundant. Localization of COLXVIII, its cleavage product endostatin, and MMP10, ADAM33, and TIMP1 was confirmed in pulmonary arteries by immunohistochemistry. ELISA for collagen XVIII/endostatin demonstrated significantly elevated plasma levels in IPAH patients compared with donors, whereas circulating MMP10, ADAM33, and TIMP1 levels were similar between the two groups. Endostatin levels were correlated with pulmonary arterial wedge pressure, and established prognostic markers of IPAH, right atrial pressure, cardiac index, 6-min walking distance, NH2-terminal pro-brain natriuretic peptide, and uric acid. Expression of unstudied collagens, MMPs, ADAMs, and TIMPs were found to be significantly altered in IPAH intima+media. Elevated levels of circulating collagen XVIII/endostatin are associated with markers of a poor prognosis. PMID:25840998

  7. Virus-specific mRNA capping enzyme encoded by hepatitis E virus.

    PubMed

    Magden, J; Takeda, N; Li, T; Auvinen, P; Ahola, T; Miyamura, T; Merits, A; Kääriäinen, L

    2001-07-01

    Hepatitis E virus (HEV), a positive-strand RNA virus, is an important causative agent of waterborne hepatitis. Expression of cDNA (encoding amino acids 1 to 979 of HEV nonstructural open reading frame 1) in insect cells resulted in synthesis of a 110-kDa protein (P110), a fraction of which was proteolytically processed to an 80-kDa protein. P110 was tightly bound to cytoplasmic membranes, from which it could be released by detergents. Immunopurified P110 catalyzed transfer of a methyl group from S-adenosylmethionine (AdoMet) to GTP and GDP to yield m(7)GTP or m(7)GDP. GMP, GpppG, and GpppA were poor substrates for the P110 methyltransferase. There was no evidence for further methylation of m(7)GTP when it was used as a substrate for the methyltransferase. P110 was also a guanylyltransferase, which formed a covalent complex, P110-m(7)GMP, in the presence of AdoMet and GTP, because radioactivity from both [alpha-(32)P]GTP and [(3)H-methyl]AdoMet was found in the covalent guanylate complex. Since both methyltransferase and guanylyltransferase reactions are strictly virus specific, they should offer optimal targets for development of antiviral drugs. Cap analogs such as m(7)GTP, m(7)GDP, et(2)m(7)GMP, and m(2)et(7)GMP inhibited the methyltransferase reaction. HEV P110 capping enzyme has similar properties to the methyltransferase and guanylyltransferase of alphavirus nsP1, tobacco mosaic virus P126, brome mosaic virus replicase protein 1a, and bamboo mosaic virus (a potexvirus) nonstructural protein, indicating there is a common evolutionary origin of these distantly related plant and animal virus families. PMID:11413290

  8. 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)

  9. 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.

  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. PMID:26795263

  11. Specificity of bacteriolytic enzyme II from a soil amoeba, Hartmannella glebae.

    PubMed Central

    Hemelt, D M; Mares, B; Upadhyay, J M

    1979-01-01

    Two bacteriolytic enzymes were produced when Hartmanella glebae was grown in the presence of both Enterobacter aerogenes and Alcaligenes faecalis. The identification of enzyme I as N-acetylmuramidase was reported earlier. Enzyme II was purified by gel filtration on a Bio-Gel A column. A recovery of 68.76% with 72.3-fold purification was obtained. It was found that 5 and 10 mM MgCl2 significantly increased the bacteriolytic activity. It is a basic protein. The cell walls of Micrococcus lysodeikticus were lysed by the enzyme, and the products of digestion were purified by Amberlite CG-120 and Sephadex G-15 chromatography to facilitate the detection of amino sugars. After reduction of the oligosaccharides with sodium borohydride and acid hydrolysis, the amino sugars were identified by paper chromatography. It was found that enzyme II cleaved the glycosidic bond between N-acetylmuramic and and N-acetylglucosamine of the peptidoglycan moiety of the cell walls. Thus, the enzyme was identified as endo-beta-N-acetylmuramidase. PMID:533270

  12. Plant rhizosphere species-specific stoichiometry and regulation of extracellular enzyme and microbial community structure

    NASA Astrophysics Data System (ADS)

    Bell, C. W.; Calderon, F.; Pendall, E.; Wallenstein, M. D.

    2012-12-01

    control soil samples) were collected on day 28, 78, and 148 (N = 4 /sample period/species). Microbial community structure was quantified using the barcoded pyrosequencing protocols. We measured the potential activity of seven hydrolytic soil enzymes to represent the degradation of C, N, and P-rich substrates. Soil microbial C:N biomass responses to specific plant rhizospheres (MBC and MBN) were measured using the chloroform fumigation extraction method followed by DOC & N analysis. Fourier Transform Infrared Spectroscopy was used to assess differences in plant and soil C chemistry. We found that species specific rhizospheres are characteristic of very different soil chemical, edaphic, and microbial properties. These plant species act as gateways that introduce variability into soil C, N, and P ecosystem functional dynamics directly facilitated by rhizosphere - microbe associations. Our results suggest that nutrient stoichiometry within plant species' rhizospheres is a useful tool for identifying intra-ecosystem functional patterns. By identifying what and how specific species rhizospheres differ among the overall plant community, we can better predict how below-ground microbial community function and subsequent ecosystem processes can be influenced by alterations in plant community shifts based on the rhizosphere effects.

  13. 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). PMID:26196069

  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. 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.

  16. Identification of testis-specific ubiquitin-conjugating enzyme in the ascidian Ciona intestinalis.

    PubMed

    Yokota, Naoto; Harada, Yoshito; Sawada, Hitoshi

    2010-07-01

    The ubiquitin-proteasome system is known to play a key role in fertilization in ascidians, sea urchins, and mammals. To obtain insights into the ubiquitin-conjugating enzymes (Ube2) involved in reproductive systems, we systematically explored Ube2 enzymes expressed in the testis of the ascidian Ciona intestinalis. Here, we report cDNA cloning and characterization of a novel type of Ube2r (Ci0100152677) that is capable of making a thiolester bond with ubiquitin. Northern analysis, whole-mount in situ hybridization and immunocytochemistry indicate that this enzyme is exclusively expressed in the testis, mainly in the germ cells during the late stage of spermatogenesis, and is localized in the sperm head and tail, suggesting possible participation in fertilization or spermatogenesis/spermiogenesis. PMID:20578064

  17. Specific Enzymatic Halogenation-From the Discovery of Halogenated Enzymes to Their Applications In Vitro and In Vivo.

    PubMed

    Weichold, Veit; Milbredt, Daniela; van Pée, Karl-Heinz

    2016-05-23

    During the last 20 years, focus has shifted from haloperoxidases to flavin-dependent and non-heme-iron halogenases because of their proven involvement in the biosynthesis of halogenated metabolites in different organisms and the regioselectivity of their reactions. During the first 10-12 years, the main research topics were the detection of halogenases as well as the elucidation of three-dimensional structures and reaction mechanisms. This Review mainly deals with studies on halogenating enzymes published between 2010 and 2015. It focusses on the elucidation of the involvement of halogenating enzymes in halometabolite biosynthesis, application of halogenases in in vivo and in vitro systems, in vivo modification of biosynthetic pathways in bacteria and plants, improvement of enzyme stability, broadening of substrate specificity, and the combination of biocatalysis with chemical synthesis to produce new compounds. PMID:27059664

  18. Rhamnogalacturonan alpha-L-rhamnopyranohydrolase. A novel enzyme specific for the terminal nonreducing rhamnosyl unit in rhamnogalacturonan regions of pectin.

    PubMed Central

    Mutter, M; Beldman, G; Schols, H A; Voragen, A G

    1994-01-01

    Two alpha-L-rhamnohydrolases with different substrate specificities were isolated from a commercial preparation produced by Aspergillus aculeatus. The first rhamnohydrolase was active toward p-nitrophenyl-alpha-L- rhamnopyranoside, naringin, and hesperidin and was termed p-nitrophenyl-alpha-L-rhamnopyranohydrolase (pnp-rhamnohydrolase). From the data collected, the enzyme seemed specific for the alpha-1,2- or alpha-1,6-linkage to beta-D-glucose. The pnp-rhamnohydrolase had a molecular mass of 87 kD (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), a pH optimum of 5.5 to 6, a temperature optimum of 60 degrees C, and a specific activity toward pnp-alpha-L-rhamnopyranoside (pnp-Rha) of 13 units mg-1 protein. The second rhamnohydrolase, on the contrary, was active toward rhamnogalacturonan (RG) fragments, releasing Rha, and was therefore termed RG-rhamnohydrolase. The RG-rhamnohydrolase had a molecular mass of 84 kD, a pH optimum of 4, a temperature optimum of 60 degrees C, and a specific activity toward RG oligomers of 60 units mg-1 protein. The RG-rhamnohydrolase liberated Rha from the nonreducing end of the RG chain and appeared specific for the alpha-1,4-linkage to alpha-D-galacturonic acid. The enzyme was hindered when this terminal Rha residue was substituted at the 4-position by a beta-D-galactose. The results so far obtained did not indicate particular preference of the enzyme for low or high molecular mass RG fragments. From the results it can be concluded that a new enzyme, an RG alpha-L-rhamnopyranohydrolase, has been isolated with high specificity toward RG regions of pectin. PMID:7972516

  19. Glucitol-specific enzymes of the phosphotransferase system in Escherichia coli. Nucleotide sequence of the gut operon.

    PubMed

    Yamada, M; Saier, M H

    1987-04-25

    The complete nucleotide sequence of the glucitol (gut) operon in Escherichia coli has been determined. The glucitol-specific Enzyme II and Enzyme III of the phosphoenolpyruvate:sugar phosphotransferase system as well as glucitol-6-phosphate dehydrogenase which are encoded by the gutA, gutB, and gutD genes of the gut operon, respectively, are predicted to consist of 506 (Mr = 54,018), 123 (Mr = 13,306), and 259 (Mr = 27,866) amino acyl residues, respectively. The hydropathic profile of the Enzyme IIgut revealed 7 or 8 long hydrophobic segments which may traverse the cell membrane as alpha-helices as well as 2 or 4 short strongly hydrophobic stretches which may traverse the membrane as beta-structure. The number of amino acyl residues in the sum of the molecular weights of the glucitol Enzyme II-III pair are nearly the same as those of the mannitol Enzyme II. The ratio of hydrophobic to hydrophilic amino acyl residues and the numbers of the hydrophobic segments are also nearly the same for both transport systems. However, no significant homology was found in the nucleotide or amino acyl sequences of the two systems. Glucitol-6-phosphate dehydrogenase was found to exhibit sequence homology with ribitol dehydrogenase. A repetitive extragenic palindromic sequence was found in the 3'-flanking region of the gutD gene, suggesting the presence of a gene downstream from the gutD gene. PMID:3553176

  20. LysK, the enzyme lysing Staphylococcus aureus cells: specific kinetic features and approaches towards stabilization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    LysK, the enzyme lysing cells of Staphylococcus aureus, can be considered as perspective antimicrobial agent. Knowledge of LysK properties and behavior would allow optimizing conditions of its storage as well as formulating strategy towards its stabilization. Reaction of LysK with substrate (suspens...

  1. Pyrimidine biosynthetic enzymes of Salmonella typhimurium, repressed specifically by growth in the presence of cytidine.

    PubMed Central

    Kelln, R A; Kinahan, J J; Foltermann, K F; O'Donovan, G A

    1975-01-01

    The repressive effects of exogenous cytidine on growing cells was examined in a specially constructed strain in which the pool sizes of endogenous uridine 5'-diphosphate and uridine 5'-triphosphate cannot be varied by the addition of uracil and/or uridine to the medium. Five enzymes of the pyrimidine biosynthetic pathway and one enzyme of the arginine biosynthetic pathway were assayed from cells grown under a variety of conditions. Cytidine repressed the synthesis of dihydroorotase (encoded by pyrC), dihydroorotate dehydrogenase (encoded by pyrD), and ornithine transcarbamylase (encoded by argI). Moreover, aspartate transcarbamylase (encoded by pyrB) became further derepressed upon cytidine addition, whereas no change occurred in the levels of the last two enzymes (encoded by pyrE and pyrF) of the pyrimidine pathway. Quantitative nucleotide pool determinations have provided evidence that any individual ribo- or deoxyribonucleoside mono-, di-, or triphosphate of cytosine or uracil is not a repressing metabolite for the pyrimidine biosynthetic enzymes. Other nucleotide derivatives or ratios must be considered. PMID:1102530

  2. NADP-malic enzyme from the C4 plant Flaveria bidentis: nucleotide substrate specificity.

    PubMed

    Ashton, A R

    1997-09-15

    NADP-malic enzyme (NADP-ME, EC 1.1.1.40) was purified to near-homogeneity from leaves of the C4 dicot Flaveria bidentis and shown to possess intrinsic NAD-dependent malic enzyme activity. The NAD-dependent activity is optimal at pH 7.5 and in the presence of Mn2+. The Km for NAD is very high (20 mM), while the Vmax is 50% greater than the Vmax with NADP under the same conditions. The NAD-dependent activity is competitively inhibited by micromolar concentrations of NADP and NADPH (Ki approximately 2 microM). This very low Ki reflects the high affinity of malic enzyme for NADP(H) under these conditions. When utilizing NADP, the Km for NADP is 1.5 microM while the Ki for NADPH is 2 microM. Chicken liver NADP-ME also has NAD-dependent activity that is inhibited by low concentrations of NADPH. These results indicate that the NAD- and NADP-dependent activities are likely catalyzed by the same active site. The use of NAD as an alternative coenzyme revealed interactions between the binding of coenzyme and metal ions on the Km values of each of the other participants in the malic enzyme reaction. Thus, the affinity of malic enzyme for the divalent metal ions Mg2+ and particularly Mn2+ as well as the other substrate L-malate is also dependent on the nucleotide coenzyme substrate. In turn, the divalent metal ion influences the affinity of the enzyme for the coenzyme as well as L-malate. With NADP as substrate the Km for Mn2+ is 4 microM, whereas with NAD the Km is 300 microM. The relatively high affinity of the enzyme for Mn2+ and low affinity for NAD required the use of metal ion buffers when determining these values because of the substantial depletion of free Mn2+ caused by binding to NADP. PMID:9308897

  3. 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.

  4. DNA cleavage and methylation specificity of the single polypeptide restriction–modification enzyme LlaGI

    PubMed Central

    Smith, Rachel M.; Diffin, Fiona M.; Savery, Nigel J.; Josephsen, Jytte; Szczelkun, Mark D.

    2009-01-01

    LlaGI is a single polypeptide restriction–modification enzyme encoded on the naturally-occurring plasmid pEW104 isolated from Lactococcus lactis ssp. cremoris W10. Bioinformatics analysis suggests that the enzyme contains domains characteristic of an mrr endonuclease, a superfamily 2 DNA helicase and a γ-family adenine methyltransferase. LlaGI was expressed and purified from a recombinant clone and its properties characterised. An asymmetric recognition sequence was identified, 5′-CTnGAyG-3′ (where n is A, G, C or T and y is C or T). Methylation of the recognition site occurred on only one strand (the non-degenerate dA residue of 5′-CrTCnAG-3′ being methylated at the N6 position). Double strand DNA breaks at distant, random sites were only observed when two head-to-head oriented, unmethylated copies of the site were present; single sites or pairs in tail-to-tail or head-to-tail repeat only supported a DNA nicking activity. dsDNA nuclease activity was dependent upon the presence of ATP or dATP. Our results are consistent with a directional long-range communication mechanism that is necessitated by the partial site methylation. In the accompanying manuscript [Smith et al. (2009) The single polypeptide restriction–modification enzyme LlaGI is a self-contained molecular motor that translocates DNA loops], we demonstrate that this communication is via 1-dimensional DNA loop translocation. On the basis of this data and that in the third accompanying manuscript [Smith et al. (2009) An Mrr-family nuclease motif in the single polypeptide restriction–modification enzyme LlaGI], we propose that LlaGI is the prototype of a new sub-classification of Restriction-Modification enzymes, named Type I SP (for Single Polypeptide). PMID:19808936

  5. NASA broadened-specification fuels combustion technology program

    NASA Technical Reports Server (NTRS)

    Fear, J. S.

    1980-01-01

    The broadened-Specification Fuels Combustion Technology program's purpose is to evolve and demonstrate the technology required to enable current and next generation high-thrust, high-bypass-ratio turbofan engines to use fuels with broadened properties and to verify the evolved technology in full scale engine tests. The three phases of the program are combustor concept screening, combustor optimization testing, and engine verification testing. Constraints for designing combustion systems are outlined and problems to be expected in the use of broadened properties fuels are listed.

  6. Detection of chitinolytic enzymes with different substrate specificity in tissues of intact sundew (Drosera rotundifolia L.): chitinases in sundew tissues.

    PubMed

    Libantová, Jana; Kämäräinen, Terttu; Moravcíková, Jana; Matusíková, Ildikó; Salaj, Jan

    2009-05-01

    The round-leaf sundew (Drosera rotundifolia L.) is a carnivorous plant expressing a wide range of chitinolytic enzymes playing role in many different processes. In this study the intact plants were analyzed for the presence of chitinase transcripts and chitinolytic activities in different organs. In situ hybridization with chitnase fragment as a probe has revealed the presence of chitinases in the mesophyll cells of leaves and vascular elements of stems of healthy, non-stressed plants. More pronounced expression was observed in cortex and stele cells of roots as well as in ovules and anthers of reproductive organs. Similarly, higher chitinase enzyme activity was typical for flowers and roots suggesting a more specific role of chitinases in these tissues. In addition to endochitinases of different substrate specificities, chitobiosidases contributed to overall chitinolytic activity of tissue extracts. The activity of chitobiosidases was again typical for flowers and roots, while their role in plant physiology remains to be elucidated. PMID:18437530

  7. 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. PMID:6193091

  8. An improved method for specificity annotation shows a distinct evolutionary divergence among the microbial enzymes of the cholylglycine hydrolase family.

    PubMed

    Panigrahi, Priyabrata; Sule, Manas; Sharma, Ranu; Ramasamy, Sureshkumar; Suresh, C G

    2014-06-01

    Bile salt hydrolases (BSHs) are gut microbial enzymes that play a significant role in the bile acid modification pathway. Penicillin V acylases (PVAs) are enzymes produced by environmental microbes, having a possible role in pathogenesis or scavenging of phenolic compounds in their microbial habitats. The correct annotation of such physiologically and industrially important enzymes is thus vital. The current methods relying solely on sequence homology do not always provide accurate annotations for these two members of the cholylglycine hydrolase (CGH) family as BSH/PVA enzymes. Here, we present an improved method [binding site similarity (BSS)-based scoring system] for the correct annotation of the CGH family members as BSH/PVA enzymes, which along with the phylogenetic information incorporates the substrate specificity as well as the binding site information. The BSS scoring system was developed through the analysis of the binding sites and binding modes of the available BSH/PVA structures with substrates glycocholic acid and penicillin V. The 198 sequences in the dataset were then annotated accurately using BSS scores as BSH/PVA enzymes. The dataset presented contained sequences from Gram-positive bacteria, Gram-negative bacteria and archaea. The clustering obtained for the dataset using the method described above showed a clear distinction in annotation of Gram-positive bacteria and Gram-negative bacteria. Based on this clustering and a detailed analysis of the sequences of the CGH family in the dataset, we could infer that the CGH genes might have evolved in accordance with the hypothesis stating the evolution of diderms and archaea from the monoderms. PMID:24644246

  9. 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

  10. 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.

  11. Stimulus specificity of prostaglandin inhibition of rabbit polymorphonuclear leukocyte lysosomal enzyme release and superoxide anion production.

    PubMed Central

    Fantone, J. C.; Marasco, W. A.; Elgas, L. J.; Ward, P. A.

    1984-01-01

    Prostaglandins (PGs) of the E series and PGI2 have been shown to inhibit acute inflammatory reactions in vivo and polymorphonuclear leukocyte (PMN), chemotaxis, lysosomal enzyme release, and superoxide anion (O-2) production in vitro. This inhibition of neutrophil stimulation by PGEs and PGI2 has been correlated with their ability to increase intracellular cyclic adenosine monophosphate (cAMP) levels. However, the mechanism(s) by which PGEs and PGI2 alter the complex biochemical and biophysical events associated with stimulus-response coupling in the neutrophil are not clear. It is reported here that both PGEs and PGI2 in micromolar concentrations inhibit formyl-methionyl-leucyl-phenylalanine (FMLP)- and zymosan-induced lysosomal enzyme secretion and superoxide anion production in a dose-dependent manner. No preincubation time of PMNs with the prostaglandins is required for inhibition. Addition of PGEs 10 seconds or later after FMLP stimulation does not alter the biologic response of the neutrophils to the stimulus, suggesting that the prostaglandin inhibition effects early events associated with stimulus-response coupling in the neutrophil. Prostaglandin inhibition of lysosomal enzyme release by the calcium ionophore A23187 was overcome by increasing the extracellular ionophore and/or calcium concentration, suggesting that PGs may modulate intracellular free calcium levels in a manner similar to that observed with platelets. Inhibition of phorbol myristate acetate (PMA)-induced neutrophil lysosomal enzyme secretion by PGEs and PGI2 was overcome by increasing concentrations of PMA. However, neither PGEs nor PGI2 altered O-2 production by PMA-treated neutrophils. These data indicate a dissociation between PMA-stimulated O-2 production and lysosomal enzyme release. These findings are consistent with the hypothesis that inhibition of neutrophil stimulation by PGEs and PGI2 is a result of increased intracellular cyclic AMP levels and modulation of calcium

  12. 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

  13. A continuous coupled spectrophotometric assay for debranching enzyme activity using reducing end-specific α-glucosidase.

    PubMed

    Do, Viet Ha; Tran, Phuong Lan; Ni, Li; Park, Kwan Hwa

    2016-01-01

    A novel continuous spectrophotometric assay to measure the activity of the debranching enzyme and α-amylase has been developed. The assay mixture comprises the debranching enzyme (GlgX from Escherichia coli) or α-amylase (PPA from porcine pancreas), a reducing end-specific α-glucosidase (MalZ), maltodextrin-branched β-cyclodextrin (Glcn-β-CD) as the substrate, and the glucose oxidase/peroxidase system (GOPOD). Due to its high reducing end specificity, the branch chains of the substrates are not hydrolyzed by MalZ. After hydrolysis by GlgX or PPA, the released maltodextrins are immediately hydrolyzed into glucose from the reducing end by MalZ, whose concentration is continuously measured by GOPOD at 510 nm in a thermostat spectrophotometer. The kinetic constants determined for GlgX (Km = 0.66 ± 0.02 mM and kcat = 76.7 ± 1.5 s(-1)) are within a reasonable range compared with those measured using high-performance anion-exchange chromatography (HPAEC). The assay procedure is convenient and sensitive, and it requires lower concentrations of enzymes and substrate compared with dinitrosalicylic acid (DNS) and HPAEC analysis. PMID:26403601

  14. Development of a Highly Sensitive and Specific Blastomycosis Antibody Enzyme Immunoassay Using Blastomyces dermatitidis Surface Protein BAD-1

    PubMed Central

    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

    2014-01-01

    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. PMID:24285817

  15. Turnaround of axoplasmic transport of selected particle-specific enzymes at an injury in control and diisopropylphosphorofluoridate-treated rats.

    PubMed

    Schmidt, R E; Yu, M J; McDougal, D B

    1980-09-01

    Reversal of direction (turnaround) of axonal transport of particle-specific enzyme activities was studied at a ligature placed on rat sciatic nerve. In the principal experiment, the ligature remained on the nerve in vivo several hours, allowing enzyme activities (acetylcholinesterase, acid phosphatase, and monoamine oxidase) to accumulate immediately proximal to the tie. The nerve was then tied a second time, proximal to the first tie, and incubated in vitro for several more hours. Accumulation of enzyme activities just distal to the second tie was measured. This second accumulation, of activities traveling in the retrograde direction, was shown to be the result of turnaround in several ways. (1) The increase in activity distal to the second tie was equal to the decrease in activity proximal to the first. (2) The increase in enzyme activities distal to the second tie was greatly reduced when the accumulation proximal to the first tie was trapped by placing a third tie between the first and second ties. (3) It was shown that the activity that accumulated distal to the second tie could not have been in retrograde motion at the time of the first tie. (4) Accumulation distal to the second tie was not a function of the length of nerve segment included between the two ties. In contrast to the consistent occurrence of turnaround of orthograde flow, turnaround of retrograde flow could not be demonstrated. Turnaround transport was blocked by incubation in the cold and in the presence of NaCN or vinblastine. The turnaround process operated on all three enzymes studied, suggesting that it operates on lysosomes and mitochondria, as well as on the endoplasmic reticulum-like material bearing acetylcholinesterase. Evidence for the participation of the transport process in the renewal of AChE in the distal portions of the axon was obtained in experiments using diisopropylphosphorofluoridate and cycloheximide. PMID:6161227

  16. A potent specific inhibitor of 6-phosphogluconate dehydrogenase of Cryptococcus neoformans and of certain other fungal enzymes.

    PubMed

    Niehaus, W G; Flynn, T

    1993-09-01

    A particular lot of the zwitterionic buffer, 2(N-morpholino) ethane sulfonic acid (MES), contained a contaminant that inhibited a number of fungal NADP-dependent dehydrogenases. Enzymes that were particularly sensitive include 6-phosphogluconate dehydrogenases from Cryptococcus neoformans and Schizophyllum commune and glucose-6-phosphate dehydrogenase from Schizophyllum commune. A number of NADP-dependent dehydrogenases of animal origin were tested and all were completely insensitive to inhibition except for rat liver 6-phosphogluconate dehydrogenase, which was 10-fold less sensitive than the Cryptococcal enzyme. The pattern of inhibition in all cases was linear competitive versus NADP. The inhibitor has been purified and identified as an ethylenesulfonic acid oligomer. This inhibitor holds promise as a model compound for the development of a specific antifungal agent. PMID:8302365

  17. Human oestrogenic 17beta-hydroxysteroid dehydrogenase specificity: enzyme regulation through an NADPH-dependent substrate inhibition towards the highly specific oestrone reduction.

    PubMed Central

    Gangloff, A; Garneau, A; Huang, Y W; Yang, F; Lin, S X

    2001-01-01

    Human oestrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD1) catalyses the final step in the biosynthesis of all active oestrogens. Here we report the steady-state kinetics for 17beta-HSD1 at 37 degrees C and pH 7.5, using a homogeneous enzyme preparation with oestrone, dehydroepiandrosterone (DHEA) or dihydrotestosterone (DHT) as substrate and NADP(H) as the cofactor. Kinetic studies made over a wide range of oestrone concentrations (10 nM-10 microM) revealed a typical substrate-inhibition phenomenon. Data analysis using the substrate-inhibition equation v=V.[s]/[K(m)+[s](1+[s]/K(i))] gave a K(m) of 0.07+/-0.01 microM, a k(cat) (for the dimer) of 1.5+/-0.1 s(-1), a specificity of 21 microM(-1) x s(-1) and a K(i) of 1.3 microM. When NADH was used instead of NADPH, substrate inhibition was no longer observed and the kinetic constants were significantly modified to 0.42+/-0.07 microM for the K(m), 0.8+/-0.04 s(-1) for the k(cat) and 1.9 microM(-1) x s(-1) for the specificity. The modification of an amino acid in the cofactor-binding site (Leu36Asp) eliminated the substrate inhibition observed in the presence of NADPH, confirming the NADPH-dependence of the phenomenon. The possible formation of an enzyme-NADP(+)-oestrone dead-end complex during the substrate-inhibition process is supported by the competitive inhibition of oestradiol oxidation by oestrone. Kinetic studies performed with either DHEA (K(m)=24+/-4 microM; k(cat)=0.47+/-0.06 s(-1); specificity=0.002 microM(-1) x s(-1)) or DHT (K(m)=26+/-6 microM; k(cat)=0.2+/-0.02 s(-1); specificity=0.0008 microM(-1) x s(-1)) in the presence of NADP(H) resulted in low specificities and no substrate inhibition. Taken together, our results demonstrate that the high specificity of 17beta-HSD1 towards oestrone is coupled with an NADPH-dependent substrate inhibition, suggesting that both the specificity and the enzyme control are provided for the cognate substrate. PMID:11336660

  18. Magnetic enzyme-linked immunosorbent assay (MELISA) for determination of specific IgG in paracoccidioidomycosis.

    PubMed

    de Camargo, Z P; Guesdon, J L; Drouhet, E; Improvisi, L

    1984-01-01

    A magnetic solid phase enzyme-linked immunosorbent assay (MELISA) for quantification of IgG antibodies to somatic and metabolic antigens of Paracoccidioides brasiliensis was developed. Activation of magnetic polyacrylamide agarose beads with concanavalin A was superior to glutaraldehyde activation, and test sensitivity was higher for somatic than for metabolic antigens. Comparative MELISA, counterimmunoelectrophoresis and erythroimmunoassay tests with sera from 33 proven cases of paracoccidioidomycosis, 14 cases of histoplasmosis and 20 normal human sera showed the MELISA could distinguish antibody levels in paracoccidioidomycosis from those in normal sera; however two sera from histoplasmosis cases cross-reacted in the MELISA. MELISA is a rapid test (5-6 h) and the results suggest it has considerable potential value for assay of anti-P. brasiliensis antibodies. PMID:6438813

  19. [Classification of alcohol metabolizing enzymes and polymorphisms--specificity in Japanese].

    PubMed

    Harada, S

    2001-04-01

    Multiple forms and gene loci of human alcohol dehydrogenase (ADH EC: 1.2.1.3) and aldehyde dehydrogenase (ALDH, EC: 1.2.1.3) in the major pathway of alcohol metabolism have been found and characterized in the last two decades. With the coenzyme NAD, these enzymes catalyze the reversible conversion of organic alcohols to ketones or aldehydes, and aldehyde to acetic acid. The ADH genes are mapped to chromosome 4p21-25, but the ALDH genes are localized at different chromosomes. The cytochrome P450 2E1 (CYP2E1) gene, which is mapped to chromosome 10q24.3-qter contributes also the conversion of ethanol to acetaldehyde. Genetic polymorphisms have been reported in these alcohol metabolizing enzymes. The metabolisms of alcohol and acetaldehyde in liver and blood after drinking alcohol are thought to be influenced by the interactive action of these enzymes. Amongst the five major classes of the ADH subunits (alpha, beta, gamma, pi, chi, sigma), beta and gamma subunits show genetic polymorphisms. Recently a new nomenclature for ALDH genes has been recommend based on divergent evolution and chromosomal mapping. Two major isoforms designated as cytosolic ALDH1 and mitochondrial ALDH2 can be distinguished by their electrophoretic and kinetic properties as well as by their subcellular localization. Mitochondrial ALDH2 is a major enzyme in the oxidation of acetaldehyde derived from ethanol metabolism. The catalytic deficiency of ALDH2 isozyme is responsible for flushing and other vasomotor symptoms caused by higher acetaldehyde levels after alcohol intake. So far, frequencies of the two alleles of ALDH2 in Mongoloid have been reported in the different population groups. The catalytic deficiency of ALDH2 is caused by a structural point mutation at amino acid position 487, where a substitution of Glu to Lys resulting from a transition of G (C) to A (T) at 1510 nucleotide from the initiation codon has occurred. Individuals deficient in ALDH2 activity refrain from excessive drinking

  20. Design specification for the core management program: COREMAP

    SciTech Connect

    Jones, D.B. , Inc., Campbell, CA )

    1991-04-01

    This report presents the design specifications for the core management program COREMAP. COREMAP is a computer code which performs fuel cycle scoping and preliminary core design calculations for light water reactors. It employs solution techniques which are compatible with existing EPRI methodologies and it includes new methodologies designed to facilitate the analysis effort. The primary neutronic and thermal-hydraulic techniques implemented in COREMAP are derived from the nodal simulation code SIMULATE-E. Code performance is improved by the development of a Spatial Collapsing Algorithm. User interaction is improved by the implementation of many user-convenient features including interactive screens for input specification, detailed error checking, and manual and automated fuel shuffle options. COREMAP is designed as a modular code system using standard data interface files. It is written entirely in FORTRAN-77 and can be implemented on any computer system supporting this language level and ASCII terminals. 16 refs., 10 figs., 7 tabs.

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

    PubMed

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

    2014-01-01

    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, transcription activator-like effectornucleases, 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 chapter, 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 (~10(12)-membered) libraries of potential nuclease substrates. PMID:25398335

  2. Engineering the glucansucrase GTFR enzyme reaction and glycosidic bond specificity: toward tailor-made polymer and oligosaccharide products.

    PubMed

    Hellmuth, Hendrik; Wittrock, Sabine; Kralj, Slavko; Dijkhuizen, Lubbert; Hofer, Bernd; Seibel, Jürgen

    2008-06-24

    Two long-standing questions about glucansucrases (EC 2.4.1.5) are how they control oligosaccharide versus polysaccharide synthesis and how they direct their glycosidic linkage specificity. This information is required for the production of tailor-made saccharides. Mutagenesis promises to be an effective tool for enzyme engineering approaches for altering the regioselectivity and acceptor substrate specificity. Therefore, we chose the most conserved motif around the transition state stabilizer in glucansucrases for a random mutagenesis of the glucansucrase GTFR of Streptococcus oralis, yielding different variants with altered reaction specificity. Modifications at position S628 achieved by saturation mutagenesis guided the reaction toward the synthesis of short chain oligosaccharides with a drastically increased yield of isomaltose (47%) or leucrose (64%). Alternatively, GTFR variant R624G/V630I/D717A exhibited a drastic switch in regioselectivity from a dextran type with mainly alpha-1,6-glucosidic linkages to a mutan type polymer with predominantly alpha-1,3-glucosidic linkages. Targeted modifications demonstrated that both mutations near the transition state stabilizer, R624G and V630I, are contributing to this alteration. It is thus shown that mutagenesis can guide the transglycosylation reaction of glucansucrase enzymes toward the synthesis of (a) various short chain oligosaccharides or (b) novel polymers with completely altered linkages, without compromising their high transglycosylation activity and efficiency. PMID:18512955

  3. Variability in intra-specific and monosporous isolates of Volvariella volvacea based on enzyme activity, ITS and RAPD.

    PubMed

    Ahlawat, O P; Gupta, P; Kamal, S; Dhar, B L

    2010-06-01

    Two parents and 15 monosporous isolates were morphologically characterized and were found to vary in their growth characteristics on malt extract agar medium. The isolates also varied in enzymes activity profile with respect to exoglucanase, endoglucanase, xylanase, laccase and polyphenol oxidase. Further in polymerase chain reaction (PCR) amplification of Internal Transcribed Spacer (ITS) region of 5.8S rDNA, an amplicon of same length (720 bp) was amplified from two parents and all the monosporous isolates, which revealed that all belong to the same species. The combined phylogenetic analysis of random amplified polymorphic DNA (RAPD) profiles obtained with five decamer primers (operon kit B) series primers also revealed intra-specific variation of 60% with in the two parent strains and their single spore isolates (SSIs). However, the variations between the parent strains and their SSIs were lesser and it was 50 and 32% in parent strains, OE-210 and OE-12, respectively. Based upon phylogenetic analysis, the isolates of parent strain, OE-210 formed 7 distinct phylogenetic clades, while of strain OE-12 formed 4 clades. The study elucidates that isolates showing variations in morphological growth characteristics and enzymes activity also showed variations in their RAPD profiles, revealed through phylogenetic analysis of RAPD profiles. It is also evident from the study that morphological characterization along with enzymes activity assay of strains is essential before their use in yield evaluation trials with final authentication from molecular analysis. PMID:23100827

  4. A specific nursing educational program in patients with Cushing's syndrome.

    PubMed

    Martínez-Momblán, M Antonia; Gómez, Carmen; Santos, Alicia; Porta, Nuria; Esteve, Julia; Úbeda, Inmaculada; Halperin, Irene; Campillo, Beatriz; Guillaumet, Montserrat; Webb, Susan M; Resmini, Eugenia

    2016-07-01

    Cushing's syndrome (CS) is a rare endocrine disease, due to cortisol hypersecretion. CS patients have comorbidities, often still present after biochemical cure. Specific nursing healthcare programs to address this disease and achieve improved health related quality of life (HRQoL) are lacking. Thus, an educational nursing intervention, through the development and promotion of specific educational tools, appears to be justified. The objective of this study is to assess the effectiveness of an educational nursing program in CS patients on HRQoL, clinical parameters, level of pain and physical activity, patterns of rest, and use of health resources. A prospective, randomized study was conducted in two reference hospitals for CS. Sixty-one patients (mean age 47 ± 12.7 years, 83.6 % females) were enrolled and divided into 2 groups: an "intervention" group where educational sessions were performed over 9 months and a "control" group, without these sessions. Specific questionnaires were used at the beginning and end of the study. After educational sessions, the intervention group had a better score in the CushingQoL questionnaire (p < 0.01), reduced level of pain (p < 0.05), improved physical activity (p < 0.01) and healthy lifestyle (p < 0.001) compared to the control group. A correlation between the CushingQoL score and reduced pain (r = 0.46, p < 0.05), improved physical activity (r = 0.89, p < 0.01), and sleep (r = 0.53, p = 0.01) was observed. This educational nursing program improved physical activity, healthy lifestyle, better sleep patterns, and reduced pain in CS patients, influencing HRQoL and reducing consumption of health resources. Moreover, the brief nature of the program suggests it as a good candidate to be used in CS patients. PMID:26400845

  5. 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.

  6. Identification of Grass-specific Enzyme That Acylates Monolignols with p-Coumarate*

    PubMed Central

    Withers, Saunia; Lu, Fachuang; Kim, Hoon; Zhu, Yimin; Ralph, John; Wilkerson, Curtis G.

    2012-01-01

    Lignin is a major component of plant cell walls that is essential to their function. However, the strong bonds that bind the various subunits of lignin, and its cross-linking with other plant cell wall polymers, make it one of the most important factors in the recalcitrance of plant cell walls against polysaccharide utilization. Plants make lignin from a variety of monolignols including p-coumaryl, coniferyl, and sinapyl alcohols to produce the three primary lignin units: p-hydroxyphenyl, guaiacyl, and syringyl, respectively, when incorporated into the lignin polymer. In grasses, these monolignols can be enzymatically preacylated by p-coumarates prior to their incorporation into lignin, and these monolignol conjugates can also be “monomer” precursors of lignin. Although monolignol p-coumarate-derived units may comprise up to 40% of the lignin in some grass tissues, the p-coumarate moiety from such conjugates does not enter into the radical coupling (polymerization) reactions of lignification. With a greater understanding of monolignol p-coumarate conjugates, grass lignins could be engineered to contain fewer pendent p-coumarate groups and more monolignol conjugates that improve lignin cleavage. We have cloned and expressed an enzyme from rice that has p-coumarate monolignol transferase activity and determined its kinetic parameters. PMID:22267741

  7. 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. PMID:26797250

  8. Specific activities of antioxidative enzymes in the cochlea of guinea pigs at different stages of development.

    PubMed

    Zelck, U; Nowak, R; Karnstedt, U; Koitschev, A; Käcker, N

    1993-01-01

    Significant activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were found in the cochleas of guinea pigs of different ages. The specific activities of SOD and GSH-Px (expressed as units/mg protein) increased significantly from fetal animals to animals 2 days old and then to 6-month-old animals. PMID:8369116

  9. Characterization of enterotoxigenic Bacteroides fragilis by a toxin-specific enzyme-linked immunosorbent assay.

    PubMed Central

    Van Tassell, R L; Lyerly, D M; Wilkins, T D

    1994-01-01

    Within the past decade, certain strains of Bacteroides fragilis have been associated with diarrhea in humans and cytotoxic activity on certain colon carcinoma cell lines. An enzyme-linked immunosorbent assay (ELISA) for detecting the enterotoxin of B. fragilis in cultures and stools was developed by using high-titer monospecific goat and rabbit antitoxins in an indirect format. The lower limit of detection for purified toxin was approximately 0.05 micrograms/ml; the linear range was from 0.05 to 10 microgram/ml. Using the ELISA to screen cultures of toxigenic and nontoxigenic strains of B. fragilis, we observed 100% correlation with 16 known toxigenic strains which had various cytotoxic activities on HT-29 cells. In addition, we found 6 of 62 previously untested strains also to be positive in both assays. Stability studies revealed that although the cytotoxic activities of crude and purified toxin preparations incubated at elevated temperatures were rapidly lost, the ELISA responses were not significantly reduced. Sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis and SDS-capillary electrophoresis showed that the purified toxin autodigested to several stable peptides. Studies on partially purified membranes from the toxigenic strains revealed the presence of several membrane-associated components which were noncytotoxic but strongly immunoreactive in the ELISA. Preliminary studies with spiked feces indicated that the ELISA may be useful for screening not only cultures for the enterotoxigenic B. fragilis but also stool specimens. Ongoing studies are focusing on determining the nature of the toxin's apparent proteolytic capabilities and investigating the feasibility of using the ELISA on stool specimens from healthy and diarrheic humans. Images PMID:8556504

  10. The ADAMTS13 metalloprotease domain: roles of subsites in enzyme activity and specificity.

    PubMed

    de Groot, Rens; Lane, David A; Crawley, James T B

    2010-10-21

    ADAMTS13 modulates von Willebrand factor (VWF) platelet-tethering function by proteolysis of the Tyr1605-Met1606 bond in the VWF A2 domain. To examine the role of the metalloprotease domain of ADAMTS13 in scissile bond specificity, we identified 3 variable regions (VR1, -2, and -3) in the ADAMTS family metalloprotease domain that flank the active site, which might be important for specificity. Eight composite sequence swaps (to residues in ADAMTS1 or ADAMTS2) and 18 single-point mutants were generated in these VRs and expressed. Swapping VR1 (E184-R193) of ADAMTS13 with that of ADAMTS1 or ADAMTS2 abolished/severely impaired ADAMTS13 function. Kinetic analysis of VR1 point mutants using VWF115 as a short substrate revealed reduced proteolytic function (k(cat)/K(m) reduced by 2- to 10-fold) as a result of D187A, R190A, and R193A substitutions. Analysis of VR2 (F216-V220) revealed a minor importance of this region. Mutants of VR3 (G236-A261) proteolysed wild-type VWF115 normally. However, using either short or full-length VWF substrates containing the P1' M1606A mutation, we identified residues within VR3 (D252-P256) that influence P1' amino acid specificity, we hypothesize, by shaping the S1' pocket. It is concluded that 2 subsites, D187-R193 and D252-P256, in the metalloprotease domain play an important role in cleavage efficiency and site specificity. PMID:20647566

  11. Exploiting enzyme specificities in digestions of chondroitin sulfates A and C: Production of well-defined hexasaccharides

    PubMed Central

    Pomin, Vitor H; Park, Younghee; Huang, Rongrong; Heiss, Christian; Sharp, Joshua S; Azadi, Parastoo; Prestegard, James H

    2012-01-01

    Interactions between proteins and glycosaminoglycans (GAGs) of the extracellular matrix are important to the regulation of cellular processes including growth, differentiation and migration. Understanding these processes can benefit greatly from the study of protein–GAG interactions using GAG oligosaccharides of well-defined structure. Materials for such studies have, however, been difficult to obtain because of challenges in synthetic approaches and the extreme structural heterogeneity in GAG polymers. Here, it is demonstrated that diversity in structures of oligosaccharides derived by limited enzymatic digestion of materials from natural sources can be greatly curtailed by a proper selection of combinations of source materials and digestive enzymes, a process aided by an improved understanding of the specificities of certain commercial preparations of hydrolases and lyases. Separation of well-defined oligosaccharides can then be accomplished by size-exclusion chromatography followed by strong anion-exchange chromatography. We focus here on two types of chondroitin sulfate (CS) as starting material (CS-A, and CS-C) and the use of three digestive enzymes with varying specificities (testicular hyaluronidase and bacterial chondroitinases ABC and C). Analysis using nuclear magnetic resonance and mass spectrometry focuses on isolated CS disaccharides and hexasaccharides. In all, 15 CS hexasaccharides have been isolated and characterized. These serve as useful contributions to growing libraries of well-defined GAG oligosaccharides that can be used in further biophysical assays. PMID:22345629

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

    PubMed

    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

    2015-10-27

    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/cm(2), 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

  13. 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.

  14. 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

  15. Dihydroflavonol 4-Reductase Genes Encode Enzymes with Contrasting Substrate Specificity and Show Divergent Gene Expression Profiles in Fragaria Species

    PubMed Central

    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. PMID:25393679

  16. Nicotinamide Adenine Dinucleotide-specific “Malic” Enzyme in Kalanchoë daigremontiana and Other Plants Exhibiting Crassulacean Acid Metabolism 1

    PubMed Central

    Dittrich, Peter

    1976-01-01

    NAD-specific “malic” enzyme (EC 1.1.1.39) has been isolated and purified 1200-fold from leaves of Kalanchoë daigremontiana. Kinetic studies of this enzyme, which is activated 14-fold by CoA, acetyl-CoA, and SO42−, suggest allosteric properties. Cofactor requirements show an absolute specificity for NAD and for Mn2+, which cannot be replaced by NADP or Mg2+. For maintaining enzyme activity in crude leaf extracts a thiol reagent, Mn2+, and PVP-40 were required. The latter could be omitted from purified preparations. By sucrose density gradient centrifugation NAD-malic enzyme could be localized in mitochondria. A survey of plants with crassulacean acid metabolism revealed the presence of NAD-malic enzyme in all 31 plants tested. Substantial levels of this enzyme (121-186 μmole/hr·mg of Chl) were detected in all members tested of the family Crassulaceae. It is proposed that NAD-malic enzyme in general supplements activity of NADP-malic enzyme present in these plants and may be specifically employed to increase internal concentrations of CO2 for recycling during cessation of gas exchange in periods of severe drought. PMID:16659473

  17. 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

  18. Determination of key residues for catalysis and RNA cleavage specificity: one mutation turns RNase II into a "SUPER-ENZYME".

    PubMed

    Barbas, Ana; Matos, Rute G; Amblar, Mónica; López-Viñas, Eduardo; Gomez-Puertas, Paulino; Arraiano, Cecília M

    2009-07-31

    RNase II is the prototype of a ubiquitous family of enzymes that are crucial for RNA metabolism. In Escherichia coli this protein is a single-stranded-specific 3'-exoribonuclease with a modular organization of four functional domains. In eukaryotes, the RNase II homologue Rrp44 (also known as Dis3) is the catalytic subunit of the exosome, an exoribonuclease complex essential for RNA processing and decay. In this work we have performed a functional characterization of several highly conserved residues located in the RNase II catalytic domain to address their precise role in the RNase II activity. We have constructed a number of RNase II mutants and compared their activity and RNA binding to the wild type using different single- or double-stranded substrates. The results presented in this study substantially improve the RNase II model for RNA degradation. We have identified the residues that are responsible for the discrimination of cleavage of RNA versus DNA. We also show that the Arg-500 residue present in the RNase II active site is crucial for activity but not for RNA binding. The most prominent finding presented is the extraordinary catalysis observed in the E542A mutant that turns RNase II into a "super-enzyme." PMID:19458082

  19. G-quadruplex RNA binding and recognition by the lysine-specific histone demethylase-1 enzyme.

    PubMed

    Hirschi, Alexander; Martin, William J; Luka, Zigmund; Loukachevitch, Lioudmila V; Reiter, Nicholas J

    2016-08-01

    Lysine-specific histone demethylase 1 (LSD1) is an essential epigenetic regulator in metazoans and requires the co-repressor element-1 silencing transcription factor (CoREST) to efficiently catalyze the removal of mono- and dimethyl functional groups from histone 3 at lysine positions 4 and 9 (H3K4/9). LSD1 interacts with over 60 regulatory proteins and also associates with lncRNAs (TERRA, HOTAIR), suggesting a regulatory role for RNA in LSD1 function. We report that a stacked, intramolecular G-quadruplex (GQ) forming TERRA RNA (GG[UUAGGG]8UUA) binds tightly to the functional LSD1-CoREST complex (Kd ≈ 96 nM), in contrast to a single GQ RNA unit ([UUAGGG]4U), a GQ DNA ([TTAGGG]4T), or an unstructured single-stranded RNA. Stabilization of a parallel-stranded GQ RNA structure by monovalent potassium ions (K(+)) is required for high affinity binding to the LSD1-CoREST complex. These data indicate that LSD1 can distinguish between RNA and DNA as well as structured versus unstructured nucleotide motifs. Further, cross-linking mass spectrometry identified the primary location of GQ RNA binding within the SWIRM/amine oxidase domain (AOD) of LSD1. An ssRNA binding region adjacent to this GQ binding site was also identified via X-ray crystallography. This RNA binding interface is consistent with kinetic assays, demonstrating that a GQ-forming RNA can serve as a noncompetitive inhibitor of LSD1-catalyzed demethylation. The identification of a GQ RNA binding site coupled with kinetic data suggests that structured RNAs can function as regulatory molecules in LSD1-mediated mechanisms. PMID:27277658

  20. Assessment of Alternative Student Aid Delivery Systems: Preliminary Specification of the Current System with Program Antecedents.

    ERIC Educational Resources Information Center

    Advanced Technology, Inc., Reston, VA.

    Specifications of the current delivery systems of the Pell Grant program, the Guaranteed Student Loan (GSL) program, and campus-based aid programs are provided. The relationship between features of the programs and delivery systems is also examined. The campus-based programs include the Supplemental Educational Opportunity Grant (SEOG) Program,…

  1. [Fibrinogen/fibrin-specific enzymes from copperhead (Agkistrodon halys halys) and cobra (Naja oxiana eichwald) snake venoms].

    PubMed

    Yunusova, E S; Sadykov, E S; Sultanalieva, N M; Shkinev, A V

    2016-03-01

    Ability of fractions of cobra's (Naja oxiana Eichwald) and copperhead snake's (Agkistrodon halys halys) venoms to hydrolyze fibrinogen/fibrin was studied. In cobra's snake a component with molecular mass of nearly 60 kDa was found to hydrolyze a-chain of fibrinogen but failed to hydrolyze casein/azocasein and fibrin. A fibrinogen-specific metalloproteinase, the enzyme was inhibited by EDTA. Cobra's venom reduced the mass of donor's fresh blood clots. The copperhead snake's venom and the fractions obtained by gel-filtration (HW-50) and ion exchange chromatography (DEAE-650) were found to hydrolyze casein/azocasein, a- and b-chains of fibrinogen/fibrin and donor's blood clots. The results from the study of the venom and proteolytically active fractions are the evidence for a thrombolytic potential in a copperhead snake's venom. PMID:27420616

  2. Detection of Francisella tularensis-specific antibodies in patients with tularemia by a novel competitive enzyme-linked immunosorbent assay.

    PubMed

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Fujita, Osamu; Uda, Akihiko; Morikawa, Shigeru; Yamada, Akio; Tanabayashi, Kiyoshi

    2013-01-01

    A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R(2) = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species. PMID:23114700

  3. Detection of Francisella tularensis-Specific Antibodies in Patients with Tularemia by a Novel Competitive Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Fujita, Osamu; Uda, Akihiko; Morikawa, Shigeru; Yamada, Akio

    2013-01-01

    A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R2 = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species. PMID:23114700

  4. Evaluation of Enzyme-Linked Immunosorbent Assays for Detection of Mycoplasma bovis-Specific Antibody in Bison Sera

    PubMed Central

    Sacco, Randy E.; Olsen, Steven C.

    2013-01-01

    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. PMID:23843427

  5. Development of Programs for Children with Specific Reading Disabilities in the German Democratic Republic.

    ERIC Educational Resources Information Center

    Raynor, Phyllis F.

    1986-01-01

    Describes a program initiated in East Germany in l962 to identify and remediate specific reading disabilities in elementary school children. Compares aspects of the program with those of programs in other nations. (FL)

  6. Glial high-affinity binding site with specificity for angiotensin II not angiotensin III: a possible N-terminal-specific converting enzyme

    SciTech Connect

    Printz, M.P.; Jennings, C.; Healy, D.P.; Kalter, V.

    1986-01-01

    Anomalous binding properties of angiotensin II to fetal rat brain primary cultures suggested a possible contribution from contaminating glia. To investigate this possibility, cultures of C6 glioma, a clonal rat cell line, were examined for the presence of angiotensin II receptors. A specific high-affinity site for (/sup 125/I)angiotensin II was measured both by traditional methodology using whole cells and by autoradiography. This site shared properties similar to that found with the brain cells, namely low ligand internalization and markedly decreased affinity for N-terminal sarcosine or arginine-angiotensin analogs. The competition rank order was angiotensin II much greater than (Sar1,Ile8)angiotensin II greater than or equal to des(Asp1,Arg2)angiotensin II. Angiotensin III did not compete for binding to the site. High-pressure liquid chromatography analysis indicated that the ligand either in the incubation or bound to the site was stable at 15 degrees C, but there was very rapid and extensive degradation by the C6 glioma cells at 37 degrees C. It is concluded that the site exhibits unusual N-terminal specificity for angiotensin with nanomolar affinity for angiotensin II. If angiotensin III is an active ligand in the brain, the site may have a converting enzyme function. Alternatively, it may form the des-Asp derivatives of angiotensin for subsequent degradation by other enzymatic pathways. Either way, it is proposed that the site may modulate the brain-angiotensin system.

  7. Content and Specifications for the Mod 1 1970 Spelling Program.

    ERIC Educational Resources Information Center

    Butler, Patricia A.

    This paper briefly describes the organization and content of the Southwest Regional Laboratory (SWRL) Mod 1 Spelling Program and its relation to the SWRL Reading Program. The Mod 1 Spelling Program includes 190 words and consists of 22 lessons. Eighteen of the 22 lessons are based on review lists which are composed individually for each…

  8. Influence of different length linker containing DHEA-7-CMO-enzyme conjugates on sensitivity and specificity of DHEA-17-CMO-antibody.

    PubMed

    Shrivastav, Tulsidas G; Chaube, Shail K; Kariya, Kiran P; Singh, Rita; Kumar, Dinesh; Pandit, Deepa; Ujawane, Pragati; Kumari, Poonam; Pandey, Bhavana

    2011-01-01

    Introduction of spacers in enzyme conjugates is known to exert an influence on the assay parameters of steroid enzyme immunoassays. We have introduced 3 to 10 atomic length linkers between enzyme and steroid moieties and studied their effects on sensitivity and specificity of dehydroepiandrosterone enzyme immunoassays. Dehydroepiandrosterone-17-carboxymethyloxime-bovine serum albumin (DHEA-17-CMO-BSA) was used as an immunogen to raise the antiserum in New Zealand white rabbits. Five enzyme conjugates were prepared using DHEA-7-CMO as carboxylic derivative of DHEA and horseradish peroxidase (HRP) as label. These were DHEA-7-CMO-HRP, DHEA-7-CMO-urea-HRP (DHEA-7-CMO-U-HRP), DHEA-7-CMO-ehylenediamine-HRP (DHEA-7-CMO-EDA-HRP), DHEA-7-CMO-carbohydrazide-HRP (DHEA-7-CMO-CH-HRP), and DHEA-7-CMO-adipic acid dihydrazide-HRP (DHEA-7-CMO-ADH-HRP). The influence of different atomic length linkers on sensitivity and specificity were studied with reference to label without linker. The results of the present investigation revealed that with incorporation of linkers, the sensitivity improves, whereas specificity only marginally improves. These differential behaviors of various linkers toward the sensitivity and specificity of assays might be due to the difference in the magnitude of overall forces of attraction between the antibody and the enzyme conjugates. PMID:21728820

  9. Specific serum immunoglobulin D, detected by antibody capture enzyme-linked immunosorbent assay (ELISA), in cytomegalovirus infection.

    PubMed Central

    Mortensen, J; Nielsen, S L; Sørensen, I; Andersen, H K

    1989-01-01

    An antibody capture enzyme-linked immunosorbent assay (ELISA) was developed for the detection of immunoglobulin D (IgD) antibodies to cytomegalovirus (CMV) in sera from blood donors and various groups of patients infected with CMV. This method has previously been found especially valuable in detecting specific antibodies of the IgM, IgE, IgA and IgG class in patients with CMV infection. Specific CMV IgD antibodies were found in 37% of CMV seropositive blood donors and in 47 (88%) of the 53 patients investigated, including bone marrow transplant and renal allograft transplant patients, patients with CMV mononucleosis, neonates with CMV infection and AIDS patients with CMV infection. The highest IgD reactivity was found in patients having either a primary post-transplant CMV infection or CMV mononucleosis. The IgD reactivity in patients with AIDS and in neonates was low. It was also found that in the acute phase of CMV infection the development of CMV antibodies of the IgD class was similar to the development of antibodies of the other classes. The maintenance of IgD activity in some patients together with the presence of CMV IgD antibodies in a great proportion of the blood donors indicates that the development of CMV IgD antibodies resembles that of the IgG class. Determination of specific IgD antibodies offered no advantage over determination of specific antibodies of the IgM, IgE and IgA classes in the diagnosis of CMV infection. PMID:2539278

  10. Peptidase specificity characterization of C- and N-terminal catalytic sites of angiotensin I-converting enzyme.

    PubMed

    Araujo, M C; Melo, R L; Cesari, M H; Juliano, M A; Juliano, L; Carmona, A K

    2000-07-25

    Quenched fluorescence peptides were used to investigate the substrate specificity requirements for recombinant wild-type angiotensin I-converting enzyme (ACE) and two full-length mutants bearing a single functional active site (N- or C-domain). We assayed two series of bradykinin-related peptides flanked by o-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp), namely, Abz-GFSPFXQ-EDDnp and Abz-GFSPFRX-EDDnp (X = natural amino acids), in which the fluorescence appeared when Abz/EDDnp are separated by substrate hydrolysis. Abz-GFSPFFQ-EDDnp was preferentially hydrolyzed by the C-domain while Abz-GFSPFQQ-EDDnp exhibits higher N-domain specificity. Internally quenched fluorescent analogues of N-acetyl-SDKP-OH were also synthesized and assayed. Abz-SDK(Dnp)P-OH, in which Abz and Dnp (2,4-dinitrophenyl) are the fluorescent donor-acceptor pair, was cleaved at the D-K(Dnp) bond with high specificity by the ACE N-domain (k(cat)/K(m) = 1.1 microM(-)(1) s(-)(1)) being practically resistant to hydrolysis by the C-domain. The importance of hydroxyl-containing amino acids at the P(2) position for N-domain specificity was shown by performing the kinetics of hydrolysis of Abz-TDK(Dnp)P-OH and Abz-YDK(Dnp)P-OH. The peptides Abz-YRK(Dnp)P-OH and Abz-FRK(Dnp)P-OH which were hydrolyzed by wild-type ACE with K(m) values of 5.1 and 4.0 microM and k(cat) values of 246 and 210 s(-)(1), respectively, have been shown to be excellent substrates for ACE. The differentiation of the catalytic specificity of the C- and N-domains of ACE seems to depend on very subtle variations on substrate-specific amino acids. The presence of a free C-terminal carboxyl group or an aromatic moiety at the same substrate position determines specific interactions with the ACE active site which is regulated by chloride and seems to distinguish the activities of both domains. PMID:10913258

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

    PubMed

    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

  12. Phloem-specific expression of Yang cycle genes and identification of novel Yang cycle enzymes in Plantago and Arabidopsis.

    PubMed

    Pommerrenig, Benjamin; Feussner, Kirstin; Zierer, Wolfgang; Rabinovych, Valentyna; Klebl, Franz; Feussner, Ivo; Sauer, Norbert

    2011-05-01

    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

  13. 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

  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

    2015-10-01

    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. PMID:26325666

  15. 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...

  16. Enzyme Informatics

    PubMed Central

    Alderson, Rosanna G.; Ferrari, Luna De; Mavridis, Lazaros; McDonagh, James L.; Mitchell, John B. O.; Nath, Neetika

    2012-01-01

    Over the last 50 years, sequencing, structural biology and bioinformatics have completely revolutionised biomolecular science, with millions of sequences and tens of thousands of three dimensional structures becoming available. The bioinformatics of enzymes is well served by, mostly free, online databases. BRENDA describes the chemistry, substrate specificity, kinetics, preparation and biological sources of enzymes, while KEGG is valuable for understanding enzymes and metabolic pathways. EzCatDB, SFLD and MACiE are key repositories for data on the chemical mechanisms by which enzymes operate. At the current rate of genome sequencing and manual annotation, human curation will never finish the functional annotation of the ever-expanding list of known enzymes. Hence there is an increasing need for automated annotation, though it is not yet widespread for enzyme data. In contrast, functional ontologies such as the Gene Ontology already profit from automation. Despite our growing understanding of enzyme structure and dynamics, we are only beginning to be able to design novel enzymes. One can now begin to trace the functional evolution of enzymes using phylogenetics. The ability of enzymes to perform secondary functions, albeit relatively inefficiently, gives clues as to how enzyme function evolves. Substrate promiscuity in enzymes is one example of imperfect specificity in protein-ligand interactions. Similarly, most drugs bind to more than one protein target. This may sometimes result in helpful polypharmacology as a drug modulates plural targets, but also often leads to adverse side-effects. Many cheminformatics approaches can be used to model the interactions between druglike molecules and proteins in silico. We can even use quantum chemical techniques like DFT and QM/MM to compute the structural and energetic course of enzyme catalysed chemical reaction mechanisms, including a full description of bond making and breaking. PMID:23116471

  17. 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. PMID:25449660

  18. Development of a sensitive and specific indirect enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen for detection of specific antibodies against Ehrlichia canis.

    PubMed

    López, Lissett; Venteo, Angel; Aguirre, Enara; García, Marga; Rodríguez, Majosé; Amusátegui, Inmaculada; Tesouro, Miguel A; Vela, Carmen; Sainz, Angel; Rueda, Paloma

    2007-11-01

    An indirect enzyme-linked immunosorbent assay (ELISA) based on baculovirus recombinant P30 protein of Ehrlichia canis and the 1BH4 anticanine IgG monoclonal antibody was developed and evaluated by examining a panel of 98 positive and 157 negative sera using the indirect fluorescent antibody (IFA) test as the reference technique. The P30-based ELISA appeared to be sensitive and specific (77.55% and 95.54%, respectively) when qualitative results (positive/negative) were compared with those of the IFA test; the coefficient of correlation (R) between the 2 tests was 0.833. Furthermore, it was possible to establish a mathematical formula for use in comparing the results of both techniques. These results indicate that recombinant P30 antigen-based ELISA is a suitable alternative of the IFA test for simple, consistent, and rapid serodiagnosis of canine ehrlichiosis. Moreover, the use of this recombinant protein as antigen offers a great advantage for antigen preparation in comparison with other techniques in which the whole E. canis organism is used as antigen. PMID:17998551

  19. 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.

  20. Influence of different length linker containing DHEA-7-cmo-enzyme conjugates on sensitivity and specificity of DHEA-3-hs-antibody.

    PubMed

    Shrivastav, Tulsidas G; Chaube, Shail K; Kariya, Kiran P; Singh, Rita; Kumar, Dinesh; Jain, Parul; Karwar, Suryakant; Uyake, Jyoti; Deshmukh, Bhavana

    2012-01-01

    The introduction of spacers in coating steroid antigen or enzyme conjugates or immunogen is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We have introduced different homobifunctional spacers having varying atomic length (3 to 10) between enzyme and dehydroepiandrosterone (DHEA) moiety and studied their effects on functional parameters such as sensitivity and specificity of DHEA enzyme immunoassays. DHEA-3-hemisuccinate-bovine serum albumin (DHEA-3-HS-BSA) was used as immunogen to raise the antiserum in New Zealand white rabbits. Five enzyme conjugates were prepared using DHEA-7-carboxymethyloxime (DHEA-7-CMO) as carboxylic derivative of DHEA and horseradish peroxidase (HRP) as an enzyme label. These were DHEA-7-CMO-HRP, DHEA-7-CMO-urea-HRP (DHEA-7-CMO-U-HRP), DHEA-7-CMO-ehylenediamine-HRP (DHEA-7-CMO-EDA-HRP), DHEA-7-CMO-carbohydrazide-HRP (DHEA-7-CMO-CH-HRP), and DHEA-7-CMO-adipic acid dihydrazide-HRP (DHEA-7-CMO-ADH-HRP). The influence of different atomic length linkers on sensitivity and specificity were studied with reference to label without linker. The results of the present investigation revealed that DHEA moiety having a 3-hemisuccinate carboxyl arm that is hydrophilic in nature and spacer arm urea that is also hydrophilic in nature when used for the link to the protein carrier and enzyme for the preparation of immunogen and enzyme conjugate respectively resulted in development of assay having comparable sensitivity and lowest ED(50) as compared to other spacers. Thus sensitivity and ED(50) of the assay depend partly on the nature of the steroid and spacer arm link to the carrier protein and the enzyme. PMID:22181816

  1. CHD5, a Brain-Specific Paralog of Mi2 Chromatin Remodeling Enzymes, Regulates Expression of Neuronal Genes

    PubMed Central

    Potts, Rebecca Casaday; Zhang, Peisu; Wurster, Andrea L.; Precht, Patricia; Mughal, Mohamed R.; Wood, William H.; Zhang, Yonqing; Becker, Kevin G.; Mattson, Mark P.; Pazin, Michael J.

    2011-01-01

    CHD5 is frequently deleted in neuroblastoma and is a tumor suppressor gene. However, little is known about the role of CHD5 other than it is homologous to chromatin remodeling ATPases. We found CHD5 mRNA was restricted to the brain; by contrast, most remodeling ATPases were broadly expressed. CHD5 protein isolated from mouse brain was associated with HDAC2, p66ß, MTA3 and RbAp46 in a megadalton complex. CHD5 protein was detected in several rat brain regions and appeared to be enriched in neurons. CHD5 protein was predominantly nuclear in primary rat neurons and brain sections. Microarray analysis revealed genes that were upregulated and downregulated when CHD5 was depleted from primary neurons. CHD5 depletion altered expression of neuronal genes, transcription factors, and brain-specific subunits of the SWI/SNF remodeling enzyme. Expression of gene sets linked to aging and Alzheimer's disease were strongly altered by CHD5 depletion from primary neurons. Chromatin immunoprecipitation revealed CHD5 bound to these genes, suggesting the regulation was direct. Together, these results indicate that CHD5 protein is found in a NuRD-like multi-protein complex. CHD5 expression is restricted to the brain, unlike the closely related family members CHD3 and CHD4. CHD5 regulates expression of neuronal genes, cell cycle genes and remodeling genes. CHD5 is linked to regulation of genes implicated in aging and Alzheimer's disease. PMID:21931736

  2. Broad-Specificity Chemiluminescence Enzyme Immunoassay for (Fluoro)quinolones: Hapten Design and Molecular Modeling Study of Antibody Recognition.

    PubMed

    Zeng, Haopeng; Chen, Jiahong; Zhang, Chijian; Huang, Xin-An; Sun, Yuanming; Xu, Zhenlin; Lei, Hongtao

    2016-04-01

    On the basis of the structural features of (fluoro)quinolones (FQs), pazufloxacin was first used as a generic immunizing hapten to raise a broad-specificity antibody. The obtained polyclonal antibody exhibited broad cross-reactivity ranging from 5.19% to 478.77% with 21 FQs. Furthermore, the antibody was able to recognize these FQs below their maximum residue limits (MRLs) in an indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA), with the limit of detection (LOD) ranging from 0.10 to 33.83 ng/mL. For simply pretreated milk samples with spiked FQs, the ic-CLEIA exhibited an excellent recovery with a range of 84.6-106.9% and an acceptable coefficient of variation below 15%, suggesting its suitability and reliability for the use of a promising tool to detect FQs. Meanwhile, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models, with statistically significant correlation coefficients (q(2)CoMFA = 0.559, r(2)CoMFA = 0.999; q(2)CoMSIA = 0.559, r(2)CoMSIA = 0.994), were established to investigate the antibody recognition mechanism. These two models revealed that in the antibody, the active cavity binding FQs' 7-position substituents worked together with another cavity (binding FQs' 1-position groups) to crucially endow the high cross-reactivity. This investigation will be significant for better exploring the recognition mechanism and for designing new haptens. PMID:26976361

  3. 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.

  4. 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

  5. Development of an equine coronavirus-specific enzyme-linked immunosorbent assay to determine serologic responses in naturally infected horses.

    PubMed

    Kooijman, Lotte J; Mapes, Samantha M; Pusterla, Nicola

    2016-07-01

    Equine coronavirus (EqCoV) infection has been documented in most reports through quantitative qPCR analysis of feces and viral genome sequencing. Although qPCR is used to detect antigen during the acute disease phase, there is no equine-specific antibody test available to study EqCoV seroprevalence in various horse populations. We developed an enzyme-linked immunosorbent assay (ELISA) targeting antibodies to the spike (S) protein of EqCoV and validated its use, using acute and convalescent sera from 83 adult horses involved in 6 outbreaks. The EqCoV S protein-based ELISA was able to reliably detect antibodies to EqCoV in naturally infected horses. The greatest seroconversion rate was observed in horses with clinical signs compatible with EqCoV infection and EqCoV qPCR detection in feces. The EqCoV S protein-based ELISA could be used effectively for seroepidemiologic studies in order to better characterize the overall infection rate of EqCoV in various horse populations. PMID:27216723

  6. An Enzyme-Mediated Methodology for the Site-Specific Radiolabeling of Antibodies Based on Catalyst-Free Click Chemistry

    PubMed Central

    Zeglis, Brian M.; Davis, Charles B.; Aggeler, Robert; Kang, Hee Chol; Chen, Aimei; Agnew, Brian J.; Lewis, Jason S.

    2013-01-01

    An enzyme- and click chemistry-mediated methodology for the site-selective radiolabeling of antibodies on the heavy chain glycans has been developed and validated. To this end, a model system based on the prostate specific membrane antigen-targeting antibody J591, the positron-emitting radiometal 89Zr, and the chelator desferrioxamine has been employed. The methodology consists of four steps: (1) the removal of sugars on the heavy chain region of the antibody to expose terminal N-acetylglucosamine residues; (2) the incorporation of azide-modified N-acetylgalactosamine monosaccharides into the glycans of the antibody; (3) the catalyst-free click conjugation of desferrioxamine-modified dibenzocyclooctynes to the azide-bearing sugars; and (4) the radiolabeling of the chelator-modified antibody with 89Zr. The site-selective labeling methodology has proven facile, reproducible, and robust, producing 89Zr-labeled radioimmunoconjguates that display high stability and immunoreactivity in vitro (>95%) in addition to high selective tumor uptake (67.5 ± 5.0 %ID/g) and tumor-to-background contrast in athymic nude mice bearing PSMA-expressing subcutaneous LNCaP xenografts. Ultimately, this strategy could play a critical role in the development of novel well-defined and highly immunoreactive radioimmunoconjugates for both the laboratory and clinic. PMID:23688208

  7. 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

    2016-01-01

    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. PMID:26729164

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

    PubMed Central

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

    2015-01-01

    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. PMID:26729164

  9. Development of a highly sensitive and specific enzyme-linked immunosorbent assay for detection of Sudan I in food samples.

    PubMed

    Han, Dan; Yu, Meng; Knopp, Dietmar; Niessner, Reinhard; Wu, Mei; Deng, Anping

    2007-08-01

    A highly selective and sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) for Sudan I was developed. Two hapten derivatives with different lengths of carboxylic spacer at the azo-bound para-position were synthesized and coupled to carrier proteins. The hapten-bovine serum albumin (BSA) conjugates were used as immunogens, while the hapten-ovalbumin (OA) conjugates were applied as coating antigens. The antisera which were obtained from four immunized rabbits were characterized in terms of sensitivity and specificity. At optimal experimental conditions it was found that IC50 and LOD values of seven pairs based on four antisera and two coating antigens were in the range of 0.3-2 ng/mL and 0.02-0.1 ng/mL, respectively. The most sensitive ELISA could be established with Sudan I-propionic acid-OA coating antigen and the antiserum which was obtained with the corresponding immunogen. The cross-reactivity values of the four antisera with Sudan II, III, and IV was estimated with 0.1-14.3%. No cross-reactivity was found with six edible colorants Sunset yellow, Amarant, Kermes, Indigotin, Bright blue and Lemon yellow, indicating high specificity for Sudan I. Six food samples were fortified with Sudan I and extracted by simple sample preparation. The methanolic extracts after dilution with methanol:water (5:95, v/v) were analyzed by the developed ELISA. Assay precision and accuracy was estimated by determination of three replicates. Acceptable recovery rates of 92.5-114% and intra-assay coefficients of variation of 5.9-24.8% were obtained. The data were validated by conventional HPLC method. As revealed, both methods were highly correlated (r = 0.9851, n = 7), demonstrating the applicability of the developed ELISA for Sudan I analysis in food samples. PMID:17622156

  10. 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. PMID:24872517

  11. Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Detecting Serum Antibodies against Mycobacterium avium subsp. paratuberculosis in Fallow Deer

    PubMed Central

    Prieto, José M.; Balseiro, Ana; Casais, Rosa; Abendaño, Naiara; Fitzgerald, Liam E.; Garrido, Joseba M.; Juste, Ramon A.

    2014-01-01

    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. PMID:24872517

  12. Educational Specifications: A Blueprint for the Future Program.

    ERIC Educational Resources Information Center

    Glass, Thomas E.

    1986-01-01

    Provides a process model for developing educational specifications--precise statements of activities, curriculum, and functions that a proposed school building will house--and highlights specific requirements and features. A graph and a relationship matrix illustrate the process. Includes 12 references. (MLF)

  13. 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.

  14. Lessons Learned about Workplace Literacy from Military Job-Specific Reading Programs.

    ERIC Educational Resources Information Center

    Philippi, Jorie W.

    For almost half a century the United States military services have incorporated formal literacy programs into job training for those enlistees who are less than fully qualified. Over the years, several successful job-specific reading programs have evolved. In 1975, the Army began the Functional Literacy (FLIT) Program, a program based on a…

  15. Field estimation of the flock-level diagnostic specificity of an enzyme-linked immunosorbent assay for Avian metapneumovirus antibodies in turkeys.

    PubMed

    Muñoz-Zanzi, Claudia; Trampel, Darrell; Hanson, Tim; Harrison, Kristen; Goyal, Sagar; Cortinas, Roberto; Lauer, Dale

    2009-03-01

    Routine serologic testing for Avian metapneumovirus (AMPV) infection of turkey flocks at slaughter is currently being used to monitor changes in the occurrence of AMPV infection in endemic areas and can also be used to detect the emergence of infection in currently unaffected areas. Because of the costs associated with false-positive results, particularly in areas that are free of AMPV infection, there is a need to obtain improved estimates of flock-level specificity (SP). The objective of this study was to estimate flock-level SP of a program to monitor AMPV infection in turkey flocks at processing using a standard enzyme-linked immunosorbent assay (ELISA). A study was carried out in which 37 AMPV-free flocks from 7 Midwest operations were followed serologically. Six percent, 3%, and 0.2% of total samples tested AMPV positive at 8 weeks, 12 weeks, and at processing, respectively. Overall, flock-level SP increased as the cutoff increased and as age increased. Flock-level SP at processing was 97%, if a cutoff of 1 was used (the flock was classified as positive if at least 1 sample tested positive), and 100%, if any other cutoff was used. Administration of antibiotics (P = 0.02) and vaccination for Bordetella avium (P = 0.08) were positively associated with the probability of (false) positive test results. These findings suggest possible cross-reactions with other infections and highlight the need to consider variable diagnostic performance depending on farm conditions. PMID:19286505

  16. 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.

  17. Software programs that address site-specific inventory characteristics issues.

    SciTech Connect

    Dare, J. H.; Cournoyer, M. E.

    2001-01-01

    The proper characterization of Hazardous, Mixed Low-Level, and Mixed Transuranic waste enhances productivity and safety. Hazardous material criteria that need to be considered include physical and health hazards inherent to the waste stream. Other factors that may influence characterization include: particulate diameter, complexing or chelating agent properties, lead, and mercury content, pressurized containers, and P-listed wastes. To meet these requirements are only a simple matter of generating a database with the proper fields. Manufactures and institutional databases bank huge sources of information, such as, work control documents, substance identification, container types, components of mixtures, physical property data, and regulatory data. In this report, utilization of commercially available software programs to take advantage of these resources in addressing waste characterization issues are presented. The application of user-friendly programs eliminates part of the tediousness associated with the complex requirements of certifying to general waste acceptance criteria with minimal impact on programmatic work. In other words, tapping into manufacturer and institutional database provides a way to take advantage of the combined expertise of these resources in managing a cost effective waste certification program as well as adding a quality assurance element to the program.

  18. WORLD-OF-WORK PROGRAM. SPECIFICS 1963-64.

    ERIC Educational Resources Information Center

    CANGEMI, JOSEPH P.; FANTINI, MARIO D.

    TWELVE CHARACTERISTIC SYMPTOMS OF STUDENT MALADJUSTMENT ARE LISTED IN AN ATTEMPT TO HELP PARENTS, TEACHERS, AND COUNSELORS TO IDENTIFY THE POTENTIAL DROPOUT. TO INSURE PROPER GUIDANCE AND SUPERVISION OF THE TRAINING PROGRAM, THE SELECTED STUDENTS WILL ATTEND A VOCATIONAL ORIENTATION WEEKEND AND WILL THEN REPORT FOR JOB INTERVIEWS, BEGINNING NEW…

  19. 13 CFR 130.350 - Specific program responsibilities.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    .... 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 business... affairs of the SBDC network to deliver effective services to the small business community, comply...

  20. 13 CFR 130.350 - Specific program responsibilities.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    .... 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 business... affairs of the SBDC network to deliver effective services to the small business community, comply...

  1. 13 CFR 130.350 - Specific program responsibilities.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    .... 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 business... affairs of the SBDC network to deliver effective services to the small business community, comply...

  2. 13 CFR 130.350 - Specific program responsibilities.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    .... 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 business... affairs of the SBDC network to deliver effective services to the small business community, comply...

  3. 75 FR 61998 - Programs for Specific Positions and Examinations (Miscellaneous)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-07

    ... INFORMATION CONTACT: Ms. Linda Watson by telephone at (202) 606-0830; by fax at (202) 606-2329; by TTY at (202) 418-3134; or by e- mail at linda.watson@opm.gov . SUPPLEMENTARY INFORMATION: On March 20, 2007, OPM published a final rule at 72 FR 12947, to revise the Administrative Law Judge Program. These...

  4. The External Quality Assurance Oversight Laboratory (EQAPOL) Proficiency Program for IFN-gamma Enzyme-Linked ImmunoSpot (IFN-γ ELISpot) assay

    PubMed Central

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

    2014-01-01

    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 the 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. PMID:24685833

  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

    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.

  6. 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.

  7. 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. PMID:26547010

  8. 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. PMID:26815172

  9. 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.

  10. Structure of Type IIL Restriction-Modification Enzyme MmeI in Complex with DNA Has Implications for Engineering New Specificities

    PubMed Central

    Callahan, Scott J.; Luyten, Yvette A.; Gupta, Yogesh K.; Wilson, Geoffrey G.; Roberts, Richard J.; Morgan, Richard D.; Aggarwal, Aneel K.

    2016-01-01

    The creation of restriction enzymes with programmable DNA-binding and -cleavage specificities has long been a goal of modern biology. The recently discovered Type IIL MmeI family of restriction-and-modification (RM) enzymes that possess a shared target recognition domain provides a framework for engineering such new specificities. However, a lack of structural information on Type IIL enzymes has limited the repertoire that can be rationally engineered. We report here a crystal structure of MmeI in complex with its DNA substrate and an S-adenosylmethionine analog (Sinefungin). The structure uncovers for the first time the interactions that underlie MmeI-DNA recognition and methylation (5’-TCCRAC-3’; R = purine) and provides a molecular basis for changing specificity at four of the six base pairs of the recognition sequence (5’-TCCRAC-3’). Surprisingly, the enzyme is resilient to specificity changes at the first position of the recognition sequence (5’-TCCRAC-3’). Collectively, the structure provides a basis for engineering further derivatives of MmeI and delineates which base pairs of the recognition sequence are more amenable to alterations than others. PMID:27082731

  11. 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...

  12. 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.

  13. Ecosystem Services Research Program: LTG 4: Ecosystem specific studies: wetlands

    EPA Science Inventory

    Includes review of ecosystem services derived from marine coastal, Great Lakes coastal, and isolated wetlands. Of particular interest is the development of guidelines to implement the 2008 EPA and ACE rules which require, for the first time, that specific ecosystem services be c...

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

    PubMed

    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

  15. 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

  16. The role of specific Smad linker region phosphorylation in TGF-β mediated expression of glycosaminoglycan synthesizing enzymes in vascular smooth muscle.

    PubMed

    Rostam, Muhamad A; Kamato, Danielle; Piva, Terence J; Zheng, Wenhua; Little, Peter J; Osman, Narin

    2016-08-01

    Hyperelongation of glycosaminoglycan chains on proteoglycans facilitates increased lipoprotein binding in the blood vessel wall and the development of atherosclerosis. Increased mRNA expression of glycosaminoglycan chain synthesizing enzymes in vivo is associated with the development of atherosclerosis. In human vascular smooth muscle, transforming growth factor-β (TGF-β) regulates glycosaminoglycan chain hyperelongation via ERK and p38 as well as Smad2 linker region (Smad2L) phosphorylation. In this study, we identified the involvement of TGF-β receptor, intracellular serine/threonine kinases and specific residues on transcription factor Smad2L that regulate glycosaminoglycan synthesizing enzymes. Of six glycosaminoglycan synthesizing enzymes, xylosyltransferase-1, chondroitin sulfate synthase-1, and chondroitin sulfotransferase-1 were regulated by TGF-β. In addition ERK, p38, PI3K and CDK were found to differentially regulate mRNA expression of each enzyme. Four individual residues in the TGF-β receptor mediator Smad2L can be phosphorylated by these kinases and in turn regulate the synthesis and activity of glycosaminoglycan synthesizing enzymes. Smad2L Thr220 was phosphorylated by CDKs and Smad2L Ser250 by ERK. p38 selectively signalled via Smad2L Ser245. Phosphorylation of Smad2L serine residues induced glycosaminoglycan synthesizing enzymes associated with glycosaminoglycan chain elongation. Phosphorylation of Smad2L Thr220 was associated with XT-1 enzyme regulation, a critical enzyme in chain initiation. These findings provide a deeper understanding of the complex signalling pathways that contribute to glycosaminoglycan chain modification that could be targeted using pharmacological agents to inhibit the development of atherosclerosis. PMID:27153775

  17. Site-Specific, Covalent Immobilization of Dehalogenase ST2570 Catalyzed by Formylglycine-Generating Enzymes and Its Application in Batch and Semi-Continuous Flow Reactors.

    PubMed

    Jian, Hui; Wang, Yingwu; Bai, Yan; Li, Rong; Gao, Renjun

    2016-01-01

    Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly) residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using formylglycine-generating enzymes encoded by a reconstructed set of the pET28a plasmid system for enzyme immobilization. The haloacid dehalogenase ST2570 from Sulfolobus tokodaii was used as a model enzyme. The C-terminal aldehyde-tagged ST2570 (ST2570CQ) exhibited significant enzymological properties, such as new free aldehyde groups, a high level of protein expression and improved enzyme activity. SBA-15 has widely been used as an immobilization support for its large surface and excellent thermal and chemical stability. It was functionalized with amino groups by aminopropyltriethoxysilane. The C-terminal aldehyde-tagged ST2570 was immobilized to SBA-15 by covalent binding. The site-specific immobilization of ST2570 avoided the chemical denaturation that occurs in general covalent immobilization and resulted in better fastening compared to physical adsorption. The site-specific immobilized ST2570 showed 3-fold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The site-specific immobilized ST2570 retained 60% of its original activity after seven cycles of batch operation, and it was superior to the ST2570 immobilized to SBA-15 by physical adsorption, which loses 40% of its original activity when used for the second time. It is remarkable that the site-specific immobilized ST2570 still retained 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. Overall, these results provide support for the industrial-scale production and application of site-specific, covalently immobilized ST2570. PMID:27409601

  18. Neural Differentiation Modulates the Vertebrate Brain Specific Splicing Program

    PubMed Central

    Madgwick, Alicia; Fort, Philippe; Hanson, Peter S.; Thibault, Philippe; Gaudreau, Marie-Claude; Lutfalla, Georges; Möröy, Tarik; Abou Elela, Sherif; Chaudhry, Bill; Elliott, David J.; Morris, Christopher M.; Venables, Julian P.

    2015-01-01

    Alternative splicing patterns are known to vary between tissues but these patterns have been found to be predominantly peculiar to one species or another, implying only a limited function in fundamental neural biology. Here we used high-throughput RT-PCR to monitor the expression pattern of all the annotated simple alternative splicing events (ASEs) in the Reference Sequence Database, in different mouse tissues and identified 93 brain-specific events that shift from one isoform to another (switch-like) between brain and other tissues. Consistent with an important function, regulation of a core set of 9 conserved switch-like ASEs is highly conserved, as they have the same pattern of tissue-specific splicing in all vertebrates tested: human, mouse and zebrafish. Several of these ASEs are embedded within genes that encode proteins associated with the neuronal microtubule network, and show a dramatic and concerted shift within a short time window of human neural stem cell differentiation. Similarly these exons are dynamically regulated in zebrafish development. These data demonstrate that although alternative splicing patterns often vary between species, there is nonetheless a core set of vertebrate brain-specific ASEs that are conserved between species and associated with neural differentiation. PMID:25993117

  19. Neural differentiation modulates the vertebrate brain specific splicing program.

    PubMed

    Madgwick, Alicia; Fort, Philippe; Hanson, Peter S; Thibault, Philippe; Gaudreau, Marie-Claude; Lutfalla, Georges; Möröy, Tarik; Abou Elela, Sherif; Chaudhry, Bill; Elliott, David J; Morris, Christopher M; Venables, Julian P

    2015-01-01

    Alternative splicing patterns are known to vary between tissues but these patterns have been found to be predominantly peculiar to one species or another, implying only a limited function in fundamental neural biology. Here we used high-throughput RT-PCR to monitor the expression pattern of all the annotated simple alternative splicing events (ASEs) in the Reference Sequence Database, in different mouse tissues and identified 93 brain-specific events that shift from one isoform to another (switch-like) between brain and other tissues. Consistent with an important function, regulation of a core set of 9 conserved switch-like ASEs is highly conserved, as they have the same pattern of tissue-specific splicing in all vertebrates tested: human, mouse and zebrafish. Several of these ASEs are embedded within genes that encode proteins associated with the neuronal microtubule network, and show a dramatic and concerted shift within a short time window of human neural stem cell differentiation. Similarly these exons are dynamically regulated in zebrafish development. These data demonstrate that although alternative splicing patterns often vary between species, there is nonetheless a core set of vertebrate brain-specific ASEs that are conserved between species and associated with neural differentiation. PMID:25993117

  20. Specifically designed physical exercise programs improve children's motor abilities.

    PubMed

    Chiodera, P; Volta, E; Gobbi, G; Milioli, M A; Mirandola, P; Bonetti, A; Delsignore, R; Bernasconi, S; Anedda, A; Vitale, M

    2008-04-01

    Physical activity in schools is declining in many countries and inactivity in childhood has become a recognized risk factor. Data from a program of professionally guided physical exercise in primary school children were collected before and after the academic year of training. Four thousand five hundred children (6-10 years) were enrolled, and conditional and coordinative motor abilities (speed, trunk flexibility, long jumping, somersault, Harre circuit test) were measured. Anthropometric measurements were focused on body mass index (BMI), weight and height. Females never showed a significant variation of BMI, whereas males in the first and fourth grades showed significant differences. On the contrary, when considering the motor abilities studied, all the comparisons were highly significant. At the end of training, both males and females did better than at the beginning, and males were constantly faster than females. Our data, generated on a large number of children, show that professionally guided programs of physical education in the primary school lead to significant progresses in the development of conditional and coordinative abilities, without altering BMI values, thus not interfering with the balanced progression of body weight and height. PMID:17490452

  1. 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.

  2. 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.

  3. Pressure effects on enzyme-catalyzed quantum tunneling events arise from protein-specific structural and dynamic changes.

    PubMed

    Hay, Sam; Johannissen, Linus O; Hothi, Parvinder; Sutcliffe, Michael J; Scrutton, Nigel S

    2012-06-13

    The rate and kinetic isotope effect (KIE) on proton transfer during the aromatic amine dehydrogenase-catalyzed reaction with phenylethylamine shows complex pressure and temperature dependences. We are able to rationalize these effects within an environmentally coupled tunneling model based on constant pressure molecular dynamics (MD) simulations. As pressure appears to act anisotropically on the enzyme, perturbation of the reaction coordinate (donor-acceptor compression) is, in this case, marginal. Therefore, while we have previously demonstrated that pressure and temperature dependences can be used to infer H-tunneling and the involvement of promoting vibrations, these effects should not be used in the absence of atomistic insight, as they can vary greatly for different enzymes. We show that a pressure-dependent KIE is not a definitive hallmark of quantum mechanical H-tunneling during an enzyme-catalyzed reaction and that pressure-independent KIEs cannot be used to exclude tunneling contributions or a role for promoting vibrations in the enzyme-catalyzed reaction. We conclude that coupling of MD calculations with experimental rate and KIE studies is required to provide atomistic understanding of pressure effects in enzyme-catalyzed reactions. PMID:22632111

  4. 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... HOUSING AND URBAN DEVELOPMENT GENERAL INTRODUCTION TO FHA PROGRAMS Minimum Property Standards § 200.936 Supplementary specific procedural requirements under HUD building products certification program for solid...

  5. 24 CFR 200.936 - Supplementary specific procedural requirements under HUD building products certification program...

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... requirements under HUD building products certification program for solid fuel type room heaters and fireplace... HOUSING AND URBAN DEVELOPMENT GENERAL INTRODUCTION TO FHA PROGRAMS Minimum Property Standards § 200.936 Supplementary specific procedural requirements under HUD building products certification program for solid...

  6. 24 CFR 200.936 - Supplementary specific procedural requirements under HUD building products certification program...

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... requirements under HUD building products certification program for solid fuel type room heaters and fireplace... HOUSING AND URBAN DEVELOPMENT GENERAL INTRODUCTION TO FHA PROGRAMS Minimum Property Standards § 200.936 Supplementary specific procedural requirements under HUD building products certification program for solid...

  7. 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... HOUSING AND URBAN DEVELOPMENT GENERAL INTRODUCTION TO FHA PROGRAMS Minimum Property Standards § 200.936 Supplementary specific procedural requirements under HUD building products certification program for solid...

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

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... requirements under HUD building products certification program for solid fuel type room heaters and fireplace... HOUSING AND URBAN DEVELOPMENT GENERAL INTRODUCTION TO FHA PROGRAMS Minimum Property Standards § 200.936 Supplementary specific procedural requirements under HUD building products certification program for solid...

  9. 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…

  10. The Relationship between Institutional, Departmental and Program-Specific Variables and the Academic Performance of Division I FBS Football Programs

    ERIC Educational Resources Information Center

    Eigenbrot, Steven C.

    2012-01-01

    This study investigated the connection between the academic evaluation of Division I FBS football programs and the various social settings that influenced these student-athletes. These social settings were classified as: institutional, departmental and program-specific. The experience of the student-athlete is thought to be impacted by all three…

  11. Crystal structures of the Helicobacter pylori MTAN enzyme reveal specific interactions between S-adenosylhomocysteine and the 5'-alkylthio binding subsite.

    PubMed

    Mishra, Vidhi; Ronning, Donald R

    2012-12-01

    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. PMID:23148563

  12. Mi2β Shows Chromatin Enzyme Specificity by Erasing a DNase I-hypersensitive Site Established by ACF*S⃞

    PubMed Central

    Ishii, Haruhiko; Du, Hansen; Zhang, Zhaoqing; Henderson, Angus; Sen, Ranjan; Pazin, Michael J.

    2009-01-01

    ATP-dependent chromatin-remodeling enzymes are linked to changes in gene expression; however, it is not clear how the multiple remodeling enzymes found in eukaryotes differ in function and work together. In this report, we demonstrate that the ATP-dependent remodeling enzymes ACF and Mi2β can direct consecutive, opposing chromatin-remodeling events, when recruited to chromatin by different transcription factors. In a cell-free system based on the immunoglobulin heavy chain gene enhancer, we show that TFE3 induces a DNase I-hypersensitive site in an ATP-dependent reaction that requires ACF following transcription factor binding to chromatin. In a second step, PU.1 directs Mi2β to erase an established DNase I-hypersensitive site, in an ATP-dependent reaction subsequent to PU.1 binding to chromatin, whereas ACF will not support erasure. Erasure occurred without displacing the transcription factor that initiated the site. Other tested enzymes were unable to erase the DNase I-hypersensitive site. Establishing and erasing the DNase I-hypersensitive site required transcriptional activation domains from TFE3 and PU.1, respectively. Together, these results provide important new mechanistic insight into the combinatorial control of chromatin structure. PMID:19158090

  13. Molecular Characterization of an rsmD-Like rRNA Methyltransferase from the Wolbachia Endosymbiont of Brugia malayi and Antifilarial Activity of Specific Inhibitors of the Enzyme

    PubMed Central

    Rana, Ajay Kumar; Chandra, Sharat; Siddiqi, Mohammad Imran

    2013-01-01

    The endosymbiotic organism Wolbachia is an attractive antifilarial drug target. Here we report on the cloning and expression of an rsmD-like rRNA methyltransferase from the Wolbachia endosymbiont of Brugia malayi, its molecular properties, and assays for specific inhibitors. The gene was found to be expressed in all the major life stages of B. malayi. The purified enzyme expressed in Escherichia coli was found to be in monomer form in its native state. The activities of the specific inhibitors (heteroaryl compounds) against the enzyme were tested with B. malayi adult and microfilariae for 7 days in vitro at various concentrations, and NSC-659390 proved to be the most potent compound (50% inhibitory concentration [IC50], 0.32 μM), followed by NSC-658343 (IC50, 4.13 μM) and NSC-657589 (IC50, 7.5 μM). On intraperitoneal administration at 5 mg/kg of body weight for 7 days to adult jirds into which B. malayi had been transplanted intraperitoneally, all the compounds killed a significant proportion of the implanted worms. A very similar result was observed in infected mastomys when inhibitors were administered. Docking studies of enzyme and inhibitors and an in vitro tryptophan quenching experiment were also performed to understand the binding mode and affinity. The specific inhibitors of the enzyme showed a higher affinity for the catalytic site of the enzyme than the nonspecific inhibitors and were found to be potent enough to kill the worm (both adults and microfilariae) in vitro as well as in vivo in a matter of days at micromolar concentrations. The findings suggest that these compounds be evaluated against other pathogens possessing a methyltransferase with a DPPY motif and warrant the design and synthesis of more such inhibitors. PMID:23733469

  14. Gene-specific amplicons from metagenomes as an alternative to directed evolution for enzyme screening: a case study using phenylacetaldehyde reductases.

    PubMed

    Itoh, Nobuya; Kazama, Miki; Takeuchi, Nami; Isotani, Kentaro; Kurokawa, Junji

    2016-06-01

    Screening gene-specific amplicons from metagenomes (S-GAM) is a highly promising technique for the isolation of genes encoding enzymes for biochemical and industrial applications. From metagenomes, we isolated phenylacetaldehyde reductase (par) genes, which code for an enzyme that catalyzes the production of various Prelog's chiral alcohols. Nearly full-length par genes were amplified by PCR from metagenomic DNA, the products of which were fused with engineered par sequences at both terminal regions of the expression vector to ensure proper expression and then used to construct Escherichia coli plasmid libraries. Sequence- and activity-based screening of these libraries identified different homologous par genes, Hpar-001 to -036, which shared more than 97% amino acid sequence identity with PAR. Comparative characterization of these active homologs revealed a wide variety of enzymatic properties including activity, substrate specificity, and thermal stability. Moreover, amino acid substitutions in these genes coincided with those of Sar268 and Har1 genes, which were independently engineered by error-prone PCR to exhibit increased activity in the presence of concentrated 2-propanol. The comparative data from both approaches suggest that sequence information from homologs isolated from metagenomes is quite useful for enzyme engineering. Furthermore, by examining the GAM-based sequence dataset derived from soil metagenomes, we easily found amino acid substitutions that increase the thermal stability of PAR/PAR homologs. Thus, GAM-based approaches can provide not only useful homologous enzymes but also an alternative to directed evolution methodologies. PMID:27419059

  15. 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.

  16. NASIS data base management system - IBM 360/370 OS MVT implementation. 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-modules, reference tables, and data sets which are common to several modules are documented separately.

  17. 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.

  18. Diversity and Strain Specificity of Plant Cell Wall Degrading Enzymes Revealed by the Draft Genome of Ruminococcus flavefaciens FD-1

    PubMed Central

    Berg Miller, Margret E.; Antonopoulos, Dionysios A.; Rincon, Marco T.; Band, Mark; Bari, Albert; Akraiko, Tatsiana; Hernandez, Alvaro; Thimmapuram, Jyothi; Henrissat, Bernard; Coutinho, Pedro M.; Borovok, Ilya; Jindou, Sadanari; Lamed, Raphael; Flint, Harry J.; Bayer, Edward A.; White, Bryan A.

    2009-01-01

    Background Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels. Methodology/Principal Findings The R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb), and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs), polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs). Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases (ORF01222, ORF03896, and ORF01315) exhibited the highest levels of up-regulation. Conclusions/Significance The genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional analysis of the genome has

  19. Pyridoxamine-5-phosphate enzyme-linked immune mass spectrometric assay substrate for linear absolute quantification of alkaline phosphatase to the yoctomole range applied to prostate specific antigen.

    PubMed

    Florentinus-Mefailoski, Angelique; Marshall, John G

    2014-11-01

    There is a need to measure proteins that are present in concentrations below the detection limits of existing colorimetric approaches with enzyme-linked immunoabsorbent assays (ELISA). The powerful enzyme alkaline phosphatase conjugated to the highly specific bacterial protein streptavidin binds to biotinylated macromolecules like proteins, antibodies, or other ligands and receptors with a high affinity. The binding of the biotinylated detection antibody, with resulting amplification of the signal by the catalytic production of reporter molecules, is key to the sensitivity of ELISA. The specificity and amplification of the signal by the enzyme alkaline phosphatase in ELISA together with the sensitivity of liquid chromatography electrospray ionization and mass spectrometry (LC-ESI-MS) to detect femtomole to picomole amounts of reporter molecules results in an ultrasensitive enzyme-linked immune mass spectrometric assay (ELIMSA). The novel ELIMSA substrate pyridoxamine-5-phosphate (PA5P) is cleaved by the enzyme alkaline phosphatase to yield the basic and hydrophilic product pyridoxamine (PA) that elutes rapidly with symmetrical peaks and a flat baseline. Pyridoxamine (PA) and (13)C PA were both observed to show a linear relationship between log ion intensity and quantity from picomole to femtomole amounts by liquid chromatography-electrospray ionization and mass spectrometry. Four independent methods, (i) internal (13)C isotope PA dilution curves, (ii) internal (13)C isotope one-point calibration, (iii) external PA standard curve, and (iv) external (13)C PA standard curve, all agreed within 1 digit in the same order of magnitude on the linear quantification of PA. Hence, a mass spectrometer can be used to robustly detect 526 ymol of the alkaline phosphatase streptavidin probe and accurately quantify zeptomole amounts of PSA against log linear absolute standard by micro electrospray on a simple ion trap. PMID:25259405

  20. [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. PMID:27024917

  1. Detection of Specific Antibodies against Tembusu Virus in Ducks by Use of an E Protein-Based Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Yin, Xiuchen; Lv, Rang; Chen, Xiaodan; Hua, Ronghong

    2013-01-01

    We developed an enzyme-linked immunosorbent assay (ELISA) using eukaryotically expressed E protein as the antigen (termed E-ELISA) to detect antibodies to tembusu virus (TMUV) in ducks. The E-ELISA did not react with antisera to other known pathogens, indicating the E protein is specific for recognizing anti-TMUV antibodies. Compared to the serum neutralization test, the specificity and sensitivity of the E-ELISA was 93.2 and 97.8%, respectively. Therefore, this E-ELISA is a sensitive and rapid method for detecting antibodies against TMUV in ducks. PMID:23616462

  2. The adaptation of mussels Crenomytilus grayanus to cadmium accumulation result in alterations in organization of microsomal enzyme-membrane complex (non-specific phosphatase).

    PubMed

    Zakhartsev; Chelomin; Belcheva

    2000-08-01

    The kinetic parameters (V(m), K(m) and slope) of membrane-bound microsomal non-specific phosphatase (NPase, with G6P as the substrate) from the digestive gland of unexposed and cadmium adapted (45 days for 100 µg Cd(2+)/l) mussels were investigated. In vivo and in vitro approaches were used. Adaptation of mussels (Crenomytilus grayanus) to cadmium resulted in a 1.6-fold increase in NPase activity. V(m) was increased by 1.6-fold, but K(m) was the same in terms of enzyme kinetics. This indicates that the total concentration of the enzymes in the digestive gland increased. Cd(2+) (1 mM) did not significantly alter the activity of the membrane-bound enzyme in vitro both for unexposed and for cadmium adapted mussels, meaning that cadmium ions are not a direct inhibitor of the membrane-bound enzyme in this concentration. The microsomal NPase activity in both unexposed and cadmium adapted mussels was inhibited by in vitro solubilization of microsomes with non-ionic detergent (Triton X100, 0.01%). This inhibition was uncompetitive for microsomes of unexposed mussels (K(m) decreased 3.1-fold). The most drastic events were observed in cadmium adapted mussels, where inhibition was mixed (K(m) decreased 7.2-fold). The simultaneous actions of detergent and cadmium ions did not alter NPase activity significantly in comparison with action of the detergent alone. The differences in the types and the extents of inhibition of the enzymes activity by membrane disordering agent (Triton X100) indicated that the enzyme-membrane complex (NPase) has been altered as a result of adaptation of mussels to cadmium accumulation. We conclude that the mussels produced a new enzyme-membrane complex, with the same K(m) as the previous complex, but with other detergent sensitivity and greater amounts. Thus, the adaptation capacity of this enzyme is reduced as result of adaptation of mussels to cadmium accumulation. PMID:10930649

  3. 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.

  4. 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

  5. A review of specification and verification methods for parallel programs, including the dataflow approach

    SciTech Connect

    Deshpande, A.K.; Kavi, K.M. . Dept. of Computer Science)

    1989-12-01

    Parallel programs are usually described informally, and these descriptions are implemented on parallel computer systems. When a program does not run correctly, it is often very difficult to determine whether the program description or the implementation is incorrect. This has led to the search for more formal descriptions of parallel programs, and proof systems for the verification of the implementations. In this paper, the authors introduce some such formal methods for the specification and verification of parallel programs. They describe a method that is based on dataflow graphs.

  6. Integrated Refractive Effects Prediction System (IREPS): Program performance specification, revision 2.0

    NASA Astrophysics Data System (ADS)

    Hitney, H. V.; Pasahow, E. W.; Obrian, M. E.

    1980-07-01

    IREPS assesses the refractive effects of the lower atmosphere for Naval electromagnetic systems operating above 100 MHz including radar, uhf and microwave communications, electronic warfare and missile guidance. This specification defines and specifies the computer program functions required by IREPS.

  7. 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

  8. 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. PMID:25360132

  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. 76 FR 35319 - Competitive and Noncompetitive Non-Formula Federal Assistance Programs-Specific Administrative...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-17

    ...The National Institute of Food and Agriculture (NIFA) is adopting as a final rule, with changes, an interim rule (published at 74 FR 45968 on September 4, 2009) containing a set of specific administrative requirements for the Beginning Farmer and Rancher Development Program (BFRDP) to supplement the Competitive and Noncompetitive Non-Formula Federal Assistance Programs--General Award......

  11. Identification and Placement Procedures Used in Florida Specific Learning Disabilities Programs.

    ERIC Educational Resources Information Center

    Taylor, Patricia Silke

    1977-01-01

    Sixteen directors of exceptional education programs, 10 supervisors of specific learning disability (SLD) programs, and 101 principals having SLD classes responded to a 14-question survey regarding the identification and placement of SLD students in Florida. Among findings were that principals were the most outspoken in what they considered…

  12. Programming for the Language Disabled Child: Booklet 3: Specific Programmatic Techniques.

    ERIC Educational Resources Information Center

    Texas Education Agency, Austin.

    Described are three specific instructional programing techniques recommended as a result of Project CHILD, a research effort to validate identification, intervention, and teacher education programs for use with language handicapped children. The three intervention models are thought of as being located at equidistant points on a continuum from…

  13. 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)

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

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 41 Public Contracts and Property Management 2 2013-07-01 2012-07-01 true Federal Specifications, Standards and Commercial Item Description Program (Federal Standardization Program). 101-29.221 Section 101-29.221 Public Contracts and Property Management Federal Property Management Regulations System FEDERAL PROPERTY MANAGEMENT REGULATIONS...

  15. 41 CFR 101-29.221 - Federal Specifications, Standards and Commercial Item Description Program (Federal...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 41 Public Contracts and Property Management 2 2010-07-01 2010-07-01 true Federal Specifications, Standards and Commercial Item Description Program (Federal Standardization Program). 101-29.221 Section 101-29.221 Public Contracts and Property Management Federal Property Management Regulations System FEDERAL PROPERTY MANAGEMENT REGULATIONS...

  16. 41 CFR 101-29.221 - Federal Specifications, Standards and Commercial Item Description Program (Federal...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 41 Public Contracts and Property Management 2 2014-07-01 2012-07-01 true Federal Specifications, Standards and Commercial Item Description Program (Federal Standardization Program). 101-29.221 Section 101-29.221 Public Contracts and Property Management Federal Property Management Regulations System FEDERAL PROPERTY MANAGEMENT REGULATIONS...

  17. 41 CFR 101-29.221 - Federal Specifications, Standards and Commercial Item Description Program (Federal...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 41 Public Contracts and Property Management 2 2011-07-01 2007-07-01 true Federal Specifications, Standards and Commercial Item Description Program (Federal Standardization Program). 101-29.221 Section 101-29.221 Public Contracts and Property Management Federal Property Management Regulations System FEDERAL PROPERTY MANAGEMENT REGULATIONS...

  18. 41 CFR 101-29.221 - Federal Specifications, Standards and Commercial Item Description Program (Federal...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 41 Public Contracts and Property Management 2 2012-07-01 2012-07-01 false Federal Specifications, Standards and Commercial Item Description Program (Federal Standardization Program). 101-29.221 Section 101-29.221 Public Contracts and Property Management Federal Property Management Regulations System FEDERAL PROPERTY MANAGEMENT REGULATIONS...

  19. 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…

  20. 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

  1. 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

  2. Influence of sporophore development, damage, storage, and tissue specificity on the enzymic formation of volatiles in mushrooms (Agaricus bisporus).

    PubMed

    Combet, Emilie; Henderson, Janey; Eastwood, Daniel C; Burton, Kerry S

    2009-05-13

    The enzymic oxidation of the polyunsaturated fatty acid-linoleic acid leads, in fungi, to the formation of a unique class of nonconjugated hydroperoxides, which are cleaved to form eight-carbon volatiles characteristic of mushroom and fungal flavor. However, the enzymes involved in this biosynthetic pathway, the bioavailability of the fatty acid substrate, and the occurrence of the reaction products (hydroperoxides and eight-carbon volatiles) are not fully understood. This study investigated the lipids, fatty acids, and hydroperoxide levels, as well as eight-carbon volatile variations in the fungal model Agaricus bisporus, according to four parameters: sporophore development, postharvest storage, tissue type, and damage. Eight-carbon volatiles were measured using solid phase microextraction and gas chromatography-mass spectrometry. Tissue disruption had a major impact on the volatile profile, both qualitatively and quantitatively; 3-octanone was identified as the main eight-carbon volatile in whole and sliced sporophore, an observation overlooked in previous studies due to the use of tissue disruption and solvent extraction for analysis. Fatty acid oxidation and eight-carbon volatile emissions decreased with sporophore development and storage, and differed according to tissue type. The release of 1-octen-3-ol and 3-octanone by incubation of sporophore tissue homogenate with free linoleic acid was inhibited by acetylsalicylic acid, providing evidence for the involvement of a heme-dioxygenase in eight-carbon volatile production. PMID:19326947

  3. 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.

  4. A localized tolerance in the substrate specificity of the fluorinase enzyme enables "last-step" 18F fluorination of a RGD peptide under ambient aqueous conditions.

    PubMed

    Thompson, Stephen; Zhang, Qingzhi; Onega, Mayca; McMahon, Stephen; Fleming, Ian; Ashworth, Sharon; Naismith, James H; Passchier, Jan; O'Hagan, David

    2014-08-18

    A strategy for last-step (18)F fluorination of bioconjugated peptides is reported that exploits an "Achilles heel" in the substrate specificity of the fluorinase enzyme. An acetylene functionality at the C-2 position of the adenosine substrate projects from the active site into the solvent. The fluorinase catalyzes a transhalogenation of 5'-chlorodeoxy-2-ethynyladenosine (ClDEA) to 5'-fluorodeoxy-2-ethynyladenosine (FDEA). Extending a polyethylene glycol linker from the terminus of the acetylene allows the presentation of bioconjugation cargo to the enzyme for (18)F labelling. The method uses an aqueous solution (H2(18)O) of [(18)F]fluoride generated by the cyclotron and has the capacity to isotopically label peptides of choice for positron emission tomography (PET). PMID:24989327

  5. 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.

  6. Story Specifications for the Second Year of the SWRL Reading Program.

    ERIC Educational Resources Information Center

    Cronnell, Bruce

    This paper describes the types of stories in the second year of the Southwest Regional Laboratory (SWRL) Reading Program and contains specifications for each type of story. The stories are described in terms of their characters and plot content, procedures for selecting new-word content, and word-usage and story-length specifications. This…

  7. 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. PMID:21883237

  8. A large database DNA sequence handling program with generalized searching specifications.

    PubMed

    Stockwell, P A

    1982-01-11

    The program described allows for the creation and manipulation of files of DNA sequence data up to very great lengths. The program uses its own paging system to load segments of the sequence into a small internal buffer so that the program does not have excessive memory requirements. The program offers a menu of functions to the user, and has been written to be forgiving of user errors. A code for the generalised specification of bases as a series of groups (i.e. A or T, Purine, etc.) has been devised and can be used in search specifications or in sequence files. Versions of the program have been developed to run with special efficiency under DIGITAL's RT11 operating system or to run under systems with a suitable implementation of FORTRAN VI. PMID:7063398

  9. The unique functional role of the C-HS hydrogen bond in the substrate specificity and enzyme catalysis of type 1 methionine aminopeptidase.

    PubMed

    Reddi, Ravikumar; Singarapu, Kiran Kumar; Pal, Debnath; Addlagatta, Anthony

    2016-07-19

    It is intriguing how nature attains recognition specificity between molecular interfaces where there is no apparent scope for classical hydrogen bonding or polar interactions. Methionine aminopeptidase (MetAP) is one such enzyme where this fascinating conundrum is at play. In this study, we demonstrate that a unique C-HS hydrogen bond exists between the enzyme methionine aminopeptidase (MetAP) and its N-terminal-methionine polypeptide substrate, which allows specific interaction between apparent apolar interfaces, imposing a strict substrate recognition specificity and efficient catalysis, a feature replicated in Type I MetAPs across all kingdoms of life. We evidence this evolutionarily conserved C-HS hydrogen bond through enzyme assays on wild-type and mutant MetAP proteins from Mycobacterium tuberculosis that show a drastic difference in catalytic efficiency. The X-ray crystallographic structure of the methionine bound protein revealed a conserved water bridge and short contacts involving the Met side-chain, a feature also observed in MetAPs from other organisms. Thermal shift assays showed a remarkable 3.3 °C increase in melting temperature for methionine bound protein compared to its norleucine homolog, where C-HS interaction is absent. The presence of C-HS hydrogen bonding was also corroborated by nuclear magnetic resonance spectroscopy through a change in chemical shift. Computational chemistry studies revealed the unique role of the electrostatic environment in facilitating the C-HS interaction. The significance of this atypical hydrogen bond is underscored by the fact that the function of MetAP is essential for any living cell. PMID:27225936

  10. Dengue virus serotyping based on envelope and membrane and nonstructural protein NS1 serotype-specific capture immunoglobulin M enzyme-linked immunosorbent assays.

    PubMed

    Shu, Pei-Yun; Chen, Li-Kuang; Chang, Shu-Fen; Su, Chien-Ling; Chien, Li-Jung; Chin, Chuan; Lin, Ting-Hsiang; Huang, Jyh-Hsiung

    2004-06-01

    Envelope and membrane (E/M) and nonstructural protein NS1 serotype-specific capture Immunoglobulin M (IgM) enzyme-linked immunosorbent assays (ELISAs) were developed to differentiate four dengue virus serotypes. A total of 93 anti-dengue virus IgM-positive serum samples collected between days 5 and 45 of illness from 59 confirmed dengue patients were analyzed. The results showed that positive serotype specificity could be identified for 86.1 and 47.6% of serum samples tested for E/M-specific IgM antibodies versus 83.3 and 42.9% of serum samples tested for NS1-specific IgM antibodies from patients with primary and secondary dengue virus infections, respectively. Dual analyses with both E/M and NS1 serotype-specific capture IgM ELISAs showed that positive serotype specificity could be correctly identified for 98.6 and 61.9% of all of the primary and secondary serum samples tested, respectively. These findings suggested that E/M and NS1 serotype-specific capture IgM ELISAs have the potential to be of use in dengue virus serotyping. PMID:15184425

  11. 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

  12. Cell-Specific Expression of Homospermidine Synthase, the Entry Enzyme of the Pyrrolizidine Alkaloid Pathway in Senecio vernalis, in Comparison with Its Ancestor, Deoxyhypusine Synthase1

    PubMed Central

    Moll, Stefanie; Anke, Sven; Kahmann, Uwe; Hänsch, Robert; Hartmann, Thomas; Ober, Dietrich

    2002-01-01

    Pyrrolizidine alkaloids (PAs) are constitutive plant defense compounds with a sporadic taxonomic occurrence. The first committed step in PA biosynthesis is catalyzed by homospermidine synthase (HSS). Recent evidence confirmed that HSS evolved by gene duplication from deoxyhypusine synthase (DHS), an enzyme involved in the posttranslational activation of the eukaryotic translation initiation factor 5A. To better understand the evolutionary relationship between these two enzymes, which are involved in completely different biological processes, we studied their tissue-specific expression. RNA-blot analysis, reverse transcriptase-PCR, and immunolocalization techniques demonstrated that DHS is constitutively expressed in shoots and roots of Senecio vernalis (Asteraceae), whereas HSS expression is root specific and restricted to distinct groups of endodermis and neighboring cortex cells located opposite to the phloem. All efforts to detect DHS by immunolocalization failed, but studies with promoter-β-glucuronidase fusions confirmed a general expression pattern, at least in young seedlings of tobacco (Nicotiana tabacum). The expression pattern for HSS differs completely from its ancestor DHS due to the adaptation of HSS to the specific requirements of PA biosynthesis. PMID:12226485

  13. Detection of Semliki Forest virus in cell culture by use of an enzyme immunoassay with peroxidase-labeled monoclonal antibodies specific for glycoproteins E1 and E2.

    PubMed Central

    van Tiel, F H; Boere, W A; Vinjé, J; Harmsen, T; Benaissa-Trouw, B J; Kraaijeveld, C A; Snippe, H

    1984-01-01

    Four noncompeting monoclonal antibodies (MA) directed against either the E1 (UM 8.64 and 8.139) or E2 (UM 8.55 and 8.73) glycoprotein of Semliki Forest virus were purified and labeled with horseradish peroxidase. Each enzyme-labeled MA was tested alone and in combination with others for its sensitivity to detect virus-infected cells. Semliki Forest virus-infected L cells seeded as monolayers in 96-well plates were screened for the virus after incubation with enzyme-labeled MA and a substrate. In this system single enzyme-labeled MA even at high dilution (10(3.0) to 10(4.5] were able to detect virus-infected cells. The sensitivity of the test could be enhanced by combining two noncompeting MA (10(4.5) to 10(5.0]. Combinations of three and four MA were less effective, due to high absorbance values for noninfected cells. The threshold of virus defection was between 10(5) and 10(6) PFU/ml. This test is sensitive and specific and therefore may be useful for diagnostic purposes. PMID:6386855

  14. Substrate specificity of the adenylation enzyme SgcC1 involved in the biosynthesis of the enediyne antitumor antibiotic C-1027.

    PubMed

    Van Lanen, Steven G; Lin, Shuangjun; Dorrestein, Pieter C; Kelleher, Neil L; Shen, Ben

    2006-10-01

    C-1027 is an enediyne antitumor antibiotic composed of a chromophore with four distinct chemical moieties, including an (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety that is derived from l-alpha-tyrosine. SgcC4, a novel aminomutase requiring no added co-factor that catalyzes the formation of the first intermediate (S)-beta-tyrosine and subsequently SgcC1 homologous to adenylation domains of nonribosomal peptide synthetases, was identified as specific for the SgcC4 product and did not recognize any alpha-amino acids. To definitively establish the substrate for SgcC1, a full kinetic characterization of the enzyme was performed using amino acid-dependent ATP-[(32)P]PP(i) exchange assay to monitor amino acid activation and electrospray ionization-Fourier transform mass spectroscopy to follow the loading of the activated beta-amino acid substrate to the peptidyl carrier protein SgcC2. The data establish (S)-beta-tyrosine as the preferred substrate, although SgcC1 shows promiscuous activity toward aromatic beta-amino acids such as beta-phenylalanine, 3-chloro-beta-tyrosine, and 3-hydroxy-beta-tyrosine, but all were <50-fold efficient. A putative active site mutant P571A adjacent to the invariant aspartic acid residue of all alpha-amino acid-specific adenylation domains known to date was prepared as a preliminary attempt to probe the substrate specificity of SgcC1; however the mutation resulted in a loss of activity with all substrates except (S)-beta-tyrosine, which was 142-fold less efficient relative to the wild-type enzyme. In total, SgcC1 is now confirmed to catalyze the second step in the biosynthesis of the (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety of C-1027, presenting downstream enzymes with an (S)-beta-tyrosyl-S-SgcC2 thioester substrate, and represents the first beta-amino acid-specific adenylation enzyme characterized biochemically. PMID:16887797

  15. Combination of Xylanase and Debranching Enzymes Specific to Wheat Arabinoxylan Improve the Growth Performance and Gut Health of Broilers.

    PubMed

    Lei, Zhao; Shao, Yuxin; Yin, Xiaonan; Yin, Dafei; Guo, Yuming; Yuan, Jianmin

    2016-06-22

    Arabinoxylan (AX) is the major antinutritional factor of wheat. This study evaluated the synergistic effects of xylanase and debranching enzymes (arabinofuranosidase [ABF] and feruloyl esterase [FAE]) on AX. During in vitro tests, the addition of ABF or FAE accelerated the hydrolysis of water-soluble AX (WE-AX) and water-insoluble AX (WU-AX) and produced more xylan oligosaccharides (XOS) than xylanase alone. XOS obtained from WE-AX stimulated greater proliferation of Lactobacillus brevis and Bacillus subtilis than did fructo-oligosaccharides (FOS) and glucose. During in vivo trials, xylanase increased the average daily growth (ADG), decreased the feed-conversion ratio (FCR), and reduced the digesta viscosity of jejunum and intestinal lesions of broilers fed a wheat-based diet on day 36. ABF or FAE additions further improved these effects. Broilers fed a combination of xylanase, ABF, and FAE exhibited the best growth. In conclusion, the synergistic effects among xylanase, ABF, and FAE increased AX degradation, which improve the growth performance and gut health of broilers. PMID:27285356

  16. Copper-modified gold electrode specific for monosaccharide detection Use in amperometric determination of phenylmercury based on invertase enzyme inhibition.

    PubMed

    Mohammadi, H; Amine, A; El Rhazi, M; Brett, C M A

    2004-04-19

    The electrochemical oxidation of mono- and disaccharides at various copper-modified electrodes is reported: glassy carbon modified at open circuit or by electrochemical deposition of copper, gold modified by electrochemical deposition, and at bulk copper electrodes. A comparative study of these four electrodes was made by linear sweep voltammetry and amperometry. The maximum oxidation peak separation between disaccharides and monosaccharides is about 200mV. After optimization, amperometric determination of monosaccharides was done at +0.30 versus Ag/AgCl in 0.15M NaOH at the copper-modified gold electrode. Using the developed method, the enzymatic activities of invertase and beta-galactosidase were determined through their reaction with sucrose and lactose, respectively. Validation was carried out by a spectrophotometric method based on 3,5-dinitrosalicylic acid, and it was shown that the proposed electrochemical method is more sensitive. The analytical utility of the copper-modified gold electrode was tested for the determination of organic mercury. Addition of phenylmercury standards to the invertase solution caused a decrease in the enzyme activity, and allowed the determination of phenylmercury in pharmaceutical samples. The concentration has been determined in the 10-55ngml(-1) range. PMID:18969385

  17. Two separate key enzymes and two pathway-specific transcription factors are involved in fusaric acid biosynthesis in Fusarium fujikuroi.

    PubMed

    Studt, Lena; Janevska, Slavica; Niehaus, Eva-Maria; Burkhardt, Immo; Arndt, Birgit; Sieber, Christian M K; Humpf, Hans-Ulrich; Dickschat, Jeroen S; Tudzynski, Bettina

    2016-03-01

    Fusaric acid (FSA) is a mycotoxin produced by several fusaria, including the rice pathogen Fusarium fujikuroi. Genes involved in FSA biosynthesis were previously identified as a cluster containing a polyketide synthase (PKS)-encoding (FUB1) and four additional genes (FUB2-FUB5). However, the biosynthetic steps leading to FSA as well as the origin of the nitrogen atom, which is incorporated into the polyketide backbone, remained unknown. In this study, seven additional cluster genes (FUB6-FUB12) were identified via manipulation of the global regulator FfSge1. The extended FUB gene cluster encodes two Zn(II)2 Cys6 transcription factors: Fub10 positively regulates expression of all FUB genes, whereas Fub12 is involved in the formation of the two FSA derivatives, i.e. dehydrofusaric acid and fusarinolic acid, serving as a detoxification mechanism. The major facilitator superfamily transporter Fub11 functions in the export of FSA out of the cell and is essential when FSA levels become critical. Next to Fub1, a second key enzyme was identified, the non-canonical non-ribosomal peptide synthetase Fub8. Chemical analyses of generated mutant strains allowed for the identification of a triketide as PKS product and the proposition of an FSA biosynthetic pathway, thereby unravelling the unique formation of a hybrid metabolite consisting of this triketide and an amino acid moiety. PMID:26662839

  18. A tRNA-dependent cysteine biosynthesis enzyme recognizes the selenocysteine-specific tRNA in Escherichia coli

    PubMed Central

    Yuan, Jing; Hohn, Michael J.; Sherrer, R. Lynn; Palioura, Sotiria; Su, Dan; Söll, Dieter

    2010-01-01

    The essential methanogen enzyme Sep-tRNA:Cys-tRNA synthase (SepCysS) converts O-phosphoseryl-tRNACys (Sep-tRNACys) into Cys-tRNACys in the presence of a sulfur donor. Likewise, Sep-tRNA:Sec-tRNA synthase (SepSecS) converts O-phosphoseryl-tRNASec (Sep-tRNASec) to selenocysteinyl-tRNASec (Sec-tRNASec) using a selenium donor. While the Sep moiety of the aminoacyl-tRNA substrates is the same in both reactions, tRNACys and tRNASec differ greatly in sequence and structure. In an Escherichia coli genetic approach that tests for formate dehydrogenase activity in the absence of selenium donor we show that SeptRNASec is a substrate for SepCysS. Since Sec and Cys are the only active site amino acids known to sustain FDH activity, we conclude that SepCysS converts Sep-tRNASec to Cys-tRNASec, and that Sep is crucial for SepCysS recognition. PMID:20493852

  19. Biochemical Studies and Ligand-bound Structures of Biphenyl Dehydrogenase from Pandoraea pnomenusa Strain B-356 Reveal a Basis for Broad Specificity of the Enzyme*

    PubMed Central

    Dhindwal, Sonali; Patil, Dipak N.; Mohammadi, Mahmood; Sylvestre, Michel; Tomar, Shailly; Kumar, Pravindra

    2011-01-01

    Biphenyl dehydrogenase, a member of short-chain dehydrogenase/reductase enzymes, catalyzes the second step of the biphenyl/polychlorinated biphenyls catabolic pathway in bacteria. To understand the molecular basis for the broad substrate specificity of Pandoraea pnomenusa strain B-356 biphenyl dehydrogenase (BphBB-356), the crystal structures of the apo-enzyme, the binary complex with NAD+, and the ternary complexes with NAD+-2,3-dihydroxybiphenyl and NAD+-4,4′-dihydroxybiphenyl were determined at 2.2-, 2.5-, 2.4-, and 2.1-Å resolutions, respectively. A crystal structure representing an intermediate state of the enzyme was also obtained in which the substrate binding loop was ordered as compared with the apo and binary forms but it was displaced significantly with respect to the ternary structures. These five structures reveal that the substrate binding loop is highly mobile and that its conformation changes during ligand binding, starting from a disorganized loop in the apo state to a well organized loop structure in the ligand-bound form. Conformational changes are induced during ligand binding; forming a well defined cavity to accommodate a wide variety of substrates. This explains the biochemical data that shows BphBB-356 converts the dihydrodiol metabolites of 3,3′-dichlorobiphenyl, 2,4,4′-trichlorobiphenyl, and 2,6-dichlorobiphenyl to their respective dihydroxy metabolites. For the first time, a combination of structural, biochemical, and molecular docking studies of BphBB-356 elucidate the unique ability of the enzyme to transform the cis-dihydrodiols of double meta-, para-, and ortho-substituted chlorobiphenyls. PMID:21880718

  20. The proteolytic enzymes of the K-1 strain of Streptomyces griseus obtained from a commercial preparation (Pronase). Specificity and immobilization of aminopeptidase.

    PubMed

    Vosbeck, K D; Greenberg, B D; Awad, W M

    1975-05-25

    We recently described the purification of two aminopeptidases from Streptomyces griseus (Vosbeck, K.D., Chow, K.-F., and Awad, W.M., Jr. (1973) J. Biol. Chem. 248, 6329-6034). An analysis of the amino acid composition reveals very little differences in the two proteins. Each protein has alanine as the NH2-terminal residue. The aminopeptidases were treated separately with acetic anhydride; as noted in the past, the presence of glycerol is required to achieve excellent yields of acetylated active derivatives (Siegel, S., and Awad, W.M., Jr. (1973 J. Biol. Chem. 248, 3233-3240). However, in the present case much higher concentrations of glycerol (50%) are needed during acetylation to obtain derivatives with completely reacted NH2-terminal residues. The epsilon-amino groups were not completely acetylated. In contrast to the native enzymes, the acetylated derivatives show an affinity for DEAE-cellulose, a property consonant with the changes in net charge. The kinetic constants for each enzyme against L-leucine-p-nitroanilide do not change significantly after acetylation. The specificities of the two aminopeptidases were examined extensively on a semiquantitative basis. The activities are not restricted by the length of substrate chains. Each enzyme shows a preference for hydrophobic residues at the ultimate and penultimate positions. Charge residues are released a slower rates. No prolidase activity is demonstrable even at high enzyme to substrate ratios; however, NH2-terminal proline residues are released readily. D-Amino acid residues at the ultimate or penultimate position substantially reduce the rate of hydrolysis; D-leucyl-D-leucine is not hydrolyzed... PMID:805135

  1. 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.

  2. 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 ...

  3. Sperm-specific expression of angiotensin-converting enzyme (ACE) is mediated by a 91-base-pair promoter containing a CRE-like element.

    PubMed Central

    Howard, T; Balogh, R; Overbeek, P; Bernstein, K E

    1993-01-01

    The gene encoding the testis isozyme of angiotensin-converting enzyme (testis ACE) is one example of the many genes expressed uniquely during spermatogenesis. This protein is expressed by developing germ cells late in their development and results from the activation of a sperm-specific promoter that is located within intron 12 of the gene encoding the somatic isozyme of ACE. In vitro transcription, DNase footprinting, gel shift assays, and transgenic mouse studies have been used to define the minimal testes ACE promoter and to characterize DNA-protein interactions mediating germ cell-specific expression. These studies show that proper cell- and stage-specific expression of testis ACE requires only a small portion of the immediate upstream sequence extending to -91. A critical motif within this core promoter is a cyclic AMP-responsive element sequence that interacts with a testis-specific transactivating factor. Since this putative cyclic AMP-responsive element has been conserved within the testis ACE promoters of different species and is found at the same site in other genes that are expressed specifically in the testis, it may provide a common mechanism for the recognition of sperm-specific promoters. Images PMID:8380220

  4. Gender-specific socioeconomic impacts of development programs in Sri Lanka.

    PubMed

    Stoeckel, J; Sirisena, N L

    1988-10-01

    Data from a Sri Lanka national sample survey -- 3597 households stratified on the basis of development program areas -- were analyzed to compare impacts of 3 national development programs and their combinations upon the occupational and income status of females and males in Sri Lanka. These programs, implemented over the last 30 years, are guaranteed price schemes that develop markets for agricultural produce, land settlement schemes that include irrigation, and rural electrification. To date, no attempt has been made to assess the gender-specific socioeconomic impacts of these individual programs and their combinations. It was hypothesized that the utilization of development program outputs will exert a gender-differential impact upon occupational and income status, but the magnitude and direction of the impacts remain to be determined. Path analysis was applied to estimate the model for each development program and their mixes for males and females separated. A multistage stratified sampling design was utilized. All of the development programs and their mixes exhibited significant effect of educational attainment upon participation in nonagricultural occupations. Rural electrification (RE) was the only program whose effect was positive; in combinations with education it accounted for 15% of the variation in occupation. Among the programs that were negatively related to male participation in nonagricultural occupations, the most important predictors were the land settlement (LS) and guarantee price scheme (GPS) programs. Each program contributed to over 1/5 of the variation in occupation net of educational attainment. RE was the only program that was not significantly related to female participation in nonhousehold occupations. All of the remaining programs exerted a positive effect upon occupation. 3 of these programs -- RE + LS, GPS, and LS + GPS -- were of almost equally high importance in predicting participation of females in nonhousehold occupations, and in

  5. 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.

  6. A single catalytic domain of the junction-resolving enzyme T7 endonuclease I is a non-specific nicking endonuclease

    PubMed Central

    Guan, Chudi; Kumar, Sanjay

    2005-01-01

    A stable heterodimeric protein containing a single correctly folded catalytic domain (SCD) of T7 endonuclease I was produced by means of a trans-splicing intein system. As predicted by a model presented earlier, purified SCD protein acts a non-specific nicking endonuclease on normal linear DNA. The SCD retains some ability to recognize and cleave a deviated DNA double-helix near a nick or a strand-crossing site. Thus, we infer that the non-specific and nicked-site cleavage activities observed for the native T7 endonuclease I (as distinct from the resolution activity) are due to uncoordinated actions of the catalytic domains. The positively charged C-terminus of T7 Endo I is essential for the enzymatic activity of SCD, as it is for the native enzyme. We propose that the preference of the native enzyme for the resolution reaction is achieved by cooperativity in the binding of its two catalytic domains when presented with two of the arms across a four-way junction or cruciform structure. PMID:16264086

  7. 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

    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

  8. 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.

  9. 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

  10. A novel mu-capture enzyme-linked immunosorbent assay based on recombinant proteins for sensitive and specific diagnosis of hemorrhagic fever with renal syndrome.

    PubMed Central

    Zöller, L G; Yang, S; Gött, P; Bautz, E K; Darai, G

    1993-01-01

    Hantavirus nucleocapsid protein has recently been identified as a major antigen inducing an early and long-lasting humoral immune response in patients with hemorrhagic fever with renal syndrome. A mu-capture enzyme-linked immunosorbent assay utilizing recombinant nucleocapsid proteins of Hantavirus strains Hantaan 76-118 (Hantaan serotype) and CG 18-20 (Puumala serotype) as diagnostic antigens and specific monoclonal antibodies as the detection system has been developed. Histidine-tailed recombinant proteins were expressed in Escherichia coli and purified in a single step by affinity chromatography on a nickel-chelate resin. The assay was evaluated with a panel of sera from patients with hemorrhagic fever with renal syndrome originating from various geographic regions. The overall sensitivity of the mu-capture enzyme-linked immunosorbent assay (both recombinant antigens) was 100%, and its specificity was also found to be 100%. Immunoglobulin M antibodies were detected as early as on day 3, and maximum titers were obtained between days 8 and 25 after onset of the disease. The assay was regularly found to be positive within 3 to 4 months but in some cases up to 2 years after the acute phase of the disease. Images PMID:8099085

  11. Systemic administration of platelet-activating factor in rat reduces specific pulmonary uptake of circulating monoclonal antibody to angiotensin-converting enzyme.

    PubMed

    Atochina, E N; Hiemisch, H H; Muzykantov, V R; Danilov, S M

    1992-01-01

    The biodistribution of radiolabeled mouse monoclonal antibody (MoAb) to angiotensin-converting enzyme (ACE) and control, nonimmune mouse IgG in platelet activating factor (PAF)-treated rats was studied. The blood level of both preparations was slightly decreased (90% of the control) in PAF-treated rats. Specific pulmonary accumulation of anti-ACE MoAb was reduced to 50% of control in contrast to a doubling in nonspecific pulmonary uptake of non-immune IgG. The changes in anti-ACE MoAb biodistribution were lung-specific and were accompanied by decrease in the pulmonary ACE activity (to 60% of control) and increase in serum ACE activity (to 170% of control). Thus anti-ACE MoAb reveals PAF-induced changes in the status of the pulmonary ACE and therefore can be used for the studies of pathology of the pulmonary endothelium. PMID:1331624

  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. Enzyme-Linked Immunosorbent Assay Specific for (1→6) Branched, (1→3)-β-d-Glucan Detection in Environmental Samples

    PubMed Central

    Milton, Donald K.; Alwis, K. Udeni; Fisette, Leslie; Muilenberg, Michael

    2001-01-01

    (1→3)-β-d-Glucans have been recognized as a potential causative agent responsible for bioaerosol-induced respiratory symptoms observed in both indoor and occupational environments. A specific enzyme immunoassay was developed to quantify (1→6) branched, (1→3)-β-d-glucans in environmental samples. The assay was based on the use of a high-affinity receptor (galactosyl ceramide) specific for (1→3)-β-d-glucans as a capture reagent and a monoclonal antibody specific for fungal cell wall β-d-glucans as a detector reagent. The assay was highly specific for (1→6) branched, (1→3)-β-d-glucans (such as that from Saccharomyces cerevisiae) and did not show any response at 200 ng/ml to curdlan, laminarin, pustulan, dextran, mannan, carboxymethyl cellulose, and endotoxins. The detection level was 0.8 ng/ml for baker's yeast glucan and Betafectin. A coefficient of variation of 7.8% was obtained for (1→3)-β-d-glucans in house dust samples. Metal working fluids spiked with (1→3)-β-d-glucans inhibited the glucan assay. Because the assay is specific for (1→6) branched, (1→3)-β-d-glucans and is sensitive and reproducible, it will be useful for the investigation of health effects from exposure to this class of biologically active molecules. PMID:11722887

  14. 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. PMID:23474534

  15. Copper-dependent metabolism of Cu,Zn-superoxide dismutase in human K562 cells. Lack of specific transcriptional activation and accumulation of a partially inactivated enzyme.

    PubMed Central

    Steinkühler, C; Carrì, M T; Micheli, G; Knoepfel, L; Weser, U; Rotilio, G

    1994-01-01

    The regulation of Cu,Zn-superoxide dismutase by copper was investigated in human K562 cells. Copper ions caused a dose- and time-dependent increase, up to 3-fold, of the steady-state level of Cu,Zu-superoxide dismutase mRNA. A comparable increase was also observed for actin and ribosomal protein L32 mRNAs, but not for metallothionein mRNA which was augmented more than 50-fold and showed a different induction pattern. The copper-induced mRNAs were actively translated as judged from their enhanced loading on polysomes, the concomitantly increased cellular protein levels and an augmented incorporation of [3H]lysine into acid-precipitable material. Cu,Zn-superoxide dismutase protein followed this general trend, as demonstrated by dose- and time-dependent increases in immunoreactive and enzymically active protein. However, a specific accumulation of Cu,Zn-superoxide dismutase was noticed in cells grown in the presence of copper, that was not detectable for other proteins. Purification of the enzyme demonstrated that Cu,Zn-superoxide dismutase was present as a reconstitutable, copper-deficient protein with high specific activity (kcat./Cu = 0.89 x 10(9) M-1.s-1) in untreated K562 cells and as a fully metallated protein with low specific activity (kcat./Cu = 0.54 x 10(9) M-1.s-1) in copper-treated cells. Pulse-chase experiments using [3H]lysine indicated that turnover rates of Cu,Zn-superoxide dismutase in K562 cells were not affected by growth in copper-enriched medium, whereas turnover of total protein was significantly enhanced as a function of metal supplementation. From these results we conclude that: (i) unlike in yeast [Carrì, Galiazzo, Ciriolo and Rotilio (1991) FEBS Lett. 278, 263-266] Cu,Zn-superoxide dismutase is not specifically regulated by copper at the transcriptional level in human K562 cells, suggesting that this type of regulation has not been conserved during the evolution of higher eukaryotes; (ii) copper ions cause an inactivation of the enzyme in

  16. Copper-dependent metabolism of Cu,Zn-superoxide dismutase in human K562 cells. Lack of specific transcriptional activation and accumulation of a partially inactivated enzyme.

    PubMed

    Steinkühler, C; Carrì, M T; Micheli, G; Knoepfel, L; Weser, U; Rotilio, G

    1994-09-15

    The regulation of Cu,Zn-superoxide dismutase by copper was investigated in human K562 cells. Copper ions caused a dose- and time-dependent increase, up to 3-fold, of the steady-state level of Cu,Zu-superoxide dismutase mRNA. A comparable increase was also observed for actin and ribosomal protein L32 mRNAs, but not for metallothionein mRNA which was augmented more than 50-fold and showed a different induction pattern. The copper-induced mRNAs were actively translated as judged from their enhanced loading on polysomes, the concomitantly increased cellular protein levels and an augmented incorporation of [3H]lysine into acid-precipitable material. Cu,Zn-superoxide dismutase protein followed this general trend, as demonstrated by dose- and time-dependent increases in immunoreactive and enzymically active protein. However, a specific accumulation of Cu,Zn-superoxide dismutase was noticed in cells grown in the presence of copper, that was not detectable for other proteins. Purification of the enzyme demonstrated that Cu,Zn-superoxide dismutase was present as a reconstitutable, copper-deficient protein with high specific activity (kcat./Cu = 0.89 x 10(9) M-1.s-1) in untreated K562 cells and as a fully metallated protein with low specific activity (kcat./Cu = 0.54 x 10(9) M-1.s-1) in copper-treated cells. Pulse-chase experiments using [3H]lysine indicated that turnover rates of Cu,Zn-superoxide dismutase in K562 cells were not affected by growth in copper-enriched medium, whereas turnover of total protein was significantly enhanced as a function of metal supplementation. From these results we conclude that: (i) unlike in yeast [Carrì, Galiazzo, Ciriolo and Rotilio (1991) FEBS Lett. 278, 263-266] Cu,Zn-superoxide dismutase is not specifically regulated by copper at the transcriptional level in human K562 cells, suggesting that this type of regulation has not been conserved during the evolution of higher eukaryotes; (ii) copper ions cause an inactivation of the enzyme in

  17. Serological differences between the multiple amine oxidases of yeasts and comparison of the specificities of the purified enzymes from Candida utilis and Pichia pastoris.

    PubMed Central

    Green, J; Haywood, G W; Large, P J

    1983-01-01

    1. Antiserum to purified methylamine oxidase of Candida boidinii formed precipitin lines (with spurs) in double-diffusion tests with crude extracts of methylamine-grown cells of the following yeast species: Candida nagoyaensis, Candida nemodendra, Hansenula minuta, Hansenula polymorpha and Pichia pinus. No cross-reaction was observed with extracts of Candida lipolytica, Candida steatolytica, Candida tropicalis, Candida utilis, Pichia pastoris, Sporobolomyces albo-rubescens, Sporopachydermia cereana or Trigonopsis variabilis. Quantitative enzyme assays enabled the relative titre of antiserum against the various methylamine oxidases to be determined. 2. The amine oxidases from two non-cross-reacting species, C. utilis and P. pastoris, were purified to near homogeneity. 3. The methylamine oxidases, despite their serological non-similarity, showed very similar catalytic properties to methylamine oxidase from C. boidinii. Their heat-stability, pH optima, molecular weights, substrate specificities and sensitivity to inhibitors are reported. 4. The benzylamine oxidases of C. utilis and P. pastoris both oxidized putrescine, and the latter enzyme failed to show any cross-reaction with antibody to C. boidinii methylamine oxidase. Benzylamine oxidase from C. boidinii itself also did not cross-react with antibody to methylamine oxidase. The heat-stability, molecular weights, substrate specificities and sensitivity to inhibitors of the benzylamine/putrescine oxidases are reported. 5. The benzylamine/putrescine oxidase of C. utilis differed only slightly from that of C. boidinii. 6. Benzylamine/putrescine oxidase from P. pastoris differed from the Candida enzymes in heat-stability, subunit molecular weight and substrate specificity. In particular it catalysed the oxidation of the primary amino groups of spermine, spermidine, lysine, ornithine and 1,2-diaminoethane, which are not substrates for either of the Candida benzylamine oxidases that have been purified. 7. Spermine and

  18. 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

  19. Prioritized Assignment to Intake Appointments for Asian Americans at an Ethnic-Specific Mental Health Program

    ERIC Educational Resources Information Center

    Akutsu, Phillip D.; Tsuru, Garyn K.; Chu, Joyce P.

    2006-01-01

    This study examined the relationship of demographic, clinical, and therapist factors to decisions about prioritized assignment to the earliest intake appointment for 983 Asian Americans who contacted an Asian-oriented, ethnic-specific mental health program. The logistic regression results showed that Asian language preference, ethnicity,…

  20. 78 FR 53791 - Draft Program-Specific Guidance About Irradiator Licenses

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-30

    .... ACTION: Draft NUREG; request for comments. SUMMARY: The U.S. Nuclear Regulatory Commission (NRC) is... NUREG-1556, Volume 6, Revision 1, ``Consolidated Guidance about Materials Licenses: Program-Specific...- 4737, or by email to pdr.resource@nrc.gov . The draft NUREG-1556, Volume 6, Revision 1, is...

  1. 78 FR 17943 - Draft Program-Specific Guidance About Fixed Gauge Licenses

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-25

    .... ACTION: Draft NUREG; request for comment. SUMMARY: The U.S. Nuclear Regulatory Commission (NRC) is... NUREG-1556, Volume 4, Revision 1, ``Consolidated Guidance About Materials Licenses: Program-Specific...- 4737, or by email to pdr.resource@nrc.gov . The draft NUREG-1556, Volume 4, Revision 1, is available...

  2. Technical Specifications for Hardware and Software, and Maintenance in Support of Computer Literacy Program. Volume II.

    ERIC Educational Resources Information Center

    District of Columbia Public Schools, Washington, DC.

    Designed for use by vendors, this guide provides an overview of the objectives for the 5-year computer literacy program to be implemented in the District of Columbia Public Schools; outlines requirements which are mandatory elements of vendors' bids unless explicitly designated "desirable"; and details specifications for computing equipment,…

  3. 43 CFR 1821.13 - What if the specific program regulations conflict with these regulations?

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 43 Public Lands: Interior 2 2012-10-01 2012-10-01 false What if the specific program regulations 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...

  4. 43 CFR 1821.13 - What if the specific program regulations conflict with these regulations?

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 43 Public Lands: Interior 2 2014-10-01 2014-10-01 false What if the specific program regulations 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...

  5. 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. 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...

  6. 42 CFR 457.1150 - Program specific review process: Impartial review.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 4 2012-10-01 2012-10-01 false Program specific review process: Impartial review... review process: Impartial review. (a) Eligibility or enrollment matter. The review of a matter described... matter under review. (b) Health services matter. The State must ensure that an enrollee has...

  7. 42 CFR 457.1150 - Program specific review process: Impartial 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: Impartial review... review process: Impartial review. (a) Eligibility or enrollment matter. The review of a matter described... matter under review. (b) Health services matter. The State must ensure that an enrollee has...

  8. 42 CFR 457.1150 - Program specific review process: Impartial review.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 4 2014-10-01 2014-10-01 false Program specific review process: Impartial review... review process: Impartial review. (a) Eligibility or enrollment matter. The review of a matter described... matter under review. (b) Health services matter. The State must ensure that an enrollee has...

  9. 42 CFR 457.1150 - Program specific review process: Impartial 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: Impartial review... review process: Impartial review. (a) Eligibility or enrollment matter. The review of a matter described... matter under review. (b) Health services matter. The State must ensure that an enrollee has...

  10. 42 CFR 457.1150 - Program specific review process: Impartial 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: Impartial review... review process: Impartial review. (a) Eligibility or enrollment matter. The review of a matter described... matter under review. (b) Health services matter. The State must ensure that an enrollee has...

  11. Star: A Dementia-Specific Training Program for Staff in Assisted Living Residences

    ERIC Educational Resources Information Center

    Teri, Linda; Huda, Piruz; Gibbons, Laura; Young, Heather; van Leynseele, June

    2005-01-01

    Purpose: This article describes, and provides data on, an innovative, comprehensive, dementia-specific training program designed to teach direct care staff in assisted living residences to improve care and reduce problems in residents with dementia. Design and Methods: STAR--which stands for Staff Training in Assisted living Residences- provides…

  12. Program for calculating total-efficiency of specific-speed characteristics of centrifugal compressors

    NASA Technical Reports Server (NTRS)

    Galvas, M. R.

    1973-01-01

    Program uses one-dimensional mean streamline analysis conducted at fixed stagnation conditions. Seven specific losses are calculated for each set of compressors geometric variables and inlet velocity diagram characteristics studied. Categories used as input information are compressor geometry, thermodynamic properties of working fluid, velocity diagram characteristics, and iteration limits.

  13. 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...

  14. The Moderation of an Early Intervention Program for Anxiety and Depression by Specific Psychological Symptoms

    PubMed Central

    Cukrowicz, Kelly C.; Smith, Phillip N.; Hohmeister, Holly C.; Joiner, Thomas E.

    2016-01-01

    The current study examined the influence of a number of psychological factors on the effectiveness of an early intervention program targeting anxiety and depression in a non-clinical sample of college students. The program was influenced by the Cognitive-Behavioral Analysis System of Psychotherapy (McCullough, 2000) delivered in a two-hour computer-based educational program. Participants completed measures of depression, anxiety, and general distress prior to the prevention program and then again eight weeks later. Additionally, participants were assessed for past Major Depression, sleep related difficulties, a number of Anxiety Disorders, and suicide ideation. Moderation of the effectiveness of the early intervention program by these factors depended on the dependent variable of interest. Specifically, the effectiveness of the intervention program on symptoms of depression was moderated by insomnia; symptoms of anxiety by past Post-Traumatic Stress Disorder and Specific Phobia as well as sleep problems related to nightmares; and symptoms of general negative affect by Social Phobia and suicide ideation. Implications are discussed. PMID:19229947

  15. The moderation of an early intervention program for anxiety and depression by specific psychological symptoms.

    PubMed

    Cukrowicz, Kelly C; Smith, Phillip N; Hohmeister, Holly C; Joiner, Thomas E

    2009-04-01

    The current study examined the influence of a number of psychological factors on the effectiveness of an early intervention program targeting anxiety and depression in a non-clinical sample of college students. The early intervention program comprised elements of the cognitive-behavioral analysis system of psychotherapy (McCullough, 2000) delivered in a 2-hour computer-based educational program. Participants completed measures of depression, anxiety, and general distress prior to the intervention program and then again 8 weeks later. Additionally, participants were assessed for past major depression, sleep related difficulties, a number of anxiety disorders, and suicide ideation. Moderation of the effectiveness of the early intervention program by these factors depended on the dependent variable of interest, specifically: the effectiveness of the intervention program on symptoms of depression was moderated by insomnia; symptoms of anxiety by past post-traumatic stress disorder (PTSD) and specific phobia as well as sleep problems related to nightmares; and symptoms of general negative affect by social phobia and suicide ideation. Implications are discussed. PMID:19229947

  16. 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. PMID:24684242

  17. 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

  18. Identification of protein-protein interactions between the TatB and TatC subunits of the twin-arginine translocase system and respiratory enzyme specific chaperones.

    PubMed

    Kuzniatsova, Lalita; Winstone, Tara M L; Turner, Raymond J

    2016-04-01

    The Twin-arginine translocation (Tat) pathway serves for translocation of fully folded proteins across the cytoplasmic membrane in bacterial and chloroplast thylakoid membranes. The Escherichia coli Tat system consists of three core components: TatA, TatB, and TatC. The TatB and TatC subunits form the receptor complex for Tat dependent proteins. The TatB protein is composed of a single transmembrane helix and cytoplasmic domain. The structure of TatC revealed six transmembrane helices. Redox Enzyme Maturation Proteins (REMPs) are system specific chaperones, which play roles in the maturation of Tat dependent respiratory enzymes. Here we applied the in vivo bacterial two-hybrid technique to investigate interaction of REMPs with the TatBC proteins, finding that all but the formate dehydrogenase REMP dock to TatB or TatC. We focused on the NarJ subfamily, where DmsD--the REMP for dimethyl sulfoxide reductase in E. coli--was previously shown to interact with TatB and TatC. We found that these REMPs interact with TatC cytoplasmic loops 1, 2 and 4, with the exception of NarJ, that only interacts with 1 and 4. An in vitro isothermal titration calorimetry study was applied to confirm the evidence of interactions between TatC fragments and DmsD chaperone. Using a peptide overlapping array, it was shown that the different NarJ subfamily REMPs interact with different regions of the TatB cytoplasmic domains. The results demonstrate a role of REMP chaperones in targeting respiratory enzymes to the Tat system. The data suggests that the different REMPs may have different mechanisms for this task. PMID:26826271

  19. Development of specific enzyme-linked immunosorbent assay for determining LH and FSH levels in tilapia, using recombinant gonadotropins.

    PubMed

    Aizen, Joseph; Kasuto, Harel; Levavi-Sivan, Berta

    2007-01-01

    We recently produced Oreochromis niloticus recombinant LH and FSH as single-chain polypeptides in the methylotrophic yeast Pichia pastoris. Glycoprotein subunit alpha was joined with tilapia (t) LHbeta or tFSHbeta mature protein-coding sequences to form a fusion gene that encodes a ;;tethered" polypeptide, in which the gonadotropin beta-subunit forms the N-terminal part and the alpha-subunit forms the C-terminal part. Recombinant (r) gonadotropins were used to develop specific and homologous competitive ELISAs for measurements of FSH and LH in the plasma and pituitary of tilapia, using primary antibodies against rtLHbeta or rtFSHbeta, respectively, and rtLHbetaalpha or rtFSHbetaalpha for the standard curves. The wells were coated with either rtLHbeta (2ng/ml) or rtFSHbeta (0.5ng/well), and the final concentrations of the antisera were 1:5000 (for tLH) or 1:50,000 (for tFSH). The sensitivity of the assay was 15.84pg/ml for tLH and 0.24pg/ml for tFSH measurements in the plasma, whereas for the measurements in the pituitary, the sensitivity was 2.43ng/ml and 1.52ng/ml for tLH and tFSH, respectively. The standard curves for tFSH and tLH paralleled those of serially diluted pituitary extracts of other cichlids, as well as of serially diluted pituitary extract of seabream, European seabass and hybrid bass. We examined plasma tFSH and tLH levels in the course of one reproductive cycle, between two successive spawnings, in three individual tilapia females. Plasma levels of both FSH and LH increased during the second day after the eggs had been removed, probably related to the vitellogenic phase. LH levels increased toward spawning, which occurred on the 11th day. FSH levels also increased on day of cycle, probably due to recruitment of a new generation of follicles for the successive spawning. The development of specific ELISAs using recombinant gonadotropins is expected to advance the study of the distinct functions of each of these important hormones. PMID:17507016

  20. 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.

  1. 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. PMID:26380981

  2. Production of Group Specific Monoclonal Antibody to Aflatoxins and its Application to Enzyme-linked Immunosorbent Assay.

    PubMed

    Kim, Sung-Hee; Cha, Sang-Ho; Karyn, Bischoff; Park, Sung-Won; Son, Seong-Wan; Kang, Hwan-Goo

    2011-06-01

    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 (R(2) > 0.99) and from 1 to 100 ng/ml (R(2) > 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

  3. A neuron-specific deletion of the microRNA-processing enzyme DICER induces severe but transient obesity in mice.

    PubMed

    Mang, Géraldine M; Pradervand, Sylvain; Du, Ngoc-Hien; Arpat, Alaaddin Bulak; Preitner, Frédéric; Wigger, Leonore; Gatfield, David; Franken, Paul

    2015-01-01

    MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression post-transcriptionally. MiRNAs are implicated in various biological processes associated with obesity, including adipocyte differentiation and lipid metabolism. We used a neuronal-specific inhibition of miRNA maturation in adult mice to study the consequences of miRNA loss on obesity development. Camk2a-CreERT2 (Cre+) and floxed Dicer (Dicerlox/lox) mice were crossed to generate tamoxifen-inducible conditional Dicer knockouts (cKO). Vehicle- and/or tamoxifen-injected Cre+;Dicerlox/lox and Cre+;Dicer+/+ served as controls. Four cohorts were used to a) measure body composition, b) follow food intake and body weight dynamics, c) evaluate basal metabolism and effects of food deprivation, and d) assess the brain transcriptome consequences of miRNA loss. cKO mice developed severe obesity and gained 18 g extra weight over the 5 weeks following tamoxifen injection, mainly due to increased fat mass. This phenotype was highly reproducible and observed in all 38 cKO mice recorded and in none of the controls, excluding possible effects of tamoxifen or the non-induced transgene. Development of obesity was concomitant with hyperphagia, increased food efficiency, and decreased activity. Surprisingly, after reaching maximum body weight, obese cKO mice spontaneously started losing weight as rapidly as it was gained. Weight loss was accompanied by lowered O2-consumption and respiratory-exchange ratio. Brain transcriptome analyses in obese mice identified several obesity-related pathways (e.g. leptin, somatostatin, and nemo-like kinase signaling), as well as genes involved in feeding and appetite (e.g. Pmch, Neurotensin) and in metabolism (e.g. Bmp4, Bmp7, Ptger1, Cox7a1). A gene cluster with anti-correlated expression in the cerebral cortex of post-obese compared to obese mice was enriched for synaptic plasticity pathways. While other studies have identified a role for miRNAs in obesity, we here

  4. 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

  5. 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

  6. 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.

  7. Post-translational control of vegetative cell separation enzymes through a direct interaction with specific inhibitor IseA in Bacillus subtilis.

    PubMed

    Yamamoto, Hiroki; Hashimoto, Masayuki; Higashitsuji, Yuhei; Harada, Hiroyuki; Hariyama, Nozomi; Takahashi, Lisa; Iwashita, Tomoaki; Ooiwa, Seika; Sekiguchi, Junichi

    2008-10-01

    Three D,L-endopeptidases, LytE, LytF and CwlS, are involved in the vegetative cell separation in Bacillus subtilis. A novel cell surface protein, IseA, inhibits the cell wall lytic activities of these d,l-endopeptidases in vitro, and IseA negatively regulates the cell separation enzymes at the post-translational level. Immunofluorescence microscopy indicated that the IseA-3xFLAG fusion protein was specifically localized at cell separation sites and poles on the vegetative cell surface in a similar manner of the d,l-endopeptidases. Furthermore, pull-down assay showed that IseA binds to the catalytic domain of LytF, indicating that IseA is localized on the cell surface through the catalytic domain of LytF. Overexpression of IseA caused a long-chained cell morphology in the exponential growth phase, indicating that IseA inhibits the cell separation D,L-endopeptidases in vivo. Besides, overexpression of IseA in a cwlO disruptant affected cell growth, implying that IseA is also involved in the cell elongation event. However, although IseA inhibits the activities of LytE, LytF, CwlS and CwlO in vitro, it is unlikely to inhibit CwlS and CwlO in vivo. This is the first demonstration that the cell separation event is post-translationally controlled through a direct interaction between cell separation enzymes and a specific novel inhibitor in bacteria. PMID:18761694

  8. NASA broad-specification fuels combustion technology program: Status and description

    NASA Technical Reports Server (NTRS)

    Fear, J. S.

    1979-01-01

    The program presented is a contracted effort to evolve and demonstrate the technology required to utilize broad-specification fuels in current and next generation commercial Conventional Takeoff and Landing aircraft engines, and to verify this technology in full-scale engine tests in 1983. The program consists of three phases: Combustor Concept Screening, Combustor Optimization Testing, and Engine Verification Testing. The development and screening of the combustion system designs for the CF6-80 engine and the JT9D-7 engine, respectively, in high-pressure sector test rigs are reported.

  9. Development of an enzyme immunoassay specific for a core protein epitope of a novel small basement membrane associated heparan sulphate proteoglycan from human kidney.

    PubMed

    Stöcker, G; Stickeler, E; Switalla, S; Fischer, D C; Greiling, H; Haubeck, H D

    1997-02-01

    Heparan sulphate proteoglycans are major components of the glomerular basement membrane and play a key role in their molecular organization and function. Moreover, their presence is essential for the maintenance of the selective permeability of the glomerular basement membrane. Recently, we have isolated and characterized a novel, small basement membrane associated heparan sulphate proteoglycan from human aorta and kidney. Using specific monoclonal antibodies we have shown that the novel heparan sulphate proteoglycan is predominantly located in the glomerular basement membrane, to a lesser extent in the basement membrane of tubuli, and also in the mesangium. Turnover or, in the course of kidney diseases, degradation of heparan sulphate proteoglycan from glomerular basement membranes may lead to urinary excretion of heparan sulphate proteoglycan. Therefore, changes in the structure and function of glomerular basement membranes may be directly detected by measuring the excretion of a component of this basement menbrane, e. g. heparan sulphate proteoglycan into urine. Here we describe the establishment of an enzyme immunoassay for the sensitive detection of the novel, small heparan sulphate proteoglycan in urine. In this assay the specific monoclonal antibody 1F10/B8, which recognizes a core protein epitope, was used to detect the polyanionic heparan sulphate proteoglycan bound to the surface of a cationic charge modified microtitre plate. This assay allows the sensitive and specific detection of the small heparan sulphate proteoglycan, which is released from the glomerular basement membrane into urine during normal turnover and also in the course of kidney diseases. PMID:9056750

  10. 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. PMID:25397998

  11. Tissue-specific pioneer factors associate with androgen receptor cistromes and transcription programs

    PubMed Central

    Pihlajamaa, Päivi; Sahu, Biswajyoti; Lyly, Lauri; Aittomäki, Viljami; Hautaniemi, Sampsa; Jänne, Olli A

    2014-01-01

    Androgen receptor (AR) binds male sex steroids and mediates physiological androgen actions in target tissues. ChIP-seq analyses of AR-binding events in murine prostate, kidney and epididymis show that in vivo AR cistromes and their respective androgen-dependent transcription programs are highly tissue specific mediating distinct biological pathways. This high order of tissue specificity is achieved by the use of exclusive collaborating factors in the three androgen-responsive tissues. We find two novel collaborating factors for AR signaling in vivo—Hnf4α (hepatocyte nuclear factor 4α) in mouse kidney and AP-2α (activating enhancer binding protein 2α) in mouse epididymis—that define tissue-specific AR recruitment. In mouse prostate, FoxA1 serves for the same purpose. FoxA1, Hnf4α and AP-2α motifs are over-represented within unique AR-binding loci, and the cistromes of these factors show substantial overlap with AR-binding events distinct to each tissue type. These licensing or pioneering factors are constitutively bound to chromatin and guide AR to specific genomic loci upon hormone exposure. Collectively, liganded receptor and its DNA-response elements are required but not sufficient for establishment of tissue-specific transcription programs. PMID:24451200

  12. Enzyme markers

    MedlinePlus

    ... or defects passed down through families (inherited) can affect how enzymes work. Some enzymes are affected by several genes. Test results are usually reported as a percentage of normal enzyme activity.

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

    PubMed Central

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

    2016-01-01

    Summary 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. PMID:26972603

  14. 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. PMID:26972603

  15. Spermatogenesis-Specific Features of the Meiotic Program in Caenorhabditis elegans

    PubMed Central

    Shakes, Diane C.; Wu, Jui-ching; Sadler, Penny L.; LaPrade, Kristen; Moore, Landon L.; Noritake, Alana; Chu, Diana S.

    2009-01-01

    In most sexually reproducing organisms, the fundamental process of meiosis is implemented concurrently with two differentiation programs that occur at different rates and generate distinct cell types, sperm and oocytes. However, little is known about how the meiotic program is influenced by such contrasting developmental programs. Here we present a detailed timeline of late meiotic prophase during spermatogenesis in Caenorhabditis elegans using cytological and molecular landmarks to interrelate changes in chromosome dynamics with germ cell cellularization, spindle formation, and cell cycle transitions. This analysis expands our understanding C. elegans spermatogenesis, as it identifies multiple spermatogenesis-specific features of the meiotic program and provides a framework for comparative studies. Post-pachytene chromatin of spermatocytes is distinct from that of oocytes in both composition and morphology. Strikingly, C. elegans spermatogenesis includes a previously undescribed karyosome stage, a common but poorly understood feature of meiosis in many organisms. We find that karyosome formation, in which chromosomes form a constricted mass within an intact nuclear envelope, follows desynapsis, involves a global down-regulation of transcription, and may support the sequential activation of multiple kinases that prepare spermatocytes for meiotic divisions. In spermatocytes, the presence of centrioles alters both the relative timing of meiotic spindle assembly and its ultimate structure. These microtubule differences are accompanied by differences in kinetochores, which connect microtubules to chromosomes. The sperm-specific features of meiosis revealed here illuminate how the underlying molecular machinery required for meiosis is differentially regulated in each sex. PMID:19696886

  16. 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. PMID:25647363

  17. Differentiation between Human Coronaviruses NL63 and 229E Using a Novel Double-Antibody Sandwich Enzyme-Linked Immunosorbent Assay Based on Specific Monoclonal Antibodies ▿

    PubMed Central

    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. PMID:21084464

  18. Detection of Human Papillomavirus Type 16-Specific T Lymphocytes by a Recombinant Vaccinia Virus-Based Enzyme-Linked Immunospot Assay▿

    PubMed Central

    Kim, Kevin H.; Greenfield, William; Shotts, Ezekiel; Nakagawa, Mayumi

    2007-01-01

    Cell-mediated immunity, particularly that induced by T cells, is thought to have a key role in controlling infection. The enzyme-linked immunospot (ELISPOT) assay has been successfully adapted to detect T-cell immune response to a variety of pathogens. However, it still remains a challenge to detect antigen-specific T cells when the numbers of circulating cells are low, such as in a local cervical infection caused by genital human papillomavirus (HPV). The goal of this study was to develop a protocol for enhanced detection of HPV-specific CD8+ T cells by examining a number of the variables involved in performing an ELISPOT assay. Since blood samples consistently positive for HPV-specific T cells are difficult to obtain, previously described human papillomavirus type 16 (HPV16) E6 52-61 (FAFRDLCIVY)-specific T-cell clone cells (13) seeded in peripheral blood mononuclear cells from an HLA-B57-positive blood donor were used. The variables examined were the amounts of primary and secondary anti-gamma interferon antibodies, amounts of antigen-presenting monocytes and recombinant vaccinia virus expressing the HPV16 E6 protein, and amounts of exogenous cytokines added (recombinant human interleukin-2 [rhIL-2] and rhIL-7). The amounts of antigen-presenting monocytes, followed by the concentration of exogenous rhIL-2, had the most pronounced and significant effects in enhancing sensitivity of the ELISPOT assay. Blood samples from six patients being monitored for abnormal Pap smear results and from 12 healthy volunteers were examined using the enhanced conditions. PMID:17314231

  19. 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

  20. 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. PMID:21084464

  1. Proficiency monitoring of monoclonal antibody cocktail-based enzyme-linked immunosorbent assay for detection of allergen-specific immunoglobulin E in dogs.

    PubMed

    Lee, Kenneth W; Blankenship, Karen; McKinney, Brennan; Kern, Gerhard; Buch, Jesse; Greenwood, Janice; Brazis, Pilar; Drouet, Laurent; Tambone, Cecilia; Faas, Rebecca; Weaver, Gareth

    2015-07-01

    The purpose of our study was to document the continued comparative proficiency of different laboratories that perform a monoclonal antibody-based enzyme-linked immunosorbent assay (macELISA) for detection of allergen-specific immunoglobulin (Ig)E in dogs. Replicate samples of 18 different sera pools were independently evaluated in a single blinded fashion by each of 16 different operators functioning in 10 different laboratories. The average intra-assay variance among reactive assay calibrators in all laboratories was 6.0% (range: 2.7-16.1%), while the average intralaboratory interassay variance was 7.5% (range: 3.9-10.9%). The overall interassay interlaboratory variance was consistent among laboratories and averaged 11.4% (range: 8.5-12.5%). All laboratories yielded similar profiles and magnitudes of responses for replicate unknown samples; dose response profiles observed in each of the laboratories were indistinguishable. Considering the positive or negative results, interassay interlaboratory concordance of results exceeded 90%. Correlation of optical density values between and among all laboratories was strong (r > 0.9, P < 0.001). Collectively, the results demonstrated that the macELISA for measuring allergen-specific canine IgE is reproducible, and documents that consistency of results can be achieved not only in an individual laboratory by differing operators but also among laboratories using the same monoclonal-based ELISA. PMID:26069227

  2. A new fluorimetric enzyme assay for the diagnosis of Niemann-Pick A/B, with specificity of natural sphingomyelinase substrate.

    PubMed

    van Diggelen, O P; Voznyi, Ya V; Keulemans, J L M; Schoonderwoerd, K; Ledvinova, J; Mengel, E; Zschiesche, M; Santer, R; Harzer, K

    2005-01-01

    6-Hexadecanoylamino-4-methylumbelliferylphosphorylcholine (HMUPC) was shown to be a specific substrate for the determination of acid (lysosomal) sphingomyelinase (ASM; gene SMPD1). Fibroblasts (n = 27) and leukocytes (n = 8) from both the A and B types of Niemann-Pick disease showed < 6% and < 10% of mean normal ASM activity, respectively. Niemann-Pick A or B patients bearing the Q292K mutation had apparently normal ASM activity with our new artificial substrate. These patients with false-normal sphingomyelinase activity, however, could readily be detected by determining the extent of inhibition of enzymatic hydrolysis of the artificial substrate HMU-PC by an unlabelled natural substrate, in particular lysosphingomyelin. This approach is generally applicable. Our novel assay for ASM combines the ease of a rapid and robust enzyme assay using a fluorogenic substrate with the specificity of an ASM assay using a natural substrate. Such assays are obviously more convenient to the diagnostic laboratory, since radiolabelled substrates are not required. PMID:16151905

  3. Mumps virus-specific antibody titers from pre-vaccine era sera: comparison of the plaque reduction neutralization assay and enzyme immunoassays.

    PubMed

    Mauldin, Jeremy; Carbone, Kathryn; Hsu, Henry; Yolken, Robert; Rubin, Steven

    2005-09-01

    Mumps virus-neutralizing antibodies are believed to be the most predictable surrogate marker of protective immunity. However, assays used to detect neutralizing antibodies, such as the plaque reduction neutralization (PRN) assay, are labor- and time-intensive and consequently are often supplanted by the more rapid and inexpensive enzyme immunoassay (EIA) technique. For virus infections for which international antibody standards exist and are bridged to clinical studies of protection (e.g., measles and rubella), the EIA has been successfully used to determine immune surrogate endpoints, yet no such international reference exists for mumps serology. Since both virus-neutralizing and nonneutralizing antibodies are measured in the EIA, in the absence of a mumps serological standard, the EIA may be prone to yielding false-positive results when utilized for assessing surrogate markers of protective immunity. Moreover, since mumps virus-specific antibody titers are generally low in comparison to antibody levels induced by other viruses and EIA procedures often employ relatively high serum dilution factors, the EIA may be prone to yielding false-negative results. To examine these issues, a PRN assay and two commercially available EIA kits were used to evaluate wild-type mumps virus serological responses in human serum samples from the pre-mumps vaccine era. Our results indicate that the PRN assay is a more sensitive and specific method of measuring serological responses to wild-type mumps virus. PMID:16145156

  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. Donor substrate specificity of 4-alpha-glucanotransferase of porcine liver glycogen debranching enzyme and complementary action to glycogen phosphorylase on debranching.

    PubMed

    Watanabe, Yumiko; Makino, Yasushi; Omichi, Kaoru

    2008-03-01

    Glycogen debranching enzyme (GDE) has both 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase activities. Here, we examined 4-alpha-glucanotransferase action of porcine liver GDE on four 6(4)-O-alpha-maltooligosyl-pyridylamino(PA)-maltooctaoses, in the presence or absence of an acceptor, maltohexaose. HPLC analysis of digested fluorogenic branched dextrins revealed that in the presence or absence of acceptor, 6(4)-O-alpha-glucosyl-PA-maltooctaose (B4/81) was liberated from 6(4)-O-alpha-maltopentaosyl-PA-maltooctaose (B4/85), 6(4)-O-alpha-maltotetraosyl-PA-maltooctaose (B4/84) and 6(4)-O-alpha-maltotriosyl-PA-maltooctaose (B4/83), whereas 6(4)-O-alpha-maltosyl-PA-maltooctaose (B4/82) was resistant to the enzyme. The fluorogenic product was further hydrolyzed by amylo-alpha-1,6-glucosidase to PA-maltooctaose (G8PA) and glucose. The ratio of the rates of 4-alpha-glucanotransferase actions on B4/85, B4/84 and B4/83 in the absence of the acceptor was 0.15, 0.42 and 1.00, respectively. The rates increased with increasing amounts of acceptor, changing the ratio of the rates to 0.09, 1.00 and 0.60 (with 0.5 mM maltohexaose) and 0.10, 1.00 and 0.58 (with 1.0 mM maltohexaose), respectively. Donor substrate specificity of GDE 4-alpha-glucanotransferase suggests complementary action of GDE and glycogen phosphorylase on glycogen degradation in the porcine liver. Glycogen phosphorylase degrades the maltooligosaccharide branches of glycogen by phosphorolysis to form maltotetraosyl branches, and phosphorolysis does not proceed further. GDE 4-alpha-glucanotransferase removes a maltotriosyl residue from the maltotetraosyl branch such that the alpha-1,6-linked glucosyl residue is retained. PMID:18174188

  6. Concerted GABAergic actions of Aplysia feeding interneurons in motor program specification.

    PubMed

    Jing, Jian; Vilim, Ferdinand S; Wu, Jin-Sheng; Park, Ji-Ho; Weiss, Klaudiusz R

    2003-06-15

    GABAergic inhibitory interneurons regulate the activity of diverse types of neural networks, but the specific roles of these interneurons in motor control are poorly understood. In the Aplysia feeding motor network, three interneurons, cerebral-buccal interneuron-3 (CBI-3) and buccal interneurons B40 and B34, are GABA-immunoreactive and evoke fast IPSPs in their postsynaptic followers. Using a combination of pharmacological experiments with GABA antagonists, agonists, and uptake inhibitors, we found that these fast IPSPs are likely mediated by GABA. Functionally, these fast IPSPs specify two parameters for ingestive motor programs elicited by the command-like interneuron CBI-2: (1) the appropriate phasing of activity of the radula closer motor neuron B8 relative to protraction-retraction, and (2) protraction duration. First, in ingestive programs, B8 activity is phased such that it fires minimally during protraction. CBI-3 and B40 exert fast inhibition to minimize B8 activity during protraction, by either acting directly on B8 (B40) or indirectly on B8 (CBI-3). Second, these ingestive programs are characterized by long protraction duration, which is promoted by B40 and B34 because hyperpolarization of either cell shortens protraction. Such effects of B40 and B34 are attributable, at least partly, to their inhibitory effects on the retraction-phase interneuron B64 whose activation terminates protraction. Consistent with a GABAergic contribution to both B8 phasing and protraction duration, blockade of GABAergic inhibition by picrotoxin increases B8 activity during protraction and shortens protraction, without disrupting the integrity of motor programs. Thus, the concerted actions of GABAergic inhibition from three Aplysia feeding interneurons contribute to the specification of multiple features that define the motor program as an ingestive one. PMID:12832553

  7. Fast high-throughput screening of angiotensin-converting enzyme insertion/deletion polymorphism by variable programmed electric field strength-based microchip electrophoresis.

    PubMed

    Sun, Yucheng; Kim, Su-Kang; Zhang, Peng; Woo, Nain; Kang, Seong Ho

    2016-08-15

    An insertion (I)/deletion (D) polymorphism in angiotensin-converting enzyme (ACE) has been associated with susceptibility to various diseases in numerous studies. Traditionally, slab gel electrophoresis (SGE) after polymerase chain reaction (PCR) has been used to genotype this ACE I/D polymorphism. In this study, single- and multi-channel microchip electrophoresis (ME) methods based on variable programmed electric field strength (PEFS) (i.e., low constant, high constant, (+)/(-) staircase, and random electric field strengths) were developed for fast high-throughput screening of this specific polymorphism. The optimum PEFS conditions were set as 470V/cm for 0-9s, 129V/cm for 9-13s, 470V/cm for 13-13.9s, 294V/cm for 13.9-16s, and 470V/cm for 16-20s for single-channel ME, and 615V/cm for 0-22.5s, 231V/cm for 22.5-28.5s, and 615V/cm for 28.5-40s for multi-channel ME, respectively. In the multi-channel PEFS-ME, target ACE I/D polymorphism DNA fragments (D=190bp and I=490bp) were identified within 25s without loss of resolving power, which was ∼300 times faster than conventional SGE. In addition, PCR products of the ACE gene from human blood samples were detected after only 10 cycles by multi-channel PEFS-ME, but not by SGE. This parallel detection multichannel-based PEFS-ME method offers a powerful tool for fast high-throughput ACE I/D polymorphism screening with high sensitivity. PMID:27322633

  8. Do Fallers and Nonfallers Equally Benefit from Balance Specific Exercise Program? A Pilot Study

    PubMed Central

    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. PMID:24228258

  9. 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

  10. Development of 5Ns chromosome-specific SCAR markers for utilization in future wheat breeding programs.

    PubMed

    Wang, J; Wang, L M; Du, W L; Chen, L G; Liu, S H; Wu, J; Zhao, J X; Yang, Q H; Chen, X H

    2014-06-01

    In previous studies, we developed a wheat-Psathyrostachys huashanica Keng disomic addition line 3-8-10-2, which exhibited high stripe rust resistance and could be used as a donor source for introducing novel disease resistance gene(s) into wheat in future breeding programs. It was identified using cytology, genomic in situ hybridization (GISH), EST-SSR, EST-STS and morphological analyses. However, these techniques are not suitable for breeding programs that require the rapid screening of large numbers of genotypes because they are highly technical and time-consuming. In this study, three Ns genome-specific SCAR markers were developed via random amplified polymorphic DNA (RAPD) markers. These SCAR markers were further validated using a complete set of wheat-P. huashanica disomic addition lines, which segregated the 5Ns disomic addition line individuals. Our results indicated that the SCAR markers associated with the 5Ns chromosome of P. huashanica and they provide a low cost, high efficiency, alternative tool for screening 5Ns chromosomes in a wheat background. These newly developed SCAR markers that species-specificity of the markers was proved by analysis of a wide range of cereal species, and specific for 5Ns chromosome, which should be useful in marker-assisted selection for wheat breeders who want to screen genotypes that may contain 5Ns chromatin. PMID:25715460

  11. 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...

  12. 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...

  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. 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...

  15. 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...

  16. Control of neuronal synapse specification by a highly dedicated alternative splicing program.

    PubMed

    Traunmüller, Lisa; Gomez, Andrea M; Nguyen, Thi-Minh; Scheiffele, Peter

    2016-05-20

    Alternative RNA splicing represents a central mechanism for expanding the coding power of genomes. Individual RNA-binding proteins can control alternative splicing choices in hundreds of RNA transcripts, thereby tuning amounts and functions of large numbers of cellular proteins. We found that the RNA-binding protein SLM2 is essential for functional specification of glutamatergic synapses in the mouse hippocampus. Genome-wide mapping revealed a markedly selective SLM2-dependent splicing program primarily consisting of only a few target messenger RNAs that encode synaptic proteins. Genetic correction of a single SLM2-dependent target exon in the synaptic recognition molecule neurexin-1 was sufficient to rescue synaptic plasticity and behavioral defects in Slm2 knockout mice. These findings uncover a highly selective alternative splicing program that specifies synaptic properties in the central nervous system. PMID:27174676

  17. 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.

  18. 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. PMID:24659758

  19. Enzyme-linked immunosorbent assays for the detection of equine antibodies specific to a recombinant Fasciola hepatica surface antigen in an endemic area.

    PubMed

    Arias, María Sol; Piñeiro, Pablo; Hillyer, George V; Francisco, Iván; Cazapal-Monteiro, Cristiana Filipa; Suárez, José Luis; Morrondo, Patrocinio; Sánchez-Andrade, Rita; Paz-Silva, Adolfo

    2012-02-01

    The utility of an enzyme-linked immunosorbent assay to determine the sensitization against the trematode Fasciola hepatica in horses from an endemic area (NW Spain) was assessed. Blood samples were collected from 536 horses and tested against a 2.9-kDa recombinant surface protein (FhrAPS) to estimate the presence of IgG antibodies. Data were analysed regarding several intrinsic (age, gender and breed) and extrinsic factors (aptitude and housing). The farm size (number of horses/farm) was also considered. Sixty percent (95% CI 56, 64) of the horses were positive to the FhrAPS-ELISA, with a significantly higher seroprevalence in the mares (67%). Foals reached the lowest percentage of sensitization against the trematode (12%), and a significant positive correlation between the seroprevalence of fasciolosis and the age of the horses was established. When considering all the factors together, the seroprevalence of fasciolosis was initially classified into two groups (nodes) regarding the age of the horses. The node composed of the horses older than 1 year was then divided into two other clusters according to their gender. The mares were finally classified and grouped into two nodes regarding their breed. We concluded that the FhrAPS-ELISA is very useful for the demonstration of specific equine IgG antibodies against F. hepatica. An elevated risk of exposition to this trematode in horses maintained in endemic areas was proven. The possible role of horses as reservoirs for F. hepatica infections is discussed. PMID:21847600

  20. Confirmation of the assignment of the low-field proton resonance of serine proteases by using specifically nitrogen-15 labeled enzyme.

    PubMed

    Bachovchin, W W

    1985-12-01

    Proton NMR spectra of serine proteases in 1H2O solutions typically show a single resonance at very low magnetic field--i.e., 14-18 ppm from dimethylsilylapentanesulfonate. This resonance has been assigned to the proton hydrogen bonded between aspartic acid-102 and histidine-57 (chymotrypsin numbering system) of the "charge-relay system" or catalytic triad of serine proteases [Robillard, G. & Shulman, R. G. (1972) J. Mol. Biol. 71, 507-511]. Since then, there have been a number of reports that have cast doubt on its correctness. In the present work we have tested this assignment using alpha-lytic protease (EC 3.4.21.12, Myxobacter alpha-lytic proteinase), a bacterial serine protease homologous to elastase, which is specifically labeled with nitrogen-15 at N delta 1 of its single histidine residue. The low-field region of the proton spectra of this labeled enzyme shows a single resonance having the properties reported [Robillard, G. & Shulman, R. G. (1974) J. Mol. Biol. 86, 519-540], which, in addition, exhibits spin-spin splitting to the nitrogen-15 label. The observation of this 15N delta 1-H coupling makes the assignment of this resonance to the charge-relay proton unequivocal. PMID:3934665

  1. Confirmation of the assignment of the low-field proton resonance of serine proteases by using specifically nitrogen-15 labeled enzyme

    SciTech Connect

    Bachovchin, W.W.

    1985-12-01

    Proton NMR spectra of serine proteases in /sup 1/H/sub 2/O solutions typically show a single resonance at very low magnetic field i.e., 14-18 ppm from dimethylsilylapentanesulfonate. This resonance has been assigned to the proton hydrogen bonded between aspartic acid-102 and histidine-57 (chymotrypsin numbering system) of the charge-relay system or catalytic triad of serine proteases. There have been a number of reports that have cast doubt on its correctness. In the present work the authors have tested this assignment using ..cap alpha..-lytic protease, a bacterial serine protease homologous to elastase, which is specifically labeled with nitrogen-15 at N/sup delta/sub 1// of its single histidine residue. The low-field region of the proton spectra of this labeled enzyme shows a single resonance having the properties reported which, in addition, exhibits spin-spin splitting to the nitrogen-15 label. The observation of this /sup 15/N-/sup delta/sub 1//-H coupling makes the assignment of this resonance to the charge-relay proton unequivocal.

  2. Antioxidant defense enzyme genes and asthma susceptibility: gender-specific effects and heterogeneity in gene-gene interactions between pathogenetic variants of the disease.

    PubMed

    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

  3. 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

  4. Tissue-Specific Signals Control Reversible Program of Localization and Functional Polarization of Macrophages

    PubMed Central

    Okabe, Yasutaka; Medzhitov, Ruslan

    2014-01-01

    SUMMARY Tissue-resident macrophages are highly heterogeneous in terms of their functions and phenotypes as a consequence of adaptation to different tissue environments. Local tissue-derived signals are thought to control functional polarization of resident macrophages; however, the identity of these signals remains largely unknown. It is also unknown whether functional heterogeneity is a result of irreversible lineage-specific differentiation or a consequence of continuous but reversible induction of diverse functional programs. Here, we identified retinoic acid as a signal that induces tissue-specific localization and functional polarization of peritoneal macrophages through the reversible induction of transcription factor GATA6. We further found that GATA6 in macrophages regulates gut IgA production through peritoneal B-1 cells. These results provide insight into the regulation of tissue-resident macrophage functional specialization by tissue-derived signals. PMID:24792964

  5. 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

  6. Biochemical determination of enzyme-bound metabolites: preferential accumulation of a programmed octaketide on the enediyne polyketide synthase CalE8.

    PubMed

    Belecki, Katherine; Townsend, Craig A

    2013-09-25

    Despite considerable interest in the enediyne family of antitumor antibiotics, assembly of their polyketide core structures in nature remains poorly understood. Discriminating methods to access enzyme-bound intermediates are critical for elucidating unresolved polyketide and nonribosomal peptide biosynthetic pathways. Here, we describe the development of broadly applicable techniques for the mild chemical release and analysis of intermediates bound to carrier proteins (CPs), providing access to these species even in sensitive systems. These techniques were applied to CalE8, the polyketide synthase (PKS) involved in calicheamicin biosynthesis, facilitating the unambiguous identification of enzyme-bound polyketides on an enediyne PKS. Moreover, these methods enabled the preparation of fully unloaded CalE8, providing a "clean slate" for reconstituted activity and allowing us to demonstrate the preferential accumulation of a PKS-bound octaketide with evidence of programmed processing control by CalE8. This intermediate, which has the expected chain length for enediyne core construction, could previously only be indirectly inferred. These studies prove that this polyketide is an authentic product of CalE8 and may be a key precursor to the enediyne core of calicheamicin, as it is the only programmed, enzyme-bound species observed for any enediyne system to date. Our experimental advances into a generally inaccessible system illustrate the utility of these techniques for investigating CP-based biosynthetic pathways. PMID:24041368

  7. Biochemical Determination of Enzyme-bound Metabolites: Preferential Accumulation of a Programmed Octaketide on the Enediyne Polyketide Synthase CalE8

    PubMed Central

    Belecki, Katherine; Townsend, Craig A.

    2013-01-01

    Despite considerable interest in the enediyne family of antitumor antibiotics, assembly of their polyketide core structures in nature remains poorly understood. Discriminating methods to access enzyme-bound intermediates are critical for elucidating unresolved polyketide and non-ribosomal peptide biosynthetic pathways. Here, we describe the development of broadly applicable techniques for the mild chemical release and analysis of intermediates bound to carrier proteins (CPs), providing access to these species even in sensitive systems. These techniques were applied to CalE8, the polyketide synthase (PKS) involved in calicheamicin biosynthesis, facilitating the unambiguous identification of enzyme-bound polyketides on an enediyne PKS. Moreover, these methods enabled the preparation of fully unloaded CalE8, providing a "clean slate" for reconstituted activity and allowing us to demonstrate the preferential accumulation of a PKS-bound octaketide with evidence of programmed processing control by CalE8. This intermediate, which has the expected chain length for enediyne core construction, could only be indirectly inferred previously. These studies prove that this polyketide is an authentic product of CalE8 and may be a key precursor to the enediyne core of calicheamicin, as it is the only programmed, enzyme-bound species observed for any enediyne system to date. Our experimental advances into a generally inaccessible system illustrate the utility of these techniques for investigating CP-based biosynthetic pathways. PMID:24041368

  8. An arterial-specific enhancer of the human endothelin converting enzyme 1 (ECE1) gene is synergistically activated by Sox17, FoxC2, and Etv2.

    PubMed

    Robinson, Ashley S; Materna, Stefan C; Barnes, Ralston M; De Val, Sarah; Xu, Shan-Mei; Black, Brian L

    2014-11-15

    Endothelin-converting enzyme-1 (Ece-1), a crucial component of the Endothelin signaling pathway, is required for embryonic development and is an important regulator of vascular tone, yet the transcriptional regulation of the ECE1 gene has remained largely unknown. Here, we define the activity and regulation of an enhancer from the human ECE1 locus in vivo. The enhancer identified here becomes active in endothelial progenitor cells shortly after their initial specification and is dependent on a conserved FOX:ETS motif, a composite binding site for Forkhead transcription factors and the Ets transcription factor Etv2, for activity in vivo. The ECE1 FOX:ETS motif is bound and cooperatively activated by FoxC2 and Etv2, but unlike other described FOX:ETS-dependent enhancers, ECE1 enhancer activity becomes restricted to arterial endothelium and endocardium by embryonic day 9.5 in transgenic mouse embryos. The ECE1 endothelial enhancer also contains an evolutionarily-conserved, consensus SOX binding site, which is required for activity in transgenic mouse embryos. Importantly, the ECE1 SOX site is bound and activated by Sox17, a transcription factor involved in endothelial cell differentiation and an important regulator of arterial identity. Moreover, the ECE1 enhancer is cooperatively activated by the combinatorial action of FoxC2, Etv2, and Sox17. Although Sox17 is required for arterial identity, few direct transcriptional targets have been identified in endothelial cells. Thus, this work has important implications for our understanding of endothelial specification and arterial subspecification. PMID:25179465

  9. Development of a sandwich enzyme-linked immunosorbent assay for the detection of CD44v3 using exon v3- and v6-specific monoclonal antibody pairs.

    PubMed

    Jeoung, Mee Hyun; Kim, Taek-Keun; Shim, Hyunbo; Lee, Sukmook

    2016-09-01

    It has been suggested that soluble CD44 levels in cancer patient sera may be closely associated with tumor progression and metastasis. However, to date, there has been limited methodology for detecting the soluble CD44 variant 3 isoform (CD44v3). Herein, using phage display technology, we isolated monoclonal antibodies specific to exon v3 or v6 of CD44 (CD44-exonv3 or CD44-exonv6) from a human synthetic antibody library. We also confirmed the specificity of antibody binding to CD44-exonv3 or -exonv6. Label-free kinetic analysis using the Octet biolayer interferometry system showed that the Kd values of the anti-CD44-exonv3 and anti-CD44-exonv6 antibodies for CD44v3-10 are approximately 1.1nM and 1.5nM, respectively. Finally, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) using the anti-CD44-exonv3 and anti-CD44-exonv6 antibody pairs. The minimum detection limit of the assay was 6.2ng/ml CD44v3-10 and the linear range was up to 125ng/ml. Intra- and inter-assay coefficients of variation were 2.2% and 2.9%, respectively. The intra- and inter-assay recoveries were 99.3% and 105.3%, respectively. Taken together, these results suggest that this novel sandwich ELISA using the anti-CD44-exonv3 and anti-CD44-exonv6 antibody pairs will be useful for the detection of soluble CD44v3 in cancer patient sera. PMID:27288967

  10. Development of a highly sensitive and specific monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detection of Sudan I in food samples.

    PubMed

    Wang, Yuzhen; Wei, Dapeng; Yang, Hong; Yang, Yuan; Xing, Weiwei; Li, Yuan; Deng, Anping

    2009-03-15

    The use of Sudan I as an additive in food products has been prohibited in the European Union and many other countries. In this study, a highly sensitive and specific monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of Sudan I in food samples was developed. The hapten derivative with a three-carbon-atom length of carboxylic spacer at the azobound para-position was synthesized and coupled to carrier proteins. The hapten-bovine serum albumin (BSA) conjugate was used as an immunogen, while the hapten-ovalbumin (OVA) conjugate was applied as a coating antigen. The mAb against Sudan I was produced by hybridoma technique and the corresponding ELISA was characterized in terms of sensitivity, specificity, precision and accuracy. At optimal experimental conditions, the standard curve was constructed in concentrations of 0.1-100 ngmL(-1). The values of IC(50) for nine standard curves were in the range of 1.1-2.0 ngmL(-1) and the LOD at a signal-to-noise ratio of 3 (S/N=3) was 0.07-0.14 ngmL(-1). The cross-reactivity values of the mAb with Sudan II, III and IV were 9.5%, 33.9% and 0.95%; no cross-reactivity was found with other six edible colorants: Lemon yellow, Bright blue, Indigotin, Kermes, Amarant and Sunset yellow, indicating the assay displays not only high sensitivity but also high specificity as well. The organic solvent effect on the assay was tested. It was observed that the ELISA was tolerated to 30% of methanol and 10% of acetonitrile without significant loss of IC(50) value. Six food samples were spiked with Sudan I and the methanolic extracts after appropriate dilution were analyzed by ELISA. Acceptable recovery rates of 88.2-110.5% and coefficients of variation of 2.5-17.4% were obtained. The ELISA for nine spiked samples was confirmed by high-performance liquid chromatography (HPLC) with a high correlation coefficient of 0.9840 (n=9). The mAb-based ELISA proven to be a feasible quantitative

  11. Improvements in Shoulder Endurance Following a Baseball-Specific Strengthening Program in High School Baseball Players

    PubMed Central

    Moore, Stephanie D.; Uhl, Tim L.; Kibler, W. Ben

    2013-01-01

    Background: The posterior shoulder muscles play key roles in maintaining shoulder function in throwing. Arm fatigue has been identified as a risk factor for shoulder and elbow pain in youth baseball pitchers. However, endurance of the posterior shoulder muscles in overhead athletes is not routinely examined or conditioned. Hypothesis: Upper extremity muscular endurance can be improved in adolescent baseball players during a 20-week preseason training program. Secondarily, strength will be improved. Finally, these improvements will be associated with maintenance of range of motion. Study Design: Cohort study. Methods: Fourteen baseball players (age, 16 ± 2 years) attended 3 supervised training sessions per week for 20 weeks. Strengthening of the upper extremity was performed with a specific progression that utilized readily available equipment. Testing was completed at baseline and at 4, 8, and 20 weeks. The posterior shoulder endurance test was performed to assess muscular endurance. Glenohumeral internal and external rotation range of motion and strength were measured. Results: Posterior shoulder endurance improved from 30 ± 14 repetitions at baseline to 66 ± 26 at 4 weeks and 88 ± 36 at 20 weeks (P < 0.05). Glenohumeral internal rotation range of motion and the glenohumeral internal/external rotation strength ratio remained similar over the course of the program. Conclusion: Implementation of a preseason training program effectively increased shoulder muscular endurance while maintaining strength ratios and range of motion throughout the 20-week program. Clinical Relevance: This program improved a key parameter known to be associated with shoulder function and injury risk. This study describes a simple clinical tool to assess muscular endurance of the posterior shoulder. PMID:24427394

  12. 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.…

  13. Human Immunodeficiency Virus-Specific Gamma Interferon Enzyme-Linked Immunospot Assay Responses Targeting Specific Regions of the Proteome during Primary Subtype C Infection Are Poor Predictors of the Course of Viremia and Set Point▿

    PubMed Central

    Gray, Clive M.; Mlotshwa, Mandla; Riou, Catherine; Mathebula, Tiyani; de Assis Rosa, Debra; Mashishi, Tumelo; Seoighe, Cathal; Ngandu, Nobubelo; van Loggerenberg, Francois; Morris, Lynn; Mlisana, Koleka; Williamson, Carolyn; Karim, Salim Abdool

    2009-01-01

    It is unknown whether patterns of human immunodeficiency virus (HIV)-specific T-cell responses during acute infection may influence the viral set point and the course of disease. We wished to establish whether the magnitude and breadth of HIV type 1 (HIV-1)-specific T-cell responses at 3 months postinfection were correlated with the viral-load set point at 12 months and hypothesized that the magnitude and breadth of HIV-specific T-cell responses during primary infection would predict the set point. Gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay responses across the complete proteome were measured in 47 subtype C HIV-1-infected participants at a median of 12 weeks postinfection. When corrected for amino acid length and individuals responding to each region, the order of recognition was as follows: Nef > Gag > Pol > Rev > Vpr > Env > Vpu > Vif > Tat. Nef responses were significantly (P < 0.05) dominant, targeted six epitopic regions, and were unrelated to the course of viremia. There was no significant difference in the magnitude and breadth of responses for each protein region with disease progression, although there was a trend of increased breadth (mean, four to seven pools) in rapid progressors. Correlation of the magnitude and breadth of IFN-γ responses with the viral set point at 12 months revealed almost zero association for each protein region. Taken together, these data demonstrate that the magnitude and breadth of IFN-γ ELISPOT assay responses at 3 months postinfection are unrelated to the course of disease in the first year of infection and are not associated with, and have low predictive power for, the viral set point at 12 months. PMID:18945774

  14. Expression of Bacillus protease (Protease BYA) from Bacillus sp. Y in Bacillus subtilis and enhancement of its specific activity by site-directed mutagenesis-improvement in productivity of detergent enzyme-.

    PubMed

    Tobe, Seiichi; Shimogaki, Hisao; Ohdera, Motoyasu; Asai, Yoshio; Oba, Kenkichi; Iwama, Masanori; Irie, Masachika

    2006-01-01

    An attempt was made to express protease BYA produced by an alkalophilic Bacillus sp. Y in Bacillus subtilis by gene engineering methods. The gene encoding protease BYA was cloned from Bacillus sp. Y, and expression vector pTA71 was constructed from the amylase promoter of Bacillus licheniformis, DNA fragments encoding the open reading frame of protease BYA, and pUB110. Protease BYA was secreted at an activity level of 5100 APU/ml in the common industrial culture medium of Bacillus subtilis transformed with pTA71. We then attempted to increase the specific activity of protease BYA by site-directed mutagenesis. Amino acid residue Ala29 next to catalytic Asp30 was replaced by one of three uncharged amino acid residues (Val29, Leu29, Ile29), and each mutant enzyme was expressed and isolated from the culture medium. Val29 mutant enzyme was secreted at an activity level of greater than 7000 APU/ml in culture medium, and its specific activity was 1.5-fold higher than that of the wild-type enzyme. Other mutant enzymes had specific activity similar to that of the original one and were less stabile than the wild-type enzyme. It can be thought that the substitution at amino acid residue 29 affects the level of activity and stability of protease BYA. PMID:16394504

  15. Double-Antigen Enzyme-Linked Immunosorbent Assay for Detection of Hepatitis E Virus-Specific Antibodies in Human or Swine Sera ▿

    PubMed Central

    Hu, Wei Ping; Lu, Yang; Precioso, Nestor Amadeo; Chen, Hsiao Ying; Howard, Teresa; Anderson, David; Guan, Ming

    2008-01-01

    A new double-antigen sandwich-based enzyme-linked immunosorbent assay (ELISA) for the detection of total antibodies (immunoglobulin G [IgG] and IgM) specific for hepatitis E virus (HEV) was developed by utilizing well-characterized recombinant protein ET2.1 and its peroxidase-labeled counterpart. Our study showed that the ELISA detected all the positive patient samples (n = 265) regardless of whether they contained IgM or IgG antibodies, or both, while it maintained an excellent specificity of 98.8% with samples from various patient or healthy control groups (total number of samples, 424). The test had a detection limit for anti-HEV IgG antibodies that was equivalent to 62 mIU/ml of the international reference. Compared with the serological status of the specimens determined on the basis of tests performed at the individual collection sites, the testing outcome generated by the new ELISA had a good agreement of 99.3%, with a kappa value of 0.985. The positive predictive value and the negative predictive value for the new test reached 98.1% and 100%, respectively. This ELISA had a positive delta value of 4.836 and a negative delta value of 3.314 (where delta is a measure of the number of standard deviations by which the cutoff is separated from the mean of the sample groups) (N. Crofts, W. Maskill, and I. D. Gust, J. Virol. Methods 22:51-59, 1988), indicating that it had an excellent ability to differentiate the infected and noninfected cohorts. Furthermore, the new design enables the detection of antibodies not only in human samples but also in pig samples. Our preliminary data showed that the ELISA could detect seroconversion in samples from pigs at as early as 14 days postinoculation. The potential utility of detecting specific antibodies in pigs will be an added advantage for managing the disease, with suggested zoonotic implications. PMID:18495846

  16. Comparison of Chain-Length Preferences and Glucan Specificities of Isoamylase-Type α-Glucan Debranching Enzymes from Rice, Cyanobacteria, and Bacteria.

    PubMed

    Kobayashi, Taiki; Sasaki, Satoshi; Utsumi, Yoshinori; Fujita, Naoko; Umeda, Kazuhiro; Sawada, Takayuki; Kubo, Akiko; Abe, Jun-Ichi; Colleoni, Christophe; Ball, Steven; Nakamura, Yasunori

    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

  17. 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

  18. 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. PMID:26099271

  19. 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

  20. 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)

  1. 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.

  2. The germination-specific lytic enzymes SleB, CwlJ1, and CwlJ2 each contribute to Bacillus anthracis spore germination and virulence.

    PubMed

    Giebel, Jonathan D; Carr, Katherine A; Anderson, Erica C; Hanna, Philip C

    2009-09-01

    The bacterial spore cortex is critical for spore stability and dormancy and must be hydrolyzed by germination-specific lytic enzymes (GSLEs), which allows complete germination and vegetative cell outgrowth. We created in-frame deletions of three genes that encode GSLEs that have been shown to be active in Bacillus anthracis germination: sleB, cwlJ1, and cwlJ2. Phenotypic analysis of individual null mutations showed that the removal of any one of these genes was not sufficient to disrupt spore germination in nutrient-rich media. This finding indicates that these genes have partially redundant functions. Double and triple deletions of these genes resulted in more significant defects. Although a small subset of DeltasleB DeltacwlJ1 spores germinate with wild-type kinetics, for the overall population there is a 3-order-of-magnitude decrease in the colony-forming efficiency compared with wild-type spores. DeltasleB DeltacwlJ1 DeltacwlJ2 spores are unable to complete germination in nutrient-rich conditions in vitro. Both DeltasleB DeltacwlJ1 and DeltasleB DeltacwlJ1 DeltacwlJ2 spores are significantly attenuated, but are not completely devoid of virulence, in a mouse model of inhalation anthrax. Although unable to germinate in standard nutrient-rich media, spores lacking SleB, CwlJ1, and CwlJ2 are able to germinate in whole blood and serum in vitro, which may explain the persistent low levels of virulence observed in mouse infections. This work contributes to our understanding of GSLE activation and function during germination. This information may result in identification of useful therapeutic targets for the disease anthrax, as well as provide insights into ways to induce the breakdown of the protective cortex layer, facilitating easier decontamination of resistant spores. PMID:19581364

  3. 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

  4. 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

  5. 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

  6. 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

  7. Enzyme-linked immunosorbent assay for detection of serum or mucosal isotype specific IgG and IgA whole virus antibody to influenza A virus in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzyme-linked immunosorbent assays (ELISA) can be used to detect isotype specific anti-influenza antibodies in biological samples to characterize the porcine immune response to influenza A virus. The isotype antibody assay is based on an indirect ELISA using whole influenza virus as antigen and dete...

  8. Evolution of Substrate Specificity within a Diverse Family of [beta/alpha]-Barrel-fold Basic Amino Acid Decarboxylases X-ray Structure Determination of Enzymes with Specificity for L-Arginine and Carboxynorspermidine

    SciTech Connect

    Deng, Xiaoyi; Lee, Jeongmi; Michael, Anthony J.; Tomchick, Diana R.; Goldsmith, Elizabeth J.; Phillips, Margaret A.

    2010-08-26

    Pyridoxal 5{prime}-phosphate (PLP)-dependent basic amino acid decarboxylases from the {beta}/{alpha}-barrel-fold class (group IV) exist in most organisms and catalyze the decarboxylation of diverse substrates, essential for polyamine and lysine biosynthesis. Herein we describe the first x-ray structure determination of bacterial biosynthetic arginine decarboxylase (ADC) and carboxynorspermidine decarboxylase (CANSDC) to 2.3- and 2.0-{angstrom} resolution, solved as product complexes with agmatine and norspermidine. Despite low overall sequence identity, the monomeric and dimeric structures are similar to other enzymes in the family, with the active sites formed between the {beta}/{alpha}-barrel domain of one subunit and the {beta}-barrel of the other. ADC contains both a unique interdomain insertion (4-helical bundle) and a C-terminal extension (3-helical bundle) and it packs as a tetramer in the asymmetric unit with the insertions forming part of the dimer and tetramer interfaces. Analytical ultracentrifugation studies confirmed that the ADC solution structure is a tetramer. Specificity for different basic amino acids appears to arise primarily from changes in the position of, and amino acid replacements in, a helix in the {beta}-barrel domain we refer to as the 'specificity helix.' Additionally, in CANSDC a key acidic residue that interacts with the distal amino group of other substrates is replaced by Leu{sup 314}, which interacts with the aliphatic portion of norspermidine. Neither product, agmatine in ADC nor norspermidine in CANSDC, form a Schiff base to pyridoxal 5{prime}-phosphate, suggesting that the product complexes may promote product release by slowing the back reaction. These studies provide insight into the structural basis for the evolution of novel function within a common structural-fold.

  9. 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/. PMID:19574295

  10. 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.

  11. Do Girls Profit More? Gender-Specific Effectiveness of a Life Skills Program against Alcohol Consumption in Early Adolescence

    ERIC Educational Resources Information Center

    Weichold, Karina; Brambosch, Anett; Silbereisen, Rainer K.

    2012-01-01

    This study investigated the effectiveness of a life skills program with regard to alcohol consumption, life skills, knowledge, and school bonding for young adolescents. The focus was on the moderating role of gender, based on the assumption that life skills programs may address specific needs of adolescent girls better than those of boys. The…

  12. Legal and Definitional Issues Affecting the Identification and Education of Adults with Specific Learning Disabilities in Adult Education Programs

    ERIC Educational Resources Information Center

    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…

  13. 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…

  14. 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…

  15. 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.…

  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. 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)

  18. Programmed electrical stimulation of the ventricle: an efficient, sensitive, and specific protocol.

    PubMed

    Fisher, J D; Kim, S G; Ferrick, K J; Artoul, S G; Fink, D; Roth, J A; Johnston, D R; Williams, H R

    1992-04-01

    A relatively simple and efficient ventricular programmed electrical stimulation (PES) protocol was developed, capable of achieving high degrees of sensitivity and specificity. In a series of 481 subjects, 1, 2, and 3 extrastimuli (ES) were used successively during sinus rhythm and ventricular pacing at two drive cycle lengths, at one or more ventricular sites, together with rapid ventricular pacing, and other maneuvers such as isoproterenol infusion. Three ES were used immediately after two ES at each drive rate, rather than returning after completion of the protocol with two ES. Using the protocol, appropriate arrhythmias could be induced in 88% of all patients with ventricular fibrillation, 84% of all patients with sustained ventricular tachycardia (91% with underlying coronary disease), and 58% of patients with severe nonsustained ventricular tachycardia. There were significant differences in inducibility between patients whose ventricular arrhythmias were due to coronary artery disease and other causes. In contrast, sustained ventricular arrhythmias (all ventricular fibrillation) could be induced in only 5% of a control group of control patients, for a specificity of 95%. The protocol described is simpler and more efficient than those that use exhaustive testing of two ES before going to three ES. Three ES during sinus rhythm proved to be the most productive step, with a higher yield ratio (true: false-positives) than two ES or three ES during pacing, especially at faster rates. Greater efficiency is also achieved by leaving the timing of an extrastimulus just beyond its effective refractory period when an additional extrastimulus is to be added, compared to protocols in which the extrastimulus is moved later in the cycle and then decremented in tandem with the additional extrastimulus. Coupling intervals less than 200 msec produced some false-positives, but fewer overall than intervals greater than or equal to 200 msec, and with yield ratios comparable to

  19. 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

  20. 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

  1. Nonstructural Protein 1-Specific Immunoglobulin M and G Antibody Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Flaviviral Infections in Humans

    PubMed Central

    Galula, Jedhan Ucat; Shen, Wen-Fan; Davis, Brent S.

    2014-01-01

    IgM antibody- and IgG antibody-capture enzyme-linked immunosorbent assays (MAC/GAC-ELISAs) targeted at envelope protein (E) of dengue viruses (DENV), West Nile virus, and Japanese encephalitis virus (JEV) are widely used as serodiagnostic tests for presumptive confirmation of viral infection. Antibodies directed against the flavivirus nonstructural protein 1 (NS1) have been proposed as serological markers of natural infections among vaccinated populations. The aim of the current study is to optimize an IgM and IgG antibody-capture ELISA (MAC/GAC-ELISA) to detect anti-NS1 antibodies and compare it with anti-E MAC/GAC-ELISA. Plasmids to express premembrane/envelope (prM/E) or NS1 proteins of six medically important flaviviruses, including dengue viruses (DENV-1 to DENV-4), West Nile virus (WNV), and Japanese encephalitis virus (JEV), were constructed. These plasmids were used for the production of prM/E-containing virus-like particles (VLPs) and secreted NS1 (sNS1) from COS-1 cells. Archived clinical specimens from patients with confirmed DENV, JEV, and WNV infections, along with naive sera, were subjected to NS1-MAC/GAC-ELISAs before or after depletion of anti-prM/E antibodies by preabsorption with or without VLPs. Human serum specimens from previously confirmed DENV infections showed significantly enhanced positive-to-negative (P/N) ratios for NS1-MAC/GAC-ELISAs after the depletion of anti-prM/E antibodies. No statistical differences in sensitivities and specificities were found between the newly developed NS1- and VLP-MAC/GAC-ELISAs. Further application of the assays to WNV- and JEV-infected serum panels showed similar results. A novel approach to perform MAC/GAC-ELISAs for NS1 antibody detection was successfully developed with great potential to differentiate antibodies elicited by the tetravalent chimeric yellow fever-17D/dengue vaccine or DENV infection. PMID:25502522

  2. Nonstructural protein 1-specific immunoglobulin M and G antibody capture enzyme-linked immunosorbent assays in diagnosis of flaviviral infections in humans.

    PubMed

    Chao, Day-Yu; Galula, Jedhan Ucat; Shen, Wen-Fan; Davis, Brent S; Chang, Gwong-Jen J

    2015-02-01

    IgM antibody- and IgG antibody-capture enzyme-linked immunosorbent assays (MAC/GAC-ELISAs) targeted at envelope protein (E) of dengue viruses (DENV), West Nile virus, and Japanese encephalitis virus (JEV) are widely used as serodiagnostic tests for presumptive confirmation of viral infection. Antibodies directed against the flavivirus nonstructural protein 1 (NS1) have been proposed as serological markers of natural infections among vaccinated populations. The aim of the current study is to optimize an IgM and IgG antibody-capture ELISA (MAC/GAC-ELISA) to detect anti-NS1 antibodies and compare it with anti-E MAC/GAC-ELISA. Plasmids to express premembrane/envelope (prM/E) or NS1 proteins of six medically important flaviviruses, including dengue viruses (DENV-1 to DENV-4), West Nile virus (WNV), and Japanese encephalitis virus (JEV), were constructed. These plasmids were used for the production of prM/E-containing virus-like particles (VLPs) and secreted NS1 (sNS1) from COS-1 cells. Archived clinical specimens from patients with confirmed DENV, JEV, and WNV infections, along with naive sera, were subjected to NS1-MAC/GAC-ELISAs before or after depletion of anti-prM/E antibodies by preabsorption with or without VLPs. Human serum specimens from previously confirmed DENV infections showed significantly enhanced positive-to-negative (P/N) ratios for NS1-MAC/GAC-ELISAs after the depletion of anti-prM/E antibodies. No statistical differences in sensitivities and specificities were found between the newly developed NS1- and VLP-MAC/GAC-ELISAs. Further application of the assays to WNV- and JEV-infected serum panels showed similar results. A novel approach to perform MAC/GAC-ELISAs for NS1 antibody detection was successfully developed with great potential to differentiate antibodies elicited by the tetravalent chimeric yellow fever-17D/dengue vaccine or DENV infection. PMID:25502522

  3. 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.

  4. 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

  5. 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 ...

  6. 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 ...

  7. Multiplicity of 3-Ketosteroid-9α-Hydroxylase Enzymes in Rhodococcus rhodochrous DSM43269 for Specific Degradation of Different Classes of Steroids ▿ †

    PubMed Central

    Petrusma, Mirjan; Hessels, Gerda; Dijkhuizen, Lubbert; van der Geize, Robert

    2011-01-01

    The well-known large catabolic potential of rhodococci is greatly facilitated by an impressive gene multiplicity. This study reports on the multiplicity of kshA, encoding the oxygenase component of 3-ketosteroid 9α-hydroxylase, a key enzyme in steroid catabolism. Five kshA homologues (kshA1 to kshA5) were previously identified in Rhodococcus rhodochrous DSM43269. These KshADSM43269 homologues are distributed over several phylogenetic groups. The involvement of these KshA homologues in the catabolism of different classes of steroids, i.e., sterols, pregnanes, androstenes, and bile acids, was investigated. Enzyme activity assays showed that all KSH enzymes with KshADSM43269 homologues are C-9 α-hydroxylases acting on a wide range of 3-ketosteroids, but not on 3-hydroxysteroids. KshA5 appeared to be the most versatile enzyme, with the broadest substrate range but without a clear substrate preference. In contrast, KshA1 was found to be dedicated to cholic acid catabolism. Transcriptional analysis and functional complementation studies revealed that kshA5 supported growth on any of the different classes of steroids tested, consistent with its broad expression induction pattern. The presence of multiple kshA genes in the R. rhodochrous DSM43269 genome, each displaying unique steroid induction patterns and substrate ranges, appears to facilitate a dynamic and fine-tuned steroid catabolism, with C-9 α-hydroxylation occurring at different levels during microbial steroid degradation. PMID:21642460

  8. Crystal structure of a 117 kDa glucansucrase fragment provides insight into evolution and product specificity of GH70 enzymes.

    PubMed

    Vujicic-Zagar, Andreja; Pijning, Tjaard; Kralj, Slavko; López, Cesar A; Eeuwema, Wieger; Dijkhuizen, Lubbert; Dijkstra, Bauke W

    2010-12-14

    Glucansucrases are large enzymes belonging to glycoside hydrolase family 70, which catalyze the cleavage of sucrose into fructose and glucose, with the concomitant transfer of the glucose residue to a growing α-glucan polymer. Among others, plaque-forming oral bacteria secrete these enzymes to produce α-glucans, which facilitate the adhesion of the bacteria to the tooth enamel. We determined the crystal structure of a fully active, 1,031-residue fragment encompassing the catalytic and C-terminal domains of GTF180 from Lactobacillus reuteri 180, both in the native state, and in complexes with sucrose and maltose. These structures show that the enzyme has an α-amylase-like (β/α)(8)-barrel catalytic domain that is circularly permuted compared to the catalytic domains of members of glycoside hydrolase families 13 and 77, which belong to the same GH-H superfamily. In contrast to previous suggestions, the enzyme has only one active site and one nucleophilic residue. Surprisingly, in GTF180 the peptide chain follows a "U"-path, such that four of the five domains are made up from discontiguous N- and C-terminal stretches of the peptide chain. Finally, the structures give insight into the factors that determine the different linkage types in the polymeric product. PMID:21118988

  9. 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...

  10. Crystal structure of a 117 kDa glucansucrase fragment provides insight into evolution and product specificity of GH70 enzymes

    PubMed Central

    Vujičić-Žagar, Andreja; Pijning, Tjaard; Kralj, Slavko; López, Cesar A.; Eeuwema, Wieger; Dijkhuizen, Lubbert; Dijkstra, Bauke W.

    2010-01-01

    Glucansucrases are large enzymes belonging to glycoside hydrolase family 70, which catalyze the cleavage of sucrose into fructose and glucose, with the concomitant transfer of the glucose residue to a growing α-glucan polymer. Among others, plaque-forming oral bacteria secrete these enzymes to produce α-glucans, which facilitate the adhesion of the bacteria to the tooth enamel. We determined the crystal structure of a fully active, 1,031-residue fragment encompassing the catalytic and C-terminal domains of GTF180 from Lactobacillus reuteri 180, both in the native state, and in complexes with sucrose and maltose. These structures show that the enzyme has an α-amylase-like (β/α)8-barrel catalytic domain that is circularly permuted compared to the catalytic domains of members of glycoside hydrolase families 13 and 77, which belong to the same GH-H superfamily. In contrast to previous suggestions, the enzyme has only one active site and one nucleophilic residue. Surprisingly, in GTF180 the peptide chain follows a “U”-path, such that four of the five domains are made up from discontiguous N- and C-terminal stretches of the peptide chain. Finally, the structures give insight into the factors that determine the different linkage types in the polymeric product. PMID:21118988

  11. Comparison of the specificities and catalytic activities of hammerhead ribozymes and DNA enzymes with respect to the cleavage of BCR-ABL chimeric L6 (b2a2) mRNA.

    PubMed

    Kuwabara, T; Warashina, M; Tanabe, T; Tani, K; Asano, S; Taira, K

    1997-08-01

    With the eventual goal of developing a treatment for chronic myelogenous leukemia (CML), attempts have been made to design hammerhead ribozymes that can specifically cleave BCR-ABL fusion mRNA. In the case of L6 BCR-ABL fusion mRNA (b2a2 type; BCR exon 2 is fused to ABL exon 2), which has no effective cleavage sites for conventional hammerhead ribozymes near the BCR-ABL junction, it has proved very difficult to cleave the chimeric mRNA specifically. Several hammerhead ribozymes with relatively long junction-recognition sequences have poor substrate-specificity. Therefore, we explored the possibility of using newly selected DNA enzymes that can cleave RNA molecules with high activity to cleave L6 BCR-ABL fusion (b2a2) mRNA. In contrast to the results with the conventional ribozymes, the newly designed DNA enzymes, having higher flexibility for selection of cleavage sites, were able to cleave this chimeric RNA molecule specifically at sites close to the junction. Cleavage occurred only within the abnormal BCR-ABL mRNA, without any cleavage of the normal ABL or BCR mRNA. Thus, these chemically synthesized DNA enzymes seem to be potentially useful for application in vivo , especially for the treatment of CML, if we can develop exogenous delivery strategies. PMID:9224607

  12. Understanding Enzymes.

    ERIC Educational Resources Information Center

    Sinnott, M. L.

    1979-01-01

    Describes the way enzymes operate through reaction energetics, and explains that most of the catalytic power of enzymes lies in the strong noncovalent forces responsible for initial binding of substrate, which are only manifested at the transition state of the reaction. (Author/GA)

  13. Soil Enzymes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The functionality and resilience of natural and managed ecosystems mainly rely on the metabolic abilities of microbial communities, the main source of enzymes in soils. Enzyme mediated reactions are critical in the decomposition of organic matter, cycling of nutrients, and in the breakdown of herbic...

  14. A novel sample preparation and on-line HPLC-DAD-MS/MS-BCD analysis for rapid screening and characterization of specific enzyme inhibitors in herbal extracts: case study of α-glucosidase.

    PubMed

    Li, D Q; Zhao, J; Xie, J; Li, S P

    2014-01-01

    Drug discovery from complex mixture like Chinese herbs is a challenge and extensive false positives make the obtainment of specific bioactive compounds difficult. In the present study, a novel sample preparation method was proposed to rapidly reveal the specific bioactive compounds from complex mixtures using α-glucosidase as a case. Firstly, aqueous and methanol extracts of 500 traditional Chinese medicines were carried out with the aim of finding new sources of α-glucosidase inhibitors. As a result, the extracts of fruit of Terminalia chebula (FTC), flowers of Rosa rugosa (FRR) and Eugenia caryophyllata (FEC) as well as husk of Punica granatum (HPG) showed high inhibition on α-glucosidase. On-line liquid chromatography-diode array detection-tandem mass spectrometry and biochemical detection (HPLC-DAD-MS/MS-BCD) was performed to rapidly screen and characterize α-glucosidase inhibitors in these four extracts. After tentative identification, most of compounds with inhibitory activity in the investigated crude extracts were found to be tannins commonly recognized as non-specific enzyme inhibitors in vitro. Subsequently, the four extracts were treated with gelatin to improve specificity of the on-line system. Finally, two compounds with specific α-glucosidase inhibition were identified as corilagin and ellagic acid. The developed method could discover specific α-glucosidase inhibitors in complex mixtures such as plant extracts, which could also be used for discovery of specific inhibitors of other enzymes. PMID:24055848

  15. 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. PMID:22748507

  16. 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

  17. Biochemical and structural characterization of a novel ubiquitin-conjugating enzyme E2 from Agrocybe aegeria reveals Ube2w family-specific properties

    PubMed Central

    Qi, Chao; Li, De-Feng; Feng, Lei; Hou, Yanjie; Sun, Hui; Wang, Da-Cheng; Liu, Wei

    2015-01-01

    Ubiquitination is a post-translational modification that is involved in myriad cellar regulation and disease pathways. The ubiquitin-conjugating enzyme (E2) is an important player in the ubiquitin transfer pathway. Although many E2 structures are available, not all E2 families have known structures, and three-dimensional structures from fungal organisms other than yeast are lacking. We report here the crystal structure of UbcA1, which is a novel ubiquitin-conjugating enzyme identified from the edible and medicinal mushroom Agrocybe aegerita and displays potential antitumor properties. The protein belongs to the Ube2w family and shows similar biochemical characteristics to human Ube2w, including monomer-dimer equilibrium in solution, α-NH2 ubiquitin-transfer activity and a mechanism to recognize backbone atoms of intrinsically disordered N-termini in substrates. Its structure displays a unique C-terminal conformation with an orientation of helix α3 that is completely different from the reported E2 structures but similar to a recently reported NMR ensemble of Ube2w. A mutagenesis study on this novel enzyme revealed that an intact C-terminus is significant for protein dimerization and enzymatic activity. As the first crystallized full-length protein of this family, UbcA1 may supersede the truncated X-ray structure of Ube2w (PDB entry 2A7L) as the representative structure of the Ube2w family. PMID:26525192

  18. 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

  19. Tertiary and quaternary structures of photoreactive Fe-type nitrile hydratase from Rhodococcus sp. N-771: roles of hydration water molecules in stabilizing the structures and the structural origin of the substrate specificity of the enzyme.

    PubMed

    Nakasako, M; Odaka, M; Yohda, M; Dohmae, N; Takio, K; Kamiya, N; Endo, I

    1999-08-01

    suggests that the heterotetramer is physiologically relevant. In addition, it was revealed that the substrate specificity of this enzyme, recognizing small aliphatic substrates rather than aromatic ones, came from the narrowness of the entrance channel from the bulk solvent to the active center. This finding may give a clue for changing the substrate specificity of the enzyme. Under the crystallization condition described here, one 1,4-dioxane molecule plugged the channel. Through spectroscopic and crystallographic experiments, we found that the molecule prevented the dissociation of the endogenous NO molecule from the active center even when the crystal was exposed to light. PMID:10433695

  20. The effect of a culture-specific education program to promote breastfeeding among Vietnamese women in Sydney.

    PubMed

    Rossiter, J C

    1994-08-01

    The rate of breastfeeding among immigrant Vietnamese women in Western countries is low compared to those in Vietnam. To counteract this trend, a language and culture specific education program was developed. An experimental design was used to test the effectiveness of this program. The sample consisted of 182 prenatal Vietnamese women. Data collection included questionnaires and interviews. Results suggested that the education program had significant effects on knowledge, attitudes, planned and actual behaviour towards breastfeeding. However, the effect did not sustain until 6 months postpartum. Implications for nursing practice and further research are discussed. PMID:7928125

  1. An Instructional Program in Prereading Skills: Needs and Specifications. Working Paper No. 78.

    ERIC Educational Resources Information Center

    Venezky, Richard L.; Chapman, Robin S.

    This working paper presents the background, justification, and development plan for a kindergarten program to identify and correct deficits in prereading skills. The program provides for individual differences in: (a) patterns of skill deficits, (b) rates of learning, and (c) degrees of assistance required. It is intended for optimal use in the…

  2. 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%…

  3. Marzano Program for Building Academic Vocabulary: Supplemental Report of Effects on Specific Subgroups (FRL & ELL Students)

    ERIC Educational Resources Information Center

    Marzano, Robert J.

    2006-01-01

    During the 2004-2005 academic year, an evaluation study was conducted of the Building Academic Vocabulary (BAV) program. The findings from this study, reported in the document "Marzano Program for Building Academic Vocabulary: Preliminary Report of the 2004-2005 Evaluation Study" (Marzano, 2005), found that students who participated in the BAV…

  4. Educational Specifications for a Comprehensive Elementary Teacher Education Program. Volume I; the Basic Report. Final Report.

    ERIC Educational Resources Information Center

    Dickson, George E.; And Others

    Chapter 1 of this report presents an overview of a project to design an innovative, behaviorally oriented undergraduate and inservice teacher education program for elementary school teachers. Chapter 2 presents the conceptual design for developing the model program, one which incorporates the concept of the multiunit school and individual research…

  5. Rationale and Specifications for a Prototype, Group-Administered End-of-Unit Assessment Instrument for the SWRL Reading Program.

    ERIC Educational Resources Information Center

    Okada, Masahito; Besel, Ronald

    This document describes the purposes, rationale, and specifications of a prototype group-administered criterion exercise to be used with blocks 1-8 of the Southwest Regional Laboratory (SWRL) Reading Program. A description of the sampling domains, sampling procedures, and the methods employed in the construction of test items is presented. Also…

  6. Gender-Specific HIV Prevention with Urban Early-Adolescent Girls: Outcomes of the Keepin' It Safe Program

    ERIC Educational Resources Information Center

    Di Noia, Jennifer; Schinke, Steven P.

    2007-01-01

    This study evaluates the efficacy of Keepin' It Safe, a theory-based, gender-specific, CD-ROM-mediated HIV prevention program for urban, early adolescent girls. Intervention effects were examined in a randomized, pretest-posttest wait-list control-group design. Changes in HIV/AIDS knowledge, protective attitudes, and skills for reducing HIV…

  7. 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,…

  8. 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.

  9. 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 Central

    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

    2015-01-01

    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 (Hbbth3/+), hereditary hemochromatosis (Hfe−/−, Hjv−/−, and Tfr2Y245X/Y245X), hypotransferrinemia (Trfhpx/hpx), heterozygous transferrin receptor 1 deficiency (Tfrc+/−) and iron refractory iron deficiency anemia (Tmprss6−/− and Tmprss6hem8/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. PMID:25425686

  10. Deazaflavin Inhibitors of Tyrosyl-DNA Phosphodiesterase 2 (TDP2) Specific for the Human Enzyme and Active against Cellular TDP2.

    PubMed

    Marchand, Christophe; Abdelmalak, Monica; Kankanala, Jayakanth; Huang, Shar-Yin; Kiselev, Evgeny; Fesen, Katherine; Kurahashi, Kayo; Sasanuma, Hiroyuki; Takeda, Shunichi; Aihara, Hideki; Wang, Zhengqiang; Pommier, Yves

    2016-07-15

    Tyrosyl-DNA phosphodiesterase 2 repairs irreversible topoisomerase II-mediated cleavage complexes generated by anticancer topoisomerase-targeted drugs and processes replication intermediates for picornaviruses (VPg unlinkase) and hepatitis B virus. There is currently no TDP2 inhibitor in clinical development. Here, we report a series of deazaflavin derivatives that selectively inhibit the human TDP2 enzyme in a competitive manner both with recombinant and native TDP2. We show that mouse, fish, and C. elegans TDP2 enzymes are highly resistant to the drugs and that key protein residues are responsible for drug resistance. Among them, human residues L313 and T296 confer high resistance when mutated to their mouse counterparts. Moreover, deazaflavin derivatives show potent synergy in combination with the topoisomerase II inhibitor etoposide in human prostate cancer DU145 cells and TDP2-dependent synergy in TK6 human lymphoblast and avian DT40 cells. Deazaflavin derivatives represent the first suitable platform for the development of potent and selective TDP2 inhibitors. PMID:27128689

  11. 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.

  12. 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

  13. The non-specific inhibition of enzymes by environmental pollutants: a study of a model system towards the development of electrochemical biosensor arrays.

    PubMed

    Young, S J; Hart, J P; Dowman, A A; Cowell, D C

    2001-12-01

    Previous research has shown that lactate dehydrogenase (LDH) was competitively inhibited by pentachlorophenol (PCP) and a modified assay produced a detection limit of 1 microM (270 microg l(-1)). This work used spectrophotometric rate-determination but in order to move towards biosensor development the selected detection method was electrochemical. The linkage of LDH to lactate oxidase (LOD) provided the electroactive species, hydrogen peroxide. This could be monitored using a screen-printed carbon electrode (SPCE) incorporating the mediator, cobalt phthalocyanine, at a potential of +300 mV (vs. Ag/AgCl). A linked LDH/LOD system was optimised with respect to inhibition by PCP. It was found that the SPCE support material, PVC, acted to reduce inhibition, possibly by combining with PCP. A cellulose acetate membrane removed this effect. Inhibition of the system was greatest at enzyme activities of 5 U ml(-1) LDH and 0.8 U ml(-1) LOD in reactions containing 246 microM pyruvate and 7.5 microM NADPH. PCP detection limits were an EC(10) of 800 nM (213 microg l(-1)) and a minimum inhibition detectable (MID) limit of 650 nM (173 microg l(-1)). The inclusion of a third enzyme, glucose dehydrogenase (GDH), provided cofactor recycling to enable low concentrations of NADPH to be incorporated within the assay. NADPH was reduced from 7.5 to 2 microM. PCP detection limits were obtained for an assay containing 5 U ml(-1) LDH, 0.8 U ml(-1) LOD and 0.1 U ml(-1) GDH with 246 microM pyruvate, 400 mM glucose and 2 microM NADPH. The EC(10) limit was 150 nM (39.9 microg l(-1)) and the MID was 100 nM (26.6 microg l(-1)). The design of the inhibition assays discussed has significance as a model for other enzymes and moves forward the possibility of an electrochemical biosensor array for pollution monitoring. PMID:11679267

  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. 75 FR 23783 - National Protection and Programs Directorate; Sector-Specific Agency Executive Management Office...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-04

    ... submissions received must include the words ``Department of Homeland Security'' and the docket number for this... the National Infrastructure Protection Plan (NIPP). Pursuant to Homeland Security Presidential... Agency: Department of Homeland Security, National Protection and Programs Directorate. Title:...

  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. PMID:22193861

  17. 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).

  18. n-Alkylboronic acid inhibitors reveal determinants of ligand specificity in the quorum-quenching and siderophore biosynthetic enzyme PvdQ.

    PubMed

    Clevenger, Kenneth D; Wu, Rui; Liu, Dali; Fast, Walter

    2014-10-28

    The enzyme PvdQ (E.C. 3.5.1.97) from Pseudomonas aeruginosa is an N-terminal nucleophile hydrolase that catalyzes the removal of an N-myristyl substituent from a biosynthetic precursor of the iron-chelating siderophore pyoverdine. Inhibitors of pyoverdine biosynthesis are potential antibiotics since iron is essential for growth and scarce in most infections. PvdQ also catalyzes hydrolytic amide bond cleavage of selected N-acyl-l-homoserine lactone quorum-sensing signals used by some Gram-negative pathogens to coordinate the transcription of virulence factors. The resulting quorum-quenching activity of PvdQ has potential applications in antivirulence therapies. To inform both inhibitor design and enzyme engineering efforts, a series of n-alkylboronic acid inhibitors of PvdQ was characterized to reveal determinants of ligand selectivity. A simple homologation series results in compounds with Ki values that span from 4.7 mM to 190 pM, with a dependence of ΔGbind values on chain length of -1.0 kcal/mol/CH2. X-ray crystal structures are determined for the PvdQ complexes with 1-ethyl-, 1-butyl-, 1-hexyl-, and 1-octylboronic acids at 1.6, 1.8, 2.0, and 2.1 Å resolution, respectively. The 1-hexyl- and 1-octylboronic acids form tetrahedral adducts with the active-site N-terminal Ser217 in the β-subunit of PvdQ, and the n-alkyl substituents are bound in the acyl-group binding site. The 1-ethyl- and 1-butylboronic acids also form adducts with Ser217 but instead form trigonal planar adducts and extend their n-alkyl substituents into an alternative binding site. These results are interpreted to propose a ligand discrimination model for PvdQ that informs the development of PvdQ-related tools and therapeutics. PMID:25290020

  19. Programmed death-1 expression on HIV-1-specific CD8+ T cells is shaped by epitope specificity, T-cell receptor clonotype usage and antigen load

    PubMed Central

    Kløverpris, Henrik N.; McGregor, Reuben; McLaren, James E.; Ladell, Kristin; Stryhn, Anette; Koofhethile, Catherine; Brener, Jacqui; Chen, Fabian; Riddell, Lynn; Graziano, Luzzi; Klenerman, Paul; Leslie, Alasdair; Buus, Søren; Price, David A.; Goulder, Philip

    2014-01-01

    Objectives: Although CD8+ T cells play a critical role in the control of HIV-1 infection, their antiviral efficacy can be limited by antigenic variation and immune exhaustion. The latter phenomenon is characterized by the upregulation of multiple inhibitory receptors, such as programmed death-1 (PD-1), CD244 and lymphocyte activation gene-3 (LAG-3), which modulate the functional capabilities of CD8+ T cells. Design and methods: Here, we used an array of different human leukocyte antigen (HLA)-B∗15 : 03 and HLA-B∗42 : 01 tetramers to characterize inhibitory receptor expression as a function of differentiation on HIV-1-specific CD8+ T-cell populations (n = 128) spanning 11 different epitope targets. Results: Expression levels of PD-1, but not CD244 or LAG-3, varied substantially across epitope specificities both within and between individuals. Differential expression of PD-1 on T-cell receptor (TCR) clonotypes within individual HIV-1-specific CD8+ T-cell populations was also apparent, independent of clonal dominance hierarchies. Positive correlations were detected between PD-1 expression and plasma viral load, which were reinforced by stratification for epitope sequence stability and dictated by effector memory CD8+ T cells. Conclusion: Collectively, these data suggest that PD-1 expression on HIV-1-specific CD8+ T cells tracks antigen load at the level of epitope specificity and TCR clonotype usage. These findings are important because they provide evidence that PD-1 expression levels are influenced by peptide/HLA class I antigen exposure. PMID:24906112

  20. Program specificity for Ptf1a in pancreas versus neural tube development correlates with distinct collaborating cofactors and chromatin accessibility.

    PubMed

    Meredith, David M; Borromeo, Mark D; Deering, Tye G; Casey, Bradford H; Savage, Trisha K; Mayer, Paul R; Hoang, Chinh; Tung, Kuang-Chi; Kumar, Manonmani; Shen, Chengcheng; Swift, Galvin H; Macdonald, Raymond J; Johnson, Jane E

    2013-08-01

    The lineage-specific basic helix-loop-helix transcription factor Ptf1a is a critical driver for development of both the pancreas and nervous system. How one transcription factor controls diverse programs of gene expression is a fundamental question in developmental biology. To uncover molecular strategies for the program-specific functions of Ptf1a, we identified bound genomic regions in vivo during development of both tissues. Most regions bound by Ptf1a are specific to each tissue, lie near genes needed for proper formation of each tissue, and coincide with regions of open chromatin. The specificity of Ptf1a binding is encoded in the DNA surrounding the Ptf1a-bound sites, because these regions are sufficient to direct tissue-restricted reporter expression in transgenic mice. Fox and Sox factors were identified as potential lineage-specific modifiers of Ptf1a binding, since binding motifs for these factors are enriched in Ptf1a-bound regions in pancreas and neural tube, respectively. Of the Fox factors expressed during pancreatic development, Foxa2 plays a major role. Indeed, Ptf1a and Foxa2 colocalize in embryonic pancreatic chromatin and can act synergistically in cell transfection assays. Together, these findings indicate that lineage-specific chromatin landscapes likely constrain the DNA binding of Ptf1a, and they identify Fox and Sox gene families as part of this process. PMID:23754747

  1. Potential application of nonstructural protein NS1 serotype-specific immunoglobulin G enzyme-linked immunosorbent assay in the seroepidemiologic study of dengue virus infection: correlation of results with those of the plaque reduction neutralization test.

    PubMed

    Shu, Pei-Yun; Chen, Li-Kuang; Chang, Shu-Fen; Yueh, Yi-Yun; Chow, Ling; Chien, Li-Jung; Chin, Chuan; Yang, Hui-Hua; Lin, Ting-Hsiang; Huang, Jyh-Hsiung

    2002-05-01

    An NS1 serotype-specific indirect enzyme-linked immunosorbent assay (ELISA) was developed to differentiate primary and secondary dengue virus infections and serotypes of primary dengue virus infection. For this report, we carried out retrospective seroepidemiologic studies on serum samples collected from residents of Liuchiu Hsiang, Pingtung County, an isolated island in southern Taiwan during 1997-1998. The results demonstrated that good correlation existed between dengue virus NS1 serotype-specific immunoglobulin G (IgG) ELISA and dengue virus plaque reduction neutralization test (PRNT). Our data suggested that NS1 serotype-specific IgG ELISA could replace PRNT for seroepidemiologic studies to differentiate Japanese encephalitis and dengue virus infections and for dengue virus serotyping. PMID:11980973

  2. A trial of a job-specific workers' health surveillance program for construction workers: study protocol

    PubMed Central

    2011-01-01

    Background Dutch construction workers are offered periodic health examinations. This care can be improved by tailoring this workers health surveillance (WHS) to the demands of the job and adjust the preventive actions to the specific health risks of a worker in a particular job. To improve the quality of the WHS for construction workers and stimulate relevant job-specific preventive actions by the occupational physician, we have developed a job-specific WHS. The job-specific WHS consists of modules assessing both physical and psychological requirements. The selected measurement instruments chosen, are based on their appropriateness to measure the workers' capacity and health requirements. They include a questionnaire and biometrical tests, and physical performance tests that measure physical functional capabilities. Furthermore, our job-specific WHS provides occupational physicians with a protocol to increase the worker-behavioural effectiveness of their counselling and to stimulate job-specific preventive actions. The objective of this paper is to describe and clarify our study to evaluate the behavioural effects of this job-specific WHS on workers and occupational physicians. Methods/Design The ongoing study of bricklayers and supervisors is a nonrandomised trial to compare the outcome of an intervention (job-specific WHS) group (n = 206) with that of a control (WHS) group (n = 206). The study includes a three-month follow-up. The primary outcome measure is the proportion of participants who have undertaken one or more of the preventive actions advised by their occupational physician in the three months after attending the WHS. A process evaluation will be carried out to determine context, reach, dose delivered, dose received, fidelity, and satisfaction. The present study is in accordance with the TREND Statement. Discussion This study will allow an evaluation of the behaviour of both the workers and occupational physician regarding the preventive actions

  3. Food Enzymes

    ERIC Educational Resources Information Center

    McBroom, Rachel; Oliver-Hoyo, Maria T.

    2007-01-01

    Many students view biology and chemistry as two unrelated, separate sciences; how these courses are generally taught in high schools may do little to change that impression. The study of enzymes provide a great opportunity for both biology and chemistry teachers to share with students the interdisciplinary nature of science. This article describes…

  4. Zinc Enzymes.

    ERIC Educational Resources Information Center

    Bertini, I.; And Others

    1985-01-01

    Discusses the role of zinc in various enzymes concerned with hydration, hydrolysis, and redox reactions. The binding of zinc to protein residues, properties of noncatalytic zinc(II) and catalytic zinc, and the reactions catalyzed by zinc are among the topics considered. (JN)

  5. Development and evaluation of an N9-specific enzyme-linked immunosorbent assay to detect antibodies in duck and chicken sera.

    PubMed

    Schmitz, Audrey; Le Bras, Marie-Odile; Louboutin, Katell; Jestin, Véronique

    2015-03-01

    A serological test for detecting N9-specific antibodies may be useful as a DIVA strategy to differentiate vaccinated from infected animals or simply for direct serological detection of infection with N9-subtype virus. The method currently recommended for the detection of antibodies against neuraminidase is neuraminidase inhibition (NI), which is a laborious method using toxic chemicals and has low sensitivity. The present study describes the development and validation of an N9-specific ELISA. Data obtained with this N9 ELISA were compared to those obtained with nucleoprotein-based ELISA, haemagglutination inhibition test using homologous antigen and NI assay. 785 sera from ducks and chickens were used, from flocks previously determined to be AI negative or from experimentally infected or immunized flocks. Sensitivity and specificity were evaluated, and a ROC curve and kappa values, which provide a comparison between methods, were calculated. The results obtained in this study indicate that the N9 based-ELISA is effective in detecting N9-specific antibodies with high specificity and with better sensitivity than the recommended NI method; using data from 177 common sera tested with N9 ELISA and NI assay both compared to NP-based ELISA, their specificity were evaluated at 93.6% and 91.5% respectively, and sensitivity at 90.8% and 39.2% respectively. PMID:25445791

  6. Comparison of capture immunoglobulin M (IgM) and IgG enzyme-linked immunosorbent assay (ELISA) and nonstructural protein NS1 serotype-specific IgG ELISA for differentiation of primary and secondary dengue virus infections.

    PubMed

    Shu, Pei-Yun; Chen, Li-Kuang; Chang, Shu-Fen; Yueh, Yi-Yun; Chow, Ling; Chien, Li-Jung; Chin, Chuan; Lin, Ting-Hsiang; Huang, Jyh-Hsiung

    2003-07-01

    We have found that NS1 serotype-specific immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) can be used to differentiate primary and secondary dengue virus infections. This is due to the fact that the NS1-specific IgG antibody cannot be detected before day 9 of illness for primary infection, so the NS1-specific IgG antibodies measured in acute-phase sera must come from previous infection. Comparison of NS1 serotype-specific IgG ELISA with envelope- and membrane-specific capture IgM and IgG ELISA in the differentiation of primary and secondary dengue virus infections showed good correlation (95.90% agreement). Most important, we have found that the serotype of the dengue virus from the majority of patients with primary infection could be correctly identified when convalescent-phase or postinfection sera were analyzed by NS1 serotype-specific IgG ELISA. These findings suggested that NS1 serotype-specific IgG ELISA could be reliably applied for serodiagnosis and seroepidemiological study of dengue virus infection. PMID:12853395

  7. 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.

  8. 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.

  9. 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.

  10. Target-specific identification and characterization of the putative gene cluster for brasilinolide biosynthesis revealing the mechanistic insights and combinatorial synthetic utility of 2-deoxy-l-fucose biosynthetic enzymes.

    PubMed

    Chiu, Hsien-Tai; Weng, Chien-Pao; Lin, Yu-Chin; Chen, Kuan-Hung

    2016-02-14

    Brasilinolides exhibiting potent immunosuppressive and antifungal activities with remarkably low toxicity are structurally characterized by an unusual modified 2-deoxy-l-fucose (2dF) attached to a type I polyketide (PK-I) macrolactone. From the pathogenic producer Nocardia terpenica (Nocardia brasiliensis IFM-0406), a 210 kb genomic fragment was identified by target-specific degenerate primers and subsequently sequenced, revealing a giant nbr gene cluster harboring genes (nbrCDEF) required for TDP-2dF biosynthesis and those for PK-I biosynthesis, modification and regulation. The results showed that the genetic and domain arrangements of nbr PK-I synthases agreed colinearly with the PK-I structures of brasilinolides. Subsequent heterologous expression of nbrCDEF in Escherichia coli accomplished in vitro reconstitution of TDP-2dF biosynthesis. The catalytic functions and mechanisms of NbrCDEF enzymes were further characterized by systematic mix-and-match experiments. The enzymes were revealed to display remarkable substrate and partner promiscuity, leading to the establishment of in vitro hybrid deoxysugar biosynthetic pathways throughout an in situ one-pot (iSOP) method. This study represents the first demonstration of TDP-2dF biosynthesis at the enzyme and molecular levels, and provides new hope for expanding the structural diversity of brasilinolides by combinatorial biosynthesis. PMID:26754528

  11. Peroxidases identified in a subtractive cDNA library approach show tissue-specific transcript abundance and enzyme activity during seed germination of Lepidium sativum

    PubMed Central

    Linkies, Ada; Schuster-Sherpa, Uta; Tintelnot, Stefanie; Leubner-Metzger, Gerhard; Müller, Kerstin

    2010-01-01

    The micropylar endosperm is a major regulator of seed germination in endospermic species, to which the close Brassicaceae relatives Arabidopsis thaliana and Lepidium sativum (cress) belong. Cress seeds are about 20 times larger than the seeds of Arabidopsis. This advantage was used to construct a tissue-specific subtractive cDNA library of transcripts that are up-regulated late in the germination process specifically in the micropylar endosperm of cress seeds. The library showed that a number of transcripts known to be up-regulated late during germination are up-regulated in the micropylar endosperm cap. Detailed germination kinetics of SALK lines carrying insertions in genes present in our library showed that the identified transcripts do indeed play roles during germination. Three peroxidases were present in the library. These peroxidases were identified as orthologues of Arabidopsis AtAPX01, AtPrx16, and AtPrxIIE. The corresponding SALK lines displayed significant germination phenotypes. Their transcripts were quantified in specific cress seed tissues during germination in the presence and absence of ABA and they were found to be regulated in a tissue-specific manner. Peroxidase activity, and particularly its regulation by ABA, also differed between radicles and micropylar endosperm caps. Possible implications of this tissue-specificity are discussed. PMID:19884228

  12. 42 CFR 457.1130 - Program specific review process: Matters subject to review.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 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... HEALTH AND HUMAN SERVICES (CONTINUED) STATE CHILDREN'S HEALTH INSURANCE PROGRAMS (SCHIPs) ALLOTMENTS...

  13. 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... HEALTH AND HUMAN SERVICES (CONTINUED) STATE CHILDREN'S HEALTH INSURANCE PROGRAMS (SCHIPs) ALLOTMENTS...

  14. 42 CFR 457.1130 - Program specific review process: Matters subject to review.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 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... HEALTH AND HUMAN SERVICES (CONTINUED) STATE CHILDREN'S HEALTH INSURANCE PROGRAMS (SCHIPs) ALLOTMENTS...

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

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 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... HEALTH AND HUMAN SERVICES (CONTINUED) STATE CHILDREN'S HEALTH INSURANCE PROGRAMS (SCHIPs) ALLOTMENTS...

  16. 42 CFR 457.1130 - Program specific review process: Matters subject to review.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 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... HEALTH AND HUMAN SERVICES (CONTINUED) STATE CHILDREN'S HEALTH INSURANCE PROGRAMS (SCHIPs) ALLOTMENTS...

  17. Technical Specifications for Conducting an Annual Assessment of Overall Payment Error in the Pell Grant Program.

    ERIC Educational Resources Information Center

    Advanced Technology, Inc., Reston, VA.

    The issues, options, and procedures for annually measuring overall payment error in the Pell Grant program are specified in detail. Guidelines for establishing a definition of Pell Grant payment error are provided, and the design issues related to error measurement are examined. A comparison is made of options for selecting a study sample and for…

  18. 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...

  19. 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…

  20. 42 CFR 457.1160 - Program specific review process: Time frames.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... HUMAN SERVICES (CONTINUED) STATE CHILDREN'S HEALTH INSURANCE PROGRAMS (SCHIPs) ALLOTMENTS AND GRANTS TO... life or health or ability to attain, maintain or regain maximum function. If the enrollee has access to... extend the 72-hour time frame by up to 14 calendar days, if the enrollee requests an extension....

  1. 42 CFR 457.1160 - Program specific review process: Time frames.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... HUMAN SERVICES (CONTINUED) STATE CHILDREN'S HEALTH INSURANCE PROGRAMS (SCHIPs) ALLOTMENTS AND GRANTS TO... life or health or ability to attain, maintain or regain maximum function. If the enrollee has access to... extend the 72-hour time frame by up to 14 calendar days, if the enrollee requests an extension....

  2. 42 CFR 457.1160 - Program specific review process: Time frames.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... HUMAN SERVICES (CONTINUED) STATE CHILDREN'S HEALTH INSURANCE PROGRAMS (SCHIPs) ALLOTMENTS AND GRANTS TO... life or health or ability to attain, maintain or regain maximum function. If the enrollee has access to... extend the 72-hour time frame by up to 14 calendar days, if the enrollee requests an extension....

  3. 42 CFR 457.1160 - Program specific review process: Time frames.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... HUMAN SERVICES (CONTINUED) STATE CHILDREN'S HEALTH INSURANCE PROGRAMS (SCHIPs) ALLOTMENTS AND GRANTS TO... life or health or ability to attain, maintain or regain maximum function. If the enrollee has access to... extend the 72-hour time frame by up to 14 calendar days, if the enrollee requests an extension....

  4. 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…

  5. Solution structure of the N-terminal amphitropic domain of Escherichia coli glucose-specific enzyme IIA in membrane-mimetic micelles

    PubMed Central

    Wang, Guangshun; Keifer, Paul A.; Peterkofsky, Alan

    2003-01-01

    The N-terminal domain of enzyme IIAGlc of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system confers amphitropism to the protein, allowing IIAGlc to shuttle between the cytoplasm and the membrane. To further understand this amphitropic protein, we have elucidated, by NMR spectroscopy, the solution structure of a synthetic peptide corresponding to the N-terminal domain of IIAGlc. In water, this peptide is predominantly disordered, consistent with previous data obtained in the absence of membranes. In detergent micelles of dihexanoylphosphatidylglycerol (DHPG) or sodium dodecylsulfate (SDS), however, residues Phe 3–Val 10 of the peptide adopt a helical conformation in the ensemble of structures calculated on the basis of NOE-derived distance restraints. The root mean square deviations for superimposing the backbone atoms of the helical region are 0.18 Å in DHPG and 0.22 Å in SDS. The structure, chemical shifts, and spin–spin coupling constants all indicate that, of the four lysines in the N-terminal domain of IIAGlc, only Lys 5 and Lys 7 in the amphipathic helical region interact with DHPG. In addition, the peptide-detergent interactions were investigated using intermolecular NOESY experiments. The aliphatic chains of anionic detergents DHPG, SDS, and 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt (DSS) all showed intermolecular NOE cross-peaks to the peptide, providing direct evidence for the putative membrane anchor of IIAGlc in binding to the membrane-mimicking micelles. PMID:12717030

  6. Task-specific enhancement of hippocampus-dependent learning in mice deficient in monoacylglycerol lipase, the major hydrolyzing enzyme of the endocannabinoid 2-arachidonoylglycerol

    PubMed Central

    Kishimoto, Yasushi; Cagniard, Barbara; Yamazaki, Maya; Nakayama, Junko; Sakimura, Kenji; Kirino, Yutaka; Kano, Masanobu

    2015-01-01

    Growing evidence indicates that the endocannabinoid system is important for the acquisition and/or extinction of learning and memory. However, it is unclear which endocannabinoid(s) play(s) a crucial role in these cognitive functions, especially memory extinction. To elucidate the physiological role of 2-arachidonoylglycerol (2-AG), a major endocannabinoid, in behavioral and cognitive functions, we conducted a comprehensive behavioral test battery in knockout (KO) mice deficient in monoacylglycerol lipase (MGL), the major hydrolyzing enzyme of 2-AG. We found age-dependent increases in spontaneous physical activity (SPA) in MGL KO mice. Next, we tested the MGL KO mice using 5 hippocampus-dependent learning paradigms (i.e., Morris water maze (MWM), contextual fear conditioning, novel object recognition test, trace eyeblink conditioning, and water-finding test). In the MWM, MGL KO mice showed normal acquisition of reference memory, but exhibited significantly faster extinction of the learned behavior. Moreover, they showed faster memory acquisition on the reversal-learning task of the MWM. In contrast, in the contextual fear conditioning, MGL KO mice tended to show slower memory extinction. In the novel object recognition and water-finding tests, MGL KO mice exhibited enhanced memory acquisition. Trace eyeblink conditioning was not altered in MGL KO mice throughout the acquisition and extinction phases. These results indicate that 2-AG signaling is important for hippocampus-dependent learning and memory, but its contribution is highly task-dependent. PMID:26082696

  7. The only active mutant of thymidylate synthase D169, a residue far from the site of methyl transfer, demonstrates the exquisite nature of enzyme specificity.

    PubMed

    Birdsall, David L; Finer-Moore, Janet; Stroud, Robert M

    2003-03-01

    Cysteine is the only variant of D169, a cofactor-binding residue in thymidylate synthase, that shows in vivo activity. The 2.4 A crystal structure of Escherichia coli thymidylate synthase D169C in a complex with dUMP and the antifolate CB3717 shows it to be an asymmetric dimer, with only one active site covalently bonded to dUMP. At the active site with covalently bound substrate, C169 S gamma adopts the roles of both carboxyl oxygens of D169, making a 3.6 A S...H[bond]N hydrogen bond to 3-NH of CB3717 and a 3.4 A water-mediated hydrogen bond to H212. Analogous hydrogen bonds formed during the enzyme reaction are important for cofactor binding and are postulated to contribute to catalysis. The C169 side chain is likely to be ionized, making it a better hydrogen bond acceptor than a neutral sulfhydryl group. At the second active site, C169 S gamma makes a shorter (3 A) hydrogen bond to the 3-NH of CB3717, CB3717 is approximately 1.5 A out of its binding site and there is no covalent bond between dUMP and the catalytic cysteine. Changes to partitioning among productive and non-productive conformations of reaction intermediates may contribute as much, if not more, to the diminished activity of this mutant than reduced stabilization of transition states. PMID:12702803

  8. Cadmium telluride quantum dots (CdTe-QDs) and enhanced ultraviolet-B (UV-B) radiation trigger antioxidant enzyme metabolism and programmed cell death in wheat seedlings.

    PubMed

    Chen, Huize; Gong, Yan; Han, Rong

    2014-01-01

    Nanoparticles (NPs) are becoming increasingly widespread in the environment. Free cadmium ions released from commonly used NPs under ultraviolet-B (UV-B) radiation are potentially toxic to living organisms. With increasing levels of UV-B radiation at the Earth's surface due to the depletion of the ozone layer, the potential additive effect of NPs and UV-B radiation on plants is of concern. In this study, we investigated the synergistic effect of CdTe quantum dots (CdTe-QDs), a common form of NP, and UV-B radiation on wheat seedlings. Graded doses of CdTe-QDs and UV-B radiation were tested, either alone or in combination, based on physical characteristics of 5-day-old seedlings. Treatments of wheat seedlings with either CdTe-QDs (200 mg/L) or UV-B radiation (10 KJ/m(2)/d) induced the activation of wheat antioxidant enzymes. CdTe-QDs accumulation in plant root cells resulted in programmed cell death as detected by DNA laddering. CdTe-QDs and UV-B radiation inhibited root and shoot growth, respectively. Additive inhibitory effects were observed in the combined treatment group. This research described the effects of UV-B and CdTe-QDs on plant growth. Furthermore, the finding that CdTe-QDs accumulate during the life cycle of plants highlights the need for sustained assessments of these interactions. PMID:25329900

  9. EZ-FIT: a practical curve-fitting microcomputer program for the analysis of enzyme kinetic data on IBM-PC compatible computers.

    PubMed

    Perrella, F W

    1988-11-01

    EZ-FIT, an interactive microcomputer software package, has been developed for the analysis of enzyme kinetic and equilibrium binding data. EZ-FIT was designed as a user-friendly menu-driven package that has the facility for data entry, editing, and filing. Data input permits the conversion of cpm, dpm, or optical density to molar per minute per milligram protein. Data can be fit to any of 14 model equations including Michaelis-Menten, Hill, isoenzyme, inhibition, dual substrate, agonist, antagonist, and modified integrated Michaelis-Menten. The program uses the Nelder-Mead simplex and Marquardt nonlinear regression algorithms sequentially. A report of the results includes the parameter estimates with standard errors, a Student t test to determine the accuracy of the parameter values, a Runs statistic test of the residuals, identification of outlying data, an Akaike information criterion test for goodness-of-fit, and, when the experimental variance is included, a chi 2 statistic test for goodness-of-fit. Several different graphs can be displayed: an X-Y, a Scatchard, an Eadie-Hofstee, a Lineweaver-Burk, a semilogarithmic, and a residual plot. A data analysis report and graphs are designed to evaluate the goodness-of-fit of the data to a particular model. PMID:3239747

  10. An accurate metalloprotein-specific scoring function and molecular docking program devised by a dynamic sampling and iteration optimization strategy.

    PubMed

    Bai, Fang; Liao, Sha; Gu, Junfeng; Jiang, Hualiang; Wang, Xicheng; Li, Honglin

    2015-04-27

    Metalloproteins, particularly zinc metalloproteins, are promising therapeutic targets, and recent efforts have focused on the identification of potent and selective inhibitors of these proteins. However, the ability of current drug discovery and design technologies, such as molecular docking and molecular dynamics simulations, to probe metal-ligand interactions remains limited because of their complicated coordination geometries and rough treatment in current force fields. Herein we introduce a robust, multiobjective optimization algorithm-driven metalloprotein-specific docking program named MpSDock, which runs on a scheme similar to consensus scoring consisting of a force-field-based scoring function and a knowledge-based scoring function. For this purpose, in this study, an effective knowledge-based zinc metalloprotein-specific scoring function based on the inverse Boltzmann law was designed and optimized using a dynamic sampling and iteration optimization strategy. This optimization strategy can dynamically sample and regenerate decoy poses used in each iteration step of refining the scoring function, thus dramatically improving both the effectiveness of the exploration of the binding conformational space and the sensitivity of the ranking of the native binding poses. To validate the zinc metalloprotein-specific scoring function and its special built-in docking program, denoted MpSDockZn, an extensive comparison was performed against six universal, popular docking programs: Glide XP mode, Glide SP mode, Gold, AutoDock, AutoDock4Zn, and EADock DSS. The zinc metalloprotein-specific knowledge-based scoring function exhibited prominent performance in accurately describing the geometries and interactions of the coordination bonds between the zinc ions and chelating agents of the ligands. In addition, MpSDockZn had a competitive ability to sample and identify native binding poses with a higher success rate than the other six docking programs. PMID:25746437

  11. 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.

  12. Public marginal willingness to trade off among water quality programs: Estimates of statewide and watershed-specific budget values

    NASA Astrophysics Data System (ADS)

    Blomquist, Glenn C.; Newsome, Michael A.; Stone, D. Brad

    2000-05-01

    A budget survey is used to elicit individuals' relative values for various public water quality programs. Because a budget constraint is incorporated explicitly and people allocate across various statewide and watershed-specific programs, marginal willingness to trade off values is revealed. These values are useful in the decision making of state and federal agencies responsible for water quality programs. We estimate values using the results of a 1997 random sample of Kentucky residents, with oversampling of residents of a small watershed in eastern Kentucky. Results show that people allocate the largest amounts to combat illegal dumping, untreated sewage, and hazardous material disposal. The lowest-ranked budget category, farming erosion, receives less than half the amount allocated to illegal dumping. We find that in the watershed, while the top two categories are the same as for the state as a whole, mining drainage and logging erosion are more important.

  13. 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.

  14. Phase variation of a Type IIG restriction-modification enzyme alters site-specific methylation patterns and gene expression in Campylobacter jejuni strain NCTC11168.

    PubMed

    Anjum, Awais; Brathwaite, Kelly J; Aidley, Jack; Connerton, Phillippa L; Cummings, Nicola J; Parkhill, Julian; Connerton, Ian; Bayliss, Christopher D

    2016-06-01

    Phase-variable restriction-modification systems are a feature of a diverse range of bacterial species. Stochastic, reversible switches in expression of the methyltransferase produces variation in methylation of specific sequences. Phase-variable methylation by both Type I and Type III methyltransferases is associated with altered gene expression and phenotypic variation. One phase-variable gene of Campylobacter jejuni encodes a homologue of an unusual Type IIG restriction-modification system in which the endonuclease and methyltransferase are encoded by a single gene. Using both inhibition of restriction and PacBio-derived methylome analyses of mutants and phase-variants, the cj0031c allele in C. jejuni strain NCTC11168 was demonstrated to specifically methylate adenine in 5'CCCGA and 5'CCTGA sequences. Alterations in the levels of specific transcripts were detected using RNA-Seq in phase-variants and mutants of cj0031c but these changes did not correlate with observed differences in phenotypic behaviour. Alterations in restriction of phage growth were also associated with phase variation (PV) of cj0031c and correlated with presence of sites in the genomes of these phages. We conclude that PV of a Type IIG restriction-modification system causes changes in site-specific methylation patterns and gene expression patterns that may indirectly change adaptive traits. PMID:26786317

  15. Phase variation of a Type IIG restriction-modification enzyme alters site-specific methylation patterns and gene expression in Campylobacter jejuni strain NCTC11168

    PubMed Central

    Anjum, Awais; Brathwaite, Kelly J.; Aidley, Jack; Connerton, Phillippa L.; Cummings, Nicola J.; Parkhill, Julian; Connerton, Ian; Bayliss, Christopher D.

    2016-01-01

    Phase-variable restriction-modification systems are a feature of a diverse range of bacterial species. Stochastic, reversible switches in expression of the methyltransferase produces variation in methylation of specific sequences. Phase-variable methylation by both Type I and Type III methyltransferases is associated with altered gene expression and phenotypic variation. One phase-variable gene of Campylobacter jejuni encodes a homologue of an unusual Type IIG restriction-modification system in which the endonuclease and methyltransferase are encoded by a single gene. Using both inhibition of restriction and PacBio-derived methylome analyses of mutants and phase-variants, the cj0031c allele in C. jejuni strain NCTC11168 was demonstrated to specifically methylate adenine in 5′CCCGA and 5′CCTGA sequences. Alterations in the levels of specific transcripts were detected using RNA-Seq in phase-variants and mutants of cj0031c but these changes did not correlate with observed differences in phenotypic behaviour. Alterations in restriction of phage growth were also associated with phase variation (PV) of cj0031c and correlated with presence of sites in the genomes of these phages. We conclude that PV of a Type IIG restriction-modification system causes changes in site-specific methylation patterns and gene expression patterns that may indirectly change adaptive traits. PMID:26786317

  16. COMPETITIVE ENZYME-LINKED IMMUNOSORBENT ASSAY BASED ON A RHOPTRY-ASSOCIATED PROTEIN 1 EPITOPE SPECIFICALLY IDENTIFIES BABESIA BOVIS-INFECTED CATTLE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The competitive ELISA (cELISA) format has proven to be an accurate, reliable, easily standardized, and high throughput method to detect hemoparasite infections. In this study, a specied-specific, broadly conserved, and tandemly repeated B-cell epitope within the C-terminus of the rhoptry associated ...

  17. 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

  18. Genomic selection needs to be carefully assessed to meet specific requirements in livestock breeding programs

    PubMed Central

    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. PMID

  19. 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. PMID

  20. Optimization of headspace solid-phase microextraction for the analysis of specific flavors in enzyme modified and natural Cheddar cheese using factorial design and response surface methodology.

    PubMed

    Januszkiewicz, Julien; Sabik, Hassan; Azarnia, Sorayya; Lee, Byong

    2008-06-27

    A headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC/MS) method was developed using experimental designs to quantify the flavor of commercial Cheddar cheese and enzyme-modified Cheddar cheese (EMCC). Seven target compounds (dimethyl disulfide, hexanal, hexanol, 2-heptanone, ethyl hexanoate, heptanoic acid, delta-decalactone) representative of different chemical families frequently present in Cheddar cheese were selected for this study. Three types of SPME fibres were tested: Carboxen/polydimethylsiloxane (CAR/PDMS), polyacrylate (PA) and Carbowax/divinylbenzene (CW/DVB). NaCl concentration and temperature, as well as extraction time were tested for their effect on the HS-SPME process. Two series of two-level full factorial designs were carried out for each fibre to determine the factors which best support the extraction of target flavors. Therefore, central composite designs (CCDs) were performed and response surface models were derived. Optimal extraction conditions for all selected compounds, including internal standards, were: 50 min at 55 degrees C in 3M NaCl for CAR/PDMS, 64 min at 62 degrees C in 6M NaCl for PA, and 37 min at 67 degrees C in 6M NaCl for CW/DVB. Given its superior sensitivity, CAR/PDMS fibre was selected to evaluate the target analytes in commercial Cheddar cheese and EMCC. With this fibre, calibration curves were linear for all targeted compounds (from 0.5 to 6 microg g(-1)), except for heptanoic acid which only showed a linear response with PA fibres. Detection limits ranged from 0.3 to 1.6 microg g(-1) and quantification limits from 0.8 to 3.6 microg g(-1). The mean repeatability value for all flavor compounds was 8.8%. The method accuracy is satisfactory with recoveries ranging from 97 to 109%. Six of the targeted flavors were detected in commercial Cheddar cheese and EMCC. PMID:18495140