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Sample records for enzymatically inactive recombinant

  1. [Enzymatic regulatory processes in gene recombination].

    PubMed

    Kovarskiĭ, V A; Profir, A V

    1988-01-01

    Recombination bistability in the system of genetic regulation in pro- and eucaryots is analysed on the basis of sigmoid kinetics of regulatory enzymes. It is shown that under an increase of either exogenic factors (temperature) or endogenic factors (concentration of molecules, which activate the enzymes) of crucial values, bistability solutions for recombination frequencies are possible. Histeresic character of the dependence of this value on the external parameters is pointed out. The role of fluctuation processes in distortion of the memory effects is discussed. On the basis of monostable solutions molecular account for the empiric Plau law is given for U-shaped dependence of recombination frequency on temperature.

  2. Non-Enzymatic Template-Directed Recombination of RNAs

    PubMed Central

    Nechaev, Sergey Y.; Lutay, Alexei V.; Vlassov, Valentin V.; Zenkova, Marina A.

    2009-01-01

    RNA non-enzymatic recombination reactions are of great interest within the hypothesis of the “RNA world”, which argues that at some stage of prebiotic life development proteins were not yet engaged in biochemical reactions and RNA carried out both the information storage task and the full range of catalytic roles necessary in primitive self-replicating systems. Here we report on the study of recombination reaction occuring between two 96 nucleotides (nts) fragments of RNAs under physiological conditions and governed by a short oligodeoxyribonucleotide template, partially complementary to sequences within each of the RNAs. Analysis of recombination products shows that ligation is predominantly template-directed, and occurs within the complementary complex with the template in “butt-to-butt” manner, in 1- or 3- nts bulges or in 2–3 nts internal loops. Minor recombination products formed in the template-independent manner are detected as well. PMID:19468339

  3. Distinction of two different classes of small-cell lung cancer cell lines by enzymatically inactive neuron-specific enolase.

    PubMed Central

    Splinter, T. A.; Verkoelen, C. F.; Vlastuin, M.; Kok, T. C.; Rijksen, G.; Haglid, K. G.; Boomsma, F.; van de Gaast, A.

    1992-01-01

    Neuron specific enolase (NSE) is widely used as a neuro-endocrine marker. However the presence of NSE in many non-neuroendocrine tissues has raised questions on the specificity of NSE. We have investigated NSE immunoreactivity (NSA-ag), gamma-enolase activity and total enolase activity in small cell lung cancer (SCLC) cell lines. During well-controlled exponential growth comparison of NSE-ag content and gamma-enolase activity with the doubling-time (Td) and NSE-ag content with gamma-enolase and total enolase activity led to a clear distinction of two types of cell line: variant cell lines plus part of the classic cell lines (type I) and the remaining classic cell lines (type II). The distinction was based upon both an abrupt 6-fold increase of gamma-enolase activity and an 18-fold increase of NSE-ag, which for the larger part was enzymatically inactive. Within each group the increase of NSE-ag content was significantly correlated with the increase of gamma-enolase activity and both NSE-ag content and gamma-enolase activity increased linearly with Td. It is concluded that gamma-enolase seems to be associated with the regulation of growth rate and that a compound with the gamma-enolase antigen but without enzyme activity can distinguish two different classes of SCLC cell lines. Furthermore the demonstration that NSE-ag can represent the active enzyme as well as an enzymatically inactive compound may explain why a controversy about neuron- or non-specificity of NSE exists. PMID:1333786

  4. Structure-Informed Design of an Enzymatically Inactive Vaccine Component for Group A Streptococcus

    PubMed Central

    Henningham, Anna; Ericsson, Daniel J.; Langer, Karla; Casey, Lachlan W.; Jovcevski, Blagojce; Chhatwal, G. Singh; Aquilina, J. Andrew; Batzloff, Michael R.; Kobe, Bostjan; Walker, Mark J.

    2013-01-01

    ABSTRACT Streptococcus pyogenes (group A Streptococcus [GAS]) causes ~700 million human infections/year, resulting in >500,000 deaths. There is no commercial GAS vaccine available. The GAS surface protein arginine deiminase (ADI) protects mice against a lethal challenge. ADI is an enzyme that converts arginine to citrulline and ammonia. Administration of a GAS vaccine preparation containing wild-type ADI, a protein with inherent enzymatic activity, may present a safety risk. In an approach intended to maximize the vaccine safety of GAS ADI, X-ray crystallography and structural immunogenic epitope mapping were used to inform vaccine design. This study aimed to knock out ADI enzyme activity without disrupting the three-dimensional structure or the recognition of immunogenic epitopes. We determined the crystal structure of ADI at 2.5 Å resolution and used it to select a number of amino acid residues for mutagenesis to alanine (D166, E220, H275, D277, and C401). Each mutant protein displayed abrogated activity, and three of the mutant proteins (those with the D166A, H275A, and D277A mutations) possessed a secondary structure and oligomerization state equivalent to those of the wild type, produced high-titer antisera, and avoided disruption of B-cell epitopes of ADI. In addition, antisera raised against the D166A and D277A mutant proteins bound to the GAS cell surface. The inactivated D166A and D277A mutant ADIs are ideal for inclusion in a GAS vaccine preparation. There is no human ortholog of ADI, and we confirm that despite limited structural similarity in the active-site region to human peptidyl ADI 4 (PAD4), ADI does not functionally mimic PAD4 and antiserum raised against GAS ADI does not recognize human PAD4. PMID:23919999

  5. Inactive enzymatic mutant proteins (phosphoglycerate mutase and enolase) as sugar binders for ribulose-1,5-bisphosphate regeneration reactors.

    PubMed

    De, Debojyoti; Dutta, Debajyoti; Kundu, Moloy; Mahato, Sourav; Schiavone, Marc T; Chaudhuri, Surabhi; Giri, Ashok; Gupta, Vidya; Bhattacharya, Sanjoy K

    2005-02-02

    BACKGROUND: Carbon dioxide fixation bioprocess in reactors necessitates recycling of D-ribulose1,5-bisphosphate (RuBP) for continuous operation. A radically new close loop of RuBP regenerating reactor design has been proposed that will harbor enzyme-complexes instead of purified enzymes. These reactors will need binders enabling selective capture and release of sugar and intermediate metabolites enabling specific conversions during regeneration. In the current manuscript we describe properties of proteins that will act as potential binders in RuBP regeneration reactors. RESULTS: We demonstrate specific binding of 3-phosphoglycerate (3PGA) and 3-phosphoglyceraldehyde (3PGAL) from sugar mixtures by inactive mutant of yeast enzymes phosphoglycerate mutase and enolase. The reversibility in binding with respect to pH and EDTA has also been shown. No chemical conversion of incubated sugars or sugar intermediate metabolites were found by the inactive enzymatic proteins. The dissociation constants for sugar metabolites are in the micromolar range, both proteins showed lower dissociation constant (Kd) for 3-phosphoglycerate (655-796 muM) compared to 3-phosphoglyceraldehyde (822-966 muM) indicating higher affinity for 3PGA. The proteins did not show binding to glucose, sucrose or fructose within the sensitivity limits of detection. Phosphoglycerate mutase showed slightly lower stability on repeated use than enolase mutants. CONCLUSIONS: The sugar and their intermediate metabolite binders may have a useful role in RuBP regeneration reactors. The reversibility of binding with respect to changes in physicochemical factors and stability when subjected to repeated changes in these conditions are expected to make the mutant proteins candidates for in-situ removal of sugar intermediate metabolites for forward driving of specific reactions in enzyme-complex reactors.

  6. Inactive enzymatic mutant proteins (phosphoglycerate mutase and enolase) as sugar binders for ribulose-1,5-bisphosphate regeneration reactors

    PubMed Central

    De, Debojyoti; Dutta, Debajyoti; Kundu, Moloy; Mahato, Sourav; Schiavone, Marc T; Chaudhuri, Surabhi; Giri, Ashok; Gupta, Vidya; Bhattacharya, Sanjoy K

    2005-01-01

    Background Carbon dioxide fixation bioprocess in reactors necessitates recycling of D-ribulose1,5-bisphosphate (RuBP) for continuous operation. A radically new close loop of RuBP regenerating reactor design has been proposed that will harbor enzyme-complexes instead of purified enzymes. These reactors will need binders enabling selective capture and release of sugar and intermediate metabolites enabling specific conversions during regeneration. In the current manuscript we describe properties of proteins that will act as potential binders in RuBP regeneration reactors. Results We demonstrate specific binding of 3-phosphoglycerate (3PGA) and 3-phosphoglyceraldehyde (3PGAL) from sugar mixtures by inactive mutant of yeast enzymes phosphoglycerate mutase and enolase. The reversibility in binding with respect to pH and EDTA has also been shown. No chemical conversion of incubated sugars or sugar intermediate metabolites were found by the inactive enzymatic proteins. The dissociation constants for sugar metabolites are in the micromolar range, both proteins showed lower dissociation constant (Kd) for 3-phosphoglycerate (655–796 μM) compared to 3-phosphoglyceraldehyde (822–966 μM) indicating higher affinity for 3PGA. The proteins did not show binding to glucose, sucrose or fructose within the sensitivity limits of detection. Phosphoglycerate mutase showed slightly lower stability on repeated use than enolase mutants. Conclusions The sugar and their intermediate metabolite binders may have a useful role in RuBP regeneration reactors. The reversibility of binding with respect to changes in physicochemical factors and stability when subjected to repeated changes in these conditions are expected to make the mutant proteins candidates for in-situ removal of sugar intermediate metabolites for forward driving of specific reactions in enzyme-complex reactors. PMID:15689239

  7. Enzymatic characterization of recombinant nitrate reductase expressed and purified from Neurospora crassa.

    PubMed

    Ringel, Phillip; Probst, Corinna; Dammeyer, Thorben; Buchmeier, Sabine; Jänsch, Lothar; Wissing, Josef; Tinnefeld, Philip; Mendel, Ralf R; Jockusch, Brigitte M; Kruse, Tobias

    2015-07-01

    We established an expression and purification procedure for recombinant protein production in Neurospora crassa (N. crassa). This Strep-tag® based system was successfully used for purifying recombinant N. crassa nitrate reductase (NR), whose enzymatic activity was compared to recombinant N. crassa NR purified from Escherichia coli. The purity of the two different NR preparations was similar but NR purified from N. crassa showed a significantly higher nitrate turnover rate. Two phosphorylation sites were identified for NR purified from the endogenous expression system. We conclude that homologous expression of N. crassa NR yields a higher active enzyme and propose that NR phosphorylation causes enhanced enzymatic activity.

  8. Enzymatic vitreolysis with recombinant tissue plasminogen activator for vitreomacular traction

    PubMed Central

    Raczyńska, Dorota; Lipowski, Paweł; Zorena, Katarzyna; Skorek, Andrzej; Glasner, Paulina

    2015-01-01

    Aims The aim of our research was to gain data about the efficacy of intravitreal injections of a recombinant tissue plasminogen activator (rTPA) in dissolving vitreoretinal tractions (VRTs). Materials and methods The study group consisted of patients of our Ophthalmology Clinic who had received an injection of rTPA (TPA Group) for an existent vitreomacular traction confirmed by optical coherence tomography and stereoscopic examinations. The control group consisted of patients who had declined treatment despite the existence of a vitreomacular traction confirmed by the same diagnostic methods. Each group consisted of 30 people (30 eyes). The observation period was 6 months. Conclusion In both groups some of the VRTs had dissolved. In the TPA group the traction dissolved in 10 patients (33.33%) and in the control group only in 5 (16.67%). It is also important to point out that the mean baseline membrane thickness was higher in the TPA group than in the control group. Observing patients in both groups we noticed that the dissolution of vitreoretinal membrane occurred most frequently in those cases where the membrane was thin. In the TPA group, the mean membrane thickness after 6 months decreased considerably. At the same time, no significant change in the membrane thickness could be observed in the control group. Observation of the retinal thickness allows us to draw the following conclusion: in the TPA group, the retinal thickness in the macular area (edema) had decreased over the study period, whereas in the control group it had increased. In those cases where the traction had dissolved, the edema of the retina decreased by the end of the 6-month period in both groups. In the TPA group, the dissolution of the membrane occurred most often within 3 months from the primary injection. Based on statistics, we can confirm that in the control group there was a decrease in visual acuity during the 6 months of the study period. At the same time, visual acuity in the TPA

  9. Enzymatic Pre-treatment of Wastewater to Minimize Recovery by Reverse Transcriptase PCR of RNA from Inactive Bacteriophages.

    PubMed

    Unnithan, Veena V; Unc, Adrian; Smith, Geoffrey B

    2015-07-01

    Quantitative viral risk assessments for wastewaters are notoriously difficult. The often considered quantitative reverse transcriptase PCR reflects poorly on virus infectivity rates leading to inaccurate risk interpretations. Various techniques focused on the degradation of the nucleic acids of non-infective viruses were previously employed. We comparatively assessed the effectiveness of such enzymatic treatments for MS2 bacteriophage in treated wastewaters. The single use of RNase A at an appropriate concentration may be as effective as the combination of RNase followed by Proteinase K and more rapid. While all tested enzymatic treatments minimized recovery of RNA (>95 %) in the absence of infective MS2, none completely eliminated the signal recovery. Selection of any enzymatic protocol for minimizing recovery of RNA from degraded, non-infective viruses should balance the methods efficacy with its expediency.

  10. Expression, purification and enzymatic characterization of a recombinant human ubiquitin-specific protease 47.

    PubMed

    Piao, Jinhua; Tashiro, Aika; Nishikawa, Minako; Aoki, Yutaka; Moriyoshi, Eiko; Hattori, Akira; Kakeya, Hideaki

    2015-12-01

    In this study, the physicochemical and enzymatic properties of recombinant human ubiquitin (Ub)-specific protease (USP) 47, a novel member of the C19 family of de-ubiquitinating enzymes (DUB), were characterized for the first time. Recombinant human USP47 was expressed in a baculovirus expression system and purified to homogeneity. The purified protein was shown to be a monomeric protein with a molecular mass of ∼146 kDa on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. USP47 released Ub from Ub-aminoacyl-4-metheylcoumaryl-7-amide and Ub-tagged granzyme B. The substitution of the potential nucleophile Cys109 with Ser severely abrogated the Ub-releasing activity of USP47, indicating that USP47 is indeed a cysteine DUB. An assay using Ub dimer substrates showed that the enzyme cleaved a variety of isopeptide bonds between 2 Ub molecules, including the Lys48- and Lys63-linked isopeptide bonds. USP47 also released a Ub moiety from Lys48- and Lys63-linked polyUb chains. Of the inhibitors tested, N-ethylmaleimide, Zn ion and Ub aldehyde revealed a dose-dependent inhibition of USP47. In this study, clear differences in the enzymatic properties between USP47 and USP7 (the most closely related proteins among DUBs) were also found. Therefore, our results suggest that USP47 may play distinct roles in Ub-mediated cellular processes via DUB activity.

  11. Enzymatic cross-linking of human recombinant elastin (HELP) as biomimetic approach in vascular tissue engineering.

    PubMed

    Bozzini, Sabrina; Giuliano, Liliana; Altomare, Lina; Petrini, Paola; Bandiera, Antonella; Conconi, Maria Teresa; Farè, Silvia; Tanzi, Maria Cristina

    2011-12-01

    The use of polymers naturally occurring in the extracellular matrix (ECM) is a promising strategy in regenerative medicine. If compared to natural ECM proteins, proteins obtained by recombinant DNA technology have intrinsic advantages including reproducible macromolecular composition, sequence and molecular mass, and overcoming the potential pathogens transmission related to polymers of animal origin. Among ECM-mimicking materials, the family of recombinant elastin-like polymers is proposed for drug delivery applications and for the repair of damaged elastic tissues. This work aims to evaluate the potentiality of a recombinant human elastin-like polypeptide (HELP) as a base material of cross-linked matrices for regenerative medicine. The cross-linking of HELP was accomplished by the insertion of cross-linking sites, glutamine and lysine, in the recombinant polymer and generating ε-(γ-glutamyl) lysine links through the enzyme transglutaminase. The cross-linking efficacy was estimated by infrared spectroscopy. Freeze-dried cross-linked matrices showed swelling ratios in deionized water (≈2500%) with good structural stability up to 24 h. Mechanical compression tests, performed at 37°C in wet conditions, in a frequency sweep mode, indicated a storage modulus of 2/3 kPa, with no significant changes when increasing number of cycles or frequency. These results demonstrate the possibility to obtain mechanically resistant hydrogels via enzymatic crosslinking of HELP. Cytotoxicity tests of cross-linked HELP were performed with human umbilical vein endothelial cells, by use of transwell filter chambers for 1-7 days, or with its extracts in the opportune culture medium for 24 h. In both cases no cytotoxic effects were observed in comparison with the control cultures. On the whole, the results suggest the potentiality of this genetically engineered HELP for regenerative medicine applications, particularly for vascular tissue regeneration.

  12. High Yield of Recombinant Protein in Shaken E. coli Cultures with Enzymatic Glucose Release Medium EnPresso B.

    PubMed

    Ukkonen, Kaisa; Neubauer, Antje; Pereira, Vinit J; Vasala, Antti

    2017-01-01

    Expression of recombinant proteins in sufficient quantities is essential for protein structure-function studies. The most commonly used method for recombinant protein production is overexpression in E. coli cultures. However, producing high yields of functional proteins in E. coli can be a challenge in conventional shaken cultures. This is often due to nonoptimal growth conditions, which result in low cell yields and insoluble or incorrectly folded target protein. To overcome the shortcomings of shake flask cultivation, we present a cultivation method based on enzymatic glucose delivery. This system mimics the fed-batch principle used in bioreactor cultivations and provides high yields of biomass and recombinant proteins in shaken cultivations.

  13. Purification, enzymatic activity and inhibitor discovery for recombinant human carbonic anhydrase XIV.

    PubMed

    Juozapaitienė, Vaida; Bartkutė, Brigita; Michailovienė, Vilma; Zakšauskas, Audrius; Baranauskienė, Lina; Satkūnė, Sandra; Matulis, Daumantas

    2016-12-20

    Human carbonic anhydrase XIV (CA XIV), a transmembrane protein, highly expressed in the central nervous system, is difficult to recombinantly express and purify in large scale for the measurements of inhibitor binding and drug design. CA XIV belongs to the family of twelve catalytically active CA isoforms in the human body. Disorders in the expression of CA XIV cause serious diseases and CA XIV has been described as a possible drug target for the treatment of epilepsy, some retinopathies, and skin tumors. In this study, the effect of different promoters, E. coli strains, and the length of recombinant CA XIV protein construct were analyzed for the production CA XIV in large scale by using affinity purification. Active site titration by inhibitors and the isothermal titration calorimery revealed over 96% purity of the protein. Enzymatic activity of the purified CA XIV was determined by following the CO2 hydration using the stopped-flow technique. Several inhibitors were discovered that exhibited selectivity towards CA XIV over other CA isoforms and could be developed as drugs. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. [Correlation of antitumor effect of recombinant sea snake basic phospholipase A2 to its enzymatic activity].

    PubMed

    Liang, Yong-Ju; Yang, Xiao-Ping; Wei, Jian-Wen; Fu, Li-Wu; Jiang, Xiao-Yu; Chen, Shang-Wu; Yang, Wen-Li

    2005-12-01

    Snake venom phospolipase A2 (PLA(2)), a large family of homologous (14 ku) soluble proteins, exerts diverse pharmacologic activities as well as enzymatic activities. So far, the structure and function of terrestrial snake PLA(2), especially the relationship of its enzymatic and pharmacologic activities have been studied extensively, but the investigation of sea snake PLA(2) are limited. This study was to investigate the in vitro and in vivo antitumor effects of recombinant sea snake basic PLA(2) (rSSBPLA(2)) and its mutants rN48 and rK4 from sea snake Lapemis hardwickii venom, and to explore the influence of 2 residues related with the enzymatic activity on the antitumor effects. Site-directed mutagenesis of the 2 conserved residues related with enzymatic activity (His48 mutated to Asn and Asp49 mutated to Lys) was performed. The inhibitory effects of rSSBPLA(2), rN48 and rK49 on proliferation of human myeloid leukemia cell line HL-60, human neuroblastoma cell line SK-N-SH, human gastric cancer cell line MGC-803, and human liver cancer cell line HepG2 were assessed by MTT assay. Their antitumor effects on sarcoma cell line S180 xenograft and EAC ascites cancer model in mice were detected. The relative enzymatic activities of rN48 and rK49 were 0 and 5% of that of rSSBPLA(2). The 50% inhibitory concentration (IC(50)) of rSSBPLA(2) for HL60, SK-N-SH, and MGC-803 cells were (45.28+/-0.09) microg/ml, (57.07+/-0.12) microg/ml, and (69.34+/-0.35) microg/ml, respectively, but it had no inhibitory effect on proliferation of HepG2 cells. rSSBPLA(2) obviously inhibited growth of S180 xenograft in miceû the inhibitory rates were 50.8%, 43.2%, 38.2%, and 55.5%, respectively, under the dose of 2 mg/kg (qd x 10), 2 mg/kg (q2d x 5), 4 mg/kg (qd x 1) and 4 mg/kg (q5d x 2). The inhibitory rate of EAC model was 33.5% under the dose of 4 mg/kg (q5d x 2). The inhibitory rates were significantly higher in test groups than in control groups (P<0.01). rN48 and rK49 had no inhibitory

  15. Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.

    PubMed Central

    Barnes, H J; Arlotto, M P; Waterman, M R

    1991-01-01

    When the cDNA encoding bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017 alpha per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017 alpha can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017 alpha enzyme. Surprisingly, it is found that E. coli contain an electron-transport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17 alpha-hydroxylase and 17,20-lyase activities of P45017 alpha. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structure-function analysis of the cytochrome P450 system. Images PMID:1829523

  16. Co-cultivation of Aspergillus nidulans Recombinant Strains Produces an Enzymatic Cocktail as Alternative to Alkaline Sugarcane Bagasse Pretreatment

    PubMed Central

    Lima, Matheus S.; Damasio, André R. de L.; Crnkovic, Paula M.; Pinto, Marcelo R.; da Silva, Ana M.; da Silva, Jean C. R.; Segato, Fernando; de Lucas, Rosymar C.; Jorge, João A.; Polizeli, Maria de L. T. de M.

    2016-01-01

    Plant materials represent a strategic energy source because they can give rise to sustainable biofuels through the fermentation of their carbohydrates. A clear example of a plant-derived biofuel resource is the sugar cane bagasse exhibiting 60–80% of fermentable sugars in its composition. However, the current methods of plant bioconversion employ severe and harmful chemical/physical pretreatments raising biofuel cost production and environmental degradation. Replacing these methods with co-cultivated enzymatic cocktails is an alternative. Here we propose a pretreatment for sugarcane bagasse using a multi-enzymatic cocktail from the co-cultivation of four Aspergillus nidulans recombinant strains. The co-cultivation resulted in the simultaneous production of GH51 arabinofuranosidase (AbfA), GH11 endo-1,4-xylanase (XlnA), GH43 endo-1,5-arabinanase (AbnA) and GH12 xyloglucan specific endo-β-1,4-glucanase (XegA). This core set of recombinant enzymes was more efficient than the alternative alkaline method in maintaining the cellulose integrity and exposing this cellulose to the following saccharification process. Thermogravimetric and differential thermal analysis revealed residual byproducts on the alkali pretreated biomass, which were not found in the enzymatic pretreatment. Therefore, the enzymatic pretreatment was residue-free and seemed to be more efficient than the applied alkaline method, which makes it suitable for bioethanol production. PMID:27199917

  17. Co-cultivation of Aspergillus nidulans Recombinant Strains Produces an Enzymatic Cocktail as Alternative to Alkaline Sugarcane Bagasse Pretreatment.

    PubMed

    Lima, Matheus S; Damasio, André R de L; Crnkovic, Paula M; Pinto, Marcelo R; da Silva, Ana M; da Silva, Jean C R; Segato, Fernando; de Lucas, Rosymar C; Jorge, João A; Polizeli, Maria de L T de M

    2016-01-01

    Plant materials represent a strategic energy source because they can give rise to sustainable biofuels through the fermentation of their carbohydrates. A clear example of a plant-derived biofuel resource is the sugar cane bagasse exhibiting 60-80% of fermentable sugars in its composition. However, the current methods of plant bioconversion employ severe and harmful chemical/physical pretreatments raising biofuel cost production and environmental degradation. Replacing these methods with co-cultivated enzymatic cocktails is an alternative. Here we propose a pretreatment for sugarcane bagasse using a multi-enzymatic cocktail from the co-cultivation of four Aspergillus nidulans recombinant strains. The co-cultivation resulted in the simultaneous production of GH51 arabinofuranosidase (AbfA), GH11 endo-1,4-xylanase (XlnA), GH43 endo-1,5-arabinanase (AbnA) and GH12 xyloglucan specific endo-β-1,4-glucanase (XegA). This core set of recombinant enzymes was more efficient than the alternative alkaline method in maintaining the cellulose integrity and exposing this cellulose to the following saccharification process. Thermogravimetric and differential thermal analysis revealed residual byproducts on the alkali pretreated biomass, which were not found in the enzymatic pretreatment. Therefore, the enzymatic pretreatment was residue-free and seemed to be more efficient than the applied alkaline method, which makes it suitable for bioethanol production.

  18. Iron Inhibits Activation-induced Cytidine Deaminase Enzymatic Activity and Modulates Immunoglobulin Class Switch DNA Recombination*

    PubMed Central

    Li, Guideng; Pone, Egest J.; Tran, Daniel C.; Patel, Pina J.; Dao, Lisa; Xu, Zhenming; Casali, Paolo

    2012-01-01

    Immunoglobulin (Ig) class switch DNA recombination (CSR) and somatic hypermutation (SHM) are critical for the maturation of the antibody response. Activation-induced cytidine deaminase (AID) initiates CSR and SHM by deaminating deoxycytidines (dCs) in switch (S) and V(D)J region DNA, respectively, to generate deoxyuracils (dUs). Processing of dUs by uracil DNA glycosylase (UNG) yields abasic sites, which are excised by apurinic/apyrimidinic endonucleases, eventually generating double strand DNA breaks, the obligatory intermediates of CSR. Here, we found that the bivalent iron ion (Fe2+, ferrous) suppressed CSR, leading to decreased number of switched B cells, decreased postrecombination Iμ-CH transcripts, and reduced titers of secreted class-switched IgG1, IgG3, and IgA antibodies, without alterations in critical CSR factors, such as AID, 14-3-3γ, or PTIP, or in general germline IH-S-CH transcription. Fe2+ did not affect B cell proliferation or plasmacytoid differentiation. Rather, it inhibited AID-mediated dC deamination in a dose-dependent fashion. The inhibition of intrinsic AID enzymatic activity by Fe2+ was specific, as shown by lack of inhibition of AID-mediated dC deamination by other bivalent metal ions, such as Zn2+, Mn2+, Mg2+, or Ni2+, and the inability of Fe2+ to inhibit UNG-mediated dU excision. Overall, our findings have outlined a novel role of iron in modulating a B cell differentiation process that is critical to the generation of effective antibody responses to microbial pathogens and tumoral cells. They also suggest a possible role of iron in dampening AID-dependent autoimmunity and neoplastic transformation. PMID:22556412

  19. High-level expression and characterization of recombinant acid urease for enzymatic degradation of urea in rice wine.

    PubMed

    Yang, Yuqing; Kang, Zhen; Zhou, Jianli; Chen, Jian; Du, Guocheng

    2015-01-01

    Ethylcarbamate, a carcinogenic compound, is formed from urea and ethanol in rice wine, and enzymatic elimination of urea is always attractive. In the present work, we amplified the acid urease gene cluster ureABCEFGD from Lactobacillus reuteri CICC6124 and constructed robust Lactococcus lactis cell factories for the production of acid urease. The titer of the recombinant acid urease was increased from 1,550 to 11,560 U/L by optimization of the cultivation process. Meanwhile, the enzyme showed satisfied properties toward urea elimination in the rice wine model system. By incubating the enzyme (50 U/L) at 20 °C for 60 h, about 95.8% of urea in rice wine was removed. Interestingly, this acid urease also exhibited activity toward ethylcarbamate. The results demonstrated that this recombinant acid urease has great potential in the elimination of urea in rice wine.

  20. Recombinant Botulinum Neurotoxin Hc Subunit (BoNT Hc) and Catalytically Inactive Clostridium botulinum Holoproteins (ciBoNT HPs) as Vaccine Candidates for the Prevention of Botulism.

    PubMed

    Webb, Robert P; Smith, Theresa J; Smith, Leonard A; Wright, Patrick M; Guernieri, Rebecca L; Brown, Jennifer L; Skerry, Janet C

    2017-09-03

    There are few available medical countermeasures against botulism and the discontinuation of the pentavalent botulinum toxoid vaccine by the Centers for Disease Control and Prevention in 2011 has resulted in the need for a safe and effective prophylactic alternative. Advances in genetic engineering have resulted in subsequent vaccine efforts being primarily focused on the production of highly purified recombinant protein antigens representing one or more domains of the botulinum neurotoxin. Recombinant subunit vaccines based on the carboxy one-third of the toxin (Hc) developed in our lab against serotypes A-F have been shown to be safe and effective. However, in response to the identification of an ever increasing number of BoNT subtypes with significant amino acid heterogeneity, we have developed catalytically inactive BoNT holoproteins (ciBoNT HPs) in an attempt to elicit greater protective immunity to address these toxin variants. Here we report the production of ciBoNT/B1 HP, ciBoNT/C1 HP, ciBoNT/E1 HP and ciBoNT/F1 HP and compare the immunological and protective abilities of ciBoNT HPs and BoNT/A Hc, BoNT/B Hc, BoNT/C Hc, BoNT/E Hc and BoNT/F Hc vaccines when challenged with homologous and heterologous toxins. Our results suggest the ciBoNT HP vaccines exhibit superior potency after single vaccinations but multiple vaccinations with BoNT/Hc antigens resulted in increased survival rates at the toxin challenge levels used.

  1. Crystal structure of an enzymatically inactive trans-sialidase-like lectin from Trypanosoma cruzi: the carbohydrate binding mechanism involves residual sialidase activity.

    PubMed

    Oppezzo, Pablo; Obal, Gonzalo; Baraibar, Martín A; Pritsch, Otto; Alzari, Pedro M; Buschiazzo, Alejandro

    2011-09-01

    Trans-sialidases are surface-located proteins in Trypanosoma cruzi that participate in key parasite-host interactions and parasite virulence. These proteins are encoded by a large multigenic family, with tandem-repeated and individual genes dispersed throughout the genome. While a large number of genes encode for catalytically active enzyme isoforms, many others display mutations that involve catalytic residues. The latter ultimately code for catalytically inactive proteins with very high similarity to their active paralogs. These inactive members have been shown to be lectins, able to bind sialic acid and galactose in vitro, although their cellular functions are yet to be fully established. We now report structural and biochemical evidence extending the current molecular understanding of these lectins. We have solved the crystal structure of one such catalytically inactive trans-sialidase-like protein, after soaking with a specific carbohydrate ligand, sialyl-α2,3-lactose. Instead of the expected trisaccharide, the binding pocket was observed occupied by α-lactose, strongly suggesting that the protein retains residual hydrolytic activity. This hypothesis was validated by enzyme kinetics assays, in comparison to fully active wild-type trans-sialidase. Surface plasmon resonance also confirmed that these trans-sialidase-like lectins are not only able to bind small oligosaccharides, but also sialylated glycoproteins, which is relevant in the physiologic scenario of parasite infection. Inactive trans-sialidase proteins appear thus to be β-methyl-galactosyl-specific lectins, evolved within an exo-sialidase scaffold, thus explaining why their lectin activity is triggered by the presence of terminal sialic acid.

  2. Detection and Quantitation of Afucosylated N-Linked Oligosaccharides in Recombinant Monoclonal Antibodies Using Enzymatic Digestion and LC-MS

    NASA Astrophysics Data System (ADS)

    Du, Yi; May, Kimberly; Xu, Wei; Liu, Hongcheng

    2012-07-01

    The presence of N-linked oligosaccharides in the CH2 domain has a significant impact on the structure, stability, and biological functions of recombinant monoclonal antibodies. The impact is also highly dependent on the specific oligosaccharide structures. The absence of core-fucose has been demonstrated to result in increased binding affinity to Fcγ receptors and, thus, enhanced antibody-dependent cellular cytotoxicity (ADCC). Therefore, a method that can specifically determine the level of oligosaccharides without the core-fucose (afucosylation) is highly desired. In the current study, recombinant monoclonal antibodies and tryptic peptides from the antibodies were digested using endoglycosidases F2 and H, which cleaves the glycosidic bond between the two primary GlcNAc residues. As a result, various oligosaccharides of either complex type or high mannose type that are commonly observed for recombinant monoclonal antibodies are converted to either GlcNAc residue only or GlcNAc with the core-fucose. The level of GlcNAc represents the sum of all afucosylated oligosaccharides, whereas the level of GlcNAc with the core-fucose represents the sum of all fucosylated oligosaccharides. LC-MS analysis of the enzymatically digested antibodies after reduction provided a quick estimate of the levels of afucosylation. An accurate determination of the level of afucosylation was obtained by LC-MS analysis of glycopeptides after trypsin digestion.

  3. Comparison of Enzymatic Traits between Native and Recombinant Glycine Sarcosine N-Methyltransferase from Methanohalophilus portucalensis FDF1T.

    PubMed

    Lai, Shu-Jung; Deng, Yu-Chen; Lai, Mei-Chin

    2016-01-01

    The halophilic methanoarchaeon Methanohalophilus portucalensis FDF1T possesses the ability to synthesize the osmolyte betaine from its precursor, glycine, in response to extracellular salt stress through a three-step transmethylation process. Analysis of recombinant glycine sarcosine N-methyltransferase (rGSMT) and recombinant sarcosine dimethylglycine N-methyltransferase (rSDMT) from Escherichia coli indicated that betaine synthesis is rate-limited by rGSMT and is constitutively activated by rSDMT. Therefore, it is of interest to purify native GSMT from Methanohalophilus portucalensis to further compare its enzymatic characteristics and kinetics with rGSMT. In this study, native GSMT was purified through DEAE ion exchange and gel filtration chromatography with 95% purity. The enzymatic characteristics of GSMT and rGSMT showed similar trends of activities that were activated by high concentrations of monovalent cations. Both were feedback-inhibited by the end product, betaine, and competitively inhibited by S-adenosylhomocysteine (SAH). Native GSMT was 2-fold more sensitive to SAH than rGSMT. Notably, comparison of the kinetic parameters illustrated that the turnover rate of glycine methylation of GSMT was promoted by potassium ions, whereas rGSMT was activated by increasing protein-glycine binding affinity. These results suggest that GSMT and rGSMT may have different levels of post-translational modifications. Our preliminary mass spectrometry evidence indicated that there was no detectable phosphosite on GSMT after the complicated purification processes, whereas purified rGSMT still possessed 23.1% of its initial phosphorylation level. We believe that a phosphorylation-mediated modification may be involved in the regulation of this energy consuming betaine synthesis pathway during the stress response in halophilic methanoarchaea.

  4. Comparison of Enzymatic Traits between Native and Recombinant Glycine Sarcosine N-Methyltransferase from Methanohalophilus portucalensis FDF1T

    PubMed Central

    Lai, Shu-Jung; Deng, Yu-Chen; Lai, Mei-Chin

    2016-01-01

    The halophilic methanoarchaeon Methanohalophilus portucalensis FDF1T possesses the ability to synthesize the osmolyte betaine from its precursor, glycine, in response to extracellular salt stress through a three-step transmethylation process. Analysis of recombinant glycine sarcosine N-methyltransferase (rGSMT) and recombinant sarcosine dimethylglycine N-methyltransferase (rSDMT) from Escherichia coli indicated that betaine synthesis is rate-limited by rGSMT and is constitutively activated by rSDMT. Therefore, it is of interest to purify native GSMT from Methanohalophilus portucalensis to further compare its enzymatic characteristics and kinetics with rGSMT. In this study, native GSMT was purified through DEAE ion exchange and gel filtration chromatography with 95% purity. The enzymatic characteristics of GSMT and rGSMT showed similar trends of activities that were activated by high concentrations of monovalent cations. Both were feedback-inhibited by the end product, betaine, and competitively inhibited by S-adenosylhomocysteine (SAH). Native GSMT was 2-fold more sensitive to SAH than rGSMT. Notably, comparison of the kinetic parameters illustrated that the turnover rate of glycine methylation of GSMT was promoted by potassium ions, whereas rGSMT was activated by increasing protein-glycine binding affinity. These results suggest that GSMT and rGSMT may have different levels of post-translational modifications. Our preliminary mass spectrometry evidence indicated that there was no detectable phosphosite on GSMT after the complicated purification processes, whereas purified rGSMT still possessed 23.1% of its initial phosphorylation level. We believe that a phosphorylation-mediated modification may be involved in the regulation of this energy consuming betaine synthesis pathway during the stress response in halophilic methanoarchaea. PMID:28036340

  5. [Properties of sucrose phosphorylase from recombinant Escherichia coli and enzymatic synthesis of alpha-arbutin].

    PubMed

    Wan, Yuejia; Ma, Jiangfeng; Xu, Rong; He, Aiyong; Jiang, Min; Chen, Kequan; Jiang, Yin

    2012-12-01

    Sucrose phosphorylase (EC 2.4.1.7, Sucrose phosphorylase, SPase) can be produced by recombinant strain Escherichia coli Rosetta(DE3)/Pet-SPase. Crude enzyme was obtained from the cells by the high pressure disruption and centrifugation. Sucrose phosphorylase was purified by Ni-NTA affinity column chromatography and desalted by ultrafiltration. The specific enzyme activity was 1.1-fold higher than that of the crude enzyme, and recovery rate was 82.7%. The purified recombinant SPase had a band of 59 kDa on SDS-PAGE. Thermostability of the enzyme was shown at temperatures up to 37 degrees C, and pH stability between pH 6.0 and 6.7. The optimum temperature and pH were 37 degrees C and 6.7, respectively. The K(m) of SPase for sucrose was 7.3 mmol/L, and Vmax was 0.2 micromol/(min x mg). Besides, alpha-arbutin was synthesized from sucrose and hydroquinone by transglucosylation with recombinant SPase. The optimal conditions for synthesis of alpha-arbutin were 200 U/mL of recombinant SPase, 20% of sucrose, and 1.6% hydroquinone at pH 6-6.5 and 25 degrees C for 21 h. Under these conditions, alpha-arbutin was obtained with a 78.3% molar yield with respect to hydroquinone, and the concentration of alpha-arbutin was about 31 g/L.

  6. Detection of contaminating enzymatic activity in plant-derived recombinant biotechnology products.

    PubMed

    Brinson, Robert G; Giulian, Gary G; Kelman, Zvi; Marino, John P

    2014-12-02

    Residual impurities in recombinantly produced protein biologics, such as host cell proteins (HCP), can potentially cause unwanted toxic or immunogenic responses in patients. Additionally, undetected impurities found in recombinant proteins used in cell culture may adversely impact basic research and biotechnology applications. Currently, the enzyme-linked immunosorbent assay (ELISA) is the standard for detection of residual HCP contamination in recombinantly produced biologics. Alternatively, two-dimensional liquid chromatography coupled to mass spectrometry is being developed as a tool for assessing this critical quality attribute. Both of these methods rely on the direct detection of HCPs and some previous knowledge of the contaminant. For contaminating enzymes, the mass level of the impurity may fall below the threshold of detection of these methods and underestimate the true impact. To address this point, here we demonstrate facile detection and characterization of contaminating phytase activity in rice-derived recombinant human serum albumin (rHSA) using a sensitive, label-free nuclear magnetic resonance (NMR) spectroscopy assay. We observed varying degrees of phytase contamination in biotechnology-grade rHSA from various manufacturers by monitoring the degradation of adenosine-5'-triphosphate and myo-inositol-1,2,3,4,5,6-hexakisphosphate by (31)P NMR. The observed lot-to-lot variability may result in irreproducible cell culture results and should be evaluated as a possible critical quality attribute in plant-derived biotherapeutics.

  7. Rapid enzymatic hydrolysis using a novel recombinant β-glucuronidase in benzodiazepine urinalysis.

    PubMed

    Morris, Ayodele A; Chester, Scot A; Strickland, Erin C; McIntire, Gregory L

    2014-10-01

    Only trace amounts of parent benzodiazepines are present in urine following extensive metabolism and conjugation. Thus, hydrolysis of glucuronides is necessary for improved detection. Enzyme hydrolysis is preferred to retain identification specificity, but can be costly and time-consuming. The assessment of a novel recombinant β-glucuronidase for rapid hydrolysis in benzodiazepine urinalysis is presented. Glucuronide controls for oxazepam, lorazepam and temazepam were treated with IMCSzyme™ recombinant β-glucuronidase. Hydrolysis efficiency was assessed at 55°C and at room temperature (RT) using the recommended optimum pH. Hydrolysis efficiency for four other benzodiazepines was evaluated solely with positive patient samples. Maximum hydrolysis of glucuronide controls at 5 min at RT (mean analyte recovery ≥ 94% for oxazepam and lorazepam and ≥ 80% for temazepam) was observed. This was considerably faster than the optimized 30 min incubation time for the abalone β-glucuronidase at 65°C. Mean analyte recovery increased at longer incubation times at 55°C for temazepam only. Total analyte in patient samples compared well to targets from abalone hydrolysis after recombinant β-glucuronidase hydrolysis at RT with no incubation. Some matrix effect, differential reactivity, conjugation variability and transformation impacting total analyte recovery were indicated. The unique potential of the IMCSzyme™ recombinant β-glucuronidase was demonstrated with fast benzodiazepine hydrolysis at RT leading to decreased processing time without the need for heat activation. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  8. Enzymatic activity and motility of recombinant Arabidopsis myosin XI, MYA1.

    PubMed

    Hachikubo, You; Ito, Kohji; Schiefelbein, John; Manstein, Dietmar J; Yamamoto, Keiichi

    2007-06-01

    We expressed recombinant Arabidopsis myosin XI (MYA1), in which the motor domain of MYA1 was connected to an artificial lever arm composed of triple helical repeats of Dictyostelium alpha-actinin, in order to understand its motor activity and intracellular function. The V(max) and K(actin) of the actin-activated Mg(2+) ATPase activity of the recombinant MYA1 were 50.7 Pi head(-1) s(-1) and 30.2 microM, respectively, at 25 degrees C. The recombinant MYA1 could translocate actin filament at the maximum velocity of 1.8 microm s(-1) at 25 degrees C in the in vitro motility assay. The value corresponded to a motility of 3.2 microm s(-1) for native MYA1 if we consider the difference in the lever arm length, and this value was very close to the velocity of cytoplasmic streaming in Arabidopsis hypocotyl epidermal cells. The extent of inhibition by ADP of the motility of MYA1 was similar to that of the well-known processive motor, myosin V, suggesting that MYA1 is a processive motor. The dissociation rate of the actin-MYA1-ADP complex induced by ATP (73.5 s(-1)) and the V(max) value of the actin-activated Mg(2+) ATPase activity revealed that MYA1 stays in the actin-bound state for about 70% of its mechanochemical cycle time. This high ratio of actin-bound states is also a characteristic of processive motors. Our results strongly suggest that MYA1 is a processive motor and involved in vesicle transport and/or cytoplasmic streaming.

  9. Enzymatic biodiesel synthesis from yeast oil using immobilized recombinant Rhizopus oryzae lipase.

    PubMed

    Duarte, Susan Hartwig; Hernández, Gonzalo Lázaro del Peso; Canet, Albert; Benaiges, Maria Dolors; Maugeri, Francisco; Valero, Francisco

    2015-05-01

    The recombinant Rhizopus oryzae lipase (1-3 positional selective), immobilized on Relizyme OD403, has been applied to the production of biodiesel using single cell oil from Candida sp. LEB-M3 growing on glycerol from biodiesel process. The composition of microbial oil is quite similar in terms of saponifiable lipids than olive oil, although with a higher amount of saturated fatty acids. The reaction was carried out in a solvent system, and n-hexane showed the best performance in terms of yield and easy recovery. The strategy selected for acyl acceptor addition was a stepwise methanol addition using crude and neutralized single cell oil, olive oil and oleic acid as substrates. A FAMEs yield of 40.6% was obtained with microbial oils lower than olive oil 54.3%. Finally in terms of stability, only a lost about 30% after 6 reutilizations were achieved.

  10. Albinism-Causing Mutations in Recombinant Human Tyrosinase Alter Intrinsic Enzymatic Activity

    PubMed Central

    Dolinska, Monika B.; Kovaleva, Elena; Backlund, Peter; Wingfield, Paul T.; Brooks, Brian P.; Sergeev, Yuri V.

    2014-01-01

    Background Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. Methodology/Principal Findings The intra-melanosomal domain of human tyrosinase (residues 19–469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. Conclusions/Significance The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure – function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1. PMID:24392141

  11. Albinism-causing mutations in recombinant human tyrosinase alter intrinsic enzymatic activity.

    PubMed

    Dolinska, Monika B; Kovaleva, Elena; Backlund, Peter; Wingfield, Paul T; Brooks, Brian P; Sergeev, Yuri V

    2014-01-01

    Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. The intra-melanosomal domain of human tyrosinase (residues 19-469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure - function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1.

  12. Purification of recombinant catalase-peroxidase HPI from E. coli and its application in enzymatic polymerization reactions.

    PubMed

    Di Gennaro, Patrizia; Bargna, Anna; Bruno, Ferdinando; Sello, Guido

    2014-02-01

    In this paper, a recombinant catalase-peroxidase HPI from Escherichia coli was prepared, purified, and used in enzymatic polymerization reactions for the production of several oligomeric products. We tested the enzyme on four different substrates, chosen as representative of phenols and anilines: phenol, 3-methoxyphenol, catechol, and aniline. The polymerization reactions were followed by SEC-HPLC analysis, and except for aniline, all the other substrates were completely converted into one or more polymerization products. Results showed that reactions performed with phenol and 3-methoxyphenol allowed the isolation of some oligomers of different weight: a 27-monomeric unit oligomer and a 23-U oligomer are the heaviest ones. Experiments performed with catechol showed the formation of oligomers of 7 U in the reaction with HPI. HPI polymerization reactions performed with aniline allowed the identification of two different oligomers, one of 4 U and one of 10 U. All the substrates have been also used in reactions catalyzed by HRP in the same reaction conditions. Several products were common to the two enzymes. This work suggests the use of HPI as an alternative enzyme in peroxidatic reactions for the production of different oligomers from phenols and other compounds.

  13. Recombinant botulinum neurotoxin Hc subunit (BoNT Hc) and catalytically inactive Clostridium botulinum holoproteins (ciBoNT HPs) as vaccine candidates for the prevention of botulism

    DTIC Science & Technology

    2017-09-03

    Centers for Disease Control and Prevention in 2011 17 has resulted in the need for a safe and effective prophylactic alternative. Advances in genetic ...18 engineering have resulted in subsequent vaccine efforts being primarily focused on the 19 production of highly purified recombinant protein...specific genetic sequences that could be mobilized into heterologous 70 microbial expression platforms to produce recombinant proteins that could be

  14. Origin of the CMS gene locus in rapeseed cybrid mitochondria: active and inactive recombination produces the complex CMS gene region in the mitochondrial genomes of Brassicaceae.

    PubMed

    Oshima, Masao; Kikuchi, Rie; Imamura, Jun; Handa, Hirokazu

    2010-01-01

    CMS (cytoplasmic male sterile) rapeseed is produced by asymmetrical somatic cell fusion between the Brassica napus cv. Westar and the Raphanus sativus Kosena CMS line (Kosena radish). The CMS rapeseed contains a CMS gene, orf125, which is derived from Kosena radish. Our sequence analyses revealed that the orf125 region in CMS rapeseed originated from recombination between the orf125/orfB region and the nad1C/ccmFN1 region by way of a 63 bp repeat. A precise sequence comparison among the related sequences in CMS rapeseed, Kosena radish and normal rapeseed showed that the orf125 region in CMS rapeseed consisted of the Kosena orf125/orfB region and the rapeseed nad1C/ccmFN1 region, even though Kosena radish had both the orf125/orfB region and the nad1C/ccmFN1 region in its mitochondrial genome. We also identified three tandem repeat sequences in the regions surrounding orf125, including a 63 bp repeat, which were involved in several recombination events. Interestingly, differences in the recombination activity for each repeat sequence were observed, even though these sequences were located adjacent to each other in the mitochondrial genome. We report results indicating that recombination events within the mitochondrial genomes are regulated at the level of specific repeat sequences depending on the cellular environment.

  15. Dilute sulfuric acid pretreatment of corn stover for enzymatic hydrolysis and efficient ethanol production by recombinant Escherichia coli FBR5 without detoxification.

    PubMed

    Avci, Ayse; Saha, Badal C; Kennedy, Gregory J; Cotta, Michael A

    2013-08-01

    A pretreatment strategy for dilute H2SO4 pretreatment of corn stover was developed for the purpose of reducing the generation of inhibitory substances during pretreatment so that a detoxification step is not required prior to fermentation while maximizing sugar yield. The optimal conditions for pretreatment of corn stover (10%, w/v) were: 0.75% H2SO4, 160°C, and 0-5 min holding time. The conditions were chosen based on maximum glucose release after enzymatic hydrolysis, minimum loss of pentose sugars and minimum formation of sugar degradation products such as furfural and hydroxymethyl furfural. The pretreated corn stover after enzymatic saccharification generated 63.2 ± 2.2 and 63.7 ± 2.3 g total sugars per L at 0 and 5 min holding time, respectively. Furfural production was 0.45 ± 0.1 and 0.87 ± 0.4 g/L, respectively. The recombinant Escherichia coli strain FBR5 efficiently fermented non-detoxified corn stover hydrolyzate if the furfural content is <0.5 g/L. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Expression of enzymatically active, recombinant barley alpha-glucosidase in yeast and immunological detection of alpha-glucosidase from seed tissue.

    PubMed

    Tibbot, B K; Henson, C A; Skadsen, R W

    1998-10-01

    An alpha-glucosidase cDNA clone derived from barley aleurone tissue was expressed in Pichia pastoris and Escherichia coli. The gene was fused with the N-terminal region of the Saccharomyces cerevisiae alpha-factor secretory peptide and placed under control of the Pichia AOX1 promoter in the vector pPIC9. Enzymatically active, recombinant alpha-glucosidase was synthesized and secreted from the yeast upon induction with methanol. The enzyme hydrolyzed maltose > trehalose > nigerose > isomaltose. Maltase activity occurred over the pH range 3.5-6.3 with an optimum at pH 4.3, classifying the enzyme as an acid alpha-glucosidase. The enzyme had a Km of 1.88 mM and Vmax of 0.054 micromol/min on maltose. The recombinant alpha-glucosidase expressed in E. coli was used to generate polyclonal antibodies. The antibodies detected 101 and 95 kDa forms of barley alpha-glucosidase early in seed germination. Their levels declined sharply later in germination, as an 81 kDa alpha-glucosidase became prominent. Synthesis of these proteins also occurred in isolated aleurones after treatment with gibberellin, and this was accompanied by a 14-fold increase in alpha-glucosidase enzyme activity.

  17. Overexpression, purification and enzymatic characterization of a recombinant plastidial glucose-6-phosphate dehydrogenase from barley (Hordeum vulgare cv. Nure) roots.

    PubMed

    Cardi, Manuela; Chibani, Kamel; Castiglia, Daniela; Cafasso, Donata; Pizzo, Elio; Rouhier, Nicolas; Jacquot, Jean-Pierre; Esposito, Sergio

    2013-12-01

    In plant cells, the plastidial glucose 6-phosphate dehydrogenase (P2-G6PDH, EC 1.1.1.49) represents one of the most important sources of NADPH. However, previous studies revealed that both native and recombinant purified P2-G6PDHs show a great instability and a rapid loss of catalytic activity. Therefore it has been difficult to describe accurately the catalytic and physico-chemical properties of these isoforms. The plastidial G6PDH encoding sequence from barley roots (Hordeum vulgare cv. Nure), devoid of a long plastidial transit peptide, was expressed as recombinant protein in Escherichia coli, either untagged or with an N-terminal his-tag. After purification from both the soluble fraction and inclusion bodies, we have explored its kinetic parameters, as well as its sensitivity to reduction. The obtained results are consistent with values determined for other P2-G6PDHs previously purified from barley roots and from other land plants. Overall, these data shed light on the catalytic mechanism of plant P2-G6PDH, summarized in a proposed model in which the sequential mechanism is very similar to the mammalian cytosolic G6PDH. This study provides a rational basis to consider the recombinant barley root P2-G6PDH as a good model for further kinetic and structural studies.

  18. Genetic Determinants for Enzymatic Digestion of Lignocellulosic Biomass Are Independent of Those for Lignin Abundance in a Maize Recombinant Inbred Population

    DOE PAGES

    Penning, Bryan W.; Sykes, Robert W.; Babcock, Nicholas C.; ...

    2014-06-27

    Biotechnological approaches to reduce or modify lignin in biomass crops are predicated on the assumption that it is the principal determinant of the recalcitrance of biomass to enzymatic digestion for biofuels production. We defined quantitative trait loci (QTL) in the Intermated B73 x 3 Mo17 recombinant inbred maize (Zea mays) population using pyrolysis molecular-beam mass spectrometry to establish stem lignin content and an enzymatic hydrolysis assay to measure glucose and xylose yield. Among five multiyear QTL for lignin abundance, two for 4-vinylphenol abundance, and four for glucose and/or xylose yield, not a single QTL for aromatic abundance and sugar yieldmore » was shared. A genome-wide association study for lignin abundance and sugar yield of the 282- member maize association panel provided candidate genes in the 11 QTL of the B73 and Mo17 parents but showed that many other alleles impacting these traits exist among this broader pool of maize genetic diversity. B73 and Mo17 genotypes exhibited large differences in gene expression in developing stem tissues independent of allelic variation. Combining these complementary genetic approaches provides a narrowed list of candidate genes. A cluster of SCARECROW-LIKE9 and SCARECROW-LIKE14 transcription factor genes provides exceptionally strong candidate genes emerging from the genome-wide association study. In addition to these and genes associated with cell wall metabolism, candidates include several other transcription factors associated with vascularization and fiber formation and components of cellular signaling pathways. Finally, these results provide new insights and strategies beyond the modification of lignin to enhance yields of biofuels from genetically modified biomass.« less

  19. Genetic Determinants for Enzymatic Digestion of Lignocellulosic Biomass Are Independent of Those for Lignin Abundance in a Maize Recombinant Inbred Population

    SciTech Connect

    Penning, Bryan W.; Sykes, Robert W.; Babcock, Nicholas C.; Dugard, Christopher K.; Held, Michael A.; Klimek, John F.; Shreve, Jacob T.; Fowler, Matthew; Ziebell, Angela; Davis, Mark F.; Decker, Stephen R.; Turner, Geoffrey B.; Mosier, Nathan S.; Springer, Nathan M.; Thimmapuram, Jyothi; Weil, Clifford F.; McCann, Maureen C.; Carpita, Nicholas C.

    2014-06-27

    Biotechnological approaches to reduce or modify lignin in biomass crops are predicated on the assumption that it is the principal determinant of the recalcitrance of biomass to enzymatic digestion for biofuels production. We defined quantitative trait loci (QTL) in the Intermated B73 x 3 Mo17 recombinant inbred maize (Zea mays) population using pyrolysis molecular-beam mass spectrometry to establish stem lignin content and an enzymatic hydrolysis assay to measure glucose and xylose yield. Among five multiyear QTL for lignin abundance, two for 4-vinylphenol abundance, and four for glucose and/or xylose yield, not a single QTL for aromatic abundance and sugar yield was shared. A genome-wide association study for lignin abundance and sugar yield of the 282- member maize association panel provided candidate genes in the 11 QTL of the B73 and Mo17 parents but showed that many other alleles impacting these traits exist among this broader pool of maize genetic diversity. B73 and Mo17 genotypes exhibited large differences in gene expression in developing stem tissues independent of allelic variation. Combining these complementary genetic approaches provides a narrowed list of candidate genes. A cluster of SCARECROW-LIKE9 and SCARECROW-LIKE14 transcription factor genes provides exceptionally strong candidate genes emerging from the genome-wide association study. In addition to these and genes associated with cell wall metabolism, candidates include several other transcription factors associated with vascularization and fiber formation and components of cellular signaling pathways. Finally, these results provide new insights and strategies beyond the modification of lignin to enhance yields of biofuels from genetically modified biomass.

  20. Genetic Determinants for Enzymatic Digestion of Lignocellulosic Biomass Are Independent of Those for Lignin Abundance in a Maize Recombinant Inbred Population.

    PubMed

    Penning, Bryan W; Sykes, Robert W; Babcock, Nicholas C; Dugard, Christopher K; Held, Michael A; Klimek, John F; Shreve, Jacob T; Fowler, Matthew; Ziebell, Angela; Davis, Mark F; Decker, Stephen R; Turner, Geoffrey B; Mosier, Nathan S; Springer, Nathan M; Thimmapuram, Jyothi; Weil, Clifford F; McCann, Maureen C; Carpita, Nicholas C

    2014-08-01

    Biotechnological approaches to reduce or modify lignin in biomass crops are predicated on the assumption that it is the principal determinant of the recalcitrance of biomass to enzymatic digestion for biofuels production. We defined quantitative trait loci (QTL) in the Intermated B73 × Mo17 recombinant inbred maize (Zea mays) population using pyrolysis molecular-beam mass spectrometry to establish stem lignin content and an enzymatic hydrolysis assay to measure glucose and xylose yield. Among five multiyear QTL for lignin abundance, two for 4-vinylphenol abundance, and four for glucose and/or xylose yield, not a single QTL for aromatic abundance and sugar yield was shared. A genome-wide association study for lignin abundance and sugar yield of the 282-member maize association panel provided candidate genes in the 11 QTL of the B73 and Mo17 parents but showed that many other alleles impacting these traits exist among this broader pool of maize genetic diversity. B73 and Mo17 genotypes exhibited large differences in gene expression in developing stem tissues independent of allelic variation. Combining these complementary genetic approaches provides a narrowed list of candidate genes. A cluster of SCARECROW-LIKE9 and SCARECROW-LIKE14 transcription factor genes provides exceptionally strong candidate genes emerging from the genome-wide association study. In addition to these and genes associated with cell wall metabolism, candidates include several other transcription factors associated with vascularization and fiber formation and components of cellular signaling pathways. These results provide new insights and strategies beyond the modification of lignin to enhance yields of biofuels from genetically modified biomass. © 2014 American Society of Plant Biologists. All Rights Reserved.

  1. Genetic Determinants for Enzymatic Digestion of Lignocellulosic Biomass Are Independent of Those for Lignin Abundance in a Maize Recombinant Inbred Population1[W][OPEN

    PubMed Central

    Penning, Bryan W.; Sykes, Robert W.; Babcock, Nicholas C.; Dugard, Christopher K.; Held, Michael A.; Klimek, John F.; Shreve, Jacob T.; Fowler, Matthew; Ziebell, Angela; Davis, Mark F.; Decker, Stephen R.; Turner, Geoffrey B.; Mosier, Nathan S.; Springer, Nathan M.; Thimmapuram, Jyothi; Weil, Clifford F.; McCann, Maureen C.; Carpita, Nicholas C.

    2014-01-01

    Biotechnological approaches to reduce or modify lignin in biomass crops are predicated on the assumption that it is the principal determinant of the recalcitrance of biomass to enzymatic digestion for biofuels production. We defined quantitative trait loci (QTL) in the Intermated B73 × Mo17 recombinant inbred maize (Zea mays) population using pyrolysis molecular-beam mass spectrometry to establish stem lignin content and an enzymatic hydrolysis assay to measure glucose and xylose yield. Among five multiyear QTL for lignin abundance, two for 4-vinylphenol abundance, and four for glucose and/or xylose yield, not a single QTL for aromatic abundance and sugar yield was shared. A genome-wide association study for lignin abundance and sugar yield of the 282-member maize association panel provided candidate genes in the 11 QTL of the B73 and Mo17 parents but showed that many other alleles impacting these traits exist among this broader pool of maize genetic diversity. B73 and Mo17 genotypes exhibited large differences in gene expression in developing stem tissues independent of allelic variation. Combining these complementary genetic approaches provides a narrowed list of candidate genes. A cluster of SCARECROW-LIKE9 and SCARECROW-LIKE14 transcription factor genes provides exceptionally strong candidate genes emerging from the genome-wide association study. In addition to these and genes associated with cell wall metabolism, candidates include several other transcription factors associated with vascularization and fiber formation and components of cellular signaling pathways. These results provide new insights and strategies beyond the modification of lignin to enhance yields of biofuels from genetically modified biomass. PMID:24972714

  2. Enzymatic production of l-citrulline by hydrolysis of the guanidinium group of l-arginine with recombinant arginine deiminase.

    PubMed

    Song, Wei; Sun, Xia; Chen, Xiulai; Liu, Dongxu; Liu, Liming

    2015-08-20

    In this study, a simple, efficient enzymatic production process for the environmentally friendly synthesis of l-citrulline from l-arginine was developed using arginine deiminase (ADI) from Lactococcus lactis. Following overexpression of L. lactis ADI in Escherichia. coli BL21 (DE3) and experimental evolution using error-prone PCR, mutant FMME106 was obtained with a Km for l-arginine of 3.5mM and a specific activity of 195.7U/mg. This mutant exhibited a maximal conversion of 92.6% and achieved a final l-citrulline concentration of 176.9g/L under optimal conditions (190g/L l-arginine, 15g/L whole-cell biocatalyst treated with 2% isopropanol for 30min, 50°C, pH 7.2, 8h). The average l-citrulline synthesis rate of 22.1g/L/h is considerably higher than that reported for other similar biocatalytic approaches, therefore the process developed in the present work has great potential for large-scale production of l-citrulline.

  3. Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid

    PubMed Central

    Huang, Yan; Su, Lingqia; Wu, Jing

    2016-01-01

    Glutamate decarboxylase (GAD) catalyzes the irreversible decarboxylation of L-glutamate to the valuable food supplement γ-aminobutyric acid (GABA). In this study, GAD from Escherichia coli K12, a pyridoxal phosphate (PLP)-dependent enzyme, was overexpressed in E. coli. The GAD produced in media supplemented with 0.05 mM soluble vitamin B6 analog pyridoxine hydrochloride (GAD-V) activity was 154.8 U mL-1, 1.8-fold higher than that of GAD obtained without supplementation (GAD-C). Purified GAD-V exhibited increased activity (193.4 U mg-1, 1.5-fold higher than that of GAD-C), superior thermostability (2.8-fold greater than that of GAD-C), and higher kcat/Km (1.6-fold higher than that of GAD-C). Under optimal conditions in reactions mixtures lacking added PLP, crude GAD-V converted 500 g L-1 monosodium glutamate (MSG) to GABA with a yield of 100%, and 750 g L-1 MSG with a yield of 88.7%. These results establish the utility of pyridoxine supplementation and lay the foundation for large-scale enzymatic production of GABA. PMID:27438707

  4. Enzymatic synthesis of S-adenosylhomocysteine: immobilization of recombinant S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum (ATCC 13032).

    PubMed

    Lozada-Ramírez, J D; Sánchez-Ferrer, A; García-Carmona, F

    2012-03-01

    Recombinant S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum (CgSAHase) was covalently bound to Eupergit® C. The maximum yield of bound protein was 91% and the catalytic efficiency was 96.9%. When the kinetic results for the immobilized enzyme were compared with those for the soluble enzyme, no decrease in the catalytic efficiency of the former was detected. Both soluble and immobilized enzymes showed similar optimum pH and temperature ranges. The reuse of immobilized CgSAHase caused a loss of synthetic activity due to NAD(+) release, although the binding to the support was sufficiently strong for up to 5 cycles with 95% conversion efficiency. The immobilized enzyme was incubated every 3 cycles with 100 μM NAD(+) to recover the loss of activity after 5 cycles. This maintained the activity for another 50 cycles. The purification of S-adenosylhomocysteine (SAH) provided an overall yield of 76% and 98% purity as determined by HPLC and NMR analyses. The results indicate the suitability of immobilized CgSAHase for synthesizing SAH and other important S-nucleosidylhomocysteine.

  5. Recombinant plasmepsin 1 from the human malaria parasite Plasmodium falciparum: Enzymatic characterization, active site inhibitor design, and structural analysis

    PubMed Central

    Liu, Peng; Marzahn, Melissa R.; Robbins, Arthur H.; Gutiérrez-de-Terán, Hugo; Rodríguez, David; McClung, Scott; Stevens, Stanley M.; Yowell, Charles A.; Dame, John B.; McKenna, Robert; Dunn, Ben M.

    2009-01-01

    A mutated form of truncated proplasmepsin 1 (proPfPM1) from the human malaria parasite Plasmodium falciparum, proPfPM1 K110pN, was generated and overexpressed in E. coli. The auto-maturation process was carried out at pH 4.0 and 4.5, and the optimal catalytic pH of the resulting mature PfPM1 was determined to be pH 5.5. This mature PfPM1 showed comparable binding affinity to peptide substrates and inhibitors with the naturally-occurring form isolated from parasites. The S3-S3’ subsite preferences of the recombinant mature PfPM1 were explored using combinatorial chemistry based peptide libraries. Based on the results, a peptidomimetic inhibitor (compound 1) was designed and yielded 5-fold selectivity for binding to PfPM1 versus the homologous human cathepsin D (hcatD). The 2.8 Å structure of the PfPMP2-compound 1 complex is reported. Modeling studies were conducted using a series of peptidomimetic inhibitors (compounds 1–6, Table 3) and three plasmepsins: the crystal structure of PfPM2, and homology derived models of PfPM1 and PfPM4. PMID:19271776

  6. Cloning of PEPC-1 from a C4 halophyte Suaeda aralocaspica without Kranz anatomy and its recombinant enzymatic activity in responses to abiotic stresses.

    PubMed

    Cheng, Gang; Wang, Lu; Lan, Haiyan

    2016-02-01

    Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of C4 photosynthetic pathway and plays an important biochemical role in higher plants and micro organisms. To gain understanding of the role of PEPC in stress adaptation in plant, we cloned PEPC gene from Suaeda aralocaspica, a C4 species without Kranz anatomy, and performed a series of experiments with PEPC gene expressed in Escherichia coli under various abiotic stresses. Results showed that, based on the homology cloning and 5'-RACE technique, the full-length cDNA sequence of PEPC (2901 bp) from S. aralocaspica was obtained, which shares the typical conserved domains to documented PEPCs and was identified as PEPC-1 in accord to the reported partial sequence (ppc-1) in S. aralocaspica. qRT-PCR analysis revealed the expression patterns of PEPC-1 and PEPC-2 (known as ppc-2, another plant type of PEPC) in S. aralocaspica, suggesting that PEPC-1 was up-regulated during seed germination and under NaCl stress, and presented higher level in chlorenchyma than other tissues, which were significantly different with PEPC-2. Afterwards, PEPC-1 was recombinant in E. coli (pET-28a-PEPC) and expressed as an approximate 110 kDa protein. Under various abiotic stresses, the recombinant E. coli strain harboring with PEPC-1 showed significant advantage in growth at 400-800 mmol L(-1) NaCl, 10-20% PEG6000, 25 and 30 °C lower temperature, 50-200 μmol L(-1) methyl viologen, and pH 5.0 and 9.0 condition, compared to control. Further analysis of the enzymatic characteristics of the recombinant PEPC-1 suggests that it was the higher enzyme activity of PEPC-1 which might confer the stress tolerance to E. coli. We speculate that over expression of PEPC-1 is probably related to regulation of oxaloacetate (OAA) in tricarboxylic acid (TCA) cycle in E. coli, which may contribute to further understanding of the physiological function of PEPC in S. aralocaspica.

  7. Exercise Responses after Inactivity

    NASA Technical Reports Server (NTRS)

    Convertino, Victor A.

    1986-01-01

    The exercise response after bed rest inactivity is a reduction in the physical work capacity and is manifested by significant decreases in oxygen uptake. The magnitude of decrease in maximal oxygen intake V(dot)O2max is related to the duration of confinement and the pre-bed-rest level of aerobic fitness; these relationships are relatively independent of age and gender. The reduced exercise performance and V(dot)O2max following bed rest are associated with various physiological adaptations including reductions in blood volume, submaximal and maximal stroke volume, maximal cardiac output, sceletal muscle tone and strength, and aerobic enzyme capacities, as well as increases in venous compliance and submaximal and maximal heart rate. This reduction in physiological capacity can be partially restored by specific countermeasures that provide regular muscular activity or orhtostatic stress or both during the bed rest exposure. The understanding of these physiological and physical responses to exercise following bed rest inactivity has important implications for the solution to safety and health problems that arise in clinical medicine, aerospace medicine, sedentary living, and aging.

  8. Exercise Responses after Inactivity

    NASA Technical Reports Server (NTRS)

    Convertino, Victor A.

    1986-01-01

    The exercise response after bed rest inactivity is a reduction in the physical work capacity and is manifested by significant decreases in oxygen uptake. The magnitude of decrease in maximal oxygen intake V(dot)O2max is related to the duration of confinement and the pre-bed-rest level of aerobic fitness; these relationships are relatively independent of age and gender. The reduced exercise performance and V(dot)O2max following bed rest are associated with various physiological adaptations including reductions in blood volume, submaximal and maximal stroke volume, maximal cardiac output, sceletal muscle tone and strength, and aerobic enzyme capacities, as well as increases in venous compliance and submaximal and maximal heart rate. This reduction in physiological capacity can be partially restored by specific countermeasures that provide regular muscular activity or orhtostatic stress or both during the bed rest exposure. The understanding of these physiological and physical responses to exercise following bed rest inactivity has important implications for the solution to safety and health problems that arise in clinical medicine, aerospace medicine, sedentary living, and aging.

  9. From Power to Inaction.

    PubMed

    Durso, Geoffrey R O; Briñol, Pablo; Petty, Richard E

    2016-12-01

    Research has shown that people who feel powerful are more likely to act than those who feel powerless, whereas people who feel ambivalent are less likely to act than those whose reactions are univalent (entirely positive or entirely negative). But what happens when powerful people also are ambivalent? On the basis of the self-validation theory of judgment, we hypothesized that power and ambivalence would interact to predict individuals' action. Because power can validate individuals' reactions, we reasoned that feeling powerful strengthens whatever reactions people have during a decision. It can strengthen univalent reactions and increase action orientation, as shown in past research. Among people who hold an ambivalent judgment, however, those who feel powerful would be less action oriented than those who feel powerless. Two experiments provide evidence for this hypothesized interactive effect of power and ambivalence on individuals' action tendencies during both positive decisions (promoting an employee; Experiment 1) and negative decisions (firing an employee; Experiment 2). In summary, when individuals' reactions are ambivalent, power increases the likelihood of inaction.

  10. One-step enzymatic hydrolysis of starch using a recombinant strain of Saccharomyces cerevisiae producing alpha-amylase, glucoamylase and pullulanase.

    PubMed

    Janse, B J; Pretorius, I S

    1995-03-01

    A recombinant strain of Saccharomyces cerevisiae was constructed that contained the genes encoding a bacterial alpha-amylase (AMY1), a yeast glucoamylase (STA2) and a bacterial pullulanase (pulA). The Bacillus amyloliquefaciens alpha-amylase and S. cerevisiae var. diastaticus glucoamylase genes were expressed in S. cerevisiae using their native promoters and the encoded enzymes secreted under direction of their native leader sequences. In contrast, the Klebsiella pneumoniae pullulanase gene was placed under the control of the yeast alcohol dehydrogenase gene promoter (ADC1P) and secreted using the yeast mating pheromone alpha-factor secretion signal (MF alpha 1S). Transcription termination of the pullulanase gene was effected by the yeast tryptophan synthase gene terminator (TRP5T), whereas termination of the glucoamylase and alpha-amylase genes was directed by their native terminators. Pullulanase (PUL1) produced by recombinant yeasts containing ADC1P MF alpha 1S pulA TRP5T (designated PUL1) was further characterized and compared to its bacterial counterpart (PulA). The different genes were introduced into S. cerevisiae in different combinations and the various amylolytic Saccharomyces transformants compared to Schwanniomyces occidentalis. Introduction of PUL1 into a S. cerevisiae strain containing both STA2 and AMY1, resulted in 99% assimilation of starch.

  11. Glutathione S-transferase can be used as a C-terminal, enzymatically active dimerization module for a recombinant protease inhibitor, and functionally secreted into the periplasm of Escherichia coli.

    PubMed Central

    Tudyka, T.; Skerra, A.

    1997-01-01

    Glutathione S-transferase (GST) from Schistosoma japonicum, which is widely used for the production of fusion proteins in the cytoplasm of Escherichia coli, was employed as a functional fusion module that effects dimer formation of a recombinant protein and confers enzymatic reporter activity at the same time. For this purpose GST was linked via a flexible spacer to the C-terminus of the thiol-protease inhibitor cystatin, whose binding properties for papain were to be studied. The fusion protein was secreted into the bacterial periplasm by means of the OmpA signal peptide to ensure formation of the two disulfide bonds in cystatin. The formation of wrong crosslinks in the oxidizing milieu was prevented by replacing three of the four exposed cysteine residues in GST. Using the tetracycline promoter for tightly controlled gene expression the soluble fusion protein could be isolated from the periplasmic protein fraction. Purification to homogeneity was achieved in one step by means of an affinity column with glutathione agarose. Alternatively, the protein was isolated via streptavidin affinity chromatography after the Strep-tag had been appended to its C terminus. The GST moiety of the fusion protein was enzymatically active and the kinetic parameters were determined using glutathione and 1-chloro-2,4-dinitrobenzene as substrates. Furthermore, strong binding activity for papain was detected in an ELISA. The signal with the cystatin-GST fusion protein was much higher than with cystatin itself, demonstrating an avidity effect due to the dimer formation of GST. The quaternary structure was further confirmed by chemical crosslinking, which resulted in a specific reaction product with twice the molecular size. Thus, engineered GST is suitable as a moderately sized, secretion-competent fusion partner that can confer bivalency to a protein of interest and promote detection of binding interactions even in cases of low affinity. PMID:9336840

  12. Ammodytoxins efficiently release arachidonic acid and induce apoptosis in a motoneuronal cell line in an enzymatic activity-dependent manner.

    PubMed

    Jenko-Pražnikar, Zala; Petan, Toni; Pungerčar, Jože

    2013-03-01

    Secreted phospholipases A2 (sPLA2s) are phospholipolytic enzymes and receptor ligands whose action affects cell death and survival. We have previously shown that ammodytoxin A (AtxA), a snake venom sPLA2, is rapidly internalized into motoneuronal NSC34 cells, inducing characteristic neurotoxic sPLA2 cell damage and apoptosis. In this study, we have analyzed the role of sPLA2 enzymatic activity, including arachidonic acid (AA) release, in the induction of motoneuronal apoptosis by AtxA and homologous recombinant sPLA2s with different enzymatic properties: an AtxA mutant (V31W) with very high enzymatic activity, enzymatically inactive S49-sPLA2 (ammodytin L, AtnL), its mutant (LW) with restored enzymatic activity, and non-toxic, enzymatically active sPLA2 (AtnI2). Addition of AA, AtxA, AtxA-V31W and AtnL-LW, but not AtnL and AtnI2, to NSC34 cells resulted in caspase-3 activation, DNA fragmentation and disruption of mitochondrial membrane potential, leading to a significant and rapid decrease in motoneuronal cell viability that was not observed in C2C12 myoblasts and HEK293 cells. AtxA, AtxA-V31W and AtnL-LW, but not AtnL and AtnI2, also liberated large amounts of AA specifically from motoneuronal cells, and this ability correlated well with the ability to induce apoptotic changes and decrease cell viability. The enzymatic activity of AtxA and similar sPLA2s is thus necessary, but not sufficient, for inducing motoneuronal apoptosis. This suggests that specific binding to the motoneuronal cell surface, followed by internalization and enzymatic activity-dependent induction of apoptosis, possibly as a consequence of extensive extra- and intracellular AA release, is necessary for Atx-induced motoneuronal cell death.

  13. A novel enzymatic approach in the production of food with low purine content using Arxula adeninivorans endogenous and recombinant purine degradative enzymes.

    PubMed

    Jankowska, Dagmara A; Trautwein-Schult, Anke; Cordes, Arno; Bode, Rüdiger; Baronian, Keith; Kunze, Gotthard

    2015-01-01

    The purine degradation pathway in humans ends with uric acid, which has low water solubility. When the production of uric acid is increased either by elevated purine intake or by impaired kidney function, uric acid will accumulate in the blood (hyperuricemia). This increases the risk of gout, a disease described in humans for at least 1000 years. Many lower organisms, such as the yeast Arxula adeninivorans, possess the enzyme, urate oxidase that converts uric acid to 5-hydroxyisourate, thus preventing uric acid accumulation. We have examined the complete purine degradation pathway in A. adeninivorans and analyzed enzymes involved. Recombinant adenine deaminase, guanine deaminase, urate oxidase and endogenous xanthine oxidoreductase have been investigated as potential additives to degrade purines in the food. Here, we review the current model of the purine degradation pathway of A. adeninivorans and present an overview of proposed enzyme system with perspectives for its further development.

  14. A novel enzymatic approach in the production of food with low purine content using Arxula adeninivorans endogenous and recombinant purine degradative enzymes

    PubMed Central

    Jankowska, Dagmara A; Trautwein-Schult, Anke; Cordes, Arno; Bode, Rüdiger; Baronian, Keith; Kunze, Gotthard

    2015-01-01

    The purine degradation pathway in humans ends with uric acid, which has low water solubility. When the production of uric acid is increased either by elevated purine intake or by impaired kidney function, uric acid will accumulate in the blood (hyperuricemia). This increases the risk of gout, a disease described in humans for at least 1000 years. Many lower organisms, such as the yeast Arxula adeninivorans, possess the enzyme, urate oxidase that converts uric acid to 5-hydroxyisourate, thus preventing uric acid accumulation. We have examined the complete purine degradation pathway in A. adeninivorans and analyzed enzymes involved. Recombinant adenine deaminase, guanine deaminase, urate oxidase and endogenous xanthine oxidoreductase have been investigated as potential additives to degrade purines in the food. Here, we review the current model of the purine degradation pathway of A. adeninivorans and present an overview of proposed enzyme system with perspectives for its further development. PMID:25513995

  15. A straightforward experimental approach to expression, purification, refolding, and enzymatic analysis of recombinant dengue virus NS2B(H)-NS3pro protease.

    PubMed

    Junaid, M; Angsuthanasombat, C; Wikberg, J E S; Ali, N; Katzenmeier, G

    2013-08-01

    Dengue virus threatens around 2.5 billion people worldwide; about 50 million become infected every year, and yet no vaccine or drug is available for prevention and/or treatment. The flaviviral NS2B-NS3pro complex is indispensable for flaviviral replication and is considered to be an important drug target. The aim of this study was to develop a simple and generally applicable experimental strategy to construct, purify, and assay a highly active recombinant NS2B(H)-NS3pro complex that would be useful for high-throughput screening of potential inhibitors. The sequence of NS2B(H)-NS3pro was generated by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro complex was expressed in E. coli predominantly as insoluble protein and purified to >95% purity by single-step immobilized metal affinity chromatography. SDS-PAGE followed by immunoblotting of the purified enzyme demonstrated the presence of the NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 37, 21, and 10 kDa bands, respectively. Kinetic parameters, Km, kcat, and kcat/Km for the fluorophore-linked protease model substrate Ac-nKRR-amc were obtained using inner-filter effect correction. The kinetic parameters Km, kcat, and kcat/Km for Ac-nKRR-amc substrate were 100 µM, 0.112 s(-1), and 1120 M(-1)·s(-1), respectively. A simplified procedure for the cloning, overexpression, and purification of the NS2B(H)-NS3pro complex was applied, and a highly active recombinant NS2B(H)-NS3pro complex was obtained that could be useful for the design of high-throughput assays aimed at flaviviral inhibitor discovery.

  16. Inactive and abandoned noncoal mines

    SciTech Connect

    Not Available

    1991-08-01

    Volume 1 outlines the environmental, health and safety problems at IAMS (Inactive, Abandoned Mine Sites), remediation technologies, remediation costs, the methodology states used in preparing state reports, and state summary tables. It also describes the broad range of policy options for remediation of problems associated with IAMS. Volume 2 gives state reports for inactive and abandoned noncoal mines for the following states: Alaska, Arizona, California, Colorado, Florida, Idaho, Minnesota, Missouri, Montana, Nevada, New Mexico, Oregon, South Carolina, South Dakota, Utah, Washington, Wisconsin and Wyoming. Volume 3 lists the State reports for the inactive and abandoned noncoal mines for the following states: Alabama, Arkansas, Illinois, Indiana, Iowa, Louisiana, Maine, Maryland, Mississippi, New York, North Carolina, Ohio, Oklahoma, Pennsylvania, Texas, and Virginia. A separate abstract is included for each of the 3 volumes of this set.

  17. Biochemical Characterization of a Recombinant UDP-glucosyltransferase from Rice and Enzymatic Production of Deoxynivalenol-3-O-β-d-glucoside

    PubMed Central

    Michlmayr, Herbert; Malachová, Alexandra; Varga, Elisabeth; Kleinová, Jana; Lemmens, Marc; Newmister, Sean; Rayment, Ivan; Berthiller, Franz; Adam, Gerhard

    2015-01-01

    Glycosylation is an important plant defense mechanism and conjugates of Fusarium mycotoxins often co-occur with their parent compounds in cereal-based food and feed. In case of deoxynivalenol (DON), deoxynivalenol-3-O-β-d-glucoside (D3G) is the most important masked mycotoxin. The toxicological significance of D3G is not yet fully understood so that it is crucial to obtain this compound in pure and sufficient quantities for toxicological risk assessment and for use as an analytical standard. The aim of this study was the biochemical characterization of a DON-inactivating UDP-glucosyltransferase from rice (OsUGT79) and to investigate its suitability for preparative D3G synthesis. Apparent Michaelis constants (Km) of recombinant OsUGT79 were 0.23 mM DON and 2.2 mM UDP-glucose. Substrate inhibition occurred at DON concentrations above 2 mM (Ki = 24 mM DON), and UDP strongly inhibited the enzyme. Cu2+ and Zn2+ (1 mM) inhibited the enzyme completely. Sucrose synthase AtSUS1 was employed to regenerate UDP-glucose during the glucosylation reaction. With this approach, optimal conversion rates can be obtained at limited concentrations of the costly co-factor UDP-glucose. D3G can now be synthesized in sufficient quantity and purity. Similar strategies may be of interest to produce β-glucosides of other toxins. PMID:26197338

  18. Biochemical Characterization of a Recombinant UDP-glucosyltransferase from Rice and Enzymatic Production of Deoxynivalenol-3-O-β-D-glucoside.

    PubMed

    Michlmayr, Herbert; Malachová, Alexandra; Varga, Elisabeth; Kleinová, Jana; Lemmens, Marc; Newmister, Sean; Rayment, Ivan; Berthiller, Franz; Adam, Gerhard

    2015-07-21

    Glycosylation is an important plant defense mechanism and conjugates of Fusarium mycotoxins often co-occur with their parent compounds in cereal-based food and feed. In case of deoxynivalenol (DON), deoxynivalenol-3-O-β-D-glucoside (D3G) is the most important masked mycotoxin. The toxicological significance of D3G is not yet fully understood so that it is crucial to obtain this compound in pure and sufficient quantities for toxicological risk assessment and for use as an analytical standard. The aim of this study was the biochemical characterization of a DON-inactivating UDP-glucosyltransferase from rice (OsUGT79) and to investigate its suitability for preparative D3G synthesis. Apparent Michaelis constants (Km) of recombinant OsUGT79 were 0.23 mM DON and 2.2 mM UDP-glucose. Substrate inhibition occurred at DON concentrations above 2 mM (Ki = 24 mM DON), and UDP strongly inhibited the enzyme. Cu2+ and Zn2+ (1 mM) inhibited the enzyme completely. Sucrose synthase AtSUS1 was employed to regenerate UDP-glucose during the glucosylation reaction. With this approach, optimal conversion rates can be obtained at limited concentrations of the costly co-factor UDP-glucose. D3G can now be synthesized in sufficient quantity and purity. Similar strategies may be of interest to produce β-glucosides of other toxins.

  19. Recombinant thermo-alkali-stable endoglucanase of Myceliopthora thermophila BJA (rMt-egl): Biochemical characteristics and applicability in enzymatic saccharification of agro-residues.

    PubMed

    Phadtare, Priya; Joshi, Swati; Satyanarayana, T

    2017-11-01

    Codon adaptation index (CAI) of a 1263bp long endoglucanase encoding gene from the thermophilic mould Myceliopthora thermophile BJA has been improved from 0.44 to 0.76 by in vitro gene synthesis. The codon optimized endoglucanase gene (Mt-egl) has been constitutively expressed in Pichia pastoris under the regulation of GAP promoter. Recombinant endoglucanase (rMt-egl), purified by size exclusion chromatography, has been confirmed to be a monomeric protein of ∼47kDa. rMt-egl is optimally active at pH 10 and 50°C, displaying stability in broad pH and temperature ranges, with a t1/2 of 60 and 15min at 90 and 100°C, respectively. This retained ∼70% of activity after 3h incubation at pH 5-12. The Km, Vmax, kcat and kcat/Km of rMt-egl were 5mgmL(-1), 20μmolesmin(-1)mg(-1), 1.02×10(3)s(-1) and 204s(-1)mg(-1)mL(-1), respectively. Homology modeling and bioinformatics analysis confirmed catalytically important role of glutamate 234 and 344. rMt-egl released high amounts of reducing sugars from wheat bran and corn cobs (421 and 382mgg(-1)), thus making it a useful biocatalyst for producing bioethanol and fine chemicals from agro-residues. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Oculocutaneous Albinism Type 1: Link between Mutations, Tyrosinase Conformational Stability, and Enzymatic Activity

    PubMed Central

    Dolinska, Monika B.; Kus, Nicole; Farney, Katie; Wingfield, Paul T.; Brooks, Brian P.; Sergeev, Yuri V.

    2017-01-01

    Summary Oculocutaneous albinism Type 1 (OCA1) is an autosomal recessive disorder caused by mutations in the tyrosinase gene. Two subtypes of OCA1 have been described: severe OCA1A with complete absence of tyrosinase activity and less severe OCA1B with residual tyrosinase activity. Here, we characterize the recombinant human tyrosinase intra-melanosomal domain and mutant variants, which mimic genetic changes in both subtypes of OCA1 patients. Proteins were prepared using site-directed mutagenesis, expressed in insect larvae, purified by chromatography, and characterized by enzymatic activities- tryptophan fluorescence, and Gibbs free energy changes. The OCA1A mutants show very low protein expression, protein yield, and are enzymatically inactive. Mutants mimicking OCA1B were biochemically similar to the wild type, but exhibited lower specific activities and protein stabilities. The results are consistent with clinical data, which indicates that OCA1A mutations inactivate tyrosinase and result in severe phenotype, while OCA1B mutations partially inactive tyrosinase and results in OCA1B albinism. PMID:27775880

  1. Enzymatic vitreous disruption.

    PubMed

    Gandorfer, A

    2008-10-01

    Enzymatic vitreous disruption refers to cleaving the vitreoretinal junction by enzymatic means, thereby inducing posterior vitreous detachment (PVD) and liquefaction of the vitreous gel. Several enzymes have been proposed in this respect, including chondroitinase, hyaluronidase, dispase, and plasmin. In an experimental setting, chondroitinase induced PVD and was helpful in removing epiretinal membranes but no further data have been reported yet. Hyaluronidase liquefies the vitreous as demonstrated in a phase III trial in diabetic patients with vitreous haemorrhage. Dispase induces PVD but also causes inner retinal damage and is now used as an animal model of proliferative vitreoretinopathy. Plasmin has the capability of both PVD induction and liquefaction. However, plasmin is highly unstable and not available for clinical use. Microplasmin (ThromboGenics Ltd, Dublin, Ireland) is a truncated form of human plasmin sharing the same catalytic activity like plasmin. Recombinant microplasmin is under clinical investigation in patients with vitreomacular traction. This review article reports on the current knowledge of enzymatic vitreous disruption and discusses details of the enzyme candidates in basic and clinical research terms.

  2. Health Risks of an Inactive Lifestyle

    MedlinePlus

    ... may develop a hormonal imbalance What are the health risks of an inactive lifestyle? Having an inactive ... the more sedentary you are, the higher your health risks are. How can I get started with ...

  3. Inactive sites and the evolution of cooperation

    NASA Astrophysics Data System (ADS)

    da Silva Júnior, E. J.; Wardil, L. L.; da Silva, J. K. L.

    2016-10-01

    Cooperation is often conditioned on environmental factors. Behaviors may be inactive due to external factors, and yet the trait itself may not change. We study the evolution of cooperation with active and inactive sites. In inactive sites cooperators behave as defectors, receiving but not providing benefits. This unintentional mimicry provides local advantage to cooperation, but also prevents the mutual reinforcement provided by clusters of active cooperators. In general, we found that cooperation is enhanced by inactivity. In particular, if most sites are inactive, cooperation survives even if the temptation to defect is very large. Interestingly, in the square lattice with pairwise comparison rule we found that cooperation is enforced by inactive sites only up to a certain limit.

  4. Obesity and Physical Inactivity in Rural America

    ERIC Educational Resources Information Center

    Patterson, Paul Daniel; Moore, Charity G.; Probst, Janice C.; Shinogle, Judith Ann

    2004-01-01

    Context and Purpose: Obesity and physical inactivity are common in the United States, but few studies examine this issue within rural populations. The present study uses nationally representative data to study obesity and physical inactivity in rural populations. Methods: Data came from the 1998 National Health Interview Survey Sample Adult and…

  5. 21 CFR 312.45 - Inactive status.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 5 2010-04-01 2010-04-01 false Inactive status. 312.45 Section 312.45 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS FOR HUMAN USE INVESTIGATIONAL NEW DRUG APPLICATION Administrative Actions § 312.45 Inactive status. (a) If...

  6. Regrets of Action and Inaction across Cultures.

    ERIC Educational Resources Information Center

    Gilovich, Thomas; Wang, Ranxiao Frances; Regan, Dennis; Nishina, Sadafumi

    2003-01-01

    Conducted five studies in three cultures considered less individualistic than the United States (China, Japan, and Russia) to investigate regrets related to action and inaction in people's lives. Respondents in all three cultures tended to regret inactions more than actions in the long term. Types of regrets (generally involving the self rather…

  7. 21 CFR 201.117 - Inactive ingredients.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Inactive ingredients. 201.117 Section 201.117 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... drug that is ordinarily used as an inactive ingredient, such as a coloring, emulsifier, excipient...

  8. Obesity and Physical Inactivity in Rural America

    ERIC Educational Resources Information Center

    Patterson, Paul Daniel; Moore, Charity G.; Probst, Janice C.; Shinogle, Judith Ann

    2004-01-01

    Context and Purpose: Obesity and physical inactivity are common in the United States, but few studies examine this issue within rural populations. The present study uses nationally representative data to study obesity and physical inactivity in rural populations. Methods: Data came from the 1998 National Health Interview Survey Sample Adult and…

  9. 21 CFR 201.117 - Inactive ingredients.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Inactive ingredients. 201.117 Section 201.117 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS: GENERAL LABELING Exemptions From Adequate Directions for Use § 201.117 Inactive ingredients. A...

  10. 21 CFR 201.117 - Inactive ingredients.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 4 2011-04-01 2011-04-01 false Inactive ingredients. 201.117 Section 201.117 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS: GENERAL LABELING Exemptions From Adequate Directions for Use § 201.117 Inactive ingredients. A...

  11. Non enzymatic upregulation of tissue factor expression by gamma-glutamyl transferase in human peripheral blood mononuclear cells.

    PubMed

    Scalise, Valentina; Balia, Cristina; Cianchetti, Silvana; Neri, Tommaso; Carnicelli, Vittoria; Zucchi, Riccardo; Franzini, Maria; Corti, Alessandro; Paolicchi, Aldo; Celi, Alessandro; Pedrinelli, Roberto

    2016-01-01

    Besides maintaining intracellular glutathione stores, gamma-glutamyltransferase(GGT) generates reactive oxygen species and activates NFkB, a redox-sensitive transcription factor key in the induction of Tissue Factor (TF) gene expression, the principal initiator of the clotting cascade. Thus, GGT might be involved in TF-mediated coagulation processes, an assumption untested insofar. Experiments were run with either equine, enzymatically active GGT or human recombinant (hr) GGT, a wheat germ-derived protein enzymatically inert because of missing post-translational glycosylation. TF Procoagulant Activity (PCA, one-stage clotting assay), TF antigen(ELISA) and TFmRNA(real-time PCR) were assessed in unpooled human peripheral blood mononuclear cell(PBMC) suspensions obtained from healthy donors through discontinuous Ficoll/Hystopaque density gradient. Equine GGT increased PCA, an effect insensitive to GGT inhibition by acivicin suggesting mechanisms independent of its enzymatic activity, a possibility confirmed by the maintained stimulation in response to hrGGT, an enzymatically inactive molecule. Endotoxin(LPS) contamination of GGT preparations was excluded by heat inactivation studies and direct determination(LAL method) of LPS concentrations <0.1 ng/mL practically devoid of procoagulant effect. Inhibition by anti-GGT antibodies corroborated that conclusion. Upregulation by hrGGT of TF antigen and mRNA and its downregulation by BAY-11-7082, a NFkB inhibitor, and N-acetyl-L-cysteine, an antioxidant, was consistent with a NFkB-driven, redox-sensitive transcriptional site of action. GGT upregulates TF expression independent of its enzymatic activity, a cytokine-like behaviour mediated by NFκB activation, a mechanism contributing to promote acute thrombotic events, a possibility in need, however, of further evaluation.

  12. Microplate-based active/inactive 1 screen for biomass degrading enzyme library purification and gene discovery

    USDA-ARS?s Scientific Manuscript database

    We present here a whole-cell and permeabilized E. coli cell 1' active/inactive microplate screen for ß-D-xylosidase, xylanase, endocellulase, and ferulic acid esterase enzyme activities which are critical for the enzymatic deconstruction of biomass for fuels and chemicals. Transformants from genomic...

  13. Temperature-Compensating Inactive Strain Gauge

    NASA Technical Reports Server (NTRS)

    Moore, Thomas C., Sr.

    1993-01-01

    Thermal contribution to output of active gauge canceled. High-temperature strain gauges include both active gauge wires sensing strains and inactive gauge wires providing compensation for thermal contributions to gauge readings. Inactive-gauge approach to temperature compensation applicable to commercially available resistance-type strain gauges operating at temperatures up to 700 degrees F and to developmental strain gauges operating at temperatures up to 2,000 degrees F.

  14. Inactive Wells: Economic and Policy Issues

    NASA Astrophysics Data System (ADS)

    Krupnick, A.

    2016-12-01

    This paper examines the economic and policy issues associated with various types of inactive oil and gas wells. It covers the costs of decommissioning wells, and compares them to the bonding requirements on these wells, looking at a large number of states. It also reviews the detailed regulations governing treatment of inactive wells by states and the federal government and compares them according to their completeness and stringency.

  15. Bovine Pancreatic Trypsin Inhibitor-Trypsin Complex as a Detection System for Recombinant Proteins

    NASA Astrophysics Data System (ADS)

    Borjigin, Jimo; Nathans, Jeremy

    1993-01-01

    Bovine pancreatic trypsin inhibitor (BPTI) binds to trypsin and anhydrotrypsin (an enzymatically inactive derivative of trypsin) with affinities of 6 x 10-14 and 1.1 x 10-13 M, respectively. We have taken advantage of the high affinity and specificity of this binding reaction to develop a protein tagging system in which biotinylated trypsin or biotinylated anhydrotrypsin is used as the reagent to detect recombinant fusion proteins into which BPTI has been inserted. Two proteins, opsin and growth hormone, were used as targets for insertional mutagenesis with BPTI. In each case, both domains of the fusion protein appear to be correctly folded. The fusion proteins can be specifically and efficiently detected by biotinylated trypsin or biotinylated anhydrotrypsin, as demonstrated by staining of transfected cells, protein blotting, affinity purification, and a mobility shift assay in SDS/polyacrylamide gels.

  16. The Kell protein of the common K2 phenotype is a catalytically active metalloprotease, whereas the rare Kell K1 antigen is inactive. Identification of novel substrates for the Kell protein.

    PubMed

    Clapéron, Audrey; Rose, Christiane; Gane, Pierre; Collec, Emmanuel; Bertrand, Olivier; Ouimet, Tanja

    2005-06-03

    The Kell blood group is a highly polymorphic system containing over 20 different antigens borne by the protein Kell, a 93-kDa type II glycoprotein that displays high sequence homology with members of the M13 family of zinc-dependent metalloproteases whose prototypical member is neprilysin. Kell K1 is an antigen expressed in 9% of the Caucasian population, characterized by a point mutation (T193M) of the Kell K2 antigen, and located within a putative N-glycosylation consensus sequence. Recently, a recombinant, non-physiological, soluble form of Kell was shown to cleave Big ET-3 to produce the mature vasoconstrictive peptide. To better characterize the enzymatic activity of the Kell protein and the possible differences introduced by antigenic point mutations affecting post-translational processing, the membrane-bound forms of the Kell K1 and Kell K2 antigens were expressed either in K562 cells, an erythroid cell line, or in HEK293 cells, a non-erythroid system, and their pharmacological profiles and enzymatic specificities toward synthetic and natural peptides were evaluated. Results presented herein reveal that the two antigens possess considerable differences in their enzymatic activities, although not in their trafficking pattern. Indeed, although both antigens are expressed at the cell surface, Kell K1 protein is shown to be inactive, whereas the Kell K2 antigen binds neprilysin inhibitory compounds such as phosphoramidon and thiorphan with high affinity, cleaves the precursors of the endothelin peptides, and inactivates members of the tachykinin family with enzymatic properties resembling those of other members of the M13 family of metalloproteases to which it belongs.

  17. Enzymatic intracrine regulation of white adipose tissue

    PubMed Central

    DiSilvestro, David; Petrosino, Jennifer; Aldoori, Ayat; Melgar-Bermudez, Emiliano; Wells, Alexandra; Ziouzenkova, Ouliana

    2015-01-01

    Abdominal fat formation has become a permanent risk factor for metabolic syndrome and various cancers in one-third of the world's population of obese and even lean patients. Formation of abdominal fat involves additional mechanisms beyond an imbalance in energy intake and expenditure, which explains systemic obesity. In this review, we briefly summarized autonomous regulatory circuits that locally produce hormones from inactive precursors or nutrients for intra-/auto-/paracrine signaling in white adipose depots. Enzymatic pathways activating steroid and thyroid hormones in adipose depots were compared with enzymatic production of retinoic acid from vitamin A. We discussed the role of intracrine circuits in fat-depot functions and strategies to reduce abdominal adiposity through thermogenic adipocytes with interrupted generation of retinoic acid. PMID:25390015

  18. Physical Inactivity, Sedentary Behavior and Chronic Diseases.

    PubMed

    González, Karimé; Fuentes, Jorge; Márquez, José Luis

    2017-05-01

    New research into physical activity suggests that it is no longer sufficient just to meet minimum levels recommended by health guidelines in order to reduce cardiovascular risk. Both physical inactivity and sedentary behavior have their own health hazards and need to be addressed separately, in order to explore their different deleterious mechanisms. The aim of this review was to define and to characterize both concepts, and their relationship with major non-communicable chronic diseases. A PubMed database search was undertaken, using the following key words: physical activity, physical inactivity, sedentarism, sedentary behavior, and non-communicable chronic disease. This literature review provides an updated view on physical inactivity and sedentary behavior, and reevaluates their prevalence and association with major non-communicable chronic disease.

  19. Generating Functional Recombinant NRPS Enzymes in the Laboratory Setting via Peptidyl Carrier Protein Engineering.

    PubMed

    Owen, Jeremy G; Calcott, Mark J; Robins, Katherine J; Ackerley, David F

    2016-11-17

    Non-ribosomal peptide synthetases (NRPSs) are modular enzymatic assembly lines where substrates and intermediates undergo rounds of transformation catalyzed by adenylation (A), condensation (C), and thioesterase (TE) domains. Central to the NRPS biosynthesis are peptidyl carrier protein (PCP) domains, small, catalytically inactive domains that shuttle substrates and intermediates between the catalytic modules and govern product release from TE domains. There is strong interest in recombination of NRPS systems to generate new chemical entities. However, the intrinsic complexity of these systems has been a major challenge. Here, we employ domain substitution and random mutagenesis to recapitulate NRPS evolution, focusing on PCP domains. Using NRPS model systems that produce two different pigmented molecules, pyoverdine and indigoidine, we found that only evolutionarily specialized recombinant PCP domains could interact effectively with the native TE domain for product release. Overall, we highlight that substituted PCP domains require very minor changes to result in functional NRPSs, and infer that positive selection pressure may improve recombinant NRPS outcomes.

  20. Elective Mutism Associated with Selective Inactivity.

    ERIC Educational Resources Information Center

    Hill, Linda; Scull, John

    1985-01-01

    Effective treatment procedures for a nine-year-old boy with elective mutism and selective inactivity included increasing the frequency of situations in which he could already speak and decreasing the frequency of those in which he seldom spoke (specifically coercive situations). (CL)

  1. Physical inactivity and obesity: A vicious circle

    PubMed Central

    Pietiläinen, Kirsi H; Kaprio, Jaakko; Borg, Patrik; Plasqui, Guy; Yki-Järvinen, Hannele; Kujala, Urho M.; Rose, Richard J; Westerterp, Klaas R; Rissanen, Aila

    2007-01-01

    Objective Physical activity (PA) begins to decline in adolescence with concomitant increase in weight. We hypothesized that a vicious circle may arise between decreasing physical activity and weight gain from adolescence to early adulthood. Research Methods and Procedures PA and self-perceived physical fitness assessed in adolescence (16-18 years) were used to predict the development of obesity (BMI≥30 kg/m2) and abdominal obesity (waist≥88 cm in females and ≥102 cm in males) at age 25 in 4240 twin individuals (90% of twins born in Finland 1975-1979). Ten 25-year-old monozygotic (MZ) twin pairs discordant for obesity (16 kg weight difference) were then carefully evaluated for current PA (triaxial accelerometer), total energy expenditure (TEE, doubly labeled water), and basal metabolic rate (BMR, indirect calorimetry). Results Physical inactivity in adolescence strongly predicted the risk of obesity (OR 3.9, 95%CI 1.4-10.9) and abdominal obesity (4.8, 1.9-12.0) at age 25, even after adjusting for baseline and current BMI. Poor physical fitness in adolescence also increased the risk of overall (5.1, 2.0-12.7) and abdominal obesity (3.2, 1.5-6.7) in adulthood. Physical inactivity was both causative and secondary to the development of obesity discordance in the MZ pairs. TEE did not differ between the MZ co-twins. PA levels were lower whereas BMR was higher in the obese co-twins. Discussion Physical inactivity in adolescence strongly and independently predicts total and especially abdominal obesity in young adulthood, favoring the development of a self-perpetuating vicious circle of obesity and physical inactivity. Physical (in)activity should be a major target of obesity prevention in the young. PMID:18239652

  2. Enzymatic approach to biodiesel production.

    PubMed

    Akoh, Casimir C; Chang, Shu-Wei; Lee, Guan-Chiun; Shaw, Jei-Fu

    2007-10-31

    The need for alternative energy sources that combine environmental friendliness with biodegradability, low toxicity, renewability, and less dependence on petroleum products has never been greater. One such energy source is referred to as biodiesel. This can be produced from vegetable oils, animal fats, microalgal oils, waste products of vegetable oil refinery or animal rendering, and used frying oils. Chemically, they are known as monoalkyl esters of fatty acids. The conventional method for producing biodiesel involves acid and base catalysts to form fatty acid alkyl esters. Downstream processing costs and environmental problems associated with biodiesel production and byproducts recovery have led to the search for alternative production methods and alternative substrates. Enzymatic reactions involving lipases can be an excellent alternative to produce biodiesel through a process commonly referred to alcoholysis, a form of transesterification reaction, or through an interesterification (ester interchange) reaction. Protein engineering can be useful in improving the catalytic efficiency of lipases as biocatalysts for biodiesel production. The use of recombinant DNA technology to produce large quantities of lipases, and the use of immobilized lipases and immobilized whole cells, may lower the overall cost, while presenting less downstream processing problems, to biodiesel production. In addition, the enzymatic approach is environmentally friendly, considered a "green reaction", and needs to be explored for industrial production of biodiesel.

  3. Enzymatic glycosylation of multivalent scaffolds.

    PubMed

    Bojarová, Pavla; Rosencrantz, Ruben R; Elling, Lothar; Křen, Vladimír

    2013-06-07

    The design of glycoclusters, glycodendrimers, glycopolymers and other complex glycostructures that mimic the multivalent carbohydrate display on the cell surface is of immense interest for diagnosis and therapy. This review presents a detailed insight into the exciting possibilities of multiple glycosylation using enzymes, particularly glycosyltransferases (EC 2.4). A representative choice of available scaffolds for the enzyme action is practically infinite and comprises synthetic polymers, carbosilane dendrimers, multiantennary glycans or hyperbranched conjugates. The introduced glyco-patterns range from common sialyl Lewis(x) and sialyl lacto-chains to chemically functionalized carbohydrate units for detection purposes. The possibilities of in vitro enzymatic production of N- and O-glycans and other natural polymers are also discussed. In harmony with their natural tasks, glycosyltransferases may in vitro complete the imperfect glycosylation pattern of proteins, recombinantly produced in pro- and eukaryotic hosts. What is more, the required enzymatic battery may be directly co-expressed with the protein, in order to elegantly accomplish the production of eukaryotic glycans. Ingenious metabolic labeling enables facile imaging of glycostructures. The boom of glycoarray technology opens vast possibilities in high-throughput screening for novel enzymes and substrate specificities as well as in the synthesis. Though there is still a long way until the Nature's ideal of multivalent glycans is achievable in the laboratory, the sketched pathways to multivalent glycostructures open tremendous possibilities for the future glycobiological research.

  4. Norovirus recombination.

    PubMed

    Bull, Rowena A; Tanaka, Mark M; White, Peter A

    2007-12-01

    RNA recombination is a significant driving force in viral evolution. Increased awareness of recombination within the genus Norovirus of the family Calicivirus has led to a rise in the identification of norovirus (NoV) recombinants and they are now reported at high frequency. Currently, there is no classification system for recombinant NoVs and a widely accepted recombinant genotyping system is still needed. Consequently, there is duplication in reporting of novel recombinants. This has led to difficulties in defining the number and types of recombinants in circulation. In this study, 120 NoV nucleotide sequences were compiled from the current GenBank database and published literature. NoV recombinants and their recombination breakpoints were identified using three methods: phylogenetic analysis, SimPlot analysis and the maximum chi2 method. A total of 20 NoV recombinant types were identified in circulation worldwide. The recombination point is the ORF1/2 overlap in all isolates except one, which demonstrated a double recombination event within the polymerase region.

  5. Substrate Specificity of Purified Recombinant Chicken β-Carotene 9',10'-Oxygenase (BCO2).

    PubMed

    Dela Seña, Carlo; Sun, Jian; Narayanasamy, Sureshbabu; Riedl, Kenneth M; Yuan, Yan; Curley, Robert W; Schwartz, Steven J; Harrison, Earl H

    2016-07-08

    Provitamin A carotenoids are oxidatively cleaved by β-carotene 15,15'-dioxygenase (BCO1) at the central 15-15' double bond to form retinal (vitamin A aldehyde). Another carotenoid oxygenase, β-carotene 9',10'-oxygenase (BCO2) catalyzes the oxidative cleavage of carotenoids at the 9'-10' bond to yield an ionone and an apo-10'-carotenoid. Previously published substrate specificity studies of BCO2 were conducted using crude lysates from bacteria or insect cells expressing recombinant BCO2. Our attempts to obtain active recombinant human BCO2 expressed in Escherichia coli were unsuccessful. We have expressed recombinant chicken BCO2 in the strain E. coli BL21-Gold (DE3) and purified the enzyme by cobalt ion affinity chromatography. Like BCO1, purified recombinant chicken BCO2 catalyzes the oxidative cleavage of the provitamin A carotenoids β-carotene, α-carotene, and β-cryptoxanthin. Its catalytic activity with β-carotene as substrate is at least 10-fold lower than that of BCO1. In further contrast to BCO1, purified recombinant chicken BCO2 also catalyzes the oxidative cleavage of 9-cis-β-carotene and the non-provitamin A carotenoids zeaxanthin and lutein, and is inactive with all-trans-lycopene and β-apocarotenoids. Apo-10'-carotenoids were detected as enzymatic products by HPLC, and the identities were confirmed by LC-MS. Small amounts of 3-hydroxy-β-apo-8'-carotenal were also consistently detected in BCO2-β-cryptoxanthin reaction mixtures. With the exception of this activity with β-cryptoxanthin, BCO2 cleaves specifically at the 9'-10' bond to produce apo-10'-carotenoids. BCO2 has been shown to function in preventing the excessive accumulation of carotenoids, and its broad substrate specificity is consistent with this. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Fire protection for inactive contaminated structures

    SciTech Connect

    Wyatt, D.M.

    1994-02-01

    In general industry and construction, destruction of an inactive/surplus facility by fire may be considered a blessing. However, in a decommissioned contaminated structure, where radiological and other hazardous materials exist, such a fire could be a major catastrophe. The losses from this type of fire are not only property (i.e., structure and its contents) but also the resulting environmental damage, required cleanup, offsite releases, and public relations and reactions. The purpose of this presentation is to (1) promote an awareness among the waste management community of fire protection engineering aspects that must be considered for inactive/surplus contaminated structures, and (2) present to the fire protection community an opportunity to become involved in the decommissioning process while promoting the DOE objectives to manage the risks associated with these structures.

  7. Exploring human inactivity in computer power consumption

    NASA Astrophysics Data System (ADS)

    Candrawati, Ria; Hashim, Nor Laily Binti

    2016-08-01

    Managing computer power consumption has become an important challenge in computer society and this is consistent with a trend where a computer system is more important to modern life together with a request for increased computing power and functions continuously. Unfortunately, previous approaches are still inadequately designed to handle the power consumption problem due to unpredictable workload of a system caused by unpredictable human behaviors. This is happens due to lack of knowledge in a software system and the software self-adaptation is one approach in dealing with this source of uncertainty. Human inactivity is handled by adapting the behavioral changes of the users. This paper observes human inactivity in the computer usage and finds that computer power usage can be reduced if the idle period can be intelligently sensed from the user activities. This study introduces Control, Learn and Knowledge model that adapts the Monitor, Analyze, Planning, Execute control loop integrates with Q Learning algorithm to learn human inactivity period to minimize the computer power consumption. An experiment to evaluate this model was conducted using three case studies with same activities. The result show that the proposed model obtained those 5 out of 12 activities shows the power decreasing compared to others.

  8. Recombinant microorganisms for increased production of organic acids

    DOEpatents

    Yi, Jian; Kleff, Susanne; Guettler, Michael V

    2013-04-30

    Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

  9. Recombinant microorganisms for increased production of organic acids

    DOEpatents

    Yi, Jian [East Lansing, MI; Kleff, Susanne [East Lansing, MI; Guettler, Michael V [Holt, MI

    2012-02-21

    Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

  10. Pretreatment and Enzymatic Hydrolysis

    SciTech Connect

    2006-06-01

    Activities in this project are aimed at overcoming barriers associated with high capital and operating costs and sub-optimal sugar yields resulting from pretreatment and subsequent enzymatic hydrolysis of biomass.

  11. Enzymatic modification of schizophyllan

    USDA-ARS?s Scientific Manuscript database

    An enzymatic method was developed for the progressive modification of the polysaccharide schizophyllan. Fungal strains Hypocrea nigricans NRRL 62555, Penicillium crustosum NRRL 62558, and Penicillium simplicissimum NRRL 62550 were previously identified as novel sources of ß-endoglucanase with specif...

  12. The Global Physical Inactivity Pandemic: An Analysis of Knowledge Production

    ERIC Educational Resources Information Center

    Piggin, Joe; Bairner, Alan

    2016-01-01

    In July 2012, "The Lancet" announced a pandemic of physical inactivity and a global call to action to effect change. The worldwide pandemic is said to be claiming millions of lives every year. Asserting that physical inactivity is pandemic is an important moment. Given the purported scale and significance of physical inactivity around…

  13. The Global Physical Inactivity Pandemic: An Analysis of Knowledge Production

    ERIC Educational Resources Information Center

    Piggin, Joe; Bairner, Alan

    2016-01-01

    In July 2012, "The Lancet" announced a pandemic of physical inactivity and a global call to action to effect change. The worldwide pandemic is said to be claiming millions of lives every year. Asserting that physical inactivity is pandemic is an important moment. Given the purported scale and significance of physical inactivity around…

  14. Enzymatic desulfurization of coal

    SciTech Connect

    Marquis, J.K. . School of Medicine); Kitchell, J.P. )

    1988-12-15

    Our current efforts to develop clean coal technology emphasize the advantages of enzymatic desulfurization techniques and have specifically addressed the potential of using partially-purified extracellular microbial enzymes or commercially available enzymes. Our work is focused on the treatment of model'' organic sulfur compounds such as dibenzothiophene (DBT) and ethylphenylsulfide (EPS). Furthermore, we are designing experiments to facilitate the enzymatic process by means of a hydrated organic solvent matrix.

  15. Enzymatic desulfurization of coal

    SciTech Connect

    Marquis, J.K. . School of Medicine); Kitchell, J.P. )

    1988-10-07

    Our current efforts to develop clean coal technology, emphasize the advantages of enzymatic desulfurization techniques and have specifically addressed the potential of using partially-purified extracellular microbial enzymes or commercially available enzymes. Our work is focused on the treatment of model'' organic sulfur compounds such as dibenzothiophene (DBT) and ethylphenylsulfide (EPS). Furthermore, we are designing experiments to facilitate the enzymatic process by means of a hydrated organic solvent matrix.

  16. Enzymatic desulfurization of coal

    SciTech Connect

    Boyer, Y.N.; Crooker, S.C.; Kitchell, J.P.; Nochur, S.V. ); Marquis, J.K. . School of Medicine)

    1989-06-16

    Our current efforts to develop clean coal technology emphasize the advantages of enzymatic desulfurization techniques and have specifically addressed the potential of using partially-purified extracellular microbial enzymes as well as commercially available enzymes. Our work is focused on the treatment of model'' organic sulfur compounds such as dibenzothiophene (DBT) and ethylphenylsulfide (EPS). Furthermore, we are designing experiments to facilitate the enzymatic process by means of a hydrated organic solvent matrix.

  17. [Approaching Physically Inactive Elderly for Physical Activity].

    PubMed

    Allmer, H; Allmer, M; Euskirchen, J; Froböse, I; Wallmann, B; Walter, T; Walschek, R

    2015-09-01

    The majority of elderly persons are still not sufficiently physically active. Therefore the aim of this study was to investigate different approaches (physical activity courses, poster, online-survey) for activating elderly to participate in physical activity. The most effective approach was target group physical activity courses with which higher course participation rates in men as well as in people with lower levels of education were achieved. Referring to the transtheoretical model (TTM) it is necessary for future analyses of target group approaches to consider more intensely the initial motivational position of physically inactive elderly.

  18. Molecular characterization of the thi3 gene involved in thiamine biosynthesis in Zea mays: cDNA sequence and enzymatic and structural properties of the recombinant bifunctional protein with 4-amino-5-hydroxymethyl-2-methylpyrimidine (phosphate) kinase and thiamine monophosphate synthase activities.

    PubMed

    Rapala-Kozik, Maria; Olczak, Mariusz; Ostrowska, Katarzyna; Starosta, Agata; Kozik, Andrzej

    2007-12-01

    A thiamine biosynthesis gene, thi3, from maize Zea mays has been identified through cloning and sequencing of cDNA and heterologous overexpression of the encoded protein, THI3, in Escherichia coli. The recombinant THI3 protein was purified to homogeneity and shown to possess two essentially different enzymatic activities of HMP(-P) [4-amino-5-hydroxymethyl-2-methylpyrimidine (phosphate)] kinase and TMP (thiamine monophosphate) synthase. Both activities were characterized in terms of basic kinetic constants, with interesting findings that TMP synthase is uncompetitively inhibited by excess of one of the substrates [HMP-PP (HMP diphosphate)] and ATP. A bioinformatic analysis of the THI3 sequence suggested that these activities were located in two distinct, N-terminal kinase and C-terminal synthase, domains. Models of the overall folds of THI3 domains and the arrangements of active centre residues were obtained with the SWISS-MODEL protein modelling server, on the basis of the known three-dimensional structures of Salmonella enterica serotype Typhimurium HMP(-P) kinase and Bacillus subtilis TMP synthase. The essential roles of Gln98 and Met134 residues for HMP kinase activity and of Ser444 for TMP synthase activity were experimentally confirmed by site-directed mutagenesis.

  19. Genetic Recombination

    ERIC Educational Resources Information Center

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  20. Genetic Recombination

    ERIC Educational Resources Information Center

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  1. A Mononuclear Non-Heme Manganese(IV)-Oxo Complex Binding Redox-Inactive Metal Ions

    SciTech Connect

    Chen, Junying; Lee, Yong-Min; Davis, Katherine M.; Wu, Xiujuan; Seo, Mi Sook; Cho, Kyung-Bin; Yoon, Heejung; Park, Young Jun; Fukuzumi, Shunichi; Pushkar, Yulia N.; Nam, Wonwoo

    2013-05-29

    Redox-inactive metal ions play pivotal roles in regulating the reactivities of high-valent metal–oxo species in a variety of enzymatic and chemical reactions. A mononuclear non-heme Mn(IV)–oxo complex bearing a pentadentate N5 ligand has been synthesized and used in the synthesis of a Mn(IV)–oxo complex binding scandium ions. The Mn(IV)–oxo complexes were characterized with various spectroscopic methods. The reactivities of the Mn(IV)–oxo complex are markedly influenced by binding of Sc3+ ions in oxidation reactions, such as a ~2200-fold increase in the rate of oxidation of thioanisole (i.e., oxygen atom transfer) but a ~180-fold decrease in the rate of C–H bond activation of 1,4-cyclohexadiene (i.e., hydrogen atom transfer). The present results provide the first example of a non-heme Mn(IV)–oxo complex binding redox-inactive metal ions that shows a contrasting effect of the redox-inactive metal ions on the reactivities of metal–oxo species in the oxygen atom transfer and hydrogen atom transfer reactions.

  2. A mononuclear non-heme manganese(IV)-oxo complex binding redox-inactive metal ions.

    PubMed

    Chen, Junying; Lee, Yong-Min; Davis, Katherine M; Wu, Xiujuan; Seo, Mi Sook; Cho, Kyung-Bin; Yoon, Heejung; Park, Young Jun; Fukuzumi, Shunichi; Pushkar, Yulia N; Nam, Wonwoo

    2013-05-01

    Redox-inactive metal ions play pivotal roles in regulating the reactivities of high-valent metal-oxo species in a variety of enzymatic and chemical reactions. A mononuclear non-heme Mn(IV)-oxo complex bearing a pentadentate N5 ligand has been synthesized and used in the synthesis of a Mn(IV)-oxo complex binding scandium ions. The Mn(IV)-oxo complexes were characterized with various spectroscopic methods. The reactivities of the Mn(IV)-oxo complex are markedly influenced by binding of Sc(3+) ions in oxidation reactions, such as a ~2200-fold increase in the rate of oxidation of thioanisole (i.e., oxygen atom transfer) but a ~180-fold decrease in the rate of C-H bond activation of 1,4-cyclohexadiene (i.e., hydrogen atom transfer). The present results provide the first example of a non-heme Mn(IV)-oxo complex binding redox-inactive metal ions that shows a contrasting effect of the redox-inactive metal ions on the reactivities of metal-oxo species in the oxygen atom transfer and hydrogen atom transfer reactions.

  3. Photoelectrochemical enzymatic biosensors.

    PubMed

    Zhao, Wei-Wei; Xu, Jing-Juan; Chen, Hong-Yuan

    2017-06-15

    Enzymatic biosensors have been valuable bioanalytical devices for analysis of diverse targets in disease diagnosis, biological and biomedical research, etc. Photoelectrochemical (PEC) bioanalysis is a recently emerged method that promptly becoming a subject of new research interests due to its attractive potential for future bioanalysis with high sensitivity and specificity. PEC enzymatic biosensors integrate the inherent sensitivities of PEC bioanalysis and the selectivity of enzymes and thus share their both advantages. Currently, PEC enzymatic biosensors have become a hot topic of significant research and the recent impetus has grown rapidly as demonstrated by increased research papers. Given the pace of advances in this area, this review will make a thorough discussion and survey on the fundamentals, sensing strategies, applications and the state of the art in PEC enzymatic biosensors, followed by future prospects based on our own opinions. We hope this work could provide an accessible introduction to PEC enzymatic biosensors for any scientist. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Enzymatic synthesis of isotopically labeled isoprenoid diphosphates.

    PubMed

    Christensen, D J; Poulter, C D

    1994-07-01

    Recombinant yeast isopentenyl diphosphate (IPP) isomerase and avian farnesyl diphosphate (FPP) synthase from overproducing strains of Escherichia coli were used to synthesize FPP from IPP and dimethylallyl diphosphate (DMAPP). [2,4,5-13C3]IPP and [2,4,5-13C3]DMAPP were synthesized from ethyl [2-13C]bromoacetate and [1,3-13C2]acetone. Thes compounds were used as substrates for enzymatic synthesis of FPP selectivity labeled at the first or third isoprene residue or at all three.

  5. Enzymatic hydrolysis of wheat arabinoxylan by a recombinant "minimal" enzyme cocktail containing beta-xylosidase and novel endo-1,4-beta-xylanase and alpha-l-arabinofuranosidase activities.

    PubMed

    Sørensen, Hanne R; Pedersen, Sven; Jørgensen, Christel T; Meyer, Anne S

    2007-01-01

    This study describes the identification of the key enzyme activities required in a "minimal" enzyme cocktail able to catalyze hydrolysis of water-soluble and water-insoluble wheat arabinoxylan and whole vinasse, a fermentation effluent resulting from industrial ethanol manufacture from wheat. The optimal arabinose-releasing and xylan-depolymerizing enzyme activities were identified from data obtained when selected, recombinant enzymes were systematically supplemented to the different arabinoxylan substrates in mixtures; this examination revealed three novel alpha-l-arabinofuranosidase activities: (i) one GH51 enzyme from Meripilus giganteus and (ii) one GH51 enzyme from Humicola insolens, both able to catalyze arabinose release from singly substituted xylose; and (iii) one GH43 enzyme from H. insolens able to catalyze the release of arabinose from doubly substituted xylose. Treatment of water-soluble and water-insoluble wheat arabinoxylan with an enzyme cocktail containing a 20%:20%:20%:40% mixture and a 25%:25%:25%:25% mixture, respectively, of the GH43 alpha-l-arabinofuranosidase from H. insolens (Abf II), the GH51 alpha-l-arabinofuranosidase from M. giganteus (Abf III), a GH10 endo-1,4-beta-xylanase from H. insolens (Xyl III), and a GH3 beta-xylosidase from Trichoderma reesei (beta-xyl) released 322 mg of arabinose and 512 mg of xylose per gram of water-soluble wheat arabinoxylan dry matter and 150 mg of arabinose and 266 mg of xylose per gram of water-insoluble wheat arabinoxylan dry matter after 24 h at pH 5, 50 degrees C. A 10%:40%:50% mixture of Abf II, Abf III, and beta-xyl released 56 mg of arabinose and 91 mg of xylose per gram of vinasse dry matter after 24 h at pH 5, 50 degrees C. The optimal dosages of the "minimal" enzyme cocktails were determined to be 0.4, 0.3, and 0.2 g enzyme protein per kilogram of substrate dry matter for the water-soluble wheat arabinoxylan, the water-insoluble wheat arabinoxylan, and the vinasse, respectively. These enzyme

  6. Impact of cysteine variants on the structure, activity, and stability of recombinant human α-galactosidase A.

    PubMed

    Qiu, Huawei; Honey, Denise M; Kingsbury, Jonathan S; Park, Anna; Boudanova, Ekaterina; Wei, Ronnie R; Pan, Clark Q; Edmunds, Tim

    2015-09-01

    Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity.

  7. Impact of cysteine variants on the structure, activity, and stability of recombinant human α-galactosidase A

    PubMed Central

    Qiu, Huawei; Honey, Denise M; Kingsbury, Jonathan S; Park, Anna; Boudanova, Ekaterina; Wei, Ronnie R; Pan, Clark Q; Edmunds, Tim

    2015-01-01

    Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity. PMID:26044846

  8. DNA recombination: the replication connection.

    PubMed

    Haber, J E

    1999-07-01

    Chromosomal double-strand breaks (DSBs) arise after exposure to ionizing radiation or enzymatic cleavage, but especially during the process of DNA replication itself. Homologous recombination plays a critical role in repair of such DSBs. There has been significant progress in our understanding of two processes that occur in DSB repair: gene conversion and recombination-dependent DNA replication. Recent evidence suggests that gene conversion and break-induced replication are related processes that both begin with the establishment of a replication fork in which both leading- and lagging-strand synthesis occur. There has also been much progress in characterization of the biochemical roles of recombination proteins that are highly conserved from yeast to humans.

  9. Enzymatic vitreous surgery.

    PubMed

    Trese, M T

    2000-06-01

    Enzymatic manipulation of the vitreous and vitreoretinal juncture is currently in the process of being evaluated in many centers around the world. The goals of such manipulation are either to disinsert the posterior hyaloid from the retina surface in an atraumatic, very clean, cleavage plane or, at this point, to try to disinsert the peripheral vitreous from the neurosensory retina. In addition, enzymatic manipulation of the central vitreous in terms of liquefaction has also been evaluated. Although this is certainly the beginning of this type of vitreal surgery, adjuvant or alternative, it does appear to be a new and exciting area of vitreoretinal surgery.

  10. Electrically inactive poly-silicon grain boundaries

    SciTech Connect

    Chen, S.P.; Kress, J.D.; Voter, A.F.; Albers, R.C.

    1996-05-01

    Structures, energies, and electronic properties of symmetric [001] tilt grain boundaries in Si have been studied using Stillinger-Weber and Tersoff classical potentials, and semi-empirical (tight-binding) electronic structure methods. The calculated lowest energy (310) grain boundary structure and electronic properties are consistent with previous TEM measurement and calculations. For the controversial (710) grain boundaries, the tight-binding calculations do not show any electronic energy levels in the band gap. This indicates that with every atom fully fourfold coordinated, the (710) grain boundary should be electrically inactive. Some high-energy metastable grain boundaries were found to be electrically active by the presence of the levels introduced in the band gap. Also, the vacancy concentration at the (310) GB was found to be enhanced by many orders of magnitude relative to bulk. The dangling bond states of the vacancies should be electrically active.

  11. Substrate Specificity of Purified Recombinant Chicken β-Carotene 9′,10′-Oxygenase (BCO2)*

    PubMed Central

    dela Seña, Carlo; Sun, Jian; Narayanasamy, Sureshbabu; Riedl, Kenneth M.; Yuan, Yan; Curley, Robert W.; Schwartz, Steven J.; Harrison, Earl H.

    2016-01-01

    Provitamin A carotenoids are oxidatively cleaved by β-carotene 15,15′-dioxygenase (BCO1) at the central 15-15′ double bond to form retinal (vitamin A aldehyde). Another carotenoid oxygenase, β-carotene 9′,10′-oxygenase (BCO2) catalyzes the oxidative cleavage of carotenoids at the 9′-10′ bond to yield an ionone and an apo-10′-carotenoid. Previously published substrate specificity studies of BCO2 were conducted using crude lysates from bacteria or insect cells expressing recombinant BCO2. Our attempts to obtain active recombinant human BCO2 expressed in Escherichia coli were unsuccessful. We have expressed recombinant chicken BCO2 in the strain E. coli BL21-Gold (DE3) and purified the enzyme by cobalt ion affinity chromatography. Like BCO1, purified recombinant chicken BCO2 catalyzes the oxidative cleavage of the provitamin A carotenoids β-carotene, α-carotene, and β-cryptoxanthin. Its catalytic activity with β-carotene as substrate is at least 10-fold lower than that of BCO1. In further contrast to BCO1, purified recombinant chicken BCO2 also catalyzes the oxidative cleavage of 9-cis-β-carotene and the non-provitamin A carotenoids zeaxanthin and lutein, and is inactive with all-trans-lycopene and β-apocarotenoids. Apo-10′-carotenoids were detected as enzymatic products by HPLC, and the identities were confirmed by LC-MS. Small amounts of 3-hydroxy-β-apo-8′-carotenal were also consistently detected in BCO2-β-cryptoxanthin reaction mixtures. With the exception of this activity with β-cryptoxanthin, BCO2 cleaves specifically at the 9′-10′ bond to produce apo-10′-carotenoids. BCO2 has been shown to function in preventing the excessive accumulation of carotenoids, and its broad substrate specificity is consistent with this. PMID:27143479

  12. Enzymatic DNA molecules

    NASA Technical Reports Server (NTRS)

    Joyce, Gerald F. (Inventor); Breaker, Ronald R. (Inventor)

    1998-01-01

    The present invention discloses deoxyribonucleic acid enzymes--catalytic or enzymatic DNA molecules--capable of cleaving nucleic acid sequences or molecules, particularly RNA, in a site-specific manner, as well as compositions including same. Methods of making and using the disclosed enzymes and compositions are also disclosed.

  13. Enzymatic Modifications of Polysaccharides

    USDA-ARS?s Scientific Manuscript database

    Polysaccharides are often modified chemically in order to improve its properties or to impart specific characteristics. Indeed quite a few commercial products are based on modified polysaccharides. In this talk, I shall describe a new set of modified polysaccharides based on enzymatic reactions. ...

  14. Nurse administrators' intentions and considerations in recruiting inactive nurses.

    PubMed

    Yu, Hsing-Yi; Tang, Fu-In; Chen, I-Ju; Yin, Teresa J C; Chen, Chu-Chieh; Yu, Shu

    2016-07-01

    To understand nurse administrators' intentions and considerations in recruiting inactive nurses and to examine predictors of intent to recruit. Few studies have provided insight into employer intentions and considerations in recruiting inactive nurses. A census survey collected data from 392 nurse administrators via a mailing method. Overall, 89.0% of nurse administrators were willing to recruit inactive nurses. Stepwise regression analysis revealed that the only predictor of nurse administrators' intention to recruit was nurse turnover rate at the hospital. Nurse administrators perceived the most important recruiting considerations were inactive nurses' cooperation with alternating shifts, health status and nursing licence. The most frequent reasons for not recruiting were an inactive nurse's lack of understanding of the medical environment and poor nursing competence. Most hospital nurse administrators were willing to recruit inactive nurses. Inactive nurses who wish to return to work should be qualified, willing to work both day and night shifts, and in good health. Nurse administrators can reduce the nursing shortage by recruiting inactive nurses. Re-entry preparation programmes should be implemented that will provide inactive nurses with knowledge of the current medical environment and the skills required to improve their nursing competence. © 2016 John Wiley & Sons Ltd.

  15. [Biosynthesis and isolation of a recombinant protein for producing genetically-engineered human proinsulin].

    PubMed

    Ivankin, A N; Mitaleva, S I; Nekliudov, A D

    1998-01-01

    Isolation of the recombinant protein from a genetically engineered Escherichia coli 1854 producer for further chemical enzymatic transformation into human insulin through proinsulin was studied. Under optimal conditions, the recombinant protein formation was more than 35% of the total cell proteins. Structures of the polypeptides obtained and purified chromatographically were confirmed by amino acid analysis. Human proinsulin was derived from the recombinant protein isolated.

  16. Characterizing inactive ribosomes in translational profiling

    PubMed Central

    Liu, Botao; Qian, Shu-Bing

    2016-01-01

    ABSTRACT The broad impact of translational regulation has emerged explosively in the last few years in part due to the technological advance in genome-wide interrogation of gene expression. During mRNA translation, the majority of actively translating ribosomes exist as polysomes in cells with multiple ribosomes loaded on a single transcript. The importance of the monosome, however, has been less appreciated in translational profiling analysis. Here we report that the monosome fraction isolated by sucrose sedimentation contains a large quantity of inactive ribosomes that do not engage on mRNAs to direct translation. We found that the elongation factor eEF2, but not eEF1A, stably resides in these non-translating ribosomes. This unique feature permits direct evaluation of ribosome status under various stress conditions and in the presence of translation inhibitors. Ribosome profiling reveals that the monosome has a similar but not identical pattern of ribosome footprints compared to the polysome. We show that the association of free ribosomal subunits minimally contributes to ribosome occupancy outside of the coding region. Our results not only offer a quantitative method to monitor ribosome availability, but also uncover additional layers of ribosome status needed to be considered in translational profiling analysis. PMID:27335722

  17. Physical inactivity and arterial stiffness in COPD

    PubMed Central

    Sievi, Noriane A; Franzen, Daniel; Kohler, Malcolm; Clarenbach, Christian F

    2015-01-01

    Background Arterial stiffness is an important predictor of cardiovascular risk besides classic cardiovascular risk factors. Previous studies showed that arterial stiffness is increased in patients with COPD compared to healthy controls and exercise training may reduce arterial stiffness. Since physical inactivity is frequently observed in patients with COPD and exercise training may improve arterial stiffness, we hypothesized that low daily physical activity may be associated with increased arterial stiffness. Methods In 123 patients with COPD (72% men; mean [standard deviation] age: 62 [7.5] years; median [quartile] forced expiratory volume in 1 second 35 [27/65] %predicted), arterial stiffness was assessed by augmentation index (AI). Daily physical activity level (PAL) was measured by an activity monitor (SenseWear Pro™) >1 week. The association between AI and PAL was investigated by univariate and multivariate regression analysis, taking into account disease-specific characteristics and comorbidities. Results Patients suffered from moderate (35%), severe (32%), and very severe (33%) COPD, and 22% were active smokers. Median (quartile) PAL was 1.4 (1.3/1.5) and mean (standard deviation) AI 26% (9.2%). PAL showed a negative association with AI (B=−9.32, P=0.017) independent of age, sex, blood pressure, and airflow limitation. Conclusion In COPD patients, a higher PAL seems to favorably influence arterial stiffness and therefore may reduce cardiovascular risk. Clinical Trial Registration http://www.ClinicalTrials.gov, NCT01527773 PMID:26392763

  18. Enzymatic Synthesis of Psilocybin.

    PubMed

    Fricke, Janis; Blei, Felix; Hoffmeister, Dirk

    2017-09-25

    Psilocybin is the psychotropic tryptamine-derived natural product of Psilocybe carpophores, the so-called "magic mushrooms". Although its structure has been known for 60 years, the enzymatic basis of its biosynthesis has remained obscure. We characterized four psilocybin biosynthesis enzymes, namely i) PsiD, which represents a new class of fungal l-tryptophan decarboxylases, ii) PsiK, which catalyzes the phosphotransfer step, iii) the methyltransferase PsiM, catalyzing iterative N-methyl transfer as the terminal biosynthetic step, and iv) PsiH, a monooxygenase. In a combined PsiD/PsiK/PsiM reaction, psilocybin was synthesized enzymatically in a step-economic route from 4-hydroxy-l-tryptophan. Given the renewed pharmaceutical interest in psilocybin, our results may lay the foundation for its biotechnological production. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Enzymatic desulfurization of coal

    SciTech Connect

    Boyer, Y.N.; Crooker, S.C.; Kitchell, J.P.; Nochur, S.V.

    1991-05-16

    The overall objective of this program was to investigate the feasibility of an enzymatic desulfurization process specifically intended for organic sulfur removal from coal. Toward that end, a series of specific objectives were defined: (1) establish the feasibility of (bio)oxidative pretreatment followed by biochemical sulfate cleavage for representative sulfur-containing model compounds and coals using commercially-available enzymes; (2) investigate the potential for the isolation and selective use of enzyme preparations from coal-utilizing microbial systems for desulfurization of sulfur-containing model compounds and coals; and (3) develop a conceptual design and economic analysis of a process for enzymatic removal of organic sulfur from coal. Within the scope of this program, it was proposed to carry out a portion of each of these efforts concurrently. (VC)

  20. [Enzymatic pancreatogenic omental bursitis].

    PubMed

    Tolstoĭ, A D; Kolupaev, I O; Sopiia, R A

    1996-01-01

    The most common causes of omental sac collections (OSC) are necrotic pancreatitis (90%) and pancreatic trauma (10%). Acute OSC is a form of local peritonitis in acute pancreatitis, subacute OSC are caused by internal pancreatic fistulas. The clinical and radiological signs, enzymatic activity of the exudate, morphological features of peritonitis were investigated. Treatment of acute OSC included conservative measures, of subacute OSC-surgical procedures.

  1. The synthetic hydroxyproline-containing collagen analogue (Gly–Pro–Hyp)10 promotes enzymatic activity of matrixmetalloproteinase-2 in vitro

    PubMed Central

    Freise, C.; Ruehl, M.; Erben, U.; Farndale, R. W.; Somasundaram, R.; Heimesaat, M. M.

    2012-01-01

    Diseases such as liver fibrosis and intestinal inflammation are characterized by accumulated components of the extracellular matrix (ECM). Given that fibrillar collagen structures were shown to serve as storage site for inactive proforms of matrixmetalloproteinases (MMPs), modulating this MMP–collagen interaction might offer a rational interventional (therapeutic) approach to enhance degradation of accumulated ECM. The synthetic triple helical collagen analogue (Gly–Pro–Hyp)10 – (GPO)10 – was shown to trigger release and enzymatic activation of collagen sequestered proMMP-2. In the presented study, we, for the first time, investigated how MMP–(GPO)10 interaction impacts cellular responses in vitro. We found that recombinant proMMP-2 induced proliferation of hepatic stellate cells (HSC), which was enhanced after addition of (GPO)10 reaching comparable levels following incubation with fully activated MMP-2. In addition, (GPO)10 induced HSC migration similar to the platelet-derived growth factor subunit-B. Further, the MMP-2-dependent invasion of HT1080 fibrosarcoma cells through an ECM membrane was enhanced after addition of (GPO)10. Since cellular proliferation and migration concomitant with matrix degradation is stimulated, we conclude that the MMP–(GPO)10 interaction also functions in a physiological environment. Thus, a potential therapeutic effect of (GPO)10 should be further tested in animal models for MMP-associated diseases such as colitis or fibrosis. PMID:24688764

  2. The synthetic hydroxyproline-containing collagen analogue (Gly-Pro-Hyp)10 promotes enzymatic activity of matrixmetalloproteinase-2 in vitro.

    PubMed

    Freise, C; Ruehl, M; Erben, U; Farndale, R W; Somasundaram, R; Heimesaat, M M

    2012-09-01

    Diseases such as liver fibrosis and intestinal inflammation are characterized by accumulated components of the extracellular matrix (ECM). Given that fibrillar collagen structures were shown to serve as storage site for inactive proforms of matrixmetalloproteinases (MMPs), modulating this MMP-collagen interaction might offer a rational interventional (therapeutic) approach to enhance degradation of accumulated ECM. The synthetic triple helical collagen analogue (Gly-Pro-Hyp)10 - (GPO)10 - was shown to trigger release and enzymatic activation of collagen sequestered proMMP-2. In the presented study, we, for the first time, investigated how MMP-(GPO)10 interaction impacts cellular responses in vitro. We found that recombinant proMMP-2 induced proliferation of hepatic stellate cells (HSC), which was enhanced after addition of (GPO)10 reaching comparable levels following incubation with fully activated MMP-2. In addition, (GPO)10 induced HSC migration similar to the platelet-derived growth factor subunit-B. Further, the MMP-2-dependent invasion of HT1080 fibrosarcoma cells through an ECM membrane was enhanced after addition of (GPO)10. Since cellular proliferation and migration concomitant with matrix degradation is stimulated, we conclude that the MMP-(GPO)10 interaction also functions in a physiological environment. Thus, a potential therapeutic effect of (GPO)10 should be further tested in animal models for MMP-associated diseases such as colitis or fibrosis.

  3. Mobile App to Reduce Inactivity in Sedentary Overweight Women.

    PubMed

    Finkelstein, Joseph; Bedra, McKenzie; Li, Xuan; Wood, Jeffrey; Ouyang, Pamela

    2015-01-01

    Recent studies demonstrated that the duration of inactivity (sedentary state) is independently associated with increased risk of cardiovascular disease. Our goal was to develop the technology that can measure the amount of inactivity in real time, remind a person that a preprogrammed period of inactivity has occurred and encourage a period of activity, and provide web-based feedback with tailored information to the participant and investigators. Once it was developed, we carried out a pilot study in a group of sedentary overweight women. The objective of the study was to assess potential of the mobile app to reduce inactivity in our target population. A randomized crossover design was employed with study subjects randomly assigned to a 4-week each "message-on" and "message-off" periods. Out of 30 enrolled subjects, 27 completed the study. The average age of particpants was 52±12; BMI: 37±6; 47% were white and 47% were African American. Overall, inactivity was significantly lower (p<0.02) during "message-on" periods (24.6%) as compared to the "message-off" periods (30.4%). We conluded that mobile app monitoring inactivity and providing a real-time notification when inactivity period exceeds healthy limits was able to significantly reduce inactivity periods in overweight sedentary women.

  4. 25 CFR 117.17 - Inactive surplus funds accounts.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 25 Indians 1 2011-04-01 2011-04-01 false Inactive surplus funds accounts. 117.17 Section 117.17 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR FINANCIAL ACTIVITIES DEPOSIT AND EXPENDITURE... COMPETENCY § 117.17 Inactive surplus funds accounts. When the balance of surplus funds to the credit of an...

  5. 25 CFR 117.17 - Inactive surplus funds accounts.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 25 Indians 1 2013-04-01 2013-04-01 false Inactive surplus funds accounts. 117.17 Section 117.17 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR FINANCIAL ACTIVITIES DEPOSIT AND EXPENDITURE... COMPETENCY § 117.17 Inactive surplus funds accounts. When the balance of surplus funds to the credit of an...

  6. 25 CFR 117.17 - Inactive surplus funds accounts.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 25 Indians 1 2014-04-01 2014-04-01 false Inactive surplus funds accounts. 117.17 Section 117.17 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR FINANCIAL ACTIVITIES DEPOSIT AND EXPENDITURE... COMPETENCY § 117.17 Inactive surplus funds accounts. When the balance of surplus funds to the credit of an...

  7. 25 CFR 117.17 - Inactive surplus funds accounts.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 25 Indians 1 2012-04-01 2011-04-01 true Inactive surplus funds accounts. 117.17 Section 117.17 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR FINANCIAL ACTIVITIES DEPOSIT AND EXPENDITURE... COMPETENCY § 117.17 Inactive surplus funds accounts. When the balance of surplus funds to the credit of an...

  8. 25 CFR 117.17 - Inactive surplus funds accounts.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false Inactive surplus funds accounts. 117.17 Section 117.17 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR FINANCIAL ACTIVITIES DEPOSIT AND EXPENDITURE... COMPETENCY § 117.17 Inactive surplus funds accounts. When the balance of surplus funds to the credit of an...

  9. 1. AERIAL VIEW, NAVAL INACTIVE SHIPS MAINTENANCE FACILITY, SINCLAIR ISLET, ...

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

    1. AERIAL VIEW, NAVAL INACTIVE SHIPS MAINTENANCE FACILITY, SINCLAIR ISLET, BREMERTON, KITSAP COUNTY, WASHINGTON WITH EX-USS HORNET CVS-12, THREE MINECRAFT ALONGSIDE TO PORT. OTHER INACTIVE SHIPS IN BACKGROUND. - U.S.S. HORNET, Puget Sound Naval Shipyard, Sinclair Inlet, Bremerton, Kitsap County, WA

  10. 25 CFR 23.63 - Appeals from inaction of official.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 25 Indians 1 2014-04-01 2014-04-01 false Appeals from inaction of official. 23.63 Section 23.63 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR HUMAN SERVICES INDIAN CHILD WELFARE ACT Appeals § 23.63 Appeals from inaction of official. A person or persons whose interests are...

  11. 25 CFR 23.63 - Appeals from inaction of official.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 25 Indians 1 2013-04-01 2013-04-01 false Appeals from inaction of official. 23.63 Section 23.63 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR HUMAN SERVICES INDIAN CHILD WELFARE ACT Appeals § 23.63 Appeals from inaction of official. A person or persons whose interests are...

  12. 25 CFR 23.63 - Appeals from inaction of official.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false Appeals from inaction of official. 23.63 Section 23.63 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR HUMAN SERVICES INDIAN CHILD WELFARE ACT Appeals § 23.63 Appeals from inaction of official. A person or persons whose interests are...

  13. 25 CFR 23.63 - Appeals from inaction of official.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 25 Indians 1 2011-04-01 2011-04-01 false Appeals from inaction of official. 23.63 Section 23.63 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR HUMAN SERVICES INDIAN CHILD WELFARE ACT Appeals § 23.63 Appeals from inaction of official. A person or persons whose interests are...

  14. 25 CFR 23.63 - Appeals from inaction of official.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 25 Indians 1 2012-04-01 2011-04-01 true Appeals from inaction of official. 23.63 Section 23.63 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR HUMAN SERVICES INDIAN CHILD WELFARE ACT Appeals § 23.63 Appeals from inaction of official. A person or persons whose interests are...

  15. Recombinant allergens

    PubMed Central

    Jutel, Marek; Solarewicz-Madejek, Katarzyna; Smolinska, Sylwia

    2012-01-01

    Allergen specific immunotherapy (SIT) is the only known causative treatment of allergic diseases. Recombinant allergen-based vaccination strategies arose from a strong need to both to improve safety and enhance efficacy of SIT. In addition, new vaccines can be effective in allergies including food allergy or atopic dermatitis, which poorly respond to the current treatment with allergen extracts. A number of successful clinical studies with both wild-type and hypoallergenic derivatives of recombinant allergens vaccines have been reported for the last decade. They showed high efficacy and safety profile as well as very strong modulation of T and B cell responses to specific allergens. PMID:23095874

  16. A downstream process allowing the efficient isolation of a recombinant amphiphilic protein from tobacco leaves.

    PubMed

    Gecchele, Elisa; Schillberg, Stefan; Merlin, Matilde; Pezzotti, Mario; Avesani, Linda

    2014-06-01

    The 65-kDa isoform of human glutamic acid decarboxylase (hGAD65) is a major autoantigen in autoimmune diabetes. The heterologous production of hGAD65 for diagnostic and therapeutic applications is hampered by low upstream productivity and the absence of a robust and efficient downstream process for product isolation. A tobacco-based platform has been developed for the production of an enzymatically-inactive form of the protein (hGAD65mut), but standard downstream processing strategies for plant-derived recombinant proteins cannot be used in this case because the product is amphiphilic. We therefore evaluated different extraction buffers and an aqueous micellar two-phase system (AMTPS) to optimize the isolation and purification of hGAD65mut from plants. We identified the extraction conditions offering the greatest selectivity for hGAD65mut over native tobacco proteins using a complex experimental design approach. Under our optimized conditions, the most efficient initial extraction and partial purification strategy achieved an overall hGAD65mut yield of 92.5% with a purification factor of 12.3 and a concentration factor of 23.8. The process also removed a significant quantity of phenols, which are major contaminants present in tobacco tissue. This is the first report describing the use of AMTPS for the partial purification of an amphiphilic recombinant protein from plant tissues and our findings could also provide a working model for the initial recovery and partial purification of hydrophobic recombinant proteins from transgenic tobacco plants.

  17. Decreasing Physical Inactivity in the Veterans Health Administration Employee Population.

    PubMed

    Schult, Tamara M; Schmunk, Sandra K; Awosika, Ebi R

    2016-12-01

    The aim of this study was to describe a comprehensive approach to decrease physical inactivity in the Veterans Health Administration (VHA) employee population. The approach included (1) initiatives to decrease physical inactivity in the workplace; (2) two operational surveys to assess system-wide service provision; and (3) two national employee surveys. From 2010 to 2012, 86 employee fitness centers were completed in VA medical centers. A grants program (2010 to 2015) funded smaller projects designed to decrease physical inactivity in the workplace. Projects involved the provision of equipment to decrease sedentary behaviors, including stability balls, treadmill and sit-to-stand desks, stairwell projects, and funding for on-site fitness classes, bicycle racks, and outdoor par courses and walking paths among others. A comprehensive approach to decrease physical inactivity in VHA employees was successful. Overall, self-reported, age-adjusted physical inactivity in VHA employees decreased from 25.3% in 2010 to 16.1% in 2015.

  18. Spectrum Recombination.

    ERIC Educational Resources Information Center

    Greenslade, Thomas B., Jr.

    1984-01-01

    Describes several methods of executing lecture demonstrations involving the recombination of the spectrum. Groups the techniques into two general classes: bringing selected portions of the spectrum together using lenses or mirrors and blurring the colors by rapid movement or foreshortening. (JM)

  19. Recombinant gonadotropins.

    PubMed

    Lathi, R B; Milki, A A

    2001-10-01

    Recombinant DNA technology makes it possible to produce large amounts of human gene products for pharmacologic applications, supplanting the need for human tissues. The genes for the alpha and beta subunits of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotropin (hCG) have been characterized and cloned. Recombinant FSH (rFSH) has been shown to be safe and effective in the treatment of fertility disorders. In comparison with the urinary gonadotropin products, human menopausal gonadotropins (HMG), and urinary follitropins (uFSH), rFSH is more potent and better tolerated by patients. Recombinant HCG appears to be as efficacious as urinary HCG with the benefit of improved local tolerance. Recombinant LH (rLH) is likely to be recommended as a supplement to rFSH for ovulation induction in hypogonadotropic women. It may also benefit in vitro fertilization patients undergoing controlled ovarian hyperstimulation with rFSH combined with pituitary suppression, with a gonadotropin-releasing hormone agonist or antagonist.

  20. Spectrum Recombination.

    ERIC Educational Resources Information Center

    Greenslade, Thomas B., Jr.

    1984-01-01

    Describes several methods of executing lecture demonstrations involving the recombination of the spectrum. Groups the techniques into two general classes: bringing selected portions of the spectrum together using lenses or mirrors and blurring the colors by rapid movement or foreshortening. (JM)

  1. An Exploratory Study of Inactive Health Information Seekers

    PubMed Central

    2014-01-01

    Purpose This study aims to identify people who do not actively seek out health information and the demographic characteristics of Inactive Seekers. The possible determinants of inactive seeking behaviors is also explored. Design and Measurements A total of 14,420 survey respondents were drawn from the 2009 Annenberg National Health Communication Survey (ANHCS) data. K-means clustering was used to discriminate Inactive Seekers from Active Seekers. The inactive information seeker group was formed based on their experience with health information seeking. The potential determinants that were tested to predict inactive seeking included the following: health condition, health service use, health media exposure, and computer/Internet activities. Results Within this national survey data, the respondents were more likely to be included in the Inactive Seekers (N=8,312, 58.5%) compared to Active Seekers (N=5,908, 41.5%). The demographic characteristics indicated that the Inactive Seekers were identified as younger, male, highly educated, White, and high household income people. The binary logistic regression results from the study model indicated that healthier people were less likely to seek out health information than their counterparts. In addition, those who were exposed to various media were almost 1.6 times more likely to seek out health information than those who were not exposed to such media. Within this study data, the statistically significant determinants identified were health condition and health media exposure while computer/Internet activities did not show strong indications in predicting inactive seeking behavior. Conclusion The development of more generalizable measures for health literacy or behavioral patterns will bolster advanced study on inactive seeking relating to knowledge of technology and health context. Further study should be directed at estimating the negative aspects of information seeking such as information ignorance or information

  2. Enzymatic temperature change indicator

    DOEpatents

    Klibanov, Alexander M.; Dordick, Jonathan S.

    1989-01-21

    A temperature change indicator is described which is composed of an enzyme and a substrate for that enzyme suspended in a solid organic solvent or mixture of solvents as a support medium. The organic solvent or solvents are chosen so as to melt at a specific temperature or in a specific temperature range. When the temperature of the indicator is elevated above the chosen, or critical temperature, the solid organic solvent support will melt, and the enzymatic reaction will occur, producing a visually detectable product which is stable to further temperature variation.

  3. Enzymatic cascade bioreactor

    DOEpatents

    Simmons, Blake A.; Volponi, Joanne V.; Ingersoll, David; Walker, Andrew

    2007-09-04

    Disclosed is an apparatus and method for continuously converting sucrose to .beta.-D-glucose. The method comprises a three stage enzymatic reactor in which an aqueous solution of sucrose is first converted into a solution of fructose and .alpha.-D-glucose by passing it through a porous, packed column containing an inert media on which invertase is immobilized. This solution is then sent through a second packed column containing glucose isomerase and finally a third packed column containing mutarotase. Solution temperature and pH are adjusted to maximize glucose output.

  4. Histone deacetylases 9 and 10 are required for homologous recombination.

    PubMed

    Kotian, Shweta; Liyanarachchi, Sandhya; Zelent, Arthur; Parvin, Jeffrey D

    2011-03-11

    We tested the role of histone deacetylases (HDACs) in the homologous recombination process. A tissue-culture based homology-directed repair assay was used in which repair of a double-stranded break by homologous recombination results in gene conversion of an inactive GFP allele to an active GFP gene. Our rationale was that hyperacetylation caused by HDAC inhibitor treatment would increase chromatin accessibility to repair factors, thereby increasing homologous recombination. Contrary to expectation, treatment of cells with the inhibitors significantly reduced homologous recombination activity. Using RNA interference to deplete each HDAC, we found that depletion of either HDAC9 or HDAC10 specifically inhibited homologous recombination. By assaying for sensitivity of cells to the interstrand cross-linker mitomycin C, we found that treatment of cells with HDAC inhibitors or depletion of HDAC9 or HDAC10 resulted in increased sensitivity to mitomycin C. Our data reveal an unanticipated function of HDAC9 and HDAC10 in the homologous recombination process.

  5. Enzymatic and non-enzymatic detoxification of 4-hydroxynonenal: Methodological aspects and biological consequences.

    PubMed

    Mol, Marco; Regazzoni, Luca; Altomare, Alessandra; Degani, Genny; Carini, Marina; Vistoli, Giulio; Aldini, Giancarlo

    2017-02-02

    4-Hydroxynonenal (HNE), an electrophilic end-product deriving from lipid peroxidation, undergoes a heterogeneous set of biotransformations including enzymatic and non-enzymatic reactions. The former mostly involve red-ox reactions on the HNE oxygenated functions (phase I metabolism) and GSH conjugations (phase II) while the latter are due to the HNE capacity to spontaneously condense with nucleophilic sites within endogenous molecules such as proteins, nucleic acids and phospholipids. The overall metabolic fate of HNE has recently attracted great interest not only because it clearly determines the HNE disposal, but especially because the generated metabolites and adducts are not inactive molecules (as initially believed) but show biological activities even more pronounced than those of the parent compound as exemplified by potent pro-inflammatory stimulus induced by GSH conjugates. Similarly, several studies revealed that the non-enzymatic reactions, initially considered as damaging processes randomly involving all endogenous nucleophilic reactants, are in fact quite selective in terms of both reactivity of the nucleophilic sites and stability of the generated adducts. Even though many formed adducts retain the expected toxic consequences, some adducts exhibit well-defined beneficial roles as documented by the protective effects of sublethal concentrations of HNE against toxic concentrations of HNE. Clearly, future investigations are required to gain a more detailed understanding of the metabolic fate of HNE as well as to identify novel targets involved in the biological activity of the HNE metabolites. These studies are and will be permitted by the continuous progress in the analytical methods for the identification and quantitation of novel HNE metabolites as well as for proteomic analyses able to offer a comprehensive picture of the HNE-induced adducted targets. On these grounds, the present review will focus on the major enzymatic and non-enzymatic HNE

  6. Three-dimensional structure of porcine procarboxypeptidase B: a structural basis of its inactivity.

    PubMed Central

    Coll, M; Guasch, A; Avilés, F X; Huber, R

    1991-01-01

    Procarboxypeptidase B is converted to enzymatically active carboxypeptidase B by limited proteolysis catalysed by trypsin, removing the long N-terminal activation segment of 95 amino acids. The three-dimensional crystal structure of procarboxypeptidase B from porcine pancreas has been determined at 2.3 A resolution and refined to a crystallographic R-factor of 0.169. The functional determinants of its enzymatic inactivity and of its activation by limited proteolysis have thus been unveiled. The activation segment folds in a globular region with an open sandwich antiparallel-alpha antiparallel-beta topology and in a C terminal alpha-helix which connects it to the enzyme moiety. The globular region (A7-A82) shields the preformed active site, and establishes specific interactions with residues important for substrate recognition. AspA41 forms a salt bridge with Arg145, which in active carboxypeptidase binds the C-terminal carboxyl group of substrate molecules. The connecting region occupies the putative extended substrate binding site. The scissile peptide bond cleaved by trypsin during activation is very exposed. Its cleavage leads to the release of the activation segment and to exposure of the substrate binding site. An open-sandwich folding has been observed in a number of other proteins and protein domains. One of them is the C-terminal fragment of L7/L12, a ribosomal protein from Escherichia coli that displays a topology similar to the activation domain of procarboxypeptidase. Images PMID:1989878

  7. Active and inactive enhancers co-operate to exert localized and long-range control of gene regulation

    PubMed Central

    Proudhon, Charlotte; Snetkova, Valentina; Raviram, Ramya; Lobry, Camille; Badri, Sana; Jiang, Tingting; Hao, Bingtao; Trimarchi, Thomas; Kluger, Yuval; Aifantis, Iannis; Bonneau, Richard; Skok, Jane A

    2016-01-01

    V(D)J recombination relies on the presence of proximal enhancers that activate the antigen receptor (AgR) loci in a lineage and stage specific manner. Unexpectedly we find that both active and inactive AgR enhancers co-operate to disseminate their effects in a localized and long-range manner. Here we demonstrate the importance of short-range contacts between active enhancers that constitute an Igk super-enhancer in B cells. Deletion of one element reduces the interaction frequency between other enhancers in the hub, which compromises the transcriptional output of each component. We further establish that in T cells long-range contact and co-operation between the inactive Igk enhancer, MiEκ and the active Tcrb enhancer, Eβ, alters enrichment of CBFβ binding in a manner that impacts Tcrb recombination. These findings underline the complexities of enhancer regulation and point to a role for localized and long-range enhancer-sharing between active and inactive elements in lineage and stage specific control. PMID:27239026

  8. Basal Ganglia Dysfunction Contributes to Physical Inactivity in Obesity.

    PubMed

    Friend, Danielle M; Devarakonda, Kavya; O'Neal, Timothy J; Skirzewski, Miguel; Papazoglou, Ioannis; Kaplan, Alanna R; Liow, Jeih-San; Guo, Juen; Rane, Sushil G; Rubinstein, Marcelo; Alvarez, Veronica A; Hall, Kevin D; Kravitz, Alexxai V

    2017-02-07

    Obesity is associated with physical inactivity, which exacerbates the health consequences of weight gain. However, the mechanisms that mediate this association are unknown. We hypothesized that deficits in dopamine signaling contribute to physical inactivity in obesity. To investigate this, we quantified multiple aspects of dopamine signaling in lean and obese mice. We found that D2-type receptor (D2R) binding in the striatum, but not D1-type receptor binding or dopamine levels, was reduced in obese mice. Genetically removing D2Rs from striatal medium spiny neurons was sufficient to reduce motor activity in lean mice, whereas restoring Gi signaling in these neurons increased activity in obese mice. Surprisingly, although mice with low D2Rs were less active, they were not more vulnerable to diet-induced weight gain than control mice. We conclude that deficits in striatal D2R signaling contribute to physical inactivity in obesity, but inactivity is more a consequence than a cause of obesity.

  9. Can neighborhoods explain racial/ethnic differences in adolescent inactivity?

    PubMed

    Richmond, Tracy K; Field, Alison E; Rich, Michael

    2007-01-01

    To determine if neighborhoods and their attributes contribute to racial/ethnic disparities in adolescent inactivity. We undertook a cross-sectional analysis of the National Longitudinal Study of Adolescent Health (n = 17,007), a nationally representative school-based study in the United States. Stratifying by gender, we used multivariate linear regression and multi-level modeling to determine whether neighborhood of residence may partially explain racial/ethnic disparities in adolescent physical inactivity, defined as hours viewing television or videos/DVDs and/or playing computer/video games each week. Participants lived in largely segregated communities. Black and Hispanic adolescent girls reported higher levels of inactivity than White adolescent girls (21 vs. 15 vs. 13 hours/week, respectively, p <0.001). Similar patterns were seen in adolescent boys, with Black adolescent males reporting a mean of 26 hours/week; Hispanic boys a mean of 20 hours/week; and White boys a mean of 17 hours/week of inactivity (p <0.001). After accounting for between-neighborhood variation, there were no residual within-neighborhood differences in inactivity between Hispanic and White adolescent girls (gamma = -0.06, p =0.93); when living in the same neighborhood Hispanic and White girls had similar levels of inactivity. Black adolescent girls and boys were found to have higher levels of inactivity no matter where they lived (gamma =7.00, p <0.001 for girls; gamma = 6.96, p <0.001 for boys). Hispanic boys had similar patterns of inactivity to White boys (gamma =-1.57, p = 0.12). In both males and females, the reported rate of violent crime in the neighborhood was associated with inactivity, despite the individual's perception of his/her neighborhood as safe not being predictive. Although inactivity varies by race/ethnicity and gender, only in Hispanic adolescent girls does neighborhood fully explain the differential use. Our findings suggest that approaches other than changing

  10. Pretreatment and enzymatic hydrolysis of corn fiber

    SciTech Connect

    Grohmann, K.; Bothast, R.J.

    1996-10-01

    Corn fiber is a co-product of the corn wet milling industry which is usually marketed as a low value animal feed ingredient. Approximately 1.2 x 10{sup 6} dry tons of this material are produced annually in the United States. The fiber is composed of kernel cell wall fractions and a residual starch which can all be potentially hydrolyzed to a mixture of glucose, xylose, arabinose and galactose. We have investigated a sequential saccharification of polysaccharides in corn fiber by a treatment with dilute sulfuric acid at 100 to 160{degrees}C followed by partial neutralization and enzymatic hydrolysis with mixed cellulose and amyloglucosidase enzymes at 45{degrees}C. The sequential treatment achieved a high (approximately 85%) conversion of all polysaccharides in the corn fiber to monomeric sugars, which were in most cases fermentable to ethanol by the recombinant bacterium Escherichia coli KOll.

  11. Fluorometric enzymatic assay of L-arginine

    NASA Astrophysics Data System (ADS)

    Stasyuk, Nataliya; Gayda, Galina; Yepremyan, Hasmik; Stepien, Agnieszka; Gonchar, Mykhailo

    2017-01-01

    The enzymes of L-arginine (further - Arg) metabolism are promising tools for elaboration of selective methods for quantitative Arg analysis. In our study we propose an enzymatic method for Arg assay based on fluorometric monitoring of ammonia, a final product of Arg splitting by human liver arginase I (further - arginase), isolated from the recombinant yeast strain, and commercial urease. The selective analysis of ammonia (at 415 nm under excitation at 360 nm) is based on reaction with o-phthalaldehyde (OPA) in the presence of sulfite in alkali medium: these conditions permit to avoid the reaction of OPA with any amino acid. A linearity range of the fluorometric arginase-urease-OPA method is from 100 nM to 6 μМ with a limit of detection of 34 nM Arg. The method was used for the quantitative determination of Arg in the pooled sample of blood serum. The obtained results proved to be in a good correlation with the reference enzymatic method and literature data. The proposed arginase-urease-OPA method being sensitive, economical, selective and suitable for both routine and micro-volume formats, can be used in clinical diagnostics for the simultaneous determination of Arg as well as urea and ammonia in serum samples.

  12. Novel Catalytically-Inactive PII Metalloproteinases from a Viperid Snake Venom with Substitutions in the Canonical Zinc-Binding Motif

    PubMed Central

    Camacho, Erika; Sanz, Libia; Escalante, Teresa; Pérez, Alicia; Villalta, Fabián; Lomonte, Bruno; Neves-Ferreira, Ana Gisele C.; Feoli, Andrés; Calvete, Juan J.; Gutiérrez, José María; Rucavado, Alexandra

    2016-01-01

    Snake venom metalloproteinases (SVMPs) play key biological roles in prey immobilization and digestion. The majority of these activities depend on the hydrolysis of relevant protein substrates in the tissues. Hereby, we describe several isoforms and a cDNA clone sequence, corresponding to PII SVMP homologues from the venom of the Central American pit viper Bothriechis lateralis, which have modifications in the residues of the canonical sequence of the zinc-binding motif HEXXHXXGXXH. As a consequence, the proteolytic activity of the isolated proteins was undetectable when tested on azocasein and gelatin. These PII isoforms comprise metalloproteinase and disintegrin domains in the mature protein, thus belonging to the subclass PIIb of SVMPs. PII SVMP homologues were devoid of hemorrhagic and in vitro coagulant activities, effects attributed to the enzymatic activity of SVMPs, but induced a mild edema. One of the isoforms presents the characteristic RGD sequence in the disintegrin domain and inhibits ADP- and collagen-induced platelet aggregation. Catalytically-inactive SVMP homologues may have been hitherto missed in the characterization of snake venoms. The presence of such enzymatically-inactive homologues in snake venoms and their possible toxic and adaptive roles deserve further investigation. PMID:27754342

  13. [Overexpression of Aspergillus candidus lactase and analysis of enzymatic properties].

    PubMed

    Zhang, Wei; Fan, Yun-liu; Yao, Bin

    2005-04-01

    The lactase gene lacb' from Aspergillus candidus was fused behind alpha-factor signal sequence in the Pichia pastoris expression vector pPIC9, then integrated into the genome of P. pastoris by recombination events. The P. pastoris recombinants for lactase overexpression were screened by enzyme activity analysis and SDS-PAGE. The lactase expressed in P. pastoris was glycosylated protein with an apparent molecular weight of 130 kD, while the deglycosylated lactase treated with Endo H had an apparent molecular weight of about 110 kD. The expression level of secreted lactase protein in recombinant P. pastoris was 6 mg/mL with enzymatic activity of 3600 U/mL in the 5 L fermenter, which was the highest among that of all kinds of recombinant strains reported now. The optimal pH and optimal temperature of the lactase are 5.2 and 60 degrees C. The Vmax, Km, and specific activity of the lactase are 3.3 micromol/min, 1.7 mmol/L and 706.5 +/- 2.6 U/mg, respectively. Compare to the lactase from Aspergillus oryzae ATCC 20423, the expressed lactase from A. candidus have better enzymatic properties including the high thermostability, high specific activity and wide pH range for enzyme reaction.

  14. Experiential versus genetic accounts of inactivity: implications for inactive individuals' self-efficacy beliefs and intentions to exercise.

    PubMed

    Beauchamp, Mark R; Rhodes, Ryan E; Kreutzer, Christiane; Rupert, James L

    2011-01-01

    The overall purpose of this study was to examine the effect of deterministic media reports, linking genetics to inactivity, in relation to inactive people's social cognitions concerning physical activity involvement. Sixty three inactive university students were randomly allocated to one of three experimental conditions (control, genetically-primed, experientially-primed) and completed measures of instrumental and affective attitudes, subjective norms, self-efficacy, and exercise intentions. One week later participants in the two experimental conditions were provided with a bogus newspaper report that either reflected a genetic explanation for physical inactivity or an experiential basis for inactivity. Shortly afterwards, participants in all three conditions completed the same measures as at pre-test. The results revealed that after controlling for baseline measures participants in the experientially-primed condition reported significantly higher levels of self-efficacy and intentions to exercise than those in the genetically-primed condition. These findings raise a cautionary flag concerning the presentation of genetic research in the media, especially with regard to inactive populations.

  15. Prevalence and factors associated with physical inactivity among Malaysian adults.

    PubMed

    Ying, Chanying; Kuay, Lim Kuang; Huey, Teh Chien; Hock, Lim Kuang; Hamid, Hamizatul Akmal Abd; Omar, Mohd Azahadi; Ahmad, Noor Ani; Cheong, Kee Chee

    2014-03-01

    Using data from the Third National Health and Morbidity Survey (NHMS III) in 2006, this study examined the association between socio-demographic factors and physical inactivity in a sample of 33,949 adults aged 18 years and above by gender. Physical activity levels were measured using the Global Physical Activity Questionnaire (GPAQ vers 1). Physical inactivity was defined as having a total physical activity level of less than 600 metabolic equivalents-minutes per week (METs-minutes/week) contributed by all three different life domains.Logistic regression analyses were conducted.The prevalence of overall physical inactivity was 43.7% (95% CI: 42.9-44.5). The mean total physical activity level was 894.2 METs-minutes/ week. The means METs-minutes/week for the domain of work, travelling, and leisure time were 518.4, 288.1, and 134.8, respectively. Multivariable logistic regression analyses indicated that females were more likely to be physically inactive than males were (aOR=1.62; 95% CI: 1.53-1.72). Among women, being a housewife (aOR = 1.78; 95% CI: 1.56-2.03), widow/divorcee (aOR = 1.23; 95% CI: 1.05-1.43), and those with no formal education (aOR = 1.20; 95% CI: 1.01-1.43) were found to be significantly associated with physical inactivity.Urban residents, older adults aged 65 years and above, private employees, nonworking group, and those with a monthly household income level of MYR5,000 and above appeared to be consistently associated with physical inactivity across men, women, and combined group (both). Specific health intervention strategies to promote physical activity should be targeted on population subgroups who are inactive.

  16. Enzymatic characterization of starch synthase III from kidney bean (Phaseolus vulgaris L.).

    PubMed

    Senoura, Takeshi; Asao, Ayako; Takashima, Yoshinori; Isono, Naoto; Hamada, Shigeki; Ito, Hiroyuki; Matsui, Hirokazu

    2007-09-01

    In plants and green algae, several starch synthase isozymes are responsible for the elongation of glucan chains in the biosynthesis of amylose and amylopectin. Multiple starch synthase isozymes, which are classified into five major classes (granule-bound starch synthases, SSI, SSII, SSIII, and SSIV) according to their primary sequences, have distinct enzymatic properties. All the starch synthase isozymes consist of a transit peptide, an N-terminal noncatalytic region (N-domain), and a C-terminal catalytic region (C-domain). To elucidate the enzymatic properties of kidney bean (Phaseolus vulgaris L.) SSIII and the function of the N-domain of kidney bean SSIII, three recombinant proteins were constructed: putative mature recombinant SSIII, recombinant kidney bean SSIII N-domain, and recombinant kidney bean SSIII C-domain. Purified recombinant kidney bean SSIII displayed high specific activities for primers as compared to the other starch synthase isozymes from kidney bean. Kinetic analysis showed that the high specific activities of recombinant kidney bean SSIII are attributable to the high k(cat) values, and that the K(m) values of recombinant kidney bean SSIII C-domain for primers were much higher than those of recombinant kidney bean recombinant SSIII. Recombinant kidney bean SSIII and recombinant kidney bean SSIII C-domain had similar chain-length specificities for the extension of glucan chains, indicating that the N-domain of kidney bean SSIII does not affect the chain-length specificity. Affinity gel electrophoresis indicated that recombinant kidney bean SSIII and recombinant kidney bean SSIII N-domain have high affinities for amylose and amylopectin. The data presented in this study provide direct evidence for the function of the N-domain of kidney bean SSIII as a carbohydrate-binding module.

  17. Enzymatic hydrolysis of spent coffee ground.

    PubMed

    Jooste, T; García-Aparicio, M P; Brienzo, M; van Zyl, W H; Görgens, J F

    2013-04-01

    Spent coffee ground (SCG) is the main residue generated during the production of instant coffee by thermal water extraction from roasted coffee beans. This waste is composed mainly of polysaccharides such as cellulose and galactomannans that are not solubilised during the extraction process, thus remaining as unextractable, insoluble solids. In this context, the application of an enzyme cocktail (mannanase, endoglucanase, exoglucanase, xylanase and pectinase) with more than one component that acts synergistically with each other is regarded as a promising strategy to solubilise/hydrolyse remaining solids, either to increase the soluble solids yield of instant coffee or for use as raw material in the production of bioethanol and food additives (mannitol). Wild fungi were isolated from both SCG and coffee beans and screened for enzyme production. The enzymes produced from the selected wild fungi and recombinant fungi were then evaluated for enzymatic hydrolysis of SCG, in comparison to commercial enzyme preparations. Out of the enzymes evaluated on SCG, the application of mannanase enzymes gave better yields than when only cellulase or xylanase was utilised for hydrolysis. The recombinant mannanase (Man1) provided the highest increments in soluble solids yield (17 %), even when compared with commercial preparations at the same protein concentration (0.5 mg/g SCG). The combination of Man1 with other enzyme activities revealed an additive effect on the hydrolysis yield, but not synergistic interaction, suggesting that the highest soluble solid yields was mainly due to the hydrolysis action of mannanase.

  18. Enzymatic assay of beta-lactamase using circular dichroism spectropolarimetry.

    PubMed

    Long, D M

    1997-05-01

    A method for measuring the rates of enzymatic hydrolysis of beta-lactam antibiotics based on circular dichroism spectropolarimetry is described. Unhydrolyzed beta-lactam antibiotics have high molar ellipticities, but the hydrolyzed compounds are circular dichroism (CD) inactive in the case of penams or have significantly different CD spectra in the case of cephems. By measuring CD at constant wavelength as a function of time for reaction mixtures containing beta-lactamase and beta-lactam antibiotics, rates of hydrolysis and steady-state enzyme kinetic constants can be derived. The method was applied to measurement of a wide range of enzymatic reaction constants for wild-type and four mutant RTEM-1 beta-lactamases. Compared to the commonly employed assay based on ultraviolet spectroscopy, the new method offers several advantages. These include the ability to measure larger enzymatic Michaelis-Menten constants, less interference from high concentrations of beta-lactamase, higher sensitivity, and potentially less interference from other uv-absorbing components of complex reaction mixtures.

  19. The Costs of Inaction with Respect to Climate Change

    SciTech Connect

    Cline, William R.

    2005-04-14

    This paper first considers the principal trade-offs in the timing of policy response to climate change. It then turns to a synoptic review of the economic literature on costs and benefits of climate change abatement. The discussion closes with an estimate of the potential economic cost that might be associated with delay in abatement, based on the analysis in the author, published elsewhere. In the specific calculations of the paper, the costs of inaction are specified in two alternative ways. In the first and likely more relevant formulation, it is assumed that inaction persists for only two decades. The costs of inaction are then calculated as the damage arising from the additional climate change above that identified in the optimal abatement path as a consequence of failing to carry out any abatement in the first two decades, net of the savings in abatement costs thereby avoided. In the second and more extreme formulation, inaction is assumed to be permanent (i.e. there is never any reduction in emissions from their baseline path). In this case the costs of inaction are again the net costs of future climate damage after deducting the savings from absence of abatement, but this time over the full time horizon of three centuries.

  20. Enzymatically active ultrathin pepsin membranes.

    PubMed

    Raaijmakers, Michiel J T; Schmidt, Thomas; Barth, Monika; Tutus, Murat; Benes, Nieck E; Wessling, Matthias

    2015-05-11

    Enzymatically active proteins enable efficient and specific cleavage reactions of peptide bonds. Covalent coupling of the enzymes permits immobilization, which in turn reduces autolysis-induced deactivation. Ultrathin pepsin membranes were prepared by facile interfacial polycondensation of pepsin and trimesoyl chloride. The pepsin membrane allows for simultaneous enzymatic conversion and selective removal of digestion products. The large water fluxes through the membrane expedite the transport of large molecules through the pepsin layers. The presented method enables the large-scale production of ultrathin, cross-linked, enzymatically active membranes.

  1. Enzymatic degradation of endomorphins.

    PubMed

    Janecka, Anna; Staniszewska, Renata; Gach, Katarzyna; Fichna, Jakub

    2008-11-01

    Centrally acting plant opiates, such as morphine, are the most frequently used analgesics for the relief of severe pain, even though their undesired side effects are serious limitation to their usefulness. The search for new therapeutics that could replace morphine has been mainly focused on the development of peptide analogs or peptidomimetics with high selectivity for one receptor type and high bioavailability, that is good blood-brain barrier permeability and enzymatic stability. Drugs, in order to be effective, must be able to reach the target tissue and to remain metabolically stable to produce the desired effects. The study of naturally occurring peptides provides a rational and powerful approach in the design of peptide therapeutics. Endogenous opioid peptides, endomorphin-1 and endomorphin-2, are two potent and highly selective mu-opioid receptor agonists, discovered only a decade ago, which display potent analgesic activity. However, extensive studies on the possible use of endomorphins as analgesics instead of morphine met with failure due to their instability. This review deals with the recent investigations that allowed determine degradation pathways of endomorphins in vitro and in vivo and propose modifications that will lead to more stable analogs.

  2. Chemo-Enzymatic Synthesis of Optically Active γ- and δ-Decalactones and Their Effect on Aphid Probing, Feeding and Settling Behavior

    PubMed Central

    Boratyński, Filip; Dancewicz, Katarzyna; Paprocka, Marlena; Gabryś, Beata; Wawrzeńczyk, Czesław

    2016-01-01

    The enantiomerically enriched γ- and δ-decalactones (4a and 4b) were prepared from corresponding racemic primary-secondary 1,4- and 1,5-diols (1a and 1b), as products of enzymatic oxidation catalyzed by different alcohol dehydrogenases. The results of biotransformations indicated that the oxidation processes catalyzed by alcohol dehydrogenase (HLADH), both isolated from horse liver and recombinant in Escherichia coli, were characterized by the highest degree of conversion with moderate enantioselectivity of the reaction. Useful, environmentally friendly extraction procedure of decalactones (4a and 4b) based on hydrodistillation using a Deryng apparatus was developed. Both racemic lactones (4a and 4b), as well as their enantiomerically enriched isomers, were tested for feeding deterrent activity against Myzus persicae. The effect of these compounds on probing, feeding and settling behavior of M. persicae was studied in vivo. The deterrent activity of decalactones (4a and 4b) against aphids depended on the size of the lactone ring and the enantiomeric purity of the compounds. δ-Decalactone (4b) appeared inactive against M. persicae while γ-decalactone (4a) restrained aphid probing at ingestional phase. Only (–)-(S)-γ-decalactone (4a) had strong and durable (i.e. lasting for at least 24 hours) limiting effect, expressed at phloem level. PMID:26741824

  3. Chemo-Enzymatic Synthesis of Optically Active γ- and δ-Decalactones and Their Effect on Aphid Probing, Feeding and Settling Behavior.

    PubMed

    Boratyński, Filip; Dancewicz, Katarzyna; Paprocka, Marlena; Gabryś, Beata; Wawrzeńczyk, Czesław

    2016-01-01

    The enantiomerically enriched γ- and δ-decalactones (4a and 4b) were prepared from corresponding racemic primary-secondary 1,4- and 1,5-diols (1a and 1b), as products of enzymatic oxidation catalyzed by different alcohol dehydrogenases. The results of biotransformations indicated that the oxidation processes catalyzed by alcohol dehydrogenase (HLADH), both isolated from horse liver and recombinant in Escherichia coli, were characterized by the highest degree of conversion with moderate enantioselectivity of the reaction. Useful, environmentally friendly extraction procedure of decalactones (4a and 4b) based on hydrodistillation using a Deryng apparatus was developed. Both racemic lactones (4a and 4b), as well as their enantiomerically enriched isomers, were tested for feeding deterrent activity against Myzus persicae. The effect of these compounds on probing, feeding and settling behavior of M. persicae was studied in vivo. The deterrent activity of decalactones (4a and 4b) against aphids depended on the size of the lactone ring and the enantiomeric purity of the compounds. δ-Decalactone (4b) appeared inactive against M. persicae while γ-decalactone (4a) restrained aphid probing at ingestional phase. Only (-)-(S)-γ-decalactone (4a) had strong and durable (i.e. lasting for at least 24 hours) limiting effect, expressed at phloem level.

  4. Parental Predictors of Physical Inactivity in Spanish Adolescents

    PubMed Central

    Sanz-Arazuri, Eva; Ponce-de-León-Elizondo, Ana; Valdemoros-San-Emeterio, María Ángeles

    2012-01-01

    The purpose of this study was to determine some parental predictors of physical inactivity in Spanish adolescents. The sample comprised 1,978 children, aged between 12 and 16 years. A quantitative and qualitative technical triangulation was employed. The study analyzed data of the parents' educational level, the importance they grant to physical-sport activities, and their physical-sport practice. Quantitative technique: a questionnaire (MACOFYD) was used to collect the data. Descriptive, bivariate, and multinomial regression analyses were employed. Statistical significance was set at p < 0.05. Qualitative technique: four discussion groups were conducted, consisting of parents, physical education teachers, teachers of other subjects, and children aged between 12 and 16 years. The results indicated that adolescents are four times more likely to be physically inactive if their parents have never exercised (odds ratio [OR] = 4.065, and = 3.487, for the fathers and mothers, respectively, p < 0.05). When parents grant “some” or “much” importance to physical-sport practice, adolescents are less likely to be physically inactive (OR = 0.185 and 0.118 respectively, p < 0.01). No significant correlation was found between adolescents' physical-sport activity and parents' educational level. However, young people reproach their parents because they emphasize academic goals more than physical-sport practice-an observation that teachers also confirm. Young people perceive their parents as being the education agents with the greatest influence over their inactive lifestyles. Many parents are unaware of their influence and, therefore, do not take responsibility, declaring that the teachers' influence is greater. Key points Parental factors significantly affect adolescent physical inactivity. Parents' physical inactivity is among the most important factors. Statistically significant results were found for gender. Being female tripled the likelihood of being sedentary. The

  5. Homogeneous, Heterogeneous, and Enzymatic Catalysis.

    ERIC Educational Resources Information Center

    Oyama, S. Ted; Somorjai, Gabor A.

    1988-01-01

    Discusses three areas of catalysis: homegeneous, heterogeneous, and enzymatic. Explains fundamentals and economic impact of catalysis. Lists and discusses common industrial catalysts. Provides a list of 107 references. (MVL)

  6. Enzymatic reactions in confined environments.

    PubMed

    Küchler, Andreas; Yoshimoto, Makoto; Luginbühl, Sandra; Mavelli, Fabio; Walde, Peter

    2016-05-05

    Within each biological cell, surface- and volume-confined enzymes control a highly complex network of chemical reactions. These reactions are efficient, timely, and spatially defined. Efforts to transfer such appealing features to in vitro systems have led to several successful examples of chemical reactions catalysed by isolated and immobilized enzymes. In most cases, these enzymes are either bound or adsorbed to an insoluble support, physically trapped in a macromolecular network, or encapsulated within compartments. Advanced applications of enzymatic cascade reactions with immobilized enzymes include enzymatic fuel cells and enzymatic nanoreactors, both for in vitro and possible in vivo applications. In this Review, we discuss some of the general principles of enzymatic reactions confined on surfaces, at interfaces, and inside small volumes. We also highlight the similarities and differences between the in vivo and in vitro cases and attempt to critically evaluate some of the necessary future steps to improve our fundamental understanding of these systems.

  7. Homogeneous, Heterogeneous, and Enzymatic Catalysis.

    ERIC Educational Resources Information Center

    Oyama, S. Ted; Somorjai, Gabor A.

    1988-01-01

    Discusses three areas of catalysis: homegeneous, heterogeneous, and enzymatic. Explains fundamentals and economic impact of catalysis. Lists and discusses common industrial catalysts. Provides a list of 107 references. (MVL)

  8. Enzymatic reactions in confined environments

    NASA Astrophysics Data System (ADS)

    Küchler, Andreas; Yoshimoto, Makoto; Luginbühl, Sandra; Mavelli, Fabio; Walde, Peter

    2016-05-01

    Within each biological cell, surface- and volume-confined enzymes control a highly complex network of chemical reactions. These reactions are efficient, timely, and spatially defined. Efforts to transfer such appealing features to in vitro systems have led to several successful examples of chemical reactions catalysed by isolated and immobilized enzymes. In most cases, these enzymes are either bound or adsorbed to an insoluble support, physically trapped in a macromolecular network, or encapsulated within compartments. Advanced applications of enzymatic cascade reactions with immobilized enzymes include enzymatic fuel cells and enzymatic nanoreactors, both for in vitro and possible in vivo applications. In this Review, we discuss some of the general principles of enzymatic reactions confined on surfaces, at interfaces, and inside small volumes. We also highlight the similarities and differences between the in vivo and in vitro cases and attempt to critically evaluate some of the necessary future steps to improve our fundamental understanding of these systems.

  9. Enzymatic synthesis of organophosphorus compounds

    NASA Astrophysics Data System (ADS)

    Kolodiazhnyi, Oleg I.

    2011-09-01

    Data on biocatalytic methods for the preparation of chiral organophosphorus compounds are generalized and described systematically. Various examples of enzymatic and microbiological synthesis of hydroxyphosphonates, aminophosphonates, phosphinites, phosphine oxides and tertiary phosphines are discussed. The bibliography includes 154 references.

  10. The economic benefits of reducing physical inactivity: an Australian example

    PubMed Central

    2011-01-01

    Background Physical inactivity has major impacts on health and productivity. Our aim was to estimate the health and economic benefits of reducing the prevalence of physical inactivity in the 2008 Australian adult population. The economic benefits were estimated as 'opportunity cost savings', which represent resources utilized in the treatment of preventable disease that are potentially available for re-direction to another purpose from fewer incident cases of disease occurring in communities. Methods Simulation models were developed to show the effect of a 10% feasible, reduction target for physical inactivity from current Australian levels (70%). Lifetime cohort health benefits were estimated as fewer incident cases of inactivity-related diseases; deaths; and Disability Adjusted Life Years (DALYs) by age and sex. Opportunity costs were estimated as health sector cost impacts, as well as paid and unpaid production gains and leisure impacts from fewer disease events associated with reduced physical inactivity. Workforce production gains were estimated by comparing surveyed participation and absenteeism rates of physically active and inactive adults, and valued using the friction cost approach. The impact of an improvement in health status on unpaid household production and leisure time were modeled from time use survey data, as applied to the exposed and non-exposed population subgroups and valued by suitable proxy. Potential costs associated with interventions to increase physical activity were not included. Multivariable uncertainty analyses and univariate sensitivity analyses were undertaken to provide information on the strength of the conclusions. Results A 10% reduction in physical inactivity would result in 6,000 fewer incident cases of disease, 2,000 fewer deaths, 25,000 fewer DALYs and provide gains in working days (114,000), days of home-based production (180,000) while conferring a AUD96 million reduction in health sector costs. Lifetime potential

  11. Recombination hotspots: Models and tools for detection.

    PubMed

    Paul, Prosenjit; Nag, Debjyoti; Chakraborty, Supriyo

    2016-04-01

    Recombination hotspots are the regions within the genome where the rate, and the frequency of recombination are optimum with a size varying from 1 to 2kb. The recombination event is mediated by the double-stranded break formation, guided by the combined enzymatic action of DNA topoisomerase and Spo 11 endonuclease. These regions are distributed non-uniformly throughout the human genome and cause distortions in the genetic map. Numerous lines of evidence suggest that the number of hotspots known in humans has increased manifold in recent years. A few facts about the hotspot evolutions were also put forward, indicating the differences in the hotspot position between chimpanzees and humans. In mice, recombination hot spots were found to be clustered within the major histocompatibility complex (MHC) region. Several models, that help explain meiotic recombination has been proposed. Moreover, scientists also developed some computational tools to locate the hotspot position and estimate their recombination rate in humans is of great interest to population and medical geneticists. Here we reviewed the molecular mechanisms, models and in silico prediction techniques of hot spot residues.

  12. Homologous recombination using bacterial artificial chromosomes.

    PubMed

    Lai, Cary; Fischer, Tobias; Munroe, Elizabeth

    2015-02-02

    This protocol introduces the technique of homologous recombination in bacteria to insert a linear DNA fragment into bacterial artificial chromosomes (BACs). Homologous recombination allows the modification of large DNA molecules, in contrast with conventional restriction endonuclease-based strategies, which cleave large DNAs into numerous fragments and are unlikely to permit the precise targeting afforded by recombination-based approaches. The method uses a phage lambda-derived recombination system (using exo, beta, and gam) as well as other enzymatic activities provided by the host (Escherichia coli). In the method described here, a DNA fragment encoding enhanced cyan fluorescent protein is inserted immediately after the start codon of the gene encoding choline acetyltransferase ("ChAT"), the final enzyme in acetylcholine biosynthesis, using homologous recombination between sequences that are present both on the introduced DNA fragment and in the target BAC. The desired recombination products are identified via positive selection for resistance to kanamycin. In principle, the resulting modified BAC could be used to produce transgenic mice that express this fluorescent protein in cholinergic neurons. The approach described here could be used to insert any DNA fragment.

  13. Watch and Wait Management of Inactive Cystic Echinococcosis - Does the Path to Inactivity Matter - Analysis of a Prospective Patient Cohort.

    PubMed

    Stojkovic, Marija; Rosenberger, Kerstin Daniela; Steudle, Franziska; Junghanss, Thomas

    2016-12-01

    Overdiagnosis and overtreatment are rarely discussed in the context of NTDs despite their relevance for patients under the care of health services with limited resources where the risks of therapy induced complications are often disproportionate to the benefit. The advantages of cyst staging-based management of patients with cystic echinococcosis (CE) are not yet fully explored. Questions are: Do inactive cysts (CE 4 and CE 5) need treatment and is there a difference between cysts which reach CE4 and CE5 naturally or by benzimidazole therapy? Analysis of long-term follow-up data from a prospective CE patient cohort of 223 patients of a national clinical center for echinococcosis. The event of interest "relapse" was defined as the reversal of a cyst from an inactive stage (CE4, CE5) back to an active stage. The watch &wait (ww) group included 30 patients with 46 inactive cysts who never received medical treatment. The benzimidazole-treated (med) group included 15 patients with 17 cysts. There was no relapse in the ww-group whereas 8/17 cysts showed relapse within 18 months after treatment in the med-group. Loss to follow-up was 15.5%. Data from the watch & wait group impressively show how stable naturally inactivated cysts are in contrast to cysts which reach inactivity through treatment with benzimidazoles. A substantial proportion of patients can be spared from treatment through cyst staging. Cysts which inactivated through a natural course do not relapse with very high likelihood. We recommend follow up of 5 years to confirm the stability of the inactive stage. Cysts driven into inactivity through benzimidazole therapy instead need careful monitoring to identify those which reactivate (around 50% within 18 months). 5 years follow-up appears safe to make a final decision on the need for further monitoring.

  14. A catalytically inactive mutant of the deubiquitylase YOD-1 enhances antigen cross-presentation

    PubMed Central

    Sehrawat, Sharvan; Koenig, Paul-Albert; Kirak, Oktay; Schlieker, Christian; Fankhauser, Manuel

    2013-01-01

    Antigen presenting cells (APCs) that express a catalytically inactive version of the deubiquitylase YOD1 (YOD1-C160S) present exogenous antigens more efficiently to CD8+ T cells, both in vitro and in vivo. Compared with controls, immunization of YOD1-C160S mice led to greater expansion of specific CD8+ T cells and showed improved control of infection with a recombinant γ-herpes virus, MHV-68, engineered to express SIINFEKL peptide, the ligand for the ovalbumin-specific TCR transgenic OT-I cells. Enhanced expansion of specific CD8+ T cells was likewise observed on infection of YOD1-C160S mice with a recombinant influenza A virus expressing SIINFEKL. YOD1-C160S APCs retained antigen longer than did control APCs. Enhanced cross-presentation by YOD1-C160S APCs was transporter associated with antigen processing (TAP1)–independent but sensitive to inclusion of inhibitors of acidification and of the proteasome. The activity of deubiquitylating enzymes may thus help control antigen-specific CD8+ T-cell responses during immunization. PMID:23243279

  15. Identification of inactive medications in narrative medical text.

    PubMed

    Breydo, Eugene M; Chu, Julia T; Turchin, Alexander

    2008-11-06

    Discontinued medications are frequently not removed from EMR medication lists - a patient safety risk. We developed an algorithm to identify inactive medications using in the text of narrative notes in the EMR. The algorithm was evaluated against manual review of 297 randomly selected notes. One in five notes documented inactive medications. Sensitivity and precision of 87.7% and 80.7%, respectively, on per-note basis and 66.3% and 80.0%, respectively, on per-medication basis. When medication names missing from the dictionary were excluded, the algorithm achieved sensitivity of 91.4%. Using real clinical data, the algorithm identified inactive medications documented in the note but still listed as active on the patients medication list in more than one in ten notes. Documentation of inactive medications is common in narrative provider notes and can be computationally extracted. This technology could be employed in real-time patient care as well as for research and quality of care monitoring.

  16. 40 CFR 256.25 - Recommendation for inactive facilities.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 26 2013-07-01 2013-07-01 false Recommendation for inactive facilities. 256.25 Section 256.25 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES GUIDELINES FOR DEVELOPMENT AND IMPLEMENTATION OF STATE SOLID WASTE MANAGEMENT PLANS Solid...

  17. 40 CFR 256.25 - Recommendation for inactive facilities.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 24 2010-07-01 2010-07-01 false Recommendation for inactive facilities. 256.25 Section 256.25 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES GUIDELINES FOR DEVELOPMENT AND IMPLEMENTATION OF STATE SOLID WASTE MANAGEMENT PLANS Solid...

  18. 40 CFR 256.25 - Recommendation for inactive facilities.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 25 2011-07-01 2011-07-01 false Recommendation for inactive facilities. 256.25 Section 256.25 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES GUIDELINES FOR DEVELOPMENT AND IMPLEMENTATION OF STATE SOLID WASTE MANAGEMENT PLANS Solid...

  19. 40 CFR 256.25 - Recommendation for inactive facilities.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 26 2012-07-01 2011-07-01 true Recommendation for inactive facilities. 256.25 Section 256.25 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES GUIDELINES FOR DEVELOPMENT AND IMPLEMENTATION OF STATE SOLID WASTE MANAGEMENT PLANS Solid...

  20. 40 CFR 256.25 - Recommendation for inactive facilities.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 25 2014-07-01 2014-07-01 false Recommendation for inactive facilities. 256.25 Section 256.25 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES GUIDELINES FOR DEVELOPMENT AND IMPLEMENTATION OF STATE SOLID WASTE MANAGEMENT PLANS Solid...

  1. Active and inactive faults in southern California viewed from Skylab

    NASA Technical Reports Server (NTRS)

    Merifield, P. M.; Lamar, D. L.

    1975-01-01

    The application is discussed of Skylab imagery along with larger scale photography and field investigations in preparing fault maps of California for use in land use planning. The images were used to assist in distinguishing active from inactive faults (by recognizing indications of recent displacement), determining the length of potentially active faults, identifying previously unmapped faults, and gaining additional information on regional tectonic history.

  2. A New Direction for Wisconsin Inactive Health Personnel.

    ERIC Educational Resources Information Center

    Wisconsin Univ., Madison. Univ. Extension.

    Supported by a contract with the United States Public Health Service, the University of Wisconsin Department of Nursing, University Extension, has provided refresher courses for inactive nurses, on a state-wide basis. This is a report of a survey of enrollees in the 18 courses offered between September 1967 and December 1968. The majority of the…

  3. Inactive Health Personnel Project in New Hampshire. Final Report.

    ERIC Educational Resources Information Center

    New Hampshire Health Careers Council, Concord.

    With the goal of more efficient utilization of existing health manpowers in New Hampshire, data were gathered on inactive medical personnel, and the feasibility of various methods of refresher training was explored. Because of New Hampshire's intrinsic characteristics of climate and scattered population and the scarcity of qualified instructors,…

  4. Home Delivery Medicament Program: access, inactivity and cardiovascular risk 1

    PubMed Central

    Araújo, Roque da Silva; Arcuri, Edna Apparecida Moura; Lopes, Victor Cauê

    2016-01-01

    ABSTRACT Objective: to verify causes of inactivity in the Home Delivery Medicament Program, as referred by users from a Primary Health Care Service in São Paulo, comparing them to the causes registered in the program and analyzing them in the theoretical model Concept of Access to Health. Methods: cross-sectional study, interviewing 111 inactive users; and documentary study in the program records. Results: half of the users did not know the condition of inactivity. Discrepancies were found between the user's and the program's information, observing different levels of agreement: Absence of physician and administrative staff member 0%; Transfer to other service 25%; Death 50%; Option to quit 50%; Address change 57% and Change in therapeutic schedule 80%. The users' feeling of accepting the program was observed. In the health access concept, inactivity can be explained in the information dimension, in the degree of asymmetry between the patient's and the health professional's knowledge, identified through the indicators: education, knowledge and information sources. Conclusions: due to the low education level, the user does not assimilate the information on the steps of the program flowchart, does not return for the assessment that guarantees its continuity. Consequently, (s)he stops receiving the medication and spends a long time without treatment, increasing the cardiovascular risk of hypertensive (92% of the sample), diabetic (44%) and dyslipidemic patients (31%). PMID:27737378

  5. Do Dopaminergic Impairments Underlie Physical Inactivity in People with Obesity?

    PubMed Central

    Kravitz, Alexxai V.; O'Neal, Timothy J.; Friend, Danielle M.

    2016-01-01

    Obesity is associated with physical inactivity, which exacerbates the negative health consequences of obesity. Despite a wide consensus that people with obesity should exercise more, there are few effective methods for increasing physical activity in people with obesity. This lack is reflected in our limited understanding of the cellular and molecular causes of physical inactivity in obesity. We hypothesize that impairments in dopamine signaling contribute to physical inactivity in people with obesity, as in classic movement disorders such as Parkinson's disease. Here, we review two lines of evidence supporting this hypothesis: (1) chronic exposure to obesogenic diets has been linked to impairments in dopamine synthesis, release, and receptor function, particularly in the striatum, and (2) striatal dopamine is necessary for the proper control of movement. Identifying the biological determinants of physical inactivity may lead to more effective strategies for increasing physical activity in people with obesity, as well as improve our understanding of why it is difficult for people with obesity to alter their levels of physical activity. PMID:27790107

  6. 30 CFR 57.5045 - Posting of inactive workings.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Posting of inactive workings. 57.5045 Section 57.5045 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND..., shall be posted against unauthorized entry and designated by signs indicating them as areas in...

  7. 30 CFR 57.5045 - Posting of inactive workings.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Posting of inactive workings. 57.5045 Section 57.5045 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND..., shall be posted against unauthorized entry and designated by signs indicating them as areas in...

  8. Distinct Recycling of Active and Inactive β1 Integrins

    PubMed Central

    Arjonen, Antti; Alanko, Jonna; Veltel, Stefan; Ivaska, Johanna

    2012-01-01

    Integrin trafficking plays an important role in cellular motility and cytokinesis. Integrins undergo constant endo/exocytic shuttling to facilitate the dynamic regulation of cell adhesion. Integrin activity toward the components of the extracellular matrix is regulated by the ability of these receptors to switch between active and inactive conformations. Several cellular signalling pathways have been described in the regulation of integrin traffic under different conditions. However, the interrelationship between integrin activity conformations and their endocytic fate have remained incompletely understood. Here, we have investigated the endocytic trafficking of active and inactive β1 integrins in cancer cells. Both conformers are endocytosed in a clathrin- and dynamin-dependent manner. The net endocytosis rate of the active β1 integrins is higher, whereas endocytosis of the inactive β1 integrin is counteracted by rapid recycling back to the plasma membrane via an ARF 6- and early endosome antigen 1-positive compartment in an Rab 4a- and actin-dependent manner. Owing to these distinct trafficking routes, the two receptor pools display divergent subcellular localization. At steady state, the inactive β1 integrin is mainly on the plasma membrane, whereas the active receptor is predominantly intracellular. These data provide new insights into the endocytic traffic of integrins and imply the possibility of a previously unappreciated crosstalk between pathways regulating integrin activity and traffic. PMID:22222055

  9. Physical Inactivity, Obesity, and Type 2 Diabetes: An Evolutionary Perspective

    ERIC Educational Resources Information Center

    Eaton, S. Boyd; Eaton, Stanley B.

    2017-01-01

    Physical inactivity (and unhealthy nutrition) has distorted body composition and, in turn, reordered the proportions of myocyte and adipocyte insulin receptors. Insulin acting on adipocyte receptors produces less glucose uptake than does comparable interaction with myocyte receptors. Accordingly, in individuals with disproportionate muscle/fat…

  10. Influence of magnetic field on enzymatic ONOO- production

    NASA Astrophysics Data System (ADS)

    Dranova, T.; Petrovskii, D.; Ershov, N.; Slepneva, I.; Stass, D.

    2017-08-01

    Enzymatic oxidation of L-arginine catalyzed by inducible nitric oxide synthase gives nitric oxide as the main product and superoxide anion as a side reaction product. Recombination of these radicals gives a very reactive species - peroxynitrite, which is involved in many biochemical processes. In the current work it was shown that such a system can be a usable model system for investigating the influence of magnetic field on enzymatic peroxynitrite formation. Using a selective fluorescent probe for peroxynitrite - coumarin boronic acid and an adopted for the experimental purpose incubation mixture, magnetic field experiments have been done at 11.7T. The averaged magnetic field effect is equal to 2.8±0.9%.

  11. Muscle activity and inactivity periods during normal daily life.

    PubMed

    Tikkanen, Olli; Haakana, Piia; Pesola, Arto J; Häkkinen, Keijo; Rantalainen, Timo; Havu, Marko; Pullinen, Teemu; Finni, Taija

    2013-01-01

    Recent findings suggest that not only the lack of physical activity, but also prolonged times of sedentary behaviour where major locomotor muscles are inactive, significantly increase the risk of chronic diseases. The purpose of this study was to provide details of quadriceps and hamstring muscle inactivity and activity during normal daily life of ordinary people. Eighty-four volunteers (44 females, 40 males, 44.1±17.3 years, 172.3±6.1 cm, 70.1±10.2 kg) were measured during normal daily life using shorts measuring muscle electromyographic (EMG) activity (recording time 11.3±2.0 hours). EMG was normalized to isometric MVC (EMG(MVC)) during knee flexion and extension, and inactivity threshold of each muscle group was defined as 90% of EMG activity during standing (2.5±1.7% of EMG(MVC)). During normal daily life the average EMG amplitude was 4.0±2.6% and average activity burst amplitude was 5.8±3.4% of EMG(MVC) (mean duration of 1.4±1.4 s) which is below the EMG level required for walking (5 km/h corresponding to EMG level of about 10% of EMG(MVC)). Using the proposed individual inactivity threshold, thigh muscles were inactive 67.5±11.9% of the total recording time and the longest inactivity periods lasted for 13.9±7.3 min (2.5-38.3 min). Women had more activity bursts and spent more time at intensities above 40% EMG(MVC) than men (p<0.05). In conclusion, during normal daily life the locomotor muscles are inactive about 7.5 hours, and only a small fraction of muscle's maximal voluntary activation capacity is used averaging only 4% of the maximal recruitment of the thigh muscles. Some daily non-exercise activities such as stair climbing produce much higher muscle activity levels than brisk walking, and replacing sitting by standing can considerably increase cumulative daily muscle activity.

  12. Human macrophages produce dimeric forms of IL-18 which can be detected with monoclonal antibodies specific for inactive IL-18.

    PubMed

    Kikkawa, S; Matsumoto, M; Shida, K; Fukumori, Y; Toyoshima, K; Seya, T

    2001-02-23

    We established two monoclonal antibodies (mAbs) which specifically recognize human 'functionally inactive' recombinant IL-18, and IL-18 protein polymorphism was examined using human monocytes and macrophages (M phi). In 6 day GM-CSF-treated M phi, an 'inactive' IL-18-recognizing mAb 21 detected the IL-18 proform (24 kDa) and a 48-kDa protein, which were gradually increased concomitant with maturation stage. Majority of the 24- and 48-kDa forms were barely detectable with other mAbs recognizing 'active' IL-18. No reagents including Toll stimulators up-regulated these IL-18 populations in M phi. The 21-recognizable IL-18 species were separated using an anion-exchanger column and their IFN gamma-inducing activity was assessed with human lymphocytes plus IL-12. Virtually no as yet known activity was detected with these IL-18 species. After processed with M phi proteases, an 18-kDa form was generated to express the IFN gamma-inducing activity, although the activity was far weaker than that of control 'active' IL-18. These observations suggested that large amounts of various IL-18 species are produced with monocyte-M phi differentiation and most of these IL-18 species are functionally 'inactive' in terms of the reported IL-18 function even after proteolytic 18-kDa conversion.

  13. Enzymatic Hydrolysis of Cellulosic Biomass

    SciTech Connect

    Yang, Bin; Dai, Ziyu; Ding, Shi-You; Wyman, Charles E.

    2011-08-22

    Biological conversion of cellulosic biomass to fuels and chemicals offers the high yields to products vital to economic success and the potential for very low costs. Enzymatic hydrolysis that converts lignocellulosic biomass to fermentable sugars may be the most complex step in this process due to substrate-related and enzyme-related effects and their interactions. Although enzymatic hydrolysis offers the potential for higher yields, higher selectivity, lower energy costs, and milder operating conditions than chemical processes, the mechanism of enzymatic hydrolysis and the relationship between the substrate structure and function of various glycosyl hydrolase components are not well understood. Consequently, limited success has been realized in maximizing sugar yields at very low cost. This review highlights literature on the impact of key substrate and enzyme features that influence performance to better understand fundamental strategies to advance enzymatic hydrolysis of cellulosic biomass for biological conversion to fuels and chemicals. Topics are summarized from a practical point of view including characteristics of cellulose (e.g., crystallinity, degree of polymerization, and accessible surface area) and soluble and insoluble biomass components (e.g., oligomeric xylan, lignin, etc.) released in pretreatment, and their effects on the effectiveness of enzymatic hydrolysis. We further discuss the diversity, stability, and activity of individual enzymes and their synergistic effects in deconstructing complex lignocellulosic biomass. Advanced technologies to discover and characterize novel enzymes and to improve enzyme characteristics by mutagenesis, post-translational modification, and over-expression of selected enzymes and modifications in lignocellulosic biomass are also discussed.

  14. Macrophage migration inhibitory factor (MIF) is rendered enzymatically inactive by myeloperoxidase-derived oxidants but retains its immunomodulatory function.

    PubMed

    Dickerhof, Nina; Schindler, Lisa; Bernhagen, Jürgen; Kettle, Anthony J; Hampton, Mark B

    2015-12-01

    Macrophage migration inhibitory factor (MIF) is an important player in the regulation of the inflammatory response. Elevated plasma MIF is found in sepsis, arthritis, cystic fibrosis and atherosclerosis. Immunomodulatory activities of MIF include the ability to promote survival and recruitment of inflammatory cells and to amplify pro-inflammatory cytokine production. MIF has an unusual nucleophilic N-terminal proline with catalytic tautomerase activity. It remains unclear whether tautomerase activity is required for MIF function, but small molecules that inhibit tautomerase activity also inhibit the pro-inflammatory activities of MIF. A prominent feature of the acute inflammatory response is neutrophil activation and production of reactive oxygen species, including myeloperoxidase (MPO)-derived hypochlorous acid and hypothiocyanous acid. We hypothesized that MPO-derived oxidants would oxidize the N-terminal proline of MIF and alter its biological activity. MIF was exposed to hypochlorous acid and hypothiocyanous acid and the oxidative modifications on MIF were examined by LC-MS/MS. Imine formation and carbamylation was observed on the N-terminal proline in response to MPO-dependent generation of hypochlorous and hypothiocyanous acid, respectively. These modifications led to a complete loss of tautomerase activity. However, modified MIF still increased CXCL-8/IL-8 production by peripheral blood mononuclear cells (PBMCs) and blocked neutrophil apoptosis, indicating that tautomerase activity is not essential for these biological functions. Pre-treatment of MIF with hypochlorous acid protected the protein from covalent modification by the MIF inhibitor 4-iodo-6-phenylpyrimidine (4-IPP). Therefore, oxidant generation at inflammatory sites may protect MIF from inactivation by more disruptive electrophiles, including drugs designed to target the tautomerase activity of MIF.

  15. Reduced Aerobic Capacity and Quality of Life in Physically Inactive Patients With Systemic Lupus Erythematosus With Mild or Inactive Disease.

    PubMed

    Pinto, Ana J; Miyake, Cintia N H; Benatti, Fabiana B; Silva, Clovis A; Sallum, Adriana M E; Borba, Eduardo; de Sá-Pinto, Ana L; Bonfá, Eloisa; Gualano, Bruno

    2016-12-01

    To compare aerobic capacity and health-related quality of life (HRQOL) in physically inactive adult systemic lupus erythematosus (A-SLE) and childhood-onset systemic lupus erythematosus (C-SLE) patients with mild/inactive disease versus healthy controls. In a cross-sectional study, 39 patients (C-SLE: n = 18, ages 9-18 years; and A-SLE: n = 21, ages 23-45 years) with inactive disease activity (Systemic Lupus Erythematosus Disease Activity Index ≤4) and low cumulative damage (Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index ≤2) and 30 healthy controls (15 children and adolescents [C-CTRL], 15 adults [A-CTRL]) matched by physical inactivity, age, sex, and body mass index (BMI) were assessed for aerobic capacity and HRQOL. All participants were considered physically inactive according to physical activity guidelines. C-SLE and A-SLE patients showed lower VO2peak (95% CI confidence interval [95% CI] -10.5, -1.2 and -11.1, -3.8, respectively) and higher time-to-exhaustion when compared with C-CTRL and A-CTRL (95% CI -2.8, 0.1 and -3.9, -1.7, respectively). C-SLE patients showed significantly lower scores in scholar functioning from the Pediatric Quality of Life Inventory questionnaire (P < 0.05) whereas A-SLE patients showed lower scores in most domains of the Short Form 36 health survey questionnaire (physical function, role-physical, bodily pain, general health, vitality, social function, and mental health) when compared with healthy controls (P < 0.05 for all). Our study provides novel data suggesting that A-SLE and C-SLE patients with mild/inactive disease have impaired aerobic capacity and HRQOL when compared with controls matched by physical inactivity, age, sex, and BMI. These findings reinforce the recommendation of physical activity in SLE treatment. © 2016, American College of Rheumatology.

  16. Enzymatic properties of a GH19 chitinase isolated from rice lacking a major loop structure involved in chitin binding.

    PubMed

    Tanaka, Jun; Fukamizo, Tamo; Ohnuma, Takayuki

    2017-05-01

    The catalytic domains of family GH19 chitinases have been found to consist of a conserved, α-helical core-region and different numbers (1-6) of loop structures, located at both ends of the substrate-binding groove and which extend over the glycon- and aglycon-binding sites. We expressed, purified and enzymatically characterized a GH19 chitinase from rice, Oryza sativa L. cv. Nipponbare (OsChia2a), lacking a major loop structure (loop III) connected to the functionally important β-stranded region. The new enzyme thus contained the five remaining loop structures (loops I, II, IV, V and C-term). The OsChia2a recombinant protein catalyzed hydrolysis of chitin oligosaccharides, (GlcNAc)n (n = 3-6), with inversion of anomeric configuration, indicating that OsChia2a correctly folded without loop III. From thermal unfolding experiments and calorimetric titrations using the inactive OsChia2a mutant (OsChia2a-E68Q), in which the catalytic residue Glu68 was mutated to glutamine, we found that the binding affinities towards (GlcNAc)n (n = 2-6) were almost proportional to the degree of polymerization of (GlcNAc)n, but were much lower than those obtained for a moss GH19 chitinase having only loop III [Ohnuma T, Sørlie M, Fukuda T, Kawamoto N, Taira T, Fukamizo T. 2011. Chitin oligosaccharide binding to a family GH19 chitinase from the moss, Bryum coronatum. FEBS J. 278:3991-4001]. Nevertheless, OsChia2a exhibited significant antifungal activity. It appears that loop III connected to the β-stranded region is important for (GlcNAc)n binding, but is not essential for antifungal activity. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. 200-Area plateau inactive miscellaneous underground storage tanks locations

    SciTech Connect

    Brevick, C.H.

    1997-12-01

    Fluor Daniel Northwest (FDNW) has been tasked by Lockheed Martin Hanford Corporation (LMHC) to incorporate current location data for 64 of the 200-Area plateau inactive miscellaneous underground storage tanks (IMUST) into the centralized mapping computer database for the Hanford facilities. The IMUST coordinate locations and tank names for the tanks currently assigned to the Hanford Site contractors are listed in Appendix A. The IMUST are inactive tanks installed in underground vaults or buried directly in the ground within the 200-East and 200-West Areas of the Hanford Site. The tanks are categorized as tanks with a capacity of less than 190,000 liters (50,000 gal). Some of the IMUST have been stabilized, pumped dry, filled with grout, or may contain an inventory or radioactive and/or hazardous materials. The IMUST have been out of service for at least 12 years.

  18. Bipartite structure of the inactive mouse X chromosome.

    PubMed

    Deng, Xinxian; Ma, Wenxiu; Ramani, Vijay; Hill, Andrew; Yang, Fan; Ay, Ferhat; Berletch, Joel B; Blau, Carl Anthony; Shendure, Jay; Duan, Zhijun; Noble, William S; Disteche, Christine M

    2015-08-07

    In mammals, one of the female X chromosomes and all imprinted genes are expressed exclusively from a single allele in somatic cells. To evaluate structural changes associated with allelic silencing, we have applied a recently developed Hi-C assay that uses DNase I for chromatin fragmentation to mouse F1 hybrid systems. We find radically different conformations for the two female mouse X chromosomes. The inactive X has two superdomains of frequent intrachromosomal contacts separated by a boundary region. Comparison with the recently reported two-superdomain structure of the human inactive X shows that the genomic content of the superdomains differs between species, but part of the boundary region is conserved and located near the Dxz4/DXZ4 locus. In mouse, the boundary region also contains a minisatellite, Ds-TR, and both Dxz4 and Ds-TR appear to be anchored to the nucleolus. Genes that escape X inactivation do not cluster but are located near the periphery of the 3D structure, as are regions enriched in CTCF or RNA polymerase. Fewer short-range intrachromosomal contacts are detected for the inactive alleles of genes subject to X inactivation compared with the active alleles and with genes that escape X inactivation. This pattern is also evident for imprinted genes, in which more chromatin contacts are detected for the expressed allele. By applying a novel Hi-C method to map allelic chromatin contacts, we discover a specific bipartite organization of the mouse inactive X chromosome that probably plays an important role in maintenance of gene silencing.

  19. Investigation of the organic matter in inactive nuclear tank liquids

    SciTech Connect

    Schenley, R.L.; Griest, W.H.

    1990-08-01

    Environmental Protection Agency (EPA) methodology for regulatory organics fails to account for the organic matter that is suggested by total organic carbon (TOC) analysis in the Oak Ridge National Laboratory (ORNL) inactive nuclear waste-tank liquids and sludges. Identification and measurement of the total organics are needed to select appropriate waste treatment technologies. An initial investigation was made of the nature of the organics in several waste-tank liquids. This report details the analysis of ORNL wastes.

  20. Physical inactivity: the "Cinderella" risk factor for noncommunicable disease prevention.

    PubMed

    Bull, Fiona C; Bauman, Adrian E

    2011-08-01

    There is strong evidence demonstrating the direct and indirect pathways by which physical activity prevents many of the major noncommunicable diseases (NCD) responsible for premature death and disability. Physical inactivity was identified as the 4th leading risk factor for the prevention of NCD, preceded only by tobacco use, hypertension, and high blood glucose levels, and accounting for more than 3 million preventable deaths globally in 2010. Physical inactivity is a global public health priority but, in most countries, this has not yet resulted in widespread recognition nor specific physical activity-related policy action at the necessary scale. Instead, physical inactivity could be described as the Cinderella of NCD risk factors, defined as "poverty of policy attention and resourcing proportionate to its importance." The pressing question is "Why is this so?" The authors identify and discuss 8 possible explanations and the need for more effective communication on the importance of physical activity in the NCD prevention context. Although not all of the issues identified will be relevant for any 1 country, it is likely that at different times and in different combinations these 8 problems continue to delay national-level progress on addressing physical inactivity in many countries. The authors confirm that there is sufficient evidence to act, and that much better use of well-planned, coherent communication strategies are needed in most countries and at the international level. Significant opportunities exist. The Toronto Charter on Physical Activity and the Seven Investments that Work are 2 useful tools to support increased advocacy on physical activity within and beyond the context of the crucial 2011 UN High-Level Meeting on NCDs.

  1. Inactivity amplifies the catabolic response of skeletal muscle to cortisol

    NASA Technical Reports Server (NTRS)

    Ferrando, A. A.; Stuart, C. A.; Sheffield-Moore, M.; Wolfe, R. R.

    1999-01-01

    Severe injury or trauma is accompanied by both hypercortisolemia and prolonged inactivity or bed rest (BR). Trauma and BR alone each result in a loss of muscle nitrogen, albeit through different metabolic alterations. Although BR alone can result in a 2-3% loss of lean body mass, the effects of severe trauma can be 2- to 3-fold greater. We investigated the combined effects of hypercortisolemia and prolonged inactivity on muscle protein metabolism in healthy volunteers. Six males were studied before and after 14 days of strict BR using a model based on arteriovenous sampling and muscle biopsy. Fractional synthesis and breakdown rates of skeletal muscle protein were also directly calculated. Each assessment of protein metabolism was conducted during a 12-h infusion of hydrocortisone sodium succinate (120 microg/kg x h), resulting in blood cortisol concentrations that mimic severe injury (approximately 31 microg/dL). After 14 days of strict BR, hypercortisolemia increased phenylalanine efflux from muscle by 3-fold (P < 0.05). The augmented negative amino acid balance was the result of an increased muscle protein breakdown (P < 0.05) without a concomitant change in muscle protein synthesis. Muscle efflux of glutamine and alanine increased significantly after bed rest due to a significant increase in de novo synthesis (P < 0.05). Thus, inactivity sensitizes skeletal muscle to the catabolic effects of hypercortisolemia. Furthermore, these effects on healthy volunteers are analogous to those seen after severe injury.

  2. Inactivity amplifies the catabolic response of skeletal muscle to cortisol

    NASA Technical Reports Server (NTRS)

    Ferrando, A. A.; Stuart, C. A.; Sheffield-Moore, M.; Wolfe, R. R.

    1999-01-01

    Severe injury or trauma is accompanied by both hypercortisolemia and prolonged inactivity or bed rest (BR). Trauma and BR alone each result in a loss of muscle nitrogen, albeit through different metabolic alterations. Although BR alone can result in a 2-3% loss of lean body mass, the effects of severe trauma can be 2- to 3-fold greater. We investigated the combined effects of hypercortisolemia and prolonged inactivity on muscle protein metabolism in healthy volunteers. Six males were studied before and after 14 days of strict BR using a model based on arteriovenous sampling and muscle biopsy. Fractional synthesis and breakdown rates of skeletal muscle protein were also directly calculated. Each assessment of protein metabolism was conducted during a 12-h infusion of hydrocortisone sodium succinate (120 microg/kg x h), resulting in blood cortisol concentrations that mimic severe injury (approximately 31 microg/dL). After 14 days of strict BR, hypercortisolemia increased phenylalanine efflux from muscle by 3-fold (P < 0.05). The augmented negative amino acid balance was the result of an increased muscle protein breakdown (P < 0.05) without a concomitant change in muscle protein synthesis. Muscle efflux of glutamine and alanine increased significantly after bed rest due to a significant increase in de novo synthesis (P < 0.05). Thus, inactivity sensitizes skeletal muscle to the catabolic effects of hypercortisolemia. Furthermore, these effects on healthy volunteers are analogous to those seen after severe injury.

  3. Bioluminescence methods for enzymatic determinations

    SciTech Connect

    Bostick, W.D.; Denton, M.S.; Dinsmore, S.R.

    1982-11-02

    An enzymatic method for continuous, on-line and rapid detection of diagnostically useful biomarkers, which are symptomatic of disease or trauma-related tissue damage, is disclosed. The method is characterized by operability on authentic samples of complex biological fluids which contain the biomarkers.

  4. Bioluminescence methods for enzymatic determinations

    DOEpatents

    Bostick, William D.; Denton, Mark S.; Dinsmore, Stanley R.

    1982-01-01

    An enzymatic method for continuous, on-line and rapid detection of diagnostically useful biomarkers, which are symptomatic of disease or trauma-related tissue damage, is disclosed. The method is characterized by operability on authentic samples of complex biological fluids which contain the biomarkers.

  5. Multifractality in intracellular enzymatic reactions.

    PubMed

    Aranda, Juan S; Salgado, Edgar; Muñoz-Diosdado, Alejandro

    2006-05-21

    Enzymatic kinetics adjust well to the Michaelis-Menten paradigm in homogeneous media with dilute, perfectly mixed reactants. These conditions are quite different from the highly structured cell plasm, so applications of the classic kinetics theory to this environment are rather limited. Cytoplasmic structure produces molecular crowding and anomalous diffusion of substances, modifying the mass action kinetic laws. The reaction coefficients are no longer constant but time-variant, as stated in the fractal kinetics theory. Fractal kinetics assumes that enzymatic reactions on such heterogeneous media occur within a non-Euclidian space characterized by a certain fractal dimension, this fractal dimension gives the dependence on time of the kinetic coefficients. In this work, stochastic simulations of enzymatic reactions under molecular crowding have been completed, and kinetic coefficients for the reactions, including the Michaelis-Menten parameter KM, were calculated. The simulations results led us to confirm the time dependence of michaelian kinetic parameter for the enzymatic catalysis. Besides, other chaos related phenomena were pointed out from the obtained KM time series, such as the emergence of strange attractors and multifractality.

  6. Enzymatic reactions on immobilised substrates.

    PubMed

    Gray, Christopher J; Weissenborn, Martin J; Eyers, Claire E; Flitsch, Sabine L

    2013-08-07

    This review gives an overview of enzymatic reactions that have been conducted on substrates attached to solid surfaces. Such biochemical reactions have become more important with the drive to miniaturisation and automation in chemistry, biology and medicine. Technical aspects such as choice of solid surface and analytical methods are discussed and examples of enzyme reactions that have been successful on these surfaces are provided.

  7. Enzymatic hydrolysis of organic phosphorus

    USDA-ARS?s Scientific Manuscript database

    Orthophosphate-releasing enzymatic hydrolysis is an alternative means for characterizing organic phosphorus (Po) in animal manure. The approach is not only simple and fast, but can also provide information difficult to obtain by other methods. Currently, commercially available phosphatases are mainl...

  8. Enzymatic Activity of Xyloglucan Xylosyltransferase 51[OPEN

    PubMed Central

    Culbertson, Alan T.; Chou, Yi-Hsiang; Smith, Adrienne L.; Young, Zachary T.; Tietze, Alesia A.; Cottaz, Sylvain

    2016-01-01

    Xyloglucan, the most abundant hemicellulosic component of the primary cell wall of flowering plants, is composed of a β-(1,4)-glucan backbone decorated with d-xylosyl residues. Three xyloglucan xylosyltransferases (XXTs) participate in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana). Two of these, XXT1 and XXT2, have been shown to be active in vitro, whereas the catalytic activity of XXT5 has yet to be demonstrated. By optimizing XXT2 expression in a prokaryotic system and in vitro activity assay conditions, we demonstrate that nonglycosylated XXT2 lacking its cytosolic amino-terminal and transmembrane domain displays high catalytic activity. Using this optimized procedure for the expression of XXT5, we report, to our knowledge for the first time, that recombinant XXT5 shows enzymatic activity in vitro, although at a significantly slower rate than XXT1 and XXT2. Kinetic analysis showed that XXT5 has a 7-fold higher Km and 9-fold lower kcat compared with XXT1 and XXT2. Activity assays using XXT5 in combination with XXT1 or XXT2 indicate that XXT5 is not specific for their products. In addition, mutagenesis experiments showed that the in vivo function and in vitro catalytic activity of XXT5 require the aspartate-serine-aspartate motif. These results demonstrate that XXT5 is a catalytically active xylosyltransferase involved in xylosylation of the xyloglucan backbone. PMID:27208276

  9. Sucrase-alpha-dextrinase in the rat. Postinsertional conversion to inactive molecular species by a carbohydrate-free diet.

    PubMed Central

    Quan, R; Gray, G M

    1993-01-01

    Absence of dietary carbohydrate decreases both activities of intestinal brush border sucrase-alpha-dextrinase. We examined the molecular mechanism causing this decrease. Adult rats were fed chow (70% CHO) or matched carbohydrate-free (CHO-free) diet for 7 d. Sucrase activity decreased by 50% in whole homogenates and brush borders. Enzyme kinetics revealed no change in sucrose affinity (CHO-free Km = 18 mM, chow Km = 21 mM), but fewer active sites (CHO-free Vmax = 2,720, chow Vmax = 5,000 mumol/min per g protein). Intraintestinal pulse-labeling of [35S]methionine in vivo revealed no differences in incorporation into sucrase. Immunoreactive sucrase protein, assayed by ELISA and rocket immunoelectrophoresis, increased twofold per milliunit of sucrase enzymatic activity in CHO-free jejunum. Total immunosucrase (St), the sum of active and inactive enzyme (St = Sa+Si), was unchanged with carbohydrate withdrawal, but > 50% of the sucrase protein became inactive. SDS-PAGE of sucrase immunoprecipitates revealed alteration of alpha, beta, and gamma subunits in CHO-free animals: (a) alpha and beta subunits migrated farther (mass change--2 kD); and (b) the alpha subunit became diffuse or was a doublet and was less abundant than the beta subunit. Rather than representing loss of sucrase protein, the decline in sucrase activity is achieved with structural subunit changes, probably involving postinsertional processing. Images PMID:8514885

  10. ERICA: leisure-time physical inactivity in Brazilian adolescents

    PubMed Central

    Cureau, Felipe Vogt; da Silva, Thiago Luiz Nogueira; Bloch, Katia Vergetti; Fujimori, Elizabeth; Belfort, Dilson Rodrigues; de Carvalho, Kênia Mara Baiocchi; de Leon, Elisa Brosina; de Vasconcellos, Mauricio Teixeira Leite; Ekelund, Ulf; Schaan, Beatriz D

    2016-01-01

    ABSTRACT OBJECTIVE To evaluate the prevalence of leisure-time physical inactivity in Brazilian adolescents and their association with geographical and sociodemographic variables. METHODS The sample was composed by 74,589 adolescents participating in the Study of Cardiovascular Risks in Adolescents (ERICA). This cross-sectional study of school basis with national scope involved adolescents aged from 12 to 17 years in Brazilian cities with more than 100 thousand inhabitants. The prevalence of leisure-time physical inactivity was categorized according to the volume of weekly practice (< 300; 0 min). The prevalences were estimated for the total sample and by sex. Poisson regression models were used to assess associated factors. RESULTS The prevalence of leisure-time physical inactivity was 54.3% (95%CI 53.4-55.2), and higher for the female sex (70.7%, 95%CI 69.5-71.9) compared to the male (38.0%, 95%CI 36.7-39.4). More than a quarter of adolescents (26.5%, 95%CI 25.8-27.3) reported not practicing physical activity in the leisure time, a condition more prevalent for girls (39.8%, 95%CI 38.8-40.9) than boys (13.4%, 95%CI 12.4-14.4). For girls, the variables that were associated with physical inactivity were: reside in the Northeast (RP = 1.13, 95%CI 1.08-1.19), Southeast (RP = 1.16, 95%CI 1.11-1.22) and South (RP = 1.12, 95%CI 1.06-1.18); have 16-17 years (RP = 1.06, 95%CI 1.12-1.15); and belong to the lower economic class (RP = 1.33, 95%CI 1.20-1.48). The same factors, except reside in the Southeast and South, were also associated with not practicing physical activity in the leisure time for the same group. In males, as well as the region, being older (p < 0.001) and declaring to be indigenous (RP = 0.37, 95%CI 0.19-0.73) were also associated with not practicing physical activities in the leisure time. CONCLUSIONS The prevalence of leisure-time physical inactivity in Brazilian adolescents is high. It presents regional variations and is associated with age and low

  11. Who needs 'lazy' workers? Inactive workers act as a 'reserve' labor force replacing active workers, but inactive workers are not replaced when they are removed.

    PubMed

    Charbonneau, Daniel; Sasaki, Takao; Dornhaus, Anna

    2017-01-01

    Social insect colonies are highly successful, self-organized complex systems. Surprisingly however, most social insect colonies contain large numbers of highly inactive workers. Although this may seem inefficient, it may be that inactive workers actually contribute to colony function. Indeed, the most commonly proposed explanation for inactive workers is that they form a 'reserve' labor force that becomes active when needed, thus helping mitigate the effects of colony workload fluctuations or worker loss. Thus, it may be that inactive workers facilitate colony flexibility and resilience. However, this idea has not been empirically confirmed. Here we test whether colonies of Temnothorax rugatulus ants replace highly active (spending large proportions of time on specific tasks) or highly inactive (spending large proportions of time completely immobile) workers when they are experimentally removed. We show that colonies maintained pre-removal activity levels even after active workers were removed, and that previously inactive workers became active subsequent to the removal of active workers. Conversely, when inactive workers were removed, inactivity levels decreased and remained lower post-removal. Thus, colonies seem to have mechanisms for maintaining a certain number of active workers, but not a set number of inactive workers. The rapid replacement (within 1 week) of active workers suggests that the tasks they perform, mainly foraging and brood care, are necessary for colony function on short timescales. Conversely, the lack of replacement of inactive workers even 2 weeks after their removal suggests that any potential functions they have, including being a 'reserve', are less important, or auxiliary, and do not need immediate recovery. Thus, inactive workers act as a reserve labor force and may still play a role as food stores for the colony, but a role in facilitating colony-wide communication is unlikely. Our results are consistent with the often cited, but never

  12. 37 CFR 11.20 - Disciplinary sanctions; Transfer to disability inactive status.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...; Transfer to disability inactive status. 11.20 Section 11.20 Patents, Trademarks, and Copyrights UNITED..., Investigations, and Proceedings § 11.20 Disciplinary sanctions; Transfer to disability inactive status. (a) Types... practitioner take and pass a professional responsibility examination. (c) Transfer to disability inactive...

  13. 38 CFR 3.372 - Initial grant following inactivity of tuberculosis.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... inactivity of tuberculosis. 3.372 Section 3.372 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF... Considerations Relative to Specific Diseases § 3.372 Initial grant following inactivity of tuberculosis. When... tuberculosis and there is satisfactory evidence that the condition was active previously but is now inactive...

  14. 38 CFR 3.372 - Initial grant following inactivity of tuberculosis.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... inactivity of tuberculosis. 3.372 Section 3.372 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF... Considerations Relative to Specific Diseases § 3.372 Initial grant following inactivity of tuberculosis. When... tuberculosis and there is satisfactory evidence that the condition was active previously but is now inactive...

  15. 38 CFR 3.372 - Initial grant following inactivity of tuberculosis.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... inactivity of tuberculosis. 3.372 Section 3.372 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF... Considerations Relative to Specific Diseases § 3.372 Initial grant following inactivity of tuberculosis. When... tuberculosis and there is satisfactory evidence that the condition was active previously but is now inactive...

  16. 38 CFR 3.372 - Initial grant following inactivity of tuberculosis.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... inactivity of tuberculosis. 3.372 Section 3.372 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF... Considerations Relative to Specific Diseases § 3.372 Initial grant following inactivity of tuberculosis. When... tuberculosis and there is satisfactory evidence that the condition was active previously but is now inactive...

  17. 38 CFR 3.372 - Initial grant following inactivity of tuberculosis.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... inactivity of tuberculosis. 3.372 Section 3.372 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF... Considerations Relative to Specific Diseases § 3.372 Initial grant following inactivity of tuberculosis. When... tuberculosis and there is satisfactory evidence that the condition was active previously but is now inactive...

  18. 37 CFR 11.20 - Disciplinary sanctions; Transfer to disability inactive status.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...; Transfer to disability inactive status. 11.20 Section 11.20 Patents, Trademarks, and Copyrights UNITED..., Investigations, and Proceedings § 11.20 Disciplinary sanctions; Transfer to disability inactive status. (a) Types... circumstances. (c) Transfer to disability inactive status. The USPTO Director, after notice and opportunity...

  19. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  20. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  1. Expression and activity of recombinant proaerolysin derived from Aeromonas hydrophila cultured from diseased channel catfish

    USDA-ARS?s Scientific Manuscript database

    Proaerolysin-coding gene was cloned from the genomic DNA of A. hydrophila and heterologously expressed in E. coli. The purified recombinant proaerolysin was inactive and could be activated by treatment with proteases, furin and trypsin, and extra-cellular proteins (ECPs, the cell-free supernatant of...

  2. Permanent recording of light helicity on optically inactive metal surfaces.

    PubMed

    Wang, Jincheng; Guo, Chunlei

    2006-12-15

    We report on an unusual permanent recording of light helicity on optically achiral metals. Following a number of circularly polarized (CP) or elliptically polarized (EP) femtosecond laser pulses, well-defined periodic surface structures are found on metal surfaces. These surface structures show different orientation when formed by left CP/EP compared with right CP/EP light. The formation of these structures is attributed to the interference between the incident light and the excited surface plasmons. To our knowledge, this is the only phenomenon that can permanently record light helicity with an optically inactive material.

  3. Recent developments in enzymatic chlorination.

    PubMed

    Murphy, Cormac D

    2006-04-01

    While the existence of chlorinated natural products has been known for over 100 years, our understanding of the enzymology of biological chlorination reactions has been limited to chloroperoxidases, which are now known not to play a significant role in chlorometabolite biosynthesis. The discoveries of new classes of halogenases, described in this Highlight, have shed new light on the mechanisms of enzymatic chlorination of aromatic and aliphatic compounds.

  4. Enzymatic synthesis of prebiotic oligosaccharides.

    PubMed

    Rabelo, Maria C; Honorato, Talita L; Gonçalves, Luciana R B; Pinto, Gustavo A S; Rodrigues, Sueli

    2006-04-01

    Prebiotic oligosaccharides are nondigestible carbohydrates that can be obtained by enzymatic synthesis. Glucosyltransferases can be used to produce these carbohydrates through an acceptor reaction synthesis. When maltose is the acceptor a trisaccharide composed of one maltose unit and one glucose unit linked by an alpha-1,6-glycosidic bond (panose) is obtained as the primer product of the dextransucrase acceptor reaction. In this work, panose enzymatic synthesis was evaluated by a central composite experimental design in which maltose and sucrose concentration were varied in a wide range of maltose/sucrose ratios in a batch reactor system. A partially purified enzyme was used in order to reduce the process costs, because enzyme purification is one of the most expensive steps in enzymatic synthesis. Even using high maltose/sucrose ratios, dextran and higher-oligosaccharide formation were not avoided. The results showed that intermediate concentrations of sucrose and high maltose concentration resulted in high panose productivity with low dextran and higher-oligosaccharide productivity.

  5. Enzymatic Processes in Marine Biotechnology.

    PubMed

    Trincone, Antonio

    2017-03-25

    In previous review articles the attention of the biocatalytically oriented scientific community towards the marine environment as a source of biocatalysts focused on the habitat-related properties of marine enzymes. Updates have already appeared in the literature, including marine examples of oxidoreductases, hydrolases, transferases, isomerases, ligases, and lyases ready for food and pharmaceutical applications. Here a new approach for searching the literature and presenting a more refined analysis is adopted with respect to previous surveys, centering the attention on the enzymatic process rather than on a single novel activity. Fields of applications are easily individuated: (i) the biorefinery value-chain, where the provision of biomass is one of the most important aspects, with aquaculture as the prominent sector; (ii) the food industry, where the interest in the marine domain is similarly developed to deal with the enzymatic procedures adopted in food manipulation; (iii) the selective and easy extraction/modification of structurally complex marine molecules, where enzymatic treatments are a recognized tool to improve efficiency and selectivity; and (iv) marine biomarkers and derived applications (bioremediation) in pollution monitoring are also included in that these studies could be of high significance for the appreciation of marine bioprocesses.

  6. Enzymatic Processes in Marine Biotechnology

    PubMed Central

    Trincone, Antonio

    2017-01-01

    In previous review articles the attention of the biocatalytically oriented scientific community towards the marine environment as a source of biocatalysts focused on the habitat-related properties of marine enzymes. Updates have already appeared in the literature, including marine examples of oxidoreductases, hydrolases, transferases, isomerases, ligases, and lyases ready for food and pharmaceutical applications. Here a new approach for searching the literature and presenting a more refined analysis is adopted with respect to previous surveys, centering the attention on the enzymatic process rather than on a single novel activity. Fields of applications are easily individuated: (i) the biorefinery value-chain, where the provision of biomass is one of the most important aspects, with aquaculture as the prominent sector; (ii) the food industry, where the interest in the marine domain is similarly developed to deal with the enzymatic procedures adopted in food manipulation; (iii) the selective and easy extraction/modification of structurally complex marine molecules, where enzymatic treatments are a recognized tool to improve efficiency and selectivity; and (iv) marine biomarkers and derived applications (bioremediation) in pollution monitoring are also included in that these studies could be of high significance for the appreciation of marine bioprocesses. PMID:28346336

  7. Elimination kinetic of recombinant somatotropin in bovine.

    PubMed

    Le Breton, Marie-Hélène; Rochereau-Roulet, Sandrine; Pinel, Gaud; Cesbron, Nora; Le Bizec, Bruno

    2009-04-01

    Bovine somatotropin (bST), also called growth hormone is a protein hormone produced by the pituitary gland and responsible directly or indirectly for various effects on growth, development and reproductive functions. Its recombinant bovine somatotropin form (rbST) is used in dairy cattle to enhance milk production. Even if the effects of treatment with rbST have been largely studied, until now analytical methods able to detect rbST were limited to immunoassays, which suffer from the impossibility to distinguish between the endogenous and the recombinant form. In this study, a sample preparation procedure based on different precipitation steps, extraction on solid phase and enzymatic digestion was used to purify rbST from serum. The detection was performed by liquid chromatography coupled to tandem mass spectrometry in positive electrospray ionization mode (LC-ESI(+)-MS/MS) allowing the unambiguous identification and quantification of rbST in serum. Samples collected from a cow treated with recombinant bovine somatotropin were analysed and for the first time, the elimination kinetic specific to recombinant somatotropin has been characterized in serum. Detection of rbST was possible from 4h 30min to 4 days after administration and concentration was found up to 10ngmL(-1) during the kinetic.

  8. Photoionization and Recombination

    NASA Technical Reports Server (NTRS)

    Nahar, Sultana N.

    2000-01-01

    Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

  9. Physical inactivity and obesity underlie the insulin resistance of aging.

    PubMed

    Amati, Francesca; Dubé, John J; Coen, Paul M; Stefanovic-Racic, Maja; Toledo, Frederico G S; Goodpaster, Bret H

    2009-08-01

    OBJECTIVE Age-associated insulin resistance may underlie the higher prevalence of type 2 diabetes in older adults. We examined a corollary hypothesis that obesity and level of chronic physical inactivity are the true causes for this ostensible effect of aging on insulin resistance. RESEARCH DESIGN AND METHODS We compared insulin sensitivity in 7 younger endurance-trained athletes, 12 older athletes, 11 younger normal-weight subjects, 10 older normal-weight subjects, 15 younger obese subjects, and 15 older obese subjects using a glucose clamp. The nonathletes were sedentary. RESULTS Insulin sensitivity was not different in younger endurance-trained athletes versus older athletes, in younger normal-weight subjects versus older normal-weight subjects, or in younger obese subjects versus older obese subjects. Regardless of age, athletes were more insulin sensitive than normal-weight sedentary subjects, who in turn were more insulin sensitive than obese subjects. CONCLUSIONS Insulin resistance may not be characteristic of aging but rather associated with obesity and physical inactivity.

  10. Alterations in protein metabolism during space flight and inactivity

    NASA Technical Reports Server (NTRS)

    Ferrando, Arny A.; Paddon-Jones, Doug; Wolfe, Robert R.

    2002-01-01

    Space flight and the accompanying diminished muscular activity lead to a loss of body nitrogen and muscle function. These losses may affect crew capabilities and health in long-duration missions. Space flight alters protein metabolism such that the body is unable to maintain protein synthetic rates. A concomitant hypocaloric intake and altered anabolic/catabolic hormonal profiles may contribute to or exacerbate this problem. The inactivity associated with bedrest also reduces muscle and whole-body protein synthesis. For this reason, bedrest provides a good model for the investigation of potential exercise and nutritional countermeasures to restore muscle protein synthesis. We have demonstrated that minimal resistance exercise preserves muscle protein synthesis throughout bedrest. In addition, ongoing work indicates that an essential amino acid and carbohydrate supplement may ameliorate the loss of lean body mass and muscle strength associated with 28 d of bedrest. The investigation of inactivity-induced alterations in protein metabolism, during space flight or prolonged bedrest, is applicable to clinical populations and, in a more general sense, to the problems associated with the decreased activity that occur with aging.

  11. Alterations in protein metabolism during space flight and inactivity

    NASA Technical Reports Server (NTRS)

    Ferrando, Arny A.; Paddon-Jones, Doug; Wolfe, Robert R.

    2002-01-01

    Space flight and the accompanying diminished muscular activity lead to a loss of body nitrogen and muscle function. These losses may affect crew capabilities and health in long-duration missions. Space flight alters protein metabolism such that the body is unable to maintain protein synthetic rates. A concomitant hypocaloric intake and altered anabolic/catabolic hormonal profiles may contribute to or exacerbate this problem. The inactivity associated with bedrest also reduces muscle and whole-body protein synthesis. For this reason, bedrest provides a good model for the investigation of potential exercise and nutritional countermeasures to restore muscle protein synthesis. We have demonstrated that minimal resistance exercise preserves muscle protein synthesis throughout bedrest. In addition, ongoing work indicates that an essential amino acid and carbohydrate supplement may ameliorate the loss of lean body mass and muscle strength associated with 28 d of bedrest. The investigation of inactivity-induced alterations in protein metabolism, during space flight or prolonged bedrest, is applicable to clinical populations and, in a more general sense, to the problems associated with the decreased activity that occur with aging.

  12. Small molecule stabilization of the KSR inactive state antagonizes oncogenic Ras signalling

    PubMed Central

    Dhawan, Neil S.; scopton, Alex P.; Dar, Arvin C.

    2016-01-01

    Deregulation of the Ras–mitogen activated protein kinase (MAPK) pathway is an early event in many different cancers and a key driver of resistance to targeted therapies1. Sustained signalling through this pathway is caused most often by mutations in K-Ras, which biochemically favours the stabilization of active RAF signalling complexes2. Kinase suppressor of Ras (KSR) is a MAPK scaffold3–5 that is subject to allosteric regulation through dimerization with RAF6,7. Direct targeting of KSR could have important therapeutic implications for cancer; however, testing this hypothesis has been difficult owing to a lack of small-molecule antagonists of KSR function. Guided by KSR mutations that selectively suppress oncogenic, but not wild-type, Ras signalling, we developed a class of compounds that stabilize a previously unrecognized inactive state of KSR. These compounds, exemplified by APS-2-79, modulate KSR-dependent MAPK signalling by antagonizing RAF heterodimerization as well as the conformational changes required for phosphorylation and activation of KSR-bound MEK (mitogen-activated protein kinase kinase). Furthermore, APS-2-79 increased the potency of several MEK inhibitors specifically within Ras-mutant cell lines by antagonizing release of negative feedback signalling, demonstrating the potential of targeting KSR to improve the efficacy of current MAPK inhibitors. These results reveal conformational switching in KSR as a druggable regulator of oncogenic Ras, and further suggest co-targeting of enzymatic and scaffolding activities within Ras–MAPK signalling complexes as a therapeutic strategy for overcoming Ras-driven cancers. PMID:27556948

  13. Catalytic inactive heme oxygenase-1 protein regulates its own expression in oxidative stress.

    PubMed

    Lin, Qing S; Weis, Sebastian; Yang, Guang; Zhuang, Tiangang; Abate, Aida; Dennery, Phyllis A

    2008-03-01

    Heme oxygenase-1 (HO-1) catalyzes the degradation of heme and forms antioxidant bile pigments as well as the signaling molecule carbon monoxide. HO-1 is inducible in response to a variety of chemical and physical stress conditions to function as a cytoprotective molecule. Therefore, it is important to maintain the basal level of HO-1 expression even when substrate availability is limited. We hypothesized that the HO-1 protein itself could regulate its own expression in a positive feedback manner, and that this positive feedback was important in the HO-1 gene induction in response to oxidative stress. In cultured NIH 3T3 cells, transfection of HO-1 cDNA or intracellular delivery of pure HO-1 protein resulted in activation of a 15-kb HO-1 promoter upstream of luciferase as visualized by bioluminescent technology and increased HO-1 mRNA and protein levels. These effects were independent of HO activity because an enzymatically inactive mutant form of HO-1 similarly activated the HO-1 promoter and incubation with HO inhibitor metalloporphyrin SnPP did not affect the promoter activation. In addition, HO-1-specific siRNA significantly reduced hemin and cadmium chloride-mediated HO-1 induction. Furthermore, deletion analyses demonstrated that the E1 and E2 distal enhancers of the HO-1 promoter are required for this HO-1 autoregulation. These experiments document feed-forward autoregulation of HO-1 in oxidative stress and suggest that HO-1 protein has a role in the induction process. We speculate that this mechanism may be useful for maintaining HO-1 expression when substrate is limited and may also serve to up-regulate other genes to promote cytoprotection and to modulate cell proliferation.

  14. Molecular and enzymatic characterization of Schistosoma mansoni thioredoxin peroxidase.

    PubMed

    Kwatia, M A; Botkin, D J; Williams, D L

    2000-10-01

    The ability of Schistosoma mansoni to escape oxidative damage from immune system-generated reactive oxygen intermediates has been extensively documented. The limiting step in the parasite's detoxification process appears to be at the level of hydrogen peroxide neutralization. In the present study, the possible role of a novel class of antioxidant enzymes, thioredoxin peroxidase (TPx), in hydrogen peroxide neutralization by schistosomes was investigated. An expressed sequence tag was characterized from the Schistosoma Genome Initiative with high similarity to TPx from other organisms. The gene encodes a polypeptide containing 2 conserved active-site cysteines and flanking amino acids, and 60-70% identity with previously characterized TPx proteins. Recombinant schistosome TPx was enzymatically active and found to have thioredoxin-dependent hydrogen peroxide reducing activity of 4500 nmol hydrogen peroxide/min/mg protein. Native TPx activity was determined to be 48.1 nmol hydrogen peroxide/min/mg protein in adult worm homogenates compared with 46.9 for glutathione peroxidase. TPx activity was precipitated from adult worm homogenates with antibodies prepared against the recombinant protein. Western blotting with antibodies made against recombinant protein showed that TPx was expressed in both male and female adult worms. This is the first demonstration of a TPx activity in schistosomes and our results suggest that TPx plays a significant role in schistosome-host interactions.

  15. Rapid enzymatic test for phenotypic HIV protease drug resistance.

    PubMed

    Hoffmann, Dieter; Assfalg-Machleidt, Irmgard; Nitschko, Hans; von der Helm, Klaus; Koszinowski, Ulrich; Machleidt, Werner

    2003-07-01

    A phenotypic resistance test based on recombinant expression of the active HIV protease in E. coli from patient blood samples was developed. The protease is purified in a rapid one-step procedure as active enzyme and tested for inhibition by five selected synthetic inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) used presently for chemotherapy of HIV-infected patients. The HPLC system used in a previous approach was replaced by a continuous fluorogenic assay suitable for high-throughput screening on microtiter plates. This reduces significantly the total assay time and allows the determination of inhibition constants (Ki). The Michaelis constant (Km) and the inhibition constant (Ki) of recombinant wild-type protease agree well with published data for cloned HIV protease. The enzymatic test was evaluated with recombinant HIV protease derived from eight HIV-positive patients scored from 'sensitive' to 'highly resistant' according to mutations detected by genotypic analysis. The measured Ki values correlate well with the genotypic resistance scores, but allow a higher degree of differentiation. The non-infectious assay enables a more rapid yet sensitive detection of HIV protease resistance than other phenotypic assays.

  16. Development and Validation of an Enzymatic Method To Determine Stevioside Content from Stevia rebaudiana.

    PubMed

    Udompaisarn, Somsiri; Arthan, Dumrongkiet; Somana, Jamorn

    2017-04-19

    An enzymatic method for specific determination of stevioside content was established. Recombinant β-glucosidase BT_3567 (rBT_3567) from Bacteroides thetaiotaomicron HB-13 exhibited selective hydrolysis of stevioside at β-1,2-glycosidic bond to yield rubusoside and glucose. Coupling of this enzyme with glucose oxidase and peroxidase allowed for quantitation of stevioside content in Stevia samples by using a colorimetric-based approach. The series of reactions for stevioside determination can be completed within 1 h at 37 °C. Stevioside determination using the enzymatic assay strongly correlated with results obtained from HPLC quantitation (r(2) = 0.9629, n = 16). The percentages of coefficient variation (CV) of within day (n = 12) and between days (n = 12) assays were lower than 5%, and accuracy ranges were 95-105%. This analysis demonstrates that the enzymatic method developed in this study is specific, easy to perform, accurate, and yields reproducible results.

  17. Biofuel cells: enhanced enzymatic bioelectrocatalysis.

    PubMed

    Meredith, Matthew T; Minteer, Shelley D

    2012-01-01

    Enzymatic biofuel cells represent an emerging technology that can create electrical energy from biologically renewable catalysts and fuels. A wide variety of redox enzymes have been employed to create unique biofuel cells that can be used in applications such as implantable power sources, energy sources for small electronic devices, self-powered sensors, and bioelectrocatalytic logic gates. This review addresses the fundamental concepts necessary to understand the operating principles of biofuel cells, as well as recent advances in mediated electron transfer- and direct electron transfer-based biofuel cells, which have been developed to create bioelectrical devices that can produce significant power and remain stable for long periods.

  18. Discovery of a proteolytic flagellin family in diverse bacterial phyla that assembles enzymatically active flagella.

    PubMed

    Eckhard, Ulrich; Bandukwala, Hina; Mansfield, Michael J; Marino, Giada; Cheng, Jiujun; Wallace, Iain; Holyoak, Todd; Charles, Trevor C; Austin, John; Overall, Christopher M; Doxey, Andrew C

    2017-09-12

    Bacterial flagella are cell locomotion and occasional adhesion organelles composed primarily of the polymeric protein flagellin, but to date have not been associated with any enzymatic function. Here, we report the bioinformatics-driven discovery of a class of enzymatic flagellins that assemble to form proteolytically active flagella. Originating by a metallopeptidase insertion into the central flagellin hypervariable region, this flagellin family has expanded to at least 74 bacterial species. In the pathogen, Clostridium haemolyticum, metallopeptidase-containing flagellin (which we termed flagellinolysin) is the second most abundant protein in the flagella and is localized to the extracellular flagellar surface. Purified flagellar filaments and recombinant flagellin exhibit proteolytic activity, cleaving nearly 1000 different peptides. With ~ 20,000 flagellin copies per  ~ 10-μm flagella this assembles the largest proteolytic complex known. Flagellum-mediated extracellular proteolysis expands our understanding of the functional plasticity of bacterial flagella, revealing this family as enzymatic biopolymers that mediate interactions with diverse peptide substrates.So far no enzymatic activity has been attributed to flagellin, the major component of bacterial flagella. Here the authors use bioinformatic analysis and identify a metallopeptidase insertion in flagellins from 74 bacterial species and show that recombinant flagellin and flagellar filaments have proteolytic activity.

  19. Social background, bullying, and physical inactivity: National study of 11- to 15-year-olds.

    PubMed

    Henriksen, P W; Rayce, S B; Melkevik, O; Due, P; Holstein, B E

    2016-10-01

    More children from lower social backgrounds are physically inactive than those from higher ones. We studied whether bullying was a mediating factor between lower social background and physical inactivity. We also examined the combined effect of low social class and exposure to bullying on physical inactivity. The Danish sample of the Health Behaviour in School-aged Children (HBSC) study 2006 included 6269 schoolchildren in three age groups: 11-, 13-, and 15-year-olds from a random sample of 80 schools. The students answered the internationally standardized HBSC questionnaire. The applied definition leaves 4.0% in the category physically inactive. The sex and age-adjusted OR (95% CI) for physical inactivity was 2.10 (1.39-3.18) among students with low social class and unclassifiable 3.53 (2.26-5.53). Exposure to bullying was associated with physical inactivity, sex and age-adjusted OR = 2.39 (1.67-3.41). Exposure to bullying did not explain the association between social class and physical inactivity. The association between social class and physical inactivity was more pronounced among participants also exposed to bullying. In conclusion, there was a significantly increased odds ratio for physical inactivity among students from lower social classes and for students exposed to bullying. There was a combined effect of low social class and bullying on physical inactivity. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. Mechanical properties and fiber type composition of chronically inactive muscles

    NASA Technical Reports Server (NTRS)

    Roy, R. R.; Zhong, H.; Monti, R. J.; Vallance, K. A.; Kim, J. A.; Edgerton, V. R.

    2000-01-01

    A role for neuromuscular activity in the maintenance of skeletal muscle properties has been well established. However, the role of activity-independent factors is more difficult to evaluate. We have used the spinal cord isolation model to study the effects of chronic inactivity on the mechanical properties of the hindlimb musculature in cats and rats. This model maintains the connectivity between the motoneurons and the muscle fibers they innervate, but the muscle unit is electrically "silent". Consequently, the measured muscle properties are activity-independent and thus the advantage of using this model is that it provides a baseline level (zero activity) from which regulatory factors that affect muscle cell homeostasis can be defined. In the present paper, we will present a brief review of our findings using the spinal cord isolation model related to muscle mechanical and fiber type properties.

  1. Mechanical properties and fiber type composition of chronically inactive muscles

    NASA Technical Reports Server (NTRS)

    Roy, R. R.; Zhong, H.; Monti, R. J.; Vallance, K. A.; Kim, J. A.; Edgerton, V. R.

    2000-01-01

    A role for neuromuscular activity in the maintenance of skeletal muscle properties has been well established. However, the role of activity-independent factors is more difficult to evaluate. We have used the spinal cord isolation model to study the effects of chronic inactivity on the mechanical properties of the hindlimb musculature in cats and rats. This model maintains the connectivity between the motoneurons and the muscle fibers they innervate, but the muscle unit is electrically "silent". Consequently, the measured muscle properties are activity-independent and thus the advantage of using this model is that it provides a baseline level (zero activity) from which regulatory factors that affect muscle cell homeostasis can be defined. In the present paper, we will present a brief review of our findings using the spinal cord isolation model related to muscle mechanical and fiber type properties.

  2. Recombination of cluster ions

    NASA Technical Reports Server (NTRS)

    Johnsen, Rainer

    1993-01-01

    Some of our recent work on molecular band emissions from recombination of molecular dimer ions (N4(+) and CO(+) CO) is discussed. Much of the experimental work was done by Y. S. Cao; the results on N4(+) recombination have been published. A brief progress report is given on our ongoing measurements of neutral products of recombination using the flowing-afterglow Langmuir-probe technique in conjunction with laser-induced fluorescence.

  3. Physical inactivity at leisure and work: a 12-month study of cardiac patients.

    PubMed

    Rogerson, Michelle C; Murphy, Barbara M; Le Grande, Michael R; Worcester, Marian U C

    2013-01-01

    Physical inactivity has been identified as a distinct health risk. However, little is known about how this can vary at leisure and work in cardiac patients. The aim of this study was to examine the prevalence and predictors of inactivity during leisure and work in the 12 months following a cardiac event in Australian cardiac patients. A total of 346 patients consecutively admitted to hospital with acute coronary syndrome or to undergo coronary artery bypass graft surgery were interviewed in hospital, and 4 and 12 months later. Leisure and occupational physical activity was measured using the Stanford Brief Activity Survey. Sociodemographic, psychosocial, and clinical data were also collected. The prevalence of leisure-time physical inactivity declined over time, with 52% inactive preevent and 29% inactive at 12 months. Approximately 50% of participants were physically inactive in their work, regardless of whether this was measured before or after the cardiac event. Logistic regression revealed that the significant predictors of leisure-time physical inactivity at 12 months were non-home ownership (OR = 2.19; P = .007) and physical inactivity in leisure-time prior to the event (OR = 2.44; P = .001). The significant predictors of occupational physical inactivity at 12 months were white-collar occupation (OR = 3.10; P < .001) and physical inactivity at work prior to the event (OR = 12.99; P < .001). Preevent physical inactivity, socioeconomic, and clinical factors predicted both leisure and work inactivity after an acute cardiac event. Effective interventions could be designed and implemented to target those most at risk of being physically inactive at work or leisure.

  4. Nutritional status and physical inactivity in moderated asthmatics

    PubMed Central

    Bruno, Andreina; Uasuf, Carina Gabriela; Insalaco, Giuseppe; Barazzoni, Rocco; Ballacchino, Antonella; Gjomarkaj, Mark; Pace, Elisabetta

    2016-01-01

    Abstract Preservation of nutritional status and of fat-free mass (FFM) and/or preventing of fat mass (FM) accumulation have a positive impact on well-being and prognosis in asthma patients. Physical inactivity is identified by World Health Organization as the fourth leading risk factor for global mortality. Physical activity (PA) may contribute to limit FM accumulation, but little information is available on the interactions between habitual PA and body composition and their association with disease severity in asthma severity. Associations between habitual PA, FM, FFM, and pulmonary function were investigated in 42 subjects (24 patients with mild-moderate asthma and 18 matched control subjects). Sensewear Armband was used to measure PA and metabolic equivalent of tasks (METs) continuously over 4 days, while body composition was measured by bioelectrical impedance analysis. Respiratory functions were also assessed in all study participants. FM and FFM were comparable in mild-moderate asthmatics and controls, but PA was lower in asthmatics and it was negatively correlated with FM and positively with the FFM marker body cell mass in all study subjects (P < 0.05). Among asthmatics, treated moderate asthmatics (ICS, n = 12) had higher FM and lower PA, METs, steps number/die, and forced expiratory volume in the 1st second (FEV1)/forced vital capacity (FVC) than in untreated intermittent asthmatics (UA, n = 12). This pilot study assesses that in mild-moderate asthma patients, lower PA is associated with higher FM and higher disease severity. The current results support enhancement of habitual PA as a potential tool to limit FM accumulation and potentially contribute to preserve pulmonary function in moderate asthma, considering the physical inactivity a strong risk factor for asthma worsening. PMID:27495092

  5. Inactivity, age, and exercise: single-muscle fiber power generation.

    PubMed

    Kim, Jong-Hee; Thompson, Ladora V

    2013-01-01

    We examined the effects of mild therapeutic exercise during a period of inactivity on size and contractile functions of myosin heavy chain (MHC) type I (n = 204) and type II (n = 419) single fibers from the medial gastrocnemius in three age groups. Young adult (5-12 mo), middle-aged (24-31 mo), and old (32-37 mo) F344BNF1 rats were assigned to one of three groups: weight-bearing control, non-weight bearing (NWB), and NWB plus exercise (NWBX). Fourteen days of hindlimb suspension were applied in NWB rats. The NWBX rats exercised on the treadmill for 15 min, four times a day, during the period of NWB. The NWBX did not improve peak power, but increased normalized power of MHC type I fibers in young adult rats. In MHC type II fibers, NWBX did not change peak power, isometric maximal force, V(max), and fiber size from young adult and middle-aged rats. NWBX did not improve peak power and isometric maximal force and showed a dramatic decline in V(max) and normalized power in the old rats. Collectively, mild treadmill exercise during a period of inactivity does not improve peak power of MHC type I or type II fiber from the gastrocnemius in young, middle-aged, and old rats. However, NWBX is beneficial in enhancing normalized power of MHC type I fibers in young adult rats, most likely due to the stimulus intensity and the ability of the individual fibers to adapt to the stimulus. In contrast, several factors, such as impaired adaptation potential, inappropriate exercise intensity, or increased susceptibility to muscle damage, may contribute to the lack of improvement in the older rats.

  6. KEPLER FLARES. I. ACTIVE AND INACTIVE M DWARFS

    SciTech Connect

    Hawley, Suzanne L.; Davenport, James R. A.; Kowalski, Adam F.; Wisniewski, John P.; Deitrick, Russell; Hilton, Eric J.; Hebb, Leslie

    2014-12-20

    We analyzed Kepler short-cadence M dwarf observations. Spectra from the Astrophysical Research Consortium 3.5 m telescope identify magnetically active (Hα in emission) stars. The active stars are of mid-M spectral type, have numerous flares, and have well-defined rotational modulation due to starspots. The inactive stars are of early M type, exhibit less starspot signature, and have fewer flares. A Kepler to U-band energy scaling allows comparison of the Kepler flare frequency distributions with previous ground-based data. M dwarfs span a large range of flare frequency and energy, blurring the distinction between active and inactive stars designated solely by the presence of Hα. We analyzed classical and complex (multiple peak) flares on GJ 1243, finding strong correlations between flare energy, amplitude, duration, and decay time, with only a weak dependence on rise time. Complex flares last longer and have higher energy at the same amplitude, and higher energy flares are more likely to be complex. A power law fits the energy distribution for flares with log E{sub K{sub p}}> 31 erg, but the predicted number of low-energy flares far exceeds the number observed, at energies where flares are still easily detectable, indicating that the power-law distribution may flatten at low energy. There is no correlation of flare occurrence or energy with starspot phase, the flare waiting time distribution is consistent with flares occurring randomly in time, and the energies of consecutive flares are uncorrelated. These observations support a scenario where many independent active regions on the stellar surface are contributing to the observed flare rate.

  7. Differential membrane fluidization by active and inactive cannabinoid analogues.

    PubMed

    Mavromoustakos, T; Papahatjis, D; Laggner, P

    2001-06-06

    The effects of the two cannabinomimetic drugs (-)-2-(6a,7,10,10a-tetrahydro-6,6,9-trimethyl-1-hydroxy-6H-dibenzo[b,d]pyranyl-2-(hexyl)-1,3-dithiolane (AMG-3) and its pharmacologically less active 1-methoxy analogue (AMG-18) on the thermotropic and structural properties of dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) liposomes have been studied by X-ray diffraction and differential scanning calorimetry (DSC). DSC data revealed that the incorporation of the drugs affect differently the thermotropic properties of DPPC. The presence of the more active drug distinctly broadened and attenuated both the pretransition and main phase transition of DPPC bilayers, while the inactive analogue had only minor effects. Small and wide angle X-ray diffraction data showed that the two cannabinoids have different effects on the lipid phase structures and on the hydrocarbon chain packing. The pharmacologically active analogue, AMG-3, was found to efficiently fluidize domains of the lipids in the L(beta)' gel phase, and to perturb the regular multibilayer lattice. In the liquid crystalline L(alpha) phase, AMG-3 was also found to cause irregularities in packing, suggesting that the drug induces local curvature. At the same concentration, the inactive AMG-18 had only minor structural effects on the lipids. At about 10-fold or higher concentrations, AMG-18 was found to produce similar but still less pronounced effects in comparison to those observed by AMG-3. The dose-dependent, different thermotropic and structural effects by the two cannabinoid analogues suggest that these may be related to their biological activity.

  8. Enforced physical inactivity increases endothelial microparticle levels in healthy volunteers.

    PubMed

    Navasiolava, Nastassia M; Dignat-George, Françoise; Sabatier, Florence; Larina, Irina M; Demiot, Claire; Fortrat, Jacques-Olivier; Gauquelin-Koch, Guillemette; Kozlovskaya, Inesa B; Custaud, Marc-Antoine

    2010-08-01

    A sedentary lifestyle has adverse effects on the cardiovascular system, including impaired endothelial functions. Subjecting healthy men to 7 days of dry immersion (DI) presented a unique opportunity to analyze the specific effects of enhanced inactivity on the endothelium. We investigated endothelial properties before, during, and after 7 days of DI involving eight subjects. Microcirculatory functions were assessed with laser Doppler in the skin of the calf. We studied basal blood flow and endothelium-dependent and -independent vasodilation. We also measured plasma levels of microparticles, a sign of cellular dysfunction, and soluble endothelial factors, reflecting the endothelial state. Basal flow and endothelium-dependent vasodilation were reduced by DI (22 + or - 4 vs. 15 + or - 2 arbitrary units and 29 + or - 6% vs. 12 + or - 6%, respectively, P < 0.05), and this was accompanied by an increase in circulating endothelial microparticles (EMPs), which was significant on day 3 (42 + or - 8 vs. 65 + or - 10 EMPs/microl, P < 0.05), whereas microparticles from other cell origins remained unchanged. Plasma soluble VEGF decreased significantly during DI, whereas VEGF receptor 1 and soluble CD62E were unchanged, indicating that the increase in EMPs was associated with a change in antiapoptotic tone rather than endothelial activation. Our study showed that extreme physical inactivity in humans induced by 7 days of DI causes microvascular impairment with a disturbance of endothelial functions, associated with a selective increase in EMPs. Microcirculatory endothelial dysfunction might contribute to cardiovascular deconditioning as well as to hypodynamia-associated pathologies. In conclusion, the endothelium should be the focus of special care in situations of acute limitation of physical activity.

  9. Acid mine drainage from inactive eastern coal operations.

    PubMed

    Erickson, P M; Ladwig, K J; Kleinmann, R L

    1985-03-01

    The most widely used technique for abatement of acid drainage from inactive surface mines and refuse disposal areas is revegetation of a soil cover applied to the waste material. Nonetheless, acid production often persists and in some cases, limits establishment of vegetation. This paper reports on several field studies intended to determine the location of pyrite oxidation zones and migration pathways of oxidation products at inactive spoil and refuse sites.Oxygen required for pyrite oxidation is believed to he provided in the gaseous state from the atmosphere. Therefore, the oxygen concentration in unsaturated mine waste should provide an estimate of the weathering tendency in the local environment. We are currently monitoring gas composition in refuse and spoil at six sites. Barren refuse appeared to be oxygenated (>2% 02) in a shallow zone extending less than 1 metre below the surface during most of the year. Preliminary data from coal spoil showed that oxygen can be available throughout the unsaturated thickness, even at a revegetated site. Gas composition varied vertically and laterally at a single site and also appeared to show seasonal dependence.Hydrologic factors are also important in acid production and transport. Discharge monitoring alone does not adequately describe the mass transport of acid products through the spoil. For example, at one reclaimed mine the mean sulfate content in six monitoring wells ranged from 24% to 240% of the mean concentration at the discharge point. Sources of recharge and relative flow rates determine the contribution of a particular zone to overall discharge quality.These basic studies of acid production and transport indicate some shortcomings of standard reclamation practices at certain sites. This information will he used to develop alternative abatement technology designed to mitigate acid production at the source.

  10. Physical inactivity post-stroke: a 3-year longitudinal study.

    PubMed

    Kunkel, Dorit; Fitton, Carolyn; Burnett, Malcolm; Ashburn, Ann

    2015-01-01

    To explore change in activity levels post-stroke. We measured activity levels using the activPAL™ in hospital and at 1, 2 and 3 years' post-stroke onset. Of the 74 participants (mean age 76 (SD 11), 39 men), 61 were assessed in hospital: 94% of time was spent in sitting/lying, 4% standing and 2% walking. Activity levels improved over time (complete cases n = 15); time spent sitting/lying decreased (p = 0.001); time spent standing, walking and number of steps increased (p = 0.001, p = 0.028 and p = 0.03, respectively). At year 3, 18% of time was spent in standing and 9% walking. Time spent upright correlated significantly with Barthel (r = 0.69 on admission, r = 0.68 on discharge, both p < 0.01) and functional ambulation category scores (r = 0.55 on admission, 0.63 on discharge, both p < 0.05); correlations remained significant at all assessment points. Depression (in hospital), left hemisphere infarction (Years 1-2), visual neglect (Year 2), poor mobility and balance (Years 1-3) correlated with poorer activity levels. People with stroke were inactive for the majority of time. Time spent upright improved significantly by 1 year post-stroke; improvements slowed down thereafter. Poor activity levels correlated with physical and psychological measures. Larger studies are indicated to identify predictors of activity levels. Implications for Rehabilitation Activity levels (measured using activPAL™ activity monitor), increased significantly by 1 year post-stroke but improvements slowed down at 2 and 3 years. People with stroke were inactive for the majority of their day in hospital and in the community. Poor activity levels correlated with physical and psychological measures. Larger studies are indicated to identify the most important predictors of activity levels.

  11. Lactose autoinduction with enzymatic glucose release: characterization of the cultivation system in bioreactor.

    PubMed

    Mayer, Sonja; Junne, Stefan; Ukkonen, Kaisa; Glazyrina, Julia; Glauche, Florian; Neubauer, Peter; Vasala, Antti

    2014-02-01

    The lactose autoinduction system for recombinant protein production was combined with enzymatic glucose release as a method to provide a constant feed of glucose instead of using glycerol as a carbon substrate. Bioreactor cultivation confirmed that the slow glucose feed does not prevent the induction by lactose. HPLC studies showed that with successful recombinant protein production only a very low amount of lactose was metabolized during glucose-limited fed-batch conditions by the Escherichia coli strain BL21(DE3)pLysS in well-aerated conditions, which are problematic for glycerol-based autoinduction systems. We propose that slow enzymatic glucose feed does not cause a full activation of the lactose operon. However recombinant PDI-A protein (A-domain of human disulfide isomerase) was steadily produced until the end of the cultivation. The results of the cultivations confirmed our earlier observations with shaken cultures showing that lactose autoinduction cultures based on enzymatic glucose feed have good scalability, and that this system can be applied also to bioreactor cultivations.

  12. Biogeography and Biodiversity in Sulfide Structures of Active and Inactive Vents at Deep-Sea Hydrothermal Fields of the Southern Mariana Trough▿ †

    PubMed Central

    Kato, Shingo; Takano, Yoshinori; Kakegawa, Takeshi; Oba, Hironori; Inoue, Kazuhiko; Kobayashi, Chiyori; Utsumi, Motoo; Marumo, Katsumi; Kobayashi, Kensei; Ito, Yuki; Ishibashi, Jun-ichiro; Yamagishi, Akihiko

    2010-01-01

    The abundance, diversity, activity, and composition of microbial communities in sulfide structures both of active and inactive vents were investigated by culture-independent methods. These sulfide structures were collected at four hydrothermal fields, both on- and off-axis of the back-arc spreading center of the Southern Mariana Trough. The microbial abundance and activity in the samples were determined by analyzing total organic content, enzymatic activity, and copy number of the 16S rRNA gene. To assess the diversity and composition of the microbial communities, 16S rRNA gene clone libraries including bacterial and archaeal phylotypes were constructed from the sulfide structures. Despite the differences in the geological settings among the sampling points, phylotypes related to the Epsilonproteobacteria and cultured hyperthermophilic archaea were abundant in the libraries from the samples of active vents. In contrast, the relative abundance of these phylotypes was extremely low in the libraries from the samples of inactive vents. These results suggest that the composition of microbial communities within sulfide structures dramatically changes depending on the degree of hydrothermal activity, which was supported by statistical analyses. Comparative analyses suggest that the abundance, activity and diversity of microbial communities within sulfide structures of inactive vents are likely to be comparable to or higher than those in active vent structures, even though the microbial community composition is different between these two types of vents. The microbial community compositions in the sulfide structures of inactive vents were similar to those in seafloor basaltic rocks rather than those in marine sediments or the sulfide structures of active vents, suggesting that the microbial community compositions on the seafloor may be constrained by the available energy sources. Our findings provide helpful information for understanding the biogeography, biodiversity and

  13. Biogeography and biodiversity in sulfide structures of active and inactive vents at deep-sea hydrothermal fields of the Southern Mariana Trough.

    PubMed

    Kato, Shingo; Takano, Yoshinori; Kakegawa, Takeshi; Oba, Hironori; Inoue, Kazuhiko; Kobayashi, Chiyori; Utsumi, Motoo; Marumo, Katsumi; Kobayashi, Kensei; Ito, Yuki; Ishibashi, Jun-ichiro; Yamagishi, Akihiko

    2010-05-01

    The abundance, diversity, activity, and composition of microbial communities in sulfide structures both of active and inactive vents were investigated by culture-independent methods. These sulfide structures were collected at four hydrothermal fields, both on- and off-axis of the back-arc spreading center of the Southern Mariana Trough. The microbial abundance and activity in the samples were determined by analyzing total organic content, enzymatic activity, and copy number of the 16S rRNA gene. To assess the diversity and composition of the microbial communities, 16S rRNA gene clone libraries including bacterial and archaeal phylotypes were constructed from the sulfide structures. Despite the differences in the geological settings among the sampling points, phylotypes related to the Epsilonproteobacteria and cultured hyperthermophilic archaea were abundant in the libraries from the samples of active vents. In contrast, the relative abundance of these phylotypes was extremely low in the libraries from the samples of inactive vents. These results suggest that the composition of microbial communities within sulfide structures dramatically changes depending on the degree of hydrothermal activity, which was supported by statistical analyses. Comparative analyses suggest that the abundance, activity and diversity of microbial communities within sulfide structures of inactive vents are likely to be comparable to or higher than those in active vent structures, even though the microbial community composition is different between these two types of vents. The microbial community compositions in the sulfide structures of inactive vents were similar to those in seafloor basaltic rocks rather than those in marine sediments or the sulfide structures of active vents, suggesting that the microbial community compositions on the seafloor may be constrained by the available energy sources. Our findings provide helpful information for understanding the biogeography, biodiversity and

  14. Enzymatic biotransformation of synthetic dyes.

    PubMed

    Rodríguez-Couto, S

    2009-11-01

    Environmental pollution by discharge of dye-containing effluents represents a serious ecological concern in many countries. Public demands for colour-free discharges to receiving waters have made decolouration of a variety of industrial wastewater a top priority. The current existing techniques for dye removal have several drawbacks such as high cost, low efficiency, use of large amounts of chemicals and formation of toxic sub-products. This has impelled the search for alternative methods such as those based on oxidative enzymes. This approach is believed to be a promising technology since it is cost-effective, environmentally friendly and does not produce sludge. Enzymatic transformation of synthetic dyes can be described as the conversion of dye molecules by enzymes into simpler and generally colourless molecules. Detailed characterisation of the metabolites produced during enzymatic transformation of synthetic dyes as well as ecotoxicity studies is of great importance to assess the effectiveness of the biodegradation process. However, most reports on the biotreatment of dyes mainly deal with decolouration and there are few reports on the reduction in toxicity or on the identification of the biodegradation products. This implies a limitation to assess their true technical potential.

  15. Enzymatic processing in microfluidic reactors.

    PubMed

    Miyazaki, Masaya; Honda, Takeshi; Yamaguchi, Hiroshi; Briones, Maria Portia P; Maeda, Hideaki

    2008-01-01

    Microreaction technology is an interdisciplinary area of science and engineering. It has attracted the attention of researchers from different fields in the past few years and consequently, several microreactors have been developed. Enzymes are organic catalysts used for the production useful substances in an environmentally friendly way, and have high potential for analytical applications. However, relatively few enzymatic processes have been commercialized because of problems in the stability of enzyme molecule, and the cost and efficiency of the reactions. Thus, there have been demands for innovation in process engineering particularly for enzymatic reactions, and microreaction devices can serve as efficient tools for the development of enzyme processes. In this review, we summarize the recent advances of microchannel reaction technologies and focus our discussion on enzyme microreactors. We discuss the manufacturing process of microreaction devices and the advantages of microreactors compared with the conventional reactors. Fundamental techniques for enzyme microreactors and important applications of this multidisciplinary technology in chemical processing are also included in our topics.

  16. Enzymatic Synthesis of Magnetic Nanoparticles

    PubMed Central

    Kolhatkar, Arati G.; Dannongoda, Chamath; Kourentzi, Katerina; Jamison, Andrew C.; Nekrashevich, Ivan; Kar, Archana; Cacao, Eliedonna; Strych, Ulrich; Rusakova, Irene; Martirosyan, Karen S.; Litvinov, Dmitri; Lee, T. Randall; Willson, Richard C.

    2015-01-01

    We report the first in vitro enzymatic synthesis of paramagnetic and antiferromagnetic nanoparticles toward magnetic ELISA reporting. With our procedure, alkaline phosphatase catalyzes the dephosphorylation of l-ascorbic-2-phosphate, which then serves as a reducing agent for salts of iron, gadolinium, and holmium, forming magnetic precipitates of Fe45±14Gd5±2O50±15 and Fe42±4Ho6±4O52±5. The nanoparticles were found to be paramagnetic at 300 K and antiferromagnetic under 25 K. Although weakly magnetic at 300 K, the room-temperature magnetization of the nanoparticles found here is considerably greater than that of analogous chemically-synthesized LnxFeyOz (Ln = Gd, Ho) samples reported previously. At 5 K, the nanoparticles showed a significantly higher saturation magnetization of 45 and 30 emu/g for Fe45±14Gd5±2O50±15 and Fe42±4Ho6±4O52±5, respectively. Our approach of enzymatically synthesizing magnetic labels reduces the cost and avoids diffusional mass-transfer limitations associated with pre-synthesized magnetic reporter particles, while retaining the advantages of magnetic sensing. PMID:25854425

  17. Enzymatic synthesis of magnetic nanoparticles.

    PubMed

    Kolhatkar, Arati G; Dannongoda, Chamath; Kourentzi, Katerina; Jamison, Andrew C; Nekrashevich, Ivan; Kar, Archana; Cacao, Eliedonna; Strych, Ulrich; Rusakova, Irene; Martirosyan, Karen S; Litvinov, Dmitri; Lee, T Randall; Willson, Richard C

    2015-04-03

    We report the first in vitro enzymatic synthesis of paramagnetic and antiferromagnetic nanoparticles toward magnetic ELISA reporting. With our procedure, alkaline phosphatase catalyzes the dephosphorylation of l-ascorbic-2-phosphate, which then serves as a reducing agent for salts of iron, gadolinium, and holmium, forming magnetic precipitates of Fe45±14Gd5±2O50±15 and Fe42±4Ho6±4O52±5. The nanoparticles were found to be paramagnetic at 300 K and antiferromagnetic under 25 K. Although weakly magnetic at 300 K, the room-temperature magnetization of the nanoparticles found here is considerably greater than that of analogous chemically-synthesized LnxFeyOz (Ln = Gd, Ho) samples reported previously. At 5 K, the nanoparticles showed a significantly higher saturation magnetization of 45 and 30 emu/g for Fe45±14Gd5±2O50±15 and Fe42±4Ho6±4O52±5, respectively. Our approach of enzymatically synthesizing magnetic labels reduces the cost and avoids diffusional mass-transfer limitations associated with pre-synthesized magnetic reporter particles, while retaining the advantages of magnetic sensing.

  18. Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana

    PubMed Central

    Alkanaimsh, Salem; Karuppanan, Kalimuthu; Guerrero, Andrés; Tu, Aye M.; Hashimoto, Bryce; Hwang, Min Sook; Phu, My L.; Arzola, Lucas; Lebrilla, Carlito B.; Dandekar, Abhaya M.; Falk, Bryce W.; Nandi, Somen; Rodriguez, Raymond L.; McDonald, Karen A.

    2016-01-01

    To optimize the expression, extraction and purification of plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), we describe the development and use of plant viral amplicon-based gene expression system; Tobacco Mosaic Virus (TMV) RNA-based overexpression vector (TRBO) to express enzymatically active FLAG-tagged plant made recombinant butyrylcholinesterase (rBChE) in Nicotiana benthamiana leaves using transient agroinfiltration. Two gene expression cassettes were designed to express the recombinant protein in either the ER or to the apoplastic compartment. Leaf homogenization was used to isolate ER-retained recombinant butyrylcholinesterase (prBChE-ER) while apoplast-targeted rBChE was isolated by either leaf homogenization (prBChE) or vacuum-extraction of apoplastic wash fluid (prBChE-AWF). rBChE from apoplast wash fluid had a higher specific activity but lower enzyme yield than leaf homogenate. To optimize the isolation and purification of total recombinant protein from leaf homogenates, an acidic extraction buffer was used. The acidic extraction buffer yielded >95% enzymatically active tetrameric rBChE as verified by Coomassie stained and native gel electrophoresis. Furthermore, when compared to human butyrylcholinesterase, the prBChE was found to be similar in terms of tetramerization and enzyme kinetics. The N-linked glycan profile of purified prBChE-ER was found to be mostly high mannose structures while the N-linked glycans on prBChE-AWF were primarily complex. The glycan profile of the prBChE leaf homogenates showed a mixture of high mannose, complex and paucimannose type N-glycans. These findings demonstrate the ability of plants to produce rBChE that is enzymatically active and whose oligomeric state is comparable to mammalian butyrylcholinesterase. The process of plant made rBChE tetramerization and strategies for improving its pharmacokinetics properties are also discussed. PMID:27379103

  19. [Physical inactivity in Galicia (Spain): trends and the impact of changes in the definition].

    PubMed

    Pérez-Ríos, Mónica; Santiago-Pérez, María I; Rodríguez-Camacho, Elena; Malvar, Alberto; Suanzes, Jorge; Hervada, Xurxo

    2015-01-01

    To estimate the prevalence of physical inactivity during leisure time in Galicia (Spain) between 2007 and 2011 and to assess the impact of including non-leisure time activities in the definition of physical inactivity. A cross-sectional study was conducted in the population aged 16 years and older (n=19,235). Physical activity was assessed by the Minnesota Questionnaire. In 2011, inactivity was estimated by including daily activities. Between 2007 and 2011, the prevalence of inactivity in Galicia remained stable (p=0.249) and close to 50%. This prevalence was higher among women and those who worked or were in education. Inactivity decreased from 47% to 16% when non-leisure time activities were included in the definition. Between 2007 and 2011 in Galicia, the prevalence of inactivity remained high and stable. This prevalence was significantly decreased when non-leisure time activities were included in the definition. Copyright © 2014 SESPAS. Published by Elsevier Espana. All rights reserved.

  20. Naïve Definitions of Action and Inaction: The Continuum, Spread, and Valence of Behaviors.

    PubMed

    McCulloch, Kathleen C; Li, Hong; Hong, Sungjin; Albarracin, Dolores

    2012-03-01

    The cohesiveness of a society depends, in part, on how its individual members manage their daily activities with respect to the goals of that society. Hence, there should be a degree of social agreement on what constitutes action and what constitutes inaction. The present research investigated the structure of action and inaction definitions, the evaluation of action versus inaction, and individual differences in these evaluations. Action-inaction ratings of behaviors and states showed more social agreement at the ends of the inaction-action continuum than at the middle, suggesting a socially shared construal of this definition. Action-inaction ratings were also shown to correlate with the valence of the rated behaviors, such that the more active the behavior the more positive its valence. Lastly, individual differences in locomotion, need for closure, and Christian religious beliefs correlated positively with a preference for action.

  1. Naïve Definitions of Action and Inaction: The Continuum, Spread, and Valence of Behaviors

    PubMed Central

    McCulloch, Kathleen C.; Li, Hong; Hong, Sungjin; Albarracin, Dolores

    2011-01-01

    The cohesiveness of a society depends, in part, on how its individual members manage their daily activities with respect to the goals of that society. Hence, there should be a degree of social agreement on what constitutes action and what constitutes inaction. The present research investigated the structure of action and inaction definitions, the evaluation of action versus inaction, and individual differences in these evaluations. Action-inaction ratings of behaviors and states showed more social agreement at the ends of the inaction-action continuum than at the middle, suggesting a socially shared construal of this definition. Action-inaction ratings were also shown to correlate with the valence of the rated behaviors, such that the more active the behavior the more positive its valence. Lastly, individual differences in locomotion, need for closure, and Christian religious beliefs correlated positively with a preference for action. PMID:23487013

  2. Calculating a checksum with inactive networking components in a computing system

    DOEpatents

    Aho, Michael E; Chen, Dong; Eisley, Noel A; Gooding, Thomas M; Heidelberger, Philip; Tauferner, Andrew T

    2015-01-27

    Calculating a checksum utilizing inactive networking components in a computing system, including: identifying, by a checksum distribution manager, an inactive networking component, wherein the inactive networking component includes a checksum calculation engine for computing a checksum; sending, to the inactive networking component by the checksum distribution manager, metadata describing a block of data to be transmitted by an active networking component; calculating, by the inactive networking component, a checksum for the block of data; transmitting, to the checksum distribution manager from the inactive networking component, the checksum for the block of data; and sending, by the active networking component, a data communications message that includes the block of data and the checksum for the block of data.

  3. Calculating a checksum with inactive networking components in a computing system

    DOEpatents

    Aho, Michael E; Chen, Dong; Eisley, Noel A; Gooding, Thomas M; Heidelberger, Philip; Tauferner, Andrew T

    2014-12-16

    Calculating a checksum utilizing inactive networking components in a computing system, including: identifying, by a checksum distribution manager, an inactive networking component, wherein the inactive networking component includes a checksum calculation engine for computing a checksum; sending, to the inactive networking component by the checksum distribution manager, metadata describing a block of data to be transmitted by an active networking component; calculating, by the inactive networking component, a checksum for the block of data; transmitting, to the checksum distribution manager from the inactive networking component, the checksum for the block of data; and sending, by the active networking component, a data communications message that includes the block of data and the checksum for the block of data.

  4. Physarum polymalic acid hydrolase: Recombinant expression and enzyme activation.

    PubMed

    Mueller, Wolfgang; Haindl, Markus; Holler, Eggehard

    2008-12-19

    As a platform for syntheses of nanoconjugates in antitumor drug delivery, polymalic acid together with its tailoring specific exohydrolase is purified from plasmodium cultures of the slime mold Physarum polycephalum, a member of the phylum myxomycota. Polymalic acid hydrolase is expressed in an inactive form that functions as a molecular adapter for polymalic acid trafficking within the plasmodium and is activated only during secretion. Activation follows specific protein tyrosine phosphorylation and dissociation from plasma membranes. Purified inactive Physarum polymalic acid hydrolase, recombinantly expressed in yeast Saccharomyces, is activated on a preparative basis by the addition of plasma membrane fragments from plasmodia of P. polycephalum. Activation of polymalic acid hydrolase and inhibition of polymalic acid synthesis by protein tyrosine phosphorylation are complementary events and could indicate a joint signal response to plasma membrane damage.

  5. beta. -Sulfopyruvate: chemical and enzymatic syntheses and enzymatic assay

    SciTech Connect

    Weinstein, C.L.; Griffith, O.W.

    1986-01-01

    BETA-Sulfopyruvic acid (2-carboxy-2-oxoethanesulfonic acid) is prepared in greater than 90% yield by reaction of bromopyruvic acid with sodium sulfite. ..beta..-(/sup 35/S)Sulfopyruvate is prepared by transamination between (/sup 35/)cysteinesulfonate (cysteate) and ..cap alpha..-ketoglutarate using mitochondrial aspartate aminotransferase isolated from rat liver. Following either chemical or enzymatic synthesis the crude reaction product is conveniently purified by chromatography on Dowex 1; ..beta..-sulfopyruvate is isolated as the stable, water-soluble dilithium salt. ..beta..-Sulfopyruvate is shown to be an alternative substrate of mitochondrial malate dehydrogenase; in the presence of 0.25 mM NADH, ..beta..-sulfopyruvate is reduced with an apparent K/sub m/ of 6.3 mM and a V/sub max/ equal to about 40% of that observed with oxaloacetate. This finding forms the basis of a convenient spectrophotometric assay of ..beta..-sulfopyruvate.

  6. Standardization and quality control in quantifying non-enzymatic oxidative protein modifications in relation to ageing and disease: Why is it important and why is it hard?

    PubMed

    Nedić, Olgica; Rogowska-Wrzesinska, Adelina; Rattan, Suresh I S

    2015-08-01

    Post-translational modifications (PTM) of proteins determine the activity, stability, specificity, transportability and lifespan of a protein. Some PTM are highly specific and regulated involving various enzymatic pathways, but there are other non-enzymatic PTM (nePTM), which occur stochastically, depend on the ternary structure of proteins and can be damaging. It is often observed that inactive and abnormal proteins accumulate in old cells and tissues. The nature, site and extent of nePTM give rise to a population of that specific protein with alterations in structure and function ranging from being fully active to totally inactive molecules. Determination of the type and the amount (abundance) of nePTM is essential for establishing connection between specific protein structure and specific biological role. This article summarizes analytical demands for reliable quantification of nePTM, including requirements for the assay performance, standardization and quality control, and points to the difficulties, uncertainties and un-resolved issues.

  7. Standardization and quality control in quantifying non-enzymatic oxidative protein modifications in relation to ageing and disease: Why is it important and why is it hard?

    PubMed Central

    Nedić, Olgica; Rogowska-Wrzesinska, Adelina; Rattan, Suresh I.S.

    2015-01-01

    Post-translational modifications (PTM) of proteins determine the activity, stability, specificity, transportability and lifespan of a protein. Some PTM are highly specific and regulated involving various enzymatic pathways, but there are other non-enzymatic PTM (nePTM), which occur stochastically, depend on the ternary structure of proteins and can be damaging. It is often observed that inactive and abnormal proteins accumulate in old cells and tissues. The nature, site and extent of nePTM give rise to a population of that specific protein with alterations in structure and function ranging from being fully active to totally inactive molecules. Determination of the type and the amount (abundance) of nePTM is essential for establishing connection between specific protein structure and specific biological role. This article summarizes analytical demands for reliable quantification of nePTM, including requirements for the assay performance, standardization and quality control, and points to the difficulties, uncertainties and un-resolved issues. PMID:25909343

  8. Recombinant Baculovirus Isolation.

    PubMed

    King, Linda A; Hitchman, Richard; Possee, Robert D

    2016-01-01

    Although there are several different methods available of making recombinant baculovirus expression vectors (reviewed in Chapter 3 ), all require a stage in which insect cells are transfected with either the virus genome alone (Bac-to-Bac(®) or BaculoDirect™, Invitrogen) or virus genome and transfer vector. In the latter case, this allows the natural process of homologous recombination to transfer the foreign gene, under control of the polyhedrin or other baculovirus gene promoter, from the transfer vector to the virus genome to create the recombinant virus. Previously, many methods required a plaque-assay to separate parental and recombinant virus prior to amplification and use of the recombinant virus. Fortunately, this step is no longer required for most systems currently available. This chapter provides an overview of the historical development of increasingly more efficient systems for the isolation of recombinant baculoviruses (Chapter 3 provides a full account of the different systems and transfer vectors available). The practical details cover: transfection of insect cells with either virus DNA or virus DNA and plasmid transfer vector; a reliable plaque-assay method that can be used to separate recombinant virus from parental (nonrecombinant) virus where this is necessary; methods for the small-scale amplification of recombinant virus; and subsequent titration by plaque-assay or real-time polymerase chain reaction (PCR). Methods unique to the Bac-to-Bac(®) system are also covered and include the transformation of bacterial cells and isolation of bacmid DNA ready for transfection of insect cells.

  9. Soluble expression and enzymatic activity evaluation of protease from reticuloendotheliosis virus.

    PubMed

    Hu, Feng; Zhao, Yan; Qi, Xiaole; Cui, Hongyu; Gao, Yulong; Gao, Honglei; Liu, Changjun; Wang, Yongqiang; Zhang, Yanping; Li, Kai; Wang, Xiaomei; Wang, Yunfeng

    2015-10-01

    The protease (PR) encoded by most retroviruses is deeply involved in the lifecycle and infection process of retroviruses by possessing the specificity necessary to correctly cleave the viral polyproteins and host cell proteins. However, as an important representative of avian retroviruses, the enzymatic properties of PR from reticuloendotheliosis virus (REV) have not been clearly documented. The recombinant PR, its mutant fused with a His-tag, and its substrate p18-p30 fused with a GST-tag were expressed in the Escherichia coli system as soluble enzymes. The soluble PR and p18-p30 were purified using Ni-NTA His Bind Resin and Glutathione Sepharose 4B, respectively. The enzymatic activity of PR was analyzed using the substrate of p18-p30. The expressed prokaryotic protease has enzyme activity that is dependent on such conditions as temperature, pH, and ions, and its activity can be inhibited by caspase inhibitor and the divalent metal ions Ca(2+) and Ni(2+). In addition, the key role of the residue Thr (amino acids 28) for the enzymatic activity of PR was identified. Furthermore, the caspase inhibitor Z-VAD-FMK was confirmed to inhibit the PR enzymatic activity of REV. For the first time, the PR of REV was expressed in the soluble form, and the optimal enzymatic reaction system in vitro was developed and preliminarily used. This study provides essential tools and information for further understanding the infection mechanism of REV and for the development of antiviral drugs treating retroviruses.

  10. 38 CFR 4.89 - Ratings for inactive nonpulmonary tuberculosis in effect on August 19, 1968.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...' Relief DEPARTMENT OF VETERANS AFFAIRS SCHEDULE FOR RATING DISABILITIES Disability Ratings Infectious Diseases, Immune Disorders and Nutritional Deficiencies § 4.89 Ratings for inactive nonpulmonary...

  11. 38 CFR 4.89 - Ratings for inactive nonpulmonary tuberculosis in effect on August 19, 1968.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...' Relief DEPARTMENT OF VETERANS AFFAIRS SCHEDULE FOR RATING DISABILITIES Disability Ratings Infectious Diseases, Immune Disorders and Nutritional Deficiencies § 4.89 Ratings for inactive nonpulmonary...

  12. 38 CFR 4.89 - Ratings for inactive nonpulmonary tuberculosis in effect on August 19, 1968.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...' Relief DEPARTMENT OF VETERANS AFFAIRS SCHEDULE FOR RATING DISABILITIES Disability Ratings Infectious Diseases, Immune Disorders and Nutritional Deficiencies § 4.89 Ratings for inactive nonpulmonary...

  13. 38 CFR 4.89 - Ratings for inactive nonpulmonary tuberculosis in effect on August 19, 1968.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...' Relief DEPARTMENT OF VETERANS AFFAIRS SCHEDULE FOR RATING DISABILITIES Disability Ratings Infectious Diseases, Immune Disorders and Nutritional Deficiencies § 4.89 Ratings for inactive nonpulmonary...

  14. 38 CFR 4.89 - Ratings for inactive nonpulmonary tuberculosis in effect on August 19, 1968.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...' Relief DEPARTMENT OF VETERANS AFFAIRS SCHEDULE FOR RATING DISABILITIES Disability Ratings Infectious Diseases, Immune Disorders and Nutritional Deficiencies § 4.89 Ratings for inactive nonpulmonary...

  15. Structural organization of the inactive X chromosome in the mouse.

    PubMed

    Giorgetti, Luca; Lajoie, Bryan R; Carter, Ava C; Attia, Mikael; Zhan, Ye; Xu, Jin; Chen, Chong Jian; Kaplan, Noam; Chang, Howard Y; Heard, Edith; Dekker, Job

    2016-07-28

    X-chromosome inactivation (XCI) involves major reorganization of the X chromosome as it becomes silent and heterochromatic. During female mammalian development, XCI is triggered by upregulation of the non-coding Xist RNA from one of the two X chromosomes. Xist coats the chromosome in cis and induces silencing of almost all genes via its A-repeat region, although some genes (constitutive escapees) avoid silencing in most cell types, and others (facultative escapees) escape XCI only in specific contexts. A role for Xist in organizing the inactive X (Xi) chromosome has been proposed. Recent chromosome conformation capture approaches have revealed global loss of local structure on the Xi chromosome and formation of large mega-domains, separated by a region containing the DXZ4 macrosatellite. However, the molecular architecture of the Xi chromosome, in both the silent and expressed regions,remains unclear. Here we investigate the structure, chromatin accessibility and expression status of the mouse Xi chromosome in highly polymorphic clonal neural progenitors (NPCs) and embryonic stem cells. We demonstrate a crucial role for Xist and the DXZ4-containing boundary in shaping Xi chromosome structure using allele-specific genome-wide chromosome conformation capture (Hi-C) analysis, an assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) and RNA sequencing. Deletion of the boundary disrupts mega-domain formation, and induction of Xist RNA initiates formation of the boundary and the loss of DNA accessibility. We also show that in NPCs, the Xi chromosome lacks active/inactive compartments and topologically associating domains (TADs), except around genes that escape XCI. Escapee gene clusters display TAD-like structures and retain DNA accessibility at promoter-proximal and CTCF-binding sites. Furthermore, altered patterns of facultative escape genes indifferent neural progenitor clones are associated with the presence of different TAD

  16. Characterization of inactive renin ("prorenin") from renin-secreting tumors of nonrenal origin. Similarity to inactive renin from kidney and normal plasma.

    PubMed Central

    Atlas, S A; Hesson, T E; Sealey, J E; Dharmgrongartama, B; Laragh, J H; Ruddy, M C; Aurell, M

    1984-01-01

    Inactive renin comprises well over half the total renin in normal human plasma. There is a direct relationship between active and inactive renin levels in normal and hypertensive populations, but the proportion of inactive renin varies inversely with the active renin level; as much as 98% of plasma renin is inactive in patients with low renin, whereas the proportion is consistently lower (usually 20-60%) in high-renin states. Two hypertensive patients with proven renin-secreting carcinomas of non-renal origin (pancreas and ovary) had high plasma active renin (119 and 138 ng/h per ml) and the highest inactive renin levels we have ever observed (5,200 and 14,300 ng/h per ml; normal range 3-50). The proportion of inactive renin (98-99%) far exceeded that found in other patients with high active renin levels. A third hypertensive patient with a probable renin-secreting ovarian carcinoma exhibited a similar pattern. Inactive renins isolated from plasma and tumors of these patients were biochemically similar to semipurified inactive renins from normal plasma or cadaver kidney. All were bound by Cibacron Blue-agarose, were not retained by pepstatin-Sepharose, and had greater apparent molecular weights (Mr) than the corresponding active forms. Plasma and tumor inactive renins from the three patients were similar in size (Mr 52,000-54,000), whereas normal plasma inactive renin had a slightly larger Mr than that from kidney (56,000 vs. 50,000). Inactive renin from each source was activated irreversibly by trypsin and reversibly by dialysis to pH 3.3 at 4 degrees C; the reversal process followed the kinetics of a first-order reaction in each instance. The trypsin-activated inactive renins were all identical to semipurified active renal renin in terms of pH optimum (pH 5.5-6.0) and kinetics with homologous angiotensinogen (Michaelis constants, 0.8-1.3 microM) and inhibition by pepstatin or by serial dilutions of renin-specific antibody. These results indicate that a markedly

  17. Enzymatic regeneration of adenosine triphosphate cofactor

    NASA Technical Reports Server (NTRS)

    Marshall, D. L.

    1974-01-01

    Regenerating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by enzymatic process which utilizes carbamyl phosphate as phosphoryl donor is technique used to regenerate expensive cofactors. Process allows complex enzymatic reactions to be considered as candidates for large-scale continuous processes.

  18. Monitoring enzymatic ATP hydrolysis by EPR spectroscopy.

    PubMed

    Hacker, Stephan M; Hintze, Christian; Marx, Andreas; Drescher, Malte

    2014-07-14

    An adenosine triphosphate (ATP) analogue modified with two nitroxide radicals is developed and employed to study its enzymatic hydrolysis by electron paramagnetic resonance spectroscopy. For this application, we demonstrate that EPR holds the potential to complement fluorogenic substrate analogues in monitoring enzymatic activity.

  19. Purification and enzymatic characterization of alcohol dehydrogenase from Arabidopsis thaliana.

    PubMed

    Cheng, Fangfang; Hu, Tao; An, Yan; Huang, Jianqin; Xu, Yingwu

    2013-08-01

    Alcohol dehydrogenases (ADH) catalyze the interconversion between alcohols and aldehydes with the reduction of nicotinamide adenine dinucleotide (NAD(+)) to NADH. In this study, for the first time we report an over-expression and purification strategy for the Arabidosis thaliana ADH (AtADH), and characterize its enzymatic properties. AtADH was expressed in an Escherichia coli system, the polyhistidine-tag was removed after the recombinant AtADH protein was purified by metal chelating affinity chromatography. Activity assays demonstrated that AtADH has distinct enzymatic properties when compared with many well-known ADHs. It held peak activity at pH 10.5 and showed broad substrate selectivity for primary and secondary alcohols. The kinetic Km parameters for both ethanol and coenzyme were in the order of mM. This relative low affinity may reflect the need of the plant to maintain a supply of NAD(+) in nature. Different from yeast ADH, AtADH showed almost the same activity for short straight chain alcohols and reduced activity for secondary alcohols. This broad spectrum in alcohol selection and the observed higher catalytic activity (high Vmax (EtOH)) may result from the requirement of the single enzyme to accommodate many substrates. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Modular enzymatically crosslinked protein polymer hydrogels for in situ gelation

    PubMed Central

    Davis, Nicolynn E.; Ding, Sheng; Forster, Ryan; Pinkas, Daniel M.; Barron, Annelise E.

    2012-01-01

    Biomaterials that mimic the extracellular matrix in both modularity and crosslinking chemistry have the potential to recapitulate the instructive signals that ultimately control cell fate. Toward this goal, modular protein polymer-based hydrogels were created through genetic engineering and enzymatic crosslinking. Animal derived tissue transglutaminase (tTG) and recombinant human transglutaminase (hTG) enzymes were used for coupling two classes of protein polymers containing either lysine or glutamine, which have the recognition substrates for enzymatic crosslinking, evenly spaced along the protein backbone. Utilizing tTG under physiological conditions, crosslinking occurred within two minutes, as determined by particle tracking microrheology. Hydrogel composition impacted the elastic storage modulus of the gel over 4-fold and also influenced microstructure and degree of swelling, but did not appreciably effect degradation by plasmin. Mouse 3T3 and primary human fibroblasts were cultured in both 2- and 3-dimensions without a decrease in cell viability and displayed spreading in 2D. The properties of these gels, which are controlled through the specific nature of the protein polymer precursors, render these gels valuable for in situ therapies. Furthermore, the modular hydrogel composition allows tailoring of mechanical and physical properties for specific tissue engineering applications. PMID:20609472

  1. Enzymatic disease of the podocyte

    PubMed Central

    Kistler, Andreas D.; Peev, Vasil; Forst, Anna-Lena; El Hindi, Shafic; Altintas, Mehmet M.

    2014-01-01

    Proteinuria is an early sign of kidney disease and has gained increasing attention over the past decade because of its close association with cardio-vascular and renal morbidity and mortality. Podocytes have emerged as the cell type that is critical in maintaining proper functioning of the kidney filter. A few genes have been identified that explain genetic glomerular failure and recent insights shed light on the pathogenesis of acquired proteinuric diseases. This review highlights the unique role of the cysteine protease cathepsin L as a regulatory rather than a digestive protease and its action on podocyte structure and function. We provide arguments why many glomerular diseases can be regarded as podocyte enzymatic disorders. PMID:20130922

  2. Synthesis of an enzymatically active FLP recombinase in vitro: search for a DNA-binding domain.

    PubMed Central

    Amin, A A; Sadowski, P D

    1989-01-01

    We have used an in vitro transcription and translation system to synthesize an enzymatically active FLP protein. The FLP mRNA synthesized in vitro by SP6 polymerase is translated efficiently in a rabbit reticulocyte lysate to produce enzymatically active FLP. Using this system, we assessed the effect of deletions and tetrapeptide insertions on the ability of the respective variant proteins synthesized in vitro to bind to the FLP recognition target site and to carry out excisive recombination. Deletions of as few as six amino acids from either the carboxy- or amino-terminal region of FLP resulted in loss of binding activity. Likewise, insertions at amino acid positions 79, 203, and 286 abolished DNA-binding activity. On the other hand, a protein with an insertion at amino acid 364 retained significant DNA-binding activity but had no detectable recombination activity. Also, an insertion at amino acid 115 had no measurable effect on DNA binding, but recombination was reduced by 95%. In addition, an insertion at amino acid 411 had no effect on DNA binding and recombination. On the basis of these results, we conclude that this approach fails to define a discrete DNA-binding domain. The possible reasons for this result are discussed. Images PMID:2664465

  3. Role of Inactive and Active Trypanosoma cruzi Trans-sialidases on T Cell Homing and Secretion of Inflammatory Cytokines

    PubMed Central

    Freire-de-Lima, Leonardo; Gentile, Luciana B.; da Fonseca, Leonardo M.; da Costa, Kelli M.; Santos Lemos, Jessica; Jacques, Lucas Rodrigues; Morrot, Alexandre; Freire-de-Lima, Célio G.; Nunes, Marise P.; Takiya, Christina M.; Previato, Jose O.; Mendonça-Previato, Lucia

    2017-01-01

    Trans-sialidase from Trypanosoma cruzi (Tc-TS) belongs to a superfamily of proteins that may have enzymatic activity. While enzymatically active members (Tc-aTS) are able to transfer sialic acid from the host cell sialyl-glycoconjugates onto the parasite or to other molecules on the host cell surface, the inactive members (Tc-iTS) are characterized by their lectinic properties. Over the last 10 years, several papers demonstrated that, individually, Tc-aTS or Tc-iTS is able to modulate several biological events. Since the genes encoding Tc-iTS and Tc-aTS are present in the same copy number, and both proteins portray similar substrate-specificities as well, it would be plausible to speculate that such molecules may compete for the same sialyl-glycan structures and govern numerous immunobiological phenomena. However, their combined effect has never been evaluated in the course of an acute infection. In this study, we investigated the ability of both proteins to modulate the production of inflammatory signals, as well as the homing of T cells to the cardiac tissue of infected mice, events that usually occur during the acute phase of T. cruzi infection. The results showed that the intravenous administration of Tc-iTS, but not Tc-aTS protected the cardiac tissue from injury caused by reduced traffic of inflammatory cells. In addition, the ability of Tc-aTS to modulate the production of inflammatory cytokines was attenuated and/or compromised when Tc-iTS was co-injected in the same proportions. These results suggest that although both proteins present structural similarities and compete for the same sialyl-glycan epitopes, they might present distinct immunomodulatory properties on T cells following T. cruzi infection. PMID:28744279

  4. Effects of Physical (In)activity on Platelet Function.

    PubMed

    Heber, Stefan; Volf, Ivo

    2015-01-01

    As platelet activation is closely related to the liberation of growth factors and inflammatory mediators, platelets play a central role in the development of CVD. Virtually all cardiovascular risk factors favor platelet hyperreactivity and, accordingly, also physical (in)activity affects platelet function. Within this paper, we will summarize and discuss the current knowledge on the impact of acute and habitual exercise on platelet function. Although there are apparent discrepancies regarding the reported effects of acute, strenuous exercise on platelet activation, a deeper analysis of the available literature reveals that the applied exercise intensity and the subjects' cardiorespiratory fitness represent critical determinants for the observed effects. Consideration of these factors leads to the summary that (i) acute, strenuous exercise can lead to platelet activation, (ii) regular physical activity and/or physical fitness diminish or prevent platelet activation in response to acute exercise, and (iii) habitual physical activity and/or physical fitness also favorably modulate platelet function at physical rest. Notably, these effects of exercise on platelet function show obvious similarities to the well-recognized relation between exercise and the risk for cardiovascular events where vigorous exercise transiently increases the risk for myocardial infarction and a physically active lifestyle dramatically reduces cardiovascular mortality.

  5. EPA Facility Registry Service (FRS): RCRA_INACTIVE

    EPA Pesticide Factsheets

    This web feature service contains location and facility identification information from EPA's Facility Registry Service (FRS) for the subset of hazardous waste facilities that link to the Resource Conservation and Recovery Act Information System (RCRAInfo). EPA's comprehensive information system in support of the Resource Conservation and Recovery Act (RCRA) of 1976 and the Hazardous and Solid Waste Amendments (HSWA) of 1984, RCRAInfo tracks many types of information about generators, transporters, treaters, storers, and disposers of hazardous waste. FRS identifies and geospatially locates facilities, sites or places subject to environmental regulations or of environmental interest. Using vigorous verification and data management procedures, FRS integrates facility data from EPA's national program systems, other federal agencies, and State and tribal master facility records and provides EPA with a centrally managed, single source of comprehensive and authoritative information on facilities. This data set contains the subset of FRS integrated facilities that link to inactive RCRAInfo hazardous waste facilities once the RCRAInfo data has been integrated into the FRS database. Additional information on FRS is available at the EPA website https://www.epa.gov/enviro/facility-registry-service-frs.

  6. Effects of Physical (In)activity on Platelet Function

    PubMed Central

    Heber, Stefan; Volf, Ivo

    2015-01-01

    As platelet activation is closely related to the liberation of growth factors and inflammatory mediators, platelets play a central role in the development of CVD. Virtually all cardiovascular risk factors favor platelet hyperreactivity and, accordingly, also physical (in)activity affects platelet function. Within this paper, we will summarize and discuss the current knowledge on the impact of acute and habitual exercise on platelet function. Although there are apparent discrepancies regarding the reported effects of acute, strenuous exercise on platelet activation, a deeper analysis of the available literature reveals that the applied exercise intensity and the subjects' cardiorespiratory fitness represent critical determinants for the observed effects. Consideration of these factors leads to the summary that (i) acute, strenuous exercise can lead to platelet activation, (ii) regular physical activity and/or physical fitness diminish or prevent platelet activation in response to acute exercise, and (iii) habitual physical activity and/or physical fitness also favorably modulate platelet function at physical rest. Notably, these effects of exercise on platelet function show obvious similarities to the well-recognized relation between exercise and the risk for cardiovascular events where vigorous exercise transiently increases the risk for myocardial infarction and a physically active lifestyle dramatically reduces cardiovascular mortality. PMID:26557653

  7. Physical inactivity, neurological disability, and cardiorespiratory fitness in multiple sclerosis.

    PubMed

    Motl, R W; Goldman, M

    2011-02-01

    We examined the associations among physical activity, neurological disability, and cardiorespiratory fitness in two studies of individuals with multiple sclerosis (MS). Study 1 included 25 women with relapsing-remitting MS (RRMS) who undertook an incremental exercise test for measuring peak oxygen (VO₂(peak) ) consumption, wore an accelerometer during a 7-day period, and completed the Godin Leisure-Time Exercise Questionnaire (GLTEQ). Study 2 was a follow-up of Study 1 and included 24 women with RRMS who completed the self-reported Expanded Disability Status Scale (EDSS), undertook an incremental exercise test, wore an accelerometer during a 7-day period, and completed the GLTEQ. Study 1 indicated that VO₂(peak) was significantly correlated with accelerometer counts (pr = 0.69) and GLTEQ scores (pr = 0.63) even after controlling for age and MS duration. Study 2 indicated that VO₂(peak) was significantly correlated with accelerometer counts (pr = 0.50), GLTEQ scores (pr = 0.59), and EDSS scores (pr = -0.43) even after controlling for age and MS duration; there was a moderate partial correlation between accelerometer counts and EDSS scores (pr = -0.43). Multiple linear regression analysis indicated that both accelerometer counts (β = 0.32) and EDSS scores (β = -0.40) had statistically significant associations with VO₂(peak). The findings indicate that physical inactivity and neurological disability might represent independent risk factors for reduced levels of cardiorespiratory fitness in this population. © 2010 John Wiley & Sons A/S.

  8. The development of low temperature inactive (Lti) baker's yeast.

    PubMed

    Gysler, C; Niederberger, P

    2002-02-01

    The construction of a novel baker's yeast variety via traditional genetic techniques is described. The phenotype was designated "Lti" ("Low temperature inactive"). Lti mutations with the desired characteristics within a genetically well-defined haploid laboratory strain of Saccharomyces cerevisiae were isolated, and two different approaches were taken to obtain baker's yeast strains, which exhibit reduced fermenting activity at refrigeration temperatures. In a first approach, a chosen Lti strain carrying mutation lti9 was combined with other laboratory strains carrying defined MAL alleles. In a second approach, the same lti mutation was introduced in the genetic background of polyploid commercial baker's yeast strains that harbor important "industrial" properties. Lti strains arising from both approaches were characterized with specifically developed screening procedures. Strains of the "academic" Lti strain family displayed between 85% and 92% of the biomass yield of a commercial reference strain, whereas strains of the "industrial" Lti strain family showed a variation between 60% and 115%. Lti strains from both families varied strongly among each other in their activity in model doughs: at 8 degrees C they displayed activities between 5% and 30%, and at 30 degrees C between 40% and 113% of a commercial reference baker's yeast strain.

  9. Neighborhood safety and physical inactivity in adults from Curitiba, Brazil.

    PubMed

    Rech, Cassiano Ricardo; Reis, Rodrigo Siqueira; Hino, Adriano Akira Ferreira; Rodriguez-Añez, Ciro Romélio; Fermino, Rogério Cesar; Gonçalves, Priscila Bezerra; Hallal, Pedro Curi

    2012-06-12

    Neighborhood safety is one of the environmental aspects that can influence physical activity. We analyzed the association between perceived neighborhood safety and physical inactivity (PI) in adults and examined effect modification according to sociodemographic variables. A cross-sectional study was conducted with 1,261 adults (62% women), age 18-69 years from Curitiba, Brazil. The perception of unsafe neighborhood was higher among women, older participants, those classified in the high socioeconomic (SES) group, overweighed and also among those reporting to have PA equipments and children. The association between perception safety of walking during the day and walking for leisure (women PR=1.12 CI95%=1.02-1.22; men PR=0.82 CI95%=0.64-1.05; interaction term PR=1.38 CI95%=1.03-1.83) and safe perception was associated with PI, just in the highest SES group (PR=1.09; CI95%=1.00-1.19; p trend=0.032) when compared with their counterparts (low SES PR=0.99; CI95%=0.90-1.04; p trend=0.785; interaction term PR=1.09; CI95%=1.03-1.15; p trend=0.007). The perception of safety in the neighborhood was associated with PI in transport, but this association varies across of sociodemographic variables.

  10. Adolescent reproductive health in Indonesia: contested values and policy inaction.

    PubMed

    Utomo, Iwu Dwisetyani; McDonald, Peter

    2009-06-01

    This study examines the changing social and political context of adolescent sexual and reproductive health policy in Indonesia. We describe how, in 2001, Indonesia was on the brink of implementing an adolescent reproductive health policy that was consistent with international agreements to which the Indonesian government was a party. Although the health of young Indonesians was known to be at risk, the opportunity for reform passed quickly with the emergence of a new competing force, Middle Eastern fundamentalist Islam. Faced with the risk of regional separatism and competing politico-religious influences, the Indonesian government retreated to the safety of inaction in this area of policy. In the absence of a supportive and committed political environment that reinforces policy specifically targeted to young people's reproductive health, extremist approaches that involve considerable health risk prevailed. The sexual and reproductive values and behaviors that are emerging among single young people in contemporary Indonesia are conditioned by a political context that allows the conflicting forces of traditional Indonesian values, Westernization, and the strong emerging force of fundamentalist Islam to compete for the allegiance of young people.

  11. [Burden of mortality associated to physical inactivity in Bogota, Colombia].

    PubMed

    Lobelo, Felipe; Pate, Rusell; Parra, Diana; Duperly, John; Pratt, Michael

    2006-12-01

    Estimates of the burden of mortality associated to physical inactivity (PI) have not been quantified for large urban centers located in developing countries. To estimate the burden of mortality due to six chronic diseases (CDZ) associated to PI and the number of potentially preventable deaths associated to reductions in the prevalence of PI. PI exposure prevalence obtained via population surveys was linked to mortality data registered during 2002 among adult (> 45 y) Bogotá residents. The strength of association between PI and disease-specific mortality was obtained from the literature. Population attributable risk (PAR) was used to calculate the CDZ mortality attributable to PI and to estimate the number of potentially preventable deaths associated to a 30 % reduction in the prevalence of PI. A 53,2 % PI exposure prevalence was associated to a PAR of 19,3 % for coronary artery disease, 24,2 % for stroke, 13,8 % for arterial hypertension, 21 % for Diabetes Mellitus, 17,9 % for colon cancer and 14,2 % for breast cancer. An estimated 7,6 % of all-cause mortality and 20,1 % of CDZ mortality could be attributed to PI. An estimated 5% of the CDZ mortality could be prevented if PI prevalence is reduced by 30 %. Conservative estimates indicate that a considerable proportion of deaths due to highly prevalent CDZ could be attributed to PI. Strategies to reduce the prevalence of PI in Bogotá could lead to progressive reductions in the burden of CDZ mortality.

  12. Hedgehog signaling pathway is inactive in colorectal cancer cell lines.

    PubMed

    Chatel, Guillaume; Ganeff, Corine; Boussif, Naima; Delacroix, Laurence; Briquet, Alexandra; Nolens, Gregory; Winkler, Rosita

    2007-12-15

    The Hedgehog (Hh) signaling pathway plays an important role in human development. Abnormal activation of this pathway has been observed in several types of human cancers, such as the upper gastro-intestinal tract cancers. However, activation of the Hh pathway in colorectal cancers is controversial. We analyzed the expression of the main key members of the Hh pathway in 7 colon cancer cell lines in order to discover whether the pathway is constitutively active in these cells. We estimated the expression of SHH, IHH, PTCH, SMO, GLI1, GLI2, GLI3, SUFU and HHIP genes by RT-PCR. Moreover, Hh ligand, Gli3 and Sufu protein levels were quantified by western blotting. None of the cell lines expressed the complete set of Hh pathway members. The ligands were absent from Colo320 and HCT116 cells, Smo from Colo205, HT29 and WiDr. GLI1 gene was not expressed in SW480 cells nor were GLI2/GLI3 in Colo205 or Caco-2 cells. Furthermore the repressive form of Gli3, characteristic of an inactive pathway, was detected in SW480 and Colo320 cells. Finally treatment of colon cancer cells with cyclopamine, a specific inhibitor of the Hh pathway, did not downregulate PTCH and GLI1 genes expression in the colorectal cells, whereas it did so in PANC1 control cells. Taken together, these results indicate that the aberrant activation of the Hh signaling pathway is not common in colorectal cancer cell lines.

  13. [Juvenile obesity and the role of physical activity and inactivity].

    PubMed

    Graf, C

    2010-07-01

    Physical activity has important health benefits. Despite of the use of different measurement instruments, a decrease in physical activity and an increase in sedentary habits has been described in children and adolescents. As a consequence, a reduction in physical performance and motor abilities and an increase in overweight and fat mass is found associated with comorbidities, e.g., ranging from insulin resistance up to the metabolic syndrome. Therefore, beside the therapeutic use of exercise in obesity programs, adequate preventive strategies are warranted. However, within this discussion, it must be taken into consideration that special subgroups are more affected by insufficient physical activity/sedentary habits, e.g., females, adolescents, ethnicity, lower socioeconomic status. In many other groups, recommendations for physical activity (1 h/day) are achieved. Hence, interventions must focus on these at-risk groups and intensified. In addition, recommendations related to physical activity and inactivity, in terms of TV consumption, must be critically analyzed as to whether the recommendations are sufficient and how they can be implemented to achieve lasting results.

  14. Resurrecting inactive antimicrobial peptides from the lipopolysaccharide trap.

    PubMed

    Mohanram, Harini; Bhattacharjya, Surajit

    2014-01-01

    Host defense antimicrobial peptides (AMPs) are a promising source of antibiotics for the treatment of multiple-drug-resistant pathogens. Lipopolysaccharide (LPS), the major component of the outer leaflet of the outer membrane of Gram-negative bacteria, functions as a permeability barrier against a variety of molecules, including AMPs. Further, LPS or endotoxin is the causative agent of sepsis killing 100,000 people per year in the United States alone. LPS can restrict the activity of AMPs inducing aggregations at the outer membrane, as observed for frog AMPs, temporins, and also in model AMPs. Aggregated AMPs, "trapped" by the outer membrane, are unable to traverse the cell wall, causing their inactivation. In this work, we show that these inactive AMPs can overcome LPS-induced aggregations while conjugated with a short LPS binding β-boomerang peptide motif and become highly bactericidal. The generated hybrid peptides exhibit activity against Gram-negative and Gram-positive bacteria in high-salt conditions and detoxify endotoxin. Structural and biophysical studies establish the mechanism of action of these peptides in LPS outer membrane. Most importantly, this study provides a new concept for the development of a potent broad-spectrum antibiotic with efficient outer membrane disruption as the mode of action.

  15. Histone acetylation controls the inactive X chromosome replication dynamics

    PubMed Central

    Casas-Delucchi, Corella S.; Brero, Alessandro; Rahn, Hans-Peter; Solovei, Irina; Wutz, Anton; Cremer, Thomas; Leonhardt, Heinrich; Cardoso, M. Cristina

    2011-01-01

    In mammals, dosage compensation between male and female cells is achieved by inactivating one female X chromosome (Xi). Late replication of Xi was proposed to be involved in the maintenance of its silenced state. Here, we show a highly synchronous replication of the Xi within 1 to 2 h during early-mid S-phase by following DNA replication in living mammalian cells with green fluorescent protein-tagged replication proteins. The Xi was replicated before or concomitant with perinuclear or perinucleolar facultative heterochromatin and before constitutive heterochromatin. Ectopic expression of the X-inactive-specific transcript (Xist) gene from an autosome imposed the same synchronous replication pattern. We used mutations and chemical inhibition affecting different epigenetic marks as well as inducible Xist expression and we demonstrate that histone hypoacetylation has a key role in controlling Xi replication. The epigenetically controlled, highly coordinated replication of the Xi is reminiscent of embryonic genome replication in flies and frogs before genome activation and might be a common feature of transcriptionally silent chromatin. PMID:21364561

  16. Mice with Catalytically Inactive Cathepsin A Display Neurobehavioral Alterations

    PubMed Central

    Calhan, O. Y.

    2017-01-01

    The lysosomal carboxypeptidase A, Cathepsin A (CathA), is a serine protease with two distinct functions. CathA protects β-galactosidase and sialidase Neu1 against proteolytic degradation by forming a multienzyme complex and activates sialidase Neu1. CathA deficiency causes the lysosomal storage disease, galactosialidosis. These patients present with a broad range of clinical phenotypes, including growth retardation, and neurological deterioration along with the accumulation of the vasoactive peptide, endothelin-1, in the brain. Previous in vitro studies have shown that CathA has specific activity against vasoactive peptides and neuropeptides, including endothelin-1 and oxytocin. A mutant mouse with catalytically inactive CathA enzyme (CathAS190A) shows increased levels of endothelin-1. In the present study, we elucidated the involvement of CathA in learning and long-term memory in 3-, 6-, and 12-month-old mice. Hippocampal endothelin-1 and oxytocin accumulated in CathAS190A mice, which showed learning impairments as well as long-term and spatial memory deficits compared with wild-type littermates, suggesting that CathA plays a significant role in learning and in memory consolidation through its regulatory role in vasoactive peptide processing. PMID:28133419

  17. Home Delivery Medicament Program: access, inactivity and cardiovascular risk.

    PubMed

    Araújo, Roque da Silva; Arcuri, Edna Apparecida Moura; Lopes, Victor Cauê

    2016-10-10

    to verify causes of inactivity in the Home Delivery Medicament Program, as referred by users from a Primary Health Care Service in São Paulo, comparing them to the causes registered in the program and analyzing them in the theoretical model Concept of Access to Health. cross-sectional study, interviewing 111 inactive users; and documentary study in the program records. half of the users did not know the condition of inactivity. Discrepancies were found between the user's and the program's information, observing different levels of agreement: Absence of physician and administrative staff member 0%; Transfer to other service 25%; Death 50%; Option to quit 50%; Address change 57% and Change in therapeutic schedule 80%. The users' feeling of accepting the program was observed. In the health access concept, inactivity can be explained in the information dimension, in the degree of asymmetry between the patient's and the health professional's knowledge, identified through the indicators: education, knowledge and information sources. due to the low education level, the user does not assimilate the information on the steps of the program flowchart, does not return for the assessment that guarantees its continuity. Consequently, (s)he stops receiving the medication and spends a long time without treatment, increasing the cardiovascular risk of hypertensive (92% of the sample), diabetic (44%) and dyslipidemic patients (31%). verificar causas de inatividade no Programa Remédio em Casa, referidas por usuários de Unidade Básica de Saúde de São Paulo, comparando-as às registradas pelo programa e analisando-as no modelo teórico Conceito de Acesso à Saúde. estudo transversal entrevistando 111 usuários inativos; e documental, nos registros do programa. metade dos usuários desconhecia a condição de inatividade. Constatadas discrepâncias nas informações usuário versus programa, observando-se diferentes níveis de concordância: Falta de médico e funcion

  18. Burden of physical inactivity and hospitalization costs due to chronic diseases

    PubMed Central

    Bielemann, Renata Moraes; da Silva, Bruna Gonçalves Cordeiro; Coll, Carolina de Vargas Nunes; Xavier, Mariana Otero; da Silva, Shana Ginar

    2015-01-01

    OBJECTIVE To evaluate the physical inactivity-related inpatient costs of chronic non-communicable diseases. METHODS This study used data from 2013, from Brazilian Unified Health System, regarding inpatient numbers and costs due to malignant colon and breast neoplasms, cerebrovascular diseases, ischemic heart diseases, hypertension, diabetes, and osteoporosis. In order to calculate the share physical inactivity represents in that, the physical inactivity-related risks, which apply to each disease, were considered, and physical inactivity prevalence during leisure activities was obtained from Pesquisa Nacional por Amostra de Domicílio (Brazil’s National Household Sample Survey). The analysis was stratified by genders and residing country regions of subjects who were 40 years or older. The physical inactivity-related hospitalization cost regarding each cause was multiplied by the respective share it regarded to. RESULTS In 2013, 974,641 patients were admitted due to seven different causes in Brazil, which represented a high cost. South region was found to have the highest patient admission rate in most studied causes. The highest prevalences for physical inactivity were observed in North and Northeast regions. The highest inactivity-related share in men was found for osteoporosis in all regions (≈ 35.0%), whereas diabetes was found to have a higher share regarding inactivity in women (33.0% to 37.0% variation in the regions). Ischemic heart diseases accounted for the highest total costs that could be linked to physical inactivity in all regions and for both genders, being followed by cerebrovascular diseases. Approximately 15.0% of inpatient costs from Brazilian Unified Health System were connected to physical inactivity. CONCLUSIONS Physical inactivity significantly impacts the number of patient admissions due to the evaluated causes and through their resulting costs, with different genders and country regions representing different shares. PMID:26487291

  19. Network social capital, social participation, and physical inactivity in an urban adult population.

    PubMed

    Legh-Jones, Hannah; Moore, Spencer

    2012-05-01

    Research on individual social capital and physical activity has tended to focus on the association among physical activity, generalized trust, and social participation. Less is known about the association between network social capital, i.e., the resources accessed through one's social connections, and physical inactivity. Using formal network measures of social capital, this study examined which specific dimension of network capital (i.e. diversity, reach and range) was associated with physical inactivity, and whether network social capital mediated the association between physical inactivity and social participation. Data came from the 2008 Montreal (Canada) Neighbourhood Networks and Healthy Aging survey, in which 2707 adults 25 years and older in 300 Montreal neighbourhoods were surveyed. Physical activity was self-reported using the International Physical Activity Questionnaire (IPAQ). IPAQ guidelines provided the basis for the physical inactivity cutoff. Network social capital was measured with a position generator instrument. Multilevel logistic methods were used to examine the association between physical inactivity and individual social capital dimensions, while adjusting for socio-demographic and -economic factors. Higher network diversity was associated with a decreased likelihood of physical inactivity. Consistent with previous findings, individuals who did not participate in any formal associations were more likely to be physically inactive compared to those with high levels of participation. Network diversity mediated the association between physical inactivity and participation. Generalized trust and the network components of reach and range were not shown associated with physical inactivity. Findings highlight the importance of social participation and network social capital and the added value of network measures in the study of social capital and physical inactivity. Population-based programs targeting physical inactivity among adults might

  20. Ethanol production from lignocellulosic biomass by recombinant Escherichia coli strain FBR5

    USDA-ARS?s Scientific Manuscript database

    Lignocellulosic biomass, upon pretreatment and enzymatic hydrolysis, generates a mixture of hexose and pentose sugars such as glucose, xylose, arabinose and galactose. Escherichia coli utilizes all these sugars well but it lacks the ability to produce ethanol from them. Recombinant ethanologenic E...

  1. Enhancement of xylose utilization from corn stover by a recombinant bacterium for ethanol production

    USDA-ARS?s Scientific Manuscript database

    Effects of substrate-selective inoculum prepared by growing on glucose, xylose, arabinose, GXA (glucose, xylose, arabinose, 1:1:1) and corn stover hydrolyzate (dilute acid pretreated and enzymatically hydrolyzed, CSH) on ethanol production from CSH by a mixed sugar utilizing recombinant Escherichia ...

  2. Enzymatic Kolbe-Schmitt reaction to form salicylic acid from phenol: enzymatic characterization and gene identification of a novel enzyme, Trichosporon moniliiforme salicylic acid decarboxylase.

    PubMed

    Kirimura, Kohtaro; Gunji, Hiroaki; Wakayama, Rumiko; Hattori, Takasumi; Ishii, Yoshitaka

    2010-04-02

    Salicylic acid decarboxylase (Sdc) can produce salicylic acid from phenol; it was found in the yeast Trichosporon moniliiforme WU-0401 and was for the first time enzymatically characterized, with the sdc gene heterologously expressed. Sdc catalyzed both reactions: decarboxylation of salicylic acid to phenol and the carboxylation of phenol to form salicylic acid without any byproducts. Both reactions were detected without the addition of any cofactors and occurred even in the presence of oxygen, suggesting that this Sdc is reversible, nonoxidative, and oxygen insensitive. Therefore, it is readily applicable in the selective production of salicylic acid from phenol, the enzymatic Kolbe-Schmitt reaction. The deduced amino acid sequence of the gene, sdc, encoding Sdc comprises 350 amino acid residues corresponding to a 40-kDa protein. The recombinant Escherichia coli BL21(DE3) expressing sdc converted phenol to salicylic acid with a 27% (mol/mol) yield at 30 degrees C for 9h.

  3. Recombination and Replication

    PubMed Central

    Syeda, Aisha H.; Hawkins, Michelle; McGlynn, Peter

    2014-01-01

    The links between recombination and replication have been appreciated for decades and it is now generally accepted that these two fundamental aspects of DNA metabolism are inseparable: Homologous recombination is essential for completion of DNA replication and vice versa. This review focuses on the roles that recombination enzymes play in underpinning genome duplication, aiding replication fork movement in the face of the many replisome barriers that challenge genome stability. These links have many conserved features across all domains of life, reflecting the conserved nature of the substrate for these reactions, DNA. PMID:25341919

  4. Dissociative recombination in aeronomy

    NASA Technical Reports Server (NTRS)

    Fox, J. L.

    1989-01-01

    The importance of dissociative recombination in planetary aeronomy is summarized, and two examples are discussed. The first is the role of dissociative recombination of N2(+) in the escape of nitrogen from Mars. A previous model is updated to reflect new experimental data on the electronic states of N produced in this process. Second, the intensity of the atomic oxygen green line on the nightside of Venus is modeled. Use is made of theoretical rate coefficients for production of O (1S) in dissociative recombination from different vibrational levels of O2(+).

  5. The landscape of recombination in African Americans.

    PubMed

    Hinch, Anjali G; Tandon, Arti; Patterson, Nick; Song, Yunli; Rohland, Nadin; Palmer, Cameron D; Chen, Gary K; Wang, Kai; Buxbaum, Sarah G; Akylbekova, Ermeg L; Aldrich, Melinda C; Ambrosone, Christine B; Amos, Christopher; Bandera, Elisa V; Berndt, Sonja I; Bernstein, Leslie; Blot, William J; Bock, Cathryn H; Boerwinkle, Eric; Cai, Qiuyin; Caporaso, Neil; Casey, Graham; Cupples, L Adrienne; Deming, Sandra L; Diver, W Ryan; Divers, Jasmin; Fornage, Myriam; Gillanders, Elizabeth M; Glessner, Joseph; Harris, Curtis C; Hu, Jennifer J; Ingles, Sue A; Isaacs, William; John, Esther M; Kao, W H Linda; Keating, Brendan; Kittles, Rick A; Kolonel, Laurence N; Larkin, Emma; Le Marchand, Loic; McNeill, Lorna H; Millikan, Robert C; Murphy, Adam; Musani, Solomon; Neslund-Dudas, Christine; Nyante, Sarah; Papanicolaou, George J; Press, Michael F; Psaty, Bruce M; Reiner, Alex P; Rich, Stephen S; Rodriguez-Gil, Jorge L; Rotter, Jerome I; Rybicki, Benjamin A; Schwartz, Ann G; Signorello, Lisa B; Spitz, Margaret; Strom, Sara S; Thun, Michael J; Tucker, Margaret A; Wang, Zhaoming; Wiencke, John K; Witte, John S; Wrensch, Margaret; Wu, Xifeng; Yamamura, Yuko; Zanetti, Krista A; Zheng, Wei; Ziegler, Regina G; Zhu, Xiaofeng; Redline, Susan; Hirschhorn, Joel N; Henderson, Brian E; Taylor, Herman A; Price, Alkes L; Hakonarson, Hakon; Chanock, Stephen J; Haiman, Christopher A; Wilson, James G; Reich, David; Myers, Simon R

    2011-07-20

    Recombination, together with mutation, gives rise to genetic variation in populations. Here we leverage the recent mixture of people of African and European ancestry in the Americas to build a genetic map measuring the probability of crossing over at each position in the genome, based on about 2.1 million crossovers in 30,000 unrelated African Americans. At intervals of more than three megabases it is nearly identical to a map built in Europeans. At finer scales it differs significantly, and we identify about 2,500 recombination hotspots that are active in people of West African ancestry but nearly inactive in Europeans. The probability of a crossover at these hotspots is almost fully controlled by the alleles an individual carries at PRDM9 (P value < 10(-245)). We identify a 17-base-pair DNA sequence motif that is enriched in these hotspots, and is an excellent match to the predicted binding target of PRDM9 alleles common in West Africans and rare in Europeans. Sites of this motif are predicted to be risk loci for disease-causing genomic rearrangements in individuals carrying these alleles. More generally, this map provides a resource for research in human genetic variation and evolution.

  6. The landscape of recombination in African Americans

    PubMed Central

    Hinch, Anjali G.; Tandon, Arti; Patterson, Nick; Song, Yunli; Rohland, Nadin; Palmer, Cameron D.; Chen, Gary K.; Wang, Kai; Buxbaum, Sarah G.; Akylbekova, Meggie; Aldrich, Melinda C.; Ambrosone, Christine B.; Amos, Christopher; Bandera, Elisa V.; Berndt, Sonja I.; Bernstein, Leslie; Blot, William J.; Bock, Cathryn H.; Boerwinkle, Eric; Cai, Qiuyin; Caporaso, Neil; Casey, Graham; Cupples, L. Adrienne; Deming, Sandra L.; Diver, W. Ryan; Divers, Jasmin; Fornage, Myriam; Gillanders, Elizabeth M.; Glessner, Joseph; Harris, Curtis C.; Hu, Jennifer J.; Ingles, Sue A.; Isaacs, Williams; John, Esther M.; Kao, W. H. Linda; Keating, Brendan; Kittles, Rick A.; Kolonel, Laurence N.; Larkin, Emma; Le Marchand, Loic; McNeill, Lorna H.; Millikan, Robert C.; Murphy, Adam; Musani, Solomon; Neslund-Dudas, Christine; Nyante, Sarah; Papanicolaou, George J.; Press, Michael F.; Psaty, Bruce M.; Reiner, Alex P.; Rich, Stephen S.; Rodriguez-Gil, Jorge L.; Rotter, Jerome I.; Rybicki, Benjamin A.; Schwartz, Ann G.; Signorello, Lisa B.; Spitz, Margaret; Strom, Sara S.; Thun, Michael J.; Tucker, Margaret A.; Wang, Zhaoming; Wiencke, John K.; Witte, John S.; Wrensch, Margaret; Wu, Xifeng; Yamamura, Yuko; Zanetti, Krista A.; Zheng, Wei; Ziegler, Regina G.; Zhu, Xiaofeng; Redline, Susan; Hirschhorn, Joel N.; Henderson, Brian E.; Taylor, Herman A.; Price, Alkes L.; Hakonarson, Hakon; Chanock, Stephen J.; Haiman, Christopher A.; Wilson, James G.; Reich, David; Myers, Simon R.

    2011-01-01

    Recombination, together with mutation, is the ultimate source of genetic variation in populations. We leverage the recent mixture of people of African and European ancestry in the Americas to build a genetic map measuring the probability of crossing-over at each position in the genome, based on about 2.1 million crossovers in 30,000 unrelated African Americans. At intervals of more than three megabases it is nearly identical to a map built in Europeans. At finer scales it differs significantly, and we identify about 2,500 recombination hotspots that are active in people of West African ancestry but nearly inactive in Europeans. The probability of a crossover at these hotspots is almost fully controlled by the alleles an individual carries at PRDM9 (P<10−245). We identify a 17 base pair DNA sequence motif that is enriched in these hotspots, and is an excellent match to the predicted binding target of African-enriched alleles of PRDM9. PMID:21775986

  7. Social determinants of physical inactivity in the Northern Manhattan Study (NOMAS)

    PubMed Central

    Willey, Joshua Z.; Paik, Myunghee C; Sacco, Ralph; Elkind, Mitchell S.V.; Boden-Albala, Bernadette

    2010-01-01

    Background Physical inactivity is an important and modifiable cardiovascular disease risk factor. Little is known about the social determinants of physical inactivity in older, urban-dwelling populations. Methods We collected socio-demographic and medical risk factor information and physical activity questionnaires in the Northern Manhattan Study. Logistic regression models were constructed to examine whether measures of social isolation, race-ethnicity, and sex were associated with physical inactivity. Results Physical inactivity was present in 40.5% of the cohort. In multivariable models adjusted for medical comorbidities, Hispanic race-ethnicity (compared to non-Hispanic white) was associated with higher odds of physical inactivity (OR 2.18, 95% CI 1.78, 2.67), while women were more likely to be inactive than men (OR 1.33, 95% CI 1.15, 1.54). Having Medicaid/being uninsured (OR 1.20, 95% CI 1.02, 1.42), and having fewer than 3 friends (1.41, 95% CI 1.15, 1.72) were also associated with physical inactivity. Conclusions Physical inactivity is common, particularly in Hispanics, women, and those who are socially isolated. Public health interventions aimed at increasing physical activity in these more sedentary groups are required. PMID:20574777

  8. Excess Medical Care Costs Associated with Physical Inactivity among Korean Adults: Retrospective Cohort Study.

    PubMed

    Min, Jin-Young; Min, Kyoung-Bok

    2016-01-18

    Physical inactivity is a major risk factor for chronic diseases and premature death. The increased health risks associated with physical inactivity may also generate a heavier economic burden to society. We estimated the direct medical costs attributable to physical inactivity among adults using data from the 2002-2010 Korean National Health Insurance Service-National Sample Cohort. A total of 68,556 adults whose reported physical activity status did not change during the study period was included for this study. Propensity scores for inactive adults were used to match 23,645 inactive groups with 23,645 active groups who had similar propensity scores. We compared medical expenditures between the two groups using generalized linear models with a gamma distribution and a log link. Direct medical costs were based on the reimbursement records of all medical facilities from 2005 to 2010. The average total medical costs for inactive individuals were $1110.5, which was estimated to be 11.7% higher than the costs for physically active individuals. With respect to specific diseases, the medical costs of inactive people were significantly higher than those of active people, accounting for approximately 8.7% to 25.3% of the excess burden. Physical inactivity is associated with considerable medical care expenditures per capita among Korean adults.

  9. 37 CFR 11.20 - Disciplinary sanctions; Transfer to disability inactive status.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...; Transfer to disability inactive status. 11.20 Section 11.20 Patents, Trademarks, and Copyrights UNITED..., Investigations, and Proceedings § 11.20 Disciplinary sanctions; Transfer to disability inactive status. (a) Types... practitioner take and pass a professional responsibility examination. (c) Transfer to disability...

  10. Modeling the minimum enzymatic requirements for optimal cellulose conversion

    NASA Astrophysics Data System (ADS)

    den Haan, R.; van Zyl, J. M.; Harms, T. M.; van Zyl, W. H.

    2013-06-01

    Hydrolysis of cellulose is achieved by the synergistic action of endoglucanases, exoglucanases and β-glucosidases. Most cellulolytic microorganisms produce a varied array of these enzymes and the relative roles of the components are not easily defined or quantified. In this study we have used partially purified cellulases produced heterologously in the yeast Saccharomyces cerevisiae to increase our understanding of the roles of some of these components. CBH1 (Cel7), CBH2 (Cel6) and EG2 (Cel5) were separately produced in recombinant yeast strains, allowing their isolation free of any contaminating cellulolytic activity. Binary and ternary mixtures of the enzymes at loadings ranging between 3 and 100 mg g-1 Avicel allowed us to illustrate the relative roles of the enzymes and their levels of synergy. A mathematical model was created to simulate the interactions of these enzymes on crystalline cellulose, under both isolated and synergistic conditions. Laboratory results from the various mixtures at a range of loadings of recombinant enzymes allowed refinement of the mathematical model. The model can further be used to predict the optimal synergistic mixes of the enzymes. This information can subsequently be applied to help to determine the minimum protein requirement for complete hydrolysis of cellulose. Such knowledge will be greatly informative for the design of better enzymatic cocktails or processing organisms for the conversion of cellulosic biomass to commodity products.

  11. Surface recombination in semiconductors

    SciTech Connect

    Langer, J.M.; Walukiewicz, W.

    1995-07-01

    We propose two general criteria for a surface defect state to act as an efficient, nonradiative recombination center. The first is that the thermal ionization energy should not deviate from the mid-gap energy by more than the relaxation energy of the defect, In this case the activation energy for the recombination is given by the barrier for the capture of the first carrier, whereas the second carrier is captured athermally. The second citerion is related to the position of the average dangling bond energy relative to the band edges. If, as in the cases of InP or InAs, it is located close to a band edge, a low surface recombination velocity is expected. However a much faster recombination is predicated and experimentally observed in the materials with the average dangling bond energy located close to the mid-gap. The relevance of these criteria for the novel wide-gap optoelectronic materials is discussed.

  12. Relationship between Physical Inactivity and Health Characteristics among Participants in an Employee Wellness Program

    PubMed Central

    Birdee, Gurjeet S.; Byrne, Daniel W.; McGown, Paula W.; Rothman, Russell L.; Rolando, Lori A.; Holmes, Marilyn C.; Yarbrough, Mary I.

    2013-01-01

    Objective To characterize factors associated with physical inactivity among employees with access to workplace wellness program. Methods We examined data on physical inactivity, defined as exercise less than once a week, from the 2010 health risk assessment (HRA) completed by employees at a major academic institution (n=16,976). Results Among employees, 18% individuals reported physical activity less than once a week. Individuals who were physically inactive as compared with physically active reported higher prevalence of cardiovascular diseases (AOR 1.36 [1.23–1.51], fair or poor health status (AOR 3.52 [2.97–4.17]) and absenteeism from work (AOR 1.59 [1.41–1.79]). Overall, physically inactive employees as compared to physically active employees reported more interest in health education programs. Conclusions Future research is needed to address barriers to physical inactivity to improve employee wellness and potentially lower health utility costs. PMID:23618884

  13. Motor-Driven (Passive) Cycling: A Potential Physical Inactivity Countermeasure?

    PubMed

    Peterman, James E; Wright, Kenneth P; Melanson, Edward L; Kram, Rodger; Byrnes, William C

    2016-09-01

    We have previously shown that motor-driven (passive) stationary cycling elevates energy expenditure (EE). This study aimed to quantify how acute passive cycling affects glucose and insulin responses to an oral glucose tolerance test (OGTT) and basic cognition compared with sitting and moderate-intensity active cycling. Twenty-four physically inactive healthy males completed three trials in randomized order involving 30-min conditions of sitting, passive cycling, and moderate-intensity cycling. During each condition, EE was measured, and participants performed cognitive tests. After each condition, a 2-h OGTT was performed. EE was significantly higher during the cycling conditions compared with sitting (1.36 ± 0.58 and 6.50 ± 1.73 kcal·min greater than sitting for passive and moderate-intensity, respectively). A significant correlation was found between body fat percentage and postsitting OGTT 2-h postplasma glucose (r = 0.30, P < 0.05); thus, participants were divided into lean (n = 11) and nonlean (n = 13) groups. In the nonlean group, compared with sitting, passive cycling lowered 2-h postplasma glucose (7.7 ± 1.3 vs 6.9 ± 1.6 mmol·L, respectively, P < 0.05), and the Matsuda whole-body insulin sensitivity index (WBISI) was higher (2.74 ± 0.86 vs 3.36 ± 1.08, P < 0.05). In addition, passive and moderate-intensity cycling had similar beneficial effects on 2-h postplasma glucose and WBISI. Cognitive performance did not significantly differ between the sitting and passive cycling conditions. Two-hour postplasma glucose was lower and WBISI after acute passive cycling was higher in nonlean participants. Given that and the increase in EE without changes in cognitive performance, we propose passive cycling as a promising intervention to counteract some of the deleterious effects of prolonged sitting in the workplace.

  14. Physical inactivity, neurological disability, and cardiorespiratory fitness in multiple sclerosis

    PubMed Central

    Motl, R. W.; Goldman, M.

    2011-01-01

    Objective We examined the associations among physical activity, neurological disability, and cardiorespiratory fitness in two studies of individuals with multiple sclerosis (MS). Method Study 1 included 25 women with relapsing-remitting MS (RRMS) who undertook an incremental exercise test for measuring peak oxygen (V̇O2peak) consumption, wore an accelerometer during a 7-day period, and completed the Godin Leisure-Time Exercise Questionnaire (GLTEQ). Study 2 was a follow-up of Study 1 and included 24 women with RRMS who completed the self-reported Expanded Disability Status Scale (EDSS), undertook an incremental exercise test, wore an accelerometer during a 7-day period, and completed the GLTEQ. Results Study 1 indicated that V̇O2peak was significantly correlated with accelerometer counts (pr = 0.69) and GLTEQ scores (pr = 0.63) even after controlling for age and MS duration. Study 2 indicated that V̇O2peak was significantly correlated with accelerometer counts (pr = 0.50), GLTEQ scores (pr = 0.59), and EDSS scores (pr = −0.43) even after controlling for age and MS duration; there was a moderate partial correlation between accelerometer counts and EDSS scores (pr = −0.43). Multiple linear regression analysis indicated that both accelerometer counts (β = 0.32) and EDSS scores (β = −0.40) had statistically significant associations with V̇O2peak. Conclusion The findings indicate that physical inactivity and neurological disability might represent independent risk factors for reduced levels of cardiorespiratory fitness in this population. PMID:21108624

  15. Effects of Physical Activity and Inactivity on Muscle Fatigue

    PubMed Central

    Bogdanis, Gregory C.

    2012-01-01

    The aim of this review was to examine the mechanisms by which physical activity and inactivity modify muscle fatigue. It is well known that acute or chronic increases in physical activity result in structural, metabolic, hormonal, neural, and molecular adaptations that increase the level of force or power that can be sustained by a muscle. These adaptations depend on the type, intensity, and volume of the exercise stimulus, but recent studies have highlighted the role of high intensity, short-duration exercise as a time-efficient method to achieve both anaerobic and aerobic/endurance type adaptations. The factors that determine the fatigue profile of a muscle during intense exercise include muscle fiber composition, neuromuscular characteristics, high energy metabolite stores, buffering capacity, ionic regulation, capillarization, and mitochondrial density. Muscle fiber-type transformation during exercise training is usually toward the intermediate type IIA at the expense of both type I and IIx myosin heavy-chain isoforms. High-intensity training results in increases of both glycolytic and oxidative enzymes, muscle capillarization, improved phosphocreatine resynthesis and regulation of K+, H+, and lactate ions. Decreases of the habitual activity level due to injury or sedentary lifestyle result in partial or even compete reversal of the adaptations due to previous training, manifested by reductions in fiber cross-sectional area, decreased oxidative capacity, and capillarization. Complete immobilization due to injury results in markedly decreased force output and fatigue resistance. Muscle unloading reduces electromyographic activity and causes muscle atrophy and significant decreases in capillarization and oxidative enzymes activity. The last part of the review discusses the beneficial effects of intermittent high-intensity exercise training in patients with different health conditions to demonstrate the powerful effect of exercise on health and well being. PMID

  16. Superstructures of wet inactive chromatin and the chromosome surface.

    PubMed

    Basu, S

    1979-01-01

    Superpacking of chromatin and the surface features of metaphase chromosomes have been studied by SiO replication of wet, unstained, and unfixed specimens in an exceedingly thin (less than or equal to nm) aqueous layer, keeping them wet. Hydrophilic Formvar substrates allow controlled thinning of the aqueous layer covering the wet specimens. Whole mounts of chromatin and chromosomes were prepared by applying a microsurface spreading method to swollen nuclei and mitotic cells at metaphase. The highest level of nucleosome folding of the inactive chromatin in chicken erythrocytes and rat liver nuclei is basically a second-order superhelical organization (width 150--200 nm, pitch distance 50--150 nm) of the elementary nucleosome filament. In unfavorable environments (as determined by ionic agents, fixative, and dehydrating agetns) this superstructure collapses into chains of superbeads and beads. Formalin (10%) apparently attacks at discrete sites of chromatin, which are then separated into superbeads. The latter consist of 4--6 nucleosomes and seemingly correspond to successive turns of an original solenoidal coil (width 30--35 nm), which forms the superhilical organization. When this organization is unfolded, eg, in 1--2 mM EDTA, DNAse-sensitive filaments (diameter 1.7 nm) are seen to be wrapped around the nucleosomes. The wet chromosomes in each metaphase spread are held to each other by smooth microtubular fibers, 20--20 nm in diameter. Before they enter into a chromsome, these fibers branch into 9--13 protofilaments, each 5 nm wide. The chromosome surface contains a dense distribution of subunits about 10--25 nm in diameter. This size distribution corresponds to that of nucleosomes and their superbeads. Distinct from this beaded chromosome surface are several smooth, 23--30-nm-diameter fibers, which are longitudinal at the centromere and seem to continue into the chromatid structure. The surface replicas of dried chromosomes do not show these features, which are

  17. Neighborhood safety and physical inactivity in adults from Curitiba, Brazil

    PubMed Central

    2012-01-01

    Background Neighborhood safety is one of the environmental aspects that can influence physical activity. We analyzed the association between perceived neighborhood safety and physical inactivity (PI) in adults and examined effect modification according to sociodemographic variables. Methods A cross-sectional study was conducted with 1,261 adults (62% women), age 18–69 years from Curitiba, Brazil. Results The perception of unsafe neighborhood was higher among women, older participants, those classified in the high socioeconomic (SES) group, overweighed and also among those reporting to have PA equipments and children. The association between perception safety of walking during the day and walking for leisure (women PR = 1.12 CI95% = 1.02–1.22; men PR = 0.82 CI95% = 0.64–1.05; interaction term PR = 1.38 CI95% = 1.03–1.83) and safe perception was associated with PI, just in the highest SES group (PR = 1.09; CI95% = 1.00–1.19; p trend = 0.032) when compared with their counterparts (low SES PR = 0.99; CI95% = 0.90–1.04; p trend = 0.785; interaction term PR = 1.09; CI95% = 1.03–1.15; p trend = 0.007). Conclusion The perception of safety in the neighborhood was associated with PI in transport, but this association varies across of sociodemographic variables. PMID:22691163

  18. Career redevelopment programmes for inactive nurses in Japan.

    PubMed

    Tanaka, Sachiko; Serizawa, Takako; Sakaguchi, Chizuru

    2008-12-01

    The purpose of this paper is to examine the challenges and problems in using career redevelopment programmes and individual hospital programmes to prepare inactive nurses to re-enter into the workforce in Japan. It is critical to supply sufficient skilled health human resources for medical care. Although, Japan has a mandatory retraining programme for supporting nurses to return to the workplace after a career break, it is unclear to what extent there are benefits to nurses from these programmes. The research of career redevelopment programme was undertaken in three administrative divisions' nurse centres in local prefecture A, B and C. A survey of nurses participating in the programme running in T Hospital was also conducted. The issues examined were the background and motivations of participants, the length of career break, the percentages returning to work and the effectiveness of each programme. The average age of participants was 40 years, ranging widely from the 20-60 years. Local prefecture A tended to have narrower age range than others, namely from the 30-50 years. The average period of career break was around eight years at two of three. Length of experience was quite varied from entry level to 20 or 30 years in nursing. Feedback from nurses in the case study T Hospital suggests that the most effective ways of providing support through the programme was to meet the need for continuing support, including working styles after return to work and using the resources programme in their own area of domicile. In the potential return of the nurse, the following are important: (i) job support system by using social resources effectively in the community level; and (ii) introduction of diverse working styles that take account of varying work-life balance, as well as childcare support, by using existing facilities or human resources.

  19. Multiphoton Assisted Recombination

    NASA Astrophysics Data System (ADS)

    Shuman, E. S.; Jones, R. R.; Gallagher, T. F.

    2008-12-01

    We have observed multiphoton assisted recombination in the presence of a 38.8 GHz microwave field. Stimulated emission of up to ten microwave photons results in energy transfer from continuum electrons, enabling recombination. The maximum electron energy loss is far greater than the 2Up predicted by the standard “simpleman’s” model. The data are well reproduced by both an approximate analytic expression and numerical simulations in which the combined Coulomb and radiation fields are taken into account.

  20. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  1. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  2. Mapping the Prevalence of Physical Inactivity in U.S. States, 1984-2015.

    PubMed

    An, Ruopeng; Xiang, Xiaoling; Yang, Yan; Yan, Hai

    2016-01-01

    Physical inactivity is a leading cause of morbidity, disability and premature mortality in the U.S. and worldwide. This study aimed to map the prevalence of physical inactivity across U.S. states over the past three decades, and estimate the over-time adjusted changes in the prevalence of physical inactivity in each state. Individual-level data (N = 6,701,954) were taken from the 1984-2015 Behavioral Risk Factor Surveillance System (BRFSS), an annually repeated cross-sectional survey of state-representative adult population. Prevalence of self-reported leisure-time physical inactivity was estimated by state and survey year, accounting for the BRFSS sampling design. Logistic regressions were performed to estimate the changes in the prevalence of physical inactivity over the study period for each state, adjusting for individual characteristics including sex, age, race/ethnicity, education, marital status, and employment status. The prevalence of leisure-time physical inactivity varied substantially across states and survey years. In general, the adjusted prevalence of physical inactivity gradually declined over the past three decades in a majority of states. However, a substantial proportion of American adults remain physically inactive. Among the 50 states and District of Columbia, 45 had over a fifth of their adult population without any leisure-time physical activity, and 8 had over 30% without physical activity in 2015. Moreover, the adjusted prevalence of physical inactivity in several states (Arizona, North Carolina, North Dakota, Utah, West Virginia, and Wyoming) remained largely unchanged or even increased (Minnesota and Ohio) over the study period. Although the prevalence of physical inactivity declined over the past three decades in a majority of states, the rates remain substantially high and vary considerably across states. Closely monitoring and tracking physical activity level using the state physical activity maps can help guide policy and program

  3. Mapping the Prevalence of Physical Inactivity in U.S. States, 1984-2015

    PubMed Central

    Xiang, Xiaoling; Yang, Yan; Yan, Hai

    2016-01-01

    Background Physical inactivity is a leading cause of morbidity, disability and premature mortality in the U.S. and worldwide. This study aimed to map the prevalence of physical inactivity across U.S. states over the past three decades, and estimate the over-time adjusted changes in the prevalence of physical inactivity in each state. Methods Individual-level data (N = 6,701,954) were taken from the 1984–2015 Behavioral Risk Factor Surveillance System (BRFSS), an annually repeated cross-sectional survey of state-representative adult population. Prevalence of self-reported leisure-time physical inactivity was estimated by state and survey year, accounting for the BRFSS sampling design. Logistic regressions were performed to estimate the changes in the prevalence of physical inactivity over the study period for each state, adjusting for individual characteristics including sex, age, race/ethnicity, education, marital status, and employment status. Results The prevalence of leisure-time physical inactivity varied substantially across states and survey years. In general, the adjusted prevalence of physical inactivity gradually declined over the past three decades in a majority of states. However, a substantial proportion of American adults remain physically inactive. Among the 50 states and District of Columbia, 45 had over a fifth of their adult population without any leisure-time physical activity, and 8 had over 30% without physical activity in 2015. Moreover, the adjusted prevalence of physical inactivity in several states (Arizona, North Carolina, North Dakota, Utah, West Virginia, and Wyoming) remained largely unchanged or even increased (Minnesota and Ohio) over the study period. Conclusions Although the prevalence of physical inactivity declined over the past three decades in a majority of states, the rates remain substantially high and vary considerably across states. Closely monitoring and tracking physical activity level using the state physical activity

  4. Recombination and chromosome segregation.

    PubMed Central

    Sherratt, David J; Søballe, Britta; Barre, François-Xavier; Filipe, Sergio; Lau, Ivy; Massey, Thomas; Yates, James

    2004-01-01

    The duplication of DNA and faithful segregation of newly replicated chromosomes at cell division is frequently dependent on recombinational processes. The rebuilding of broken or stalled replication forks is universally dependent on homologous recombination proteins. In bacteria with circular chromosomes, crossing over by homologous recombination can generate dimeric chromosomes, which cannot be segregated to daughter cells unless they are converted to monomers before cell division by the conserved Xer site-specific recombination system. Dimer resolution also requires FtsK, a division septum-located protein, which coordinates chromosome segregation with cell division, and uses the energy of ATP hydrolysis to activate the dimer resolution reaction. FtsK can also translocate DNA, facilitate synapsis of sister chromosomes and minimize entanglement and catenation of newly replicated sister chromosomes. The visualization of the replication/recombination-associated proteins, RecQ and RarA, and specific genes within living Escherichia coli cells, reveals further aspects of the processes that link replication with recombination, chromosome segregation and cell division, and provides new insight into how these may be coordinated. PMID:15065657

  5. What’s New in Enzymatic Halogenations

    PubMed Central

    Fujimori, Danica Galoniæ; Walsh, Christopher T.

    2007-01-01

    Summary The halogenation of thousands of natural products occurs during biosynthesis and often confers important functional properties. While haloperoxidases had been the default paradigm for enzymatic incorporation of halogens, via X+ equivalents into organic scaffolds, a combination of microbial genome sequencing, enzymatic studies and structural biology have provided deep new insights into enzymatic transfer of halide equivalents in three oxidation states. These are: (1) the halide ions (X−) abundant in nature, (2) halogen atoms (X•), and (3) the X+ equivalents. The mechanism of halogen incorporation is tailored to the electronic demands of specific substrates and involves enzymes with distinct redox coenzyme requirements. PMID:17881282

  6. Enzymatic Vitrectomy and Pharmacologic Vitreodynamics.

    PubMed

    Shah, Ankoor R; Trese, Michael T

    2016-01-01

    The field of vitreoretinal surgery has evolved substantially over the last several decades. Scientific advances have improved our understanding of disease pathophysiology, and new surgical adjuncts and techniques have decreased surgical time and improved patient outcomes. Pharmacologic agents have recently been developed for intraocular use in order to enhance vitreous removal and even as a nonsurgical treatment for pathology due to an abnormal vitreoretinal interface. Plasmin can successfully cause vitreous liquefaction and induce a posterior vitreous detachment. Additionally, ocriplasmin has been approved for symptomatic vitreomacular adhesion and others appear to be promising for pharmacologic manipulation of the vitreous. The ability to induce vitreous liquefaction and a complete posterior vitreous detachment (PVD) with a single intravitreal injection has potential implications for the management of multiple vitreoretinopathies. Enzymatic vitrectomy may help to reduce vitreous viscosity, thereby facilitating removal during vitrectomy and reducing surgical time, especially when using smaller-gauge vitrectomy instruments. The induction of a PVD also has the potential to reduce intraoperative complications. As we improve our understanding of the molecular flux in the vitreous cavity, pharmacologic vitreodynamics will likely become more important as it may allow for improved manipulation of intravitreal molecules.

  7. Enzymatic Reactions in Microfluidic Devices

    NASA Astrophysics Data System (ADS)

    Ristenpart, W. D.; Wan, J.; Stone, H. A.

    2008-11-01

    We establish simple scaling laws for enzymatic reactions in microfluidic devices, and we demonstrate that kinetic parameters obtained conventionally using multiple stop-flow experiments may instead be extracted from a single microfluidic experiment. Introduction of an enzyme and substrate species in different arms of a Y-shaped channel allows the two species to diffuse across the parallel streamlines and to begin reacting. Measurements of the product concentration versus distance down the channel provide information about the kinetics of the reaction. In the limit where the enzyme is much larger (and thus less diffusive) than the substrate, we show that near the entrance the total amount of product (P) formed varies as a power law in the distance x down the channel. For reactions that follow standard Michaelis-Menten kinetics, the power law takes the form P˜(Vmax/Km) x^5/2, where Vmax and Km are the maximum reaction rate and Michaelis constant respectively. If a large excess of substrate is used, then Km is identified by measuring Vmax far downstream where the different species are completely mixed by diffusion. Numerical simulations and experiments using the bioluminescent reaction between luciferase and ATP as a model system are both shown to accord with the model. We discuss the implications for significant savings in the amount of time and enzyme required for determination of kinetic parameters.

  8. Enzymatic Processing of Platinated RNAs

    PubMed Central

    Chapman, Erich G.; DeRose, Victoria J.

    2011-01-01

    The broadly prescribed anti-tumor drug cisplatin coordinates to DNA, altering the activity of cellular proteins whose functions rely upon sensing DNA structure. Cisplatin is also known to coordinate to RNA, but the effects of RNA-Pt adducts on the large number of proteins that process the transcriptome are currently unknown. In an effort to address how platination of an RNA alters the function of RNA processing enzymes, we have determined the influence of [Pt(NH3)2]2+-RNA adducts on the activities of 3’ → 5’ and 5’→3’ phosphodiesterases, a purine-specific endoribonuclease, and a reverse transcriptase. Single Pt(II) adducts on RNA oligonucleotides of form (5’-U6-XY-U5-3’: XY=GG, GA, AG, GU) are found to block exonucleolytic digestion. Similar disruption of endonucleolytic cleavage is observed, except for the platinated XY= GA RNA where RNase U2 uniquely tolerates platinum modification. Platinum adducts formed with a more complex RNA prevent reverse transcription, providing evidence that platination is capable of interfering with RNA’s role in relaying sequence information. The observed disruptions in enzymatic activity point to the possibility that cellular RNA processing may be similarly affected, which could contribute to the cell-wide effects of platinum anti-tumor drugs. Additionally, we show that thiourea reverses cisplatin-RNA adducts, providing a chemical tool for use in future studies regarding cisplatin targeting of cellular RNAs. PMID:20099814

  9. Substitution of Glu-59 by Val in amidase from Pseudomonas aeruginosa results in a catalytically inactive enzyme.

    PubMed

    Karmali, A; Tata, R; Brown, P R

    2000-09-01

    A mutant strain, KLAM59, of Pseudomonas aeruginosa has been isolated that synthesizes a catalytically inactive amidase. The mutation in the amidase gene has been identified (Glu59Val) by direct sequencing of PCR-amplified mutant gene and confirmed by sequencing the cloned PCR-amplified gene. The wild-type and altered amidase genes were cloned into an expression vector and both enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide followed by gel filtration chromatography. The mutant enzyme was catalytically inactive, and it was detected in column fractions by monoclonal antibodies previously raised against the wild-type enzyme using an ELISA sandwich method. The recombinant wild-type and mutant enzymes were purified with a final recovery of enzyme in the range of 70-80%. The wild-type and mutant enzymes behaved differently on the affinity column as shown by their elution profiles. The molecular weights of the purified wild-type and mutant amidases were found to be 210,000 and 78,000 Dalton, respectively, by gel filtration chromatography. On the other hand, the mutant enzyme ran as a single protein band on SDS-PAGE and native PAGE with a M(r) of 38,000 and 78,000 Dalton, respectively. These data suggest that the substitution Glu59Val was responsible for the dimeric structure of the mutant enzyme as opposed to the hexameric form of the wild-type enzyme. Therefore, the Glu59 seems to be a critical residue in the maintenance of the native quaternary structure of amidase.

  10. Positive mood + action = negative mood + inaction: effects of general action and inaction concepts on decisions and performance as a function of affect.

    PubMed

    Albarracin, Dolores; Hart, William

    2011-08-01

    General action and inaction concepts have been shown to produce broad, goal-mediated effects on cognitive and motor activity irrespective of the type of activity. The current research tested a model in which action and inaction goals interact with the valence of incidental moods to guide behavior. Over four experiments, participants' moods were manipulated to be positive (happy), neutral, or negative (angry or sad), and then general action, inaction, and neutral concepts were primed. In Experiment 1, action primes increased intellectual performance when participants experienced a positive (happy) or neutral mood, whereas inaction primes increased performance when participants experienced a negative (angry) mood. Including a control-prime condition, Experiments 2 and 3 replicated these results measuring the number of general interest articles participants were willing to read and participants' memory for pictures of celebrities. Experiment 4 replicated the results comparing happiness with sadness and suggested that the effect of the prime's adoption was automatic. Overall, the findings supported an interactive model by which action concepts and positive affect produce the same increases in active behavior as inaction concepts and negative affect.

  11. Positive Mood + Action = Negative Mood + Inaction: Effects of General Action and Inaction Concepts on Decisions and Performance as a Function of Affect

    PubMed Central

    Albarracin, Dolores; Hart, William

    2013-01-01

    General action and inaction concepts have been shown to produce broad, goal-mediated effects on cognitive and motor activity irrespective of the type of activity. The current research tested a model in which action and inaction goals interact with the valence of incidental moods to guide behavior. Over four experiments, participants’ moods were manipulated to be positive (happy), neutral, or negative (angry or sad), and then general action, inaction, and neutral concepts were primed. In Experiment 1, action primes increased intellectual performance when participants experienced a positive (happy) or neutral mood, whereas inaction primes increased performance when participants experienced a negative (angry) mood. Including a control-prime condition, Experiments 2 and 3 replicated these results measuring the number of general interest articles participants were willing to read and participants’ memory for pictures of celebrities. Experiment 4 replicated the results comparing happiness with sadness and suggested that the effect of the prime’s adoption was automatic. Overall, the findings supported an interactive model by which action concepts and positive affect produce the same increases in active behavior as inaction concepts and negative affect. PMID:21859209

  12. IMPORTANCE OF ENZYMATIC BIOTRANSFORMATION IN IMMUNOTOXICOLOGY

    EPA Science Inventory

    Many immunotoxic compounds, such as benzene and other organic solvents, pesticides, mycotoxins and polycyclic aromatic hydrocarbons, can alter immune function only after undergoing enzyme-mediated reactions within various tissues. In the review that follows, the role of enzymatic...

  13. IMPORTANCE OF ENZYMATIC BIOTRANSFORMATION IN IMMUNOTOXICOLOGY

    EPA Science Inventory

    Many immunotoxic compounds, such as benzene and other organic solvents, pesticides, mycotoxins and polycyclic aromatic hydrocarbons, can alter immune function only after undergoing enzyme-mediated reactions within various tissues. In the review that follows, the role of enzymatic...

  14. Motor-Driven (Passive) Cycling: A Potential Physical Inactivity Countermeasure?

    PubMed Central

    Peterman, James E.; Wright, Kenneth P.; Melanson, Edward L.; Kram, Rodger; Byrnes, William C.

    2016-01-01

    We have previously shown that motor-driven (passive) stationary cycling elevates energy expenditure (EE). Purpose To quantify how acute passive cycling affects glucose and insulin responses to an oral glucose tolerance test (OGTT) and basic cognition compared to sitting and moderate-intensity active cycling. Methods Twenty-four physically inactive healthy males completed three trials in randomized order involving 30-minute conditions of sitting, passive cycling and moderate-intensity cycling. During each condition, EE was measured and participants performed cognitive tests. Following each condition, a 2-hour OGTT was performed. Results EE was significantly higher during the cycling conditions compared to sitting (1.36±0.58 and 6.50±1.73 kcal·min−1 greater than sitting for passive and moderate-intensity, respectively). A significant correlation was found between body fat percentage and post-sitting OGTT 2-h post plasma glucose (r2=0.30, p<0.05) so participants were divided into lean (n=11) and non-lean (n=13) groups. In the non-lean group, compared to sitting, passive cycling lowered 2-h post plasma glucose (7.7±1.3 vs. 6.9±1.6mmol·L−1, respectively, p<0.05) and Matsuda whole body insulin sensitivity index (WBISI) was higher (2.74±0.86 vs. 3.36±1.08, p<0.05). Additionally, passive and moderate-intensity cycling had similar beneficial effects on 2-h post plasma glucose and WBISI. Cognitive performance did not significantly differ between the sitting and passive cycling conditions. Conclusion 2-h post plasma glucose was lower and WBISI following acute passive cycling was higher in non-lean participants. Given that and the increase in EE without changes in cognitive performance, we propose passive cycling as a promising intervention to counteract some of the deleterious effects of prolonged sitting in the workplace. PMID:27054677

  15. Physical Inactivity Differentially Alters Dietary Oleate and Palmitate Trafficking

    PubMed Central

    Bergouignan, Audrey; Trudel, Guy; Simon, Chantal; Chopard, Angèle; Schoeller, Dale A.; Momken, Iman; Votruba, Susanne B.; Desage, Michel; Burdge, Graham C.; Gauquelin-Koch, Guillemette; Normand, Sylvie; Blanc, Stéphane

    2009-01-01

    OBJECTIVE— Obesity and diabetes are characterized by the incapacity to use fat as fuel. We hypothesized that this reduced fat oxidation is secondary to a sedentary lifestyle. RESEARCH DESIGN AND METHODS— We investigated the effect of a 2-month bed rest on the dietary oleate and palmitate trafficking in lean women (control group, n = 8) and the effect of concomitant resistance/aerobic exercise training as a countermeasure (exercise group, n = 8). Trafficking of stable isotope–labeled dietary fats was combined with muscle gene expression and magnetic resonance imaging–derived muscle fat content analyses. RESULTS— In the control group, bed rest increased the cumulative [1-13C]oleate and [d31]palmitate appearance in triglycerides (37%, P = 0.009, and 34%, P = 0.016, respectively) and nonesterified fatty acids (NEFAs) (37%, P = 0.038, and 38%, P = 0.002) and decreased muscle lipoprotein lipase (P = 0.043) and fatty acid translocase CD36 (P = 0.043) mRNA expressions. Plasma NEFA-to-triglyceride ratios for [1-13C]oleate and [d31]palmitate remained unchanged, suggesting that the same proportion of tracers enters the peripheral tissues after bed rest. Bed rest did not affect [1-13C]oleate oxidation but decreased [d31]palmitate oxidation by −8.2 ± 4.9% (P < 0.0001). Despite a decreased spontaneous energy intake and a reduction of 1.9 ± 0.3 kg (P = 0.001) in fat mass, exercise training did not mitigate these alterations but partially maintained fat-free mass, insulin sensitivity, and total lipid oxidation in fasting and fed states. In both groups, muscle fat content increased by 2.7% after bed rest and negatively correlated with the reduction in [d31]palmitate oxidation (r2 = 0.48, P = 0.003). CONCLUSIONS— While saturated and monounsaturated fats have similar plasma trafficking and clearance, physical inactivity affects the partitioning of saturated fats toward storage, likely leading to an accumulation of palmitate in muscle fat. PMID:19017764

  16. Maternal inactivity: 45-year trends in mothers' use of time.

    PubMed

    Archer, Edward; Lavie, Carl J; McDonald, Samantha M; Thomas, Diana M; Hébert, James R; Taverno Ross, Sharon E; McIver, Kerry L; Malina, Robert M; Blair, Steven N

    2013-12-01

    To examine 45-year trends in time use and physical activity energy expenditure (PAEE) in a nationally representative sample of US mothers. We quantified time allocation to physical activity (PA), sedentary behaviors (SED), and PAEE from 1965 to 2010 in mothers with older children (MOC) (>5 to ≤18 years) and mothers with younger children (MYC) (≤5 years). Physical activity was the sum of time allocated to housework, child care, laundry, food preparation, postmeal cleanup, and exercise. Sedentary behavior was the sum of time spent in a vehicle and using screen-based media. Physical activity energy expenditure was calculated using body weights from national surveys and metabolic equivalents. From 1965 to 2010, the time allocated to PA decreased by 11.1 h/wk (from 32.0 to 20.9 h/wk) in MOC and by 13.9 h/wk (from 43.6 to 29.7 h/wk) in MYC. The time spent in SED increased by 7.0 h/wk in MOC (from 17.7 to 24.7 h/wk) and increased by 5.7 h/wk in MYC (from 17.0 to 22.7 h/wk). Physical activity energy expenditure decreased by 1237.6 kcal/wk (176.8 kcal/d) in MOC (from 5835.3 to 4597.7 kcal/wk), and in MYC, PAEE decreased by 1572.5 kcal/wk (224.6 kcal/d), from 7690.5 to 6118.0 kcal/wk. There was a significant reallocation of time by mothers from PA (eg, housework) to SED (eg, watching television) between 1965 and 2010. Given the essential role of PA for health and the potential for the intergenerational transmission of obesity and obesogenic behaviors, these results suggest that maternal inactivity may be an important target for the primary prevention of chronic noncommunicable diseases and obesity. Copyright © 2013 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.

  17. Analytical techniques for characterizing enzymatic biofuel cells.

    PubMed

    Moehlenbrock, Michael J; Arechederra, Robert L; Sjöholm, Kyle H; Minteer, Shelley D

    2009-12-01

    Enzymatic biofuel cells, which replace expensive metal catalysts with enzymes, are still in an early stage of development. This article details the analytical techniques that are often employed for evaluating and characterizing enzymatic biofuel cells and their corresponding bioanodes and biocathodes. (To listen to a podcast about this feature, please go to the Analytical Chemistry multimedia page at pubs.acs.org/page/ancham/audio/index.html.).

  18. Physical inactivity among older adults across Europe based on the SHARE database.

    PubMed

    Gomes, Marcos; Figueiredo, Daniela; Teixeira, Laetitia; Poveda, Verónica; Paúl, Constança; Santos-Silva, Alice; Costa, Elísio

    2017-01-20

    Regular physical activity is one of the key components of a healthy lifestyle. It is associated with better physical and cognitive functioning in later life and with increased life expectancy. The purpose of this study was to evaluate the prevalence of, and factors related to, physical inactivity among older adults across Europe. In this cross-sectional analysis, we used data from participants aged 55 or older in Wave 4 of the Survey of Health, Ageing, and Retirement in Europe (SHARE) database, a multidisciplinary and cross-national panel database covering health, socioeconomic status, and social and family networks. Individuals included in this study were classified as physically active or physically inactive. Clinical, psychosocial and sociodemographic variables were evaluated for their association with physical inactivity. From the total of 58,489 individuals in SHARE, we selected 19,298 people age 55 or older (mean age 67.8 ± 8.9 years; 11,430 (59.2%) female). The overall prevalence of inactivity among individuals age 55 or older in the 16 included countries was 12.5%. The prevalence of physical inactivity varied between countries, ranging from 4.9% (Sweden) to 29% (Portugal). Increasing age, depression, physical limitations, poor sense of meaning in life, social support and memory loss were significant variables associated with physical inactivity. Physical inactivity can be explained by physical, cognitive and psychological conditions. Interventions aimed at promoting physical activity among older people are needed to address this diversity of factors.

  19. Influence of physical inactivity in psychophysiological state of breast cancer survivors.

    PubMed

    Ariza-García, A; Galiano-Castillo, N; Cantarero-Villanueva, I; Fernández-Lao, C; Díaz-Rodríguez, L; Arroyo-Morales, M

    2013-11-01

    Physical inactivity has been postulated as mediator of the relationship between cancer-related symptoms and psychoneurobiological alterations. The aim of the study was to evaluate the influence of physical inactivity level on mood state, fitness level as well as on salivary markers of the hypothalamic-pituitary-adrenal axis (cortisol) and the SNS (α-amylase) in breast cancer survivors. One hundred and eight breast cancer survivors (stages I-IIIa) participated in this cross-sectional study. Data were gathered on the following: Minnesota Leisure Time Physical Activity Questionnaire, profile of mood state, 6-min walk test, force handgrip, blood pressure, salivary cortisol concentration and salivary α-amylase activity. For our analysis, two groups were formed based on physical activity level measured as energy expenditure during diary leisure activities of the participants at the moment of the study, a physical inactivity level group (<3 METs × h/week) and an adequate physical activity level group (>3 METs × h/week). Fitness level was significantly higher in the active than the inactive group, while anger, fatigue, depression, confusion, mood disturbance, diastolic blood pressure and salivary α-amylase activity were significantly greater in the inactive than the active group. These results suggest that physical inactivity induces a worse psychoneurobiological state in inactive than in active breast cancer survivors. © 2013 John Wiley & Sons Ltd.

  20. Inactive nurses in Taiwan: human capital, intention to return to hospital nursing, and incentives for returning.

    PubMed

    Yu, Hsing-Yi; Tang, Fu-In; Chen, I-Ju; Yin, Teresa J C; Chen, Chu-Chieh; Yu, Shu

    2016-04-01

    To investigate inactive nurses' human capital, intention to return to hospital nursing and incentives for returning. Few studies have discussed the loss of human capital with regard to inactive nurses and how to attract them to return to clinical work. Systematic random sampling was used, with 328 subjects completing the mailed questionnaires, resulting in a response rate of 25.4%. Inactive nurses not only had moderate to high human capital (average years of nursing experience was 10.29, with moderate to high levels of nursing professional commitment and nursing competence) and were young. Forty-three percent of subjects reported intending to return to hospital nursing. Sufficient nurse staffing, greater safety in the working environment, and re-entry preparation programmes were incentives for returning. Recruiting inactive nurses back to hospital work is vital and feasible as inactive nurses had a moderate to high degree of human capital. The most feasible way is offering reasonable working conditions, in particular, providing sufficient staffing, a safe working environment and re-entry preparation programmes. The findings confirm the human capital of inactive nurses and provide concrete directions for nursing managers to follow when recruiting inactive nurses to hospital nursing. © 2015 John Wiley & Sons Ltd.

  1. Reversible Activation of Halophilic β-lactamase from Methanol-Induced Inactive Form: Contrast to Irreversible Inactivation of Non-Halophilic Counterpart.

    PubMed

    Tokunaga, Hiroko; Maeda, Junpei; Arakawa, Tsutomu; Tokunaga, Masao

    2017-06-01

    Effects of a water-miscible organic solvent, methanol, on the structure and activity of halophilic β-lactamase derived from Chromohalobacter sp.560 (HaBla), were investigated by means of circular dichroism (CD) measurement and enzymatic activity determination. Beta-lactamase activity was enhanced about 1.2-fold in the presence of 10-20% methanol. CD measurement of HaBla revealed different structures depending on the methanol concentration: native-like active form (Form I) in 10-20% methanol and methanol-induced inactive form at higher concentration (Form II in 40-60% and Form III in 75-80% methanol). Incubation of HaBla with 40% methanol led to the complete loss of activity within ~80 min accompanied by the formation of Form II, whose activity was recovered promptly up to ~80% of full activity upon dilution of the methanol concentration to 10%. In addition, when the protein concentration was sufficiently high (e.g., 0.7 mg/ml), HaBla activity of Form III in 75% methanol could be recovered in the same way (with slightly slower recovery rate), upon dilution of the methanol concentration. In contrast, non-halophilic β-lactamase from Escherichia coli K12 strain MG1655 (EcBla) was irreversibly denatured in the presence of 40% methanol. HaBla showed remarkable ability to renature from the methanol-induced inactive states.

  2. Screen for reactivation of MeCP2 on the inactive X chromosome identifies the BMP/TGF-β superfamily as a regulator of XIST expression.

    PubMed

    Sripathy, Smitha; Leko, Vid; Adrianse, Robin L; Loe, Taylor; Foss, Eric J; Dalrymple, Emily; Lao, Uyen; Gatbonton-Schwager, Tonibelle; Carter, Kelly T; Payer, Bernhard; Paddison, Patrick J; Grady, William M; Lee, Jeannie T; Bartolomei, Marisa S; Bedalov, Antonio

    2017-02-14

    Rett syndrome (RS) is a debilitating neurological disorder affecting mostly girls with heterozygous mutations in the gene encoding the methyl-CpG-binding protein MeCP2 on the X chromosome. Because restoration of MeCP2 expression in a mouse model reverses neurologic deficits in adult animals, reactivation of the wild-type copy of MeCP2 on the inactive X chromosome (Xi) presents a therapeutic opportunity in RS. To identify genes involved in MeCP2 silencing, we screened a library of 60,000 shRNAs using a cell line with a MeCP2 reporter on the Xi and found 30 genes clustered in seven functional groups. More than half encoded proteins with known enzymatic activity, and six were members of the bone morphogenetic protein (BMP)/TGF-β pathway. shRNAs directed against each of these six genes down-regulated X-inactive specific transcript (XIST), a key player in X-chromosome inactivation that encodes an RNA that coats the silent X chromosome, and modulation of regulators of this pathway both in cell culture and in mice demonstrated robust regulation of XIST. Moreover, we show that Rnf12, an X-encoded ubiquitin ligase important for initiation of X-chromosome inactivation and XIST transcription in ES cells, also plays a role in maintenance of the inactive state through regulation of BMP/TGF-β signaling. Our results identify pharmacologically suitable targets for reactivation of MeCP2 on the Xi and a genetic circuitry that maintains XIST expression and X-chromosome inactivation in differentiated cells.

  3. Regulation of Meiotic Recombination

    SciTech Connect

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  4. Chronic Recreational Physical Inactivity and Epithelial Ovarian Cancer Risk: Evidence from the Ovarian Cancer Association Consortium

    PubMed Central

    Cannioto, Rikki; LaMonte, Michael J.; Risch, Harvey A.; Hong, Chi-Chen; Sucheston-Campbell, Lara E.; Eng, Kevin H.; Szender, J. Brian; Chang-Claude, Jenny; Schmalfeldt, Barbara; Klapdor, Ruediger; Gower, Emily; Minlikeeva, Albina N.; Zirpoli, Gary; Bandera, Elisa V.; Berchuck, Andrew; Cramer, Daniel; Doherty, Jennifer A.; Edwards, Robert P.; Fridley, Brooke L.; Goode, Ellen L.; Goodman, Marc T.; Hogdall, Estrid; Hosono, Satoyo; Jensen, Allan; Jordan, Susan; Kjaer, Susanne K.; Matsuo, Keitaro; Ness, Roberta B.; Olsen, Catherine M.; Olson, Sara H.; Pearce, Celeste Leigh; Pike, Malcolm C.; Rossing, Mary Anne; Szamreta, Elizabeth A.; Thompson, Pamela J.; Tseng, Chiu-Chen; Vierkant, Robert A.; Webb, Penelope M.; Wentzensen, Nicolas; Wicklund, Kristine G.; Winham, Stacey J.; Wu, Anna H.; Modugno, Francesmary; Schildkraut, Joellen M.; Terry, Kathryn L.; Kelemen, Linda E.; Moysich, Kirsten B.

    2016-01-01

    Background Despite a large body of literature evaluating the association between recreational physical activity and epithelial ovarian cancer (EOC) risk, the extant evidence is inconclusive and little is known about the independent association between recreational physical inactivity and EOC risk. We conducted a pooled analysis of nine studies from the Ovarian Cancer Association Consortium (OCAC) to investigate the association between chronic recreational physical inactivity and EOC risk. Methods In accordance with the 2008 Physical Activity Guidelines for Americans, women reporting no regular, weekly recreational physical activity were classified as inactive. Multivariable logistic regression was utilized to estimate the odds ratios (OR) and 95% confidence intervals (CI) for the association between inactivity and EOC risk overall and by subgroups based upon histotype, menopausal status, race and body mass index (BMI). Results The current analysis included data from 8,309 EOC patients and 12,612 controls. We observed a significant positive association between inactivity and EOC risk (OR=1.34, 95% CI: 1.14-1.57) and similar associations were observed for each histotype. Conclusions In this large pooled analysis examining the association between recreational physical inactivity and EOC risk, we observed consistent evidence of an association between chronic inactivity and all EOC histotypes. Impact These data add to the growing body of evidence suggesting that inactivity is an independent risk factor for cancer. If the apparent association between inactivity and EOC risk is substantiated, additional work via targeted interventions should be pursued to characterize the dose of activity required to mitigate the risk of this highly fatal disease. PMID:27197285

  5. Prevalence of Irritable Bowel Syndrome–like Symptoms in Japanese Patients with Inactive Inflammatory Bowel Disease

    PubMed Central

    Tomita, Toshihiko; Kato, Yu; Takimoto, Mayu; Yamasaki, Takahisa; Kondo, Takashi; Kono, Tomoaki; Tozawa, Katsuyuki; Yokoyama, Yoko; Ikehara, Hisatomo; Ohda, Yoshio; Oshima, Tadayuki; Fukui, Hirokazu; Tanaka, Shigemi; Shima, Masayuki; Watari, Jiro; Miwa, Hiroto

    2016-01-01

    Background/Aims Few studies are available that have investigated the risk factors for overlapping irritable bowel syndrome (IBS)-like symptoms in patients with inactive inflammatory bowel disease (IBD). The present study has 3 objectives: (1) to assess the prevalence of IBS-like symptoms in Japanese patients with inactive IBD using Rome III criteria, (2) to examine the relationship of IBS-like symptoms to health related quality of life (HR-QOL), and (3) to investigate associations for developing IBS-like symptoms in patients with inactive IBD. Methods IBS-like symptoms were evaluated using the Rome III questionnaire for functional gastrointestinal disorders. HR-QOL and hospital anxiety and depression scale were evaluated. Results IBS-like symptoms were found in 17.5% (7/40) of patients with inactive ulcerative colitis, 27.1% (29/107) of patients with inactive Crohn’s disease (CD), and 5.3% (23/438) of healthy control subjects. The QOL level was significantly lower and anxiety score was significantly higher in inactive CD patients with IBS-like symptoms than in those without such symptoms (P = 0.003, P = 0.009). Use of anti-anxiety drugs was associated with the presence of IBS symptoms (P = 0.045). HR-QOL score was lower and anxiety score was higher in patients with inactive ulcerative colitis, but the difference was not statistically significant. Conclusions The prevalence of IBS-like symptoms in inactive IBD patients was significantly higher than in healthy controls. Inactive CD patients with IBS-like symptoms has low QOL and anxiety; suggesting that anxiety may be associated with symptom development in such patients. PMID:27193973

  6. Thrombolytic efficacy and enzymatic activity of rt-PA-loaded echogenic liposomes.

    PubMed

    Bader, Kenneth B; Bouchoux, Guillaume; Peng, Tao; Klegerman, Melvin E; McPherson, David D; Holland, Christy K

    2015-08-01

    Echogenic liposomes (ELIP), that can encapsulate both recombinant tissue-type plasminogen activator (rt-PA) and microbubbles, are under development to improve the treatment of thrombo-occlusive disease. However, the enzymatic activity, thrombolytic efficacy, and stable cavitation activity generated by this agent has yet to be evaluated and compared to another established ultrasound-enhanced thrombolytic scheme. A spectrophotometric method was used to compare the enzymatic activity of the rt-PA incorporated into ELIP (t-ELIP) to that of rt-PA. An in vitro flow model was employed to measure the thrombolytic efficacy and dose of ultraharmonic emissions from stable cavitation for 120-kHz ultrasound exposure of three treatment schemes: rt-PA, rt-PA and the perfluorocarbon-filled microbubble Definity(®), and t-ELIP. The enzymatic activity of rt-PA incorporated into t-ELIP was 28 % that of rt-PA. Thrombolytic efficacy of t-ELIP or rt-PA and Definity(®) was equivalent when the dose of t-ELIP was adjusted to produce comparable enzymatic activity. Sustained bubble activity was nucleated from Definity but not from t-ELIP exposed to 120-kHz ultrasound. These results emphasize the advantages of encapsulating a thrombolytic and the importance of incorporating an insoluble gas required to promote sustained, stable cavitation activity.

  7. Chemo-Enzymatic Synthesis of Linear and Branched Cationic Peptides: Evaluation as Gene Carriers.

    PubMed

    Ageitos, Jose Manuel; Chuah, Jo-Ann; Numata, Keiji

    2015-07-01

    Cationic peptides such as poly(l-lysine) and poly(l-arginine) are important tools for gene delivery since they can efficiently condense DNA. It is difficult to produce cationic peptides by recombinant bacterial expression, and its chemical synthesis requires several steps of protection/deprotection and toxic agents. Chemo-enzymatic synthesis of peptides is a clean chemistry technique that allows fast production under mild conditions. With the aim to simplify the production of cationic peptides, the present work develops an enzymatic reaction which enables the synthesis of linear cationic peptides and, through terminal functionalization with tris(2-aminoethyl)amine, of branched cationic peptide conjugates, which show improved DNA complex formation. Cytotoxicity and transfection efficiency of all the chemo-enzymatically synthesized cationic peptides are evaluated for their novel use as gene delivery agents. Synthesized peptides exhibit transfection efficiencies comparable to previously reported monodisperse peptides. Chemo-enzymatic synthesis opens the door for efficient production of cationic peptides for their use as gene delivery carriers. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Watch and Wait Management of Inactive Cystic Echinococcosis – Does the Path to Inactivity Matter – Analysis of a Prospective Patient Cohort

    PubMed Central

    Stojkovic, Marija; Rosenberger, Kerstin Daniela; Steudle, Franziska; Junghanss, Thomas

    2016-01-01

    Background Overdiagnosis and overtreatment are rarely discussed in the context of NTDs despite their relevance for patients under the care of health services with limited resources where the risks of therapy induced complications are often disproportionate to the benefit. The advantages of cyst staging-based management of patients with cystic echinococcosis (CE) are not yet fully explored. Questions are: Do inactive cysts (CE 4 and CE 5) need treatment and is there a difference between cysts which reach CE4 and CE5 naturally or by benzimidazole therapy? Methodology/Principal findings Analysis of long-term follow-up data from a prospective CE patient cohort of 223 patients of a national clinical center for echinococcosis. The event of interest “relapse” was defined as the reversal of a cyst from an inactive stage (CE4, CE5) back to an active stage. The watch &wait (ww) group included 30 patients with 46 inactive cysts who never received medical treatment. The benzimidazole-treated (med) group included 15 patients with 17 cysts. There was no relapse in the ww-group whereas 8/17 cysts showed relapse within 18 months after treatment in the med-group. Loss to follow-up was 15.5%. Conclusions Data from the watch & wait group impressively show how stable naturally inactivated cysts are in contrast to cysts which reach inactivity through treatment with benzimidazoles. A substantial proportion of patients can be spared from treatment through cyst staging. Cysts which inactivated through a natural course do not relapse with very high likelihood. We recommend follow up of 5 years to confirm the stability of the inactive stage. Cysts driven into inactivity through benzimidazole therapy instead need careful monitoring to identify those which reactivate (around 50% within 18 months). 5 years follow-up appears safe to make a final decision on the need for further monitoring. PMID:27992434

  9. A Pedigree-Based Map of Recombination in the Domestic Dog Genome

    PubMed Central

    Campbell, Christopher L.; Bhérer, Claude; Morrow, Bernice E.; Boyko, Adam R.; Auton, Adam

    2016-01-01

    Meiotic recombination in mammals has been shown to largely cluster into hotspots, which are targeted by the chromatin modifier PRDM9. The canid family, including wolves and dogs, has undergone a series of disrupting mutations in this gene, rendering PRDM9 inactive. Given the importance of PRDM9, it is of great interest to learn how its absence in the dog genome affects patterns of recombination placement. We have used genotypes from domestic dog pedigrees to generate sex-specific genetic maps of recombination in this species. On a broad scale, we find that placement of recombination events in dogs is consistent with that in mice and apes, in that the majority of recombination occurs toward the telomeres in males, while female crossing over is more frequent and evenly spread along chromosomes. It has been previously suggested that dog recombination is more uniform in distribution than that of humans; however, we found that recombination in dogs is less uniform than in humans. We examined the distribution of recombination within the genome, and found that recombination is elevated immediately upstream of the transcription start site and around CpG islands, in agreement with previous studies, but that this effect is stronger in male dogs. We also found evidence for positive crossover interference influencing the spacing between recombination events in dogs, as has been observed in other species including humans and mice. Overall our data suggests that dogs have similar broad scale properties of recombination to humans, while fine scale recombination is similar to other species lacking PRDM9. PMID:27591755

  10. A Pedigree-Based Map of Recombination in the Domestic Dog Genome.

    PubMed

    Campbell, Christopher L; Bhérer, Claude; Morrow, Bernice E; Boyko, Adam R; Auton, Adam

    2016-09-02

    Meiotic recombination in mammals has been shown to largely cluster into hotspots, which are targeted by the chromatin modifier PRDM9. The canid family, including wolves and dogs, has undergone a series of disrupting mutations in this gene, rendering PRDM9 inactive. Given the importance of PRDM9 it is of great interest to learn how its absence in the dog genome affects patterns of recombination placement. We have used genotypes from domestic dog pedigrees to generate sex-specific genetic maps of recombination in this species. On a broad scale, we find that placement of recombination events in dogs is consistent with that in mice and apes, in that the majority of recombination occurs toward the telomeres in males, while female crossing over is more frequent and evenly spread along chromosomes. It has been previously suggested that dog recombination is more uniform in distribution than that of humans, however, we found that recombination in dogs is less uniform than humans. We examined the distribution of recombination within the genome, and find that recombination is elevated immediately upstream of the transcription start site, and around CpG islands, in agreement with previous studies, but find that this effect is stronger in male dogs. We also find evidence for positive crossover interference influencing the spacing between recombination events in dogs, as has been observed in other species including humans and mice. Overall our data suggests that dogs have similar broad scale properties of recombination to humans, while fine-scale recombination is similar to other species lacking PRDM9.

  11. Simple enzymatic procedure for l‐carnosine synthesis: whole‐cell biocatalysis and efficient biocatalyst recycling

    PubMed Central

    Heyland, Jan; Antweiler, Nicolai; Lutz, Jochen; Heck, Tobias; Geueke, Birgit; Kohler, Hans‐Peter E.; Blank, Lars M.; Schmid, Andreas

    2010-01-01

    Summary β‐Peptides and their derivates are usually stable to proteolysis and have an increased half‐life compared with α‐peptides. Recently, β‐aminopeptidases were described as a new enzyme class that enabled the enzymatic degradation and formation of β‐peptides. As an alternative to the existing chemical synthesis routes, the aim of the present work was to develop a whole‐cell biocatalyst for the synthesis and production of β‐peptides using this enzymatic activity. For the optimization of the reaction system we chose the commercially relevant β,α‐dipeptide l‐carnosine (β‐alanine‐l‐histidine) as model product. We were able to show that different recombinant yeast and bacteria strains, which overexpress a β‐peptidase, could be used directly as whole‐cell biocatalysts for the synthesis of l‐carnosine. By optimizing relevant reaction conditions for the best‐performing recombinant Escherichia coli strain, such as pH and substrate concentrations, we obtained high l‐carnosine yields of up to 71%. Long‐time as well as biocatalyst recycling experiments indicated a high stability of the developed biocatalyst for at least five repeated batches. Application of the recombinant E. coli in a fed‐batch process enabled the accumulation of l‐carnosine to a concentration of 3.7 g l−1. PMID:21255308

  12. Production of recombinant Agaricus bisporus tyrosinase in Saccharomyces cerevisiae cells.

    PubMed

    Lezzi, Chiara; Bleve, Gianluca; Spagnolo, Stefano; Perrotta, Carla; Grieco, Francesco

    2012-12-01

    It has been demonstrated that Agaricus bisporus tyrosinase is able to oxidize various phenolic compounds, thus being an enzyme of great importance for a number of biotechnological applications. The tyrosinase-coding PPO2 gene was isolated by reverse-transcription polymerase chain reaction (RT-PCR) using total RNA extracted from the mushroom fruit bodies as template. The gene was sequenced and cloned into pYES2 plasmid, and the resulting pY-PPO2 recombinant vector was then used to transform Saccharomyces cerevisiae cells. Native polyacrylamide gel electrophoresis followed by enzymatic activity staining with L-3,4-dihydroxyphenylalanine (L-DOPA) indicated that the recombinant tyrosinase is biologically active. The recombinant enzyme was overexpressed and biochemically characterized, showing that the catalytic constants of the recombinant tyrosinase were higher than those obtained when a commercial tyrosinase was used, for all the tested substrates. The present study describes the recombinant production of A. bisporus tyrosinase in active form. The produced enzyme has similar properties to the one produced in the native A. bisporus host, and its expression in S. cerevisiae provides good potential for protein engineering and functional studies of this important enzyme.

  13. Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens

    PubMed Central

    Li, Tiejun; Chen, Li; Sun, Yuncheng; Yuan, Jian; Graham, Michael; French, Peter

    2015-01-01

    RNA interference (RNAi) libraries screens have become widely used for small RNA (sRNA) therapeutic targets development. However, conventional enzymatically libraries, typically prepared using the type 2 restriction enzyme MmeI, produce sRNAs between 18 and 20 bp, much shorter than the usual lengths of 19–23 bp. Here we develop a size unbiased representative enzymatically generated RNAi (SURER) library, which employs type 3 restriction modification enzyme EcoP15I to produce sRNAs ranging from 19 to 23 bp using a group of rationally designed linkers, which can completely mimic the length of sRNAs naturally generated by Dicer enzyme in living cells, and the screening results of SURER libraries showed high recombination rate and knockdown efficiency. SURER library provides a useful tool for RNAi therapeutics screening in a fast and simple way. PMID:25493330

  14. Costameric proteins in human skeletal muscle during muscular inactivity

    PubMed Central

    Anastasi, Giuseppe; Cutroneo, Giuseppina; Santoro, Giuseppe; Arco, Alba; Rizzo, Giuseppina; Bramanti, Placido; Rinaldi, Carmen; Sidoti, Antonina; Amato, Aldo; Favaloro, Angelo

    2008-01-01

    normal, or slightly increased, compared with that in normal skeletal muscle fibres. We also observed a lower level of α7B- and β1D-integrin mRNA and a normal, or slightly higher than normal, level of α7A-integrin mRNA in the skeletal muscle fibres of the patients with sensitive-motor polyneuropathy, compared with those in the skeletal muscle of normal patients. Additionally, transmission electron microscopy of transverse sections of skeletal muscle fibres indicated that the normal muscle fibre architecture was disrupted, with no myosin present inside the actin hexagons. Based on our results, we hypothesize that skeletal muscle inactivity, such as that found after denervation, could result in a reorganization of the costameres, with α7B-integrin being replaced by α7A-integrin. In this way, the viability of the skeletal muscle fibre is maintained. It will be interesting to clarify, by future experimentation, the mechanisms that lead to the down-regulation of integrins and agrin in muscular dystrophies. PMID:18537849

  15. Using a novel environmental quality measure to understand population-level physical inactivity

    EPA Science Inventory

    Physical inactivity has been associated with numerous adverse health outcomes including obesity, heart disease, and depression, and is considered a major contributor to all-cause mortality worldwide. Understanding the role of the overall ambient environment in population inactivi...

  16. Patterns of association between environmental quality and physical inactivity vary across the rural-urban continuum

    EPA Science Inventory

    Physical inactivity has been associated with numerous adverse health outcomes including obesity, heart disease, and depression, and is considered a major contributor to all-cause mortality worldwide. Many studies have shown associations between specific environmental features (la...

  17. A new light on DNA replication from the inactive X chromosome.

    PubMed

    Aladjem, Mirit I; Fu, Haiqing

    2014-06-01

    While large portions of the mammalian genome are known to replicate sequentially in a distinct, tissue-specific order, recent studies suggest that the inactive X chromosome is duplicated rapidly via random, synchronous DNA synthesis at numerous adjacent regions. The rapid duplication of the inactive X chromosome was observed in high-resolution studies visualizing DNA replication patterns in the nucleus, and by allele-specific DNA sequencing studies measuring the extent of DNA synthesis. These studies conclude that inactive X chromosomes complete replication earlier than previously thought and suggest that the strict order of DNA replication detected in the majority of genomic regions is not preserved in non-transcribed, "silent" chromatin. These observations alter current concepts about the regulation of DNA replication in non-transcribed portions of the genome in general and in the inactive X-chromosome in particular.

  18. Increasing and decreasing motor and cognitive output: a model of general action and inaction goals.

    PubMed

    Albarracín, Dolores; Handley, Ian M; Noguchi, Kenji; McCulloch, Kathleen C; Li, Hong; Leeper, Joshua; Brown, Rick D; Earl, Allison; Hart, William P

    2008-09-01

    General action and inaction goals can influence the amount of motor or cognitive output irrespective of the type of behavior in question, with the same stimuli producing trivial and important motor and cognitive manifestations normally viewed as parts of different systems. A series of experiments examined the effects of instilling general action and inaction goals using word primes, such as "action" and "rest." The first 5 experiments showed that the same stimuli influenced motor output, such as doodling on a piece of paper and eating, as well as cognitive output, such as recall and problem solving. The last 2 experiments supported the prediction that these diverse effects can result from the instigation of general action and inaction goals. Specifically, these last 2 studies confirmed that participants were motivated to achieve active or inactive states and that attaining them decreased the effects of the primes on behavior.

  19. General Action and Inaction Goals: Their Behavioral, Cognitive, and Affective Origins and Influences.

    PubMed

    Albarracin, Dolores; Hepler, Justin; Tannenbaum, Melanie

    2011-04-01

    Since the 1970s, researchers on motivation and behavior have taken the stance that important human behaviors are determined by specific attitudes, intentions, and goals. In the present article, we review evidence suggesting that, in addition to specific motivational constructs, general goals of action and inaction are also vital determinants of many important human behaviors. This research examines the effects of these goals on motor behavior, cognitive performance, and political participation. Furthermore, we connect these general action and inaction goals with other important areas in psychology, including affect, approach/avoidance, energization, material resources, mindsets, and power. Finally, we conclude with a discussion of individual and regional/cultural differences in action and inaction. Overall, general goals for action and inaction are shown to influence a vast array of important behaviors, suggesting that in addition to considering specific attitudes, intentions, and goals, researchers may gain important insight into human behavior by considering general motivations.

  20. General Action and Inaction Goals: Their Behavioral, Cognitive, and Affective Origins and Influences

    PubMed Central

    Albarracin, Dolores; Hepler, Justin; Tannenbaum, Melanie

    2013-01-01

    Since the 1970s, researchers on motivation and behavior have taken the stance that important human behaviors are determined by specific attitudes, intentions, and goals. In the present article, we review evidence suggesting that, in addition to specific motivational constructs, general goals of action and inaction are also vital determinants of many important human behaviors. This research examines the effects of these goals on motor behavior, cognitive performance, and political participation. Furthermore, we connect these general action and inaction goals with other important areas in psychology, including affect, approach/avoidance, energization, material resources, mindsets, and power. Finally, we conclude with a discussion of individual and regional/cultural differences in action and inaction. Overall, general goals for action and inaction are shown to influence a vast array of important behaviors, suggesting that in addition to considering specific attitudes, intentions, and goals, researchers may gain important insight into human behavior by considering general motivations. PMID:23766569

  1. Being Active and Impulsive: The Role of Goals for Action and Inaction in Self-Control

    PubMed Central

    Hepler, Justin; Albarracin, Dolores; McCulloch, Kathleen C.; Noguchi, Kenji

    2013-01-01

    Although self-control often requires behavioral inaction (i.e., not eating a piece of cake), the process of inhibiting impulsive behavior is commonly characterized as cognitively active (i.e., actively exerting self-control). Two experiments examined whether motivation for action or inaction facilitates self-control behavior in the presence of tempting stimuli. Experiment 1 used a delay discounting task to assess the ability to delay gratification with respect to money. Experiment 2 used a Go/No-Go task to assess the ability to inhibit a dominant but incorrect motor response to the words “condom” and “sex”. The results demonstrate that goals for inaction promote self-control, whereas goals for action promote impulsive behavior. These findings are discussed in light of recent evidence suggesting that goals for action and inaction modulate physiological resources that promote behavioral execution. PMID:23766548

  2. Being Active and Impulsive: The Role of Goals for Action and Inaction in Self-Control.

    PubMed

    Hepler, Justin; Albarracin, Dolores; McCulloch, Kathleen C; Noguchi, Kenji

    2012-12-01

    Although self-control often requires behavioral inaction (i.e., not eating a piece of cake), the process of inhibiting impulsive behavior is commonly characterized as cognitively active (i.e., actively exerting self-control). Two experiments examined whether motivation for action or inaction facilitates self-control behavior in the presence of tempting stimuli. Experiment 1 used a delay discounting task to assess the ability to delay gratification with respect to money. Experiment 2 used a Go/No-Go task to assess the ability to inhibit a dominant but incorrect motor response to the words "condom" and "sex". The results demonstrate that goals for inaction promote self-control, whereas goals for action promote impulsive behavior. These findings are discussed in light of recent evidence suggesting that goals for action and inaction modulate physiological resources that promote behavioral execution.

  3. 37 CFR 11.19 - Disciplinary jurisdiction; Jurisdiction to transfer to disability inactive status.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... inactive status. (1) Grounds for discipline include: (i) Conviction of a serious crime; (ii) Discipline on... appearing before any Federal program or agency; (iii) Failure to comply with any order of a Court...

  4. Increasing and Decreasing Motor and Cognitive Output: A Model of General Action and Inaction Goals

    PubMed Central

    Albarracín, Dolores; Handley, Ian M.; Noguchi, Kenji; McCulloch, Kathleen C.; Li, Hong; Leeper, Joshua; Brown, Rick D.; Earl, Allison; Hart, William P.

    2013-01-01

    General action and inaction goals can influence the amount of motor or cognitive output irrespective of the type of behavior in question, with the same stimuli producing trivial and important motor and cognitive manifestations normally viewed as parts of different systems. A series of experiments examined the effects of instilling general action and inaction goals using word primes, such as “action” and “rest.” The first 5 experiments showed that the same stimuli influenced motor output, such as doodling on a piece of paper and eating, as well as cognitive output, such as recall and problem solving. The last 2 experiments supported the prediction that these diverse effects can result from the instigation of general action and inaction goals. Specifically, these last 2 studies confirmed that participants were motivated to achieve active or inactive states and that attaining them decreased the effects of the primes on behavior. PMID:18729691

  5. Evidence for a chemoautotrophically based food web at inactive hydrothermal vents (Manus Basin)

    NASA Astrophysics Data System (ADS)

    Erickson, K. L.; Macko, S. A.; Van Dover, C. L.

    2009-09-01

    Hydrothermal vents are ephemeral systems. When venting shuts down, sulfide-dependent taxa die off, and non-vent taxa can colonize the hard substrata. In Manus Basin (Papua New Guinea), where hydrothermally active and inactive sites are interspersed, hydroids, cladorhizid sponges, barnacles, bamboo corals, and other invertebrate types may occupy inactive sites. Carbon and nitrogen isotopic compositions of animals occupying inactive sites are consistent with nutritional dependence on either chemoautotrophically or photosynthetically produced organic material, but sulfur isotopic compositions of these animals point to a chemoautotrophic source of sulfur from dissolved sulfide in vent fluids rather than sulfur derived from seawater sulfate through photosynthesis. Given that suspension-feeding and micro-carnivorous invertebrates are the biomass dominants at inactive sites, the primary source of chemoautotrophic nutrition is likely suspended particulates and organisms delivered from nearby active vents.

  6. Evidence for a Chemoautotrophically Based Food Web at Inactive Hydrothermal Vents (Manus Basin)

    NASA Astrophysics Data System (ADS)

    van Dover, C. L.; Erickson, K.; Macko, S.

    2008-12-01

    Hydrothermal vents are ephemeral systems. When venting shuts down, sulfide-dependent taxa die off, and non-vent taxa can colonize the hard substrata. In Manus Basin (Papua New Guinea), where active and inactive sulfide mounds are interspersed, hydroids, cladorhizid sponges, barnacles, and bamboo sponges, and other invertebrate types may occupy inactive sulfide mounds. Carbon and nitrogen isotopic compositions of animals occupying inactive sulfide mounds are consistent with nutritional dependence on either chemoautotrophically or photosynthetically produced organic material, but sulfur isotopic compositions of these animals point to a chemoautotrophic source of sulfur from dissolved sulfide in vent fluids rather than sulfur derived from seawater sulfate through photosynthesis. Given that suspension-feeding and micro- carnivorous invertebrates are the biomass dominants at inactive sulfide mounds, the primary source of chemoautotrophic nutrition is likely suspended particulates and organisms delivered from nearby active vents.

  7. Patterns of association between environmental quality and physical inactivity vary across the rural-urban continuum

    EPA Science Inventory

    Physical inactivity has been associated with numerous adverse health outcomes including obesity, heart disease, and depression, and is considered a major contributor to all-cause mortality worldwide. Many studies have shown associations between specific environmental features (la...

  8. Using a novel environmental quality measure to understand population-level physical inactivity

    EPA Science Inventory

    Physical inactivity has been associated with numerous adverse health outcomes including obesity, heart disease, and depression, and is considered a major contributor to all-cause mortality worldwide. Understanding the role of the overall ambient environment in population inactivi...

  9. Are U.S. Teens Now as Inactive as 60-Year-Olds?

    MedlinePlus

    ... news/fullstory_166666.html Are U.S. Teens Now as Inactive as 60-Year-Olds? The lack of exercise more ... Here's some compelling evidence that Americans have become a sedentary bunch: Research suggests that the average teen ...

  10. SEMINAR PUBLICATION: MANAGING ENVIRONMENTAL PROBLEMS AT INACTIVE AND ABANDONED METALS MINE SITES

    EPA Science Inventory

    Environmental problems associated with abandoned and inactive mines are addressed along with some approaches to resolving those problems, including case studies demonstrating technologies that have worked. New technologies being investigated are addressed also.

  11. The economic burden of physical inactivity: a global analysis of major non-communicable diseases.

    PubMed

    Ding, Ding; Lawson, Kenny D; Kolbe-Alexander, Tracy L; Finkelstein, Eric A; Katzmarzyk, Peter T; van Mechelen, Willem; Pratt, Michael

    2016-09-24

    The pandemic of physical inactivity is associated with a range of chronic diseases and early deaths. Despite the well documented disease burden, the economic burden of physical inactivity remains unquantified at the global level. A better understanding of the economic burden could help to inform resource prioritisation and motivate efforts to increase levels of physical activity worldwide. Direct health-care costs, productivity losses, and disability-adjusted life-years (DALYs) attributable to physical inactivity were estimated with standardised methods and the best data available for 142 countries, representing 93·2% of the world's population. Direct health-care costs and DALYs were estimated for coronary heart disease, stroke, type 2 diabetes, breast cancer, and colon cancer attributable to physical inactivity. Productivity losses were estimated with a friction cost approach for physical inactivity related mortality. Analyses were based on national physical inactivity prevalence from available countries, and adjusted population attributable fractions (PAFs) associated with physical inactivity for each disease outcome and all-cause mortality. Conservatively estimated, physical inactivity cost health-care systems international $ (INT$) 53·8 billion worldwide in 2013, of which $31·2 billion was paid by the public sector, $12·9 billion by the private sector, and $9·7 billion by households. In addition, physical inactivity related deaths contribute to $13·7 billion in productivity losses, and physical inactivity was responsible for 13·4 million DALYs worldwide. High-income countries bear a larger proportion of economic burden (80·8% of health-care costs and 60·4% of indirect costs), whereas low-income and middle-income countries have a larger proportion of the disease burden (75·0% of DALYs). Sensitivity analyses based on less conservative assumptions led to much higher estimates. In addition to morbidity and premature mortality, physical inactivity is

  12. Oxidative Weathering and Microbial Diversity of an Inactive Seafloor Hydrothermal Sulfide Chimney.

    PubMed

    Li, Jiangtao; Cui, Jiamei; Yang, Qunhui; Cui, Guojie; Wei, Bingbing; Wu, Zijun; Wang, Yong; Zhou, Huaiyang

    2017-01-01

    When its hydrothermal supply ceases, hydrothermal sulfide chimneys become inactive and commonly experience oxidative weathering on the seafloor. However, little is known about the oxidative weathering of inactive sulfide chimneys, nor about associated microbial community structures and their succession during this weathering process. In this work, an inactive sulfide chimney and a young chimney in the early sulfate stage of formation were collected from the Main Endeavor Field of the Juan de Fuca Ridge. To assess oxidative weathering, the ultrastructures of secondary alteration products accumulating on the chimney surface were examined and the presence of possible Fe-oxidizing bacteria (FeOB) was investigated. The results of ultrastructure observation revealed that FeOB-associated ultrastructures with indicative morphologies were abundantly present. Iron oxidizers primarily consisted of members closely related to Gallionella spp. and Mariprofundus spp., indicating Fe-oxidizing species likely promote the oxidative weathering of inactive sulfide chimneys. Abiotic accumulation of Fe-rich substances further indicates that oxidative weathering is a complex, dynamic process, alternately controlled by FeOB and by abiotic oxidization. Although hydrothermal fluid flow had ceased, inactive chimneys still accommodate an abundant and diverse microbiome whose microbial composition and metabolic potential dramatically differ from their counterparts at active vents. Bacterial lineages within current inactive chimney are dominated by members of α-, δ-, and γ-Proteobacteria and they are deduced to be closely involved in a diverse set of geochemical processes including iron oxidation, nitrogen fixation, ammonia oxidation and denitrification. At last, by examining microbial communities within hydrothermal chimneys at different formation stages, a general microbial community succession can be deduced from early formation stages of a sulfate chimney to actively mature sulfide

  13. Oxidative Weathering and Microbial Diversity of an Inactive Seafloor Hydrothermal Sulfide Chimney

    PubMed Central

    Li, Jiangtao; Cui, Jiamei; Yang, Qunhui; Cui, Guojie; Wei, Bingbing; Wu, Zijun; Wang, Yong; Zhou, Huaiyang

    2017-01-01

    When its hydrothermal supply ceases, hydrothermal sulfide chimneys become inactive and commonly experience oxidative weathering on the seafloor. However, little is known about the oxidative weathering of inactive sulfide chimneys, nor about associated microbial community structures and their succession during this weathering process. In this work, an inactive sulfide chimney and a young chimney in the early sulfate stage of formation were collected from the Main Endeavor Field of the Juan de Fuca Ridge. To assess oxidative weathering, the ultrastructures of secondary alteration products accumulating on the chimney surface were examined and the presence of possible Fe-oxidizing bacteria (FeOB) was investigated. The results of ultrastructure observation revealed that FeOB-associated ultrastructures with indicative morphologies were abundantly present. Iron oxidizers primarily consisted of members closely related to Gallionella spp. and Mariprofundus spp., indicating Fe-oxidizing species likely promote the oxidative weathering of inactive sulfide chimneys. Abiotic accumulation of Fe-rich substances further indicates that oxidative weathering is a complex, dynamic process, alternately controlled by FeOB and by abiotic oxidization. Although hydrothermal fluid flow had ceased, inactive chimneys still accommodate an abundant and diverse microbiome whose microbial composition and metabolic potential dramatically differ from their counterparts at active vents. Bacterial lineages within current inactive chimney are dominated by members of α-, δ-, and γ-Proteobacteria and they are deduced to be closely involved in a diverse set of geochemical processes including iron oxidation, nitrogen fixation, ammonia oxidation and denitrification. At last, by examining microbial communities within hydrothermal chimneys at different formation stages, a general microbial community succession can be deduced from early formation stages of a sulfate chimney to actively mature sulfide

  14. Light saturation response of inactive photosystem II reaction centers in spinach.

    PubMed

    Chylla, R A; Whitmarsh, J

    1990-07-01

    The effective absorption cross section of inactive photosystem II (PS II) centers, which is the product of the effective antenna size and the quantum yield for photochemistry, was investigated by comparing the light saturation curves of inactive PS II and active reaction centers in intact chloroplasts and thylakoid membranes of spinach (Spinacia oleracea). Inactive PS II centers are defined as the impaired PS II reaction centers that require greater than 50 ms for the reoxidation of QA (-) subsequent to a single turnover flash. Active reaction centers are defined as the rapidly turning over PS II centers (recovery time less than 50 ms) and all of the PS I centers. The electrochromic shift, measured by the flash-induced absorbance increase at 518 nm, was used to probe the activity of the reaction centers. Light saturation curves were generated for inactive PS II centers and active reaction centers by measuring the extent of the absorbance increase at 518 nm induced by red actinic flashes of variable energy. The light saturation curves show that inactive PS II centers required over twice as many photons as active reaction centers to achieve the same yield. The ratio of the flash energy required for 50% saturation for active reaction centers (PS II active + PS I) compared to inactive PS II centers was 0.45±0.04 in intact chloroplasts, and 0.54±0.11 in thylakoid membranes. Analysis of the light saturation curves using a Poisson statistical model in which the ratio of the antenna size of active PS II centers to that of PS I is considered to range from 1 to 1.5, indicates that the effective absorption cross section of inactive PS II centers was 0.54-0.37 times that of active PS II centers. If the quantum yield for photochemistry is assumed to be one, we estimate that the antenna system serving the inactive PS II centers contains approx. 110 chlorophyll molecules.

  15. Prevalence and Correlates of Physical Inactivity among Older Adults in Rio Grande do Sul, Brazil

    PubMed Central

    Souza, Adelle M. R.; Fillenbaum, Gerda G.; Blay, Sergio L.

    2015-01-01

    Background Current information on the epidemiology of physical inactivity among older adults is lacking, making it difficult to target the inactive and to plan for interventions to ameliorate adverse effects. Objectives To present statewide representative findings on the prevalence of physical inactivity among older community residents, its correlates and associated health service use. Methods A representative non-institutionalized random sample of 6963 individuals in Rio Grande do Sul, Brazil, aged ≥60 years, was interviewed face-to-face. Information was obtained on demographic characteristics, social resources, health conditions and behaviors, health service use, and physical inactivity. Controlled logistic regression was used to determine the association of physical inactivity with these characteristics. Results Overall, 62% reported no regular physical activity. Physical inactivity was significantly more prevalent among women, older persons, those with lower education and income, Afro-Brazilians (73%; White: 61%; “other”: 64%), those no longer married, and was associated with multiple individual health conditions and impaired activities of daily living (ADL). In adjusted analyses, associations remained for sociodemographic characteristics, social participation, impaired self-rated health, ADL, vision, and depression (odds ratios (OR) 1.2–1.7). Physically inactive respondents were less likely to report outpatient visits (OR 0.81), but more likely to be hospitalized (OR 1.41). Conclusions Physical inactivity is highly prevalent, particularly among Afro -Brazilians. It is associated with adverse sociodemographic characteristics; lack of social interaction; and poor self-rated health, ADL, vision, and depression; although not with other health conditions. Self-care may be neglected, resulting in hospitalization. PMID:25700161

  16. Plasmodium falciparum SERA5 plays a non-enzymatic role in the malarial asexual blood-stage lifecycle

    PubMed Central

    Stallmach, Robert; Kavishwar, Manoli; Withers-Martinez, Chrislaine; Hackett, Fiona; Collins, Christine R; Howell, Steven A; Yeoh, Sharon; Knuepfer, Ellen; Atid, Avshalom J; Holder, Anthony A; Blackman, Michael J

    2015-01-01

    The malaria parasite Plasmodium falciparum replicates in an intraerythrocytic parasitophorous vacuole (PV). The most abundant P. falciparum PV protein, called SERA5, is essential in blood stages and possesses a papain-like domain, prompting speculation that it functions as a proteolytic enzyme. Unusually however, SERA5 possesses a Ser residue (Ser596) at the position of the canonical catalytic Cys of papain-like proteases, and the function of SERA5 or whether it performs an enzymatic role is unknown. In this study, we failed to detect proteolytic activity associated with the Ser596-containing parasite-derived or recombinant protein. However, substitution of Ser596 with a Cys residue produced an active recombinant enzyme with characteristics of a cysteine protease, demonstrating that SERA5 can bind peptides. Using targeted homologous recombination in P. falciparum, we substituted Ser596 with Ala with no phenotypic consequences, proving that SERA5 does not perform an essential enzymatic role in the parasite. We could also replace an internal segment of SERA5 with an affinity-purification tag. In contrast, using almost identical targeting constructs, we could not truncate or C-terminally tag the SERA5 gene, or replace Ser596 with a bulky Arg residue. Our findings show that SERA5 plays an indispensable but non-enzymatic role in the P. falciparum blood-stage life cycle. PMID:25599609

  17. A novel computational method to simulate non-enzymatic self-replication. [Abstract only

    NASA Technical Reports Server (NTRS)

    Navarro-Gonzalez, Rafael; Reggia, James A.; Wu, Jayoung; Chou, Hui-Hsien

    1994-01-01

    Non-enzymatic, template-directed synthesis of oligonucleotides has been extensively studied in the laboratory as a model to understand the kind of chemical processes that might have contributed to the origin of life on Earth. Several oligonucleotides have been shown to catalyze the synthesis of their complements from activated mononucleotides; however, a restricted number of them have been found to self-replicate. Recently we developed an efficient modified cellular automata method that supports the study of self-replicating oligonucleotides. With this method the oligonucleotide molecules are represented as active cells imbedded in a two-dimensional array of inactive cells symbolizing the environment. Random movements and probability-governed chemical reactions occurring in a cellular space can effectively simulate the experimental behavior observed in self-directed replication of oligonucleotides.

  18. Recombinant renewable polyclonal antibodies.

    PubMed

    Ferrara, Fortunato; D'Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew R M

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.

  19. The dissociative recombination of ?

    NASA Astrophysics Data System (ADS)

    Laubé, S.; Lehfaoui, L.; Rowe, B. R.; Mitchell, J. B. A.

    1998-09-01

    The dissociative recombination rate coefficient for 0953-4075/31/18/016/img2 has been measured at 300 K using a flowing afterglow Langmuir probe-mass spectrometer apparatus. A value of 0953-4075/31/18/016/img3 has been found.

  20. Introduction to dissociative recombination

    NASA Technical Reports Server (NTRS)

    Guberman, Steven L.; Mitchell, J. Brian A.

    1989-01-01

    Dissociative recombination (DR) of molecular ions with electrons has important consequences in many areas of physical science. Ab-initio calculations coupled with resonant scattering theory and multichannel quantum defect studies have produced detailed results illuminating the role of ion vibrational excitation, the quantum yields of the DR products, and the role of Rydberg states. The theoretical and experimental results are discussed.

  1. Recombinant renewable polyclonal antibodies

    PubMed Central

    Ferrara, Fortunato; D’Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew RM

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products. PMID:25530082

  2. Recombinant DNA for Teachers.

    ERIC Educational Resources Information Center

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  3. Recombineering linear BACs.

    PubMed

    Chen, Qingwen; Narayanan, Kumaran

    2015-01-01

    Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.

  4. Recombinant DNA for Teachers.

    ERIC Educational Resources Information Center

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  5. [Physical inactivity and associated factors in adults, São Paulo, Brazil].

    PubMed

    Zanchetta, Luane Margarete; Barros, Marilisa Berti de Azevedo; César, Chester Luiz Galvão; Carandina, Luana; Goldbaum, Moisés; Alves, Maria Cecília Goi Porto

    2010-09-01

    To analyze the prevalence of overall and leisure time physical inactivity and associated factors and types of exercises or sports modalities according to schooling in 2,050 adults from 18 to 59 years of age - state of São Paulo, Brazil. Population-based cross-sectional study with a stratified sample of clusters performed in multiple stages. Physical inactivity was determined using the short version of the International Physical Activity Questionnaire - IPAQ and by a question on the regular practice of leisure time physical activity. Data analysis took the sample design into account. Prevalence of physical inactivity during leisure was higher among women. Poisson multiple regression model in man indicated that overall sedentarism was lower among single and separated men, students and without car in the household. Leisure physical inactivity was greater among men over forty years, among those with less schooling and full-time students. Overall physical inactivity was more prevalent among woman with more schooling, with less qualified occupations and widows. Leisure physical inactivity decreased with age and schooling. Among modalities practiced for leisure, walking was more prevalent among women and football was more prevalent among men. Most modalities were directly associated with schooling; approximately 25% of the individuals with more than 12 years of schooling practiced walking. These results suggest that interventions and public policies to promote physical activity should consider differences in gender and socioeconomic status as well as the preferences for different modalities and the context in which the physical activity is practiced.

  6. Ocular surface characteristics and impression cytology in patients with active versus inactive Thyroid Eye Disease.

    PubMed

    Xu, Nuo; Huang, Danping; Yang, Huasheng; Lai, Zhaoguang; Luo, Quan

    2012-06-01

    To compare the clinical findings, tear film function and impression cytology between patients with active and inactive Thyroid Eye Disease (TED). A total of 56 patients with TED and 30 controls were recruited in this prospective observational cohort study. TED patients were divided into active TED and inactive TED types according to a seven-point modified formulation of the Clinical Activity Score (CAS). All participants underwent full eye examinations including Ocular Surface Disease Index (OSDI) score, tear film break-up time (TBUT), fluorescein staining and Schirmer I test. Thirty nine patients with thyroid-associated orbitopathy (TAO) received Nelson's grade with conjunctival impression cytology. Proptosis, palpebral fissure width and lagophthalmos were assessed. Ocular surface parameters including proptosis, palpebral fissure width and lagophthalmos did not differ between active and inactive TED patients (P>0.05). Both active and inactive TED patients obtained higher fluorescein staining scores, lower TBUT scores and significantly lower Schirmer test scores than those of controls (P<0.001 for all). Additionally, the TBUT score was significantly lower and the OSDI score significantly higher in the active TED group compared with those in the inactive TED group (P<0.001 for both). Impression cytology revealed a higher proportion of grade 2-3 changes in the active TED group compared with the inactive TED group (P<0.001). Orbital inflammation in TED patients may lead to decreased tear film stability and ocular surface squamous metaplasia.

  7. The inactive X chromosome is epigenetically unstable and transcriptionally labile in breast cancer

    PubMed Central

    Chaligné, Ronan; Popova, Tatiana; Mendoza-Parra, Marco-Antonio; Saleem, Mohamed-Ashick M.; Gentien, David; Ban, Kristen; Piolot, Tristan; Leroy, Olivier; Mariani, Odette

    2015-01-01

    Disappearance of the Barr body is considered a hallmark of cancer, although whether this corresponds to genetic loss or to epigenetic instability and transcriptional reactivation is unclear. Here we show that breast tumors and cell lines frequently display major epigenetic instability of the inactive X chromosome, with highly abnormal 3D nuclear organization and global perturbations of heterochromatin, including gain of euchromatic marks and aberrant distributions of repressive marks such as H3K27me3 and promoter DNA methylation. Genome-wide profiling of chromatin and transcription reveal modified epigenomic landscapes in cancer cells and a significant degree of aberrant gene activity from the inactive X chromosome, including several genes involved in cancer promotion. We demonstrate that many of these genes are aberrantly reactivated in primary breast tumors, and we further demonstrate that epigenetic instability of the inactive X can lead to perturbed dosage of X-linked factors. Taken together, our study provides the first integrated analysis of the inactive X chromosome in the context of breast cancer and establishes that epigenetic erosion of the inactive X can lead to the disappearance of the Barr body in breast cancer cells. This work offers new insights and opens up the possibility of exploiting the inactive X chromosome as an epigenetic biomarker at the molecular and cytological levels in cancer. PMID:25653311

  8. Childhood adversities and socioeconomic position as predictors of leisure-time physical inactivity in early adulthood.

    PubMed

    Kestilä, Laura; Mäki-Opas, Tomi; Kunst, Anton E; Borodulin, Katja; Rahkonen, Ossi; Prättälä, Ritva

    2015-02-01

    Limited knowledge exists on how childhood social, health-related and economic circumstances predict adult physical inactivity. Our aim was a) to examine how various childhood adversities and living conditions predict leisure-time physical inactivity in early adulthood and b) to find out whether these associations are mediated through the respondent's own education. Young adults aged 18-29 were used from the Health 2000 Study of the Finnish. The cross-sectional data were based on interviews and questionnaires including retrospective information on childhood circumstances. The analyses were carried out on 68% of the original sample (N = 1894). The outcome measure was leisure-time physical inactivity. Only a few of the 11 childhood adversities were related with physical activity in early adulthood. Having been bullied at school was associated with physical inactivity independently of the other childhood circumstances and the respondent's own education. Low parental education predicted leisure-time physical inactivity in men and the association was mediated by the respondent's own education. Respondents with only primary or vocational education were more likely to be physically inactive during leisure-time compared with those with secondary or higher education. There is some evidence that few specific childhood adversities, especially bullying at school, have long-lasting effects on physical activity levels.

  9. Motives for Physical Activity among Active and Inactive Persons in Their Mid-Thirties

    PubMed Central

    Aaltonen, Sari; Rottensteiner, Mirva; Kaprio, Jaakko; Kujala, Urho M

    2013-01-01

    The purpose of this study was to examine the motives for leisure time physical activity among active and inactive men and women in their mid-thirties. We used both cross-sectional and longitudinal designs. Altogether, 2308 participants (mean age 33.9 years, 53.4 % women) were identified from the population-based FinnTwin16 Cohort. Physically active and inactive individuals were identified on the basis of their leisure time MET hours/day. We evaluated participants’ physical activity motivation with a modified version of the Recreational Exercise Motivation Measure. Comparisons between active and inactive individuals were analysed using the Wald test for equality of means, and effect sizes were calculated as Cohen’s d. Motives related to mastery, physical fitness, social aspect of physical activity, psychological state, enjoyment, willingness to be fitter/look better than others and appearance were significantly more important for the active than inactive participants. Conforming to others’ expectations was the only item on which the inactive persons scored higher than active persons. The longitudinal results for physical activity were parallel to the cross-sectional results. This study supports to the view that motivation factors differ between active and inactive persons, and that intrinsic motives are associated with consistent leisure time physical activity. PMID:23331765

  10. Review: Enzymatic Hydrolysis of Cellulosic Biomass

    SciTech Connect

    Yang, Bin; Dai, Ziyu; Ding, Shi-You; Wyman, Charles E.

    2011-07-16

    Biological conversion of cellulosic biomass to fuels and chemicals offers the high yields to products vital to economic success and the potential for very low costs. Enzymatic hydrolysis that converts lignocellulosic biomass to fermentable sugars may be the most complex step in this process due to substrate-related and enzyme-related effects and their interactions. Although enzymatic hydrolysis offers the potential for higher yields, higher selectivity, lower energy costs, and milder operating conditions than chemical processes, the mechanism of enzymatic hydrolysis and the relationship between the substrate structure and function of various glycosyl hydrolase components are not well understood. Consequently, limited success has been realized in maximizing sugar yields at very low cost. This review highlights literature on the impact of key substrate and enzyme features that influence performance to better understand fundamental strategies to advance enzymatic hydrolysis of cellulosic biomass for biological conversion to fuels and chemicals. Topics are summarized from a practical point of view including characteristics of cellulose (e.g., crystallinity, degree of polymerization, and accessible surface area) and soluble and insoluble biomass components (e.g., oligomeric xylan, lignin, etc.) released in pretreatment, and their effects on the effectiveness of enzymatic hydrolysis. We further discuss the diversity, stability, and activity of individual enzymes and their synergistic effects in deconstructing complex lignocellulosic biomass. Advanced technologies to discover and characterize novel enzymes and to improve enzyme characteristics by mutagenesis, post-translational modification, and over-expression of selected enzymes and modifications in lignocellulosic biomass are also discussed.

  11. Structural Perspective on Enzymatic Halogenation

    PubMed Central

    2008-01-01

    Simple halogen substituents frequently afford key structural features that account for the potency and selectivity of natural products, including antibiotics and hormones. For example, when a single chlorine atom on the antibiotic vancomycin is replaced by hydrogen, the resulting antibacterial activity decreases by up to 70% (HarrisC. M.; KannanR.; KopeckaH.; HarrisT. M.J. Am. Chem. Soc.1985, 107, 6652−6658). This Account analyzes how structure underlies mechanism in halogenases, the molecular machines designed by nature to incorporate halogens into diverse substrates. Traditional synthetic methods of integrating halogens into complex molecules are often complicated by a lack of specificity and regioselectivity. Nature, however, has developed a variety of elegant mechanisms for halogenating specific substrates with both regio- and stereoselectivity. An improved understanding of the biological routes toward halogenation could lead to the development of novel synthetic methods for the creation of new compounds with enhanced functions. Already, researchers have co-opted a fluorinase from the microorganism Streptomyces cattleya to produce 18F-labeled molecules for use in positron emission tomography (PET) (DengH.; CobbS. L.; GeeA. D.; LockhartA.; MartarelloL.; McGlincheyR. P.; O’HaganD.; OnegaM.Chem. Commun.2006, 652−654). Therefore, the discovery and characterization of naturally occurring enzymatic halogenation mechanisms has become an active area of research. The catalogue of known halogenating enzymes has expanded from the familiar haloperoxidases to include oxygen-dependent enzymes and fluorinases. Recently, the discovery of a nucleophilic halogenase that catalyzes chlorinations has expanded the repertoire of biological halogenation chemistry (DongC.; HuangF.; DengH.; SchaffrathC.; SpencerJ. B.; O’HaganD.; NaismithJ. H.Nature2004, 427, 561−56514765200). Structural characterization has provided a basis toward a mechanistic understanding of the specificity

  12. Genomic homologous recombination in planta.

    PubMed Central

    Gal, S; Pisan, B; Hohn, T; Grimsley, N; Hohn, B

    1991-01-01

    A system for monitoring intrachromosomal homologous recombination in whole plants is described. A multimer of cauliflower mosaic virus (CaMV) sequences, arranged such that CaMV could only be produced by recombination, was integrated into Brassica napus nuclear DNA. This set-up allowed scoring of recombination events by the appearance of viral symptoms. The repeated homologous regions were derived from two different strains of CaMV so that different recombinant viruses (i.e. different recombination events) could be distinguished. In most of the transgenic plants, a single major virus species was detected. About half of the transgenic plants contained viruses of the same type, suggesting a hotspot for recombination. The remainder of the plants contained viruses with cross-over sites distributed throughout the rest of the homologous sequence. Sequence analysis of two recombinant molecules suggest that mismatch repair is linked to the recombination process. Images PMID:2026150

  13. Accelerated progression of chronic lymphocytic leukemia in Eμ-TCL1 mice expressing catalytically inactive RAG1.

    PubMed

    Nganga, Vincent K; Palmer, Victoria L; Naushad, Hina; Kassmeier, Michele D; Anderson, Dirk K; Perry, Greg A; Schabla, Nathan M; Swanson, Patrick C

    2013-05-09

    Chronic lymphocytic leukemia (CLL) is a prevalent B-cell neoplasia that is often preceded by a more benign monoclonal CD5(+) B-cell lymphocytosis. We previously generated transgenic mice expressing catalytically inactive RAG1 (dominant-negative recombination activating gene 1 [dnRAG1] mice) that develop an early-onset indolent CD5(+) B-cell lymphocytosis attributed to a defect in secondary V(D)J rearrangements initiated to edit autoreactive B-cell receptor (BCR) specificity. Hypothesizing that CD5(+) B cells in these animals represent potential CLL precursors, we crossed dnRAG1 mice with CLL-prone Eμ-TCL1 mice to determine whether dnRAG1 expression in Eμ-TCL1 mice accelerates CLL onset. Consistent with this hypothesis, CD5(+) B-cell expansion and CLL progression occurred more rapidly in double-transgenic mice compared with Eμ-TCL1 mice. Nevertheless, CD5(+) B cells in the 2 mouse strains exhibited close similarities in phenotype, immunoglobulin gene usage, and mutation status, and expression of genes associated with immune tolerance and BCR signaling. Gene expression profiling further revealed a potential role for prolactin signaling in regulating BCR editing. These results suggest a model in which benign accumulation of CD5(+) B cells can be initiated through a failure to successfully edit autoreactive BCR specificity and may, in turn, progress to CLL upon introduction of additional genetic mutations.

  14. Accelerated progression of chronic lymphocytic leukemia in Eμ-TCL1 mice expressing catalytically inactive RAG1

    PubMed Central

    Nganga, Vincent K.; Palmer, Victoria L.; Naushad, Hina; Kassmeier, Michele D.; Anderson, Dirk K.; Perry, Greg A.; Schabla, Nathan M.

    2013-01-01

    Chronic lymphocytic leukemia (CLL) is a prevalent B-cell neoplasia that is often preceded by a more benign monoclonal CD5+ B-cell lymphocytosis. We previously generated transgenic mice expressing catalytically inactive RAG1 (dominant-negative recombination activating gene 1 [dnRAG1] mice) that develop an early-onset indolent CD5+ B-cell lymphocytosis attributed to a defect in secondary V(D)J rearrangements initiated to edit autoreactive B-cell receptor (BCR) specificity. Hypothesizing that CD5+ B cells in these animals represent potential CLL precursors, we crossed dnRAG1 mice with CLL-prone Eμ-TCL1 mice to determine whether dnRAG1 expression in Eμ-TCL1 mice accelerates CLL onset. Consistent with this hypothesis, CD5+ B-cell expansion and CLL progression occurred more rapidly in double-transgenic mice compared with Eμ-TCL1 mice. Nevertheless, CD5+ B cells in the 2 mouse strains exhibited close similarities in phenotype, immunoglobulin gene usage, and mutation status, and expression of genes associated with immune tolerance and BCR signaling. Gene expression profiling further revealed a potential role for prolactin signaling in regulating BCR editing. These results suggest a model in which benign accumulation of CD5+ B cells can be initiated through a failure to successfully edit autoreactive BCR specificity and may, in turn, progress to CLL upon introduction of additional genetic mutations. PMID:23502221

  15. DT-Diaphorase as a Bifunctional Enzyme Label That Allows Rapid Enzymatic Amplification and Electrochemical Redox Cycling.

    PubMed

    Kang, Cheolho; Kang, Juyeon; Lee, Nam-Sihk; Yoon, Young Ho; Yang, Haesik

    2017-08-01

    The most common enzyme labels in enzyme-linked immunosorbent assays are alkaline phosphatase and horseradish peroxidase, which, however, have some limitations for use in electrochemical immunosensors. This Article reports that the small and thermostable DT-diaphorase (DT-D) and electrochemically inactive 4-nitroso-1-naphthol (4-NO-1-N) can be used as a bifunctional enzyme label and a rapidly reacting substrate, respectively, for electrochemical immunosensors. This enzyme-substrate combination allows high signal amplification via rapid enzymatic amplification and electrochemical redox cycling. DT-D can convert an electrochemically inactive nitroso or nitro compound into an electrochemically active amine compound, which can then be involved in electrochemical-chemical (EC) and electrochemical-enzymatic (EN) redox cycling. Six nitroso and nitro compounds are tested in terms of signal-to-background ratio. Among them, 4-NO-1-N exhibits the highest signal-to-background ratio. The electrochemical immunosensor using DT-D and 4-NO-1-N detects parathyroid hormone (PTH) in phosphate-buffered saline containing bovine serum albumin over a wide range of concentrations with a low detection limit of 2 pg/mL. When the PTH concentration in clinical serum samples is measured using the developed immunosensor, the calculated concentrations are in good agreement with the concentrations obtained using a commercial instrument. Thus, the use of DT-D as an enzyme label is highly promising for sensitive electrochemical detection and point-of-care testing.

  16. Structural and Functional Insights into the Catalytic Inactivity of the Major Fraction of Buffalo Milk Xanthine Oxidoreductase

    PubMed Central

    Gadave, Kaustubh S.; Panda, Santanu; Singh, Surender; Kalra, Shalini; Malakar, Dhruba; Mohanty, Ashok K.; Kaushik, Jai K.

    2014-01-01

    Background Xanthine oxidoreductase (XOR) existing in two interconvertible forms, xanthine dehydrogenase (XDH) and xanthine oxidase (XO), catabolises xanthine to uric acid that is further broken down to antioxidative agent allantoin. XOR also produces free radicals serving as second messenger and microbicidal agent. Large variation in the XO activity has been observed among various species. Both hypo and hyper activity of XOR leads to pathophysiological conditions. Given the important nutritional role of buffalo milk in human health especially in south Asia, it is crucial to understand the functional properties of buffalo XOR and the underlying structural basis of variations in comparison to other species. Methods and Findings Buffalo XO activity of 0.75 U/mg was almost half of cattle XO activity. Enzymatic efficiency (kcat/Km) of 0.11 sec−1 µM−1 of buffalo XO was 8–10 times smaller than that of cattle XO. Buffalo XOR also showed lower antibacterial activity than cattle XOR. A CD value (Δε430 nm) of 46,000 M−1 cm−1 suggested occupancy of 77.4% at Fe/S I centre. Buffalo XOR contained 0.31 molybdenum atom/subunit of which 48% existed in active sulfo form. The active form of XO in buffalo was only 16% in comparison to ∼30% in cattle. Sequencing revealed 97.4% similarity between buffalo and cattle XOR. FAD domain was least conserved, while metal binding domains (Fe/S and Molybdenum) were highly conserved. Homology modelling of buffalo XOR showed several variations occurring in clusters, especially close to FAD binding pocket which could affect NAD+ entry in the FAD centre. The difference in XO activity seems to be originating from cofactor deficiency, especially molybdenum. Conclusion A major fraction of buffalo milk XOR exists in a catalytically inactive form due to high content of demolybdo and desulfo forms. Lower Fe/S content and structural factors might be contributing to lower enzymatic efficiency of buffalo XOR in a minor way. PMID:24498153

  17. The prognosis of hepatitis B inactive carriers in Japan: a multicenter prospective study.

    PubMed

    Taida, Takashi; Arai, Makoto; Kanda, Tatsuo; Hige, Shuhei; Ueno, Yoshiyuki; Imazeki, Fumio; Izumi, Namiki; Tanaka, Eiji; Shinkai, Noboru; Yoshioka, Kentaro; Nakamoto, Yasunari; Nishiguchi, Shuhei; Tsuge, Masataka; Abe, Masanori; Sata, Michio; Yatsuhashi, Hiroshi; Ido, Akio; Kita, Kazuhiko; Azemoto, Ryousaku; Kitsukawa, Yoshio; Goto, Nobuaki; Yokosuka, Osamu

    2017-01-01

    Hepatitis B e antigen (HBeAg)-negative inactive carriers, the majority of hepatitis B virus (HBV) carriers, are considered to have a good prognosis. The definition of the inactive HBV carrier state has been based on HBV DNA and alanine aminotransferase (ALT) levels. Here we conducted a prospective study involving 18 hospitals to clarify the prognosis of HBeAg-negative inactive carriers. Three hundred eighty-eight HBeAg-negative inactive carriers at the baseline were observed prospectively from January 2011 to November 2015. We evaluated the primary end point, defined as the development of cirrhosis, hepatocellular carcinoma (HCC), or liver-related death. Also, we analyzed the factors associated with inactive carrier dropout and markedly increased levels of ALT or HBV DNA or both during the follow-up period. At the baseline, the mean age was 57.5 ± 13.1 years and 42 % of patients were male. No individual developed cirrhosis, HCC, or liver-related death during the follow-up period (1035 ± 252 days). Loss of inactive carrier status was seen in 75 patients (19.3 %). Factors associated with failure to meet the inactive carrier criteria in the multivariate analysis were the levels of ALT (hazard ratio 1.13, 95 % confidence interval 1.07-1.19, p < 0.001), HBV DNA (hazard ratio 2.70, 95 % confidence interval 1.63-4.49, p < 0.001), and γ-glutamyl transpeptidase (hazard ratio 1.01, 95 % confidence interval 1.00-1.02, p = 0.003) at the baseline. Most inactive carriers in Japan had a good prognosis. However, despite the short observation period, some patients had loss of IC status. The long-term prognosis of inactive carriers remains unclear; therefore, careful follow-up of inactive carriers is needed.

  18. Flavocytochrome b2-Based Enzymatic Method of L-Lactate Assay in Food Products

    PubMed Central

    Smutok, Halyna

    2013-01-01

    L-lactate, a key metabolite of the anaerobic glycolytic pathway, plays an important role as a biomarker in medicine, in the nutritional sector and food quality control. For these reasons, there is a need for very specific, sensitive, and simple analytical methods for the accurate L-lactate measuring. A new highly selective enzymatic method for L-lactate determination based on the use of flavocytochrome b2 (EC 1.1.2.3; FC b2) isolated from the recombinant strain of the yeast Hansenula polymorpha has been developed. A proposed enzymatic method exploits an enzymatic oxidation of L-lactate to pyruvate coupled with nitrotetrazolium blue (NTZB) reduction to a colored product, formazan. The maximal absorption peak of the colored product is near λ = 525 nm and the linear range is observed in the interval 0.005–0.14 mM of L-lactate. The main advantages of the proposed method when compared to the LDH-based routine approaches are a higher sensitivity (2.0 μM of L-lactate), simple procedure of analysis, usage of inexpensive, nontoxic reagents, and small amount of the enzyme. Enzymatic oxidation of L-lactate catalyzed by flavocytochrome b2 and coupled with formazan production from nitrotetrazolium blue was shown to be used for L-lactate assay in food samples. A high correlation between results of the proposed method and reference ones proves the possibility to use flavocytochrome b2-catalysed reaction for enzymatic measurement of L-lactate in biotechnology and food chemistry. PMID:24223505

  19. A simplified mathematical model of directional DNA site-specific recombination by serine integrases

    PubMed Central

    Zhao, Jia; Stark, W. Marshall; Colloms, Sean D.; Ebenhöh, Oliver

    2017-01-01

    Serine integrases catalyse site-specific recombination to integrate and excise bacteriophage genomes into and out of their host's genome. These enzymes exhibit remarkable directionality; in the presence of the integrase alone, recombination between attP and attB DNA sites is efficient and irreversible, giving attL and attR products which do not recombine further. However, in the presence of the bacteriophage-encoded recombination directionality factor (RDF), integrase efficiently promotes recombination between attL and attR to re-form attP and attB. The DNA substrates and products of both reactions are approximately isoenergetic, and no cofactors (such as adenosine triphosphate) are required for recombination. The thermodynamic driving force for directionality of these reactions is thus enigmatic. Here, we present a minimal mathematical model which can explain the directionality and regulation of both ‘forward’ and ‘reverse’ reactions. In this model, the substrates of the ‘forbidden’ reactions (between attL and attR in the absence of RDF, attP and attB in the presence of RDF) are trapped as inactive protein–DNA complexes, ensuring that these ‘forbidden’ reactions are extremely slow. The model is in good agreement with the observed in vitro kinetics of recombination by ϕC31 integrase, and defines core features of the system necessary and sufficient for directionality. PMID:28077763

  20. Exploring the Linkage between Activity-Friendly Zoning, Inactivity, and Cancer Incidence in the United States.

    PubMed

    Nicholson, Lisa M; Leider, Julien; Chriqui, Jamie F

    2017-03-07

    Background: Physical activity (PA) protects against cancer and enhances cancer survivorship. Given high inactivity rates nationwide, population-level physical activity facilitators are needed. Several authoritative bodies have recognized that zoning and planning helps create activity-friendly environments. This study examined the association between activity-friendly zoning, inactivity, and cancer in 478 of the most populous U.S. counties.Methods: County geocodes linked county-level data: cancer incidence and smoking (State Cancer Profiles), inactivity (Behavioral Risk Factor Surveillance System), 11 zoning measures (compiled by the study team), and covariates (from the American Community Survey and NAVTEQ). For each zoning measure, single mediation regression models and Sobel tests examined whether activity-friendly zoning was associated with reduced cancer incidence, and whether inactivity mediated those associations. All models were clustered on state with robust SEs and significance at the P < 0.05 level.Results: Zoning for crosswalks, bike-pedestrian connectivity, and bike-pedestrian trails/paths were associated with reduced cancer incidence (β between -0.71 and -1.27, P < 0.05), about 1 case per 100,000 for each 10 percentage-point increase in county population exposure to zoning. Except for crosswalks, each association was mediated by inactivity. However, county smoking attenuated these results, with only crosswalks remaining significant. Results were similar for males (with zoning for bike-pedestrian connectivity, street connectivity, and bike-pedestrian trails/paths), but not females, alone.Conclusions: Zoning can help to create activity-friendly environments that support decreased inactivity, and possibly reduced cancer incidence.Impact: Given low physical activity levels nationwide, cross-sectoral collaborations with urban planning can inform cancer prevention and public health efforts to decrease inactivity and cancer. Cancer Epidemiol Biomarkers Prev

  1. Adult physical inactivity prevalence in the Muslim world: Analysis of 38 countries

    PubMed Central

    Kahan, David

    2015-01-01

    Objective Physical inactivity surveillance informs policy and treatment options toward meeting the World Health Organization's (WHO) goal of a 10% reduction in its prevalence by 2025. We currently do not know the aggregate prevalence for Muslim-majority countries, many of which have extremely high rates of comorbidities associated with physical inactivity. Method Based on data for 163, 556 persons in 38 Muslim countries that were collected by the Global Physical Activity Questionnaire and the International Physical Activity Questionnaire, unweighted and weighted physical inactivity prevalence estimates were calculated. I used two-proportion Z tests to determine gender and ethnic differences within the sample and between the sample and 94 non-Muslim countries and odds ratios to determine the magnitude of significant differences. Results Total physical inactivity prevalence was 32.3% (95% CI: 31.9, 32.7). Prevalence among males and females was 28.8% and 35.5%, respectively. Prevalence among non-Arabs and Arabs was 28.6% and 43.7%, respectively. Females and Arabs were more likely physically inactive than their respective counterparts [OR = 1.36 (1.33, 1.39) and OR = 1.94 (1.90, 1.98)]. Muslim countries were more likely physically inactive [OR = 1.23 (1.22, 1.25)] than non-Muslim ones, which was primarily due to the influence of Arabs [OR = 2.01 (1.97, 2.04)], and in particular female Arabs [OR = 2.22 (2.17, 2.27)]. Conclusion Physical inactivity prevalence in the Muslim world is higher than non-Muslim countries and the difference is primarily due to higher rates among Arabs. PMID:26844051

  2. Motivating patients to exercise: translating high blood pressure into equivalent risk of inactivity.

    PubMed

    Li, Chu-Shiu; Liu, Chwen-Chi; Tsai, Min-Kuang; Tai, Ya-Ping; Wai, Jackson Pui Man; Tsao, Chwen-Keng; Wen, Chi-Pang

    2015-02-01

    Even with the 2008 physical activity guidelines for Americans and the strong epidemiological evidence, physicians are not routinely emphasizing the importance of exercise. We try to explore an innovative way to communicate the benefits of physical activity in a term familiar to patients. A cohort of 470, 163 adults from a medical screening program in Taiwan were recruited between 1994 and 2008. Their vital status was followed up by matching with the National Death File. Individuals were classified as 'inactive', 'low active', or 'fully active', with 'fully active' meeting the current exercise recommendation of 150  min per week or more. Cox proportional model was used to calculate the hazard ratio. More than one-half of the cohort was inactive (54%), with one-quarter fully active (24%). One in seven was hypertensive (14%), defined as SBP at least 140  mmHg. Among the hypertensive individuals, mortality risks were increased by 37% for the inactive. Inactive individuals had higher all-cause mortality than active ones across all blood pressure (BP) levels. At 110-119  mmHg, the inactive had a risk as high as the risk at 155  mmHg, an increased mortality risk equivalent to a risk of BP increase of 41.2 mmHg. The mortality risk of being inactive was equivalent to an increase of around 40  mmHg in SBP or 20  mmHg in DBP, a number relevant to hypertensive patients. Appreciating this relationship may convince the inactive to start exercising, a behavior as important as controlling BP.

  3. Active-site mutants of beta-lactamase: use of an inactive double mutant to study requirements for catalysis.

    PubMed

    Dalbadie-McFarland, G; Neitzel, J J; Richards, J H

    1986-01-28

    We have studied the catalytic activity and some other properties of mutants of Escherichia coli plasmid-encoded RTEM beta-lactamase (EC 3.5.2.6) with all combinations of serine and threonine residues at the active-site positions 70 and 71. (All natural beta-lactamases have conserved serine-70 and threonine-71.) From the inactive double mutant Ser-70----Thr, Thr-71----Ser [Dalbadie-McFarland, G., Cohen, L. W., Riggs, A. D., Morin, C., Itakura, K., & Richards, J. H. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6409-6413], an active revertant, Thr-71----Ser (i.e., residue 70 in the double mutant had changed from threonine to the serine conserved at position 70 in the wild-type enzyme), was isolated by an approach that allows identification of active revertants in the absence of a background of wild-type enzyme. This mutant (Thr-71----Ser) has about 15% of the catalytic activity of wild-type beta-lactamase. The other possible mutant involving serine and threonine residues at positions 70 and 71 (Ser-70----Thr) shows no catalytic activity. The primary nucleophiles of a serine or a cysteine residue [Sigal, I. S., Harwood, B. G., & Arentzen, R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7157-7160] at position 70 thus seem essential for enzymatic activity. Compared to wild-type enzyme, all three mutants show significantly reduced resistance to proteolysis; for the active revertant (Thr-71----Ser), we have also observed reduced thermal stability and reduced resistance to denaturation by urea.

  4. Site directed recombination

    DOEpatents

    Jurka, Jerzy W.

    1997-01-01

    Enhanced homologous recombination is obtained by employing a consensus sequence which has been found to be associated with integration of repeat sequences, such as Alu and ID. The consensus sequence or sequence having a single transition mutation determines one site of a double break which allows for high efficiency of integration at the site. By introducing single or double stranded DNA having the consensus sequence flanking region joined to a sequence of interest, one can reproducibly direct integration of the sequence of interest at one or a limited number of sites. In this way, specific sites can be identified and homologous recombination achieved at the site by employing a second flanking sequence associated with a sequence proximal to the 3'-nick.

  5. Preparation of chain-end clickable recombinant protein and its bio-orthogonal modification.

    PubMed

    Wang, Lin; Jiang, Rui; Wang, Lin; Liu, Yang; Sun, Xue-Long

    2016-04-01

    Introducing unique functional group into protein is an attractive approach for site-selective protein modification applications. In this report, we systemically investigated four site-selective strategies to introduce azide functionality into recombinant thrombomodulin (TM456), via direct recombinant expression with unnatural amino acid, chemical, and enzymatic modification for its bio-orthogonal modification application. First, a straightforward recombinant method to express TM456 with azide functionality near C-terminus by replacing methionine with azidohomoanlanine from methionine auxotroph Escherichia coli cell was investigated. Next, a sortase-mediated ligation (SML) method to incorporate azide functionality into the C-terminus of recombinant TM456 was demonstrated. The third is to add azide functionality to the N-terminal amine of recombinant TM456via amidation chemistry, and the fourth is tyrosine selective three-component Mannich reaction to introduce azide functionality to recombinant TM456. Overall, SML of recombinant protein affords the highest overall yield for incorporating azide functionality into the C-terminus recombinant TM456 since the key protein expression step uses natural amino acids. Also, single site modification facilitates the highest TM456 activity.

  6. The recombination epoch revisited

    NASA Technical Reports Server (NTRS)

    Krolik, Julian H.

    1989-01-01

    Previous studies of cosmological recombination have shown that this process produces as a by-product a highly superthermal population of Ly-alpha photons which retard completion of recombination. Cosmological redshifting was thought to determine the frequency distribution of the photons, while two-photon decay of hydrogen's 2s state was thought to control their numbers. It is shown here that frequency diffusion due to photon scattering dominate the cosmological redshift in the frequency range near line center which fixes the ratio of ground state to excited state population, while incoherent scattering into the far-red damping wing effectively destroys Ly-alpha photons as a rate which is competitive with two-photon decay. The former effect tends to hold back recombination, while the latter tends to accelerate it; the net results depends on cosmological parameters, particularly the combination Omega(b) h/sq rt (2q0), where Omega(b) is the fraction of the critical density provided by baryons.

  7. Recombinant human milk proteins.

    PubMed

    Lönnerdal, Bo

    2006-01-01

    Human milk provides proteins that benefit newborn infants. They not only provide amino acids, but also facilitate the absorption of nutrients, stimulate growth and development of the intestine, modulate immune function, and aid in the digestion of other nutrients. Breastfed infants have a lower prevalence of infections than formula-fed infants. Since many women in industrialized countries choose not to breastfeed, and an increasing proportion of women in developing countries are advised not to breastfeed because of the risk of HIV transmission, incorporation of recombinant human milk proteins into infant foods is likely to be beneficial. We are expressing human milk proteins known to have anti-infective activity in rice. Since rice is a normal constituent of the diet of infants and children, limited purification of the proteins is required. Lactoferrin has antimicrobial and iron-binding activities. Lysozyme is an enzyme that is bactericidal and also acts synergistically with lactoferrin. These recombinant proteins have biological activities identical to their native counterparts. They are equally resistant to heat processing, which is necessary for food applications, and to acid and proteolytic enzymes which are needed to maintain their biological activity in the gastrointestinal tract of infants. These recombinant human milk proteins may be incorporated into infant formulas, baby foods and complementary foods, and used with the goal to reduce infectious diseases.

  8. Strand invasion promoted by recombination protein of coliphage

    NASA Astrophysics Data System (ADS)

    Rybalchenko, Nataliya; Golub, Efim I.; Bi, Baoyuan; Radding, Charles M.

    2004-12-01

    Studies of phage in vivo have indicated that its own recombination enzymes, protein and exonuclease, are capable of catalyzing two dissimilar pathways of homologous recombination that are widely distributed in nature: single-strand annealing and strand invasion. The former is an enzymatic splicing of overlapping ends of broken homologous DNA molecules, whereas the latter is characterized by the formation of a three-stranded synaptic intermediate and subsequent strand exchange. Previous studies in vitro have shown that protein has annealing activity, and that exonuclease, acting on branched substrates, can produce a perfect splice that requires only ligation for completion. The present study shows that protein can initiate strand invasion in vitro, as evidenced both by the formation of displacement loops (D-loops) in superhelical DNA and by strand exchange between colinear single-stranded and double-stranded molecules. Thus, protein can catalyze steps that are central to both strand annealing and strand invasion pathways of recombination. These observations add protein to a set of diverse proteins that appear to promote recognition of homology by a unitary mechanism governed by the intrinsic dynamic properties of base pairs in DNA. genetic recombination | phage λ

  9. Production and physicochemical properties of recombinant Lactobacillus plantarum tannase.

    PubMed

    Curiel, José Antonio; Rodríguez, Héctor; Acebrón, Iván; Mancheño, José Miguel; De Las Rivas, Blanca; Muñoz, Rosario

    2009-07-22

    Tannase is an enzyme with important biotechnological applications in the food industry. Previous studies have identified the tannase encoding gene in Lactobacillus plantarum and also have reported the description of the purification of recombinant L. plantarum tannase through a protocol involving several chromatographic steps. Here, we describe the high-yield production of pure recombinant tannase (17 mg/L) by a one-step affinity procedure. The purified recombinant tannase exhibits optimal activity at pH 7 and 40 degrees C. Addition of Ca(2+) to the reaction mixture greatly increased tannase activity. The enzymatic activity of tannase was assayed against 18 simple phenolic acid esters. Only esters derived from gallic acid and protocatechuic acid were hydrolyzed. In addition, tannase activity was also assayed against the tannins tannic acid, gallocatechin gallate, and epigallocatechin gallate. Despite L. plantarum tannase representing a novel family of tannases, which shows no significant similarity to tannases from fungal sources, both families of enzymes shared similar substrate specificity range. The physicochemical characteristics exhibited by L. plantarum recombinant tannase make it an adequate alternative to the currently used fungal tannases.

  10. Expression and purification of biologically active recombinant human paraoxonase 1 from a Drosophila S2 stable cell line.

    PubMed

    Yun, Hyeongseok; Yu, Jiyeon; Kim, Sumi; Lee, Nari; Lee, Jinhee; Lee, Sungrae; Kim, Nam Doo; Yu, Chiho; Rho, Jaerang

    2017-03-01

    Many pesticides and chemical warfare nerve agents are highly toxic organophosphorus compounds (OPs), which inhibit acetylcholinesterase activity. Human paraoxonase 1 (PON1) has demonstrated significant potential for use as a catalytic bioscavenger capable of hydrolyzing a broad range of OPs. However, there are several limitations to the use of human PON1 as a catalytic bioscavenger, including the relatively difficult purification of PON1 from human plasma and its dependence on the presence of hydrophobic binding partners to maintain stability. Therefore, research efforts to efficiently produce recombinant human PON1 are necessary. In this study, we developed a Drosophila S2 stable cell line expressing recombinant human PON1. The recombinant human PON1 was fused with the human immunoglobulin Fc domain (PON1-hFc) to improve protein stability and purification efficiency. We purified the recombinant human PON1-hFc from the S2 stable cell line and characterized its enzymatic properties for OP hydrolysis. We purified the recombinant human PON1-hFc from the S2 stable cell line and characterized its enzymatic properties for OP hydrolysis compared with those of the recombinant human PON1 derived from E. coli. We observed that the recombinant human PON1-hFc is functionally more stable for OP hydrolyzing activities compared to the recombinant human PON1. The catalytic efficiency of the recombinant PON1-hFc towards diisopropyl fluorophosphate (DFP, 0.26 × 10(6) M(-1) min(-1)) and paraoxon hydrolysis (0.015 × 10(6) M(-1) min(-1)) was 1.63- and 1.24-fold higher, respectively, than the recombinant human PON1. Thus, we report that the recombinant PON1-hFc exerts hydrolytic activity against paraoxon and DFP. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Cell biology of mitotic recombination.

    PubMed

    Lisby, Michael; Rothstein, Rodney

    2015-03-02

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination.

  12. Cell Biology of Mitotic Recombination

    PubMed Central

    Lisby, Michael; Rothstein, Rodney

    2015-01-01

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination. PMID:25731763

  13. The proportion of youths’ physical inactivity attributable to neighbourhood built environment features

    PubMed Central

    2013-01-01

    Objective We investigated the independent association between several neighbourhood built environment features and physical inactivity within a national sample of Canadian youth, and estimated the proportion of inactivity within the population that was attributable to these built environment features. Methods This was a cross-sectional study of 6626 youth aged 11–15 years from 272 schools across Canada. Participants resided within 1 km of their school. Walkability, outdoor play areas (parks, wooded areas, yards at home, cul-de-sacs on roads), recreation facilities, and aesthetics were measured objectively within each school neighbourhood using geographic information systems. Physical inactivity (<5 days/week of 60 minutes of moderate-to-vigorous physical activity) was assessed by questionnaire. Multilevel logistic regression analyses, which controlled for several covariates, examined relationships between built environment features and physical inactivity. Results The final regression model indicated that, by comparison to youth living in the least walkable neighbourhoods, the risks for physical inactivity were 28-44% higher for youth living in neighbourhoods in the remaining three walkability quartiles. By comparison to youth living in neighbourhoods with the highest density of cul-de-sacs, risks for physical inactivity were 28-32% higher for youth living in neighbourhoods in the lowest two quartiles. By comparison to youth living in neighbourhoods with the least amount of park space, risks for physical inactivity were 28-37% higher for youth living in the neighbourhoods with a moderate to high (quartiles 2 and 3) park space. Population attributable risk estimates suggested that 23% of physical inactivity within the population was attributable to living in walkable neighbourhoods, 16% was attributable to living in neighbourhoods with a low density of cul-de-sacs, and 15% was attributable to living in neighbourhoods with a moderate to high amount of park

  14. Enzymatic preparation of nanocrystalline and microcrystalline cellulose

    Treesearch

    Sarah R. Anderson; Dominic Esposito; William Gillette; J.Y. Zhu; Ulrich Baxa; Scott E. Mcneil

    2014-01-01

    Traditional cellulose nanocrystal (CNC) production methods use harsh chemicals, are energetically expensive, and result in a hydrophilic sulfate surface chemistry with limited utility. Enzymatic production of CNCs is a less expensive alternative production method that eliminates the need for harsh chemicals and requires much less energy for mechanical fibrillation and...

  15. pH & Rate of Enzymatic Reactions.

    ERIC Educational Resources Information Center

    Clariana, Roy B.

    1991-01-01

    A quantitative and inexpensive way to measure the rate of enzymatic reaction is provided. The effects of different pH levels on the reaction rate of an enzyme from yeast are investigated and the results graphed. Background information, a list of needed materials, directions for preparing solutions, procedure, and results and discussion are…

  16. Enhanced enzymatic hydrolysis of cellulose in microgels.

    PubMed

    Chang, Aiping; Wu, Qingshi; Xu, Wenting; Xie, Jianda; Wu, Weitai

    2015-07-04

    A cellulose-based microgel, where an individual microgel contains approximately one cellulose chain on average, is synthesized via free radical polymerization of a difunctional small-molecule N,N'-methylenebisacrylamide in cellulose solution. This microgelation leads to a low-ordered cellulose, favoring enzymatic hydrolysis of cellulose to generate glucose.

  17. Ultrasonic acceleration of enzymatic processing of cotton

    USDA-ARS?s Scientific Manuscript database

    Enzymatic bio-processing of cotton generates significantly less hazardous wastewater effluents, which are readily biodegradable, but it also has several critical shortcomings that impede its acceptance by industries: expensive processing costs and slow reaction rates. It has been found that the intr...

  18. Frank Westheimer's Early Demonstration of Enzymatic Specificity

    ERIC Educational Resources Information Center

    Ault, Addison

    2008-01-01

    In this article I review one of the most significant accomplishments of Frank H. Westheimer, one of the most respected chemists of the 20th century. This accomplishment was a series of stereospecific enzymatic oxidation and reduction experiments that led chemists to recognize what we now call the enantiotopic and diastereotopic relationships of…

  19. Enzymatic mechanisms of biological magnetic sensitivity.

    PubMed

    Letuta, Ulyana G; Berdinskiy, Vitaly L; Udagawa, Chikako; Tanimoto, Yoshifumi

    2017-10-01

    Primary biological magnetoreceptors in living organisms is one of the main research problems in magnetobiology. Intracellular enzymatic reactions accompanied by electron transfer have been shown to be receptors of magnetic fields, and spin-dependent ion-radical processes can be a universal mechanism of biological magnetosensitivity. Magnetic interactions in intermediate ion-radical pairs, such as Zeeman and hyperfine (HFI) interactions, in accordance with proposed strict quantum mechanical theory, can determine magnetic-field dependencies of reactions that produce biologically important molecules needed for cell growth. Hyperfine interactions of electrons with nuclear magnetic moments of magnetic isotopes can explain the most important part of biomagnetic sensitivities in a weak magnetic field comparable to the Earth's magnetic field. The theoretical results mean that magnetic-field dependencies of enzymatic reaction rates in a weak magnetic field that can be independent of HFI constant a, if H < a, and are determined by the rate constant of chemical transformations in the enzyme active site. Both Zeeman and HFI interactions predict strong magnetic-field dependence in weak magnetic fields and magnetic-field independence of enzymatic reaction rate constants in strong magnetic fields. The theoretical results can explain the magnetic sensitivity of E. coli cell and demonstrate that intracellular enzymatic reactions are primary magnetoreceptors in living organisms. Bioelectromagnetics. 38:511-521, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  20. Frank Westheimer's Early Demonstration of Enzymatic Specificity

    ERIC Educational Resources Information Center

    Ault, Addison

    2008-01-01

    In this article I review one of the most significant accomplishments of Frank H. Westheimer, one of the most respected chemists of the 20th century. This accomplishment was a series of stereospecific enzymatic oxidation and reduction experiments that led chemists to recognize what we now call the enantiotopic and diastereotopic relationships of…

  1. pH & Rate of Enzymatic Reactions.

    ERIC Educational Resources Information Center

    Clariana, Roy B.

    1991-01-01

    A quantitative and inexpensive way to measure the rate of enzymatic reaction is provided. The effects of different pH levels on the reaction rate of an enzyme from yeast are investigated and the results graphed. Background information, a list of needed materials, directions for preparing solutions, procedure, and results and discussion are…

  2. Engineering of vault nanocapsules with enzymatic and fluorescent properties.

    PubMed

    Kickhoefer, Valerie A; Garcia, Yvette; Mikyas, Yeshi; Johansson, Erik; Zhou, Jing C; Raval-Fernandes, Sujna; Minoofar, Payam; Zink, Jeffrey I; Dunn, Bruce; Stewart, Phoebe L; Rome, Leonard H

    2005-03-22

    One of the central issues facing the emerging field of nanotechnology is cellular compatibility. Nanoparticles have been proposed for diagnostic and therapeutic applications, including drug delivery, gene therapy, biological sensors, and controlled catalysis. Viruses, liposomes, peptides, and synthetic and natural polymers have been engineered for these applications, yet significant limitations continue to prevent their use. Avoidance of the body's natural immune system, lack of targeting specificity, and the inability to control packaging and release are remaining obstacles. We have explored the use of a naturally occurring cellular nanoparticle known as the vault, which is named for its morphology with multiple arches reminiscent of cathedral ceilings. Vaults are 13-MDa ribonucleoprotein particles with an internal cavity large enough to sequester hundreds of proteins. Here, we report a strategy to target and sequester biologically active materials within the vault cavity. Attachment of a vault-targeting peptide to two proteins, luciferase and a variant of GFP, resulted in their sequestration within the vault cavity. The targeted proteins confer enzymatic and fluorescent properties on the recombinant vaults, both of which can be detected by their emission of light. The modified vaults are compatible with living cells. The ability to engineer vault particles with designed properties and functionalities represents an important step toward development of a biocompatible nanocapsule.

  3. Efficient enzymatic production of rebaudioside A from stevioside.

    PubMed

    Wang, Yu; Chen, Liangliang; Li, Yan; Li, Yangyang; Yan, Ming; Chen, Kequan; Hao, Ning; Xu, Lin

    2015-01-01

    Stevioside and rebaudioside A are the chief diterpene glycosides present in the leaves of Stevia rebaudiana. Rebaudioside A imparts a desirable sweet taste, while stevioside produces a residual bitter aftertaste. Enzymatic synthesis of rebaudioside A from stevioside can increase the ratio of rebaudioside A to stevioside in steviol glycoside products, providing a conceivable strategy to improve the organoleptic properties of steviol glycoside products. Here, we demonstrate the efficient conversion of stevioside to rebaudioside A by coupling the activities of recombinant UDP-glucosyltransferase UGT76G1 from S. rebaudiana and sucrose synthase AtSUS1 from Arabidopsis thaliana. The conversion occurred via regeneration of UDP-glucose by AtSUS1. UDP was applicable as the initial material instead of UDP-glucose for UDP-glucose recycling. The amount of UDP could be greatly reduced in the reaction mixture. Rebaudioside A yield in 30 h with 2.4 mM stevioside, 7.2 mM sucrose, and 0.006 mM UDP was 78%.

  4. Enzymatically catalyzed HES conjugation using microbial transglutaminase: Proof of feasibility.

    PubMed

    Besheer, Ahmed; Hertel, Thomas C; Kressler, Jörg; Mäder, Karsten; Pietzsch, Markus

    2009-11-01

    Polymer-drug and polymer-protein conjugates are promising candidates for the delivery of therapeutic agents. PEGylation, using poly(ethylene glycol) for the conjugation, is now the gold standard in this field, and some PEGylated proteins have successfully reached the market. Hydroxyethyl starch (HES) is a water-soluble, biodegradable derivative of starch that is currently being investigated as a substitute for PEG. So far, only chemical methods have been suggested for HES conjugation; however, these may have detrimental effects on proteins. Here, we report an enzymatic method for HES conjugation using a recombinant microbial transglutaminase (rMTG). The latter catalyzes the acyl transfer between the gamma-carboxamide group of a glutaminyl residue (acyl donors) and a variety of primary amines (acyl acceptors), including the amino group of lysine. HES was modified with N-carbobenzyloxy glutaminyl glycine (Z-QG) and hexamethylene diamine (HMDA) to act as acyl donor and acyl acceptor, respectively. Using (1)H NMR, the degree of modification with Z-QG and HMDA was found to be 4.6 and 3.9 mol%, respectively. Using SDS-PAGE, it was possible to show that the modified HES successfully coupled to test compounds, proving that it is accepted as a substrate by rMTG. Finally, the process described in this study is a simple, mild approach to produce fully biodegradable polymer-drug and polymer-protein conjugates. (c) 2009 Wiley-Liss, Inc. and the American Pharmacists Association

  5. Cyanide detoxification by recombinant bacterial rhodanese.

    PubMed

    Cipollone, Rita; Ascenzi, Paolo; Frangipani, Emanuela; Visca, Paolo

    2006-05-01

    Cyanide is a major environmental pollutant of the chemical and metallurgical industries. Although extremely toxic, cyanide can enzymatically be converted to the less toxic thiocyanate by rhodaneses (thiosulfate:cyanide sulfurtransferases, EC 2.8.1.1). We engineered a genetic system to express high levels of recombinant Pseudomonas aeruginosa rhodanese (r-RhdA) in Escherichia coli, and used this organism to test the role of r-RhdA in cyanide detoxification. Inducible expression of the rhdA gene under the control of the hybrid T7-lacO promoter yielded active r-RhdA over a 4-h period, though r-RhdA-expressing E. coli showed decreased viability starting from 1 h post-induction. At this time, Western blot analysis and enzymatic assay showed r-RhdA partition between the cytoplasm (95%) and the periplasm (5%). The accessibility of thiosulfate to r-RhdA was a limiting step for the sulfur transfer reaction in the cellular system, but cyanide conversion to thiocyanate could be increased upon permeabilization of the bacterial membrane. Specific r-RhdA activity was higher in the whole-cell assay than in the in vitro assay with pure enzyme (2154 vs. 816 micromol min-1 mg-1 r-RhdA, respectively), likely reflecting enzyme stability. The r-RhdA-dependent cyanide detoxification resulted in increased resistance of r-RhdA overexpressing E. coli to 5 mM cyanide. Bacterial survival was paralleled by release of thiocyanate into the medium. Our results indicate that cyanide detoxification by engineered E. coli cells is feasible under laboratory conditions, and suggest that microbial rhodaneses may contribute to cyanide transformation in natural environments.

  6. Prevalence and variables associated with physical inactivity in individuals with high and low socioeconomic status.

    PubMed

    Reis, Helena França Correia dos; Ladeia, Ana Marice Teixeira; Passos, Everton Carvalho; Santos, Flávio Guilherme de Oliveira; Wasconcellos, Larissa Tapioca de; Correia, Luís Cláudio Lemos; Menezes, Marta Silva; Santos, Renata Dáttoli Gouvêa; Bomfim, Victor Guerrero do; Rocha, Mário de Seixas

    2009-03-01

    Studies that considered only the leisure physical activity found that the physical inactivity is higher among lower-income individuals. There is a possibility that this association shows modifications, when considering transportation, work and domestic activities. To determine whether there is a difference between the prevalence of physical inactivity between individuals of high and low socioeconomic levels. The sample consisted of individuals of both sexes, aged 18 or older, from two groups of different socioeconomic levels. The low socioeconomic level (LSEL) group consisted of the parents of students from a public school. The high socioeconomic level (HSEL) group consisted of the parents of students from a private College. The International Physical Activity Questionnaire (IPAQ) was used to determine the level of physical activity. A total of 91 individuals were evaluated in the LSEL group and 59 in the HSEL group. In the LSEL group, 42.9% (39) of the individuals were classified as insufficiently active, compared to 57.6% (34) of individuals in the HSEL group. Taking as a parameter of physical inactivity the time of weekly physical activity < 150 minutes, there was a decrease in the classification of inactivity in both groups, although with the maintenance of higher inactivity among individuals of HSEL (49.2% vs 28.6%; p= 0.01). The individuals of HSEL are more sedentary than the individuals of LSEL.

  7. Association between Natural Resources for Outdoor Activities and Physical Inactivity: Results from the Contiguous United States

    PubMed Central

    Jiang, Yan; Yuan, Yongping; Neale, Anne; Jackson, Laura; Mehaffey, Megan

    2016-01-01

    Protected areas including national/state parks and recreational waters are excellent natural resources that promote physical activity and interaction with Nature, which can relieve stress and reduce disease risk. Despite their importance, however, their contribution to human health has not been properly quantified. This paper seeks to evaluate quantitatively how national/state parks and recreational waters are associated with human health and well-being, taking into account of the spatial dependence of environmental variables for the contiguous U.S., at the county level. First, we describe available natural resources for outdoor activities (ANROA), using national databases that include features from the Protected Areas Database, NAVSTREETS, and ATTAINSGEO 305(b) Waters. We then use spatial regression techniques to explore the association of ANROA and socioeconomic status factors on physical inactivity rates. Finally, we use variance analysis to analyze ANROA’s influence on income-related health inequality. We found a significantly negative association between ANROA and the rate of physical inactivity: ANROA and the spatial effect explained 69%, nationwide, of the variation in physical inactivity. Physical inactivity rate showed a strong spatial dependence—influenced not only by its own in-county ANROA, but also by that of its neighbors ANROA. Furthermore, community groups at the same income level and with the highest ANROA, always had the lowest physical inactivity rate. This finding may help to guide future land use planning and community development that will benefit human health and well-being. PMID:27548195

  8. The Effect of Whole-body Vibration on Muscle Activity in Active and Inactive Subjects.

    PubMed

    Lienhard, K; Vienneau, J; Friesenbichler, B; Nigg, S; Meste, O; Nigg, B M; Colson, S S

    2015-06-01

    The purpose of this study was to compare lower limb muscle activity between physically active and inactive individuals during whole-body vibration exercises. Additionally, transmissibility of the vertical acceleration to the head was quantified. 30 active and 28 inactive participants volunteered to stand in a relaxed (20°) and a squat (60°) position on a side-alternating WBV platform that induced vibrations at 16 Hz and 4 mm amplitude. Surface electromyography (sEMG) was measured in selected lower limb muscles and was normalized to the corresponding sEMG recorded during a maximal voluntary contraction. The vertical acceleration on the head was evaluated and divided by the vertical platform acceleration to obtain transmissibility values. Control trials without vibration were also assessed. The outcomes of this study showed that (1) WBV significantly increased muscle activity in the active (absolute increase: +7%, P <0.05) and inactive participants (+8%, P <0.05), (2) with no differences in sEMG increases between the groups (P>0.05). However, (3), transmissibility to the head was greater in the active (0.080) than the inactive participants (0.065, P <0.05). In conclusion, inactive individuals show similar responses in sEMG due to WBV as their active counterparts, but are at lower risk for potential side-effects of vibration exposure. © Georg Thieme Verlag KG Stuttgart · New York.

  9. A theory for fluidelastic instability of tube-support-plate inactive modes

    SciTech Connect

    Cai, Y.; Chen, S.S. ); Chandra, S. )

    1990-01-01

    Fluidelastic instability of loosely supported tubes, vibrating in a tube support plate (TSP)-inactive mode, is suspected to be one of the main causes of tube failures in some operating steam generators and heat exchangers. A mathematical model, which incorporates all motion-dependent fluid-forces based on the unsteady flow theory, is presented here for fluidelastic instability of loosely supported tubes exposed to nonuniform crossflow. In the unstable region associated with a TSP-inactive mode, the tube motion can be described by two linear models: TSP-inactive mode when tubes do not contact with the TSP and TSP-active mode when tubes contact the TSP. A bilinear model, consisting of these linear models, is presented in this paper to simulate the characteristics of fluidelastic instability of loosely supported tubes in the stable and unstable region associated with TSP-inactive modes. The analytical results are compared with published experimental data; they agree reasonably well. The prediction procedure presented for fluidelastic instability response of loosely supported tubes is applicable in the stable regions of TSP-inactive mode. 25 refs., 10 figs., 2 tabs.

  10. Monthly estimates of leisure-time physical inactivity--United States, 1994.

    PubMed

    1997-05-09

    Physical inactivity increases the risk for heart disease, diabetes, colon cancer, high blood pressure, obesity, osteoporosis, muscle and joint disorders, and symptoms of anxiety and depression. However, approximately one third of adults in the United States report no leisure-time physical activity, and rates of inactivity have been higher in January than in June. Among adults, the prevalence of leisure-time physical inactivity is highest among those who are older, Hispanic, and residing in southern states. A national health objective for the year 2000 is to reduce to < or = 15% the proportion of persons reporting no leisure-time physical activity (objecive 1.5). To assist in monitoring efforts to achieve this objective, CDC analyzed data from the 1994 Behavioral Risk Factor Surveillance system (BRFSS) and estimated for each month the proportion of adults from selected demographic groups who reported no leisure-time physical activity. The findings indicate seasonal patterns in the prevalence of reported leisure-time physical inactivity; however, monthly rates of inactivity were higher and more stable among older persons, Hispanics, and residents of southern states.

  11. Daily energy expenditure in active and inactive persons with spinal cord injury.

    PubMed

    Yamasaki, M; Irizawa, M; Komura, T; Kikuchi, K; Sasaki, H; Kai, K; Ohdoko, K

    1992-12-01

    Daily energy expenditure (DEE) was evaluated seventeen male subjects with spinal cord injury (SSCI), who had active (N = 9) and inactive (N = 8) lifestyles, and in seven normal males. DEE was estimated from the mean 24-hr heart rate and heart rate-energy expenditure relationship determined in an arm cranking exercise. The DEE of SSCI on active days did not differ from those of normal subjects. On inactive days, SSCI had significantly lower DEE than on active days and than normal subjects. In contrast, the mean 24-hr heart rates of SSCI on active days and inactive SSCI were significantly greater than those of normal subjects, suggesting that SSCI, particularly inactive SSCI, exhibited a slight degree of tachycardia when compared to normal subjects. On inactive days, the DEE was fairly independent of the level of spinal cord injury. However, on active days, there was a clear tendency for SSCI with a low lesion level to have a higher DEE. Even if a SSCI with a high lesion level engaged in active sports, his DEE was relatively small. This lower DEE was largely attributable to the smaller functional muscle mass due to the limitation of physical activity.

  12. Physical inactivity and anthropometric measures in schoolchildren from Paranavaí, Paraná, Brazil

    PubMed Central

    Guilherme, Flávio Ricardo; Molena-Fernandes, Carlos Alexandre; Guilherme, Vânia Renata; Fávero, Maria Teresa Martins; dos Reis, Eliane Josefa Barbosa; Rinaldi, Wilson

    2015-01-01

    OBJECTIVE: To investigate the association between physical inactivity and anthropometric measures in schoolchildren from Paranavaí-Parana, Brazil. METHODS: Cross-sectional survey, carried out in July and August 2013. Sample of 566 students (287 boys and 279 girls) from 6th to 9th grade, aged 10 to 14 years, from public and private schools of Paranavaí - PR, Southern Brazil. The variables analyzed were: time of weekly physical activity through a questionnaire (physical inactivity <300 minutes/week), body mass index (BMI) and waist circumference (WC). In the statistical analysis, the U Mann-Whitney and Student's t tests were used for comparison between genders. To identify factors associated with insufficient levels of physical activity, univariate and multivariate logistic regression analysis was applied and expressed in Odds ratio (OR) and 95% confidence interval (95%CI). RESULTS: There was an association between physical inactivity and anthropometric measurements for BMI (p<0.001) and WC (p<0.001), with a prevalence rate of 56.1% and 52.7% of inactive adolescents, respectively. In the multivariate analysis, there was significant association of physical inactivity and overweight (OR 1.8, 95%CI: 1.1-3.0) and with increased waist circumference (OR 2.8, 95%CI: 1.4-3.8). CONCLUSIONS: Inadequate levels of physical activity is a determining factor for overweight and abdominal adiposity. Accordingly, preventive measures should be taken, especially in schools, emphasizing the importance of exercise for body composition control and weight reduction. PMID:25623726

  13. Association between Natural Resources for Outdoor Activities and Physical Inactivity: Results from the Contiguous United States.

    PubMed

    Jiang, Yan; Yuan, Yongping; Neale, Anne; Jackson, Laura; Mehaffey, Megan

    2016-08-17

    Protected areas including national/state parks and recreational waters are excellent natural resources that promote physical activity and interaction with Nature, which can relieve stress and reduce disease risk. Despite their importance, however, their contribution to human health has not been properly quantified. This paper seeks to evaluate quantitatively how national/state parks and recreational waters are associated with human health and well-being, taking into account of the spatial dependence of environmental variables for the contiguous U.S., at the county level. First, we describe available natural resources for outdoor activities (ANROA), using national databases that include features from the Protected Areas Database, NAVSTREETS, and ATTAINSGEO 305(b) Waters. We then use spatial regression techniques to explore the association of ANROA and socioeconomic status factors on physical inactivity rates. Finally, we use variance analysis to analyze ANROA's influence on income-related health inequality. We found a significantly negative association between ANROA and the rate of physical inactivity: ANROA and the spatial effect explained 69%, nationwide, of the variation in physical inactivity. Physical inactivity rate showed a strong spatial dependence-influenced not only by its own in-county ANROA, but also by that of its neighbors ANROA. Furthermore, community groups at the same income level and with the highest ANROA, always had the lowest physical inactivity rate. This finding may help to guide future land use planning and community development that will benefit human health and well-being.

  14. The influence of the inactives subset generation on the performance of machine learning methods

    PubMed Central

    2013-01-01

    Background A growing popularity of machine learning methods application in virtual screening, in both classification and regression tasks, can be observed in the past few years. However, their effectiveness is strongly dependent on many different factors. Results In this study, the influence of the way of forming the set of inactives on the classification process was examined: random and diverse selection from the ZINC database, MDDR database and libraries generated according to the DUD methodology. All learning methods were tested in two modes: using one test set, the same for each method of inactive molecules generation and using test sets with inactives prepared in an analogous way as for training. The experiments were carried out for 5 different protein targets, 3 fingerprints for molecules representation and 7 classification algorithms with varying parameters. It appeared that the process of inactive set formation had a substantial impact on the machine learning methods performance. Conclusions The level of chemical space limitation determined the ability of tested classifiers to select potentially active molecules in virtual screening tasks, as for example DUDs (widely applied in docking experiments) did not provide proper selection of active molecules from databases with diverse structures. The study clearly showed that inactive compounds forming training set should be representative to the highest possible extent for libraries that undergo screening. PMID:23561266

  15. Enzymatic biotransformation of terpenes as bioactive agents.

    PubMed

    Sultana, Nighat; Saify, Zafar Saeed

    2013-12-01

    The plant-derived terpenoids are considered to be the most potent anticancer, anti-inflammatory and anticarcinogenic compounds known. Enzymatic biotransformation is a very useful approach to expand the chemical diversity of natural products. Recent enzymatic biotransformation studies on terpenoids have resulted in the isolation of novel compounds. 14-hydroxy methyl caryophyllene oxide produced from caryophyllene oxide showed a potent inhibitory activity against the butyryl cholinesterase enzyme, and was found to be more potent than parent caryophyllene oxide. The metabolites 3β,7β-dihydroxy-11-oxo-olean-12-en-30-oic acid, betulin, betulonic acid, argentatin A, incanilin, 18β glycyrrhetinic acid, 3,11-dioxo-olean-12-en-30-oic acid produced from 18β glycyrrhetinic acid were screened against the enzyme lipoxygenase. 3,11-Dioxo-olean-12-en-30-oic acid, was found to be more active than the parent compound. The metabolites 3β-hydroxy sclareol 18α-hydroxy sclareol, 6α,18α-dihydroxy sclareol, 11S,18α-dihydroxy sclareol, and 1β-hydroxy sclareol and 11S,18α-dihydroxy sclareol produced from sclareol were screened for antibacterial activity. 1β-Hydroxy sclareol was found to be more active than parent sclareol. There are several reports on natural product enzymatic biotransformation, but few have been conducted on terpenes. This review summarizes the classification, advantages and agents of enzymatic transformation and examines the potential role of new enzymatically transformed terpenoids and their derivatives in the chemoprevention and treatment of other diseases.

  16. Destroying God's Temple? Physical Inactivity, Poor Diet, Obesity, and Other "Sin" Behaviors.

    PubMed

    Faries, Mark D; McClendon, Megan; Jones, Eric J

    2017-02-17

    On average, our participants (N = 112), who self-proclaimed to be Christians, believed that physically inactive lifestyles, unhealthy eating, overeating, and being obese destroy the body, God's temple. However, these beliefs were less definitive, than those of other common "sin" behaviors, such as drug use, smoking, and excessive drinking of alcohol. In addition, destroying the body with physical inactivity or poor diet was not necessarily viewed as sinful. Subsequently, these beliefs did not relate to self-reported physical activity, dietary behavior, or body mass index. It is possible that inactivity, poor dietary habits, and obesity are not internalized into the spiritual perspective as destroying the body, God's temple, in the same way as other "sin" behaviors.

  17. Discrimination of active and inactive sand from remote sensing - Kelso dunes, Mojave Desert, California

    NASA Technical Reports Server (NTRS)

    Paisley, Elizabeth C. I.; Lancaster, Nicholas; Gaddis, Lisa R.; Greeley, Ronald

    1991-01-01

    Landsat TM images, field data, and laboratoray reflectance spectra were examined for the Kelso dunes, Mojave Desert, California to assess the use of visible and near-infrared (VNIR) remote sensing data to discriminate aeolian sand populations on the basis of spectral brightness. Results show that areas of inactive sand have a larger percentage of dark, fine-grained materials compared to those composed of active sand, which contain less dark fines and a higher percentage of quartz sand-size grains. Both areas are spectrally distinct in the VNIR, suggesting that VNIR spectral data can be used to discriminate active and inactive sand populations in the Mojave Desert. Analysis of laboratory spectra was complicated by the presence of magnetite in the active sands, which decreases their laboratory reflectance values to those of inactive sands. For this application, comparison of TM and laboratory spectra suggests that less than 35 percent vegetation cover does not influence the TM spectra.

  18. Discrimination of active and inactive sand from remote sensing - Kelso dunes, Mojave Desert, California

    NASA Technical Reports Server (NTRS)

    Paisley, Elizabeth C. I.; Lancaster, Nicholas; Gaddis, Lisa R.; Greeley, Ronald

    1991-01-01

    Landsat TM images, field data, and laboratoray reflectance spectra were examined for the Kelso dunes, Mojave Desert, California to assess the use of visible and near-infrared (VNIR) remote sensing data to discriminate aeolian sand populations on the basis of spectral brightness. Results show that areas of inactive sand have a larger percentage of dark, fine-grained materials compared to those composed of active sand, which contain less dark fines and a higher percentage of quartz sand-size grains. Both areas are spectrally distinct in the VNIR, suggesting that VNIR spectral data can be used to discriminate active and inactive sand populations in the Mojave Desert. Analysis of laboratory spectra was complicated by the presence of magnetite in the active sands, which decreases their laboratory reflectance values to those of inactive sands. For this application, comparison of TM and laboratory spectra suggests that less than 35 percent vegetation cover does not influence the TM spectra.

  19. Mental health, places and people: a multilevel analysis of economic inactivity and social deprivation.

    PubMed

    Fone, David L; Dunstan, Frank

    2006-09-01

    Using data on 24,975 respondents to the Welsh Health Survey 1998 aged 17-74 years, we investigated associations between individual mental health status measured using the SF-36 instrument, social class, economic inactivity and the electoral division Townsend deprivation score. In a multilevel modelling analysis, we found mental health was significantly associated with the Townsend score after adjusting for composition, and this effect was strongest in respondents who were economically inactive. Further contextual effects were shown by significant random variability in the slopes of the relation between mental health and economic inactivity at the electoral division level. Our results suggest that the places in which people live affect their mental health, supporting NHS policy that multi-agency planning to reduce inequalities in mental health status should address the wider determinants of health, as well as services for individual patients.

  20. Synthesis of the milk oligosaccharide 2'-fucosyllactose using recombinant bacterial enzymes.

    PubMed

    Albermann, C; Piepersberg, W; Wehmeier, U F

    2001-08-23

    The enzymatic synthesis of GDP-beta-L-fucose and its enzymatic transfer reaction using recombinant enzymes from bacterial sources was examined. The GDP-D-mannose 4,6-dehydratase and the GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase from Escherichia coli K-12, respectively, were used to catalyse the conversion of GDP-alpha-D-mannose to GDP-beta-L-fucose with 78% yield. For the transfer of the L-fucose to an acceptor, we cloned and overproduced the alpha-(1-->2)-fucosyltransferase (FucT2) protein from Helicobacter pylori. We were able to synthesise 2'-fucosyllactose using the overproduced FucT2 enzyme, enzymatically synthesised GDP-L-fucose and lactose. The isolation of 2'-fucosyllactose was accomplished by anion-exchange chromatography and gel filtration to give 65% yield.

  1. Preliminary evaluation of an improved enzymatic assay method for measuring potassium concentrations in serum.

    PubMed

    Ota, Eri; Sakasegawa, Shin-ichi; Ueda, Shigeru; Konishi, Kenji; Akimoto, Masaru; Tateishi, Takiko; Kawano, Miki; Hokazono, Eisaku; Kayamori, Yuzo

    2015-06-15

    K(+) has important physiological functions. K(+) concentrations in serum are generally determined using ion-selective electrodes (ISEs), though measurement using reagents in aqueous medium is also useful. K(+) concentrations were measured using recombinant inosine 5'-monophosphate dehydrogenase (IMPDH), which was activated only by K(+) and NH4(+). Exogenous NH4(+) and endogenous NH4(+) were eliminated using glutamine synthase. Regression analysis of the enzymatic assay (y) vs. the ISE method (x) gave the following relation: y=1.03x+0.09 (n=54, Sy,x=0.06 mmol/l). The linear range was up to 12 mmol/l when 1 U/ml IMPDH was used. Advantages of the proposed assay method are: (i) the measured range is wider than that of existing enzymatic methods; (ii) the conditions for K(+) determination can be maintained constant, regardless of the amount of NH4(+) in the analyte and reagents; and (iii) the elimination system is simpler because the recombinant IMPDH is stimulated by only K(+) and NH4(+) and is unaffected by biological materials. Copyright © 2015. Published by Elsevier B.V.

  2. Orientation Dependence in Homologous Recombination

    PubMed Central

    Yamamoto, K.; Takahashi, N.; Fujitani, Y.; Yoshikura, H.; Kobayashi, I.

    1996-01-01

    Homologous recombination was investigated in Escherichia coli with two plasmids, each carrying the homologous region (two defective neo genes, one with an amino-end deletion and the other with a carboxyl-end deletion) in either direct or inverted orientation. Recombination efficiency was measured in recBC sbcBC and recBC sbcA strains in three ways. First, we measured the frequency of cells carrying neo(+) recombinant plasmids in stationary phase. Recombination between direct repeats was much more frequent than between inverted repeats in the recBC sbcBC strain but was equally frequent in the two substrates in the recBC sbcA strain. Second, the fluctuation test was used to exclude bias by a rate difference between the recombinant and parental plasmids and led to the same conclusion. Third, direct selection for recombinants just after transformation with or without substrate double-strand breaks yielded essentially the same results. Double-strand breaks elevated recombination in both the strains and in both substrates. These results are consistant with our previous findings that the major route of recombination in recBC sbcBC strains generates only one recombinant DNA from two DNAs and in recBC sbcA strains generates two recombinant DNAs from two DNAs. PMID:8722759

  3. A Trivalent Enzymatic System for Uricolytic Therapy of HPRT Deficiency and Lesch-Nyhan Disease.

    PubMed

    Ronda, Luca; Marchetti, Marialaura; Piano, Riccardo; Liuzzi, Anastasia; Corsini, Romina; Percudani, Riccardo; Bettati, Stefano

    2017-07-01

    Because of the evolutionary loss of the uricolytic pathway, humans accumulate poorly soluble urate as the final product of purine catabolism. Restoration of uricolysis through enzyme therapy is a promising treatment for severe hyperuricemia caused by deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). To this end, we studied the effect of PEG conjugation on the activity and stability of the enzymatic complement required for conversion of urate into the more soluble (S)-allantoin. We produced in recombinant form three zebrafish enzymes required in the uricolytic pathway. We carried out a systematic study of the effect of PEGylation on the function and stability of the three enzymes by varying PEG length, chemistry and degree of conjugation. We assayed in vitro the uricolytic activity of the PEGylated enzymatic triad. We defined conditions that allow PEGylated enzymes to retain native-like enzymatic activity even after lyophilization or prolonged storage. A combination of the three enzymes in an appropriate ratio allowed efficient conversion of urate to (S)-allantoin with no accumulation of intermediate metabolites. Pharmaceutical restoration of the uricolytic pathway is a viable approach for the treatment of severe hyperuricemia.

  4. [Inequalities in physical inactivity according educational level in Spain, 1987 and 2007].

    PubMed

    Maestre-Miquel, Clara; Martínez, David; Polonio, Begoña; Astasio, Paloma; Santos, Juana; Regidor, Enrique

    2014-12-01

    To compare the magnitude of inequalities in the frequency of physical inactivity in Spain in 1987 and 2007, and assess whether the magnitude of inequality varies depending on the wealth of the area of residence. Descriptive cross-sectional study, national scope. Data from the National Health Survey, 1987 and 2007, adult population between 25-64 years: 30,000 individuals (1987) and 29,478 (2006/7). Main outcomes variable, leisure-time physical inactivity; exposure factor, educational level. An analysis was made of the prevalence and association using odds ratio (OR). Adjustment for socioeconomic variables: age, marital status, employment status, social class of head of household, and household income. Physical inactivity prevalence decreased in the two decades. There were more than three times more inactive males among those with primary education or less, compared to those with university studies. The magnitude of inequalities has increased in time: in a 45-64 years old man with primary school education: OR 2.43 (1.91-3.09) in 1987, to OR 2.77 (2.17-3.54) in 2007, adjusted for all socioeconomic variables. The prevalence of physical inactivity decreased between 1987 and 2007, and the largest decreases were in individuals with university studies. The gap in the differences in prevalence and OR of leisure-time physical inactivity has increased over time. It's necessary to contribute, with health education strategies and equity promotion are needed to help reduce the inequalities in risk behaviors. Copyright © 2013 Elsevier España, S.L.U. All rights reserved.

  5. Changes in diagnosed diabetes, obesity, and physical inactivity prevalence in US counties, 2004-2012.

    PubMed

    Geiss, Linda S; Kirtland, Karen; Lin, Ji; Shrestha, Sundar; Thompson, Ted; Albright, Ann; Gregg, Edward W

    2017-01-01

    Recent studies suggest that prevalence of diagnosed diabetes in the United States reached a plateau or slowed around 2008, and that this change coincided with obesity plateaus and increases in physical activity. However, national estimates can obscure important variations in geographic subgroups. We examine whether a slowing or leveling off in diagnosed diabetes, obesity, and leisure time physical inactivity prevalence is also evident across the 3143 counties of the United States. We used publicly available county estimates of the age-adjusted prevalence of diagnosed diabetes, obesity, and leisure-time physical inactivity, which were generated by the Centers for Disease Control and Prevention (CDC). Using a Bayesian multilevel regression that included random effects by county and year and applied cubic splines to smooth these estimates over time, we estimated the average annual percentage point change (APPC) from 2004 to 2008 and from 2008 to 2012 for diabetes, obesity, and physical inactivity prevalence in each county. Compared to 2004-2008, the median APPCs for diabetes, obesity, and physical inactivity were lower in 2008-2012 (diabetes APPC difference = 0.16, 95%CI 0.14, 0.18; obesity APPC difference = 0.65, 95%CI 0.59, 0.70; physical inactivity APPC difference = 0.43, 95%CI 0.37, 0.48). APPCs and APPC differences between time periods varied among counties and U.S. regions. Despite improvements, levels of these risk factors remained high with most counties merely slowing rather than reversing, which suggests that all counties would likely benefit from reductions in these risk factors. The diversity of trajectories in the prevalence of these risk factors across counties underscores the continued need to identify high risk areas and populations for preventive interventions. Awareness of how these factors are changing might assist local policy makers in targeting and tracking the impact of efforts to reduce diabetes, obesity and physical inactivity.

  6. Changes in diagnosed diabetes, obesity, and physical inactivity prevalence in US counties, 2004-2012

    PubMed Central

    Kirtland, Karen; Lin, Ji; Shrestha, Sundar; Thompson, Ted; Albright, Ann; Gregg, Edward W.

    2017-01-01

    Recent studies suggest that prevalence of diagnosed diabetes in the United States reached a plateau or slowed around 2008, and that this change coincided with obesity plateaus and increases in physical activity. However, national estimates can obscure important variations in geographic subgroups. We examine whether a slowing or leveling off in diagnosed diabetes, obesity, and leisure time physical inactivity prevalence is also evident across the 3143 counties of the United States. We used publicly available county estimates of the age-adjusted prevalence of diagnosed diabetes, obesity, and leisure-time physical inactivity, which were generated by the Centers for Disease Control and Prevention (CDC). Using a Bayesian multilevel regression that included random effects by county and year and applied cubic splines to smooth these estimates over time, we estimated the average annual percentage point change (APPC) from 2004 to 2008 and from 2008 to 2012 for diabetes, obesity, and physical inactivity prevalence in each county. Compared to 2004–2008, the median APPCs for diabetes, obesity, and physical inactivity were lower in 2008–2012 (diabetes APPC difference = 0.16, 95%CI 0.14, 0.18; obesity APPC difference = 0.65, 95%CI 0.59, 0.70; physical inactivity APPC difference = 0.43, 95%CI 0.37, 0.48). APPCs and APPC differences between time periods varied among counties and U.S. regions. Despite improvements, levels of these risk factors remained high with most counties merely slowing rather than reversing, which suggests that all counties would likely benefit from reductions in these risk factors. The diversity of trajectories in the prevalence of these risk factors across counties underscores the continued need to identify high risk areas and populations for preventive interventions. Awareness of how these factors are changing might assist local policy makers in targeting and tracking the impact of efforts to reduce diabetes, obesity and physical inactivity. PMID

  7. Linking geology and microbiology: inactive pockmarks affect sediment microbial community structure.

    PubMed

    Haverkamp, Thomas H A; Hammer, Øyvind; Jakobsen, Kjetill S

    2014-01-01

    Pockmarks are geological features that are found on the bottom of lakes and oceans all over the globe. Some are active, seeping oil or methane, while others are inactive. Active pockmarks are well studied since they harbor specialized microbial communities that proliferate on the seeping compounds. Such communities are not found in inactive pockmarks. Interestingly, inactive pockmarks are known to have different macrofaunal communities compared to the surrounding sediments. It is undetermined what the microbial composition of inactive pockmarks is and if it shows a similar pattern as the macrofauna. The Norwegian Oslofjord contains many inactive pockmarks and they are well suited to study the influence of these geological features on the microbial community in the sediment. Here we present a detailed analysis of the microbial communities found in three inactive pockmarks and two control samples at two core depth intervals. The communities were analyzed using high-throughput amplicon sequencing of the 16S rRNA V3 region. Microbial communities of surface pockmark sediments were indistinguishable from communities found in the surrounding seabed. In contrast, pockmark communities at 40 cm sediment depth had a significantly different community structure from normal sediments at the same depth. Statistical analysis of chemical variables indicated significant differences in the concentrations of total carbon and non-particulate organic carbon between 40 cm pockmarks and reference sample sediments. We discuss these results in comparison with the taxonomic classification of the OTUs identified in our samples. Our results indicate that microbial communities at the sediment surface are affected by the water column, while the deeper (40 cm) sediment communities are affected by local conditions within the sediment.

  8. The Association of Ambient Air Pollution and Physical Inactivity in the United States

    PubMed Central

    Roberts, Jennifer D.; Voss, Jameson D.; Knight, Brandon

    2014-01-01

    Background Physical inactivity, ambient air pollution and obesity are modifiable risk factors for non-communicable diseases, with the first accounting for 10% of premature deaths worldwide. Although community level interventions may target each simultaneously, research on the relationship between these risk factors is lacking. Objectives After comparing spatial interpolation methods to determine the best predictor for particulate matter (PM2.5; PM10) and ozone (O3) exposures throughout the U.S., we evaluated the cross-sectional association of ambient air pollution with leisure-time physical inactivity among adults. Methods In this cross-sectional study, we assessed leisure-time physical inactivity using individual self-reported survey data from the Centers for Disease Control and Prevention's 2011 Behavioral Risk Factor Surveillance System. These data were combined with county-level U.S. Environmental Protection Agency air pollution exposure estimates using two interpolation methods (Inverse Distance Weighting and Empirical Bayesian Kriging). Finally, we evaluated whether those exposed to higher levels of air pollution were less active by performing logistic regression, adjusting for demographic and behavioral risk factors, and after stratifying by body weight category. Results With Empirical Bayesian Kriging air pollution values, we estimated a statistically significant 16–35% relative increase in the odds of leisure-time physical inactivity per exposure class increase of PM2.5 in the fully adjusted model across the normal weight respondents (p-value<0.0001). Evidence suggested a relationship between the increasing dose of PM2.5 exposure and the increasing odds of physical inactivity. Conclusions In a nationally representative, cross-sectional sample, increased community level air pollution is associated with reduced leisure-time physical activity particularly among the normal weight. Although our design precludes a causal inference, these results provide

  9. Insulin-induced reactivation of an inactive herpes simplex thymidine kinase gene.

    PubMed Central

    Clough, D W; Morse, B S; Kucherlapati, R S; Davidson, R L

    1984-01-01

    A line of mouse cells transformed with ultraviolet-irradiated herpes simplex virus type 1 and containing a methylated and inactive viral thymidine kinase (TK) gene was treated with insulin in an attempt to induce expression of the inactive gene. Insulin was found to be capable of inducing the inactive TK gene in these cells. The induction of the TK+ phenotype was dose dependent (from 1-100 micrograms of insulin per ml), and the TK activity induced was shown to be of viral origin. Analysis of the methylation pattern of the viral TK gene by using the methylation-sensitive restriction endonucleases Sma I, Hpa II, and Hha I revealed that the active viral TK gene in the parental transformed cells was hypomethylated, whereas the inactive TK gene in the uninduced TK- cells was methylated. The active TK gene in three insulin-induced TK+ lines also was methylated, but the methylation patterns in the insulin-induced lines all were different from the uninduced TK- line. These data suggest that extensive hypomethylation of the inactive TK gene is not required for insulin induction. Four other transformed lines containing an inactive viral TK gene were tested for insulin inducibility, but insulin was unable to induce expression of the TK gene in any of the other lines. Thus, insulin inducibility does not seem to be a function of the viral TK gene itself. These results suggest that insulin inducibility of the viral TK gene may be a reflection of the region of the host genome into which the TK gene was integrated. Images PMID:6322172

  10. Light saturation curves show competence of the water splitting complex in inactive Photosystem II reaction centers.

    PubMed

    Nedbal, L; Gibas, C; Whitmarsh, J

    1991-12-01

    Photosystem II complexes of higher plants are structurally and functionally heterogeneous. While the only clearly defined structural difference is that Photosystem II reaction centers are served by two distinct antenna sizes, several types of functional heterogeneity have been demonstrated. Among these is the observation that in dark-adapted leaves of spinach and pea, over 30% of the Photosystem II reaction centers are unable to reduce plastoquinone to plastoquinol at physiologically meaningful rates. Several lines of evidence show that the impaired reaction centers are effectively inactive, because the rate of oxidation of the primary quinone acceptor, QA, is 1000 times slower than in normally active reaction centers. However, there are conflicting opinions and data over whether inactive Photosystem II complexes are capable of oxidizing water in the presence of certain artificial electron acceptors. In the present study we investigated whether inactive Photosystem II complexes have a functional water oxidizing system in spinach thylakoid membranes by measuring the flash yield of water oxidation products as a function of flash intensity. At low flash energies (less that 10% saturation), selected to minimize double turnovers of reaction centers, we found that in the presence of the artificial quinone acceptor, dichlorobenzoquinone (DCBQ), the yield of proton release was enhanced 20±2% over that observed in the presence of dimethylbenzoquinone (DMBQ). We argue that the extra proton release is from the normally inactive Photosystem II reaction centers that have been activated in the presence of DCBQ, demonstrating their capacity to oxidize water in repetitive flashes, as concluded by Graan and Ort (Biochim Biophys Acta (1986) 852: 320-330). The light saturation curves indicate that the effective antenna size of inactive reaction centers is 55±12% the size of active Photosystem II centers. Comparison of the light saturation dependence of steady state oxygen evolution

  11. Recombinant Collagenlike Proteins

    NASA Technical Reports Server (NTRS)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  12. Poly(ADP-ribose) polymerase 1 is inhibited by a histone H2A variant, MacroH2A, and contributes to silencing of the inactive X chromosome.

    PubMed

    Nusinow, Dmitri A; Hernández-Muñoz, Inmaculada; Fazzio, Thomas G; Shah, Girish M; Kraus, W Lee; Panning, Barbara

    2007-04-27

    Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that is involved in modulating chromatin structure, regulation of gene expression, and sensing DNA damage. Here, we report that PARP-1 enzymatic activity is inhibited by macroH2A, a vertebrate histone H2A variant that is enriched on facultative heterochromatin. MacroH2A family members have a large C-terminal non-histone domain (NHD) and H2A-like histone domain. MacroH2A1.2 and PARP-1 interact in vivo and in vitro via the NHD. The NHD of each macroH2A family member was sufficient to inhibit PARP-1 enzymatic activity in vitro. The NHD of macroH2A1.2 was a mixed inhibitor of PARP-1 catalytic activity, with affects on both catalytic activity and the substrate binding affinity of PARP-1. Depletion of PARP-1 by RNA interference caused reactivation of a reporter gene on the inactive X chromosome, demonstrating that PARP-1 participates in the maintenance of silencing. These results suggest that one function of macroH2A in gene silencing is to inhibit PARP-1 enzymatic activity, and this may affect PARP-1 association with chromatin.

  13. Systemic endothelial function is preserved in men with both active and inactive variant angina pectoris.

    PubMed

    Ito, K; Akita, H; Kanazawa, K; Yamada, S; Shiga, N; Terashima, M; Matsuda, Y; Takai, E; Iwai, C; Takaoka, H; Yokoyama, M

    1999-12-01

    To test the hypothesis that coronary spasm could be a coronary manifestation of systemic endothelial dysfunction and that the activity of coronary spasm could influence systemic endothelial function, we examined brachial flow-mediated, endothelium-dependent vasodilation and nitroglycerin-induced endothelium-independent vasodilation with high-resolution ultrasound in 11 men with variant angina pectoris (6 active and 5 inactive) without established coronary atherosclerosis. Endothelium-dependent vasodilation in peripheral circulation was preserved in men with active and inactive variant angina pectoris, suggesting that systemic endothelial dysfunction is not involved in either the pathogenesis or the activity of coronary spasm.

  14. Associations between physical inactivity and sedentary behaviors among adolescents in 10 cities in China

    PubMed Central

    2014-01-01

    Background Studies in western countries have revealed that excessive sedentary behavior is a major risk factor for physical inactivity in adolescents. This study was performed to investigate the association between sedentary behavior and physical inactivity in Chinese adolescents using a large-scale cross-sectional survey design. Methods This study was part of the 2011 Chinese Youth Risk Behavior Survey. Between March and September 2011, 10,214 11–18-year-olds were recruited for survey participation in 18 schools in 10 cities in China. Demographic and socioeconomic characteristics, and the prevalences of physical inactivity and sedentary behaviors, were examined. Correlations between sedentary behavior and physical inactivity were analyzed using baseline logistic regression. Results Among the final 9,901 students, physical inactivity (~80%) and sedentary behaviors (television viewing, 43%; computer use, 30.2%) were prevalent. More male than female students reported sedentary behaviors (television viewing > 2 h: 5.5% vs. 3.9%; computer use > 2 h: 7.2% vs. 3.5%; both p < 0.05), but more males were physically active than females (25.1% vs.14.6%; p < 0.05). Television viewing was associated with lower odds of no physical activity (No PA) in males [0–2 h: adjusted odds ratio (AOR) = 0.81, 95% confidence interval (CI) = 0.68–0.96; >4 h: OR = 0.34, 95% CI = 0.18–0.64], but not in females. A similar pattern between insufficient physical activity and >4 h TV viewing (AOR = 0.42, 95% CI = 0.23–0.76) and >4 h computer use (AOR = 0.49, 95% CI = 0.30–0.78) was observed in males. In females, 0–2 h daily computer use was associated with higher odds of physical inactivity (No PA: AOR = 1.42, 95% CI = 1.10–1.82; Insufficient PA: AOR = 1.58, 95% CI = 1.24–2.01), while TV viewing was not associated with No PA or Insufficient PA. The probability of physical inactivity significantly increased with

  15. "Inactive" ingredients in pharmaceutical products: update (subject review). American Academy of Pediatrics Committee on Drugs.

    PubMed

    1997-02-01

    Because of an increasing number of reports of adverse reactions associated with pharmaceutical excipients, in 1985 the Committee on Drugs issued a position statement recommending that the Food and Drug Administration mandate labeling of over-the-counter and prescription formulations to include a qualitative list of inactive ingredients. However, labeling of inactive ingredients remains voluntary. Adverse reactions continue to be reported, although some are no longer considered clinically significant, and other new reactions have emerged. The original statement, therefore, has been updated and its information expanded.

  16. Expression of MTAP inhibits tumor-related phenotypes in HT1080 cells via a mechanism unrelated to its enzymatic function.

    PubMed

    Tang, Baiqing; Kadariya, Yuwaraj; Chen, Yibai; Slifker, Michael; Kruger, Warren D

    2014-11-11

    Methylthioadenosine Phosphorylase (MTAP) is a tumor suppressor gene that is frequently deleted in human cancers and encodes an enzyme responsible for the catabolism of the polyamine byproduct 5'deoxy-5'-methylthioadenosine (MTA). To elucidate the mechanism by which MTAP inhibits tumor formation, we have reintroduced MTAP into MTAP-deleted HT1080 fibrosarcoma cells. Expression of MTAP resulted in a variety of phenotypes, including decreased colony formation in soft-agar, decreased migration, decreased in vitro invasion, increased matrix metalloproteinase production, and reduced ability to form tumors in severe combined immunodeficiency mice. Microarray analysis showed that MTAP affected the expression of genes involved in a variety of processes, including cell adhesion, extracellular matrix interaction, and cell signaling. Treatment of MTAP-expressing cells with a potent inhibitor of MTAP's enzymatic activity (MT-DADMe-ImmA) did not result in a MTAP- phenotype. This finding suggests that MTAP's tumor suppressor function is not the same as its known enzymatic function. To confirm this, we introduced a catalytically inactive version of MTAP, D220A, into HT1080 cells and found that this mutant was fully capable of reversing the soft agar colony formation, migration, and matrix metalloproteinase phenotypes. Our results show that MTAP affects cellular phenotypes in HT1080 cells in a manner that is independent of its known enzymatic activity. Copyright © 2015 Tang et al.

  17. Expression of MTAP Inhibits Tumor-Related Phenotypes in HT1080 Cells via a Mechanism Unrelated to Its Enzymatic Function

    PubMed Central

    Tang, Baiqing; Kadariya, Yuwaraj; Chen, Yibai; Slifker, Michael; Kruger, Warren D.

    2014-01-01

    Methylthioadenosine Phosphorylase (MTAP) is a tumor suppressor gene that is frequently deleted in human cancers and encodes an enzyme responsible for the catabolism of the polyamine byproduct 5′deoxy-5′-methylthioadenosine (MTA). To elucidate the mechanism by which MTAP inhibits tumor formation, we have reintroduced MTAP into MTAP-deleted HT1080 fibrosarcoma cells. Expression of MTAP resulted in a variety of phenotypes, including decreased colony formation in soft-agar, decreased migration, decreased in vitro invasion, increased matrix metalloproteinase production, and reduced ability to form tumors in severe combined immunodeficiency mice. Microarray analysis showed that MTAP affected the expression of genes involved in a variety of processes, including cell adhesion, extracellular matrix interaction, and cell signaling. Treatment of MTAP-expressing cells with a potent inhibitor of MTAP’s enzymatic activity (MT-DADMe-ImmA) did not result in a MTAP− phenotype. This finding suggests that MTAP’s tumor suppressor function is not the same as its known enzymatic function. To confirm this, we introduced a catalytically inactive version of MTAP, D220A, into HT1080 cells and found that this mutant was fully capable of reversing the soft agar colony formation, migration, and matrix metalloproteinase phenotypes. Our results show that MTAP affects cellular phenotypes in HT1080 cells in a manner that is independent of its known enzymatic activity. PMID:25387827

  18. Functional, Responsive Materials Assembled from Recombinant Oleosin

    NASA Astrophysics Data System (ADS)

    Hammer, Daniel

    Biological cells are surrounded by a plasma membrane made primarily of phospholipids that form a bilayer. This membrane is permselective and compartmentalizes the cell. A simple form of artificial cell is the vesicle, in which a phospholipid bilayer membrane surrounds an aqueous solution. However, there is no a priori reason why a membrane needs to be made of phospholipids. It could be made of any surfactant that forms a bilayer. We have assembled membranes and other structures from the recombinant plant protein oleosin. The ability to assemble from a recombinant protein means that every molecule is identical, we have complete control over the sequence, and hence can build in designer functionality with high fidelity, including adhesion and enzymatic activity. Such incorporation is trivial using the tools of molecular biology. We find that while many variants of oleosin make membranes, others make micelles and sheets. We show how the type of supramolecular structure can be altered by the conditions of solvent, such as ionic strength, and the architecture of the surfactant itself. We show that protease cleavable domains can be incorporated within oleosin, and be engineered to protect other functional domains such as adhesive motifs, to make responsive materials whose activity and shape depend on the action of proteases. We will also present the idea of making ``Franken''-oleosins, where large domains of native oleosin are replaced with domains from other functional proteins, to make hybrids conferred by the donor protein. Thus, we can view oleosin as a template upon which a vast array of designer functionalities can be imparted..

  19. Enzymic properties of recombinant BACE2.

    PubMed

    Kim, Yong-Tae; Downs, Deborah; Wu, Shili; Dashti, Azar; Pan, Yujun; Zhai, Peng; Wang, Xinjuan; Zhang, Xuejun C; Lin, Xinli

    2002-11-01

    BACE2 (Memapsin 1) is a membrane-bound aspartic protease that is highly homologous with BACE1 (Memapsin 2). While BACE1 processes the amyloid precursor protein (APP) at a key step in generating the beta-amyloid peptide and presumably causes Alzheimer's disease (AD), BACE2 has not been demonstrated to be directly involved in APP processing, and its physiological functions remain to be determined. In vivo, BACE2 is expressed as a precursor protein containing pre-, pro-, protease, transmembrane, and cytosolic domains/peptides. To determine the enzymatic properties of BACE2, two variants of its pro-protease domain, pro-BACE2-T1 (PB2-T1) and pro-BACE2-T2 (PB2-T2), were constructed. They have been expressed in Escherichia coli as inclusion bodies, refolded and purified. These two recombinant proteins have the same N terminus but differ at their C-terminal ends: PB2-T1 ends at Pro466, on the boundary of the postulated transmembrane domain, and PB2-T2 ends at Ser431, close to the homologous ends of other aspartic proteases such as pepsin. While PB2-T1 shares similar substrate specificities with BACE1 and other 'general' aspartic proteases, the specificity of PB2-T2 is more constrained, apparently preferring to cleave at the NH2-terminal side of paired basic residues. Unlike other 'typical' aspartic proteases, which are active only under acidic conditions, the recombinant BACE2, PB2-T1, was active at a broad pH range. In addition, pro-BACE2 can be processed at its in vivo maturation site by BACE1.

  20. Enzymatic induction of supramolecular order and bioactivity

    NASA Astrophysics Data System (ADS)

    Yang, Chengbiao; Ren, Xinrui; Ding, Dan; Wang, Ling; Yang, Zhimou

    2016-05-01

    We showed in this study that enzymatic triggering is a totally different pathway for the preparation of self-assembling nanomaterials to the heating-cooling process. Because the molecules were under lower energy levels and the molecular conformation was more ordered during the enzymatic triggeration under mild conditions, nanomaterials with higher supramolecular order could be obtained through biocatalytic control. In this study, nanoparticles were obtained by an enzymatic reaction and nanofibers were observed through the heating-cooling process. We observed a distinct trough at 318 nm from the CD spectrum of a particle sample but not a fiber sample, suggesting the long range arrangement of molecules and helicity in the nanoparticles. The nanoparticles with higher supramolecular order possessed much better potency as a protein vaccine adjuvant because it accelerated the DC maturation and elicited stronger T-cells cytokine production than the nanofibers. Our study demonstrated that biocatalytic triggering is a useful method for preparing supramolecular nanomaterials with higher supramolecular order and probably better bioactivity.We showed in this study that enzymatic triggering is a totally different pathway for the preparation of self-assembling nanomaterials to the heating-cooling process. Because the molecules were under lower energy levels and the molecular conformation was more ordered during the enzymatic triggeration under mild conditions, nanomaterials with higher supramolecular order could be obtained through biocatalytic control. In this study, nanoparticles were obtained by an enzymatic reaction and nanofibers were observed through the heating-cooling process. We observed a distinct trough at 318 nm from the CD spectrum of a particle sample but not a fiber sample, suggesting the long range arrangement of molecules and helicity in the nanoparticles. The nanoparticles with higher supramolecular order possessed much better potency as a protein vaccine

  1. Making recombinant extracellular matrix proteins.

    PubMed

    Ruggiero, Florence; Koch, Manuel

    2008-05-01

    A variety of approaches to understand extracellular matrix protein structure and function require production of recombinant proteins. Moreover, the expression of heterologous extracellular matrix proteins, in particular collagens, using the recombinant technology is of major interest to the biomedical industry. Although extracellular matrix proteins are large, modular and often multimeric, most of them have been successfully produced in various expression systems. This review provides important factors, including the design of the construct, the cloning strategies, the expression vectors, the transfection method and the host cell systems, to consider in choosing a reliable and cost-effective way to make recombinant extracellular matrix proteins. Advantages and drawbacks of each system have been appraised. Protocols that may ease efficient recombinant production of extracellular matrix are described. Emphasis is placed on the recombinant collagen production. Members of the collagen superfamily exhibit specific structural features and generally require complex post-translational modifications to retain full biological activity that make more arduous their recombinant production.

  2. Expression of recombinant antibodies.

    PubMed

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with "human-like" post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  3. Dissociative recombination of HCl+

    NASA Astrophysics Data System (ADS)

    Larson, Åsa; Fonseca dos Santos, Samantha; E. Orel, Ann

    2017-08-01

    The dissociative recombination of HCl+, including both the direct and indirect mechanisms, is studied. For the direct process, the relevant electronic states are calculated ab initio by combining electron scattering calculations to obtain resonance positions and autoionization widths with multi-reference configuration interaction calculations of the ion and Rydberg states. The cross section for the direct dissociation along electronic resonant states is computed by solution of the time-dependent Schrödinger equation. For the indirect process, an upper bound value for the cross section is obtained using a vibrational frame transformation of the elements of the scattering matrix at energies just above the ionization threshold. Vibrational excitations of the ionic core from the ground vibrational state, v = 0 , to the first three excited vibrational states, v = 1 , v = 2 , and v = 3 , are considered. Autoionization is neglected and the effect of the spin-orbit splitting of the ionic potential energy upon the indirect dissociative recombination cross section is considered. The calculated cross sections are compared to measurements.

  4. Recombinant electric storage battery

    SciTech Connect

    Flicker, R.P.; Fenstermacher, S.

    1989-10-10

    This patent describes a recombinant storage battery. It comprises: a plurality of positive plates containing about 2 to 4 percent of antimony based upon the total weight of the alloy and positive active material, and essentially antimony free negative plates in a closed case; a fibrous sheet plate separator between adjacent ones of the plates, and a body of an electrolyte to which the sheet separators are inert absorbed by each of the separators and maintained in contact with each of the adjacent ones of the plates. Each of the separator sheets comprising first fibers which impart to the sheet a given absorbency greater than 90 percent relative to the electrolyte and second fibers which impart to the sheet a different absorbency less than 80 percent relative to the electrolyte. The first and second fibers being present in such proportions that each of the sheet separators has an absorbency with respect to the electrolyte of from 75 to 95 percent and the second fibers being present in such proportions that the battery has a recombination rate adequate to compensate for gassing.

  5. Expression of Recombinant Antibodies

    PubMed Central

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications. PMID:23908655

  6. Revocation of Office of Generic Drug's interim policy statement on inactive ingredients. Food and Drug Administration, HHS. Notice.

    PubMed

    1999-04-30

    The Food and Drug Administration (FDA) is revoking an interim policy statement on inactive ingredients in parenteral, ophthalmic, otic, and topical generic drug products (Interim Inactive Ingredient Policy). These generic drug products are the subjects of abbreviated new drug applications (ANDA's). The Interim Inactive Ingredient Policy was issued as a memorandum from the Acting Director of the Center for Drug Evaluation and Research's (CDER's) Office of Generic Drugs, FDA, to CDER's Associate Director for Science and Medical Affairs, FDA. FDA is taking this action because the Interim Inactive Ingredient Policy no longer represents current agency policy.

  7. Germline methylation patterns determine the distribution of recombination events in the dog genome.

    PubMed

    Berglund, Jonas; Quilez, Javier; Arndt, Peter F; Webster, Matthew T

    2014-12-19

    The positive-regulatory domain containing nine gene, PRDM9, which strongly associates with the location of recombination events in several vertebrates, is inferred to be inactive in the dog genome. Here, we address several questions regarding the control of recombination and its influence on genome evolution in dogs. First, we address whether the association between CpG islands (CGIs) and recombination hotspots is generated by lack of methylation, GC-biased gene conversion (gBGC), or both. Using a genome-wide dog single nucleotide polymorphism data set and comparisons of the dog genome with related species, we show that recombination-associated CGIs have low CpG mutation rates, and that CpG mutation rate is negatively correlated with recombination rate genome wide, indicating that nonmethylation attracts the recombination machinery. We next use a neighbor-dependent model of nucleotide substitution to disentangle the effects of CpG mutability and gBGC and analyze the effects that loss of PRDM9 has on these rates. We infer that methylation patterns have been stable during canid genome evolution, but that dog CGIs have experienced a drastic increase in substitution rate due to gBGC, consistent with increased levels of recombination in these regions. We also show that gBGC is likely to have generated many new CGIs in the dog genome, but these mostly occur away from genes, whereas the number of CGIs in gene promoter regions has not increased greatly in recent evolutionary history. Recombination has a major impact on the distribution of CGIs that are detected in the dog genome due to the interaction between methylation and gBGC. The results indicate that germline methylation patterns are the main determinant of recombination rates in the absence of PRDM9. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  8. Germline Methylation Patterns Determine the Distribution of Recombination Events in the Dog Genome

    PubMed Central

    Berglund, Jonas; Quilez, Javier; Arndt, Peter F.; Webster, Matthew T.

    2015-01-01

    The positive-regulatory domain containing nine gene, PRDM9, which strongly associates with the location of recombination events in several vertebrates, is inferred to be inactive in the dog genome. Here, we address several questions regarding the control of recombination and its influence on genome evolution in dogs. First, we address whether the association between CpG islands (CGIs) and recombination hotspots is generated by lack of methylation, GC-biased gene conversion (gBGC), or both. Using a genome-wide dog single nucleotide polymorphism data set and comparisons of the dog genome with related species, we show that recombination-associated CGIs have low CpG mutation rates, and that CpG mutation rate is negatively correlated with recombination rate genome wide, indicating that nonmethylation attracts the recombination machinery. We next use a neighbor-dependent model of nucleotide substitution to disentangle the effects of CpG mutability and gBGC and analyze the effects that loss of PRDM9 has on these rates. We infer that methylation patterns have been stable during canid genome evolution, but that dog CGIs have experienced a drastic increase in substitution rate due to gBGC, consistent with increased levels of recombination in these regions. We also show that gBGC is likely to have generated many new CGIs in the dog genome, but these mostly occur away from genes, whereas the number of CGIs in gene promoter regions has not increased greatly in recent evolutionary history. Recombination has a major impact on the distribution of CGIs that are detected in the dog genome due to the interaction between methylation and gBGC. The results indicate that germline methylation patterns are the main determinant of recombination rates in the absence of PRDM9. PMID:25527838

  9. Production of MAG via enzymatic glycerolysis

    NASA Astrophysics Data System (ADS)

    Jamlus, Norul Naziraa Ahmad; Derawi, Darfizzi; Salimon, Jumat

    2015-09-01

    Enzymatic glycerolysis of a medium chain methyl ester, methyl laurate was performed using lipase Candida antarctica (Novozyme 435) for 6 hours at 55°C. The percentage of components mixture of product were determined by using gas chromatography technique. The enzymatic reaction was successfully produced monolaurin (45.9 %), dilaurin (47.1 %) and trilaurin (7.0 %) respectively. Thin layer chromatography (TLC) plate also showed a good separation of component spots. Fourier transformation infra-red (FTIR) spectrum showed the presence of ester carbonyl at wavenumber 1739.99 cm-1 and hydrogen bonded O-H at 3512.03 cm-1. The product is potentially to be used as emulsifier and additive in food industry, pharmaceutical, as well as antibacterial.

  10. Enzymatic hydrolysis of poly(ethylene furanoate).

    PubMed

    Pellis, Alessandro; Haernvall, Karolina; Pichler, Christian M; Ghazaryan, Gagik; Breinbauer, Rolf; Guebitz, Georg M

    2016-10-10

    The urgency of producing new environmentally-friendly polyesters strongly enhanced the development of bio-based poly(ethylene furanoate) (PEF) as an alternative to plastics like poly(ethylene terephthalate) (PET) for applications that include food packaging, personal and home care containers and thermoforming equipment. In this study, PEF powders of various molecular weights (6, 10 and 40kDa) were synthetized and their susceptibility to enzymatic hydrolysis was investigated for the first time. According to LC/TOF-MS analysis, cutinase 1 from Thermobifida cellulosilytica liberated both 2,5-furandicarboxylic acid and oligomers of up to DP4. The enzyme preferentially hydrolyzed PEF with higher molecular weights but was active on all tested substrates. Mild enzymatic hydrolysis of PEF has a potential both for surface functionalization and monomers recycling.

  11. A Networks Approach to Modeling Enzymatic Reactions.

    PubMed

    Imhof, P

    2016-01-01

    Modeling enzymatic reactions is a demanding task due to the complexity of the system, the many degrees of freedom involved and the complex, chemical, and conformational transitions associated with the reaction. Consequently, enzymatic reactions are not determined by precisely one reaction pathway. Hence, it is beneficial to obtain a comprehensive picture of possible reaction paths and competing mechanisms. By combining individually generated intermediate states and chemical transition steps a network of such pathways can be constructed. Transition networks are a discretized representation of a potential energy landscape consisting of a multitude of reaction pathways connecting the end states of the reaction. The graph structure of the network allows an easy identification of the energetically most favorable pathways as well as a number of alternative routes.

  12. Graphene based enzymatic bioelectrodes and biofuel cells.

    PubMed

    Karimi, Anahita; Othman, Ali; Uzunoglu, Aytekin; Stanciu, Lia; Andreescu, Silvana

    2015-04-28

    The excellent electrical conductivity and ease of functionalization make graphene a promising material for use in enzymatic bioelectrodes and biofuel cells. Enzyme based biofuel cells have attracted substantial interest due to their potential to harvest energy from organic materials. This review provides an overview of the functional properties and applications of graphene in the construction of biofuel cells as alternative power sources. The review covers the current state-of-the-art research in graphene based nanomaterials (physicochemical properties and surface functionalities), the role of these parameters in enhancing electron transfer, the stability and activity of immobilized enzymes, and how enhanced power density can be achieved. Specific examples of enzyme immobilization methods, enzyme loading, stability and function on graphene, functionalized graphene and graphene based nanocomposite materials are discussed along with their advantages and limitations. Finally, a critical evaluation of the performance of graphene based enzymatic biofuel cells, the current status, challenges and future research needs are provided.

  13. Graphene based enzymatic bioelectrodes and biofuel cells

    NASA Astrophysics Data System (ADS)

    Karimi, Anahita; Othman, Ali; Uzunoglu, Aytekin; Stanciu, Lia; Andreescu, Silvana

    2015-04-01

    The excellent electrical conductivity and ease of functionalization make graphene a promising material for use in enzymatic bioelectrodes and biofuel cells. Enzyme based biofuel cells have attracted substantial interest due to their potential to harvest energy from organic materials. This review provides an overview of the functional properties and applications of graphene in the construction of biofuel cells as alternative power sources. The review covers the current state-of-the-art research in graphene based nanomaterials (physicochemical properties and surface functionalities), the role of these parameters in enhancing electron transfer, the stability and activity of immobilized enzymes, and how enhanced power density can be achieved. Specific examples of enzyme immobilization methods, enzyme loading, stability and function on graphene, functionalized graphene and graphene based nanocomposite materials are discussed along with their advantages and limitations. Finally, a critical evaluation of the performance of graphene based enzymatic biofuel cells, the current status, challenges and future research needs are provided.

  14. Cephalopod alcohol dehydrogenase: purification and enzymatic characterization.

    PubMed

    Rosario Fernández, M; Jörnvall, H; Moreno, A; Kaiser, R; Parés, X

    1993-08-16

    Octopus, squid and cuttle-fish organs were examined for alcohol dehydrogenase activity. Only one form was detectable, with properties typical of mammalian class III alcohol dehydrogenase. The corresponding protein was purified from octopus and enzymatically characterized. Ion-exchange and affinity chromatography produced a pure protein in excellent yield (73%) after 1600-fold purification. Enzymatic parameters with several substrates were similar to those for the human class III alcohol dehydrogenase, demonstrating a largely conserved function of the enzyme through wide lines of divergence covering vertebrates, cephalopods and bacteria. The results establish the universal occurrence of class III alcohol dehydrogenase and its strictly conserved functional properties in separate living forms. The absence of other alcohol dehydrogenases in cephalopods is compatible with the emergence of the ethanol-active class I type at a later stage, in lineages leading to vertebrates.

  15. Biofunctional Properties of Enzymatic Squid Meat Hydrolysate

    PubMed Central

    Choi, Joon Hyuk; Kim, Kyung-Tae; Kim, Sang Moo

    2015-01-01

    Squid is one of the most important commercial fishes in the world and is mainly utilized or consumed as sliced raw fish or as processed products. The biofunctional activities of enzymatic squid meat hydrolysate were determined to develop value-added products. Enzymatic squid hydrolysate manufactured by Alcalase effectively quenched 1,1-diphenyl-2-picrylhydrazyl radical, hydroxyl radical, and hydrogen peroxide radical with IC50 values of 311, 3,410, and 111.5 μg/mL, respectively. Angiotensin I-converting enzyme inhibitory activity of squid hydrolysate was strong with an IC50 value of 145.1 μg/mL, while tyrosinase inhibitory activity with an IC50 value of 4.72 mg/mL was moderately low. Overall, squid meat hydrolysate can be used in food or cosmetic industries as a bioactive ingredient and possibly be used in the manufacture of seasoning, bread, noodle, or cosmetics. PMID:25866752

  16. Enzymatic Decontamination: From Concept To Commercialization

    DTIC Science & Technology

    2004-12-01

    granules are: • Particle size: 600-1000 microns • Dissolution rate: 1 kg in 500 gallons of water in 5 minutes • Enzyme activity : TBD (expected protein...enzymatic activity in a variety of tissues could catalytically detoxify diisopropylfluorophosphate (DFP). More recently, a variety of enzymes with activity ...large areas for retrograde and resupply operations, and to reconstitute individual equipment, vehicles, and weapon platforms. The objective of

  17. Molecular crowding and protein enzymatic dynamics.

    PubMed

    Echeverria, Carlos; Kapral, Raymond

    2012-05-21

    The effects of molecular crowding on the enzymatic conformational dynamics and transport properties of adenylate kinase are investigated. This tridomain protein undergoes large scale hinge motions in the course of its enzymatic cycle and serves as prototype for the study of crowding effects on the cyclic conformational dynamics of proteins. The study is carried out at a mesoscopic level where both the protein and the solvent in which it is dissolved are treated in a coarse grained fashion. The amino acid residues in the protein are represented by a network of beads and the solvent dynamics is described by multiparticle collision dynamics that includes effects due to hydrodynamic interactions. The system is crowded by a stationary random array of hard spherical objects. Protein enzymatic dynamics is investigated as a function of the obstacle volume fraction and size. In addition, for comparison, results are presented for a modification of the dynamics that suppresses hydrodynamic interactions. Consistent with expectations, simulations of the dynamics show that the protein prefers a closed conformation for high volume fractions. This effect becomes more pronounced as the obstacle radius decreases for a given volume fraction since the average void size in the obstacle array is smaller for smaller radii. At high volume fractions for small obstacle radii, the average enzymatic cycle time and characteristic times of internal conformational motions of the protein deviate substantially from their values in solution or in systems with small density of obstacles. The transport properties of the protein are strongly affected by molecular crowding. Diffusive motion adopts a subdiffusive character and the effective diffusion coefficients can change by more than an order of magnitude. The orientational relaxation time of the protein is also significantly altered by crowding.

  18. New starch preparations resistant to enzymatic digestion.

    PubMed

    Jochym, Kamila; Kapusniak, Janusz; Barczynska, Renata; Sliżewska, Katarzyna

    2012-03-15

    New starch preparations were produced by thermolysis of potato starch in the presence of inorganic (hydrochloric) and organic (citric and tartaric) acids under controlled conditions. The starch preparations were physicochemically and structurally characterised and analysed for their resistance to enzymatic digestion in vitro. The content of resistant fraction in dextrin D1, obtained by heating starch acidified with hydrochloric and citric acids, determined by the AOAC 2001.03 and pancreatin-gravimetric methods was similar (~200 g kg⁻¹). In the case of dextrin D3, obtained by heating starch acidified with hydrochloric and tartaric acids, the result of determination by the pancreatin-gravimetric method was almost four times higher than that obtained with the AOAC 2001.03 method. The enzymatic tests revealed that dextrin D3 obtained with excess tartaric acid can be classified as RS4, which can only be partially determined by enzymatic-gravimetric methods. Tartaric acid at high concentration had a significantly stronger influence on starch hydrolysis than citric acid. This was confirmed by chromatographic analysis of dextrins and chemical investigation of the reducing power. The results confirmed the possibility of applying dextrins, prepared under specific conditions, as soluble dietary fibre. Copyright © 2011 Society of Chemical Industry.

  19. Enzymatic transformation of nonfood biomass to starch.

    PubMed

    You, Chun; Chen, Hongge; Myung, Suwan; Sathitsuksanoh, Noppadon; Ma, Hui; Zhang, Xiao-Zhou; Li, Jianyong; Zhang, Y-H Percival

    2013-04-30

    The global demand for food could double in another 40 y owing to growth in the population and food consumption per capita. To meet the world's future food and sustainability needs for biofuels and renewable materials, the production of starch-rich cereals and cellulose-rich bioenergy plants must grow substantially while minimizing agriculture's environmental footprint and conserving biodiversity. Here we demonstrate one-pot enzymatic conversion of pretreated biomass to starch through a nonnatural synthetic enzymatic pathway composed of endoglucanase, cellobiohydrolyase, cellobiose phosphorylase, and alpha-glucan phosphorylase originating from bacterial, fungal, and plant sources. A special polypeptide cap in potato alpha-glucan phosphorylase was essential to push a partially hydrolyzed intermediate of cellulose forward to the synthesis of amylose. Up to 30% of the anhydroglucose units in cellulose were converted to starch; the remaining cellulose was hydrolyzed to glucose suitable for ethanol production by yeast in the same bioreactor. Next-generation biorefineries based on simultaneous enzymatic biotransformation and microbial fermentation could address the food, biofuels, and environment trilemma.

  20. Extracellular enzymatic activity of Microsporum canis isolates.

    PubMed

    Papini, R; Mancianti, F

    The enzymatic activity of 70 feline and canine Microsporum canis isolates was determined by the Api-Zym test. The liquid phase of cultures, inoculated into Tryptic Soy Broth, was used to examine 19 enzymes. Considerable differences were observed among the extracellular enzymatic patterns. All the isolates produced alkaline phosphatase and beta-glucosidase, while lipase (C14), trypsin, chymotrypsin, beta-glucuronidase, and alpha-fucosidase activity was never revealed. Esterase (C4) activity was present in 57 samples (81%), esterase lipase (C8) in 31 (44%), leucine arylamidase in 35 (50%), valine arylamidase and cystine arylamidase in 7 (10%), acid phosphatase in 64 (91%), naphthol-AS-BI-phosphohydrolase in 60 (86%), alpha-galactosidase in 5 (7%), beta-galactosidase in 6 (8%), alpha-glucosidase in 25 (36%), N-acetyl-beta-glucosaminidase in 41 (58%), and alpha-mannosidase in 51 (73%). The beta-galactosidase activity of M. canis has not been reported previously. Remarkable variations of intensity for each enzymatic activity were also detected. It is believed that these results could provide basic data for further investigations on the pathogenic role of enzymes secreted by M. canis.

  1. Microbial Enzymatic Degradation of Biodegradable Plastics.

    PubMed

    Roohi; Bano, Kulsoom; Kuddus, Mohammed; Zaheer, Mohammed R; Zia, Qamar; Khan, Mohammed F; Ashraf, Ghulam Md; Gupta, Anamika; Aliev, Gjumrakch

    2017-01-01

    The renewable feedstock derived biodegradable plastics are important in various industries such as packaging, agricultural, paper coating, garbage bags and biomedical implants. The increasing water and waste pollution due to the available decomposition methods of plastic degradation have led to the emergence of biodegradable plastics and biological degradation with microbial (bacteria and fungi) extracellular enzymes. The microbes utilize biodegradable polymers as the substrate under starvation and in unavailability of microbial nutrients. Microbial enzymatic degradation is suitable from bioremediation point of view as no waste accumulation occurs. It is important to understand the microbial interaction and mechanism involved in the enzymatic degradation of biodegradable plastics under the influence of several environmental factors such as applied pH, thermo-stability, substrate molecular weight and/or complexity. To study the surface erosion of polymer film is another approach for hydrolytic degradation characteristion. The degradation of biopolymer is associated with the production of low molecular weight monomer and generation of carbon dioxide, methane and water molecule. This review reported the degradation study of various existing biodegradable plastics along with the potent degrading microbes (bacteria and fungi). Patents available on plastic biodegradation with biotechnological significance is also summarized in this paper. This paper assesses that new disposal technique should be adopted for the degradation of polymers and further research is required for the economical production of biodegradable plastics along with their enzymatic degradation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  2. Enzymatic transformation of nonfood biomass to starch

    PubMed Central

    You, Chun; Chen, Hongge; Myung, Suwan; Sathitsuksanoh, Noppadon; Ma, Hui; Zhang, Xiao-Zhou; Li, Jianyong; Zhang, Y.-H. Percival

    2013-01-01

    The global demand for food could double in another 40 y owing to growth in the population and food consumption per capita. To meet the world’s future food and sustainability needs for biofuels and renewable materials, the production of starch-rich cereals and cellulose-rich bioenergy plants must grow substantially while minimizing agriculture’s environmental footprint and conserving biodiversity. Here we demonstrate one-pot enzymatic conversion of pretreated biomass to starch through a nonnatural synthetic enzymatic pathway composed of endoglucanase, cellobiohydrolyase, cellobiose phosphorylase, and alpha-glucan phosphorylase originating from bacterial, fungal, and plant sources. A special polypeptide cap in potato alpha-glucan phosphorylase was essential to push a partially hydrolyzed intermediate of cellulose forward to the synthesis of amylose. Up to 30% of the anhydroglucose units in cellulose were converted to starch; the remaining cellulose was hydrolyzed to glucose suitable for ethanol production by yeast in the same bioreactor. Next-generation biorefineries based on simultaneous enzymatic biotransformation and microbial fermentation could address the food, biofuels, and environment trilemma. PMID:23589840

  3. A Classroom Demonstration of Rayleigh Light Scattering in Optically Active and Inactive Systems.

    ERIC Educational Resources Information Center

    Pecina, Monica Avalos; Smith, Charles A.

    1999-01-01

    Argues that the concept of optical activity is vague to students because it is difficult for instructors to demonstrate the phenomenon in the classroom. Presents a demonstration that allows students to observe and manipulate the optical path of polarized light through optically inactive and active solutions. (CCM)

  4. Past-Year Sexual Inactivity among Older Married Persons and Their Partners

    ERIC Educational Resources Information Center

    Karraker, Amelia; DeLamater, John

    2013-01-01

    Family scholars have focused on the onset of sexual activity early in the life course, but little is known about the cessation of sexual activity in relationships in later life. We use event-history analysis techniques and logistic regression to identify the correlates of sexual inactivity among older married men and women. We analyze data for…

  5. Artist-Teachers' In-Action Mental Models While Teaching Visual Arts

    ERIC Educational Resources Information Center

    Russo-Zimet, Gila

    2017-01-01

    Studies have examined the assumption that teachers have previous perceptions, beliefs and knowledge about learning (Cochran-Smith & Villegas, 2015). This study presented the In-Action Mental Model of twenty leading artist-teachers while teaching Visual Arts in three Israeli art institutions of higher Education. Data was collected in two…

  6. Variables associated with active and inactive behavior during the after-school period.

    PubMed

    Wickel, Eric

    2013-05-01

    This study analyzed time-use interviews to report levels of active and inactive behavior during the after-school period (3-6 pm). Interviews were conducted on random days from three separate seasons during third and fourth grade. Youth with at least two interviews during third (356 completed 2 interviews; 506 completed 3 interviews [9 yrs; 50% boys]) and fourth (186 completed 2 interviews; 768 completed 3 interviews [10 yrs; 50% boys]) grade were included to report levels of moderate-to-vigorous physical activity (MVPA), light physical activity, inactive screen time, inactive nonscreen time, and travel by location and who the activity was undertaken with. Reporting time outside the home and with peers (single or group) was related to higher levels of MVPA. While inside the home, screen and nonscreen proportions were comparable (38% and 40%, respectively), despite unique patterns (screen: boys > girls; nonscreen: girls > boys). Reporting time with both parents was associated with more nonscreen time; whereas reporting time with peer groups was associated with lower screen time. Understanding active and inactive patterns of children's behavior outside of school hours can be very important in contributing toward the development of innovative interventions for increasing physical activity.

  7. Past-Year Sexual Inactivity among Older Married Persons and Their Partners

    ERIC Educational Resources Information Center

    Karraker, Amelia; DeLamater, John

    2013-01-01

    Family scholars have focused on the onset of sexual activity early in the life course, but little is known about the cessation of sexual activity in relationships in later life. We use event-history analysis techniques and logistic regression to identify the correlates of sexual inactivity among older married men and women. We analyze data for…

  8. Teacher Educators' In-Action Mental Models in Different Teaching Situations

    ERIC Educational Resources Information Center

    Mevorach, Miriam; Strauss, Sidney

    2012-01-01

    In previous studies on teachers' cognition, we discovered that teachers' teaching can be described via a general in-action mental model (IAMM) concerning the structure of the mind and the roles of teaching in fostering children's learning. The purpose of our study was to examine teacher educators' IAMM regarding student teachers' minds and…

  9. Exploring Socio-Ecological Factors Influencing Active and Inactive Spanish Students in Years 12 and 13

    ERIC Educational Resources Information Center

    Devís-Devís, José; Beltrán-Carrillo, Vicente J.; Peiró-Velert, Carmen

    2015-01-01

    This paper explores socio-ecological factors and their interplay that emerge from a qualitative study and influence adolescents' physical activity and sport participation. A total of 13 boys and 7 girls active and inactive adolescents, from years 12 and 13 and different types of school (state and private), participated in semi-structured…

  10. 9 CFR 105.4 - Termination of licenses and permits for inactivity.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Termination of licenses and permits for inactivity. 105.4 Section 105.4 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND...

  11. 9 CFR 105.4 - Termination of licenses and permits for inactivity.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Termination of licenses and permits for inactivity. 105.4 Section 105.4 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND...

  12. Results of myringoplasty operations in active and inactive ears in adults.

    PubMed

    Mills, Robert; Thiel, Gundula; Mills, Nadtaya

    2013-09-01

    To determine whether the presence of aural discharge at the time of surgery adversely affects the success rate of myringoplasty operations. Case series comparing the success rate of surgery in active and inactive ears. Data pertaining to 268 operations involving repair of the tympanic membrane without ossiculoplasty carried out in Edinburgh, United Kingdom, between 1999 and 2009 by one senior surgeon and trainees working under his supervision were collected prospectively. Other factors that might potentially influence the outcome of the surgery were investigated using logistical regression analysis. The main outcome measure was number of patients with an intact tympanic membrane 6 months after surgery in the two groups (active and inactive). Of the 268 operations carried out, 203 were successful, with an intact tympanic membrane, 6 months postoperatively, 43 had persistent perforations, and 22 patients were lost to follow-up before 6 months. The success rates for closure of the perforation at 6 months after surgery were 83% for inactive and 82% for active ears (P = .9). There is no clinically significant difference in the success rate for myringoplasty in patients whose ears are active or inactive at the time of surgery. Copyright © 2013 The American Laryngological, Rhinological and Otological Society, Inc.

  13. Effects of inactivity on myosin heavy chain composition and size of rat soleus fibers

    NASA Technical Reports Server (NTRS)

    Grossman, E. J.; Roy, R. R.; Talmadge, R. J.; Zhong, H.; Edgerton, V. R.

    1998-01-01

    Myosin heavy chain (MHC) and fiber size properties of the adult rat soleus were determined after 4-60 days of complete inactivity, i.e., lumbar spinal cord isolation. Soleus atrophy was rapid and progressive, i.e., 25% and 64% decrease in weight and 33% and 75% decrease in fiber size after 4 and 60 days of inactivity, respectively. Changes in MHC occurred at a slower rate than the atrophic response. After 15 days there was de novo expression of type IIx MHC (approximately 10%). By 60 days, type IIx MHC accounted for 33% of the total MHC content, and 7% of the fibers contained only type IIx MHC. The relative amount of type I MHC was reduced from 93% in control to 49% after 60 days of inactivity. Therefore, the effects of 60 days of inactivity suggest that during this time period at least 75% of fiber size and approximately 40% of type I MHC composition of the adult rat soleus can be attributed to activation-related events.

  14. Low cardiorespiratory fitness and physical inactivity as predictors of mortality in men with type 2 diabetes.

    PubMed

    Wei, M; Gibbons, L W; Kampert, J B; Nichaman, M Z; Blair, S N

    2000-04-18

    Although physical activity is recommended as a basic treatment for patients with diabetes, its long-term association with mortality in these patients is unknown. To evaluate the association of low cardiorespiratory fitness and physical inactivity with mortality in men with type 2 diabetes. Prospective cohort study. Preventive medicine clinic. 1263 men (50+/-10 years of age) with type 2 diabetes who received a thorough medical examination between 1970 and 1993 and were followed for mortality up to 31 December 1994. Cardiorespiratory fitness measured by a maximal exercise test, self-reported physical inactivity at baseline, and subsequent death determined by using the National Death Index. During an average follow-up of 12 years, 180 patients died. After adjustment for age, baseline cardiovascular disease, fasting plasma glucose level, high cholesterol level, overweight, current smoking, high blood pressure, and parental history of cardiovascular disease, men in the low-fitness group had an adjusted risk for all-cause mortality of 2.1 (95% CI, 1.5 to 2.9) compared with fit men. Men who reported being physically inactive had an adjusted risk for mortality that was 1.7-fold (CI, 1.2-fold to 2.3-fold) higher than that in men who reported being physically active. Low cardiorespiratory fitness and physical inactivity are independent predictors of all-cause mortality in men with type 2 diabetes. Physicians should encourage patients with type 2 diabetes to participate in regular physical activity and improve cardiorespiratory fitness.

  15. 9 CFR 105.4 - Termination of licenses and permits for inactivity.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Termination of licenses and permits for inactivity. 105.4 Section 105.4 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS...

  16. 9 CFR 105.4 - Termination of licenses and permits for inactivity.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Termination of licenses and permits for inactivity. 105.4 Section 105.4 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS...

  17. 9 CFR 105.4 - Termination of licenses and permits for inactivity.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Termination of licenses and permits for inactivity. 105.4 Section 105.4 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS...

  18. Short term clinical effect of active and inactive Salvadora persica miswak on dental plaque and gingivitis.

    PubMed

    Sofrata, Abier; Brito, Fernanda; Al-Otaibi, Meshari; Gustafsson, Anders

    2011-10-11

    Salvadora persica shrub has been used traditionally in folk medicine for different medical condition treatments. The habitual use of Salvadora persica roots (chewing sticks) for dental hygiene is still wildly spread throughout parts of Asia, Africa, and Middle. It is one of the most important species with its reported strong antibacterial, antifungal, and antiviral effects. Mechanical removal of dental plaque is regarded as an effective mean of controlling progression of periodontal disease. To evaluate the effect of active and inactive miswak on dental plaque, subgingival microbiota and gingival inflammation in patients with gingivitis. In this double blinded randomized controlled trial 68 gingivitis patients were randomly assigned to either active or inactive miswak group, and were instructed to use only issued miswaks for oral hygiene during 3 weeks experimental period. Registration of plaque, gingival inflammation, and plaque samples were taken at baseline and on completion of the study. Plaque samples were analyzed by DNA-DNA hybridization technique. Active miswak significantly reduced dental plaque (p = 0.007). There were no differences between active and inactive miswak in reduction of approximal plaque and composition of subgingival microbiota. Miswak has an overall effect on dental plaque and gingival inflammation scores. Similar results were achieved by active and inactive miswak in difficult to reach areas, indicating miswak has limited chemical effects on this study population. Therefore, miswak can be used as a dental hygiene method in conjunction with interproximal cleaning aides. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  19. 38 CFR 3.375 - Determination of inactivity (complete arrest) in tuberculosis.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... inactivity (complete arrest) in tuberculosis. 3.375 Section 3.375 Pensions, Bonuses, and Veterans' Relief...) in tuberculosis. (a) Pulmonary tuberculosis. A veteran shown to have had pulmonary tuberculosis will...) Nonpulmonary disease. Determination of complete arrest of nonpulmonary tuberculosis requires absence of...

  20. 38 CFR 3.375 - Determination of inactivity (complete arrest) in tuberculosis.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... inactivity (complete arrest) in tuberculosis. 3.375 Section 3.375 Pensions, Bonuses, and Veterans' Relief...) in tuberculosis. (a) Pulmonary tuberculosis. A veteran shown to have had pulmonary tuberculosis will...) Nonpulmonary disease. Determination of complete arrest of nonpulmonary tuberculosis requires absence of...