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Sample records for enzymatically inactive recombinant

  1. Enzymatically Inactive Procaspase 1 stabilizes the ASC Pyroptosome and Supports Pyroptosome Spreading during Cell Division.

    PubMed

    Stein, Robert; Kapplusch, Franz; Heymann, Michael Christian; Russ, Susanne; Staroske, Wolfgang; Hedrich, Christian Michael; Rösen-Wolff, Angela; Hofmann, Sigrun Ruth

    2016-08-26

    Caspase-1 is a key player during the initiation of pro-inflammatory innate immune responses, activating pro-IL-1β in so-called inflammasomes. A subset of patients with recurrent febrile episodes and systemic inflammation of unknown origin harbor mutations in CASP1 encoding caspase-1. CASP1 variants result in reduced enzymatic activity of caspase-1 and impaired IL-1β secretion. The apparent paradox of reduced IL-1β secretion but systemic inflammation led to the hypothesis that CASP1 mutations may result in variable protein interaction clusters, thus activating alternative signaling pathways. To test this hypothesis, we established and characterized an in vitro system of transduced immortalized murine macrophages expressing either WT or enzymatically inactive (p.C284A) procaspase-1 fusion reporter proteins. Macrophages with variant p.C284A caspase-1 did not secrete IL-1β and exhibited reduced inflammatory cell death, referred to as pyroptosis. Caspase-1 and apoptosis-associated speck-like protein containing a CARD (ASC) formed cytosolic macromolecular complexes (so-called pyroptosomes) that were significantly increased in number and size in cells carrying the p.C284A caspase-1 variant compared with WT caspase-1. Furthermore, enzymatically inactive caspase-1 interacted with ASC longer and with increased intensity compared with WT caspase-1. Applying live cell imaging, we documented for the first time that pyroptosomes containing enzymatically inactive variant p.C284A caspase-1 spread during cell division. In conclusion, variant p.C284A caspase-1 stabilizes pyroptosome formation, potentially enhancing inflammation by two IL-1β-independent mechanisms: pyroptosomes convey an enhanced inflammatory stimulus through the recruitment of additional proteins (such as RIP2, receptor interacting protein kinase 2), which is further amplified through pyroptosome and cell division. PMID:27402835

  2. Expression of Enzymatically Inactive Wasp Venom Phospholipase A1 in Pichia pastoris

    PubMed Central

    Borodina, Irina; Jensen, Bettina M.; Wagner, Tim; Hachem, Maher A.; Søndergaard, Ib; Poulsen, Lars K.

    2011-01-01

    Wasp venom allergy is the most common insect venom allergy in Europe. It is manifested by large local reaction or anaphylactic shock occurring after a wasp sting. The allergy can be treated by specific immunotherapy with whole venom extracts. Wasp venom is difficult and costly to obtain and is a subject to composition variation, therefore it can be advantageous to substitute it with a cocktail of recombinant allergens. One of the major venom allergens is phospholipase A1, which so far has been expressed in Escherichia coli and in insect cells. Our aim was to produce the protein in secreted form in yeast Pichia pastoris, which can give high yields of correctly folded protein on defined minimal medium and secretes relatively few native proteins simplifying purification. Residual amounts of enzymatically active phospholipase A1 could be expressed, but the venom protein had a deleterious effect on growth of the yeast cells. To overcome the problem we introduced three different point mutations at the critical points of the active site, where serine137, aspartate165 or histidine229 were replaced by alanine (S137A, D165A and H229A). All the three mutated forms could be expressed in P. pastoris. The H229A mutant did not have any detectable phospholipase A1 activity and was secreted at the level of several mg/L in shake flask culture. The protein was purified by nickel-affinity chromatography and its identity was confirmed by MALDI-TOF mass spectrometry. The protein could bind IgE antibodies from wasp venom allergic patients and could inhibit the binding of wasp venom to IgE antibodies specific for phospholipase A1 as shown by Enzyme Allergo-Sorbent Test (EAST). Moreover, the recombinant protein was allergenic in a biological assay as demonstrated by its capability to induce histamine release of wasp venom-sensitive basophils. The recombinant phospholipase A1 presents a good candidate for wasp venom immunotherapy. PMID:21731687

  3. Expression of enzymatically inactive wasp venom phospholipase A1 in Pichia pastoris.

    PubMed

    Borodina, Irina; Jensen, Bettina M; Wagner, Tim; Hachem, Maher A; Søndergaard, Ib; Poulsen, Lars K

    2011-01-01

    Wasp venom allergy is the most common insect venom allergy in Europe. It is manifested by large local reaction or anaphylactic shock occurring after a wasp sting. The allergy can be treated by specific immunotherapy with whole venom extracts. Wasp venom is difficult and costly to obtain and is a subject to composition variation, therefore it can be advantageous to substitute it with a cocktail of recombinant allergens. One of the major venom allergens is phospholipase A1, which so far has been expressed in Escherichia coli and in insect cells. Our aim was to produce the protein in secreted form in yeast Pichia pastoris, which can give high yields of correctly folded protein on defined minimal medium and secretes relatively few native proteins simplifying purification.Residual amounts of enzymatically active phospholipase A1 could be expressed, but the venom protein had a deleterious effect on growth of the yeast cells. To overcome the problem we introduced three different point mutations at the critical points of the active site, where serine137, aspartate165 or histidine229 were replaced by alanine (S137A, D165A and H229A). All the three mutated forms could be expressed in P. pastoris. The H229A mutant did not have any detectable phospholipase A1 activity and was secreted at the level of several mg/L in shake flask culture. The protein was purified by nickel-affinity chromatography and its identity was confirmed by MALDI-TOF mass spectrometry. The protein could bind IgE antibodies from wasp venom allergic patients and could inhibit the binding of wasp venom to IgE antibodies specific for phospholipase A1 as shown by Enzyme Allergo-Sorbent Test (EAST). Moreover, the recombinant protein was allergenic in a biological assay as demonstrated by its capability to induce histamine release of wasp venom-sensitive basophils.The recombinant phospholipase A1 presents a good candidate for wasp venom immunotherapy. PMID:21731687

  4. Active and Inactive Transplacement of the M26 Recombination Hotspot in Schizosaccharomyces Pombe

    PubMed Central

    Virgin, J. B.; Metzger, J.; Smith, G. R.

    1995-01-01

    The ade6-M26 mutation of the fission yeast Schizosaccharomyces pombe creates a meiotic recombination hotspot that elevates ade6 intragenic recombination ~10-15-fold. A heptanucleotide sequence including the M26 point mutation is required but not sufficient for hotspot activity. We studied the effects of plasmid and chromosomal context on M26 hotspot activity. The M26 hotspot was inactive on a multicopy plasmid containing M26 embedded within 3.0 or 5.9 kb of ade6 DNA. Random S. pombe genomic fragments totaling ~7 Mb did not activate the M26 hotspot on a plasmid. M26 hotspot activity was maintained when 3.0-, 4.4-, and 5.9-kb ade6-M26 DNA fragments, with various amounts of non-S. pombe plasmid DNA, were integrated at the ura4 chromosomal locus, but only in certain configurations relative to the ura4 gene and the cointegrated plasmid DNA. Several integrations created new M26-independent recombination hotspots. In all cases the non-ade6 DNA was located >1 kb from the M26 site, and in some cases >2 kb. Because the chromosomal context effect was transmitted over large distances, and did not appear to be mediated by a single discrete DNA sequence element, we infer that the local chromatin structure has a pronounced effect on M26 hotspot activity. PMID:8536980

  5. [Enzymatic control of homologous recombination in Escherichia coli cells and hyper-recombination].

    PubMed

    Bakhlanova, I V; Dudkina, A V; Baĭtin, D M

    2013-01-01

    The RecA protein is a major enzyme of homologous recombination in bacterial cell. Forming a right-handed helical filament on ssDNA, it provides a homology search between two DNA molecules and homologous strand exchange. The RecA protein not only defends the cell from exposure to ionizing radiation and UV-irradiation, but also ensures the recombination process in the course of normal cell growth. A number of wild-type or mutant RecA proteins demonstrate increased recombinogenic properties in vitro and in vivo as compared with the wild-type RecA protein from Escherichia coli, which leads to hyper-recombination. The hyper-rec activity of RecA proteins during the recombination process in many depends on the filamentation dynamics on ssDNA and DNA-transferase properties. Changes in filamentation and DNA-transferase abilities of RecA protein may be the result of not only specific amino-acid substitutions, but also the functioning of the cell enzymatic apparatus, including such proteins as RecO, RecR, RecF, RecX, DinI, SSB, PsiB. To date, the function of each of these proteins is identified at the molecular level. However, the role of some of them in the cell metabolism remains to be seen. Increase in recombination in vivo is not always useful for a cell and faces various limitations. Moreover, in the bacterial cell some mechanisms are activated, that cause genomic reorganization, directed to suppress the expression of hyper-active RecA protein. The ways of hyper-active RecA protein regulation are very interesting, and they are studied in different model systems. PMID:23808153

  6. 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. PMID:25914160

  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. Generation of Functional RNAs from Inactive Oligonucleotide Complexes by Non-enzymatic Primer Extension

    PubMed Central

    2014-01-01

    The earliest genomic RNAs had to be short enough for efficient replication, while simultaneously serving as unfolded templates and effective ribozymes. A partial solution to this paradox may lie in the fact that many functional RNAs can self-assemble from multiple fragments. Therefore, in early evolution, genomic RNA fragments could have been significantly shorter than unimolecular functional RNAs. Here, we show that unstable, nonfunctional complexes assembled from even shorter 3′-truncated oligonucleotides can be stabilized and gain function via non-enzymatic primer extension. Such short RNAs could act as good templates due to their minimal length and complex-forming capacity, while their minimal length would facilitate replication by relatively inefficient polymerization reactions. These RNAs could also assemble into nascent functional RNAs and undergo conversion to catalytically active forms, by the same polymerization chemistry used for replication that generated the original short RNAs. Such phenomena could have substantially relaxed requirements for copying efficiency in early nonenzymatic replication systems. PMID:25521912

  9. 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

  10. Overexpression and Enzymatic Assessment of Antigenic Fragments of Hyaluronidase Recombinant Protein From Streptococcus pyogenes

    PubMed Central

    Sadoogh Abbasian, Shabnam; Ghaznavi Rad, Ehsanollah; Akbari, Neda; Zolfaghari, Mohammad Reza; pakzad, Iraj; Abtahi, Hamid

    2014-01-01

    Background: Hyaluronidase catalyzes the hydrolysis of hyaluronan polymers to N-acetyl-D-glucosamine and D-glucuronic acid. This enzyme is a dimer of identical subunits. Hyaluronidase has different pharmaceutical and medical applications. Previously, we produced a recombinant hyaluronidase antigenic fragment of Streptococcus pyogenes. Objectives: This study aimed to improve the protein production and purity of hyaluronidase recombinant protein from S. pyogenes. In addition, the enzymatic activity of this protein was investigated. Materials and Methods: The expression of hyaluronidase antigenic fragments was optimized using IPTG concentration, time of induction, temperature, culture, and absorbance of 0.6-0.8-1 at 600 nm. Afterwards, the expressed proteins were purified and the enzymatic activity was assessed by turbid metric method. Results: Data indicated that maximum protein is produced in OD = 0.8, 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), 37ºC, NB 1.5x, without glucose, incubated for overnight. The enzymatic activity of the recombinant protein was similar to the commercial form of hyaluronidase. Conclusions: The results showed that an antigenic fragment of the recombinant hyaluronidase protein from S. pyogenes has a considerable enzymatic activity. It can be suggested to use it for medical purposes. In addition, applications of bioinformatics software would facilitate the production of a smaller protein with same antigenic properties and enzymatic activity. PMID:25789122

  11. 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

  12. Expression studies on the ba3 quinol oxidase from Paracoccus denitrificans. A bb3 variant is enzymatically inactive.

    PubMed

    Zickermann, I; Tautu, O S; Link, T A; Korn, M; Ludwig, B; Richter, O M

    1997-06-15

    Expression of the quinol oxidase from Paracoccus denitrificans has been examined using a polyclonal antibody directed against subunit II and a promoter probe vector carrying the promoter region of the qox operon. Under aerobic conditions nitrate and nitrite act as specific inducers of the expression. To obtain an enzymatically competent quinol oxidase complex, an intact ctaB gene is required, which constitutes part of the cta operon coding for the aa3 cytochrome c oxidase of P. denitrificans. Deletion of ctaB leads to a change in heme composition of the quinol oxidase with heme b replacing the high-spin heme a of the binuclear center, causing loss of electron transport activity. PMID:9219517

  13. 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.

  14. 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.

  15. 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. PMID:27199917

  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. Iron inhibits activation-induced cytidine deaminase enzymatic activity and modulates immunoglobulin class switch DNA recombination.

    PubMed

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

    2012-06-15

    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 (Fe(2+), ferrous) suppressed CSR, leading to decreased number of switched B cells, decreased postrecombination Iμ-C(H) 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 I(H)-S-C(H) transcription. Fe(2+) 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 Fe(2+) was specific, as shown by lack of inhibition of AID-mediated dC deamination by other bivalent metal ions, such as Zn(2+), Mn(2+), Mg(2+), or Ni(2+), and the inability of Fe(2+) 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.

  18. 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.

  19. 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.

  20. [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.

  1. 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. PMID:25217555

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

    PubMed

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

    2014-12-01

    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.

  3. The 67-kD elastin/laminin-binding protein is related to an enzymatically inactive, alternatively spliced form of beta-galactosidase.

    PubMed Central

    Hinek, A; Rabinovitch, M; Keeley, F; Okamura-Oho, Y; Callahan, J

    1993-01-01

    We and others have previously shown that a 67-kD cell surface elastin/laminin-binding protein (EBP) is responsible for cell adhesion to elastin and laminin and for mediating the process of elastin fiber assembly, but the nature of this protein was unknown. In this report we provide evidence that a 67-kD catalytically inactive form of beta-galactosidase produced by alternative splicing demonstrates immunological and functional similarity and sequence homology to the 67-kD EBP, suggesting that the two might be the same. Antibody prepared to a synthetic peptide, N-Ac-GSPSAQDEASPL, corresponding to a frame-shift-generated sequence unique to the alternatively spliced form of human beta-galactosidase, also recognized sheep EBP both on Western blotting and in aortic tissue. Furthermore, this synthetic peptide (S-GAL) binds to elastin and laminin, but not to fibronectin, collagen I, or collagen III. Moreover, both tropoelastin and laminin which bind to S-GAL peptide affinity columns can be specifically eluted from them with an excess of free S-GAL peptides. In addition, sequence homology among this splice variant of human beta-galactosidase, sheep EBP, and NH2-terminal sequences of some elastases suggests that these proteins share a common ligand-binding motif that has not been previously recognized. Images PMID:8383699

  4. Enzymatic activity and substrate specificity of the recombinant tomato β-galactosidase 1.

    PubMed

    Eda, Masahiro; Ishimaru, Megumi; Tada, Toshiji; Sakamoto, Tatsuji; Kotake, Toshihisa; Tsumuraya, Yoichi; Mort, Andrew J; Gross, Kenneth C

    2014-10-15

    The open reading frame of tomato β-galactosidase 1 was expressed in yeast, and the enzymatic properties and substrate specificity were investigated. The enzyme had peak activity at pH 5.0 and 40-50°C. TBG1 was active on β-(1,3)- and β-(1,6)-galactobiose and lactose. TBG1 released galactose from lupin galactan, tomato fruit alkali soluble pectin, arabinogalactan, gum arabic and methyl β-(1,6)-galactohexaoside, but not from labeled β-(1,4)-galactoheptaose. TBG1 was assessed for its ability to degrade three galactosyl-containing cell wall fractions purified from different development and ripening stages of tomato fruit. TBG1 released galactose from all of the fractions from all of the stages tested. TBG1 activity was highest on the hemicellulose fraction at the 10 and 20d after pollination stage. This result is not correlated the with TBG1 expression pattern. TBG1 might act on a small but specific set of polysaccharide containing galactose.

  5. Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei

    PubMed Central

    Liu, Peng; Robbins, Arthur H.; Marzahn, Melissa R.; McClung, Scott H.; Yowell, Charles A.; Stevens, Stanley M.; Dame, John B.; Dunn, Ben M.

    2015-01-01

    The rodent malaria parasite Plasmodium berghei is a practical model organism for experimental studies of human malaria. Plasmepsins are a class of aspartic proteinase isoforms that exert multiple pathological effects in malaria parasites. Plasmepsins residing in the food vacuole (FV) of the parasite hydrolyze hemoglobin in red blood cells. In this study, we cloned PbPM4, the FV plasmepsin gene of P. berghei that encoded an N-terminally truncated pro-segment and the mature enzyme from genomic DNA. We over-expressed this PbPM4 zymogen as inclusion bodies (IB) in Escherichia coli, and purified the protein following in vitro IB refolding. Auto-maturation of the PbPM4 zymogen to mature enzyme was carried out at pH 4.5, 5.0, and 5.5. Interestingly, we found that the PbPM4 zymogen exhibited catalytic activity regardless of the presence of the pro-segment. We determined the optimal catalytic conditions for PbPM4 and studied enzyme kinetics on substrates and inhibitors of aspartic proteinases. Using combinatorial chemistry-based peptide libraries, we studied the active site preferences of PbPM4 at subsites S1, S2, S3, S1’, S2’ and S3’. Based on these results, we designed and synthesized a selective peptidomimetic compound and tested its inhibition of PbPM4, seven FV plasmepsins from human malaria parasites, and human cathepsin D (hcatD). We showed that this compound exhibited a >10-fold selectivity to PbPM4 and human malaria parasite plasmepsin 4 orthologs versus hcatD. Data from this study furthesr our understanding of enzymatic characteristics of the plasmepsin family and provides leads for anti-malarial drug design. PMID:26510189

  6. Recombinant Cyclodextrinase from Thermococcus kodakarensis KOD1: Expression, Purification, and Enzymatic Characterization

    PubMed Central

    Lv, Xiaomin

    2015-01-01

    A gene encoding a cyclodextrinase from Thermococcus kodakarensis KOD1 (CDase-Tk) was identified and characterized. The gene encodes a protein of 656 amino acid residues with a molecular mass of 76.4 kDa harboring four conserved regions found in all members of the α-amylase family. A recombinant form of the enzyme was purified by ion-exchange chromatography, and its catalytic properties were examined. The enzyme was active in a broad range of pH conditions (pHs 4.0–10.0), with an optimal pH of 7.5 and a temperature optimum of 65°C. The purified enzyme preferred to hydrolyze β-cyclodextrin (CD) but not α- or γ-CD, soluble starch, or pullulan. The final product from β-CD was glucose. The Vmax and Km values were 3.13 ± 0.47 U mg−1 and 2.94 ± 0.16 mg mL−1 for β-CD. The unique characteristics of CDase-Tk with a low catalytic temperature and substrate specificity are discussed, and the starch utilization pathway in a broad range of temperatures is also proposed. PMID:25688178

  7. Vaccinia virus B1 kinase: phenotypic analysis of temperature-sensitive mutants and enzymatic characterization of recombinant proteins.

    PubMed Central

    Rempel, R E; Traktman, P

    1992-01-01

    The vaccinia virus B1 gene encodes a 34-kDa protein with homology to protein kinases. In L cells infected nonpermissively with mutants containing lesions in the B1 gene (ts2 and ts25), the infectious cycle arrests prior to DNA replication. In this report, we demonstrate that DNA synthesis ceases when cultures infected with these mutants at 32 degrees C are shifted to the nonpermissive temperature (39.5 degrees C) in the midst of DNA replication. We also show that B1 protein is synthesized transiently during the early phase of infection, even when the progression to later stages of gene expression is prevented. Although wild-type (wt) B1 is stable, the ts B1 proteins are markedly labile in both L and BSC40 cells at both permissive and nonpermissive temperatures. These results suggest that the ts phenotype of the mutants is complex and may in part reflect a temperature-dependent requirement for kinase activity, an induction of temperature sensitivity in B1 substrates under nonpermissive conditions, and/or ts complementation by host factors. To facilitate biochemical analyses, recombinant wt B1, ts2 B1, and ts25 B1 were produced in Escherichia coli. The wt protein was able to phosphorylate serine and threonine residues on several exogenous substrates in vitro. The activity of ts25 B1 was 3% that of the wt enzyme, and no detectable kinase activity was associated with ts2 B1. In light of the inactivity of the ts2 B1 protein in vitro and its extreme lability in vivo, we attempted to isolate a vaccinia virus B1 null mutant by targeted interruption of the B1 gene at 32 degrees C. No null mutants were isolated. These results indicate that the B1 protein kinase provides a vital function which cannot be supplied by the host or circumvented by incubation at 32 degrees C. Images PMID:1602551

  8. 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.

  9. 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.

  10. 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.

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

    DOE PAGESBeta

    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.; et al

    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

  12. 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.

  13. 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

    2014-06-27

    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 × 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.

  14. 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

  15. 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

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

    PubMed

    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

  17. 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

  18. Expression and Characterization of Recombinant, Tetrameric and Enzymatically Active Influenza Neuraminidase for the Setup of an Enzyme-Linked Lectin-Based Assay

    PubMed Central

    Prevato, Marua; Ferlenghi, Ilaria; Bonci, Alessandra; Uematsu, Yasushi; Anselmi, Giulia; Giusti, Fabiola; Bertholet, Sylvie; Legay, Francois; Telford, John Laird; Settembre, Ethan C.; Maione, Domenico; Cozzi, Roberta

    2015-01-01

    Developing a universal influenza vaccine that induces broad spectrum and longer-term immunity has become an important potentially achievable target in influenza vaccine research and development. Hemagglutinin (HA) and neuraminidase (NA) are the two major influenza virus antigens. Although antibody responses against influenza virus are mainly directed toward HA, NA is reported to be more genetically stable; hence NA-based vaccines have the potential to be effective for longer time periods. NA-specific immunity has been shown to limit the spread of influenza virus, thus reducing disease symptoms and providing cross-protection against heterosubtypic viruses in mouse challenge experiments. The production of large quantities of highly pure and stable NA could be beneficial for the development of new antivirals, subunit-based vaccines, and novel diagnostic tools. In this study, recombinant NA (rNA) was produced in mammalian cells at high levels from both swine A/California/07/2009 (H1N1) and avian A/turkey/Turkey/01/2005 (H5N1) influenza viruses. Biochemical, structural, and immunological characterizations revealed that the soluble rNAs produced are tetrameric, enzymatically active and immunogenic, and finally they represent good alternatives to conventionally used sources of NA in the Enzyme-Linked Lectin Assay (ELLA). PMID:26280677

  19. Targeting Inactive Enzyme Conformation

    PubMed Central

    Liu, Sijiu; Zeng, Li-Fan; Wu, Li; Yu, Xiao; Xue, Ting; Gunawan, Andrea M.; Ya-Qiu, Long; Zhang, Zhong-Yin

    2009-01-01

    There has been considerable interest in protein tyrosine phosphatase 1B (PTP1B) as a therapeutic target for diabetes, obesity, as well as cancer. Identifying inhibitory compounds with good bioavailability is a major challenge of drug discovery programs targeted toward PTPs. Most current PTP active site-directed pharmacophores are negatively charged pTyr mimetics which cannot readily enter the cell. This lack of cell permeability limits the utility of such compounds in signaling studies and further therapeutic development. We identify aryl diketoacids as novel pTyr surrogates and show that neutral amide-linked aryl diketoacid dimers also exhibit excellent PTP inhibitory activity. Kinetic studies establish that these aryl diketoacid derivatives act as noncompetitive inhibitors of PTP1B. Crystal structures of ligand-bound PTP1B reveal that both the aryl diketoacid and its dimeric derivative bind PTP1B at the active site, albeit with distinct modes of interaction, in the catalytically inactive, WPD loop open conformation. Furthermore, dimeric aryl diketoacids are cell permeable and enhance insulin signaling in hepatoma cells, suggesting that targeting the inactive conformation may provide a unique opportunity for creating active site-directed PTP1B inhibitors with improved pharmacological properties. PMID:19012396

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. Further evidence for involvement of a noncanonical function of uracil DNA glycosylase in class switch recombination.

    PubMed

    Begum, Nasim A; Stanlie, Andre; Doi, Tomomitsu; Sasaki, Yoko; Jin, Hai Wei; Kim, Yong Sung; Nagaoka, Hitoshi; Honjo, Tasuku

    2009-02-24

    Activation-induced cytidine deaminase (AID) introduces DNA cleavage in the Ig gene locus to initiate somatic hypermutation (SHM) and class switch recombination (CSR) in B cells. The DNA deamination model assumes that AID deaminates cytidine (C) on DNA and generates uridine (U), resulting in DNA cleavage after removal of U by uracil DNA glycosylase (UNG). Although UNG deficiency reduces CSR efficiency to one tenth, we reported that catalytically inactive mutants of UNG were fully proficient in CSR and that several mutants at noncatalytic sites lost CSR activity, indicating that enzymatic activity of UNG is not required for CSR. In this report we show that CSR activity by many UNG mutants critically depends on its N-terminal domain, irrespective of their enzymatic activities. Dissociation of the catalytic and CSR activity was also found in another UNG family member, SMUG1, and its mutants. We also show that Ugi, a specific peptide inhibitor of UNG, inhibits CSR without reducing DNA cleavage of the S (switch) region, confirming dispensability of UNG in DNA cleavage in CSR. It is therefore likely that UNG is involved in a repair step after DNA cleavage in CSR. Furthermore, requirement of the N terminus but not enzymatic activity of UNG mutants for CSR indicates that the UNG protein structure is critical. The present findings support our earlier proposal that CSR depends on a noncanonical function of the UNG protein (e.g., as a scaffold for repair enzymes) that might be required for the recombination reaction after DNA cleavage.

  5. 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…

  6. 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...

  7. 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…

  8. Health and economic costs of physical inactivity.

    PubMed

    Kruk, Joanna

    2014-01-01

    Physical inactivity has reached epidemic levels in developed countries and is being recognized as a serious public health problem. Recent evidence shows a high percentages of individuals worldwide who are physically inactive, i.e. do not achieve the WHO's present recommendation of 150 minutes of moderate to vigorous intensity per week in addition to usual activities. Living in sedentary lifestyle is one of the leading causes of deaths and a high risk factor for several chronic diseases, like cancer, cardiovascular disease, diabetes type 2, and osteoporosis. This article summarizes evidence for relative risk of the civilization diseases attributable to physical inactivity and the most important conclusions available from the recent investigations computing the economic costs specific to physical inactivity. The findings provide health and economic arguments needed for people to understand the meaning of a sedentary lifestyle. This may be also useful for public health policy in the creation of programmes for prevention of physical inactivity.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. Enzyme Inhibition by Allosteric Capture of an Inactive Conformation

    PubMed Central

    Lee, Gregory M.; Shahian, Tina; Baharuddin, Aida; Gable, Jonathan E.; Craik, Charles S.

    2011-01-01

    All members of the human herpesvirus protease family are active as weakly associating dimers, but inactive as monomers. A small molecule allosteric inhibitor of Kaposi’s sarcoma-associated herpesvirus protease (KSHV Pr) traps the enzyme in an inactive monomeric state where the C-terminal helices are unfolded and the hydrophobic dimer interface is exposed. NMR titration studies demonstrate that the inhibitor binds to KSHV Pr monomers with low μM affinity. A 2.0 Å resolution X-ray crystal structure of a C-terminal truncated KSHV Pr-inhibitor complex locates the binding pocket at the dimer interface and displays significant conformational perturbations at the active site, 15 Å from the allosteric site. NMR and CD data suggest that the small molecule inhibits human cytomegalovirus protease (HCMV Pr) via a similar mechanism. As all HHV proteases are functionally and structurally homologous, the inhibitor represents a class of compounds that may be developed into broad-spectrum therapeutics which allosterically regulate enzymatic activity by disrupting protein-protein interactions. PMID:21723875

  11. 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

  12. 29 CFR 1404.6 - Inactive status.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Arbitrators; Admission and Retention § 1404.6 Inactive status. A member of the Roster who continues to meet... longer than two (2) years and the arbitrator has not paid the annual listing fee, the arbitrator will... heath, vacation, schedule or other reasons. (b) Arbitrators whose schedules do not permit cases to...

  13. 24 CFR 214.200 - Inactive status.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 24 Housing and Urban Development 2 2012-04-01 2012-04-01 false Inactive status. 214.200 Section 214.200 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued... DEVELOPMENT MORTGAGE AND LOAN INSURANCE PROGRAMS UNDER NATIONAL HOUSING ACT AND OTHER AUTHORITIES...

  14. 24 CFR 214.200 - Inactive status.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 24 Housing and Urban Development 2 2011-04-01 2011-04-01 false Inactive status. 214.200 Section 214.200 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued... DEVELOPMENT MORTGAGE AND LOAN INSURANCE PROGRAMS UNDER NATIONAL HOUSING ACT AND OTHER AUTHORITIES...

  15. 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..., flavoring, lubricant, preservative, or solvent, in the preparation of other drugs shall be exempt...

  16. 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

    2016-07-01

    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

  17. [Physical inactivity as public health problem].

    PubMed

    Kovacić, Luka

    2007-01-01

    The aim of the paper is to present the public health aspects of physical inactivity, and the importance of sport, recreation and other types of physical activity to health of population. Reduction of physical inactivity in today's every day life became great public health concern. The causes of such situation are linked to the pattern of modern life, sedentary type of work, autoimmunization in production, passive role of citizens in sports, and others. Adequate physical activity is very important means in prevention of many health problems, like obesity, arterial hypertension, diabetes, hearth diseases, atherosclerosis, osteoporosis and others. Physical activity and regular exercises in elderly increase independency from others in every day living, increase physical condition and reduce accidents, improve mental health, satisfaction with life; reduce hypertension and quantity of drugs. Physical inactivity increases economic burden of the country. The results of the Croatian Health Survey from 2003, done on representative sample of Croatian adult population shows that 44% of men and 30% of women are physically inactive. Situation in cities is even worse. In Zagreb 85% of men and 45% of women are inactive. The criteria for physical active person were walking three times a week for 30 minutes at least. The solution of the problem for the future is to pay more and more intensive attention to develop comprehensive community programs. More support should be given in construction of facilities for sport and recreation, during wintertime and for persons with special needs. The key role and responsibility for the promotion of physical activity of citizens will have primary health services (family practitioners, public health services), nongovernmental organization and media.

  18. The inactive form of recA protein: the 'compact' structure.

    PubMed Central

    Ruigrok, R W; Bohrmann, B; Hewat, E; Engel, A; Kellenberger, E; DiCapua, E

    1993-01-01

    When recA protein is enzymatically inactive in vitro, it adopts a more compact helical polymer form than that of the active protein polymerized onto DNA in the presence of ATP. Here we describe some aspects of this structure. By cryo-electron microscopy, a pitch of 76 A is found for both the self-polymer and the inactive complex with ssDNA. A smaller pitch of 64 A is observed in conventional electron micrographs. The contour length of complexes with ssDNA was used to estimate the binding stoichiometry in the compact complex, 6 +/- 1 nt/recA. In addition, the compact structure was observed in vivo in Escherichia coli: inclusion bodies produced upon induction of recA expression in an overproducing strain have a fibrous morphology with the structural parameters of the compact polymer. Images PMID:8428597

  19. Characterisation of the influence of genetic variations on the enzyme activity of a recombinant human glycine N-acyltransferase.

    PubMed

    van der Sluis, Rencia; Badenhorst, Christoffel P S; van der Westhuizen, Francois H; van Dijk, Alberdina A

    2013-02-25

    Human glycine N-acyltransferase (human GLYAT) detoxifies a wide range of endogenous and xenobiotic metabolites, including benzoate and salicylate. Significant inter-individual variation exists in glycine conjugation capacity. The molecular basis for this variability is not known. To investigate the influence of single nucleotide polymorphisms (SNPs) in the GLYAT coding sequence on enzyme activity, we expressed and characterised a recombinant human GLYAT. Site-directed mutagenesis was used to generate six non-synonymous SNP variants of the enzyme (K16N; S17T; R131H; N156S; F168L; R199C). The variants were expressed, purified, and enzymatically characterised. The enzyme activities of the K16N, S17T and R131H variants were similar to that of the wild-type, whereas the N156S variant was more active, the F168L variant less active, and the R199C variant was inactive. We also generated an E227Q mutant, which lacks the catalytic residue proposed by Badenhorst et al. (2012). This mutant was inactive compared to the wild-type recombinant human GLYAT. A molecular model of human GLYAT containing coenzyme A (CoA) was generated which revealed that the inactivity of the R199C variant could be due to the substitution of the highly conserved Arg(199) and destabilisation of an α-loop-α motif which is important for substrate binding in the GNAT superfamily. The finding that SNP variations in the human GLYAT gene influence the kinetic properties of the enzyme may explain some of the inter-individual variation in glycine conjugation capacity, which is relevant to the metabolism of xenobiotics such as aspirin and the industrial solvent xylene, and to the treatment of some metabolic disorders. PMID:23237781

  20. 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.

  1. 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.

  2. 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.

  3. Physical inactivity, excess adiposity and premature mortality.

    PubMed

    Katzmarzyk, P T; Janssen, I; Ardern, C I

    2003-11-01

    The purpose of this report is to review the evidence that physical inactivity and excess adiposity are related to an increased risk of all-cause mortality, and to better identify the independent contributions of each to all-cause mortality rates. A variance-based method of meta-analysis was used to summarize the relationships from available studies. The summary relative risk of all-cause mortality for physical activity from the 55 analyses (31 studies) that included an index of adiposity as a covariate was 0.80 [95% confidence interval (CI) 0.78-0.821, whereas it was 0.82 [95% CI 0.80-0.84] for the 44 analyses (26 studies) that did not include an index of adiposity. Thus, physically active individuals have a lower risk of mortality by comparison to physically inactive peers, independent of level of adiposity. The summary relative risk of all-cause mortality for an elevated body mass index (BMI) from the 25 analyses (13 studies) that included physical activity as a covariate was 1.23 [95% CI 1.18-1.29], and it was 1.24 [95% CI 1.21-1.28] for the 81 analyses (36 studies) that did not include physical activity as a covariate. Studies that used a measure of adiposity other than the BMI show similar relationships with mortality, and stratified analyses indicate that both physical inactivity and adiposity are important determinants of mortality risk.

  4. 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.

  5. Recombinant microorganisms for increased production of organic acids

    DOEpatents

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

    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.

  6. [Development of complex enzymatic preparations of pactinases and celulases for sugar beet marc digestion].

    PubMed

    Bushina, E V; Rozhkova, A M; Zorov, I N; Satrutdinov, A D; Bekkarevich, A O; Koshelev, A V; Okunev, O N; Sinitsyn, A P

    2012-01-01

    Complex enzymatic preparations demonstrating activities homologous to pectinlyase A and heterologous to endo-1,4-beta-glucanase from Penicilliumverruculosum and beta-glycosidase from Aspergillusniger have been obtained on the basis of recombinant strains of the fungus Penicilliumcanescens. Two approaches were utilized: development of an enzymatic preparation on the basis of a new strain, which produced all three enzymes, and development of an enzymatic preparation via combined cultivation of three strains, each of which produced one of the enzymes.

  7. 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…

  8. 30 CFR 57.5045 - Posting of inactive workings.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Posting of inactive workings. 57.5045 Section 57.5045 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND... Posting of inactive workings. Inactive workings in which radon daughter concentrations are above 1.0...

  9. 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)

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Redox potential tuning by redox-inactive cations in nature's water oxidizing catalyst and synthetic analogues.

    PubMed

    Krewald, Vera; Neese, Frank; Pantazis, Dimitrios A

    2016-04-28

    The redox potential of synthetic oligonuclear transition metal complexes has been shown to correlate with the Lewis acidity of a redox-inactive cation connected to the redox-active transition metals of the cluster via oxo or hydroxo bridges. Such heterometallic clusters are important cofactors in many metalloenzymes, where it is speculated that the redox-inactive constituent ion of the cluster serves to optimize its redox potential for electron transfer or catalysis. A principal example is the oxygen-evolving complex in photosystem II of natural photosynthesis, a Mn4CaO5 cofactor that oxidizes water into dioxygen, protons and electrons. Calcium is critical for catalytic function, but its precise role is not yet established. In analogy to synthetic complexes it has been suggested that Ca(2+) fine-tunes the redox potential of the manganese cluster. Here we evaluate this hypothesis by computing the relative redox potentials of substituted derivatives of the oxygen-evolving complex with the cations Sr(2+), Gd(3+), Cd(2+), Zn(2+), Mg(2+), Sc(3+), Na(+) and Y(3+) for two sequential transitions of its catalytic cycle. The theoretical approach is validated with a series of experimentally well-characterized Mn3AO4 cubane complexes that are structural mimics of the enzymatic cluster. Our results reproduce perfectly the experimentally observed correlation between the redox potential and the Lewis acidities of redox-inactive cations for the synthetic complexes. However, it is conclusively demonstrated that this correlation does not hold for the oxygen evolving complex. In the enzyme the redox potential of the cluster only responds to the charge of the redox-inactive cations and remains otherwise insensitive to their precise identity, precluding redox-tuning of the metal cluster as a primary role for Ca(2+) in biological water oxidation.

  17. Enzymatic characteristics of the c-Raf-1 protein kinase.

    PubMed

    Force, T; Bonventre, J V; Heidecker, G; Rapp, U; Avruch, J; Kyriakis, J M

    1994-02-15

    The c-Raf-1 protein kinase plays a central role in the mitogenic response of cells to growth factors, cytokines, and many oncogenes. Despite the critical importance of this enzyme, very little is known of its biochemical properties or mechanisms of regulation. In these experiments, we used the only candidate physiologic substrate identified as yet for c-Raf-1, mitogen-activated protein kinase kinase (MAPKK), to examine enzymatic characteristics and candidate modulators of c-Raf-1, c-Raf-1 was purified from Sf9 cells infected with recombinant baculovirus encoding a histidine-tagged c-Raf-1. The Km values of c-Raf-1 for ATP and MAPKK were 11.6 microM and 0.8 microM, respectively, and the stoichiometry of phosphorylation of MAPKK by c-Raf-1 was 1.67 mol of phosphate per mol of MAPKK. In contrast to prior reports, Mg2+ was the preferred cation at Mg2+ and Mn2+ concentrations > 5 mM. c-Raf-1 substrate specificity was extremely restricted, consistent with the identification of only one candidate physiologic substrate to date and highlighting the necessity of using MAPKK rather than artificial substrates in c-Raf-1 activity assays. Of multiple potential substrates tested, the only one phosphorylated to > 20% of the level of MAPKK phosphorylation was myelin basic protein (22%). Heat-denatured MAPKK was phosphorylated at only 2% the level of native MAPKK, indicating that the restricted substrate specificity may be due to tertiary-structural requirements. We also examined whether c-Raf-1 activity is modulated by lipid binding to the cysteine finger region in its regulatory domain. Of multiple mitogen-stimulated or cell-membrane lipids tested, only phosphatidylserine and diacylglycerol in the presence of Ca2+ (2.5 mM) increased c-Raf-1 kinase activity significantly (1.5-fold). The increase is probably not of physiologic significance because it was about two orders of magnitude less than the stimulation of protein kinase C by these lipids. On gel-filtration chromatography, the

  18. Enzymatic characteristics of the c-Raf-1 protein kinase.

    PubMed Central

    Force, T; Bonventre, J V; Heidecker, G; Rapp, U; Avruch, J; Kyriakis, J M

    1994-01-01

    The c-Raf-1 protein kinase plays a central role in the mitogenic response of cells to growth factors, cytokines, and many oncogenes. Despite the critical importance of this enzyme, very little is known of its biochemical properties or mechanisms of regulation. In these experiments, we used the only candidate physiologic substrate identified as yet for c-Raf-1, mitogen-activated protein kinase kinase (MAPKK), to examine enzymatic characteristics and candidate modulators of c-Raf-1, c-Raf-1 was purified from Sf9 cells infected with recombinant baculovirus encoding a histidine-tagged c-Raf-1. The Km values of c-Raf-1 for ATP and MAPKK were 11.6 microM and 0.8 microM, respectively, and the stoichiometry of phosphorylation of MAPKK by c-Raf-1 was 1.67 mol of phosphate per mol of MAPKK. In contrast to prior reports, Mg2+ was the preferred cation at Mg2+ and Mn2+ concentrations > 5 mM. c-Raf-1 substrate specificity was extremely restricted, consistent with the identification of only one candidate physiologic substrate to date and highlighting the necessity of using MAPKK rather than artificial substrates in c-Raf-1 activity assays. Of multiple potential substrates tested, the only one phosphorylated to > 20% of the level of MAPKK phosphorylation was myelin basic protein (22%). Heat-denatured MAPKK was phosphorylated at only 2% the level of native MAPKK, indicating that the restricted substrate specificity may be due to tertiary-structural requirements. We also examined whether c-Raf-1 activity is modulated by lipid binding to the cysteine finger region in its regulatory domain. Of multiple mitogen-stimulated or cell-membrane lipids tested, only phosphatidylserine and diacylglycerol in the presence of Ca2+ (2.5 mM) increased c-Raf-1 kinase activity significantly (1.5-fold). The increase is probably not of physiologic significance because it was about two orders of magnitude less than the stimulation of protein kinase C by these lipids. On gel-filtration chromatography, the

  19. 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.

  20. Enzymatic activity of poliovirus RNA polymerase mutants with single amino acid changes in the conserved YGDD amino acid motif.

    PubMed

    Jablonski, S A; Luo, M; Morrow, C D

    1991-09-01

    RNA-dependent RNA polymerases contain a highly conserved region of amino acids with a core segment composed of the amino acids YGDD which have been hypothesized to be at or near the catalytic active site of the molecule. Six mutations in this conserved YGDD region of the poliovirus RNA-dependent RNA polymerase were made by using oligonucleotide site-directed DNA mutagenesis of the poliovirus cDNA to substitute A, C, M, P, S, or V for the amino acid G. The mutant polymerase genes were expressed in Escherichia coli, and the purified RNA polymerases were tested for in vitro enzyme activity. Two of the mutant RNA polymerases (those in which the glycine residue was replaced with alanine or serine) exhibited in vitro enzymatic activity ranging from 5 to 20% of wild-type activity, while the remaining mutant RNA polymerases were inactive. Alterations in the in vitro reaction conditions by modification of temperature, metal ion concentration, or pH resulted in no significant differences in the activities of the mutant RNA polymerases relative to that of the wild-type enzyme. An antipeptide antibody directed against the wild-type core amino acid segment containing the YGDD region of the poliovirus polymerase reacted with the wild-type recombinant RNA polymerase and to a limited extent with the two enzymatically active mutant polymerases; the antipeptide antibody did not react with the mutant RNA polymerases which did not have in vitro enzyme activity. These results are discussed in the context of secondary-structure predictions for the core segment containing the conserved YGDD amino acids in the poliovirus RNA polymerase. PMID:1651402

  1. Characterizing inactive ribosomes in translational profiling.

    PubMed

    Liu, Botao; Qian, Shu-Bing

    2016-01-01

    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

  2. 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)

  3. 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

  4. Sarcopenia and Physical Inactivity in Patients With Chronic Kidney Disease

    PubMed Central

    Hirai, Keiji; Ookawara, Susumu; Morishita, Yoshiyuki

    2016-01-01

    Sarcopenia and physical inactivity synergistically progress in patients with chronic kidney disease (CKD) and are strong predictors of mortality in this population. Exercise training and essential amino acids and vitamin D supplements may contribute to improving sarcopenia and physical inactivity in CKD patients. PMID:27570755

  5. Sarcopenia and Physical Inactivity in Patients With Chronic Kidney Disease.

    PubMed

    Hirai, Keiji; Ookawara, Susumu; Morishita, Yoshiyuki

    2016-05-01

    Sarcopenia and physical inactivity synergistically progress in patients with chronic kidney disease (CKD) and are strong predictors of mortality in this population. Exercise training and essential amino acids and vitamin D supplements may contribute to improving sarcopenia and physical inactivity in CKD patients. PMID:27570755

  6. 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)

  7. Enzymatic gallic acid esterification.

    PubMed

    Weetal, H H

    1985-02-01

    Gallic acid esters of n-propyl and amyl alcohols have been produced by enzymatic synthesis in organic solvents using immobilized tannase. Studies indicate that maximum esterification of gallic acid occurs with amyl alcohol. The enzyme shows broad alcohol specificity. However, the enzyme exhibits absolute specificity for the acid portion of the ester. Studies were carried out on K(m), V(max), pH, and temperature optima.

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

    PubMed

    Wagschal, Kurt; Lee, Charles C

    2012-04-01

    We present here a whole-cell and permeabilized E. coli cell 1° active/inactive microplate screen for β-d-xylosidase, xylanase, endoglucanase, and ferulic acid esterase enzyme activities, which are critical for the enzymatic deconstruction of biomass for fuels and chemicals. Transformants from genomic or mutagenesis-derived libraries are screened using fluorophore-tagged substrate/enzyme activity pairs that are assayed directly in the protein expression host growth media using a minimum of specialized equipment and supplies. PMID:22285853

  9. Different enzymatic oscillations in vivo caused by the stereoisomers of an aminopeptidase inhibitor, bestatin.

    PubMed

    Aoyagi, T; Wada, T; Yamamoto, K; Kojima, F; Nagai, M; Harada, S; Umezawa, H

    1984-08-01

    The present study was undertaken to compare the enzymatic oscillations induced in three major organs, spleen, kidney, and liver, of mice by the active and inactive forms of an aminopeptidase inhibitor, bestatin. Although bestatin caused sine curve-type oscillations of enzymatic activities in spleen and kidney, its isomer, possessing no enzyme-inhibiting actions and no biological actions, did not show any typical enzymatic changes. Such typical oscillations were not elicited by either one of those two agents in liver, in spite of the apparent difference in the type of oscillations induced by them in this organ. These observations were taken to indicate that the enzymatic oscillations in vivo caused by bestatin are closely related to its aminopeptidase-inhibiting actions seen in vitro and that the immunomodifying actions of this agent are based on its effects on enzymes sensitive to it, possibly involving immunoresponsive cells.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. Activation of Inactive Nitrogenase by Acid-Treated Component I

    PubMed Central

    Nagatani, H. H.; Shah, Vinod K.; Brill, Winston J.

    1974-01-01

    When Azotobacter vinelandii was derepressed for nitrogenase synthesis in a N-free medium containing tungstate instead of molybdate, an inactive component I was synthesized. Although this inactive component I could be activated in vivo upon addition of molybdate to the medium, it could not be activated in vitro when molybdate was added to the extracts. Activation occurred, however, when an acid-treated component I was added to extracts of cells derepressed in medium containing tungstate. Acid treatment completely abolished component I activity. Mutant strains UW45 and UW10 were unable to fix N2. Both strains synthesized normal levels of component II but produced inactive component I. Acid-treated component I activated inactive component I in extracts of mutant strain UW45 but not mutant strain UW10. This activating factor could be obtained from N2-fixing Klebsiella pneumoniae, Clostridium pasteurianum, and Rhodospirillum rubrum. PMID:4218230

  15. 1. AERIAL VIEW OF BOTH ACTIVE AND INACTIVE FLUMES, TAKEN ...

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

    1. AERIAL VIEW OF BOTH ACTIVE AND INACTIVE FLUMES, TAKEN FROM EAST, NOTE STOCK SHELTERS IN BACKGROUND AND HAYSTACKS AND STORAGE IN FOREGROUND - Grant-Kohrs Ranch, Flumes, Highway 10, Deer Lodge, Powell County, MT

  16. THE PRINTO CRITERIA FOR CLINICALLY INACTIVE DISEASE IN JUVENILE DERMATOMYOSITIS

    PubMed Central

    Lazarevic, Dragana; Pistorio, Angela; Palmisani, Elena; Miettunen, Paivi; Ravelli, Angelo; Pilkington, Clarissa; Wulffraat, Nico; Malattia, Clara; Garay, Stella; Hofer, Michael; Quartier, Pierre; Dolezalova, Pavla; Calvo, Inmaculada; Ferriani, Virginia; Ganser, Gerd; Kasapcopur, Ozgur; Melo-Gomes, Jose Antonio; Reed, Ann M.; Wierzbowska, Malgorzata; Rider, Lisa G.; Martini, Alberto; Ruperto, Nicolino

    2016-01-01

    Objectives To develop data-driven criteria for clinically inactive disease on and off therapy for juvenile dermatomyositis (JDM). Methods The PRINTO database contains 275 patients with active JDM evaluated prospectively up to 24 months. Thirty-eight patients off therapy at 24 months were defined as clinically inactive and included in the reference group. These were compared with a random sample of 76 patients who had active disease at study baseline. Individual measures of muscle strength/endurance, muscle enzymes, physician's and parent's global disease activity/damage evaluations, inactive disease criteria derived from literature, and other ad hoc criteria, were evaluated for sensitivity, specificity, and Cohen's kappa agreement. Results The individual measures that best characterised inactive disease (sensitivity and specificity >0.8 and Cohen's K>0.8) were manual muscle testing (MMT)≥78, physician global assessment of muscle activity=0, physician global assessment of overall disease activity (PhyGloVAS) ≤0.2, the Childhood Myositis Assessment Scale (CMAS) ≥48, the Disease Activity Score (DAS) ≤3 and the Myositis Disease Activity Assessment Visual Analogue Scale (MYOACT) ≤0.2. The best combination of variables to classify a patient as being in a status of inactive disease on or off therapy is at least 3 out of 4 of the following criteria: creatine kinase ≤150, CMAS≥48, MMT≥78, and PhyGloVAS≤0.2. After 24 months, 30/31 (96.8%) patients were inactive off therapy and 69/145 (47.6%) were inactive on-therapy. Conclusion PRINTO established data-driven criteria, with clearly evidence-based cut off values, to identify JDM patients with clinically inactive disease. These criteria can be used in clinical trials, in research and in clinical practice. PMID:22736096

  17. Prevalence of physical inactivity in Iran: a systematic review

    PubMed Central

    Fakhrzadeh, Hossein; Djalalinia, Shirin; Mirarefin, Mojdeh; Arefirad, Tahereh; Asayesh, Hamid; Safiri, Saeid; Samami, Elham; Mansourian, Morteza; Shamsizadeh, Morteza; Qorbani, Mostafa

    2016-01-01

    Introduction: Physical inactivity is one of the most important risk factors for chronic diseases, including cardiovascular disease, cancer, and stroke. We aim to conduct a systematic review of the prevalence of physical inactivity in Iran. Methods: We searched international databases; ISI, PubMed/Medline, Scopus, and national databases Irandoc, Barakat knowledge network system, and Scientific Information Database (SID). We collected data for outcome measures of prevalence of physical inactivity by sex, age, province, and year. Quality assessment and data extraction has been conducted independently by two independent research experts. There were no limitations for time and language. Results: We analyzed data for prevalence of physical inactivity in Iranian population. According to our search strategy we found 254 records; of them 185 were from international databases and the remaining 69 were obtained from national databases after refining the data, 34 articles that met eligible criteria remained for data extraction. From them respectively; 9, 20, 2 and 3 studies were at national, provincial, regional and local levels. The estimates for inactivity ranged from approximately 30% to almost 70% and had considerable variation between sexes and studied sub-groups. Conclusion: In Iran, most of studies reported high prevalence of physical inactivity. Our findings reveal a heterogeneity of reported values, often from differences in study design, measurement tools and methods, different target groups and sub-population sampling. These data do not provide the possibility of aggregation of data for a comprehensive inference. PMID:27777692

  18. 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

  19. 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.

  20. [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.

  1. A highly immunogenic recombinant and truncated protein of the secreted aspartic proteases family (rSap2t) of Candida albicans as a mucosal anticandidal vaccine.

    PubMed

    Sandini, Silvia; La Valle, Roberto; Deaglio, Silvia; Malavasi, Fabio; Cassone, Antonio; De Bernardis, Flavia

    2011-07-01

    Sap2 (secreted aspartyl proteinase2) is a member of the Sap family of Candida albicans, a human opportunistic pathogen, which acts as a virulence factor in experimental animal models of mucosal candidiasis. The C. albicans SAP2 was subcloned into vector pDS56-RBSII-6xhis, under the control of an inducible promoter to produce a truncated 6xhis-tagged, enzymatically inactive Sap2, lacking the N-terminus 76 amino acids (rSap2t). This recombinant protein was purified to homogeneity by one-step nickel-chelate affinity chromatography and used to immunize intravaginally oophorectomized estradiol-treated rats. These animals raised local anti-rSap2t immunoglobulin G (IgG) and IgA antibodies and were protected from the challenge of a highly vaginopathic strain of the fungus. Protection was possibly due to the specific antibodies as suggested by the passive transfer of immune vaginal fluid and the protective effects of passive vaccination with anti-rSap2t IgM and IgG monoclonal antibodies. Hence, this new recombinant proteinase constitutes a novel tool to investigate mechanisms of anti-Candida protection at the vaginal level and as vaccination against vaginal candidiasis, a common, frequently recurrent and sometimes antimycotic-refractory infection in women.

  2. 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. PMID:24968689

  3. 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.

  4. 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. PMID:26741824

  5. 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

  6. Inactive conformation enhances binding function in physiological conditions

    PubMed Central

    Yakovenko, Olga; Tchesnokova, Veronika; Sokurenko, Evgeni V.; Thomas, Wendy E.

    2015-01-01

    Many receptors display conformational flexibility, in which the binding pocket has an open inactive conformation in the absence of ligand and a tight active conformation when bound to ligand. Here we study the bacterial adhesin FimH to address the role of the inactive conformation of the pocket for initiating binding by comparing two variants: a wild-type FimH variant that is in the inactive state when not bound to its target mannose, and an engineered activated variant that is always in the active state. Not surprisingly, activated FimH has a longer lifetime and higher affinity, and bacteria expressing activated FimH bound better in static conditions. However, bacteria expressing wild-type FimH bound better in flow. Wild-type and activated FimH demonstrated similar mechanical strength, likely because mechanical force induces the active state in wild-type FimH. However, wild-type FimH displayed a faster bond association rate than activated FimH. Moreover, the ability of different FimH variants to mediate adhesion in flow reflected the fraction of FimH in the inactive state. These results demonstrate a new model for ligand-associated conformational changes that we call the kinetic-selection model, in which ligand-binding selects the faster-binding inactive state and then induces the active state. This model predicts that in physiological conditions for cell adhesion, mechanical force will drive a nonequilibrium cycle that uses the fast binding rate of the inactive state and slow unbinding rate of the active state, for a higher effective affinity than is possible at equilibrium. PMID:26216967

  7. 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

  8. 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)

  9. 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.

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

    PubMed

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

    2013-02-14

    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 -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 crosspresentation 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 antigenspecific CD8(+) T-cell responses during immunization.

  11. 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...

  12. 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...

  13. 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

  14. 5 CFR 1650.15 - Abandonment of inactive accounts.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 5 Administrative Personnel 3 2011-01-01 2011-01-01 false Abandonment of inactive accounts. 1650.15 Section 1650.15 Administrative Personnel FEDERAL RETIREMENT THRIFT INVESTMENT BOARD METHODS OF WITHDRAWING FUNDS FROM THE THRIFT SAVINGS PLAN Post-Employment Withdrawals § 1650.15 Abandonment of...

  15. 5 CFR 1650.15 - Abandonment of inactive accounts.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 5 Administrative Personnel 3 2010-01-01 2010-01-01 false Abandonment of inactive accounts. 1650.15 Section 1650.15 Administrative Personnel FEDERAL RETIREMENT THRIFT INVESTMENT BOARD METHODS OF WITHDRAWING FUNDS FROM THE THRIFT SAVINGS PLAN Post-Employment Withdrawals § 1650.15 Abandonment of...

  16. 5 CFR 1650.15 - Abandonment of inactive accounts.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 5 Administrative Personnel 3 2013-01-01 2013-01-01 false Abandonment of inactive accounts. 1650.15 Section 1650.15 Administrative Personnel FEDERAL RETIREMENT THRIFT INVESTMENT BOARD METHODS OF WITHDRAWING FUNDS FROM THE THRIFT SAVINGS PLAN Post-Employment Withdrawals § 1650.15 Abandonment of...

  17. 5 CFR 1650.15 - Abandonment of inactive accounts.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 5 Administrative Personnel 3 2012-01-01 2012-01-01 false Abandonment of inactive accounts. 1650.15 Section 1650.15 Administrative Personnel FEDERAL RETIREMENT THRIFT INVESTMENT BOARD METHODS OF WITHDRAWING FUNDS FROM THE THRIFT SAVINGS PLAN Post-Employment Withdrawals § 1650.15 Abandonment of...

  18. 5 CFR 1650.15 - Abandonment of inactive accounts.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 5 Administrative Personnel 3 2014-01-01 2014-01-01 false Abandonment of inactive accounts. 1650.15 Section 1650.15 Administrative Personnel FEDERAL RETIREMENT THRIFT INVESTMENT BOARD METHODS OF WITHDRAWING FUNDS FROM THE THRIFT SAVINGS PLAN Post-Employment Withdrawals § 1650.15 Abandonment of...

  19. Identification of Inactive Medications in Narrative Medical Text

    PubMed Central

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

    2008-01-01

    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 patient’s 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. PMID:18999079

  20. 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

  1. 11 CFR 9033.6 - Determination of inactive candidacy.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...) General. The Commission may, on the basis of the factors listed in 11 CFR 9033.6(b) below, make a... ineligible as provided in 11 CFR 9033.5. (b) Factors considered. In making its determination of inactive... in 11 CFR 9033.10(b) and will advise the candidate of the date on which active campaigning in...

  2. 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,…

  3. 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... affected, or whose ability to protect such interests is impeded by the failure of an official to act on...

  4. 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...

  5. 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...

  6. 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...

  7. 11 CFR 9033.6 - Determination of inactive candidacy.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...) General. The Commission may, on the basis of the factors listed in 11 CFR 9033.6(b) below, make a... ineligible as provided in 11 CFR 9033.5. (b) Factors considered. In making its determination of inactive... in 11 CFR 9033.10(b) and will advise the candidate of the date on which active campaigning in...

  8. Signaling Active CD95 Receptor Molecules Trigger Co-translocation of Inactive CD95 Molecules into Lipid Rafts*

    PubMed Central

    Lang, Isabell; Fick, Andrea; Schäfer, Viktoria; Giner, Tina; Siegmund, Daniela; Wajant, Harald

    2012-01-01

    The capability of soluble CD95L trimers to trigger CD95-associated signaling pathways is drastically increased by oligomerization. The latter can be achieved, for example, by antibodies recognizing a N-terminal epitope tag in recombinant CD95L variants or by genetic engineering-enforced formation of hexamers. Using highly sensitive and accurate binding studies with recombinant CD95L variants equipped with a Gaussia princeps luciferase reporter domain, we found that oligomerization of CD95L has no major effect on CD95 occupancy. This indicates that the higher activity of oligomerized CD95L trimers is not related to an avidity-related increase in apparent affinity and points instead to a crucial role of aggregation of initially formed trimeric CD95L-CD95 complexes in CD95 activation. Furthermore, binding of soluble CD95L trimers was found to be insufficient to increase the association of CD95 with the lipid raft-containing membrane fraction. However, when Gaussia princeps luciferase-CD95L trimers were used as tracers to “mark” inactive CD95 molecules, increased association of these inactive receptors was observed upon activation of the remaining CD95 molecules by help of highly active hexameric Fc-CD95L or membrane CD95L. Moreover, in cells expressing endogenous CD95 and chimeric CD40-CD95 receptors, triggering of CD95 signaling via endogenous CD95 resulted in co-translocation of CD40-CD95 to the lipid raft fraction, whereas vice versa activation of CD95-associated pathways with Fc-CD40L via CD40-CD95 resulted in co-translocation of endogenous CD95. In sum, this shows that signaling-active CD95 molecules not only enhance their own association with the lipid raft-containing membrane fraction but also those of inactive CD95 molecules. PMID:22645131

  9. Regulation of Deinococcus radiodurans RecA Protein Function via Modulation of Active and Inactive Nucleoprotein Filament States*

    PubMed Central

    Ngo, Khanh V.; Molzberger, Eileen T.; Chitteni-Pattu, Sindhu; Cox, Michael M.

    2013-01-01

    The RecA protein of Deinococcus radiodurans (DrRecA) has a central role in genome reconstitution after exposure to extreme levels of ionizing radiation. When bound to DNA, filaments of DrRecA protein exhibit active and inactive states that are readily interconverted in response to several sets of stimuli and conditions. At 30 °C, the optimal growth temperature, and at physiological pH 7.5, DrRecA protein binds to double-stranded DNA (dsDNA) and forms extended helical filaments in the presence of ATP. However, the ATP is not hydrolyzed. ATP hydrolysis of the DrRecA-dsDNA filament is activated by addition of single-stranded DNA, with or without the single-stranded DNA-binding protein. The ATPase function of DrRecA nucleoprotein filaments thus exists in an inactive default state under some conditions. ATPase activity is thus not a reliable indicator of DNA binding for all bacterial RecA proteins. Activation is effected by situations in which the DNA substrates needed to initiate recombinational DNA repair are present. The inactive state can also be activated by decreasing the pH (protonation of multiple ionizable groups is required) or by addition of volume exclusion agents. Single-stranded DNA-binding protein plays a much more central role in DNA pairing and strand exchange catalyzed by DrRecA than is the case for the cognate proteins in Escherichia coli. The data suggest a mechanism to enhance the efficiency of recombinational DNA repair in the context of severe genomic degradation in D. radiodurans. PMID:23729671

  10. 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.

  11. Short-term Physical Inactivity Impairs Vascular Function

    PubMed Central

    Nosova, Emily V.; Yen, Priscilla; Chong, Karen C.; Alley, Hugh F.; Stock, Eveline O.; Quinn, Alex; Hellmann, Jason; Conte, Michael S.; Owens, Christopher D.; Spite, Matthew; Grenon, S. Marlene

    2014-01-01

    Introduction Sedentarism, also termed physical inactivity, is an independent risk factor for cardiovascular diseases. Mechanisms thought to be involved include insulin resistance, dyslipidemia, hypertension, and increased inflammation. It is unknown whether changes in vascular and endothelial function also contribute to this excess risk. We hypothesized that short-term exposure to inactivity would lead to endothelial dysfunction, arterial stiffening and increased vascular inflammation. Methods Five healthy subjects (4 males and 1 female) underwent 5 days of bed rest (BR) to simulate inactivity. Measurements of vascular function [flow-mediated vasodilation (FMD) to evaluate endothelial function; applanation tonometry to assess arterial resistance], inflammation and metabolism were made before BR, daily during BR and after 2 recovery days. Subjects maintained an isocaloric diet throughout. Results Bed rest led to significant decreases in brachial artery and femoral artery FMD [Brachial: 11 ± 3% pre-BR vs. 9 ± 2% end-BR, P=0.04; Femoral: 4 ± 1% vs. 2 ± 1%, P=0.04]. The central augmentation index increased with BR [−4 ± 9% vs. 5 ± 11%, P=0.03]. Diastolic blood pressure (DBP) increased [58 ± 7 mmHg vs. 62 ± 7 mmHg, P=0.02], while neither systolic blood pressure nor heart rate changed. 15-HETE, an arachidonic acid metabolite, increased but the other inflammatory and metabolic biomarkers were unchanged. Conclusions Our findings show that acute exposure to sedentarism results in decreased endothelial function, arterial stiffening, increased DBP, and an increase in 15-HETE. We speculate that inactivity promotes a vascular “deconditioning” state characterized by impaired endothelial function, leading to arterial stiffness and increased arterial tone. Although physiologically significant, the underlying mechanisms and clinical relevance of these findings need to be further explored. PMID:24630521

  12. 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.

  13. 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.

  14. The pandemic of physical inactivity: global action for public health.

    PubMed

    Kohl, Harold W; Craig, Cora Lynn; Lambert, Estelle Victoria; Inoue, Shigeru; Alkandari, Jasem Ramadan; Leetongin, Grit; Kahlmeier, Sonja

    2012-07-21

    Physical inactivity is the fourth leading cause of death worldwide. We summarise present global efforts to counteract this problem and point the way forward to address the pandemic of physical inactivity. Although evidence for the benefits of physical activity for health has been available since the 1950s, promotion to improve the health of populations has lagged in relation to the available evidence and has only recently developed an identifiable infrastructure, including efforts in planning, policy, leadership and advocacy, workforce training and development, and monitoring and surveillance. The reasons for this late start are myriad, multifactorial, and complex. This infrastructure should continue to be formed, intersectoral approaches are essential to advance, and advocacy remains a key pillar. Although there is a need to build global capacity based on the present foundations, a systems approach that focuses on populations and the complex interactions among the correlates of physical inactivity, rather than solely a behavioural science approach focusing on individuals, is the way forward to increase physical activity worldwide.

  15. Recombinant protein production technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant protein production is an important technology for antibody production, biochemical activity study, and structural determination during the post-genomic era. Limiting factors in recombinant protein production include low-level protein expression, protein precipitation, and loss of protein...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  17. 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

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

    PubMed

    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

    2016-02-01

    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 socioeconomic

  19. 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

  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. 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.

  2. 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.

  3. 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...

  4. Determination of enamel protein synthesized by recombined mouse molar tooth germs in organ culture.

    PubMed

    Baba, T; Terashima, T; Oida, S; Sasaki, S

    1996-02-01

    Epithelial-mesenchymal interaction is a prerequisite for tooth morphogenesis. To study this interaction, inner enamel epithelium and dental papilla mesenchyme of molar tooth germs from a 16.5-day mouse embryo were dissociated enzymatically and cultured alone or after recombination. Characteristic matrix protein synthesized and secreted by recombined tooth germ was determined quantitatively by enzyme-linked immunosorbent assay. The protein was detected in the culture of recombined tooth germ but not of dissociated enamel epithelium alone. The amount of enamel protein increased until 8 days in culture. Morphological differentiation of the recombined epithelial rudiment into ameloblasts and enamel protein production were confirmed.

  5. 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.

  6. Expression of human recombinant granzyme A zymogen and its activation by the cysteine proteinase cathepsin C.

    PubMed

    Kummer, J A; Kamp, A M; Citarella, F; Horrevoets, A J; Hack, C E

    1996-04-19

    Human granzyme A is one of the serine proteinases present in the granules of cytotoxic T lymphocytes and natural killer cells. Granzymes are synthesized as inactive proenzymes with an amino-terminal prodipeptide, which is processed during transport of granzymes to the cytotoxic granules, where they are stored as active proteinases. In this study, we explored the possibility of producing recombinant granzymes. Recombinant human granzyme A zymogen was expressed in several eukaryotic cell lines (HepG2, Jurkat, and COS-1) after infection with a recombinant vaccinia virus containing full-length granzyme A cDNA. Immunoblot analysis of cell lysates showed that all infected cells produced a disulfide-linked homodimer of identical molecular weight as natural granzyme A. Infected HepG2 cells produced the largest amount of this protease (approximately 160 times more than lymphokine activated killer (LAK) cells). The recombinant protein only had high mannose type oligosaccharides as did the natural protein. Although infected HepG2 and COS cells contained high granzyme A antigen levels, lysates from these cells did not show any granzyme A proteolytic activity. However, the inactive proenzyme could be converted into active granzyme A by incubation with the thiol proteinase cathepsin C (dipeptidyl peptidase I). This study is the first to demonstrate expression of an active recombinant human cytotoxic lymphocyte proteinase and conversion of inactive progranzyme A into an active enzyme by cathepsin C. We suggest that a similar approach can be used for the production of other granzymes and related proteinases.

  7. Proteolytically Inactive Insulin-Degrading Enzyme Inhibits Amyloid Formation Yielding Non-Neurotoxic Aβ Peptide Aggregates

    PubMed Central

    de Tullio, Matias B.; Castelletto, Valeria; Hamley, Ian W.; Martino Adami, Pamela V.; Morelli, Laura; Castaño, Eduardo M.

    2013-01-01

    Insulin-degrading enzyme (IDE) is a neutral Zn2+ peptidase that degrades short peptides based on substrate conformation, size and charge. Some of these substrates, including amyloid β (Aβ) are capable of self-assembling into cytotoxic oligomers. Based on IDE recognition mechanism and our previous report of the formation of a stable complex between IDE and intact Aβ in vitro and in vivo, we analyzed the possibility of a chaperone-like function of IDE. A proteolytically inactive recombinant IDE with Glu111 replaced by Gln (IDEQ) was used. IDEQ blocked the amyloidogenic pathway of Aβ yielding non-fibrillar structures as assessed by electron microscopy. Measurements of the kinetics of Aβ aggregation by light scattering showed that 1) IDEQ effect was promoted by ATP independent of its hydrolysis, 2) end products of Aβ-IDEQ co-incubation were incapable of “seeding” the assembly of monomeric Aβ and 3) IDEQ was ineffective in reversing Aβ aggregation. Moreover, Aβ aggregates formed in the presence of IDEQ were non-neurotoxic. IDEQ had no conformational effects upon insulin (a non-amyloidogenic protein under physiological conditions) and did not disturb insulin receptor activation in cultured cells. Our results suggest that IDE has a chaperone-like activity upon amyloid-forming peptides. It remains to be explored whether other highly conserved metallopeptidases have a dual protease-chaperone function to prevent the formation of toxic peptide oligomers from bacteria to mammals. PMID:23593132

  8. Physical Inactivity and Incidence of Obesity among South Australian Adults

    PubMed Central

    Montgomerie, Alicia M.; Chittleborough, Catherine R.; Taylor, Anne W.

    2014-01-01

    The aim of this paper is to examine the association of physical inactivity with incidence of obesity in the South Australian adult population. Two representative data sources were used – the South Australian Monitoring and Surveillance System (SAMSS), a monthly surveillance system, and the North West Adelaide Health Study (NWAHS), a biomedical cohort study. There were 75.3% (n = 12873) SAMSS participants and 72.8% (n = 1521) of NWAHS participants that were not obese at baseline. The cumulative incidence of obesity for SAMSS participants from the previous year to the current year was 2.7%. The cumulative incidence of obesity for NWAHS participants between baseline and stage 3 was 14.4%. Physical inactivity was associated with incident obesity (RR 1.48, 95% CI 1.14–1.90 [SAMSS] and RR 1.41, 95% CI 1.03–1.93 [NWAHS]). This association remained, but was attenuated after adjustment for chronic conditions, risk factors and socio-demographic factors. However, physical activity should be continued to be encouraged in the population for its known additional health benefits. PMID:25383626

  9. 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.

  10. An Inactive Geminin Mutant That Binds Cdt1

    PubMed Central

    Suchyta, Marissa; Miotto, Benoit; McGarry, Thomas J.

    2015-01-01

    The initiation of DNA replication is tightly regulated in order to ensure that the genome duplicates only once per cell cycle. In vertebrate cells, the unstable regulatory protein Geminin prevents a second round of DNA replication by inhibiting the essential replication factor Cdt1. Cdt1 recruits mini-chromosome maintenance complex (MCM2-7), the replication helicase, into the pre-replication complex (pre-RC) at origins of DNA replication. The mechanism by which Geminin inhibits MCM2-7 loading by Cdt1 is incompletely understood. The conventional model is that Geminin sterically hinders a direct physical interaction between Cdt1 and MCM2-7. Here, we describe an inactive missense mutant of Geminin, GemininAWA, which binds to Cdt1 with normal affinity yet is completely inactive as a replication inhibitor even when added in vast excess. In fact, GemininAWA can compete with GemininWT for binding to Cdt1 and prevent it from inhibiting DNA replication. GemininAWA does not inhibit the loading of MCM2-7 onto DNA in vivo, and in the presence of GemininAWA, nuclear DNA is massively over-replicated within a single S phase. We conclude that Geminin does not inhibit MCM loading by simple steric interference with a Cdt1-MCM2-7 interaction but instead works by a non-steric mechanism, possibly by inhibiting the histone acetyltransferase HBO1. PMID:25988259

  11. Spontaneous and cisplatin-induced recombination in Escherichia coli.

    PubMed

    Nowosielska, Anetta; Calmann, Melissa A; Zdraveski, Zoran; Essigmann, John M; Marinus, M G

    2004-07-01

    To measure cisplatin (cis-diaminodichloroplatinum(II))-induced recombination, we have used a qualitative intrachromosomal assay utilizing duplicate inactive lac operons containing non-overlapping deletions and selection for Lac+ recombinants. The two operons are separated by one Mb and conversion of one of them yields the Lac+ phenotype. Lac+ formation for both spontaneous and cisplatin-induced recombination requires the products of the recA, recBC, ruvA, ruvB, ruvC, priA and polA genes. Inactivation of the recF, recO, recR and recJ genes decreased cisplatin-induced, but not spontaneous, recombination. The dependence on PriA and RecBC suggests that recombination is induced following stalling or collapse of replication forks at DNA lesions to form double strand breaks. The lack of recombination induction by trans-DDP suggests that the recombinogenic lesions for cisplatin are purine-purine intrastrand crosslinks. PMID:15177181

  12. 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.

  13. Binding of Nickel to Testicular Glutamate–Ammonia Ligase Inhibits Its Enzymatic Activity

    PubMed Central

    SUN, YINGBIAO; OU, YOUNG; CHENG, MIN; RUAN, YIBING; VAN DER HOORN, FRANS A.

    2016-01-01

    SUMMARY Exposure to nickel has been shown to cause damage to the testis in several animal models. It is not known if the testis expresses protein(s) that can bind nickel. To test this, we used a nickel-binding assay to isolate testicular nickel-binding proteins. We identified glutamate–ammonia ligase (GLUL) as a prominent nickel-binding protein by mass spectrometry. Protein analysis and reverse transcriptase polymerase chain reaction showed that GLUL is expressed in the testis, predominantly in interstitial cells. We determined that GLUL has a higher affinity for nickel than for its regular co-factor manganese. We produced an enzymatically active, recombinant GLUL protein. Upon binding, nickel interferes with the manganese-catalyzed enzymatic activity of recombinant GLUL protein. We also determined that GLUL activity in testes of animals exposed to nickel sulfate is reduced. Our results identify testicular GLUL as the first testicular protein shown to be affected by nickel exposure. PMID:21254280

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

    PubMed

    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

  15. 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

  16. 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.

  17. 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. PMID:26454139

  18. Human development, occupational structure and physical inactivity among 47 low and middle income countries

    PubMed Central

    Atkinson, Kaitlin; Lowe, Samantha; Moore, Spencer

    2015-01-01

    This study aimed to (a) assess the relationship between a person's occupational category and their physical inactivity, and (b) analyze the association among country-level variables and physical inactivity. The World Health Survey (WHS) was administered in 2002–2003 among 47 low- and middle-income countries (n = 196,742). The International Physical Activity Questionnaire (IPAQ) was used to collect verbal reports of physical activity and convert responses into measures of physical inactivity. Economic development (GDP/c), degree of urbanization, and the Human Development Index (HDI) were used to measure country-level variables and physical inactivity. Multilevel logistic regression analysis was used to examine the association among country-level factors, individual occupational status, and physical inactivity. Overall, the worldwide prevalence of physical inactivity in 2002–2003 was 23.7%. Individuals working in the white-collar industry compared to agriculture were 84% more likely to be physically inactive (OR: 1.84, CI: 1.73–1.95). Among low- and middle-income countries increased HDI values were associated with decreased levels of physical inactivity (OR: 0.98, CI: 0.97–0.99). This study is one of the first to adjust for within-country differences, specifically occupation while analyzing physical inactivity. As countries experience economic development, changes are also seen in their occupational structure, which result in increased countrywide physical inactivity levels. PMID:26844185

  19. The role of enzymatic activity in inhibition of the extrinsic tenase complex by phospholipase A2 isoenzymes from Naja nigricollis venom.

    PubMed

    Kini, R M; Evans, H J

    1995-12-01

    Three phospholipase A2 isoenzymes from Naja nigricollis venom inhibit the extrinsic tenase complex. We examined the role of their enzymatic activity in this inhibition by studying the effects of native and His-modified enzymes. Only CM-IV of the His-modified, catalytically inactive proteins showed significant inhibition of the activity of the complex. This indicates that strongly anticoagulant CM-IV inhibits the complex by both enzymatic and nonenzymatic mechanisms, whereas the weakly anticoagulant isoenzymes, CM-I and CM-II, inhibit primarily by catalytic degradation of phospholipids. This indicates a functional difference in the mode of inhibition between strongly and weakly anticoagulant phospholipase A2 enzymes.

  20. 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

  1. 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

  2. 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. PMID:24215862

  3. TALE proteins bind to both active and inactive chromatin.

    PubMed

    Scott, James N F; Kupinski, Adam P; Kirkham, Christopher M; Tuma, Roman; Boyes, Joan

    2014-02-15

    TALE (transcription activator-like effector) proteins can be tailored to bind to any DNA sequence of choice and thus are of immense utility for genome editing and the specific delivery of transcription activators. However, to perform these functions, they need to occupy their sites in chromatin. In the present study, we have systematically assessed TALE binding to chromatin substrates and find that in vitro TALEs bind to their target site on nucleosomes at the more accessible entry/exit sites, but not at the nucleosome dyad. We show further that in vivo TALEs bind to transcriptionally repressed chromatin and that transcription increases binding by only 2-fold. These data therefore imply that TALEs are likely to bind to their target in vivo even at inactive loci.

  4. Reactivation of the inactive X chromosome in development and reprogramming.

    PubMed

    Ohhata, Tatsuya; Wutz, Anton

    2013-07-01

    In mammals, one of the two X chromosomes of female cells is inactivated for dosage compensation between the sexes. X chromosome inactivation is initiated in early embryos by the noncoding Xist RNA. Subsequent chromatin modifications on the inactive X chromosome (Xi) lead to a remarkable stability of gene repression in somatic cell lineages. In mice, reactivation of genes on the Xi accompanies the establishment of pluripotent cells of the female blastocyst and the development of primordial germ cells. Xi reactivation also occurs when pluripotency is established during the reprogramming of somatic cells to induced pluripotent stem cells. The mechanism of Xi reactivation has attracted increasing interest for studying changes in epigenetic patterns and for improving methods of cell reprogramming. Here, we review recent advances in the understanding of Xi reactivation during development and reprogramming and illustrate potential clinical applications.

  5. 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.

  6. Recombinant baculovirus isolation.

    PubMed

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

    2007-01-01

    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. Additionally, many systems require a plaque-assay to separate parental and recombinant virus prior to amplification and use of the recombinant virus. 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. 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.

  7. Genetic recombination. [Escherichia coli

    SciTech Connect

    Stahl, F.W.

    1987-02-01

    The molecular pathways of gene recombination are explored and compared in studies of the model organisms, Escherichia coli and phase lambda. In the discussion of data from these studies it seems that recombination varies with the genetic idiosyncrasies of the organism and may also vary within a single organism.

  8. 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.

  9. 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

  10. Enzymatically triggered rupture of polymersomes.

    PubMed

    Jang, Woo-Sik; Park, Seung Chul; Reed, Ellen H; Dooley, Kevin P; Wheeler, Samuel F; Lee, Daeyeon; Hammer, Daniel A

    2016-01-28

    Polymersomes are robust vesicles made from amphiphilic block co-polymers. Large populations of uniform giant polymersomes with defined, entrapped species can be made by templating of double-emulsions using microfluidics. In the present study, a series of two enzymatic reactions, one inside and the other outside of the polymersome, were designed to induce rupture of polymersomes. We measured how the kinetics of rupture were affected by altering enzyme concentration. These results suggest that protocells with entrapped enzymes can be engineered to secrete contents on cue.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. Multiple Peroxisomal Enzymatic Deficiency Disorders

    PubMed Central

    Vamecq, Joseph; Draye, Jean-Pierre; Van Hoof, François; Misson, Jean-Paul; Evrard, Philippe; Verellen, Gaston; Eyssen, Hendrik J.; Van Eldere, Johan; Schutgens, Ruud B. H.; Wanders, Ronald J. A.; Roels, Frank; Goldfischer, Sidney L.

    1986-01-01

    Biologic, morphologic, and biochemical investigations performed in 2 patients demonstrate multiple peroxisomal deficiencies in the cerebrohepatorenal syndrome of Zellweger (CHRS) and neonatal adrenoleukodystrophy (NALD). Very long chain fatty acids, abnormal bile acids, including bile acid precursors (di- and trihydroxycoprostanoic acids), and C29-dicarboxylic acid accumulated in plasma in both patients. Generalized hyperaminoaciduria was also present. Peroxisomes could not be detected in CHRS liver and kidney; however, in the NALD patient, small and sparse cytoplasmic bodies resembling altered peroxisomes were found in hepatocytes. Hepatocellular and Kupffer cell lysosomes were engorged with ferritin and contained clefts and trilaminar structures believed to represent very long chain fatty acids. Enzymatic deficiencies reflected the peroxisomal defects. Hepatic glycolate oxidase and palmitoyl-CoA oxidase activities were deficient. No particle-bound catalase was found in cultured fibroblasts, and ether glycerolipid (plasmalogen) biosynthesis was markedly reduced. Administration of phenobarbital and clofibrate, an agent that induces peroxisomal proliferation and enzymatic activities, to the NALD patient did not bring about any changes in plasma metabolites, liver peroxisome population, or oxidizing activities. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5 PMID:2879480

  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 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.

  18. 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

    2015-01-01

    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

  19. 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

    2015-01-01

    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.

  20. 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. PMID:20214593

  1. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  4. An antiapoptotic role for telomerase RNA in human immune cells independent of telomere integrity or telomerase enzymatic activity

    PubMed Central

    Gazzaniga, Francesca S.

    2014-01-01

    Telomerase is a ribonucleoprotein complex that adds telomeric DNA to the ends of linear chromosomes. It contains two core canonical components: the essential RNA component, hTR, which provides the template for DNA synthesis, and the reverse transcriptase protein component, hTERT. Low telomerase activity in circulating peripheral blood mononuclear cells has been associated with a variety of diseases. It is unknown, however, whether telomerase, in addition to its long-term requirement for telomere maintenance, is also necessary for short-term immune cell proliferation and survival. We report that overexpression of enzymatically inactive hTR mutants protected against dexamethasone-induced apoptosis in stimulated CD4 T cells. Furthermore, hTR knockdown reproducibly induced apoptosis in the absence of any detectable telomere shortening or DNA damage response. In contrast, hTERT knockdown did not induce apoptosis. Strikingly, overexpression of hTERT protein caused apoptosis that was rescued by overexpression of enzymatically inactive hTR mutants. Hence, we propose that hTR can function as a noncoding RNA that protects from apoptosis independent of its function in telomerase enzymatic activity and long-term telomere maintenance in normal human immune cells. These results imply that genetic or environmental factors that alter hTR levels can directly affect immune cell function to influence health and disease. PMID:25320237

  5. Recombinant bovine rhodanese: purification and comparison with bovine liver rhodanese.

    PubMed

    Miller, D M; Kurzban, G P; Mendoza, J A; Chirgwin, J M; Hardies, S C; Horowitz, P M

    1992-06-24

    Recombinant bovine rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) has been purified to homogeneity from Escherichia coli BL21(DE3) by cation-exchange chromatography. Recombinant and bovine liver rhodanese coelectrophorese under denaturing conditions, with an apparent subunit molecular weight of 33,000. The amino terminal seven residues of the recombinant protein are identical to those of the bovine enzyme, indicating that E. coli also removes the N-terminal methionine. The Km for thiosulfate is the same for the two proteins. The specific activity of the recombinant enzyme is 12% higher (816 IU/mg) than that of the bovine enzyme (730 IU/mg). The two proteins are indistinguishable as to their ultraviolet absorbance and their intrinsic fluorescence. The ability of the two proteins to refold from 8 M urea to enzymatically active species was similar both for unassisted refolding, and when folding was assisted either by the detergent, lauryl maltoside or by the E. coli chaperonin system composed of cpn60 and cpn10. Bovine rhodanese is known to have multiple electrophoretic forms under native conditions. In contrast, the recombinant protein has only one form, which comigrates with the least negatively charged of the bovine liver isoforms. This is consistent with the retention of the carboxy terminal residues in the recombinant protein that are frequently removed from the bovine liver protein.

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. Activated recombinant adenovirus proteinases

    SciTech Connect

    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.

  17. 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.

  18. 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

  19. 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...

  20. 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...

  1. 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...

  2. 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...

  3. 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...

  4. 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...

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

    PubMed

    Min, Jin-Young; Min, Kyoung-Bok

    2016-01-01

    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. PMID:26797622

  6. 10 CFR 40.2a - Coverage of inactive tailings sites.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Coverage of inactive tailings sites. (a) Prior to the completion of the remedial action, the Commission will not require a license pursuant to 10 CFR chapter I for possession of residual radioactive... 10 Energy 1 2011-01-01 2011-01-01 false Coverage of inactive tailings sites. 40.2a Section...

  7. 10 CFR 40.2a - Coverage of inactive tailings sites.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Coverage of inactive tailings sites. (a) Prior to the completion of the remedial action, the Commission will not require a license pursuant to 10 CFR chapter I for possession of residual radioactive... 10 Energy 1 2012-01-01 2012-01-01 false Coverage of inactive tailings sites. 40.2a Section...

  8. 10 CFR 40.2a - Coverage of inactive tailings sites.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Coverage of inactive tailings sites. (a) Prior to the completion of the remedial action, the Commission will not require a license pursuant to 10 CFR chapter I for possession of residual radioactive... 10 Energy 1 2010-01-01 2010-01-01 false Coverage of inactive tailings sites. 40.2a Section...

  9. 10 CFR 40.2a - Coverage of inactive tailings sites.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Coverage of inactive tailings sites. (a) Prior to the completion of the remedial action, the Commission will not require a license pursuant to 10 CFR chapter I for possession of residual radioactive... 10 Energy 1 2014-01-01 2014-01-01 false Coverage of inactive tailings sites. 40.2a Section...

  10. Predictors of physical inactivity among elderly malaysians: recommendations for policy planning.

    PubMed

    Kaur, Jasvindar; Kaur, Gurpreet; Ho, Bee Kiau; Yao, Weng Keong; Salleh, Mohmad; Lim, Kuang Hock

    2015-04-01

    Physical inactivity is the fourth leading risk factor for global mortality. Regular moderate-intensity physical activity has significant benefits for health. To determine the socioeconomic predictors of physical inactivity among elderly Malaysian population. A nationwide community-based survey was conducted among 4831 respondents aged ≥60 years with a face-to-face questionnaire. The prevalence of physical inactivity among the elderly was 88.0%, highest in respondents aged older than 80 years (95.4%), females (90.1%), other Bumiputra (92.2%), earning household income less than RM1000 (87.9%), and residing in urban locality (88.4%). In the multivariate model, the predictors of physical inactivity were only sex, ethnicity, locality, and age group (adjusted odds ratio = 1.3-3.6). The predictors of physical inactivity can identify the risk factors to develop policies that will reduce the public health burden of noncommunicable diseases. PMID:24425796

  11. 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

  12. The prognosis and management of inactive HBV carriers.

    PubMed

    Invernizzi, Federica; Viganò, Mauro; Grossi, Glenda; Lampertico, Pietro

    2016-01-01

    Patients with chronic hepatitis B virus (HBV) infection lacking the serum hepatitis B e antigen (HBeAg) and with antibodies against HBeAg (anti-HBe), are the prevalent subgroup of HBV carriers worldwide. The prognosis of these patients is different from inactive carriers (ICs), who are characterized by persistently normal serum alanine aminotransferase (ALT) and low (<2000 IU/ml) serum HBV DNA levels, a serological profile that may also be intermittently observed in patients with HBeAg-negative chronic hepatitis. This is why a confirmed diagnosis of IC requires quarterly ALT and HBV DNA measurements for at least 1 year, while a single-point detection of combined HBsAg <1000 IU/ml and HBV DNA <2000 IU/ml has a robust predictive value for the diagnosis of IC. Characteristically, ICs have minimal or no histological lesions of the liver corresponding to liver stiffness values on Fibroscan of <5 kPa. Antiviral treatment is not indicated in ICs since the prognosis for the progression of liver disease is favourable if there are no cofactors of liver damage such as alcohol abuse, excess weight or co-infection with the hepatitis C virus or delta virus. Moreover, spontaneous HBsAg loss frequently occurs (1-1.9% per year) in these patients while the development of hepatocellular carcinoma (HCC) is rare, at least in Caucasian patients. However, an emerging issue reinforcing the need for clinical surveillance of ICs is the risk of HBV reactivation in patients who undergo immunosuppressive therapy without receiving appropriate antiviral prophylaxis. After diagnosis, management of ICs includes monitoring of ALT and HBV DNA every 12 months with periodic measurement of serum HBsAg levels to identify viral clearance. PMID:26725905

  13. 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

  14. 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

  15. 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.

  16. Light-driven Enzymatic Decarboxylation.

    PubMed

    Köninger, Katharina; Grote, Marius; Zachos, Ioannis; Hollmann, Frank; Kourist, Robert

    2016-01-01

    Oxidoreductases belong to the most-applied industrial enzymes. Nevertheless, they need external electrons whose supply is often costly and challenging. Recycling of the electron donors NADH or NADPH requires the use of additional enzymes and sacrificial substrates. Interestingly, several oxidoreductases accept hydrogen peroxide as electron donor. While being inexpensive, this reagent often reduces the stability of enzymes. A solution to this problem is the in situ generation of the cofactor. The continuous supply of the cofactor at low concentration drives the reaction without impairing enzyme stability. This paper demonstrates a method for the light-catalyzed in situ generation of hydrogen peroxide with the example of the heme-dependent fatty acid decarboxylase OleTJE. The fatty acid decarboxylase OleTJE was discovered due to its unique ability to produce long-chain 1-alkenes from fatty acids, a hitherto unknown enzymatic reaction. 1-alkenes are widely used additives for plasticizers and lubricants. OleTJE has been shown to accept electrons from hydrogen peroxide for the oxidative decarboxylation. While addition of hydrogen peroxide damages the enzyme and results in low yields, in situ generation of the cofactor circumvents this problem. The photobiocatalytic system shows clear advantages regarding enzyme activity and yield, resulting in a simple and efficient system for fatty acid decarboxylation.

  17. Light-driven Enzymatic Decarboxylation

    PubMed Central

    Köninger, Katharina; Grote, Marius; Zachos, Ioannis; Hollmann, Frank; Kourist, Robert

    2016-01-01

    Oxidoreductases belong to the most-applied industrial enzymes. Nevertheless, they need external electrons whose supply is often costly and challenging. Recycling of the electron donors NADH or NADPH requires the use of additional enzymes and sacrificial substrates. Interestingly, several oxidoreductases accept hydrogen peroxide as electron donor. While being inexpensive, this reagent often reduces the stability of enzymes. A solution to this problem is the in situ generation of the cofactor. The continuous supply of the cofactor at low concentration drives the reaction without impairing enzyme stability. This paper demonstrates a method for the light-catalyzed in situ generation of hydrogen peroxide with the example of the heme-dependent fatty acid decarboxylase OleTJE. The fatty acid decarboxylase OleTJE was discovered due to its unique ability to produce long-chain 1-alkenes from fatty acids, a hitherto unknown enzymatic reaction. 1-alkenes are widely used additives for plasticizers and lubricants. OleTJE has been shown to accept electrons from hydrogen peroxide for the oxidative decarboxylation. While addition of hydrogen peroxide damages the enzyme and results in low yields, in situ generation of the cofactor circumvents this problem. The photobiocatalytic system shows clear advantages regarding enzyme activity and yield, resulting in a simple and efficient system for fatty acid decarboxylation. PMID:27286035

  18. Enzymatic modification of flaxseed fibers.

    PubMed

    Maijala, Pekka; Mäkinen, Marliina; Galkin, Sari; Fagerstedt, Kurt; Härkäsalmi, Tiina; Viikari, Liisa

    2012-11-01

    Flaxseed (Linum usitatissimum L.) fibers were modified by oxidoreductive and cellulolytic enzymes. The lignin amount and intrinsic plant peroxidase activity was evaluated by histochemical and spectrophotometric assays. Peroxidase activity was not found from bast fibers. The flaxseed fibers were further separated and treated with laccase to conjugate the model compounds, that is, the hydrophobic gallate molecules on fiber surfaces. Laccase was able to slowly oxidize fiber-conjugated phenolics, but no fundamental changes in fiber cell surface structure or notable coupling of the applied hydrophobic gallate molecules onto the fibers occurred, as revealed by Fourier transform infrared spectroscopy. The reactivity of the mature fibers was further investigated using cellulolytic enzymes. Cellobiohydrolase (CBH) and endoglucanase (EG)-rich enzyme preparations were applied to reach a hydrolysis degree of 1-6% (of the fiber dry matter) using a standard enzyme dosage. The CBH mixture altered the fiber surface morphology distinctly, and SEM images illustrated fibers in which the cellulose fibrils seemed to be loosened and partially hydrolyzed. In contrast, the effect of the EG-rich preparation without CBH activity was notable on the fiber surface, polishing the surfaces. The cellulolytic treatments were potentially interesting for specific enzymatic modifications of flax fiber surfaces, whereas the approach to use oxidoreductive enzyme treatments on mature linseed fibers offered little potential, obviously due to the low lignin content of the fibers.

  19. Light-driven Enzymatic Decarboxylation.

    PubMed

    Köninger, Katharina; Grote, Marius; Zachos, Ioannis; Hollmann, Frank; Kourist, Robert

    2016-01-01

    Oxidoreductases belong to the most-applied industrial enzymes. Nevertheless, they need external electrons whose supply is often costly and challenging. Recycling of the electron donors NADH or NADPH requires the use of additional enzymes and sacrificial substrates. Interestingly, several oxidoreductases accept hydrogen peroxide as electron donor. While being inexpensive, this reagent often reduces the stability of enzymes. A solution to this problem is the in situ generation of the cofactor. The continuous supply of the cofactor at low concentration drives the reaction without impairing enzyme stability. This paper demonstrates a method for the light-catalyzed in situ generation of hydrogen peroxide with the example of the heme-dependent fatty acid decarboxylase OleTJE. The fatty acid decarboxylase OleTJE was discovered due to its unique ability to produce long-chain 1-alkenes from fatty acids, a hitherto unknown enzymatic reaction. 1-alkenes are widely used additives for plasticizers and lubricants. OleTJE has been shown to accept electrons from hydrogen peroxide for the oxidative decarboxylation. While addition of hydrogen peroxide damages the enzyme and results in low yields, in situ generation of the cofactor circumvents this problem. The photobiocatalytic system shows clear advantages regarding enzyme activity and yield, resulting in a simple and efficient system for fatty acid decarboxylation. PMID:27286035

  20. [Telomere Recombination in Normal Mammalian Cells].

    PubMed

    Zhdanova, N S; Rubtsov, N B

    2016-01-01

    Two mechanisms of telomere length maintenance are known to date. The first includes the use of a special enzymatic telomerase complex to solve the problems that arise during the replication of linear DNA in a normal diploid and part of tumor cells. Alternative lengthening of telomeres (ALT), which is based on the homologous recombination of telomere DNA, represents the second mechanism. Until recently, ALT was assumed to be expressed only in 15-20% of tumors lacking active telomerase and, together with telomerase reactivation represented one of two possibilities to overcome the replicative senescence observed in somatic mammalian cells due to aging or during cell culturing in vitro. Previously described sporadic cases of combinations of the two mechanisms of telomere length maintenance in several cell lines in vitro were attributed to the experimental design rather than to a real biological phenomenon, since active cellular division without active telomerase was considered to be the "gold standard" of ALT. The present review describes the morphological and functional reorganizations of mammalian telomeres observed with ALT activation, as well as recently observed,and well-documented cases of combinations between ALT-like and telomerase-dependent mechanisms in mammalian cells. The possible role of telomere recombination in telomerase-dependent cells is discussed.

  1. Meiotic recombination mechanisms.

    PubMed

    Grelon, Mathilde

    2016-01-01

    Meiosis is a specialized cell division at the origin of the haploid cells that eventually develop into the gametes. It therefore lies at the heart of Mendelian heredity. Recombination and redistribution of the homologous chromosomes arising during meiosis constitute an important source of genetic diversity, conferring to meiosis a particularly important place in the evolution and the diversification of the species. Our understanding of the molecular mechanisms governing meiotic recombination has considerably progressed these last decades, benefiting from complementary approaches led on various model species. An overview of these mechanisms will be provided as well as a discussion on the implications of these recent discoveries. PMID:27180110

  2. 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

  3. 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...

  4. Chemo-Enzymatic Synthesis of Oligoglycerol Derivatives.

    PubMed

    Singh, Abhishek K; Nguyen, Remi; Galy, Nicolas; Haag, Rainer; Sharma, Sunil K; Len, Christophe

    2016-08-09

    A cleaner and greener method has been developed and used to synthesize 14 different functionalized oligomer derivatives of glycerol in moderate 29%-39% yields over three steps. After successive regioselective enzymatic acylation of the primary hydroxyl groups, etherification or esterification of the secondary hydroxyl groups and chemoselective enzymatic saponification, the target compounds can efficiently be used as versatile building blocks in organic and supramolecular chemistry.

  5. Chemo-Enzymatic Synthesis of Oligoglycerol Derivatives.

    PubMed

    Singh, Abhishek K; Nguyen, Remi; Galy, Nicolas; Haag, Rainer; Sharma, Sunil K; Len, Christophe

    2016-01-01

    A cleaner and greener method has been developed and used to synthesize 14 different functionalized oligomer derivatives of glycerol in moderate 29%-39% yields over three steps. After successive regioselective enzymatic acylation of the primary hydroxyl groups, etherification or esterification of the secondary hydroxyl groups and chemoselective enzymatic saponification, the target compounds can efficiently be used as versatile building blocks in organic and supramolecular chemistry. PMID:27517886

  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. Concerted and differential actions of two enzymatic domains underlie Rad5 contributions to DNA damage tolerance.

    PubMed

    Choi, Koyi; Batke, Sabrina; Szakal, Barnabas; Lowther, Jonathan; Hao, Fanfan; Sarangi, Prabha; Branzei, Dana; Ulrich, Helle D; Zhao, Xiaolan

    2015-03-11

    Many genome maintenance factors have multiple enzymatic activities. In most cases, how their distinct activities functionally relate with each other is unclear. Here we examined the conserved budding yeast Rad5 protein that has both ubiquitin ligase and DNA helicase activities. The Rad5 ubiquitin ligase activity mediates PCNA poly-ubiquitination and subsequently recombination-based DNA lesion tolerance. Interestingly, the ligase domain is embedded in a larger helicase domain comprising seven consensus motifs. How features of the helicase domain influence ligase function is controversial. To clarify this issue, we use genetic, 2D gel and biochemical analyses and show that a Rad5 helicase motif important for ATP binding is also required for PCNA poly-ubiquitination and recombination-based lesion tolerance. We determine that this requirement is due to a previously unrecognized contribution of the motif to the PCNA and ubiquitination enzyme interaction, and not due to its canonical role in supporting helicase activity. We further show that Rad5's helicase-mediated contribution to replication stress survival is separable from recombination. These findings delineate how two Rad5 enzymatic domains concertedly influence PCNA modification, and unveil their discrete contributions to stress tolerance.

  8. Effect of tamoxifen on the enzymatic activity of human cytochrome CYP2B6.

    PubMed

    Sridar, Chitra; Kent, Ute M; Notley, Lisa M; Gillam, Elizabeth M J; Hollenberg, Paul F

    2002-06-01

    Tamoxifen is primarily used in the treatment of breast cancer. It has been approved as a chemopreventive agent for individuals at high risk for this disease. Tamoxifen is metabolized to a number of different products by cytochrome P450 enzymes. The effect of tamoxifen on the enzymatic activity of bacterially expressed human cytochrome CYP2B6 in a reconstituted system has been investigated. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of purified CYP2B6 was inactivated by tamoxifen in a time- and concentration-dependent manner. Enzymatic activity was lost only in samples that were incubated with both tamoxifen and NADPH. The inactivation was characterized by a K(I) of 0.9 microM, a k(inact) of 0.02 min(-1), and a t(1/2) of 34 min. The loss in the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity did not result in a similar percentage loss in the reduced carbon monoxide spectrum, suggesting that the heme moiety was not the major site of modification. The activity of CYP2B6 was not recovered after removal of free tamoxifen using spin column gel filtration. The loss in activity seemed to be due to a modification of the CYP2B6 and not reductase because adding fresh reductase back to the inactivated samples did not restore enzymatic activity. A reconstituted system containing purified CYP2B6, NADPH-reductase, and NADPH-generating system was found to catalyze tamoxifen metabolism to 4-OH-tamoxifen, 4'-OH-tamoxifen, and N-desmethyl-tamoxifen as analyzed by high-performance liquid chromatography analysis. Preliminary studies showed that tamoxifen had no effect on the activities of CYP1B1 and CYP3A4, whereas CYP2D6 and CYP2C9 exhibited a 25% loss in enzymatic activity. PMID:12023523

  9. 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…

  10. Recombineering Pseudomonas syringae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we report the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecE...

  11. 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.

  12. 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

  13. 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. PMID:23766548

  14. 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.

  15. 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.

  16. 2014 consensus statement from the first Economics of Physical Inactivity Consensus (EPIC) conference (Vancouver).

    PubMed

    Davis, Jennifer C; Verhagen, Evert; Bryan, Stirling; Liu-Ambrose, Teresa; Borland, Jeff; Buchner, David; Hendriks, Marike R C; Weiler, Richard; Morrow, James R; van Mechelen, Willem; Blair, Steven N; Pratt, Mike; Windt, Johann; al-Tunaiji, Hashel; Macri, Erin; Khan, Karim M

    2014-06-01

    This article describes major topics discussed from the 'Economics of Physical Inactivity Consensus Workshop' (EPIC), held in Vancouver, Canada, in April 2011. Specifically, we (1) detail existing evidence on effective physical inactivity prevention strategies; (2) introduce economic evaluation and its role in health policy decisions; (3) discuss key challenges in establishing and building health economic evaluation evidence (including accurate and reliable costs and clinical outcome measurement) and (4) provide insight into interpretation of economic evaluations in this critically important field. We found that most methodological challenges are related to (1) accurately and objectively valuing outcomes; (2) determining meaningful clinically important differences in objective measures of physical inactivity; (3) estimating investment and disinvestment costs and (4) addressing barriers to implementation. We propose that guidelines specific for economic evaluations of physical inactivity intervention studies are developed to ensure that related costs and effects are robustly, consistently and accurately measured. This will also facilitate comparisons among future economic evidence.

  17. 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

  18. 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...

  19. 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.

  20. 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...

  1. 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

  2. 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

  3. 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

  4. Physical Inactivity and Related Barriers: A Study in a Community Dwelling of Older Brazilians

    PubMed Central

    Gobbi, Sebastião; Sebastião, Emerson; Papini, Camila Bosquiero; Nakamura, Priscila Missaki; Valdanha Netto, Américo; Gobbi, Lilian Teresa Bucken; Kokubun, Eduardo

    2012-01-01

    This study sought to investigate the prevalence of physical inactivity and related barriers in older Brazilian adults. A cross-sectional, population-based study was conducted, and a stratified random sampling procedure was used. A total of 359 older adults were interviewed. The long version of the International Physical Activity Questionnaire (IPAQ) and the Questionnaire of Barriers to Physical Activity Practice were used to assess physical activity level and barriers, respectively. No statistically significant difference was observed on the prevalence of physical inactivity in either gender or age groups. Regarding barriers, the proportion of 9 out of 22 barriers was statistically significant between men and women. Self-reported physical inactivity/activity in older Brazilian adults continues to be a concern. Uncommonly, older males reported a higher prevalence of physical inactivity compared to their counterparts. Additionally, physical inactivity prevalence continued to increase with the aging process. Yet, personal barriers such as lack of time and poor health were strongly associated with physical inactivity. The results of this study may help health professionals and public policy makers to better address the issues related to a healthy lifestyle among older adults and promote physical activity among Brazilian older adults and in other countries with similar characteristics. PMID:23209906

  5. 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

  6. Proteolytically inactive insulin-degrading enzyme inhibits amyloid formation yielding non-neurotoxic aβ peptide aggregates.

    PubMed

    de Tullio, Matias B; Castelletto, Valeria; Hamley, Ian W; Martino Adami, Pamela V; Morelli, Laura; Castaño, Eduardo M

    2013-01-01

    Insulin-degrading enzyme (IDE) is a neutral Zn(2+) peptidase that degrades short peptides based on substrate conformation, size and charge. Some of these substrates, including amyloid β (Aβ) are capable of self-assembling into cytotoxic oligomers. Based on IDE recognition mechanism and our previous report of the formation of a stable complex between IDE and intact Aβ in vitro and in vivo, we analyzed the possibility of a chaperone-like function of IDE. A proteolytically inactive recombinant IDE with Glu111 replaced by Gln (IDEQ) was used. IDEQ blocked the amyloidogenic pathway of Aβ yielding non-fibrillar structures as assessed by electron microscopy. Measurements of the kinetics of Aβ aggregation by light scattering showed that 1) IDEQ effect was promoted by ATP independent of its hydrolysis, 2) end products of Aβ-IDEQ co-incubation were incapable of "seeding" the assembly of monomeric Aβ and 3) IDEQ was ineffective in reversing Aβ aggregation. Moreover, Aβ aggregates formed in the presence of IDEQ were non-neurotoxic. IDEQ had no conformational effects upon insulin (a non-amyloidogenic protein under physiological conditions) and did not disturb insulin receptor activation in cultured cells. Our results suggest that IDE has a chaperone-like activity upon amyloid-forming peptides. It remains to be explored whether other highly conserved metallopeptidases have a dual protease-chaperone function to prevent the formation of toxic peptide oligomers from bacteria to mammals. PMID:23593132

  7. 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.

  8. 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.

  9. 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

  10. 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.

  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. Chloroplast-Based Expression of Recombinant Proteins by Gateway® Cloning Technology.

    PubMed

    Gottschamel, Johanna; Lössl, Andreas

    2016-01-01

    Plastid transformation for the expression of recombinant proteins and entire enzymatic pathways has become a promising tool for plant biotechnology in the past decade. Several improvements of the technology have turned plant plastids into robust and dependable expression platforms for multiple high value compounds. In this chapter, we describe our current methodology based on Gateway(®) recombinant cloning, which we have adapted for plastid transformation. We describe the steps required for cloning, biolistic transformation, identification, and regeneration of transplastomic plant lines and Western blot analysis. PMID:26614278

  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. 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

  15. 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.

  16. 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

    2016-01-01

    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

  17. 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

  18. Dielectronic recombination theory

    SciTech Connect

    LaGattuta, K.J.

    1991-12-31

    A theory now in wide use for the calculation of dielectronic recombination cross sections ({sigma}{sup DR}) and rate coefficients ({alpha}{sup DR}) was one introduced originally by Feshbach for nuclear physics applications, and then later adapted for atomic scattering problems by Hahn. In the following, we briefly review this theory in a very general form, which allows one to account for the effects of overlapping and interacting resonances, as well as continuum-continuum coupling. An extension of our notation will then also allow for the inclusion of the effects of direct radiative recombination, along with a treatment of the interference between radiative and dielectronic recombination. Other approaches to the calculation of {sigma}{sup DR} have been described by Fano and by Seaton. We will not consider those theories here. Calculations of {alpha}{sup DR} have progressed considerably over the last 25 years, since the early work of Burgess. Advances in the reliability of theoretical predictions have also been promoted recently b a variety of direct laboratory measurements of {sigma}{sup DR}. While the measurements of {sigma}{sup DR} for {delta}n {ne} 0 excitations have tended to agree very well with calculations, the case of {delta}n = 0 has been much problematic. However, by invoking a mechanism originally proposed by Jacobs, which takes into account the effect of stray electric fields on high Rydberg states (HRS) participating in the DR process, new calculations have improved the agreement between theory and experiment for these cases. Nevertheless, certain discrepancies still remain.

  19. 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.

  20. 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%. PMID:26264414

  1. Pathway Engineered Enzymatic de novo Purine Nucleotide Synthesis

    PubMed Central

    Schultheisz, Heather L.; Szymczyna, Blair R.; Scott, Lincoln G.; Williamson, James R.

    2009-01-01

    A general method for isotopic labeling of the purine base moiety of nucleotides and RNA has been developed through biochemical pathway engineering in vitro. A synthetic scheme was designed and implemented utilizing recombinant enzymes from the pentose phosphate and de novo purine synthesis pathways, with regeneration of folate, aspartate, glutamine, ATP, and NADPH cofactors, in a single-pot reaction. Syntheses proceeded quickly and efficiently in comparison to chemical methods with isolated yields up to 66% for 13C, 15N enriched ATP and GTP. The scheme is robust and flexible, requiring only serine, NH4+, glucose and CO2 as stoichiometric precursors in labeled form. Using this approach, U-13C- GTP, U-13C,15N- GTP, 13C2,8- ATP and U-15N- GTP were synthesized on a millimole scale, and the utility of the isotope labeling is illustrated in NMR spectra of HIV-2 transactivation region (TAR) RNA containing 13C 2,8-adenosine and 15N-1,3,7,9,2-guanosine. Pathway engineering in vitro permits complex synthetic cascades to be effected expanding the applicability of enzymatic synthesis. PMID:18707057

  2. Innovation by homologous recombination.

    PubMed

    Trudeau, Devin L; Smith, Matthew A; Arnold, Frances H

    2013-12-01

    Swapping fragments among protein homologs can produce chimeric proteins with a wide range of properties, including properties not exhibited by the parents. Computational methods that use information from structures and sequence alignments have been used to design highly functional chimeras and chimera libraries. Recombination has generated proteins with diverse thermostability and mechanical stability, enzyme substrate specificity, and optogenetic properties. Linear regression, Gaussian processes, and support vector machine learning have been used to model sequence-function relationships and predict useful chimeras. These approaches enable engineering of protein chimeras with desired functions, as well as elucidation of the structural basis for these functions.

  3. 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…

  4. Starch: chemistry, microstructure, processing and enzymatic degradation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Starch is recognized as one of the most abundant and important commodities containing value added attributes for a vast number of industrial applications. Its chemistry, structure, property and susceptibility to various chemical, physical and enzymatic modifications offer a high technological value ...

  5. Inhibition of enzymatic cellulolysis by phenolic compounds.

    PubMed

    Tejirian, Ani; Xu, Feng

    2011-03-01

    Phenolics derived from lignin and other plant components can pose significant inhibition on enzymatic conversion of cellulosic biomass materials to useful chemicals. Understanding the mechanism of such inhibition is of importance for the development of viable biomass conversion technologies. In native plant cell wall, most of the phenolics and derivatives are found in polymeric lignin. When biomass feedstocks are pretreated (prior to enzymatic hydrolysis), simple or oligomeric phenolics and derivatives are often generated from lignin modification/degradation, which can inhibit biomass-converting enzymes. To further understand how such phenolic substances may affect cellulase reaction, we carried out a comparative study on a series of simple and oligomeric phenolics representing or mimicking the composition of lignin or its degradation products. Consistent to previous studies, we observed that oligomeric phenolics could exert more inhibition on enzymatic cellulolysis than simple phenolics. Oligomeric phenolics could inactivate cellulases by reversibly complexing them. Simple and oligomeric phenolics could also inhibit enzymatic cellulolysis by adsorbing onto cellulose. Individual cellulases showed different susceptibility toward these inhibitions. Polyethylene glycol and tannase could respectively bind and degrade the studied oligomeric phenolics, and by doing so mitigate the oligomeric phenolic's inhibition on cellulolysis. PMID:22112906

  6. 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…

  7. Ultrasonic acceleration of enzymatic processing of cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  8. 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..

  9. High level expression in Escherichia coli of soluble, enzymatically active schistosomal hypoxanthine/guanine phosphoribosyltransferase and trypanosomal ornithine decarboxylase.

    PubMed Central

    Craig, S P; Yuan, L; Kuntz, D A; McKerrow, J H; Wang, C C

    1991-01-01

    The bacterial alkaline phosphatase (phoA) promoter and signal peptide have been used previously to control recombinant expression and secretion of eukaryotic proteins in Escherichia coli. Other reports have shown that this expression system can generate relatively modest levels of active hypoxanthine/guanine phosphoribosyltransferase (HPRT; hypoxanthine phosphoribosyltransferase; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), which carries part of the signal peptide but remains in the cytosol of the bacteria. Herein, the phoA promoter without its associated signal peptide is used to regulate expression of the HPRT of Schistosoma mansoni and the ornithine decarboxylase (ODC; L-ornithine carboxy-lyase, EC 4.1.1.17) of Trypanosoma brucei, two enzymes that have been identified as potential targets for antiparasitic chemotherapy. The levels of recombinant expression range from 20% to 60% of the total bacterial protein, and the majority of both recombinant enzymes was soluble. The specific activity for the recombinant trypanosomal ODC was one-third to two-thirds that of the authentic native enzyme and yields were predicted to be 15-30 mg of active enzyme per liter of bacterial culture. The specific activity for the recombinant schistosomal HPRT was equivalent to that for the native enzyme purified from schistosomes and up to 10 mg of enzymatically active HPRT has been purified from a 0.5-liter culture of treated bacteria. These results represent a break-through in recombinant expression of HPRT and ODC. Images PMID:2006185

  10. Expression and in vitro assembly of recombinant glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.

    PubMed Central

    Diruggiero, J; Robb, F T

    1995-01-01

    The gdhA gene, encoding the hexameric glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus furiosus, was expressed in Escherichia coli by using the pET11-d system. The recombinant GDH was soluble and constituted 15% of the E. coli cell extract. The N-terminal amino acid sequence of the recombinant protein was identical to the sequence of the P. furiosus enzyme, except for the presence of an initial methionine which was absent from the enzyme purified from P. furiosus. By molecular exclusion chromatography we showed that the recombinant GDH was composed of equal amounts of monomeric and hexameric forms. Heat treatment of the recombinant protein triggered in vitro assembly of inactive monomers into hexamers, resulting in increased GDH activity. The specific activity of the recombinant enzyme, purified by heat treatment and affinity chromatography, was equivalent to that of the native enzyme from P. furiosus. The recombinant GDH displayed a slightly lower level of thermostability, with a half-life of 8 h at 100 degrees C, compared with 10.5 h for the enzyme purified from P. furiosus. PMID:7887598

  11. Bioinspired enzymatic synthesis of silica nanocrystals provided by recombinant silicatein from the marine sponge Latrunculia oparinae.

    PubMed

    Shkryl, Yury N; Bulgakov, Victor P; Veremeichik, Galina N; Kovalchuk, Svetlana N; Kozhemyako, Valery B; Kamenev, Dmitrii G; Semiletova, Irina V; Timofeeva, Yana O; Shchipunov, Yury A; Kulchin, Yury N

    2016-01-01

    The process of silica formation in marine sponges is thought to be mediated by a family of catalytically active structure-directing enzymes called silicateins. It has been demonstrated in biomimicking syntheses that silicateins facilitated the formation of amorphous SiO2. Here, we present evidence that the silicatein LoSiLA1 from the marine sponge Latrunculia oparinae catalyzes the in vitro synthesis of hexa-tetrahedral SiO2 crystals of 200–300 nm. This was possible in the presence of the silica precursor tetrakis-(2-hydroxyethyl)-orthosilicate that is completely soluble in water and biocompatible, experiences hydrolysis–condensation at neutral pH and ambient conditions.

  12. 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.

  13. 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. PMID:16546698

  14. Establishment of X chromosome inactivation and epigenomic features of the inactive X depend on cellular contexts.

    PubMed

    Vallot, Céline; Ouimette, Jean-François; Rougeulle, Claire

    2016-09-01

    X chromosome inactivation (XCI) is an essential epigenetic process that ensures X-linked gene dosage equilibrium between sexes in mammals. XCI is dynamically regulated during development in a manner that is intimately linked to differentiation. Numerous studies, which we review here, have explored the dynamics of X inactivation and reactivation in the context of development, differentiation and diseases, and the phenotypic and molecular link between the inactive status, and the cellular context. Here, we also assess whether XCI is a uniform mechanism in mammals by analyzing epigenetic signatures of the inactive X (Xi) in different species and cellular contexts. It appears that the timing of XCI and the epigenetic signature of the inactive X greatly vary between species. Surprisingly, even within a given species, various Xi configurations are found across cellular states. We discuss possible mechanisms underlying these variations, and how they might influence the fate of the Xi.

  15. 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.

  16. Dual-wavelength polymer laser based on an active/inactive/active sandwich-like structure

    NASA Astrophysics Data System (ADS)

    Zhai, Tianrui; Wu, Xiaofeng; Wang, Meng; Tong, Fei; Li, Songtao; Ma, Yanbin; Deng, Jinxiang; Zhang, Xinping

    2016-09-01

    Dual-wavelength laser emission is achieved by using an active/inactive/active sandwich-like structure, which can be conveniently fabricated using spin coating technique. Poly [(9, 9-dioctylfluorenyl-2, 7-diyl)-alt-co-(1, 4-benzo-(2, 1', 3) -thiadiazole)] and polyvinyl alcohol are employed as the active and the inactive materials, respectively. Two laser wavelengths are simultaneously observed, which are attributed to the difference of the surrounding refractive index of two active waveguides in the sandwich-like structure. Each wavelength is controlled by the respective waveguide structure, meaning that multi-wavelength laser can be designed by stacking the active/inactive layer pair. These results provide more flexibility to design compact laser sources.

  17. Role of Active and Inactive Cytotoxic Immune Response in Human Immunodeficiency Virus Dynamics

    PubMed Central

    Toro Zapata, Hernan Dario; Caicedo Casso, Angelica Graciela; Bichara, Derdei; Lee, Sunmi

    2014-01-01

    Objectives Mathematical models can be helpful to understand the complex dynamics of human immunodeficiency virus infection within a host. Most of work has studied the interactions of host responses and virus in the presence of active cytotoxic immune cells, which decay to zero when there is no virus. However, recent research highlights that cytotoxic immune cells can be inactive but never be depleted. Methods We propose a mathematical model to investigate the human immunodeficiency virus dynamics in the presence of both active and inactive cytotoxic immune cells within a host. We explore the impact of the immune responses on the dynamics of human immunodeficiency virus infection under different disease stages. Results Standard mathematical and numerical analyses are presented for this new model. Specifically, the basic reproduction number is computed and local and global stability analyses are discussed. Conclusion Our results can give helpful insights when designing more effective drug schedules in the presence of active and inactive immune responses. PMID:24955306

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. Linking Geology and Microbiology: Inactive Pockmarks Affect Sediment Microbial Community Structure

    PubMed Central

    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. PMID:24475066

  3. 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

  4. 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.

  5. Lysostaphin: use of a recombinant bactericidal enzyme as a mastitis therapeutic.

    PubMed

    Oldham, E R; Daley, M J

    1991-12-01

    A recombinant mucolytic protein, lysostaphin, was evaluated as a potential intramammary therapeutic for Staphylococcus aureus mastitis in dairy cattle. Lysostaphin, a product of Staphylococcus simulans, enzymatically degrades the cell wall of Staphylococcus aureus and is bactericidal. Thirty Holstein-Freisian dairy cattle in their first lactation were infected with Staphylococcus aureus (Newbould 305, ATCC 29740) in all quarters. Infections were established and monitored for somatic cell counts and Staphylococcus aureus colony-forming units 3 wk prior to subsequent treatment. Infected animals were injected through the teat canal with a single dose of recombinant lysostaphin (dose response 1 to 500 mg) or after three successive p.m. milkings with 100 mg of recombinant lysostaphin in 60 ml of sterile phosphate-buffered saline. Animals were considered cured if the milk remained free of Staphylococcus aureus for a total of 28 milkings after last treatment. Kinetic analysis of immunologically active recombinant lysostaphin demonstrated that a minimum bactericidal concentration was maintained in the milk for up to 36 to 48 h after a single infusion of 100 mg of recombinant lysostaphin. The cure rate of quarters receiving recombinant lysostaphin (100 mg in sterile phosphate-buffered saline, administered over three consecutive p.m. milkings) was 20% compared with 29% for sodium cephapirin in saline and 57% for a commercial antibiotic formulation, respectively. An improved formulation of recombinant lysostaphin may prove to be an effective alternative to antibiotic therapy for bovine mastitis.

  6. From physical inactivity to immobilization: Dissecting the role of oxidative stress in skeletal muscle insulin resistance and atrophy.

    PubMed

    Pierre, Nicolas; Appriou, Zephyra; Gratas-Delamarche, Arlette; Derbré, Frédéric

    2016-09-01

    In the literature, the terms physical inactivity and immobilization are largely used as synonyms. The present review emphasizes the need to establish a clear distinction between these two situations. Physical inactivity is a behavior characterized by a lack of physical activity, whereas immobilization is a deprivation of movement for medical purpose. In agreement with these definitions, appropriate models exist to study either physical inactivity or immobilization, leading thereby to distinct conclusions. In this review, we examine the involvement of oxidative stress in skeletal muscle insulin resistance and atrophy induced by, respectively, physical inactivity and immobilization. A large body of evidence demonstrates that immobilization-induced atrophy depends on the chronic overproduction of reactive oxygen and nitrogen species (RONS). On the other hand, the involvement of RONS in physical inactivity-induced insulin resistance has not been investigated. This observation outlines the need to elucidate the mechanism by which physical inactivity promotes insulin resistance.

  7. 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

  8. "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.

  9. Unraveling recombination rate evolution using ancestral recombination maps

    PubMed Central

    Munch, Kasper; Schierup, Mikkel H; Mailund, Thomas

    2014-01-01

    Recombination maps of ancestral species can be constructed from comparative analyses of genomes from closely related species, exemplified by a recently published map of the human-chimpanzee ancestor. Such maps resolve differences in recombination rate between species into changes along individual branches in the speciation tree, and allow identification of associated changes in the genomic sequences. We describe how coalescent hidden Markov models are able to call individual recombination events in ancestral species through inference of incomplete lineage sorting along a genomic alignment. In the great apes, speciation events are sufficiently close in time that a map can be inferred for the ancestral species at each internal branch - allowing evolution of recombination rate to be tracked over evolutionary time scales from speciation event to speciation event. We see this approach as a way of characterizing the evolution of recombination rate and the genomic properties that influence it. PMID:25043668

  10. 37 CFR 11.58 - Duties of disciplined or resigned practitioner, or practitioner on disability inactive status.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... OF OTHERS BEFORE THE UNITED STATES PATENT AND TRADEMARK OFFICE Investigations and Disciplinary... practitioner transferred to disability inactive status does not: (i) Communicate directly in writing,...

  11. Characterization of Enzymatic Activity of MlrB and MlrC Proteins Involved in Bacterial Degradation of Cyanotoxins Microcystins

    PubMed Central

    Dziga, Dariusz; Zielinska, Gabriela; Wladyka, Benedykt; Bochenska, Oliwia; Maksylewicz, Anna; Strzalka, Wojciech; Meriluoto, Jussi

    2016-01-01

    Bacterial degradation of toxic microcystins produced by cyanobacteria is a common phenomenon. However, our understanding of the mechanisms of these processes is rudimentary. In this paper several novel discoveries regarding the action of the enzymes of the mlr cluster responsible for microcystin biodegradation are presented using recombinant proteins. In particular, the predicted active sites of the recombinant MlrB and MlrC were analyzed using functional enzymes and their inactive muteins. A new degradation intermediate, a hexapeptide derived from linearized microcystins by MlrC, was discovered. Furthermore, the involvement of MlrA and MlrB in further degradation of the hexapeptides was confirmed and a corrected biochemical pathway of microcystin biodegradation has been proposed. PMID:26999203

  12. 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

  13. Primordial magnetogenesis before recombination

    NASA Astrophysics Data System (ADS)

    Fabre, Ophélia; Shankaranarayanan, S.

    2016-04-01

    The origin of large magnetic fields in the Universe remains currently unknown. We investigate here a mechanism before recombination based on known physics. The source of the vorticity is due to the changes in the photon distribution function caused by the fluctuations in the background photons. We show that the magnetic field generated in the MHD limit, due to the Coulomb scattering, is of the order 10-49 G on a coherence scale of 10 kpc. We explicitly show that the magnetic fields generated from this process are sustainable and are not erased by resistive diffusion. We compare the results with current observations and discuss the implications. Our seed magnetic fields are generated on small scales whereas the main mechanisms studied in the literature are on scale bigger than 1 Mpc. However, compared to more exotic theories generating seed magnetic fields on similar scales, the strength of our fields are generally smaller.

  14. Recombinant Human Erythropoietin

    PubMed Central

    Bartels, Claudia; Späte, Kira; Krampe, Henning

    2008-01-01

    Treatment of multiple sclerosis (MS) is still unsatisfactory and essentially non-existing for the progressive course of the disease. Recombinant human erythropoietin (EPO) may be a promising neuroprotective/neuroregenerative treatment of MS. In the nervous system, EPO acts anti-apoptotic, antioxidative, anti-inflammatory, neurotrophic and plasticity-modulating. Beneficial effects have been shown in animal models of various neurological and psychiatric diseases, including different models of experimental autoimmune encephalomyelitis. EPO is also effective in human brain disease, as shown in double-blind placebo-controlled clinical studies on ischemic stroke and chronic schizophrenia. An exploratory study on chronic progressive MS yielded lasting improvement in motor and cognitive performance upon high-dose long-term EPO treatment. PMID:21180577

  15. Recombinant glucose uptake system

    DOEpatents

    Ingrahm, Lonnie O.; Snoep, Jacob L.; Arfman, Nico

    1997-01-01

    Recombinant organisms are disclosed that contain a pathway for glucose uptake other than the pathway normally utilized by the host cell. In particular, the host cell is one in which glucose transport into the cell normally is coupled to PEP production. This host cell is transformed so that it uses an alternative pathway for glucose transport that is not coupled to PEP production. In a preferred embodiment, the host cell is a bacterium other than Z. mobilis that has been transformed to contain the glf and glk genes of Z. mobilis. By uncoupling glucose transport into the cell from PEP utilization, more PEP is produced for synthesis of products of commercial importance from a given quantity of biomass supplied to the host cells.

  16. The recombination of genetic material

    SciTech Connect

    Low, K.B.

    1988-01-01

    Genetic recombination is the major mechanism by which new arrangements of genetic elements are produced in all living organisms, from the simplest bacterial viruses to humans. This volume presents an overview of the types of recombination found in prokaryotes and eukaryotes.

  17. 38 CFR 3.375 - Determination of inactivity (complete arrest) in tuberculosis.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 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...

  18. 38 CFR 4.89 - Ratings for inactive nonpulmonary tuberculosis in effect on August 19, 1968.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... nonpulmonary tuberculosis in effect on August 19, 1968. 4.89 Section 4.89 Pensions, Bonuses, and Veterans... tuberculosis in effect on August 19, 1968. Public Law 90-493 repealed section 356 of title 38, United States Code which provided graduated ratings for inactive tuberculosis. The repealed section, however,...

  19. 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...

  20. 38 CFR 4.89 - Ratings for inactive nonpulmonary tuberculosis in effect on August 19, 1968.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... nonpulmonary tuberculosis in effect on August 19, 1968. 4.89 Section 4.89 Pensions, Bonuses, and Veterans... tuberculosis in effect on August 19, 1968. Public Law 90-493 repealed section 356 of title 38, United States Code which provided graduated ratings for inactive tuberculosis. The repealed section, however,...

  1. 38 CFR 4.89 - Ratings for inactive nonpulmonary tuberculosis in effect on August 19, 1968.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... nonpulmonary tuberculosis in effect on August 19, 1968. 4.89 Section 4.89 Pensions, Bonuses, and Veterans... tuberculosis in effect on August 19, 1968. Public Law 90-493 repealed section 356 of title 38, United States Code which provided graduated ratings for inactive tuberculosis. The repealed section, however,...

  2. 38 CFR 4.89 - Ratings for inactive nonpulmonary tuberculosis in effect on August 19, 1968.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... nonpulmonary tuberculosis in effect on August 19, 1968. 4.89 Section 4.89 Pensions, Bonuses, and Veterans... tuberculosis in effect on August 19, 1968. Public Law 90-493 repealed section 356 of title 38, United States Code which provided graduated ratings for inactive tuberculosis. The repealed section, however,...

  3. 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...

  4. 38 CFR 4.89 - Ratings for inactive nonpulmonary tuberculosis in effect on August 19, 1968.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... nonpulmonary tuberculosis in effect on August 19, 1968. 4.89 Section 4.89 Pensions, Bonuses, and Veterans... tuberculosis in effect on August 19, 1968. Public Law 90-493 repealed section 356 of title 38, United States Code which provided graduated ratings for inactive tuberculosis. The repealed section, however,...

  5. 38 CFR 3.375 - Determination of inactivity (complete arrest) in tuberculosis.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 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...

  6. 38 CFR 3.375 - Determination of inactivity (complete arrest) in tuberculosis.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 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...

  7. Physical inactivity as the culprit of metabolic inflexibility: evidence from bed-rest studies.

    PubMed

    Bergouignan, Audrey; Rudwill, Floriane; Simon, Chantal; Blanc, Stéphane

    2011-10-01

    Although it is no longer debatable that sedentary behaviors are an actual cause of many metabolic diseases, the physiology of physical inactivity has been poorly investigated for this purpose. Along with microgravity, the physiological adaptations to spaceflights require metabolic adaptations to physical inactivity, and that is exceedingly well-simulated during the ground-based microgravity bed-rest analogs. Bed rest thus represents a unique model to investigate the mechanisms by which physical inactivity leads to the development of current societal chronic diseases. For decades, however, clinicians and physiologists working in space research have worked separately without taking full awareness of potential strong mutual questioning. This review summarizes the data collected over the last 60 years on metabolic adaptations to bed rest in healthy subjects. Our aim is to provide evidence that supports the hypothesis that physical inactivity per se is one of the primary causes in the development of metabolic inflexibility. This evidence will focus on four main tenants of metabolic inflexiblity: 1) insulin resistance, 2) impaired lipid trafficking and hyperlipidemia, 3) a shift in substrate use toward glucose, and 4) a shift in muscle fiber type and ectopic fat storage. Altogether, this hypothesis places sedentary behaviors upstream on the list of factors involved in metabolic inflexibility, which is considered to be a primary impairment in several metabolic disorders such as obesity, insulin resistance, and type 2 diabetes mellitus. PMID:21836047

  8. Emotional Outlook on Life Predicts Increases in Physical Activity among Initially Inactive Men

    ERIC Educational Resources Information Center

    Baruth, Meghan; Lee, Duck-Chul; Sui, Xuemei; Church, Timothy S.; Marcus, Bess H.; Wilcox, Sara; Blair, Steven N.

    2011-01-01

    This study examined the relationship between emotional outlook on life and change in physical activity among inactive adults in the Aerobics Center Longitudinal Study. A total of 2,132 sedentary adults completed a baseline medical examination and returned for a follow-up examination at least 6 months later. Participants self-reported physical…

  9. 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.

  10. Coronary Heart Disease Risk between Active and Inactive Women with Multiple Sclerosis.

    ERIC Educational Resources Information Center

    Slawta, Jennifer N.; McCubbin, Jeffrey A.; Wilcox, Anthony R.; Fox, Susan D.; Nalle, Darek J.; Anderson, Gail

    2002-01-01

    Investigated whether abdominal fat accumulation and levels of triglyceride, high-density lipoprotein cholesterol, and glucose differed between 123 active and inactive women with multiple sclerosis (MS). Results indicated that low-to-moderate leisure time physical activity significantly related to less abdominal fat accumulation, lower triglyceride…

  11. Social Cognitive Correlates of Physical Activity in Inactive Adults with Multiple Sclerosis

    ERIC Educational Resources Information Center

    Dlugonski, Deirdre; Wojcicki, Thomas R.; McAuley, Edward; Motl, Robert W.

    2011-01-01

    Persons with multiple sclerosis (MS) are often physically inactive. This observation has prompted the search for modifiable constructs derived from established theories that act as correlates of physical activity. This study investigated self efficacy, outcome expectations, impediments, and goal setting as correlates of physical activity in…

  12. 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...

  13. 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...

  14. 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...

  15. 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...

  16. 9 CFR 105.4 - Termination of licenses and permits for inactivity.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS SUSPENSION, REVOCATION, OR TERMINATION OF BIOLOGICAL LICENSES OR PERMITS § 105.4 Termination of licenses and permits for inactivity. (a) If a biological product has not been prepared by a licensee, or imported by...

  17. 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…

  18. 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…

  19. 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…

  20. Physical inactivity and obesity: Using a novel environmental quality measure to control confounding

    EPA Science Inventory

    Physical inactivity is well-established as a contributor to obesity prevalence in the US. Many aspects of the ambient environment (e.g., air pollution, food deserts, neighborhood socioeconomics) have also been associated with obesity. Yet, controlling for the overall ambient envi...

  1. Bimolecular recombination in organic photovoltaics.

    PubMed

    Lakhwani, Girish; Rao, Akshay; Friend, Richard H

    2014-01-01

    The recombination of electrons and holes is a major loss mechanism in photovoltaic devices that controls their performance. We review scientific literature on bimolecular recombination (BR) in bulk heterojunction organic photovoltaic devices to bring forward existing ideas on the origin and nature of BR and highlight both experimental and theoretical work done to quantify its extent. For these systems, Langevin theory fails to explain BR, and recombination dynamics turns out to be dependent on mobility, temperature, electric field, charge carrier concentration, and trapped charges. Relationships among the photocurrent, open-circuit voltage, fill factor, and morphology are discussed. Finally, we highlight the recent emergence of a molecular-level picture of recombination, taking into account the spin and delocalization of charges. Together with the macroscopic picture of recombination, these new insights allow for a comprehensive understanding of BR and provide design principles for future materials and devices.

  2. Enzymatic polymerization of dihydroquercetin using bilirubin oxidase.

    PubMed

    Khlupova, M E; Vasil'eva, I S; Shumakovich, G P; Morozova, O V; Chertkov, V A; Shestakova, A K; Kisin, A V; Yaropolov, A I

    2015-02-01

    Dihydroquercetin (or taxifolin) is one of the most famous flavonoids and is abundant in Siberian larch (Larix sibirica). The oxidative polymerization of dihydroquercetin (DHQ) using bilirubin oxidase as a biocatalyst was investigated and some physicochemical properties of the products were studied. DHQ oligomers (oligoDHQ) with molecular mass of 2800 and polydispersity of 8.6 were obtained by enzymatic reaction under optimal conditions. The oligomers appeared to be soluble in dimethylsulfoxide, dimethylformamide, and methanol. UV-visible spectra of oligoDHQ in dimethylsulfoxide indicated the presence of highly conjugated bonds. The synthesized oligoDHQ was also characterized by FTIR and (1)H and (13)C NMR spectroscopy. Comparison of NMR spectra of oligoDHQ with DHQ monomer and the parent flavonoids revealed irregular structure of a polymer formed via the enzymatic oxidation of DHQ followed by nonselective radical polymerization. As compared with the monomer, oligoDHQ demonstrated higher thermal stability and high antioxidant activity.

  3. 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

  4. 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.

  5. Reactor optimization for enzymatic hydrolysis of cellulose

    SciTech Connect

    Lee, Y.H.; Gharpuray, M.M.; Fan, L.T.

    1982-01-01

    Enzymatic hydrolysis of cellulose furnishes sugar which can be subsequently fermented to ethanol. The production of such sugar at relatively low cost is essential for commercially viable production of ethanol. Many processes have been developed for converting cellulosic materials to sugar, and their economic feasibility has been analyzed; however, relatively little has been done to optimize such processes. A comprehensive mechanistic kinetic model for enzymatic degradation was established previously; it takes into account the structure of cellulose, mode of action of celluloytic enzyme, and mode of interaction between the enzyme and the cellulosic substrate. In the present work this model has been applied to the optimal design of cellulose hydrloysis reactors. Both batch and continously stirred reactors have been considered for this purpose. The fractional contributions of various cost parameters to the production cost have been estimated. The sensitivity of sugar cost to the important cost parameters, such as raw material and enzyme costs, have been examined. 8 figures, 7 tables.

  6. 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.

  7. 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. PMID:26854948

  8. 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. PMID:25832672

  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. Microbial antigenic variation mediated by homologous DNA recombination

    PubMed Central

    Vink, Cornelis; Rudenko, Gloria; Seifert, H. Steven

    2012-01-01

    Pathogenic microorganisms employ numerous molecular strategies in order to delay or circumvent recognition by the immune system of their host. One of the most widely used strategies of immune evasion is antigenic variation, in which immunogenic molecules expressed on the surface of a microorganism are continuously modified. As a consequence, the host is forced to constantly adapt its humoral immune response against this pathogen. An antigenic change thus provides the microorganism with an opportunity to persist and/or replicate within the host (population) for an extended period of time or to effectively infect a previously infected host. In most cases, antigenic variation is caused by genetic processes that lead to modification of the amino acid sequence of a particular antigen or to alterations in the expression of biosynthesis genes that induce changes in expression of a variant antigen. Here, we will review antigenic variation systems that rely on homologous DNA recombination and which are found in a wide range of cellular, human pathogens, including bacteria (such as Neisseria spp., Borrelia spp., Treponema pallidum and Mycoplasma spp.), fungi (like Pneumocystis carinii) and parasites (such as the African trypanosome Trypanosoma brucei). Specifically, the various DNA recombination-based antigenic variation systems will be discussed with a focus on the employed mechanisms of recombination, the DNA substrates, and the enzymatic machinery involved. PMID:22212019

  11. 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. PMID:22476233

  12. Retroviral recombination during reverse transcription.

    PubMed

    Goodrich, D W; Duesberg, P H

    1990-03-01

    After mixed infection, up to half of related retroviruses are recombinants. During infection, retroviral RNA genomes are first converted to complementary DNA (cDNA) and then to double-stranded DNA. Thus recombination could occur during reverse transcription, by RNA template switching, or after reverse transcription, by breakage and reunion of DNA. It has not been possible to distinguish between these two potential mechanisms of recombination because both single-stranded cDNA and double-stranded proviral DNA exist in infected cells during the eclipse period. Therefore we have analyzed for recombinant molecules among cDNA products transcribed in vitro from RNA of disrupted virions. Since recombinants from allelic parents can only be distinguished from parental genomes by point mutations, we have examined the cDNAs from virions with distinct genetic structures for recombinant-specific size and sequence markers. The parents share a common internal allele that allows homology-directed recombination, but each contains specific flanking sequences. One parent is a synthetically altered Harvey murine sarcoma virus RNA that lacks a retroviral 3' terminus but carries a Moloney murine retrovirus-derived envelope gene (env) fragment 3' of its transforming ras gene. The other parent is intact Moloney virus. Using a Harvey-specific 5' primer and a Moloney-specific 3' primer, we have found recombinant cDNAs with the polymerase chain reaction, proving directly that retroviruses can recombine during reverse transcription unassisted by cellular enzymes, probably by template switching during cDNA synthesis. The recombinants that were obtained in vitro were identical with those obtained in parallel experiments in vivo.

  13. Staphylococcus simulans Recombinant Lysostaphin: Production, Purification, and Determination of Antistaphylococcal Activity.

    PubMed

    Boksha, I S; Lavrova, N V; Grishin, A V; Demidenko, A V; Lyashchuk, A M; Galushkina, Z M; Ovchinnikov, R S; Umyarov, A M; Avetisian, L R; Chernukha, M Iu; Shaginian, I A; Lunin, V G; Karyagina, A S

    2016-05-01

    Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by Sigma-Aldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity. PMID:27297900

  14. Why are Jupiter-family comets active and asteroids in cometary-like orbits inactive?. How hydrostatic compression leads to inactivity

    NASA Astrophysics Data System (ADS)

    Gundlach, B.; Blum, J.

    2016-05-01

    Context. Surveys in the visible and near-infrared spectral range have revealed the presence of low-albedo asteroids in cometary-like orbits (ACOs). In contrast to Jupiter-family comets (JFCs), ACOs are inactive, but possess similar orbital parameters. Aims: In this work, we discuss why ACOs are inactive, whereas JFCs show gas-driven dust activity, although both belong to the same class of primitive solar system bodies. Methods: We hypothesize that ACOs and JFCs have formed under the same physical conditions, namely by the gravitational collapse of ensembles of ice and dust aggregates. We use the memory effect of dust-aggregate layers under gravitational compression to discuss under which conditions the gas-driven dust activity of these bodies is possible. Results: Owing to their smaller sizes, JFCs can sustain gas-driven dust activity much longer than the bigger ACOs, whose sub-surface regions possess an increased tensile strength, due to gravitational compression of the material. The increased tensile strength leads to the passivation against dust activity after a relatively short time of activity. Conclusions: The gravitational-collapse model of the formation of planetesimals, together with the gravitational compression of the sub-surface material simultaneously, explains the inactivity of ACOs and the gas-driven dust activity of JFCs. Their initially larger sizes means that ACOs possess a higher tensile strength of their sub-surface material, which leads to a faster termination of gas-driven dust activity. Most objects with radii larger than 2 km have already lost their activity due to former gravitational compression of their current surface material.

  15. Criticality and Adaptivity in Enzymatic Networks.

    PubMed

    Steiner, Paul J; Williams, Ruth J; Hasty, Jeff; Tsimring, Lev S

    2016-09-01

    The contrast between the stochasticity of biochemical networks and the regularity of cellular behavior suggests that biological networks generate robust behavior from noisy constituents. Identifying the mechanisms that confer this ability on biological networks is essential to understanding cells. Here we show that queueing for a limited shared resource in broad classes of enzymatic networks in certain conditions leads to a critical state characterized by strong and long-ranged correlations between molecular species. An enzymatic network reaches this critical state when the input flux of its substrate is balanced by the maximum processing capacity of the network. We then consider enzymatic networks with adaptation, when the limiting resource (enzyme or cofactor) is produced in proportion to the demand for it. We show that the critical state becomes an attractor for these networks, which points toward the onset of self-organized criticality. We suggest that the adaptive queueing motif that leads to significant correlations between multiple species may be widespread in biological systems. PMID:27602735

  16. 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.

  17. Criticality and Adaptivity in Enzymatic Networks.

    PubMed

    Steiner, Paul J; Williams, Ruth J; Hasty, Jeff; Tsimring, Lev S

    2016-09-01

    The contrast between the stochasticity of biochemical networks and the regularity of cellular behavior suggests that biological networks generate robust behavior from noisy constituents. Identifying the mechanisms that confer this ability on biological networks is essential to understanding cells. Here we show that queueing for a limited shared resource in broad classes of enzymatic networks in certain conditions leads to a critical state characterized by strong and long-ranged correlations between molecular species. An enzymatic network reaches this critical state when the input flux of its substrate is balanced by the maximum processing capacity of the network. We then consider enzymatic networks with adaptation, when the limiting resource (enzyme or cofactor) is produced in proportion to the demand for it. We show that the critical state becomes an attractor for these networks, which points toward the onset of self-organized criticality. We suggest that the adaptive queueing motif that leads to significant correlations between multiple species may be widespread in biological systems.

  18. 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

  19. Physical Inactivity Among Adults Aged 50 Years and Older - United States, 2014.

    PubMed

    Watson, Kathleen B; Carlson, Susan A; Gunn, Janelle P; Galuska, Deborah A; O'Connor, Ann; Greenlund, Kurt J; Fulton, Janet E

    2016-01-01

    Physical activity can help delay, prevent, or manage many of the chronic diseases for which adults aged ≥50 years are at risk (1-3). These diseases can impact the length and quality of life, as well as the long-term ability to live independently.* All adults aged ≥50 years, with or without chronic disease, gain health benefits by avoiding inactivity (2,3). To examine the prevalence of inactivity by selected demographic characteristics and chronic disease status in mid-life and older adults, CDC analyzed data on adults aged ≥50 years from the 2014 Behavioral Risk Factor Surveillance System (BRFSS). Overall, 27.5% of adults aged ≥50 years reported no physical activity outside of work during the past month. Inactivity prevalence significantly increased with increasing age and was 25.4% among adults aged 50-64 years, 26.9% among those aged 65-74 years, and 35.3% among those aged ≥75 years. Inactivity prevalence was significantly higher among women than men, among Hispanics and non-Hispanic blacks than among non-Hispanic whites, and among adults who reported ever having one or more of seven selected chronic diseases than among those not reporting one. Inactivity prevalence significantly increased with decreasing levels of education and increasing body mass index. To help adults with and without chronic disease start or maintain an active lifestyle, communities can implement evidence-based strategies, such as creating or enhancing access to places for physical activity, designing communities and streets to encourage physical activity, and offering programs that address specific barriers to physical activity. PMID:27632143

  20. Impact of Physical Inactivity on Mortality in Patients With Heart Failure.

    PubMed

    Doukky, Rami; Mangla, Ashvarya; Ibrahim, Zeina; Poulin, Marie-France; Avery, Elizabeth; Collado, Fareed M; Kaplan, Jonathan; Richardson, DeJuran; Powell, Lynda H

    2016-04-01

    The impact of physical inactivity on heart failure (HF) mortality is unclear. We analyzed data from the HF Adherence and Retention Trial (HART) which enrolled 902 patients with New York Heart Association class II/III HF, with preserved or reduced ejection fraction, who were followed for 36 months. On the basis of mean self-reported weekly exercise duration, patients were classified into inactive (0 min/week) and active (≥1 min/week) groups and then propensity score matched according to 34 baseline covariates in 1:2 ratio. Sedentary activity was determined according to self-reported daily television screen time (<2, 2 to 4, >4 h/day). The primary outcome was all-cause death. Secondary outcomes were cardiac death and HF hospitalization. There were 196 inactive patients, of whom 171 were propensity matched to 342 active patients. Physical inactivity was associated with greater risk of all-cause death (hazard ratio [HR] 2.01, confidence interval [CI] 1.47 to 3.00; p <0.001) and cardiac death (HR 2.01, CI 1.28 to 3.17; p = 0.002) but no significant difference in HF hospitalization (p = 0.548). Modest exercise (1 to 89 min/week) was associated with a significant reduction in the rate of death (p = 0.003) and cardiac death (p = 0.050). Independent of exercise duration and baseline covariates, television screen time (>4 vs <2 h/day) was associated with all-cause death (HR 1.65, CI 1.10 to 2.48; p = 0.016; incremental chi-square = 6.05; p = 0.049). In conclusion, in patients with symptomatic chronic HF, physical inactivity is associated with higher all-cause and cardiac mortality. Failure to exercise and television screen time are additive in their effects on mortality. Even modest exercise was associated with survival benefit. PMID:26853954

  1. Body image emotions, perceptions, and cognitions distinguish physically active and inactive smokers

    PubMed Central

    Contreras, Gisèle A.; Sabiston, Catherine M.; O'Loughlin, Erin K.; Bélanger, Mathieu; O'Loughlin, Jennifer

    2015-01-01

    Objectives To determine if body image emotions (body-related shame and guilt, weight-related stress), perceptions (self-perceived overweight), or cognitions (trying to change weight) differ between adolescents characterized by smoking and physical activity (PA) behavior. Methods Data for this cross-sectional analysis were collected in 2010–11 and were available for 1017 participants (mean (SD) age = 16.8 (0.5) years). Participants were categorized according to smoking and PA status into four groups: inactive smokers, inactive non-smokers, active smokers and active non-smokers. Associations between body image emotions, perceptions and cognitions, and group membership were estimated in multinomial logistic regression. Results Participants who reported body-related shame were less likely (OR (95% CI) = 0.52 (0.29–0.94)) to be in the active smoker group than the inactive smoker group; those who reported body-related guilt and those trying to gain weight were more likely (2.14 (1.32–3.48) and 2.49 (1.22–5.08), respectively) to be in the active smoker group than the inactive smoker group; those who were stressed about weight and those perceiving themselves as overweight were less likely to be in the active non-smoker group than the inactive smoker group (0.79 (0.64–0.97) and 0.41 (0.19–0.89), respectively). Conclusion Body image emotions and cognitions differentiated the active smoker group from the other three groups. PMID:26844062

  2. Impact of Physical Inactivity on Mortality in Patients With Heart Failure.

    PubMed

    Doukky, Rami; Mangla, Ashvarya; Ibrahim, Zeina; Poulin, Marie-France; Avery, Elizabeth; Collado, Fareed M; Kaplan, Jonathan; Richardson, DeJuran; Powell, Lynda H

    2016-04-01

    The impact of physical inactivity on heart failure (HF) mortality is unclear. We analyzed data from the HF Adherence and Retention Trial (HART) which enrolled 902 patients with New York Heart Association class II/III HF, with preserved or reduced ejection fraction, who were followed for 36 months. On the basis of mean self-reported weekly exercise duration, patients were classified into inactive (0 min/week) and active (≥1 min/week) groups and then propensity score matched according to 34 baseline covariates in 1:2 ratio. Sedentary activity was determined according to self-reported daily television screen time (<2, 2 to 4, >4 h/day). The primary outcome was all-cause death. Secondary outcomes were cardiac death and HF hospitalization. There were 196 inactive patients, of whom 171 were propensity matched to 342 active patients. Physical inactivity was associated with greater risk of all-cause death (hazard ratio [HR] 2.01, confidence interval [CI] 1.47 to 3.00; p <0.001) and cardiac death (HR 2.01, CI 1.28 to 3.17; p = 0.002) but no significant difference in HF hospitalization (p = 0.548). Modest exercise (1 to 89 min/week) was associated with a significant reduction in the rate of death (p = 0.003) and cardiac death (p = 0.050). Independent of exercise duration and baseline covariates, television screen time (>4 vs <2 h/day) was associated with all-cause death (HR 1.65, CI 1.10 to 2.48; p = 0.016; incremental chi-square = 6.05; p = 0.049). In conclusion, in patients with symptomatic chronic HF, physical inactivity is associated with higher all-cause and cardiac mortality. Failure to exercise and television screen time are additive in their effects on mortality. Even modest exercise was associated with survival benefit.

  3. Cold enzymatic bleaching of fluid whey.

    PubMed

    Campbell, R E; Drake, M A

    2013-01-01

    Chemical bleaching of fluid whey and retentate with hydrogen peroxide (HP) alone requires high concentrations (100-500 mg of HP/kg) and recent studies have demonstrated that off-flavors are generated during chemical bleaching that carry through to spray-dried whey proteins. Bleaching of fluid whey and retentate with enzymes such as naturally present lactoperoxidase or an exogenous commercial peroxidase (EP) at cold temperatures (4°C) may be a viable alternative to traditional chemical bleaching of whey. The objective of this study was to determine the optimum level of HP for enzymatic bleaching (both lactoperoxidase and EP) at 4°C and to compare bleaching efficacy and sensory characteristics to HP chemical bleaching at 4°C. Selected treatments were subsequently applied for whey protein concentrate with 80% protein (WPC80) manufacture. Fluid Cheddar whey and retentate (80% protein) were manufactured in triplicate from pasteurized whole milk. The optimum concentration of HP (0 to 250 mg/kg) to activate enzymatic bleaching at 4°C was determined by quantifying the loss of norbixin. In subsequent experiments, bleaching efficacy, descriptive sensory analysis, and volatile compounds were monitored at selected time points. A control with no bleaching was also evaluated. Enzymatic bleaching of fluid whey and retentate at 4°C resulted in faster bleaching and higher bleaching efficacy (color loss) than bleaching with HP alone at 250 mg/kg. Due to concentrated levels of naturally present lactoperoxidase, retentate bleached to completion (>80% norbixin destruction in 30 min) faster than fluid whey at 4°C (>80% norbixin destruction in 12h). In fluid whey, the addition of EP decreased bleaching time. Spray-dried WPC80 from bleached wheys, regardless of bleaching treatment, were characterized by a lack of sweet aromatic and buttery flavors, and the presence of cardboard flavor concurrent with higher relative abundance of 1-octen-3-ol and 1-octen-3-one. Among enzymatically

  4. Synthesizing a Cellulase like Chimeric Protein by Recombinant Molecular Biology Techniques

    PubMed Central

    Banerjee, Hirendra Nath; Krauss, Christopher; Smith, Valerie; Mahaffey, Kelly; Boston, Ava

    2016-01-01

    In order to meet the Renewable Fuels Standard demands for 30 billion gallons of biofuels by the end of 2020, new technologies for generation of cellulosic ethanol must be exploited. Breaking down cellulose by cellulase enzyme is very important for this purpose but this is not thermostable and degrades at higher temperatures in bioreactors. Towards creation of a more ecologically friendly method of rendering bioethanol from cellulosic waste, we attempted to produce recombinant higher temperature resistant cellulases for use in bioreactors. The project involved molecular cloning of genes for cellulose-degrading enzymes based on bacterial source, expressing the recombinant proteins in E. coli and optimizing enzymatic activity. We were able to generate in vitro bacterial expression systems to produce recombinant His-tag purified protein which showed cellulase like activity. PMID:27468362

  5. Recombinant human elastin-like magnetic microparticles for drug delivery and targeting.

    PubMed

    Ciofani, Gianni; Genchi, Giada Graziana; Guardia, Pablo; Mazzolai, Barbara; Mattoli, Virgilio; Bandiera, Antonella

    2014-05-01

    Bioinspired recombinant polypeptides represent a highly promising tool in biomedical research, being protein intrinsic constituents of both cells and their natural matrices. In this regard, a very interesting model is represented by polypeptides inspired by elastin, which naturally confers rubber-like elasticity to tissues, and is able to undergo wide deformations without rupture. In this paper, a microparticle system based on a recombinant human elastin-like polypeptide (HELP) is reported for drug delivery applications. HELP microparticles are prepared through a water-in-oil emulsion of an aqueous solution of recombinant polypeptide in isoctane, followed by enzymatic cross-linking. Superparamagnetic iron oxide nanoparticles are introduced in this system with the purpose of conferring magnetic properties to the microspheres, and thus controlling their targeting and tracking as drug vectors. The obtained microparticles are characterized in terms of morphology, structure, magnetic properties, drug release, and magnetic drivability, showing interesting and promising results for further biomedical applications. PMID:24318291

  6. Enhancement of xylose utilization from corn stover by a recombinant Escherichia coli strain for ethanol production.

    PubMed

    Saha, Badal C; Qureshi, Nasib; Kennedy, Gregory J; Cotta, Michael A

    2015-08-01

    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 coli (strain FBR5) were investigated. The initial ethanol productivity was faster for the seed grown on xylose followed by GXA, CSH, glucose and arabinose. Arabinose grown seed took the longest time to complete the fermentation. Delayed saccharifying enzyme addition in simultaneous saccharification and fermentation of dilute acid pretreated CS by the recombinant E. coli strain FBR5 allowed the fermentation to finish in a shorter time than adding the enzyme simultaneously with xylose grown inoculum. Use of substrate selective inoculum and fermenting pentose sugars first under glucose limited condition helped to alleviate the catabolite repression of the recombinant bacterium on ethanol production from lignocellulosic hydrolyzate.

  7. Places where preschoolers are (in)active: An observational study on Latino preschoolers and their parents using objective measures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To combat the disproportionately higher risk of childhood obesity in Latino preschool-aged children, multilevel interventions targeting physical (in)activity are needed. These require the identification of environmental and psychosocial determinants of physical (in)activity for this ethnic group. Th...

  8. 42 CFR 407.32 - Prejudice to enrollment rights because of Federal Government misrepresentation, inaction, or error.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... Government misrepresentation, inaction, or error. 407.32 Section 407.32 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES MEDICARE PROGRAM SUPPLEMENTARY MEDICAL INSURANCE... enrollment rights because of Federal Government misrepresentation, inaction, or error. If an...

  9. 42 CFR 407.32 - Prejudice to enrollment rights because of Federal Government misrepresentation, inaction, or error.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Government misrepresentation, inaction, or error. 407.32 Section 407.32 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES MEDICARE PROGRAM SUPPLEMENTARY MEDICAL INSURANCE... enrollment rights because of Federal Government misrepresentation, inaction, or error. If an...

  10. 42 CFR 407.32 - Prejudice to enrollment rights because of Federal Government misrepresentation, inaction, or error.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Government misrepresentation, inaction, or error. 407.32 Section 407.32 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES MEDICARE PROGRAM SUPPLEMENTARY MEDICAL INSURANCE... enrollment rights because of Federal Government misrepresentation, inaction, or error. If an...

  11. Probing kinetic drug binding mechanism in voltage-gated sodium ion channel: open state versus inactive state blockers.

    PubMed

    Pal, Krishnendu; Gangopadhyay, Gautam

    2015-01-01

    The kinetics and nonequilibrium thermodynamics of open state and inactive state drug binding mechanisms have been studied here using different voltage protocols in sodium ion channel. We have found that for constant voltage protocol, open state block is more efficient in blocking ionic current than inactive state block. Kinetic effect comes through peak current for mexiletine as an open state blocker and in the tail part for lidocaine as an inactive state blocker. Although the inactivation of sodium channel is a free energy driven process, however, the two different kinds of drug affect the inactivation process in a different way as seen from thermodynamic analysis. In presence of open state drug block, the process initially for a long time remains entropy driven and then becomes free energy driven. However in presence of inactive state block, the process remains entirely entropy driven until the equilibrium is attained. For oscillating voltage protocol, the inactive state blocking is more efficient in damping the oscillation of ionic current. From the pulse train analysis it is found that inactive state blocking is less effective in restoring normal repolarisation and blocks peak ionic current. Pulse train protocol also shows that all the inactive states behave differently as one inactive state responds instantly to the test pulse in an opposite manner from the other two states. PMID:26274618

  12. Probing kinetic drug binding mechanism in voltage-gated sodium ion channel: open state versus inactive state blockers

    PubMed Central

    Pal, Krishnendu; Gangopadhyay, Gautam

    2015-01-01

    The kinetics and nonequilibrium thermodynamics of open state and inactive state drug binding mechanisms have been studied here using different voltage protocols in sodium ion channel. We have found that for constant voltage protocol, open state block is more efficient in blocking ionic current than inactive state block. Kinetic effect comes through peak current for mexiletine as an open state blocker and in the tail part for lidocaine as an inactive state blocker. Although the inactivation of sodium channel is a free energy driven process, however, the two different kinds of drug affect the inactivation process in a different way as seen from thermodynamic analysis. In presence of open state drug block, the process initially for a long time remains entropy driven and then becomes free energy driven. However in presence of inactive state block, the process remains entirely entropy driven until the equilibrium is attained. For oscillating voltage protocol, the inactive state blocking is more efficient in damping the oscillation of ionic current. From the pulse train analysis it is found that inactive state blocking is less effective in restoring normal repolarisation and blocks peak ionic current. Pulse train protocol also shows that all the inactive states behave differently as one inactive state responds instantly to the test pulse in an opposite manner from the other two states. PMID:26274618

  13. 42 CFR 407.32 - Prejudice to enrollment rights because of Federal Government misrepresentation, inaction, or error.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... enrollment rights because of Federal Government misrepresentation, inaction, or error. If an individual's..., misrepresentation, on inaction of a Federal employee or any person authorized by the Federal Government to act in... 42 Public Health 2 2013-10-01 2013-10-01 false Prejudice to enrollment rights because of...

  14. 42 CFR 407.32 - Prejudice to enrollment rights because of Federal Government misrepresentation, inaction, or error.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... enrollment rights because of Federal Government misrepresentation, inaction, or error. If an individual's..., misrepresentation, on inaction of a Federal employee or any person authorized by the Federal Government to act in... 42 Public Health 2 2011-10-01 2011-10-01 false Prejudice to enrollment rights because of...

  15. Urban-Rural Differences in Overweight Status and Physical Inactivity among US Children Aged 10-17 Years

    ERIC Educational Resources Information Center

    Liu, Jihong; Bennett, Kevin J.; Harun, Nusrat; Probst, Janice C.

    2008-01-01

    Context: Few studies have examined the prevalence of overweight status and physical inactivity among children and adolescents living in rural America. Purpose: We examined urban and rural differences in the prevalence of overweight status and physical inactivity among US children. Methods: Data were drawn from the 2003 National Survey of…

  16. Delayed recombination and standard rulers

    SciTech Connect

    De Bernardis, Francesco; Melchiorri, Alessandro; Bean, Rachel; Galli, Silvia; Silk, Joseph I.; Verde, Licia

    2009-02-15

    Measurements of baryonic acoustic oscillations (BAOs) in galaxy surveys have been recognized as a powerful tool for constraining dark energy. However, this method relies on the knowledge of the size of the acoustic horizon at recombination derived from cosmic microwave background (CMB) anisotropy measurements. This estimate is typically derived assuming a standard recombination scheme; additional radiation sources can delay recombination altering the cosmic ionization history and the cosmological inferences drawn from CMB and BAO data. In this paper we quantify the effect of delayed recombination on the determination of dark energy parameters from future BAO surveys such as the Baryon Oscillation Spectroscopic Survey and the Wide-Field Multi-Object Spectrograph. We find the impact to be small but still not negligible. In particular, if recombination is nonstandard (to a level still allowed by CMB data), but this is ignored, future surveys may incorrectly suggest the presence of a redshift-dependent dark energy component. On the other hand, in the case of delayed recombination, adding to the analysis one extra parameter describing deviations from standard recombination does not significantly degrade the error bars on dark energy parameters and yields unbiased estimates. This is due to the CMB-BAO complementarity.

  17. Analysis of interchromosomal mitotic recombination.

    PubMed

    McGill, C B; Shafer, B K; Higgins, D R; Strathern, J N

    1990-07-01

    A novel synthetic locus is described that provides a simple assay system for characterizing mitotic recombinants. The locus consists of the TRP1 and HIS3 genes inserted into chromosome III of S. cerevisiae between the CRY1 and MAT loci. Defined trp1 and his3 alleles have been generated that allow the selection of interchromosomal recombinants in this interval. Trp+ or His+ recombinants can be divided into several classes based on coupling of the other alleles in the interval. The tight linkage of the CRY1 and MAT loci, combined with the drug resistance and cell type phenotypes that they respectively control, facilitates the classification of the recombinants without resorting to tetrad dissection. We present the distribution of spontaneous recombinants among the classes defined by this analysis. The data suggest that the recombination intermediate can have regions of symmetric strand exchange and that co-conversion tracts can extend over 1-3 kb. Continuous conversion tracts are favored over discontinuous tracts. The distribution among the classes defined by this analysis is altered in recombinants induced by UV irradiation.

  18. An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

    PubMed

    Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

    2015-09-01

    A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments. PMID:26021569

  19. Aspergillus: sex and recombination.

    PubMed

    Varga, János; Szigeti, Gyöngyi; Baranyi, Nikolett; Kocsubé, Sándor; O'Gorman, Céline M; Dyer, Paul S

    2014-12-01

    The genus Aspergillus is one of the most widespread groups of fungi on Earth, comprised of about 300-350 species with very diverse lifestyles. Most species produce asexual propagula (conidia) on conidial heads. Despite their ubiquity, a sexual cycle has not yet been identified for most of the aspergilli. Where sexual reproduction is present, species exhibit either homothallic (self fertile) or heterothallic (obligate outcrossing) breeding systems. A parasexual cycle has also been described in some Aspergillus species. As in other fungi, sexual reproduction is governed by mating-type (MAT) genes, which determine sexual identity and are involved in regulating later stages of sexual development. Previous population genetic studies have indicated that some supposedly asexual aspergilli exhibit evidence of a recombining population structure, suggesting the presence of a cryptic sexual cycle. In addition, genome analyses have revealed networks of genes necessary for sexual reproduction in several Aspergillus species, again consistent with latent sexuality in these fungi. Knowledge of MAT gene presence has then successfully been applied to induce sexual reproduction between MAT1-1 and MAT1-2 isolates of certain supposedly asexual aspergilli. Recent progress in understanding the extent and significance of sexual reproduction is described here, with special emphasis on findings that are relevant to clinically important aspergilli.

  20. Aspergillus: sex and recombination.

    PubMed

    Varga, János; Szigeti, Gyöngyi; Baranyi, Nikolett; Kocsubé, Sándor; O'Gorman, Céline M; Dyer, Paul S

    2014-12-01

    The genus Aspergillus is one of the most widespread groups of fungi on Earth, comprised of about 300-350 species with very diverse lifestyles. Most species produce asexual propagula (conidia) on conidial heads. Despite their ubiquity, a sexual cycle has not yet been identified for most of the aspergilli. Where sexual reproduction is present, species exhibit either homothallic (self fertile) or heterothallic (obligate outcrossing) breeding systems. A parasexual cycle has also been described in some Aspergillus species. As in other fungi, sexual reproduction is governed by mating-type (MAT) genes, which determine sexual identity and are involved in regulating later stages of sexual development. Previous population genetic studies have indicated that some supposedly asexual aspergilli exhibit evidence of a recombining population structure, suggesting the presence of a cryptic sexual cycle. In addition, genome analyses have revealed networks of genes necessary for sexual reproduction in several Aspergillus species, again consistent with latent sexuality in these fungi. Knowledge of MAT gene presence has then successfully been applied to induce sexual reproduction between MAT1-1 and MAT1-2 isolates of certain supposedly asexual aspergilli. Recent progress in understanding the extent and significance of sexual reproduction is described here, with special emphasis on findings that are relevant to clinically important aspergilli. PMID:25118872

  1. A Mononuclear Nonheme Iron(III)-Peroxo Complex Binding Redox-Inactive Metal Ions†

    PubMed Central

    Lee, Yong-Min; Bang, Suhee; Kim, Yun Mi; Cho, Jaeheung; Hong, Seungwoo; Nomura, Takashi; Ogura, Takashi; Troeppner, Oliver; Ivanović-Burmazović, Ivana

    2014-01-01

    Redox-inactive metal ions that function as Lewis acids play pivotal roles in modulating reactivities of oxygen-containing metal complexes in a variety of biological and biomimetic reactions, including dioxygen activation/formation and functionalization of organic substrates. Mononuclear nonheme iron(III)-peroxo species are invoked as active oxygen intermediates in the catalytic cycles of dioxygen activation by nonheme iron enzymes and their biomimetic compounds. Here, we report mononuclear nonheme iron(III)-peroxo complexes binding redox-inactive metal ions, [(TMC)FeIII(O2)]+-M3+ (M3+ = Sc3+ and Y3+; TMC = 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane), which are characterized spectroscopically as a ‘side-on’ iron(III)-peroxo complex binding a redox-inactive metal ion, (TMC)FeIII-(μ,η2:η2-O2)-M3+ (2-M). While an iron(III)-peroxo complex, [(TMC)FeIII(O2)]+, does not react with electron donors (e.g., ferrocene), one-electron reduction of the iron(III)-peroxo complexes binding redox-inactive metal ions occurs readily upon addition of electron donors, resulting in the generation of an iron(IV)-oxo complex, [(TMC)FeIV(O)]2+ (4), via heterolytic O-O bond cleavage of the peroxide ligand. The rates of the conversion of 2-M to 4 are found to depend on the Lewis acidity of the redox-inactive metal ions and the oxidation potential of the electron donors. We have also determined the fundamental electron-transfer properties of 2-M, such as the reduction potential and the reorganization energy in electron-transfer reaction. Based on the results presented herein, we have proposed a mechanism for the reactions of 2-M and electron donors; the reduction of 2-M to the reduced species, (TMC)FeII-(O2)-M3+ (2’-M), is the rate-determining step, followed by heterolytic O-O bond cleavage of the reduced species to form 4. The present results provide a biomimetic example demonstrating that redox-inactive metal ions bound to an iron(III)-peroxo intermediate play a

  2. Controlled release from recombinant polymers.

    PubMed

    Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

    2014-09-28

    Recombinant polymers provide a high degree of molecular definition for correlating structure with function in controlled release. The wide array of amino acids available as building blocks for these materials lend many advantages including biorecognition, biodegradability, potential biocompatibility, and control over mechanical properties among other attributes. Genetic engineering and DNA manipulation techniques enable the optimization of structure for precise control over spatial and temporal release. Unlike the majority of chemical synthetic strategies used, recombinant DNA technology has allowed for the production of monodisperse polymers with specifically defined sequences. Several classes of recombinant polymers have been used for controlled drug delivery. These include, but are not limited to, elastin-like, silk-like, and silk-elastinlike proteins, as well as emerging cationic polymers for gene delivery. In this article, progress and prospects of recombinant polymers used in controlled release will be reviewed.

  3. Controlled Release from Recombinant Polymers

    PubMed Central

    Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

    2014-01-01

    Recombinant polymers provide a high degree of molecular definition for correlating structure with function in controlled release. The wide array of amino acids available as building blocks for these materials lend many advantages including biorecognition, biodegradability, potential biocompatibility, and control over mechanical properties among other attributes. Genetic engineering and DNA manipulation techniques enable the optimization of structure for precise control over spatial and temporal release. Unlike the majority of chemical synthetic strategies used, recombinant DNA technology has allowed for the production of monodisperse polymers with specifically defined sequences. Several classes of recombinant polymers have been used for controlled drug delivery. These include, but are not limited to, elastin-like, silk-like, and silk-elastinlike proteins, as well as emerging cationic polymers for gene delivery. In this article, progress and prospects of recombinant polymers used in controlled release will be reviewed. PMID:24956486

  4. Three Decades of Recombinant DNA.

    ERIC Educational Resources Information Center

    Palmer, Jackie

    1985-01-01

    Discusses highlights in the development of genetic engineering, examining techniques with recombinant DNA, legal and ethical issues, GenBank (a national database of nucleic acid sequences), and other topics. (JN)

  5. Recombinant DNA means and method

    SciTech Connect

    Alford, B.L.; Mao, J.I.; Moir, D.T.; Taunton-Rigby, A.; Vovis, G.F.

    1987-05-19

    This patent describes a transformed living cell selected from the group consisting of fungi, yeast and bacteria, and containing genetic material derived from recombinant DNA material and coding for bovine rennin.

  6. Recombination system for storage batteries

    SciTech Connect

    Bopp, B.; Ledjeff, K.; Winsel, A.

    1983-03-29

    A recombination system for catalytic oxidation of hydrogen in storage battery gases includes a gas supply duct which makes it possible for the combustible gas flowing through it to aspirate from the ambient the necessary combustion air, following the principle of a bunsen burner, and to entrain it to the recombination catalyst. In case of over-supply of gas, an acid separator positioned in the gas supply pipe counteracts the gas aspiration by means of its flow impedance and thereby makes the recombination system safe from overload. It can also be connected following a conventional recombiner, thereby increasing its effectiveness, by receiving the excess hydrogen from same and reacting it with the aid of the air aspiration.

  7. Influenza Vaccine, Inactivated or Recombinant

    MedlinePlus

    ... die from flu, and many more are hospitalized.Flu vaccine can:keep you from getting flu, make flu ... inactivated or recombinant influenza vaccine?A dose of flu vaccine is recommended every flu season. Children 6 months ...

  8. Recombination device for storage batteries

    DOEpatents

    Kraft, Helmut; Ledjeff, Konstantin

    1985-01-01

    A recombination device including a gas-tight enclosure connected to receive he discharge gases from a rechargeable storage battery. Catalytic material for the recombination of hydrogen and oxygen to form water is supported within the enclosure. The enclosure is sealed from the atmosphere by a liquid seal including two vertical chambers interconnected with an inverted U-shaped overflow tube. The first chamber is connected at its upper portion to the enclosure and the second chamber communicates at its upper portion with the atmosphere. If the pressure within the enclosure differs as overpressure or vacuum by more than the liquid level, the liquid is forced into one of the two chambers and the overpressure is vented or the vacuum is relieved. The recombination device also includes means for returning recombined liquid to the battery and for absorbing metal hydrides.

  9. Recombination device for storage batteries

    DOEpatents

    Kraft, H.; Ledjeff, K.

    1984-01-01

    A recombination device including a gas-tight enclosure connected to receive the discharge gases from a rechargeable storage battery. Catalytic material for the recombination of hydrogen and oxygen to form water is supported within the enclosure. The enclosure is sealed from the atmosphere by a liquid seal including two vertical chambers interconnected with an inverted U-shaped overflow tube. The first chamber is connected at its upper portion to the enclosure and the second chamber communicates at its upper portion with the atmosphere. If the pressure within the enclosure differs as overpressure or vacuum by more than the liquid level, the liquid is forced into one of the two chambers and the overpressure is vented or the vacuum is relieved. The recombination device also includes means for returning recombined liquid to the battery and for absorbing metal hydrides.

  10. Comparative performance of enzymatic and combined alkaline-enzymatic pretreatments on methane production from ensiled sorghum forage.

    PubMed

    Rollini, Manuela; Sambusiti, Cecilia; Musatti, Alida; Ficara, Elena; Retinò, Isabella; Malpei, Francesca

    2014-12-01

    This study investigated the effect of enzymatic and combined alkaline-enzymatic pretreatments on chemical composition and methane production from ensiled sorghum forage. Four commercial enzymatic preparations were tested and the two yielding the highest sugars release were added to evaluate any hydrolytic effect on both untreated and alkaline pretreated samples. In the combined alkaline-enzymatic pretreatment trials, the highest sugar release was found with Primafast and BGL preparations (added at a final concentration 0.12 and 0.20 mL/g TS, respectively), with a total monomeric content of 12 and 6.5 g/L. Fibre composition analysis confirmed that the combined alkaline-enzymatic pretreatment led to cellulose (up to 32 %) and hemicelluloses (up to 56 %) solubilisation, compared to the enzymatic pretreatment alone. BMP tests were performed on both untreated and pretreated samples, and time courses of methane production were fitted. Both enzymatic and combined alkaline-enzymatic pretreatment led to a methane production increase (304 and 362 mL CH4/g VS), compared to that of untreated sorghum (265 mL CH4/g VS), as  +15 and  +37 %, respectively. Moreover, higher specific methane production rates, compared to that of untreated sorghum (20.31 mL CH4/g VS/d), were obtained by applying the enzymatic and combined alkaline-enzymatic pretreatment (33.94 and 31.65 mL CH4/g VS/d), respectively.

  11. Expression in high yield of pig alpha 1 beta 1 Na,K-ATPase and inactive mutants D369N and D807N in Saccharomyces cerevisiae.

    PubMed

    Pedersen, P A; Rasmussen, J H; Jøorgensen, P L

    1996-02-01

    Studies of structure-function relationships in Na,K-ATPase require high yield expression of inactive mutations in cells without endogenous Na,K-ATPase activity. In this work we developed a host/vector system for expression of fully active pig Na,K-ATPase as well as the inactive mutations D369N and D807N at high levels in Saccharomyces cerevisiae. The alpha 1- and beta 1-subunit cDNAs were inserted into a single 2-microns-based plasmid with a high and regulatable copy number and strong galactose-inducible promoters allowing for stoichiometric alterations of gene dosage. The protease-deficient host strain was engineered to express high levels of GAL4 transactivating protein, thereby causing a 10-fold increase in expression to 32,500 +/- 3,000 [3H]ouabain sites/cell. In one bioreactor run 150-200 g of yeast were produced with 54 +/- 5 micrograms of Na,K-pump protein/g of cells. Through purification in membrane bound form the activity of the recombinant Na,K-ATPase was increased to 42-50 pmol/mg of protein. The Na,K dependence of ATP hydrolysis and the molar activity (4,500-7,000 min-1) were close to those of native pig kidney Na,K-ATPase. Mutations to the phosphorylation site (D369N) or presumptive cation sites (D807N), both devoid of Na,K-ATPase activity, were expressed in the yeast membrane at the same alpha-subunit concentration and [3H]ouabain binding capacity as the wild type Na,K-ATPase. The high yield and absence of endogenous activity allowed assay of [3H]ATP binding at equilibrium, demonstrating a remarkable 18-fold increase in affinity for ATP in consequence of reducing the negative charge at the phosphorylation site (D369N).

  12. Continuous enzymatic liquefaction of starch for saccharification

    SciTech Connect

    Carr, M.E.; Black, L.T.; Bagby, M.O.

    1982-01-01

    A process was explored for continuous enzymatic liquefaction of corn starch at high concentration and subsequent saccharification to glucose. The process appears to be quite efficient for conversion of starch to glucose and enzymatic liquefaction and should be readily adaptable to industrial fermentation processes. Preliminary work indicated that milled corn or other cereal grains also can be suitably converted by such a process. Essentially, the process involved incorporation of a thermostable, bacterial alpha-amylase for liquefaction and, subsequently, of a glucoamylase into the continuous mixer under conditions conductive to rapid enzymatic hydrolyses. Also studied was the effect on substrate liquefaction of variables such as starch concentration (40-70%), level of alpha-amylase (0.14-0.4%, dry starch basis), temperature (70-100 degrees C), pH (5.8-7.1), and residence time (6 and 12 minutes). The degree of liquefaction was assessed by determining 1) the Brookfield viscosity, 2) the amount of reducing groups, and 3) the rate and extent of glucose formed after glucoamylase treatment. Best liquefaction processing conditions were achieved by using 50-60% starch concentration, at 95 degrees C, with 0.4% alpha-amylase, and a 6 minute residence period in the mixer. Under these conditions, rates and extents of glucose obtained after glucoamylase treatment approached those obtained in longer laboratory batch liquefactions. The amount of glucose formed in 24 hours with the use of 0.4% glucoamylase was 86% of theory after a 6-min continuous liquefaction, compared to 90% for a 30-min laboratory batch liquefaction (95 degrees C, 0.4% alpha-amylase). (Refs. 15).

  13. Mutations affecting enzymatic activity in liver arginase

    SciTech Connect

    Vockley, J.G.; Tabor, D.E.; Goodman, B.K.

    1994-09-01

    The hydrolysis of arginine to ornithine and urea is catalyzed by arginase in the last step of the urea cycle. We examined a group of arginase deficient patients by PCR-SSCP analysis to characterize the molecular basis of this disorder. A heterogeneous population of nonsense mutations, microdeletions, and missense mutations has been identified in our cohort. Microdeletions which introduce premature stop codons downstream of the deletion and nonsense mutations result in no arginase activity. These mutations occur randomly along the gene. The majority of missense mutations identified appear to occur in regions of high cross-species homology. To test the effect of these missense mutations on arginase activity, site-directed mutagenesis was used to re-create the patient mutations for in vivo expression studies in a prokaryotic fusion-protein expression system. Of 4 different missense mutations identified in 6 individuals, only one was located outside of a conserved region. The three substitution mutations within the conserved regions had a significant effect on enzymatic activity (0-3.1 nmole/30min, normal is 1300-1400 nmoles/30min, as determined by in vitro arginase assay), while the fourth mutation, a T to S substitution, did not. In addition, site-directed mutagenesis was utilized to create mutations not in residues postulated to play a significant role in the enzymatic function or active site formation in manganese-binding proteins such as arginase. We have determined that the substitution of glycine for a histidine residue, located in a very highly conserved region of exon 3, and the substitution of a histidine and an aspartic acid residue within a similarly conserved region in exon 4, totally abolishes enzymatic activity. Mutations substituting glycine for an additional histidine and aspartic acid residue in exon 4 and two aspartic acid residues in exon 7 have also been created. We are currently in the process of characterizing these mutations.

  14. Microwave-mediated enzymatic modifications of DNA.

    PubMed

    Das, Rakha Hari; Ahirwar, Rajesh; Kumar, Saroj; Nahar, Pradip

    2015-02-15

    Here we report microwave-induced specific cleavage, ligation, dephosphorylation, and phosphorylation of nucleic acids catalyzed by restriction endonucleases, T4 DNA ligase, T4 polynucleotide kinase, and calf intestinal alkaline phosphatase. The microwave-mediated method has dramatically reduced the reaction time to 20 to 50s. In control experiments, the same reactions failed to give the desired reaction products when carried out in the same time periods but without microwave irradiation. Because the microwave method is rapid, it could be a useful alternative to the time-consuming conventional procedure for enzymatic modification of DNA. PMID:25447491

  15. [Enzymatic utilization of cotton soap stock].

    PubMed

    Davranov, K D; Guliamova, K A; Alimova, B Kh; Turapova, N M

    2000-01-01

    Enzymatic hydrolysis of neutral fat of cotton oil soap stock with a nonspecific lipase produced by Oospora lactis F-500 was designed. The culture liquid and a preparation of enzyme obtained by precipitation with isopropanol from a filtrate of the culture liquid were used. Utilization of cotton oil soap stock as the only source of carbon during cultivation of the fungus was studied. The rate of hydrolysis of soap stock fat strongly depended on the way of biological conversion of cotton oil soap stock. The most effective utilization was observed during cultivation of the fungus in the medium containing soap stock.

  16. Enzymatic Catalytic Beds For Oxidation Of Alcohols

    NASA Technical Reports Server (NTRS)

    Jolly, Clifford D.; Schussel, Leonard J.

    1993-01-01

    Modules containing beds of enzymatic material catalyzing oxidation of primary alcohols and some other organic compounds developed for use in wastewater-treatment systems of future spacecraft. Designed to be placed downstream of multifiltration modules, which contain filters and sorbent beds removing most of non-alcoholic contaminants but fail to remove significant amounts of low-molecular-weight, polar, nonionic compounds like alcohols. Catalytic modules also used on Earth to oxidize primary alcohols and other compounds in wastewater streams and industrial process streams.

  17. Enzymatic Biofuel Cells on Porous Nanostructures.

    PubMed

    Wen, Dan; Eychmüller, Alexander

    2016-09-01

    Biofuel cells (BFCs) that utilize enzymes as catalysts represent a new sustainable and renewable energy technology. Numerous efforts have been directed to improve the performance of the enzymatic BFCs (EBFCs) with respect to power output and operational stability for further applications in portable power sources, self-powered electrochemical sensing, implantable medical devices, etc. The latest advances in EBFCs based on porous nanoarchitectures over the past 5 years are detailed here. Porous matrices from carbon, noble metals, and polymers promote the development of EBFCs through the electron transfer and mass transport benefits. Some key issues regarding how these nanostructured porous media improve the performance of EBFCs are also discussed. PMID:27377976

  18. Moyamoya syndrome occurred in a girl with an inactive systemic lupus erythematosus.

    PubMed

    Lee, Yun-Jin; Yeon, Gyu Min; Nam, Sang Ook; Kim, Su Yung

    2013-12-01

    We report the case of a 17-year-old Korean girl with systemic lupus erythematosus (SLE) who presented with sudden weakness of the right-sided extremities and dysarthria. Oral prednisolone was being taken to control SLE. Results of clinical and laboratory examinations did not show any evidence of antiphospholipid syndrome or thromboembolic disease nor SLE activity. Cerebral angiography showed stenosis of the left internal carotid artery and right anterior cerebral artery with accompanying collateral circulation (moyamoya vessels). After the patient underwent bypass surgery on the left side, she recovered from the neurological problems and did not experience any additional ischemic attack during the 14-month follow-up period. This case represents an unusual association between moyamoya syndrome and inactive SLE (inactive for a relatively long interval of 2 years) in a young girl.

  19. Prevalence and predictors of physical inactivity in a slum in Brazil.

    PubMed

    Alves, João Guilherme Bezerra; Figueiroa, José Natal; Alves, Lucas Victor

    2011-02-01

    The aim of this study was to ascertain the prevalence of physical inactivity and examine the role of potential predictors in a very low-income adult population in a slum located in Recife city, northeast of Brazil. A cross-sectional study was conducted with 1,176 subjects aged 20-60 years residing in a slum. Using the short version of the International Physical Activity Questionnaire, 307 (26.1%) study participants-97 (23.8%) men and 210 (27.3%) women-have a low physical activity score (MET-minutes per week). Increased age was associated with physical inactivity only in people without overweight/obesity. Low physical activity was less common (i.e., respondents were more active) than in other Brazilian population-based studies. These results suggest that the relationship between physical activity and socioeconomic level is more complex and depends on the internal characteristics of the community.

  20. Influence of radiative processes on the ignition of deuterium-tritium plasma containing inactive impurities

    NASA Astrophysics Data System (ADS)

    Gus'kov, S. Yu.; Sherman, V. E.

    2016-08-01

    The degree of influence of radiative processes on the ignition of deuterium-tritium (DT) plasma has been theoretically studied as dependent on the content of inactive impurities in plasma. The analytic criterion of plasma ignition in inertial confinement fusion (ICF) targets is modified taking into account the absorption of intrinsic radiation from plasma in the ignition region. The influence of radiative processes on the DT plasma ignition has been analytically and numerically studied for plasma that contains a significant fraction of inactive impurities either as a result of DT fuel mixing with ICF target ablator material or as a result of using light metal DT-hydrides as solid noncryogenic fuel. It has been shown that the effect of the absorption of intrinsic radiation leads to lower impurity-induced increase in the ignition energy as compared to that calculated in the approximation of optically transparent ignition region.

  1. Driven to Be Inactive?—The Genetics of Physical Activity

    PubMed Central

    Moore-Harrison, Trudy; Lightfoot, J. Timothy

    2014-01-01

    The health implications of physical inactivity, including its integral role in promoting obesity, are well known and have been well documented. Physical activity is a multifactorial behavior with various factors playing a role in determining individual physical activity levels. Research using both human and animal models in the past several years has clearly indicated that genetics is associated with physical activity. Furthermore, researchers have identified several significant and suggestive genomic quantitative trait loci associated with physical activity. To date, the identities of the causal genes underlying physical activity regulation are unclear, with few strong candidate genes. The current research provides a foundation from which future confirmatory research can be launched as well as determination of the mechanisms through which the genetic factors act. The application of this knowledge could significantly augment the information available for physical activity behavior change interventions resulting in more efficient programs for those predisposed to be inactive. PMID:21036329

  2. The effects of exergaming on physical activity among inactive children in a physical education classroom.

    PubMed

    Fogel, Victoria A; Miltenberger, Raymond G; Graves, Rachel; Koehler, Shannon

    2010-01-01

    Childhood obesity, which is due in part to lack of physical activity, is a serious concern that requires the attention of the behavioral community. Although excessive video game play has been noted in the literature as a contributor to childhood obesity, newer video gaming technology, called exergaming, has been designed to capitalize on the reinforcing effects of video games to increase physical activity in children. This study evaluated the effects of exergaming on physical activity among 4 inactive children in a physical education (PE) classroom. Results showed that exergaming produced substantially more minutes of physical activity and more minutes of opportunity to engage in physical activity than did the standard PE program. In addition, exergaming was socially acceptable to both the students and the PE teacher. Exergaming appears to hold promise as a method for increasing physical activity among inactive children and might be a possible intervention for childhood obesity.

  3. Nuclear stellar kinematics of hard X-ray selected AGNs with matched inactive galaxies

    NASA Astrophysics Data System (ADS)

    Lin, Ming-Yin; Davies, Richard; Burtscher, Leonard; Rosario, David

    2016-08-01

    In a matched sample of local, 14-195 keV selected active galactic nuclei (AGN) and inactive galaxies, we investigate the spatially resolved stellar kinematics and distributions on the scale of 10-300 pc. Here we present first results on part of the sample. We use a simple model to look for non-circular motions in the observed stellar velocity fields of both AGNs and inactive galaxies. Combining the luminosity profile with larger scale data, we decompose the large-scale disk, bulge and nuclear components. And with the stellar velocity dispersion, we search for the evidence of dynamically cold nuclear stellar populations distinct from the bulge, and study the nuclear K-band stellar mass to light ratios. The key goal of this study is to understand the role of nuclear star formation in the AGN fueling process.

  4. Revegetation and rock cover for stabilization of inactive uranium mill tailings disposal sites. Final report

    SciTech Connect

    Beedlow, P.A.

    1984-05-01

    Guidelines for using vegetation and rock to protect inactive uranium mill tailings from erosion were developed by Pacific Northwest Laboratory as part of the Department of Energy's Uranium Mill Tailings Remedial Action Project (UMTRAP) Technology Development program. Information on soils, climate, and vegetation were collected for 20 inactive tailings sites in the western United States. Sites were grouped according to similarities in climate and vegetation. Soil loss for those sites was characterized using the Universal Soil Loss Equation. Test plots were used to evaluate (1) the interaction between vegetation and sealant barrier systems and (2) the effects of surface rock on soil water and vegetation. Lysimeter and simulation studies were used to direct and support field experiments. 49 references, 17 figures, 16 tables.

  5. Biodiesel production from microalgae oil catalyzed by a recombinant lipase.

    PubMed

    Huang, Jinjin; Xia, Ji; Jiang, Wei; Li, Ying; Li, Jilun

    2015-03-01

    A recombinant Rhizomucor miehei lipase was constructed and expressed in Pichia pastoris. The target enzyme was termed Lipase GH2 and it can be used as a free enzyme for catalytic conversion of microalgae oil mixed with methanol or ethanol for biodiesel production in an n-hexane solvent system. Conversion rates of two major types of biodiesel, fatty acid methyl ester (FAME) and fatty acid ethyl ester (FAEE), reached maximal values (>90%) after 24h. The process of FAME production is generally more simple and economical than that of FAEE production, even though the two processes show similar conversion rates. In spite of the damaging effect of ethanol on enzyme activity, we successfully obtained ethyl ester by the enzymatic method. Our findings indicate that Lipase GH2 is a useful catalyst for conversion of microalgae oil to FAME or FAEE, and this system provides efficiency and reduced costs in biodiesel production.

  6. Biodiesel production from microalgae oil catalyzed by a recombinant lipase.

    PubMed

    Huang, Jinjin; Xia, Ji; Jiang, Wei; Li, Ying; Li, Jilun

    2015-03-01

    A recombinant Rhizomucor miehei lipase was constructed and expressed in Pichia pastoris. The target enzyme was termed Lipase GH2 and it can be used as a free enzyme for catalytic conversion of microalgae oil mixed with methanol or ethanol for biodiesel production in an n-hexane solvent system. Conversion rates of two major types of biodiesel, fatty acid methyl ester (FAME) and fatty acid ethyl ester (FAEE), reached maximal values (>90%) after 24h. The process of FAME production is generally more simple and economical than that of FAEE production, even though the two processes show similar conversion rates. In spite of the damaging effect of ethanol on enzyme activity, we successfully obtained ethyl ester by the enzymatic method. Our findings indicate that Lipase GH2 is a useful catalyst for conversion of microalgae oil to FAME or FAEE, and this system provides efficiency and reduced costs in biodiesel production. PMID:25585254

  7. Engineering, expression, purification, and production of recombinant thermolysin.

    PubMed

    Inouye, Kuniyo; Kusano, Masayuki; Hashida, Yasuhiko; Minoda, Masashi; Yasukawa, Kiyoshi

    2007-01-01

    Thermolysin [EC 3.4.24.27] is a thermostable neutral zinc metalloproteinase originally identified in the culture broth of Bacillus thermoproteolyticus Rokko. Since the discovery in 1962, the enzyme has been extensively studied regarding its structure and catalytic mechanism. Today, thermolysin is a representative of zinc metalloproteinase and an attractive target in protein engineering to understand the catalytic mechanism, thermostability, and halophilicity. Thermolysin is used in industry, especially for the enzymatic synthesis of N-carbobenzoxy L-Asp-L-Phe methyl ester (ZDFM), a precursor of an artificial sweetener, aspartame. Generation of genetically engineered thermolysin with higher activity in the synthesis of ZDFM has been highly desired. In accordance with the expansion of studies on thermolysin, various strategies for its expression and purification have been devised and successfully used. In this review, we aim to outline recombinant thermolysins associated with their engineering, expression, purification, and production.

  8. Delayed recombination and cosmic parameters

    SciTech Connect

    Galli, Silvia; Melchiorri, Alessandro; Bean, Rachel; Silk, Joseph

    2008-09-15

    Current cosmological constraints from cosmic microwave background anisotropies are typically derived assuming a standard recombination scheme, however additional resonance and ionizing radiation sources can delay recombination, altering the cosmic ionization history and the cosmological inferences drawn from the cosmic microwave background data. We show that for recent observations of the cosmic microwave background anisotropy, from the Wilkinson microwave anisotropy probe satellite mission (WMAP) 5-year survey and from the arcminute cosmology bolometer array receiver experiment, additional resonance radiation is nearly degenerate with variations in the spectral index, n{sub s}, and has a marked effect on uncertainties in constraints on the Hubble constant, age of the universe, curvature and the upper bound on the neutrino mass. When a modified recombination scheme is considered, the redshift of recombination is constrained to z{sub *}=1078{+-}11, with uncertainties in the measurement weaker by 1 order of magnitude than those obtained under the assumption of standard recombination while constraints on the shift parameter are shifted by 1{sigma} to R=1.734{+-}0.028. From the WMAP5 data we obtain the following constraints on the resonance and ionization sources parameters: {epsilon}{sub {alpha}}<0.39 and {epsilon}{sub i}<0.058 at 95% c.l.. Although delayed recombination limits the precision of parameter estimation from the WMAP satellite, we demonstrate that this should not be the case for future, smaller angular scales measurements, such as those by the Planck satellite mission.

  9. Ethanol production by recombinant hosts

    DOEpatents

    Ingram, Lonnie O.; Beall, David S.; Burchhardt, Gerhard F. H.; Guimaraes, Walter V.; Ohta, Kazuyoshi; Wood, Brent E.; Shanmugam, Keelnatham T.

    1995-01-01

    Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

  10. Ethanol production by recombinant hosts

    DOEpatents

    Fowler, David E.; Horton, Philip G.; Ben-Bassat, Arie

    1996-01-01

    Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

  11. Delayed recombination and cosmic parameters

    NASA Astrophysics Data System (ADS)

    Galli, Silvia; Bean, Rachel; Melchiorri, Alessandro; Silk, Joseph

    2008-09-01

    Current cosmological constraints from cosmic microwave background anisotropies are typically derived assuming a standard recombination scheme, however additional resonance and ionizing radiation sources can delay recombination, altering the cosmic ionization history and the cosmological inferences drawn from the cosmic microwave background data. We show that for recent observations of the cosmic microwave background anisotropy, from the Wilkinson microwave anisotropy probe satellite mission (WMAP) 5-year survey and from the arcminute cosmology bolometer array receiver experiment, additional resonance radiation is nearly degenerate with variations in the spectral index, ns, and has a marked effect on uncertainties in constraints on the Hubble constant, age of the universe, curvature and the upper bound on the neutrino mass. When a modified recombination scheme is considered, the redshift of recombination is constrained to z*=1078±11, with uncertainties in the measurement weaker by 1 order of magnitude than those obtained under the assumption of standard recombination while constraints on the shift parameter are shifted by 1σ to R=1.734±0.028. From the WMAP5 data we obtain the following constraints on the resonance and ionization sources parameters: γα<0.39 and γi<0.058 at 95% c.l.. Although delayed recombination limits the precision of parameter estimation from the WMAP satellite, we demonstrate that this should not be the case for future, smaller angular scales measurements, such as those by the Planck satellite mission.

  12. Arctic Ground Squirrels Limit Bone Loss during the Prolonged Physical Inactivity Associated with Hibernation.

    PubMed

    Wojda, Samantha J; Gridley, Richard A; McGee-Lawrence, Meghan E; Drummer, Thomas D; Hess, Ann; Kohl, Franziska; Barnes, Brian M; Donahue, Seth W

    2016-01-01

    Prolonged disuse (e.g., physical inactivity) typically results in increased bone porosity, decreased mineral density, and decreased bone strength, leading to increased fracture risk in many mammals. However, bears, marmots, and two species of ground squirrels have been shown to preserve macrostructural bone properties and bone strength during long seasons of hibernation while they remain mostly inactive. Some small hibernators (e.g., 13-lined ground squirrels) show microstructural bone loss (i.e., osteocytic osteolysis) during hibernation, which is not seen in larger hibernators (e.g., bears and marmots). Arctic ground squirrels (Urocitellus parryii) are intermediate in size between 13-lined ground squirrels and marmots and are perhaps the most extreme rodent hibernator, hibernating for up to 8 mo annually with body temperatures below freezing. The goal of this study was to quantify the effects of hibernation and inactivity on cortical and trabecular bone properties in arctic ground squirrels. Cortical bone geometrical properties (i.e., thickness, cross-sectional area, and moment of inertia) at the midshaft of the femur were not different in animals sampled over the hibernation and active seasons. Femoral ultimate stress tended to be lower in hibernators than in summer animals, but toughness was not affected by hibernation. The area of osteocyte lacunae was not different between active and hibernating animals. There was an increase in osteocytic lacunar porosity in the hibernation group due to increased lacunar density. Trabecular bone volume fraction in the proximal tibia was unexpectedly greater in the hibernation group than in the active group. This study shows that, similar to other hibernators, arctic ground squirrels are able to preserve many bone properties during hibernation despite being physically inactive for up to 8 mo.

  13. Investigation of the organic matter in inactive nuclear tank liquids. Environmental Restoration Program

    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.

  14. Pyridinediimine Iron Complexes with Pendant Redox-Inactive Metals Located in the Secondary Coordination Sphere.

    PubMed

    Delgado, Mayra; Ziegler, Joshua M; Seda, Takele; Zakharov, Lev N; Gilbertson, John D

    2016-01-19

    A series of pyridinediimine (PDI) iron complexes that contain a pendant 15-crown-5 located in the secondary coordination sphere were synthesized and characterized. The complex Fe((15c5)PDI)(CO)2 (2) was shown in both the solid state and solution to encapsulate redox-inactive metal ions. Modest shifts in the reduction potential of the metal-ligand scaffold were observed upon encapsulation of either Na(+) or Li(+).

  15. Inactive end cell assembly for fuel cells for improved electrolyte management and electrical contact

    DOEpatents

    Yuh, Chao-Yi; Farooque, Mohammad; Johnsen, Richard

    2007-04-10

    An assembly for storing electrolyte in a carbonate fuel cell is provided. The combination of a soft, compliant and resilient cathode current collector and an inactive anode part including a foam anode in each assembly mitigates electrical contact loss during operation of the fuel cell stack. In addition, an electrode reservoir in the positive end assembly and an electrode sink in the negative end assembly are provided, by which ribbed and flat cathode members inhibit electrolyte migration in the fuel cell stack.

  16. Food reward in active compared to inactive men: Roles for gastric emptying and body fat.

    PubMed

    Horner, Katy M; Finlayson, Graham; Byrne, Nuala M; King, Neil A

    2016-06-01

    Habitual exercise could contribute to weight management by altering processes of food reward via the gut-brain axis. We investigated hedonic processes of food reward in active and inactive men and characterised relationships with gastric emptying and body fat. Forty-four men (active: n=22; inactive: n=22, BMI range 21-36kg/m(2); percent fat mass range 9-42%) were studied. Participants were provided with a standardised fixed breakfast and an ad libitum lunch meal 5h later. Explicit liking, implicit wanting and preference among high-fat, low-fat, sweet and savoury food items were assessed immediately post-breakfast (fed state) and again pre-lunch (hungry state) using the Leeds Food Preference Questionnaire. Gastric emptying was assessed by (13)C-octanoic acid breath test. Active individuals exhibited a lower liking for foods overall and a greater implicit wanting for low-fat savoury foods in the fed state, compared to inactive men. Differences in the fed state remained significant after adjusting for percent fat mass. Active men also had a greater increase in liking for savoury foods in the interval between breakfast and lunch. Faster gastric emptying was associated with liking for savoury foods and with an increase in liking for savoury foods in the postprandial interval. In contrast, greater implicit wanting for high-fat foods was associated with slower gastric emptying. These associations were independent of each other, activity status and body fat. In conclusion, active and inactive men differ in processes of food reward. The rate of gastric emptying may play a role in the association between physical activity status and food reward, via the gut-brain axis.

  17. Arctic Ground Squirrels Limit Bone Loss during the Prolonged Physical Inactivity Associated with Hibernation.

    PubMed

    Wojda, Samantha J; Gridley, Richard A; McGee-Lawrence, Meghan E; Drummer, Thomas D; Hess, Ann; Kohl, Franziska; Barnes, Brian M; Donahue, Seth W

    2016-01-01

    Prolonged disuse (e.g., physical inactivity) typically results in increased bone porosity, decreased mineral density, and decreased bone strength, leading to increased fracture risk in many mammals. However, bears, marmots, and two species of ground squirrels have been shown to preserve macrostructural bone properties and bone strength during long seasons of hibernation while they remain mostly inactive. Some small hibernators (e.g., 13-lined ground squirrels) show microstructural bone loss (i.e., osteocytic osteolysis) during hibernation, which is not seen in larger hibernators (e.g., bears and marmots). Arctic ground squirrels (Urocitellus parryii) are intermediate in size between 13-lined ground squirrels and marmots and are perhaps the most extreme rodent hibernator, hibernating for up to 8 mo annually with body temperatures below freezing. The goal of this study was to quantify the effects of hibernation and inactivity on cortical and trabecular bone properties in arctic ground squirrels. Cortical bone geometrical properties (i.e., thickness, cross-sectional area, and moment of inertia) at the midshaft of the femur were not different in animals sampled over the hibernation and active seasons. Femoral ultimate stress tended to be lower in hibernators than in summer animals, but toughness was not affected by hibernation. The area of osteocyte lacunae was not different between active and hibernating animals. There was an increase in osteocytic lacunar porosity in the hibernation group due to increased lacunar density. Trabecular bone volume fraction in the proximal tibia was unexpectedly greater in the hibernation group than in the active group. This study shows that, similar to other hibernators, arctic ground squirrels are able to preserve many bone properties during hibernation despite being physically inactive for up to 8 mo. PMID:27082526

  18. Using gender-based analyses to understand physical inactivity among women in Yellowstone County, Montana.

    PubMed

    Duin, Diane K; Golbeck, Amanda L; Keippel, April Ennis; Ciemins, Elizabeth; Hanson, Hillary; Neary, Tracy; Fink, Heather

    2015-08-01

    Physical inactivity contributes to many health problems. Gender, the socially constructed roles and activities deemed appropriate for men and women, is an important factor in women's physical inactivity. To better understand how gender influences participation in leisure-time physical activity, a gender analysis was conducted using sex-disaggregated data from a county-wide health assessment phone survey and a qualitative analysis of focus group transcripts. From this gender analysis, several gender-based constraints emerged, including women's roles as caregivers, which left little time or energy for physical activity, women's leisure time activities and hobbies, which were less active than men's hobbies, and expectations for women's appearance that made them uncomfortable sweating in front of strangers. Gender-based opportunities included women's enjoyment of activity as a social connection, less rigid gender roles for younger women, and a sense of responsibility to set a good example for their families. The gender analysis was used to gain a deeper understanding of gender-based constraints and opportunities related to physical activity. This understanding is being used in the next step of our research to develop a gender-specific intervention to promote physical activity in women that addresses the underlying causes of physical inactivity through accommodation or transformation of those gender norms.

  19. Effects of exercise and inactivity on intravascular volume and cardiovascular control mechanisms

    NASA Astrophysics Data System (ADS)

    Convertino, Victor A.

    Exercise, inactivity and confinement have been used as effective tools to assess the contributions of vascular volume and baroreflexes to orthostatic hypotension associated with exposure to microgravity. Prolonged exposure to bedrest, physical inactivity, or wheelchair confinement removes baroreceptor unloading caused by regular upright standing and induces attenuation of cardiovascular baroreflex responses. The magnitude of reduced baroreflex sensitivity following bedrest or wheelchair confinement is related to the degree of orthostatic hypotension. Reduction in vascular volume caused by bedrest or progressive hypovolemia does not affect carotid-cardiac baroreflex function. In contrast, intense exercise that increases arterial baroreceptor loading causes an acute increase in carotid baroreceptor sensitivity and has been associated with enhanced orthostatic stability following exposure to simulated microgravity. Endurance exercise training designed to enhance orthostatic stability was associated with increased blood volume and vasoconstrictive reserve, but no change in the carotid baroreflex response. Therefore, using models of exercise, inactivity and confinement, integrated and redundant roles for vascular volume and cardiovascular baroreflexes have been demonstrated as probable underlying mechanisms that contribute independently to the development of orthostatic hypotension following spaceflight. These data suggest that loading of arterial baroreceptors may be necessary to maintain baroreflex function.

  20. A new electrochemically active-inactive switching aptamer molecular beacon to detect thrombin directly in solution.

    PubMed

    Cheng, Guifang; Shen, Bijun; Zhang, Fan; Wu, Jikui; Xu, Ying; He, Pingang; Fang, Yuzhi

    2010-06-15

    A new electrochemical aptamer molecular beacon (MB) was designed by the carminic acid (CA) covalently linking at the each end of a special single-stranded stem-loop shaped oligonucleotide and named as CAs-MB. CA is an electrochemically active molecule and two CA molecules at the ends of molecular beacon stem were closed enough to associate each other to be as CA dimer. The dimer was electrochemically inactive. It separated into two CA monomers and produced the electrochemical signal while CAs-MB combined with target. In this protocol, the detection strategy of CAs-MB for thrombin is based on electrochemical active-inactive switching between monomer and dimer forms of CA. In order to enhance the electrochemical signal, magnetic nanobeads (MNB) was applied by connecting CAs-MB with MNB through a duplex of DNA. With the magnetic enrichment, the detection limit for thrombin reached to 42.4 pM. The experiment results showed that this type of electrochemical active-inactive switching aptamer molecular beacon allowed the direct detection of target proteins in the solution with no requirement of removing uncombined CAs-MB. Besides, CAs-MB/MNB can be easily regenerated by using 2M NaCl solution to cleave the thrombin from the aptasensor. PMID:20378327

  1. The Relationship Between Neighborhood Socioeconomic Characteristics and Physical Inactivity Among Adolescents Living in Boston, Massachusetts

    PubMed Central

    Molnar, Beth E.; Cradock, Angie; Kawachi, Ichiro

    2014-01-01

    Objectives. We sought to determine whether the socioeconomic environment was associated with no participation in physical activity among adolescents in Boston, Massachusetts. Methods. We used cross-sectional data from 1878 urban adolescents living in 38 neighborhoods who participated in the 2008 Boston Youth Survey, a biennial survey of high school students (aged 14–19 years). We used multilevel multiple regression models to determine the association between neighborhood-level exposures of economic deprivation, social fragmentation, social cohesion, danger and disorder, and students’ reports of no participation in physical activity in the previous week. Results. High social fragmentation within the residential neighborhood was associated with an increased likelihood of being inactive (odds ratio = 1.53; 95% confidence interval = 1.14, 2.05). No other neighborhood exposures were associated with physical inactivity. Conclusions. Social fragmentation might be an important correlate of physical inactivity among youths living in urban settings. Interventions might be needed to assist youths living in unstable neighborhoods to be physically active. PMID:25211727

  2. Physical inactivity is an independent risk factor for hip fracture in the elderly.

    PubMed Central

    Coupland, C; Wood, D; Cooper, C

    1993-01-01

    STUDY OBJECTIVE--To test the hypothesis that physical inactivity is an independent risk factor for hip fracture in the elderly. DESIGN--Population based, case-control study. SETTING--Metropolitan borough of Newcastle upon Tyne. PARTICIPANT--A total of 197 patients aged 50 years and over, resident in Newcastle, and admitted consecutively with a hip fracture, and 382 community controls, matched by age and sex, who had not suffered a hip fracture. MEASUREMENTS AND MAIN RESULTS--Validated methods were used to assess customary physical activity. Information on body build, cigarette smoking, and alcohol consumption was also obtained. Grip strength was measured. Physical inactivity was strongly associated with the risk of hip fracture in men and women. Subjects who did not regularly weight-bear, perform muscle-loading activities such as climbing stairs, and perform productive activities such as gardening, were all more than twice as likely to sustain a hip fracture, when compared with subjects at the higher end of the activity spectrum. These increases in risk remained after adjusting for body mass index, smoking, alcohol consumption, and dependence in daily living activities. CONCLUSIONS--Customary physical inactivity is an independent determinant of hip fracture in the elderly. Strategies to improve the day to day activity of elderly people require urgent exploration. PMID:8120496

  3. A new electrochemically active-inactive switching aptamer molecular beacon to detect thrombin directly in solution.

    PubMed

    Cheng, Guifang; Shen, Bijun; Zhang, Fan; Wu, Jikui; Xu, Ying; He, Pingang; Fang, Yuzhi

    2010-06-15

    A new electrochemical aptamer molecular beacon (MB) was designed by the carminic acid (CA) covalently linking at the each end of a special single-stranded stem-loop shaped oligonucleotide and named as CAs-MB. CA is an electrochemically active molecule and two CA molecules at the ends of molecular beacon stem were closed enough to associate each other to be as CA dimer. The dimer was electrochemically inactive. It separated into two CA monomers and produced the electrochemical signal while CAs-MB combined with target. In this protocol, the detection strategy of CAs-MB for thrombin is based on electrochemical active-inactive switching between monomer and dimer forms of CA. In order to enhance the electrochemical signal, magnetic nanobeads (MNB) was applied by connecting CAs-MB with MNB through a duplex of DNA. With the magnetic enrichment, the detection limit for thrombin reached to 42.4 pM. The experiment results showed that this type of electrochemical active-inactive switching aptamer molecular beacon allowed the direct detection of target proteins in the solution with no requirement of removing uncombined CAs-MB. Besides, CAs-MB/MNB can be easily regenerated by using 2M NaCl solution to cleave the thrombin from the aptasensor.

  4. Using buriedness to improve discrimination between actives and inactives in docking.

    PubMed

    O'Boyle, Noel M; Brewerton, Suzanne C; Taylor, Robin

    2008-06-01

    A continuing problem in protein-ligand docking is the correct relative ranking of active molecules versus inactives. Using the ChemScore scoring function as implemented in the GOLD docking software, we have investigated the effect of scaling hydrogen bond, metal-ligand, and lipophilic interactions based on the buriedness of the interaction. Buriedness was measured using the receptor density, the number of protein heavy atoms within 8.0 A. Terms in the scaling functions were optimized using negative data, represented by docked poses of inactive molecules. The objective function was the mean rank of the scores of the active poses in the Astex Diverse Set (Hartshorn et al. J. Med. Chem., 2007, 50, 726) with respect to the docked poses of 99 inactives. The final four-parameter model gave a substantial improvement in the average rank from 18.6 to 12.5. Similar results were obtained for an independent test set. Receptor density scaling is available as an option in the recent GOLD release. PMID:18533645

  5. Effect of non-enzymatic proteins on enzymatic hydrolysis and simultaneous saccharification and fermentation of different lignocellulosic materials.

    PubMed

    Wang, Hui; Kobayashi, Shinichi; Mochidzuki, Kazuhiro

    2015-08-01

    Non-enzymatic proteins were added during hydrolysis of cellulose and simultaneous saccharification and fermentation (SSF) of different biomass materials. Bovine serum albumin (BSA), a model non-enzymatic protein, increased cellulose and xylose conversion efficiency and also enhanced the ethanol yield during SSF of rice straw subjected to varied pretreatments. Corn steep liquor, yeast extract, and peptone also exerted a similar effect as BSA and enhanced the enzymatic hydrolysis of rice straw. Compared to the glucose yields obtained after enzymatic hydrolysis of rice straw in the absence of additives, the glucose yields after 72h of hydrolysis increased by 12.7%, 13.5%, and 13.7% after addition of the corn steep liquor, yeast extract, and peptone, respectively. This study indicated the use of BSA as an alternative to intensive pretreatment of lignocellulosic materials for enhancing enzymatic digestibility. The utilization of non-enzymatic protein additives is promising for application in glucose and ethanol production from lignocellulosic materials.

  6. Enzymatic and bacterial conversions during sourdough fermentation.

    PubMed

    Gänzle, Michael G

    2014-02-01

    Enzymatic and microbial conversion of flour components during bread making determines bread quality. Metabolism of sourdough microbiota and the activity of cereal enzymes are interdependent. Acidification, oxygen consumption, and thiols accumulation by microbial metabolism modulate the activity of cereal enzymes. In turn, cereal enzymes provide substrates for bacterial growth. This review highlights the role of cereal enzymes and the metabolism of lactic acid bacteria in conversion of carbohydrates, proteins, phenolic compounds and lipids. Heterofermentative lactic acid bacteria prevailing in wheat and rye sourdoughs preferentially metabolise sucrose and maltose; the latter is released by cereal enzymes during fermentation. Sucrose supports formation of acetate by heterofermentative lactobacilli, and the formation of exopolysaccharides. The release of maltose and glucose by cereal enzymes during fermentation determines the exopolysaccharide yield in sourdough fermentations. Proteolysis is dependent on cereal proteases. Peptidase activities of sourdough lactic acid bacteria determine the accumulation of (bioactive) peptides, amino acids, and amino acid metabolites in dough and bread. Enzymatic conversion and microbial metabolism of phenolic compounds is relevant in sorghum and millet containing high levels of phenolic compounds. The presence of phenolic compounds with antimicrobial activity in sorghum selects for fermentation microbiota that are resistant to the phenolic compounds.

  7. Dual Enzymatic Detection by Bulk Electrogenerated Chemiluminescence.

    PubMed

    de Poulpiquet, Anne; Diez-Buitrago, Beatriz; Dumont Milutinovic, Milena; Sentic, Milica; Arbault, Stéphane; Bouffier, Laurent; Kuhn, Alexander; Sojic, Neso

    2016-06-21

    The combination of enzymes, as recognition elements for specific analytes, and of electrogenerated chemiluminescence (ECL) as a readout method has proven to be a valuable strategy for sensitive and specific analytical detection. However, ECL is intrinsically a 2D process which could potentially limit the analysis of inhomogeneous samples. Here, we show how a bulk ECL signal, generated by thousands of carbon microbeads remotely addressed via bipolar electrochemistry, are implemented as a powerful tool for the concomitant ECL sensing and imaging of two enzymatic substrates. We selected two enzymes (glucose dehydrogenase and choline oxidase) that react with their respective model substrates and produce in situ chemical species (β-nicotinamide adenine dinucleotide (NADH) and H2O2) acting as coreactants for the ECL emission of different luminophores ([Ru(bpy)3](2+) at λ = 620 nm and luminol at λ = 425 nm, respectively). Both enzymes are spatially separated in the same capillary. We demonstrate thus the simultaneous quantitative determination of both glucose and choline over a wide concentration range. The originality of this remote approach is to provide a global chemical view through one single ECL image of inhomogeneous samples such as a biochemical concentration gradient in a capillary configuration. Finally, we report the first proof-of-concept of dual biosensing based on this bulk ECL method for the simultaneous imaging of both enzymatic analytes at distinct wavelengths. PMID:27213503

  8. Categorization of some oscillatory enzymatic reactions

    SciTech Connect

    Schreiber, I.; Hung, Y.F.; Ross, J.

    1996-05-16

    We investigate the categorization of two or more proposed reaction mechanisms for each of the following oscillatory enzymatic reactions: (1) the peroxidase-oxidase reaction; (2) glycolytic oscillations; (3) oscillations of cyclic AMP in smile mold cells; (4) enzymatic pH oscillations; (5) calcium spiking in cytosol. We use prior work in stoichiometric network analysis and categorization of oscillatory reactions to identify in each proposed reaction mechanism essential and nonessential species, the specific role of each essential species, the connectivity of the essential species, including the identification of the reactions leading to oscillatory instabilities, and the category. For each model, we predict the result of several experiments including relative amplitudes, quench amplitudes, phase shifts, and sign symbolic concentration shifts and compare them with those from available experiments. These and several other experiments such as bifurcation analysis, phase response curves, entrainment experiments, qualitative and quantitative pulsed species response, delay experiments, and external periodic perturbation provide stringent tests of proposed reaction mechanisms, and appropriate ones are suggested to discriminate among competing mechanisms for a given reaction. We find the necessity for introducing a new subcategory in our categorization of oscillatory reactions. 45 refs., 6 figs., 6 tabs.

  9. Enzymatically Controlled Vacancies in Nanoparticle Crystals.

    PubMed

    Barnaby, Stacey N; Ross, Michael B; Thaner, Ryan V; Lee, Byeongdu; Schatz, George C; Mirkin, Chad A

    2016-08-10

    In atomic systems, the mixing of metals results in distinct phase behavior that depends on the identity and bonding characteristics of the atoms. In nanoscale systems, the use of oligonucleotides as programmable "bonds" that link nanoparticle "atoms" into superlattices allows for the decoupling of atom identity and bonding. While much research in atomic systems is dedicated to understanding different phase behavior of mixed metals, it is not well understood on the nanoscale how changes in the nanoscale "bond" affect the phase behavior of nanoparticle crystals. In this work, the identity of the atom is kept the same, but the chemical nature of the bond is altered, which is not possible in atomic systems, through the use of DNA and RNA bonding elements. These building blocks assemble into single crystal nanoparticle superlattices with mixed DNA and RNA bonding elements throughout. The nanoparticle crystals can be dynamically changed through the selective and enzymatic hydrolysis of the RNA bonding elements, resulting in superlattices that retain their crystalline structure and habit, while incorporating up to 35% random vacancies generated from the nanoparticles removed. Therefore, the bonding elements of nanoparticle crystals can be enzymatically and selectively addressed without affecting the nature of the atom.

  10. Recycling of cellulosic fibers by enzymatic process.

    PubMed

    Shojaei, K M; Dadashian, F; Montazer, M

    2012-02-01

    In this research, enzymatic treatment as an environmental friendly process has been used for recycling process of old cellulosic wastes such as cotton, viscose, and lyocell. Cellulase hydrolyses cellulosic chains and shortens cellulosic fibers. This study investigates to detect the optimum enzyme concentration and time of treatments for suitable changes of length and weight loss. The main purposes of this article are shortening of cellulosic fibers and evaluating of enzymatic treatment in different kind of cellulosic fibers. According to the data of experiments, with the increase of enzyme concentration and the treatment time, the length and weight loss percentage of the cellulosic fibers has been decreased. The length and weight loss percentage of treated viscose is more than that of lyocell and cotton fibers. Optimized condition, reaction time, and enzyme concentration have been determined by mean length of treated cellulosic samples. Suitable longitudinal distribution of fiber for papermaking industries is in the range of 0 to 4 mm. Optimum enzyme concentration and treatment time for recycling cotton, lyocell, and viscose fibers are 2% and 48 h for cotton and lyocell and 0.5% and 48 h for viscose, respectively. According to the data of experiment, the length of treated fibers is appropriate for its usage as a raw material in papermaking industries.

  11. Determination of lactose by an enzymatic method.

    PubMed

    Kleyn, D H

    1985-10-01

    The general methodology used for the determination of lactose in milk is considered, namely, polarimetry, gravimetry, infrared, colorimetry, gas-liquid chromatography, and high pressure liquid chromatography. The criteria for selecting an ideal analytical method followed by the relevance of most of these criteria in enzymatic methodology are discussed. The principle of the Boehringer-Mannheim method is presented, i.e., lactose is hydrolyzed to glucose and beta-galactose in the presence of beta-galactosidase and water. beta-Galactose is then oxidized by nicotinamide-adenine dinucleotide to galactonic acid in the presence of beta-galactose dehydrogenase. The amount of reduced nicotinamide-adenine dinucleotide formed is stoichiometric with the amount of lactose and is measured at 340 nm in a spectrophotometer possessing a slit width of less than or equal to 10 nm. The results of a recent Association of Official Analytical Chemists collaborative study of the B-M method are presented. From the overall mean of results on all samples, determinations by the enzymatic method averaged .49% lower than by the Association of Official Analytical Chemists gravimetric method. Standard deviations were similar for three sets of blind duplicates, which ranged between 3.67 and 4.55% lactose. F-Values revealed that variations between means obtained by laboratories differed significantly as compared with variations within laboratory means. The method has received Official First Action recognition by Association of Official Analytical Chemists.

  12. Enzymatic and bacterial conversions during sourdough fermentation.

    PubMed

    Gänzle, Michael G

    2014-02-01

    Enzymatic and microbial conversion of flour components during bread making determines bread quality. Metabolism of sourdough microbiota and the activity of cereal enzymes are interdependent. Acidification, oxygen consumption, and thiols accumulation by microbial metabolism modulate the activity of cereal enzymes. In turn, cereal enzymes provide substrates for bacterial growth. This review highlights the role of cereal enzymes and the metabolism of lactic acid bacteria in conversion of carbohydrates, proteins, phenolic compounds and lipids. Heterofermentative lactic acid bacteria prevailing in wheat and rye sourdoughs preferentially metabolise sucrose and maltose; the latter is released by cereal enzymes during fermentation. Sucrose supports formation of acetate by heterofermentative lactobacilli, and the formation of exopolysaccharides. The release of maltose and glucose by cereal enzymes during fermentation determines the exopolysaccharide yield in sourdough fermentations. Proteolysis is dependent on cereal proteases. Peptidase activities of sourdough lactic acid bacteria determine the accumulation of (bioactive) peptides, amino acids, and amino acid metabolites in dough and bread. Enzymatic conversion and microbial metabolism of phenolic compounds is relevant in sorghum and millet containing high levels of phenolic compounds. The presence of phenolic compounds with antimicrobial activity in sorghum selects for fermentation microbiota that are resistant to the phenolic compounds. PMID:24230468

  13. Mapping the Reaction Coordinates of Enzymatic Defluorination

    PubMed Central

    Chan, Peter W. Y.; Yakunin, Alexander F.; Edwards, Elizabeth A.; Pai, Emil F.

    2011-01-01

    The carbon-fluorine bond is the strongest covalent bond in organic chemistry, yet fluoroacetate dehalogenases can readily hydrolyze this bond under mild physiological conditions. Elucidating the molecular basis of this rare biocatalytic activity will provide the fundamental chemical insights of how this formidable feat is achieved. Here, we present a series of high-resolution (1.15–1.80 Å) crystal structures of a fluoroacetate dehalogenase, capturing snapshots along the defluorination reaction: the free enzyme, enzyme-fluoroacetate Michaelis complex, glycolyl-enzyme covalent intermediate and enzyme-product complex. We demonstrate that enzymatic defluorination requires a halide pocket that not only supplies three hydrogen bonds to stabilize the fluoride ion, but is also finely tailored for the smaller fluorine halogen atom to establish selectivity towards fluorinated substrates. We have further uncovered dynamics near the active site which may play pivotal roles in enzymatic defluorination. These findings may ultimately lead to the development of novel defluorinases that will enable the biotransformation of more complex fluorinated organic compounds, which in turn will assist the synthesis, detoxification, biodegradation, disposal, recycling and regulatory strategies for the growing markets of organofluorines across major industrial sectors. PMID:21510690

  14. Enzymatic activities in coniferous leaf litter

    SciTech Connect

    Spalding, B.P.

    1980-07-01

    Assays for measuring the activities of cellulase, xylanase, mannase, amylase, ..beta..-glucosidase, invertase, and protease employing buffered suspensions of ground coniferous and deciduous leaf litter exhibited zero-order kinetics. Only a small percentage of the whole-litter activities of invertase, ..beta..-glucosidase, and protease were extractable into 0.05M potassium acetate, pH 5.0; however, extractable activities of cellulase and xylanase represented from 39 to 174% of the whole-litter activities indicating their soluble exocellar nature. Extractable protease and amylase activities were best correlated with the average daily rates of CO/sub 2/ evolution in a group of 90 leaf litter samples equally representing 18 coniferous species. Enzymatic activities were readily detectable in extracts of all samples but classification of the samples by species provided little differentiation in the distribution of either enzymatic activities or rates of CO/sub 2/ evolution. Mannase, cellulase, and xylanase activities were well-correlated with each other in all samples. Assays attempting to measure a pool of readily-metabolizable substances in litter by extractable reducing substances, ninhydrin-positive substances, glucose, and phenolics failed to show correlation coefficients >0.41 with rates of CO/sub 2/ evolution. Addition of D-(+)-catechin to litter extracts, up to levels equivalent to those observed in the group of samples, did not inhibit any carbohydrase thus suggesting the lack of inhibition of litter-decomposing enzymes by the concentrations of phenolics present in these coniferous leaf litters.

  15. Mapping the Reaction Coordinates of Enzymatic Defluorination

    SciTech Connect

    Chan, Peter W.Y.; Yakunin, Alexander F.; Edwards, Elizabeth A.; Pai, Emil F.

    2011-09-28

    The carbon-fluorine bond is the strongest covalent bond in organic chemistry, yet fluoroacetate dehalogenases can readily hydrolyze this bond under mild physiological conditions. Elucidating the molecular basis of this rare biocatalytic activity will provide the fundamental chemical insights into how this formidable feat is achieved. Here, we present a series of high-resolution (1.15-1.80 {angstrom}) crystal structures of a fluoroacetate dehalogenase, capturing snapshots along the defluorination reaction: the free enzyme, enzyme-fluoroacetate Michaelis complex, glycolyl-enzyme covalent intermediate, and enzyme-product complex. We demonstrate that enzymatic defluorination requires a halide pocket that not only supplies three hydrogen bonds to stabilize the fluoride ion but also is finely tailored for the smaller fluorine halogen atom to establish selectivity toward fluorinated substrates. We have further uncovered dynamics near the active site which may play pivotal roles in enzymatic defluorination. These findings may ultimately lead to the development of novel defluorinases that will enable the biotransformation of more complex fluorinated organic compounds, which in turn will assist the synthesis, detoxification, biodegradation, disposal, recycling, and regulatory strategies for the growing markets of organofluorines across major industrial sectors.

  16. Is Moderate Red Wine Consumption Safe in Inactive Inflammatory Bowel Disease?

    PubMed Central

    Swanson, Garth R.; Tieu, Vanessa; Shaikh, Maliha; Forsyth, Chris; Keshavarzian, Ali

    2011-01-01

    Background Alcohol consumption is a potential trigger for inflammatory bowel disease (IBD) flare because of alcohol-induced oxidative stress and its deleterious effects on gut barrier function. Additionally, we have recently shown that alcohol consumption is associated with more symptoms in IBD. However, it is not known whether moderate daily alcohol consumption can modify IBD disease activity. To test what effects alcohol may have on patients with IBD, we evaluated the effect of moderate daily red wine for 1 week on two factors associated with recurrent IBD disease activity: intestinal permeability and stool calprotectin. Methods To assess the effects of moderate daily alcohol consumption on intestinal permeability and inflammation, we recruited 21 patients: 8 with inactive ulcerative colitis (UC), 6 with inactive Crohn's disease (CD), and 7 healthy controls. All participants with IBD completed a validated questionnaire on disease activity (Crohn's disease activity index or ulcerative colitis clinical activity index), to confirm they had inactive disease. All subjects then underwent a baseline assessment that included a blood draw, urine collection after sugar challenge, and stool collection. Subjects then consumed 1–3 glasses of red wine a day for 1 week (approx. 0.4 g EtOH/kg), and repeated the three measures. Results No subjects flared during the study. Moderate alcohol consumption did not significantly change either clinical disease activity scores or C-reactive protein. In contrast to healthy subjects, daily consumption of red wine significantly (1) decreased stool calprotectin in IBD subjects from baseline (p = 0.001) and (2) increased intestinal permeability as measured by urinary lactulose/mannitol excretion (marker of small bowel permeability) in CD (p = 0.028) or urinary sucralose secretion (marker of large bowel permeability) in UC (p = 0.012). Conclusions One week of moderate consumption of red wine in inactive IBD was associated with a significant

  17. Effects of optical and geometrical properties on YORP effect for inactive satellites

    NASA Astrophysics Data System (ADS)

    Albuja, A.; Scheeres, D.

    2014-09-01

    With the increasing number of space debris in Earth orbit, it is important to understand the dynamics of these objects. Initial studies have demonstrated that the Yarkovsky, O'Keefe, Radzievskii, Paddack (YORP) effect on inactive satellite needs to be further explored as it could be noticeably affecting the rotational dynamics of these Earth orbiting objects. The YORP effect is created by torques resulting from light and thermal energy being re-emitted from the surface of a body. This effect has been well studied and observed to affect the spin states of asteroids. The purpose of this paper is to further investigate YORP in the realm of large inactive Geosynchronous Earth Orbit (GEO) satellites. The forces that cause the YORP effect are highly dependent on the optical, thermal and geometrical properties of the facets making up the surface of the body being analyzed. This paper focuses on exploring the effect of these properties on the YORP effect for inactive satellite. Two different satellite models that represent bus types of inactive satellites in GEO are used for this study. By varying the optical, thermal and geometrical properties of these models, in a manner that remains consistent with realistic satellite parameters, we can understand the relationship between these properties and the torques created by YORP. Having this knowledge allows for better understanding of the possible attitude states (spin rate and obliquity) for uncontrolled satellites in GEO. This information can then be used to make predictions of the long-term behavior of the rotation rate and obliquity of these objects. Categories of potential final states for defunct GEO satellites can then be created based on geometrical and optical properties (e.g. spin up continuously, spin down continuously, etc.). This allows the population of inactive GEO satellites to be studied in a more general sense and final attitude states for these objects can be quickly identified. Furthermore, an understanding

  18. Preferential binding of allosteric modulators to active and inactive conformational states of metabotropic glutamate receptors

    PubMed Central

    Yanamala, Naveena; Tirupula, Kalyan C; Klein-Seetharaman, Judith

    2008-01-01

    Metabotropic glutamate receptors (mGluRs) are G protein coupled receptors that play important roles in synaptic plasticity and other neuro-physiological and pathological processes. Allosteric mGluR ligands are particularly promising drug targets because of their modulatory effects – enhancing or suppressing the response of mGluRs to glutamate. The mechanism by which this modulation occurs is not known. Here, we propose the hypothesis that positive and negative modulators will differentially stabilize the active and inactive conformations of the receptors, respectively. To test this hypothesis, we have generated computational models of the transmembrane regions of different mGluR subtypes in two different conformations. The inactive conformation was modeled using the crystal structure of the inactive, dark state of rhodopsin as template and the active conformation was created based on a recent model of the light-activated state of rhodopsin. Ligands for which the nature of their allosteric effects on mGluRs is experimentally known were docked to the modeled mGluR structures using ArgusLab and Autodock softwares. We find that the allosteric ligand binding pockets of mGluRs are overlapping with the retinal binding pocket of rhodopsin, and that ligands have strong preferences for the active and inactive states depending on their modulatory nature. In 8 out of 14 cases (57%), the negative modulators bound the inactive conformations with significant preference using both docking programs, and 6 out of 9 cases (67%), the positive modulators bound the active conformations. Considering results by the individual programs only, even higher correlations were observed: 12/14 (86%) and 8/9 (89%) for ArgusLab and 10/14 (71%) and 7/9 (78%) for AutoDock. These findings strongly support the hypothesis that mGluR allosteric modulation occurs via stabilization of different conformations analogous to those identified in rhodopsin where they are induced by photochemical isomerization

  19. Recombination at the DNA level. Abstracts

    SciTech Connect

    Not Available

    1984-01-01

    Abstracts of papers in the following areas are presented: (1) chromosome mechanics; (2) yeast systems; (3) mammalian homologous recombination; (4) transposons; (5) Mu; (6) plant transposons/T4 recombination; (7) topoisomerase, resolvase, and gyrase; (8) Escherichia coli general recombination; (9) recA; (10) repair; (11) eucaryotic enzymes; (12) integration and excision of bacteriophage; (13) site-specific recombination; and (14) recombination in vitro. (ACR)

  20. Macrobenthos community structure and trophic relationships within active and inactive Pacific hydrothermal sediments

    NASA Astrophysics Data System (ADS)

    Levin, Lisa A.; Mendoza, Guillermo F.; Konotchick, Talina; Lee, Raymond

    2009-09-01

    Hydrothermal fluids passing through sediments create a habitat hypothesized to influence the community structure of infaunal macrobenthos. Here we characterize the density, biomass, species composition, diversity, distributions, lifestyle, and nutritional sources of macroinfauna in hydrothermal sediments in NE and SW Pacific settings, and draw comparisons in search of faunal attributes characteristic of this habitat. There is increasing likelihood that seafloor massive sulfide deposits, associated with active and inactive hydrothermal venting, will be mined commercially. This creates a growing imperative for a more thorough understanding of the structure, dynamics, and resilience of the associated sediment faunas, and has stimulated the research presented here. Macrobenthic assemblages were studied at Manus Basin (1430-1634 m, Papua New Guinea [PNG]) as a function of location (South Su vs. Solwara 1), and hydrothermal activity (active vs. inactive), and at Middle Valley (2406-2411 m, near Juan de Fuca Ridge) as a function of habitat (active clam bed, microbial mat, hot mud, inactive background sediment). The studies conducted in PNG formed part of the environmental impact assessment work for the Solwara 1 Project of Nautilus Minerals Niugini Limited. We hypothesized that hydrothermally active sites should support (a) higher densities and biomass, (b) greater dominance and lower diversity, (c) a higher fraction of deposit feeders, and (d) greater isotopic evidence for chemosynthetic food sources than inactive sites. Manus Basin macrofauna generally had low density (<1000 ind. m -2) and low biomass (0.1-1.07 g m -2), except for the South Su active site, which had higher density (3494 ind. m -2) and biomass (11.94 g m -2), greater dominance (R1D=76%), lower diversity and more spatial (between-core) homogeneity than the Solwara 1 and South Su inactive sites. Dominant taxa at Manus Basin were Spionidae ( Prionospio sp.) in active sediments, and tanaids and deposit

  1. PROGENITORS OF RECOMBINING SUPERNOVA REMNANTS

    SciTech Connect

    Moriya, Takashi J.

    2012-05-01

    Usual supernova remnants have either ionizing plasma or plasma in collisional ionization equilibrium, i.e., the ionization temperature is lower than or equal to the electron temperature. However, the existence of recombining supernova remnants, i.e., supernova remnants with ionization temperature higher than the electron temperature, has been recently confirmed. One suggested way to have recombining plasma in a supernova remnant is to have a dense circumstellar medium at the time of the supernova explosion. If the circumstellar medium is dense enough, collisional ionization equilibrium can be established in the early stage of the evolution of the supernova remnant and subsequent adiabatic cooling, which occurs after the shock wave gets out of the dense circumstellar medium, makes the electron temperature lower than the ionization temperature. We study the circumstellar medium around several supernova progenitors and show which supernova progenitors can have a circumstellar medium dense enough to establish collisional ionization equilibrium soon after the explosion. We find that the circumstellar medium around red supergiants (especially massive ones) and the circumstellar medium dense enough to make Type IIn supernovae can establish collisional ionization equilibrium soon after the explosion and can evolve to become recombining supernova remnants. Wolf-Rayet stars and white dwarfs have the possibility to be recombining supernova remnants but the fraction is expected to be very small. As the occurrence rate of the explosions of red supergiants is much higher than that of Type IIn supernovae, the major progenitors of recombining supernova remnants are likely to be red supergiants.

  2. Progenitors of Recombining Supernova Remnants

    NASA Astrophysics Data System (ADS)

    Moriya, Takashi J.

    2012-05-01

    Usual supernova remnants have either ionizing plasma or plasma in collisional ionization equilibrium, i.e., the ionization temperature is lower than or equal to the electron temperature. However, the existence of recombining supernova remnants, i.e., supernova remnants with ionization temperature higher than the electron temperature, has been recently confirmed. One suggested way to have recombining plasma in a supernova remnant is to have a dense circumstellar medium at the time of the supernova explosion. If the circumstellar medium is dense enough, collisional ionization equilibrium can be established in the early stage of the evolution of the supernova remnant and subsequent adiabatic cooling, which occurs after the shock wave gets out of the dense circumstellar medium, makes the electron temperature lower than the ionization temperature. We study the circumstellar medium around several supernova progenitors and show which supernova progenitors can have a circumstellar medium dense enough to establish collisional ionization equilibrium soon after the explosion. We find that the circumstellar medium around red supergiants (especially massive ones) and the circumstellar medium dense enough to make Type IIn supernovae can establish collisional ionization equilibrium soon after the explosion and can evolve to become recombining supernova remnants. Wolf-Rayet stars and white dwarfs have the possibility to be recombining supernova remnants but the fraction is expected to be very small. As the occurrence rate of the explosions of red supergiants is much higher than that of Type IIn supernovae, the major progenitors of recombining supernova remnants are likely to be red supergiants.

  3. Electron-Beam Recombination Lasers

    NASA Astrophysics Data System (ADS)

    Rhoades, Robert Lewis

    1992-01-01

    The first known instance of electron-beam pumping of the 546.1 nm mercury laser is reported. This has been achieved using high-energy electrons to create intense ionization in a coaxial diode chamber containing a mixture of noble gases with a small amount of mercury vapor. Also reported are the results of a study of the 585.3 nm neon laser in He:Ne:Ar mixtures under similar experimental conditions. Both of these lasers are believed to be predominantly pumped by recombination. For the mercury laser, kinetic processes in the partially ionized plasma following the excitation pulse of high-energy electrons should favor the production of atomic mercury ions and molecular ions containing mercury. Subsequent recombination with electrons heavily favors the production of the 7^3S and 6^3 D states of Hg, of which 7^3S is the upper level of the reported laser. For the neon laser, the dominant recombining ion has been previously shown to be Ne_2^{+}. One of the dominant roles of helium in recombination lasers is inferred from the data for the neon laser at low helium concentrations. Helium appears to be necessary for the rapid relaxation of the electron energy which then increases the reaction rates for all known recombination processes thus increasing the pump rate into the upper state.

  4. Characterisation of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties

    PubMed Central

    Mertsalov, Ilya B.; Novikov, Boris N.; Scott, Hilary; Dangott, Lawrence; Panin, Vladislav M.

    2016-01-01

    CMP-sialic acid synthetase (CSAS) is a key enzyme of the sialylation pathway. CSAS produces the activated sugar donor, CMP-sialic acid, which serves as a substrate for sialyltransferases to modify glycan termini with sialic acid. Unlike other animal CMP-Sia synthetases that normally localize in the nucleus, Drosophila melanogaster CSAS (DmCSAS) localizes in the cell secretory compartment, predominantly in the Golgi, which suggests that this enzyme has properties distinct from those of its vertebrate counterparts. To test this hypothesis, we purified recombinant DmCSAS and characterised its activity in vitro. Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity for N-acetylneuraminic acid as a substrate, shows preference for lower pH and can function with a broad range of metal cofactors. When tested at a pH corresponding to the Golgi compartment, the enzyme showed significant activity with several metal cations, including Zn2+, Fe2+, Co2+ and Mn2+, while the activity with Mg2+ was found to be low. Protein sequence analysis and site-specific mutagenesis identified an aspartic acid residue that is necessary for enzymatic activity and predicted to be involved in coordinating a metal cofactor. DmCSAS enzymatic activity was found to be essential in vivo for rescuing the phenotype of DmCSAS mutants. Finally, our experiments revealed a steep dependence of the enzymatic activity on temperature. Taken together, our results indicate that DmCSAS underwent evolutionary adaptation to pH and ionic environment different from that of counterpart synthetases in vertebrates. Our data also suggest that environmental temperatures can regulate Drosophila sialylation, thus modulating neural transmission. PMID:27114558

  5. [Heterologous expression and enzymatic analysis of Streptomyces griseus trypsin in Streptomyces lividans].

    PubMed

    Ma, Tengbo; Ling, Zhenmin; Kang, Zhen; Li, Jianghua; Du, Guocheng; Chen, Jian

    2013-04-01

    Trypsin as an important serine protease has been widely used in food, pharmaceutical and tanning industries. In this study, we successfully expressed trypsin (cloning from Streptomyces griseus ATCC10137) in Streptomyces lividans TK24 and comparatively investigated its enzymatic properties. Specifically, applying S. griseus ATCC 10137 genome as template, we obtained the sprT gene and sub-cloned it into the expression plasmid pIJ86, generating the recombinant strain S. lividans TK24/pIJ86-sprT. When cultivated in R2YE and SELF, the activity of rSGT reached 9.21 U/mL and 8.61 U/mL, respectively. Meanwhile, the results of the enzymatic analysis showed that rSGT exhibited a higher acid tolerance and a higher specificity to hydrolyze amide bonds compared with bovine trypsin (BT). In addition, Zn2+ and organic solvents up-regulated esterase and amidase of rSGT. Taken together, the results obtained herein provide meaningful information for further modification of rSGT and its industrial application.

  6. Enzymatic Activity Analysis and Catalytic Essential Residues Identification of Brucella abortus Malate Dehydrogenase

    PubMed Central

    Han, Xiangan; Tong, Yongliang; Tian, Mingxing; Zhang, Yuxi; Sun, Xiaoqing; Wang, Shaohui; Qiu, Xusheng; Ding, Chan; Yu, Shengqing

    2014-01-01

    Malate dehydrogenase (MDH) plays important metabolic roles in bacteria. In this study, the recombinant MDH protein (His-MDH) of Brucella abortus was purified and its ability to catalyze the conversion of oxaloacetate (OAA) to L-malate (hereon referred to as MDH activity) was analyzed. Michaelis Constant (Km) and Maximum Reaction Velocity (Vmax) of the reaction were determined to be 6.45 × 10−3 M and 0.87 mM L−1 min−1, respectively. In vitro studies showed that His-MDH exhibited maximal MDH activity in pH 6.0 reaction buffer at 40°C. The enzymatic activity was 100%, 60%, and 40% inhibited by Cu2+, Zn2+, and Pb2+, respectively. In addition, six amino acids in the MDH were mutated to investigate their roles in the enzymatic activity. The results showed that the substitutions of amino acids Arg 89, Asp 149, Arg 152, His 176, or Thr 231 almost abolished the activity of His-MDH. The present study will help to understand MDH's roles in B. abortus metabolism. PMID:24895685

  7. Pretreatment and enzymatic hydrolysis of lignocellulosic biomass

    NASA Astrophysics Data System (ADS)

    Corredor, Deisy Y.

    The performance of soybean hulls and forage sorghum as feedstocks for ethanol production was studied. The main goal of this research was to increase fermentable sugars' yield through high-efficiency pretreatment technology. Soybean hulls are a potential feedstock for production of bio-ethanol due to their high carbohydrate content (≈50%) of nearly 37% cellulose. Soybean hulls could be the ideal feedstock for fuel ethanol production, because they are abundant and require no special harvesting and additional transportation costs as they are already in the plant. Dilute acid and modified steam-explosion were used as pretreatment technologies to increase fermentable sugars yields. Effects of reaction time, temperature, acid concentration and type of acid on hydrolysis of hemicellulose in soybean hulls and total sugar yields were studied. Optimum pretreatment parameters and enzymatic hydrolysis conditions for converting soybean hulls into fermentable sugars were identified. The combination of acid (H2SO4, 2% w/v) and steam (140°C, 30 min) efficiently solubilized the hemicellulose, giving a pentose yield of 96%. Sorghum is a tropical grass grown primarily in semiarid and dry parts of the world, especially in areas too dry for corn. The production of sorghum results in about 30 million tons of byproducts mainly composed of cellulose, hemicellulose, and lignin. Forage sorghum such as brown midrib (BMR) sorghum for ethanol production has generated much interest since this trait is characterized genetically by lower lignin concentrations in the plant compared with conventional types. Three varieties of forage sorghum and one variety of regular sorghum were characterized and evaluated as feedstock for fermentable sugar production. Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM) and X-Ray diffraction were used to determine changes in structure and chemical composition of forage sorghum before and after pretreatment and enzymatic hydrolysis

  8. Enzymatic Activity of the Soybean Ecto-Apyrase GS52 Is Essential for Stimulation of Nodulation1[W][OA

    PubMed Central

    Tanaka, Kiwamu; Nguyen, Cuong T.; Libault, Marc; Cheng, Jianlin; Stacey, Gary

    2011-01-01

    Nitrogen is an essential nutrient for plant growth. In the Rhizobium-legume symbiosis, root nodules are the sites of bacterial nitrogen fixation, in which atmospheric nitrogen is converted into a form that plants can utilize. While recent studies suggested an important role for the soybean (Glycine max) ecto-apyrase GS52 in rhizobial root hair infection and root nodule formation, precisely how this protein impacts the nodulation process remains undetermined. In this study, the biochemical characteristics of the GS52 enzyme were investigated. Computer modeling of the GS52 apyrase structure identified key amino acid residues important for catalytic activity, which were subsequently mutagenized. Although the GS52 enzyme exhibited broad substrate specificity, its activity on pyrimidine nucleotides and diphosphate nucleotides was significantly higher than on ATP. This result was corroborated by structural modeling of GS52, which predicted a low specificity for the adenine base within the substrate-binding pocket of the enzyme. The wild-type enzyme and its inactive mutant forms were expressed in soybean roots in order to evaluate the importance of GS52 enzymatic activity for nodulation. The results indicated a clear correlation between GS52 enzymatic activity and nodule number. Altogether, our study indicates that the catalytic activity of the GS52 apyrase, likely acting on extracellular nucleotides, is critical for rhizobial infection and nodulation. PMID:21346172

  9. Inhibition of enzymatic browning in foods and beverages.

    PubMed

    McEvily, A J; Iyengar, R; Otwell, W S

    1992-01-01

    Enzymatic browning is a major factor contributing to quality loss in foods and beverages. Sulfiting agents are used commonly to control browning; however, several negative attributes associated with sulfites have created the need for functional alternatives. Recent advances in the development of nonsulfite inhibitors of enzymatic browning are reviewed. The review focuses on compositions that are of practical relevance to food use.

  10. Enzymatic Products from Modified Soybean Oil Containing Hydrazinoester

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We use soybean oil to produce new, non-petroleum based products. The starting material is the ene reaction product of soybean oil and diethyl azodicarboxylate (DEAD), which can then be hydrolyzed chemically and enzymatically. Chemical hydrolysis gives hydrazino-fatty acids, whereas enzymatic hydro...

  11. Enzymatic browning and its control in fresh-cut produce

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzymatic browning of damaged tissues of fruits and vegetables during postharvest handling and processing degrades the sensory properties and nutritional value and discourages the consumer purchase of fresh-cut products. Consequently, enzymatic browning results in significant economic losses for the...

  12. Current Drive in Recombining Plasma

    SciTech Connect

    P.F. Schmit and N.J. Fisch

    2012-05-15

    The Langevin equations describing the average collisional dynamics of suprathermal particles in nonstationary plasma remarkably admit an exact analytical solution in the case of recombining plasma. The current density produced by arbitrary particle fluxes is derived including the effect of charge recombination. Since recombination has the effect of lowering the charge density of the plasma, thus reducing the charged particle collisional frequencies, the evolution of the current density can be modified substantially compared to plasma with fixed charge density. The current drive efficiency is derived and optimized for discrete and continuous pulses of current, leading to the discovery of a nonzero "residual" current density that persists indefinitely under certain conditions, a feature not present in stationary plasmas.

  13. The Dissociative Recombination of OH(+)

    NASA Technical Reports Server (NTRS)

    Guberman, Steven L.

    1995-01-01

    Theoretical quantum chemical calculations of the cross sections and rates for the dissociative recombination of the upsilon = 0 level of the ground state of OH(+) show that recombination occurs primarily along the 2 (2)Pi diabatic route. The products are 0((1)D) and a hot H atom with 6.1 eV kinetic energy. The coupling to the resonances is very small and the indirect recombination mechanism plays only a minor role. The recommended value for the rate coefficient is (6.3 +/- 0.7) x 10(exp -9)x (T(e)/1300)(exp -0.48) cu.cm/s for 10 less than T(e) less than 1000 K.

  14. Recombinant snake venom prothrombin activators.

    PubMed

    Lövgren, Ann

    2013-01-01

    Three prothrombin activators; ecarin, which was originally isolated from the venom of the saw-scaled viper Echis carinatus, trocarin from the rough-scaled snake Tropidechis carinatus, and oscutarin from the Taipan snake Oxyuranus scutellatus, were expressed in mammalian cells with the purpose to obtain recombinant prothrombin activators that could be used to convert prothrombin to thrombin. We have previously reported that recombinant ecarin can efficiently generate thrombin without the need for additional cofactors, but does not discriminate non-carboxylated prothrombin from biologically active γ-carboxylated prothrombin. Here we report that recombinant trocarin and oscutarin could not efficiently generate thrombin without additional protein co-factors. We confirm that both trocarin and oscutarin are similar to human coagulation Factor X (FX), explaining the need for additional cofactors. Sequencing of a genomic fragment containing 7 out of the 8 exons coding for oscutarin further confirmed the similarity to human FX. PMID:23111318

  15. 38 CFR 4.88c - Ratings for inactive nonpulmonary tuberculosis initially entitled after August 19, 1968.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Infectious Diseases, Immune Disorders and Nutritional Deficiencies § 4.88c Ratings for inactive nonpulmonary... active tuberculosis 100 Thereafter: Rate residuals under the specific body system or systems...

  16. 38 CFR 4.88c - Ratings for inactive nonpulmonary tuberculosis initially entitled after August 19, 1968.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Infectious Diseases, Immune Disorders and Nutritional Deficiencies § 4.88c Ratings for inactive nonpulmonary... active tuberculosis 100 Thereafter: Rate residuals under the specific body system or systems...

  17. 38 CFR 4.88c - Ratings for inactive nonpulmonary tuberculosis initially entitled after August 19, 1968.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Infectious Diseases, Immune Disorders and Nutritional Deficiencies § 4.88c Ratings for inactive nonpulmonary... active tuberculosis 100 Thereafter: Rate residuals under the specific body system or systems...

  18. 38 CFR 4.88c - Ratings for inactive nonpulmonary tuberculosis initially entitled after August 19, 1968.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Infectious Diseases, Immune Disorders and Nutritional Deficiencies § 4.88c Ratings for inactive nonpulmonary... active tuberculosis 100 Thereafter: Rate residuals under the specific body system or systems...

  19. 38 CFR 4.88c - Ratings for inactive nonpulmonary tuberculosis initially entitled after August 19, 1968.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Infectious Diseases, Immune Disorders and Nutritional Deficiencies § 4.88c Ratings for inactive nonpulmonary... active tuberculosis 100 Thereafter: Rate residuals under the specific body system or systems...

  20. The proximal J kappa germline-transcript promoter facilitates receptor editing through control of ordered recombination.

    PubMed

    Vettermann, Christian; Timblin, Greg A; Lim, Vivian; Lai, Ernest C; Schlissel, Mark S

    2015-01-01

    V(D)J recombination creates antibody light chain diversity by joining a Vκ gene segment with one of four Jκ segments. Two Jκ germline-transcript (GT) promoters control Vκ-Jκ joining, but the mechanisms that govern Jκ choice are unclear. Here, we show in gene-targeted mice that the proximal GT promoter helps targeting rearrangements to Jκ1 by preventing premature DNA breaks at Jκ2. Consequently, cells lacking the proximal GT promoter show a biased utilization of downstream Jκ segments, resulting in a diminished potential for receptor editing. Surprisingly, the proximal--in contrast to the distal--GT promoter is transcriptionally inactive prior to Igκ recombination, indicating that its role in Jκ choice is independent of classical promoter function. Removal of the proximal GT promoter increases H3K4me3 levels at Jκ segments, suggesting that this promoter could act as a suppressor of recombination by limiting chromatin accessibility to RAG. Our findings identify the first cis-element critical for Jκ choice and demonstrate that ordered Igκ recombination facilitates receptor editing.

  1. Recombinant Nepenthesin II for Hydrogen/Deuterium Exchange Mass Spectrometry.

    PubMed

    Yang, Menglin; Hoeppner, Morgan; Rey, Martial; Kadek, Alan; Man, Petr; Schriemer, David C

    2015-07-01

    The pitcher secretions of the Nepenthes genus of carnivorous plants contain a proteolytic activity that is very useful for hydrogen/deuterium exchange mass spectrometry (HX-MS). Our efforts to reconstitute pitcher fluid activity using recombinant nepenthesin I (one of two known aspartic proteases in the fluid) revealed a partial cleavage profile and reduced enzymatic stability in certain HX-MS applications. We produced and characterized recombinant nepenthesin II to determine if it complemented nepenthesin I in HX-MS applications. Nepenthesin II shares many properties with nepenthesin I, such as fast digestion at reduced temperature and pH, and broad cleavage specificity, but in addition, it cleaves C-terminal to tryptophan. Neither enzyme reproduces the C-terminal proline cleavage we observed in the natural extract. Nepenthesin II is considerably more resistant to chemical denaturants and reducing agents than nepenthesin I, and it possesses a stability profile that is similar to that of pepsin. Higher stability combined with the slightly broader cleavage specificity makes nepenthesin II a useful alternative to pepsin and a more complete replacement for pitcher fluid in HX-MS applications. PMID:25993527

  2. Recent development of miniatured enzymatic biofuel cell

    NASA Astrophysics Data System (ADS)

    Song, Yin; Penmatsa, Varun; Wang, Chunlei

    2011-06-01

    Enzymatic biofuel cells (EBFCs) that oxidize biological fuels using enzyme-modified electrodes are considered a promising candidate for implantable power sources. However, there are still challenges to overcome before biofuel cells become competitive in any practical applications. Currently, the short lifespan of the catalytic enzymes and poor power density are the most critical issues in developing EBFCs. In this paper, we will review the recent development of biofuel cells and highlight the progress in Carbon-microelectromechanical system (C-MEMS) based micro biofuel cells by both computational modeling and experimental work. Also, our effort on utilizing a covalent immobilization technique for the attachment of enzymes onto the substrate which is expected to increase the enzyme loading efficiency and the power density of devices is discussed in this paper.

  3. Zinc oxide inverse opal enzymatic biosensor

    NASA Astrophysics Data System (ADS)

    You, Xueqiu; Pikul, James H.; King, William P.; Pak, James J.

    2013-06-01

    We report ZnO inverse opal- and nanowire (NW)-based enzymatic glucose biosensors with extended linear detection ranges. The ZnO inverse opal sensors have 0.01-18 mM linear detection range, which is 2.5 times greater than that of ZnO NW sensors and 1.5 times greater than that of other reported ZnO sensors. This larger range is because of reduced glucose diffusivity through the inverse opal geometry. The ZnO inverse opal sensors have an average sensitivity of 22.5 μA/(mM cm2), which diminished by 10% after 35 days, are more stable than ZnO NW sensors whose sensitivity decreased by 10% after 7 days.

  4. Enzymatic Hydrogen Production from Starch and Water

    SciTech Connect

    Zhang, Y.-H. Percival; Evans, Barbara R; Mielenz, Jonathan R; Hopkins, Robert C.; Adams, Michael W. W.

    2007-01-01

    A novel enzymatic reaction was conducted for producing hydrogen from starch and water at 30oC. The overall reaction comprised of 13 enzymes, 1 cofactor (NADP+), and phosphate was driven by energy stored in carbohydrate starch according to the overall stoichiometry stoichiometric reaction of C6H10O5 (l) + 7 H2O (l) --> 12 H2 (g) + 6 CO2 (g). It is spontaneous and unidirectional because of negative Gibbs free energy and the removal of gaseous products from the aqueous reaction solution. With technology improvement and integration with fuel cells, this technology would be suitable for mobile applications and also solve the challenges associated with hydrogen storage, distribution, and infrastructure in a hydrogen economy.

  5. Heavy atom isotope effects on enzymatic reactions

    NASA Astrophysics Data System (ADS)

    Paneth, Piotr

    1994-05-01

    The theory of isotope effects, which has proved to be extremely useful in providing geometrical details of transition states in a variety of chemical reactions, has recently found an application in studies of enzyme-catalyzed reactions. These reactions are multistep in nature with few steps being partially rate-limiting, thus interpretation of these isotope effects is more complex. The theoretical framework of heavy-atom isotope effects on enzymatic reactions is critically analyzed on the basis of recent results of: carbon kinetic isotope effects on carbonic anhydrase and catalytic antibodies; multiple carbon, deuterium isotope effects on reactions catalyzed by formate decarboxylase; oxygen isotope effects on binding processes in reactions catalyzed by pyruvate kinase; and equilibrium oxygen isotope effect on binding an inhibitor to lactate dehydrogenase. The advantages and disadvantages of reaction complexity in learning details of formal and molecular mechanisms are discussed in the examples of reactions catalyzed by phosphoenolpyruvate carboxylase, orotidine decarboxylase and glutamine synthetase.

  6. Isothermal Titration Calorimetry to Characterize Enzymatic Reactions.

    PubMed

    Mazzei, Luca; Ciurli, Stefano; Zambelli, Barbara

    2016-01-01

    Isothermal titration calorimetry (ITC) is a technique that measures the heat released or absorbed during a chemical reaction as an intrinsic probe to characterize any chemical process that involves heat changes spontaneously occurring during the reaction. The general features of this method to determine the kinetic and thermodynamic parameters of enzymatic reactions (kcat, KM, ΔH) are described and discussed here together with some detailed applications to specific cases. ITC does not require any modification or labeling of the system under analysis, can be performed in solution, and needs only small amounts of enzyme. These properties make ITC an invaluable, powerful, and unique tool to extend the knowledge of enzyme kinetics to drug discovery.

  7. Thermal stability of bioactive enzymatic papers.

    PubMed

    Khan, Mohidus Samad; Li, Xu; Shen, Wei; Garnier, Gil

    2010-01-01

    The thermal stability of two enzymes adsorbed on paper, alkaline phosphatase (ALP) and horseradish peroxidase (HRP), was measured using a colorimetric technique quantifying the intensity of the product complex. The enzymes adsorbed on paper retained their functionality and selectivity. Adsorption on paper increased the enzyme thermal stability by 2-3 orders of magnitude compared to the same enzyme in solution. ALP and HRP enzymatic papers had half-lives of 533 h and 239 h at 23 degrees C, respectively. The thermal degradation of adsorbed enzyme was found to follow two sequential first-order reactions, indication of a reaction system. A complex pattern of enzyme was printed on paper using a thermal inkjet printer. Paper and inkjet printing are ideal material and process to manufacture low-cost-high volume bioactive surfaces.

  8. Structural basis of enzymatic benzene ring reduction.

    PubMed

    Weinert, Tobias; Huwiler, Simona G; Kung, Johannes W; Weidenweber, Sina; Hellwig, Petra; Stärk, Hans-Joachim; Biskup, Till; Weber, Stefan; Cotelesage, Julien J H; George, Graham N; Ermler, Ulrich; Boll, Matthias

    2015-08-01

    In chemical synthesis, the widely used Birch reduction of aromatic compounds to cyclic dienes requires alkali metals in ammonia as extremely low-potential electron donors. An analogous reaction is catalyzed by benzoyl-coenzyme A reductases (BCRs) that have a key role in the globally important bacterial degradation of aromatic compounds at anoxic sites. Because of the lack of structural information, the catalytic mechanism of enzymatic benzene ring reduction remained obscure. Here, we present the structural characterization of a dearomatizing BCR containing an unprecedented tungsten cofactor that transfers electrons to the benzene ring in an aprotic cavity. Substrate binding induces proton transfer from the bulk solvent to the active site by expelling a Zn(2+) that is crucial for active site encapsulation. Our results shed light on the structural basis of an electron transfer process at the negative redox potential limit in biology. They open the door for biological or biomimetic alternatives to a basic chemical synthetic tool.

  9. Fungal biodegradation and enzymatic modification of lignin

    PubMed Central

    Dashtban, Mehdi; Schraft, Heidi; Syed, Tarannum A.; Qin, Wensheng

    2010-01-01

    Lignin, the most abundant aromatic biopolymer on Earth, is extremely recalcitrant to degradation. By linking to both hemicellulose and cellulose, it creates a barrier to any solutions or enzymes and prevents the penetration of lignocellulolytic enzymes into the interior lignocellulosic structure. Some basidiomycetes white-rot fungi are able to degrade lignin efficiently using a combination of extracellular ligninolytic enzymes, organic acids, mediators and accessory enzymes. This review describes ligninolytic enzyme families produced by these fungi that are involved in wood decay processes, their molecular structures, biochemical properties and the mechanisms of action which render them attractive candidates in biotechnological applications. These enzymes include phenol oxidase (laccase) and heme peroxidases [lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP)]. Accessory enzymes such as H2O2-generating oxidases and degradation mechanisms of plant cell-wall components in a non-enzymatic manner by production of free hydroxyl radicals (·OH) are also discussed. PMID:21968746

  10. Cascade enzymatic reactions for efficient carbon sequestration.

    PubMed

    Xia, Shunxiang; Zhao, Xueyan; Frigo-Vaz, Benjamin; Zheng, Wenyun; Kim, Jungbae; Wang, Ping

    2015-04-01

    Thermochemical processes developed for carbon capture and storage (CCS) offer high carbon capture capacities, but are generally hampered by low energy efficiency. Reversible cascade enzyme reactions are examined in this work for energy-efficient carbon sequestration. By integrating the reactions of two key enzymes of RTCA cycle, isocitrate dehydrogenase and aconitase, we demonstrate that intensified carbon capture can be realized through such cascade enzymatic reactions. Experiments show that enhanced thermodynamic driving force for carbon conversion can be attained via pH control under ambient conditions, and that the cascade reactions have the potential to capture 0.5 mol carbon at pH 6 for each mole of substrate applied. Overall it manifests that the carbon capture capacity of biocatalytic reactions, in addition to be energy efficient, can also be ultimately intensified to approach those realized with chemical absorbents such as MEA.

  11. Extracellular enzymatic activities of Bipolaris sorokiniana isolates.

    PubMed

    Geimba, Mercedes P; Brandelli, Adriano

    2002-01-01

    Several enzymatic activities were investigated in six isolates of the fungus Bipolaris sorokiniana, originating from different areas of Brazil. Among the glycosidases studied, beta-glucosidase, beta-N-acetylglucosaminidase, beta-xylosidase, cellobiohydrolase, and chitobiohydrolase were the major activities. In some isolates, beta-glucuronidase, beta-galactosidase, and alpha-mannosidase activities were also present. Polysaccharide-hydrolyzing enzymes, such as pectin lyase and carboxymethyl cellulase were detected in significant amounts, and their activities were variable among the different isolates. Other enzymes, namely phosphatases, proteinases and phenol oxidase, were also examined, showing variable amounts depending on the isolate. The pH dependence of all enzymes tested was investigated. Endoproteinase, carboxymethyl cellulase, and phenoloxidase had maximum activity in the pH range of 6-8, whilst all other enzymes showed maximum activity at pH 4-6.

  12. First purification of the antiquitin protein and demonstration of its enzymatic activity.

    PubMed

    Tang, Wai-Kwan; Cheng, Christopher H K; Fong, Wing-Ping

    2002-04-10

    Antiquitin is an evolutionarily conserved protein believed to play a role in the regulation of cellular turgor. Based on sequence analysis, this protein is classified as a member of the aldehyde dehydrogenase superfamily. All previous studies on antiquitin have been confined to the nucleotide level, and the protein has never been purified and characterized. In the present investigation, the antiquitin protein was purified for the first time. An acetaldehyde-oxidizing protein was isolated from the liver of black seabream (Mylio macrocephalus) by chromatographies on alpha-cyanocinnamate Sepharose and Affi-gel Blue agarose, followed by ammonium sulfate precipitation. The purified protein was identified as antiquitin by the first 18 N-terminal amino acid residues which showed 83.3% identity with the deduced sequence of human antiquitin. Electrophoretic mobility studies indicated that black seabream antiquitin is a tetramer with a subunit molecular mass of 57.5 kDa. Kinetic analysis of the purified protein indicated that it catalyzes the oxidation of acetaldehyde with K(m) and V(max) values of 2.0 mM and 1.3 U/mg, respectively. The longer aliphatic propionaldehyde and the aromatic benzaldehyde are also substrates of the purified enzyme. The enzyme is highly specific towards NAD+ as the coenzyme and is totally inactive towards NADP+. Maximal enzymatic activity was found at about pH 9-10. PMID:11959129

  13. Urease immobilized polymer hydrogel: Long-term stability and enhancement of enzymatic activity.

    PubMed

    Kutcherlapati, S N Raju; Yeole, Niranjan; Jana, Tushar

    2016-02-01

    A method has been developed in which an enzyme namely urease was immobilized inside hydrogel matrix to study the stability and enzymatic activity in room temperature (∼27-30°C). This urease coupled hydrogel (UCG) was obtained by amine-acid coupling reaction and this procedure is such that it ensured the wider opening of mobile flap of enzyme active site. A systematic comparison of urea-urease assay and the detailed kinetic data clearly revealed that the urease shows activity for more than a month when stored at ∼27-30°C in case of UCG whereas it becomes inactive in case of free urease (enzyme in buffer solution). The aqueous microenvironment inside the hydrogel, unusual morphological features and thermal behaviour were believed to be the reasons for unexpected behaviour. UCG displayed enzyme activity at basic pH and up to 60°C. UCG showed significant enhancement in activity against thermal degradation compared to free urease. In summary, this method is a suitable process to stabilize the biomacromolecules in standard room temperature for many practical uses.

  14. Urease immobilized polymer hydrogel: Long-term stability and enhancement of enzymatic activity.

    PubMed

    Kutcherlapati, S N Raju; Yeole, Niranjan; Jana, Tushar

    2016-02-01

    A method has been developed in which an enzyme namely urease was immobilized inside hydrogel matrix to study the stability and enzymatic activity in room temperature (∼27-30°C). This urease coupled hydrogel (UCG) was obtained by amine-acid coupling reaction and this procedure is such that it ensured the wider opening of mobile flap of enzyme active site. A systematic comparison of urea-urease assay and the detailed kinetic data clearly revealed that the urease shows activity for more than a month when stored at ∼27-30°C in case of UCG whereas it becomes inactive in case of free urease (enzyme in buffer solution). The aqueous microenvironment inside the hydrogel, unusual morphological features and thermal behaviour were believed to be the reasons for unexpected behaviour. UCG displayed enzyme activity at basic pH and up to 60°C. UCG showed significant enhancement in activity against thermal degradation compared to free urease. In summary, this method is a suitable process to stabilize the biomacromolecules in standard room temperature for many practical uses. PMID:26520823

  15. Enzymatic Hydrolysis of Hydrotropic Pulps at Different Substrate Loadings.

    PubMed

    Denisova, Marina N; Makarova, Ekaterina I; Pavlov, Igor N; Budaeva, Vera V; Sakovich, Gennady V

    2016-03-01

    Enzymatic hydrolysis of cellulosic raw materials to produce nutrient broths for microbiological synthesis of ethanol and other valuable products is an important field of modern biotechnology. Biotechnological processing implies the selection of an effective pretreatment technique for raw materials. In this study, the hydrotropic treatment increased the reactivity of the obtained substrates toward enzymatic hydrolysis by 7.1 times for Miscanthus and by 7.3 times for oat hulls. The hydrotropic pulp from oat hulls was more reactive toward enzymatic hydrolysis compared to that from Miscanthus, despite that the substrates had similar compositions. As the initial substrate loadings were raised during enzymatic hydrolysis of the hydrotropic Miscanthus and oat hull pulps, the concentration of reducing sugars increased by 34 g/dm(3) and the yield of reducing sugars decreased by 31 %. The findings allow us to predict the efficiency of enzymatic hydrolysis of hydrotropic pulps from Miscanthus and oat hulls when scaling up the process by volume. PMID:26634840

  16. Selenium incorporation using recombinant techniques

    SciTech Connect

    Walden, Helen

    2010-04-01

    An overview of techniques for recombinant incorporation of selenium and subsequent purification and crystallization of the resulting labelled protein. Using selenomethionine to phase macromolecular structures is common practice in structure determination, along with the use of selenocysteine. Selenium is consequently the most commonly used heavy atom for MAD. In addition to the well established recombinant techniques for the incorporation of selenium in prokaryal expression systems, there have been recent advances in selenium labelling in eukaryal expression, which will be discussed. Tips and things to consider for the purification and crystallization of seleno-labelled proteins are also included.

  17. The Enzymatic Oxidation of Graphene Oxide

    PubMed Central

    Kotchey, Gregg P.; Allen, Brett L.; Vedala, Harindra; Yanamala, Naveena; Kapralov, Alexander A.; Tyurina, Yulia Y.; Klein-Seetharaman, Judith; Kagan, Valerian E.; Star, Alexander

    2011-01-01

    Two-dimensional graphitic carbon is a new material with many emerging applications, and studying its chemical properties is an important goal. Here, we reported a new phenomenon – the enzymatic oxidation of a single layer of graphitic carbon by horseradish peroxidase (HRP). In the presence of low concentrations of hydrogen peroxide (~40 µM), HRP catalyzed the oxidation of graphene oxide, which resulted in the formation of holes on its basal plane. During the same period of analysis, HRP failed to oxidize chemically reduced graphene oxide (RGO). The enzymatic oxidation was characterized by Raman, UV-Vis, EPR and FT-IR spectroscopy, TEM, AFM, SDS-PAGE, and GC-MS. Computational docking studies indicated that HRP was preferentially bound to the basal plane rather than the edge for both graphene oxide and RGO. Due to the more dynamic nature of HRP on graphene oxide, the heme active site of HRP was in closer proximity to graphene oxide compared to RGO, thereby facilitating the oxidation of the basal plane of graphene oxide. We also studied the electronic properties of the reduced intermediate product, holey reduced graphene oxide (hRGO), using field-effect transistor (FET) measurements. While RGO exhibited a V-shaped transfer characteristic similar to a single layer of graphene that was attributed to its zero band gap, hRGO demonstrated a p-type semiconducting behavior with a positive shift in the Dirac points. This p-type behavior rendered hRGO, which can be conceptualized as interconnected graphene nanoribbons, as a potentially attractive material for FET sensors. PMID:21344859

  18. Role of trypsin-like cleavage at arginine 192 in the enzymatic and cytotonic activities of Escherichia coli heat-labile enterotoxin.

    PubMed Central

    Grant, C C; Messer, R J; Cieplak, W

    1994-01-01

    Previous studies of cholera toxin and Escherichia coli heat-labile enterotoxin have suggested that proteolytic cleavage plays an important role in the expression of ADP-ribosyltransferase activity and toxicity. Specifically, several studies have implicated a trypsin-like cleavage at arginine 192, which lies within an exposed region subtended by a disulfide bond in the intact A subunit, in toxicity. To investigate the role of this modification in the enzymatic and cytotonic properties of heat-labile enterotoxin, the response of purified, recombinant A subunit to tryptic activation and the effect of substituting arginine 192 with glycine on the activities of the holotoxin were examined. The recombinant A subunit of heat-labile enterotoxin exhibited significant levels of ADP-ribosyltransferase activity that were only nominally increased (approximately twofold) by prior limited trypsinolysis. The enzymatic activity also did not appear to be affected by auto-ADP-ribosylation that occurs during the high-level synthesis of the recombinant A subunit in E. coli. A mutant form of the holotoxin containing the arginine 192-to-glycine substitution exhibited levels of cytotonic activity for CHO cells that were similar to that of the untreated, wild-type holotoxin but exhibited a marked delay in the ability to increase intracellular levels of cyclic AMP in Caco-2 cells. The results indicate that trypsin-like cleavage of the A subunit of E. coli heat-labile enterotoxin at arginine 192 is not requisite to the expression of enzymatic activity by the A subunit and further reveal that this modification, although it enhances the biological and enzymatic activities of the toxin, is not absolutely required for the enterotoxin to elicit cytotonic effects. Images PMID:7927684

  19. Spinal atypical protein kinase C activity is necessary to stabilize inactivity-induced phrenic motor facilitation.

    PubMed

    Strey, Kristi A; Nichols, Nicole L; Baertsch, Nathan A; Broytman, Oleg; Baker-Herman, Tracy L

    2012-11-14

    The neural network controlling breathing must establish rhythmic motor output at a level adequate to sustain life. Reduced respiratory neural activity elicits a novel form of plasticity in circuits driving the diaphragm known as inactivity-induced phrenic motor facilitation (iPMF), a rebound increase in phrenic inspiratory output observed once respiratory neural drive is restored. The mechanisms underlying iPMF are unknown. Here, we demonstrate in anesthetized rats that spinal mechanisms give rise to iPMF and that iPMF consists of at least two mechanistically distinct phases: (1) an early, labile phase that requires atypical PKC (PKCζ and/or PKCι/λ) activity to transition to a (2) late, stable phase. Early (but not late) iPMF is associated with increased interactions between PKCζ/ι and the scaffolding protein ZIP (PKCζ-interacting protein)/p62 in spinal regions associated with the phrenic motor pool. Although PKCζ/ι activity is necessary for iPMF, spinal atypical PKC activity is not necessary for phrenic long-term facilitation (pLTF) following acute intermittent hypoxia, an activity-independent form of spinal respiratory plasticity. Thus, while iPMF and pLTF both manifest as prolonged increases in phrenic burst amplitude, they arise from distinct spinal cellular pathways. Our data are consistent with the hypotheses that (1) local mechanisms sense and respond to reduced respiratory-related activity in the phrenic motor pool and (2) inactivity-induced increases in phrenic inspiratory output require local PKCζ/ι activity to stabilize into a long-lasting iPMF. Although the physiological role of iPMF is unknown, we suspect that iPMF represents a compensatory mechanism, assuring adequate motor output in a physiological system in which prolonged inactivity ends life. PMID:23152633

  20. Potential clinical translation of juvenile rodent inactivity models to study the onset of childhood obesity

    PubMed Central

    Roberts, Michael D.; Company, Joseph M.; Brown, Jacob D.; Toedebusch, Ryan G.; Padilla, Jaume; Jenkins, Nathan T.; Laughlin, M. Harold

    2012-01-01

    According to the latest data from the Center for Disease Control and Prevention 17%, or 12.5 million, of children and adolescents aged 2–19 years in the United States are obese. Physical inactivity is designated as one of the actual causes of US deaths and undoubtedly contributes to the obesity epidemic in children and adults. Examining the effects of inactivity on physiological homeostasis during youth is crucial given that 58% of children between the ages 6–11 yr old fail to obtain the recommended 60 min/day of physical activity and 92% of adolescents fail to achieve this goal [Troiano et al. Med Sci Sports Exerc. 40, 2008]. Nonetheless, invasive mechanistic studies in children linking diminished physical activity with metabolic maladies are lacking for obvious ethical reasons. The rodent wheel lock (WL) model was adopted by our laboratory and others to study how different organ systems of juvenile rats respond to a cessation of daily physical activity. Our WL model houses rats in cages equipped with voluntary running wheels starting at 28 days of age. After a certain period of voluntary running (3 to 6 wk), the wheels are locked, thus preventing the rats' primary source of physical activity. The studies discussed herein suggest that obesity-associated maladies including skeletal muscle insulin resistance, hypothalamic leptin resistance, fatty acid oxidation impairments in skeletal muscle and adipose tissue, nonalcoholic fatty liver disease, and endothelial dysfunction are initiated in juvenile animals that are restrained from voluntary exercise via WL. The use of the juvenile rodent WL or other inactivity models will continue to provide a powerful clinical translational tool that can be used for primordial prevention of human childhood obesity. PMID:22696577

  1. Potential clinical translation of juvenile rodent inactivity models to study the onset of childhood obesity.

    PubMed

    Roberts, Michael D; Company, Joseph M; Brown, Jacob D; Toedebusch, Ryan G; Padilla, Jaume; Jenkins, Nathan T; Laughlin, M Harold; Booth, Frank W

    2012-08-01

    According to the latest data from the Center for Disease Control and Prevention 17%, or 12.5 million, of children and adolescents aged 2-19 years in the United States are obese. Physical inactivity is designated as one of the actual causes of US deaths and undoubtedly contributes to the obesity epidemic in children and adults. Examining the effects of inactivity on physiological homeostasis during youth is crucial given that 58% of children between the ages 6-11 yr old fail to obtain the recommended 60 min/day of physical activity and 92% of adolescents fail to achieve this goal [Troiano et al. Med Sci Sports Exerc. 40, 2008]. Nonetheless, invasive mechanistic studies in children linking diminished physical activity with metabolic maladies are lacking for obvious ethical reasons. The rodent wheel lock (WL) model was adopted by our laboratory and others to study how different organ systems of juvenile rats respond to a cessation of daily physical activity. Our WL model houses rats in cages equipped with voluntary running wheels starting at 28 days of age. After a certain period of voluntary running (3 to 6 wk), the wheels are locked, thus preventing the rats' primary source of physical activity. The studies discussed herein suggest that obesity-associated maladies including skeletal muscle insulin resistance, hypothalamic leptin resistance, fatty acid oxidation impairments in skeletal muscle and adipose tissue, nonalcoholic fatty liver disease, and endothelial dysfunction are initiated in juvenile animals that are restrained from voluntary exercise via WL. The use of the juvenile rodent WL or other inactivity models will continue to provide a powerful clinical translational tool that can be used for primordial prevention of human childhood obesity. PMID:22696577

  2. Inflammatory, autoimmune, chronic diseases: bad diet and physical inactivity are causes or effects?

    PubMed

    Gracia, M C

    2006-01-01

    It is now well established that most chronic diseases, especially those identified as inflammatory, are statistically correlated with some typical dietary excesses and physical inactivity. But do really these habits cause the diseases, or they result from them? Current opinion favours the first option, but fails to explain why the satisfaction of eating, naturally evolved in our brains to produce health, apparently induces countless millions of people to eat unrestrictedly until becoming mortally sick, whereas trying to keep a theoretically healthy diet is most often a real torture. The inverse explanation makes much more sense: since inflammation produces much heat, calorie-rich diets are required. An inflamed digestive tract lacks digestive power and is easily irritated or damaged by solid objects, therefore requiring a refined, concentrated, low-fibre diet. And inflamed or merely sick organisms are easily exhausted by physical effort, hence physical inactivity. This study confirms that, most probably, the primary causes of inflammatory diseases are always external inflammatory agents, like infectious micro-organisms or toxic substances, of which a particularly ubiquitous example is nicotine. High-calorie/low-fibre diets and physical inactivity are direct consequences of generalised inflammation. Inversely, in most cases, physical exercise and moderation in eating, by themselves, cannot substantially suppress inflammations, but they can prevent them from being further reinforced by the neural reward system. Moreover, diets and exercise causing important suffering will usually do more harm than good, especially to children and young people, not to mention pregnant or nursing women. Only the identification and elimination of the inflammatory agents can efficiently prevent and cure inflammatory diseases, and currently nicotine, absorbed intentionally or passively, from tobacco or other sources, must be considered the chief suspect because of its inflammatory power

  3. The trophic influence of tetrodotoxin-inactive nerves on normal and reinnervated rat skeletal muscles.

    PubMed Central

    Bray, J J; Hubbard, J I; Mills, R G

    1979-01-01

    1. Nerve impulses in the rat sciatic nerve were blocked for long periods by tetrodotoxin (TTX) released from capillary implants. The TTX capillaries did not block axonal transport, nor did they cause any sign of nerve degeneration. 2. A comparison of the effects of TTX paralysis and denervation was made on both extensor digitorium longus (e.d.l.) and soleus muscles over 21 days, a time when the products of nerve degeneration were unlikely to contribute to the changes associated with denervation. The resting membrane potential of TTX-paralysed muscles was significantly different (P less than 0.005) from that of the denervated muscles at all periods and at 21 days the decrease that can be attributed to inactivity was 61% (e.d.l.) and 49% (soleus) of that which follows denervation. This disparity was even more pronounced for the ACh receptor density where the increase in receptors due to inactivity was only 34% (e.d.l.) and 21% (soleus) of that due to denervation. 3. A similar comparison was made on muscles which had been reinnervated by TTX-inactive nerves. These muscles were found to have a significantly higher resting membrane potential and lower ACh receptor density than the denervated muscles (P less than 0.05). 4. The experiments on reinnervated muscles preclude the possibility that nerve degeneration products are solely responsible for the difference between the TTX-paralysed and denervated muscles and suggest that the difference can be attributed to the trophic influence of the nerve. 5. An observed increase in the m.e.p.p. frequency of the TTX-paralysed muscles indicated that nerve action potentials play a role in regulating the spontaneous release from nerve terminals. PMID:94092

  4. Potential clinical translation of juvenile rodent inactivity models to study the onset of childhood obesity.

    PubMed

    Roberts, Michael D; Company, Joseph M; Brown, Jacob D; Toedebusch, Ryan G; Padilla, Jaume; Jenkins, Nathan T; Laughlin, M Harold; Booth, Frank W

    2012-08-01

    According to the latest data from the Center for Disease Control and Prevention 17%, or 12.5 million, of children and adolescents aged 2-19 years in the United States are obese. Physical inactivity is designated as one of the actual causes of US deaths and undoubtedly contributes to the obesity epidemic in children and adults. Examining the effects of inactivity on physiological homeostasis during youth is crucial given that 58% of children between the ages 6-11 yr old fail to obtain the recommended 60 min/day of physical activity and 92% of adolescents fail to achieve this goal [Troiano et al. Med Sci Sports Exerc. 40, 2008]. Nonetheless, invasive mechanistic studies in children linking diminished physical activity with metabolic maladies are lacking for obvious ethical reasons. The rodent wheel lock (WL) model was adopted by our laboratory and others to study how different organ systems of juvenile rats respond to a cessation of daily physical activity. Our WL model houses rats in cages equipped with voluntary running wheels starting at 28 days of age. After a certain period of voluntary running (3 to 6 wk), the wheels are locked, thus preventing the rats' primary source of physical activity. The studies discussed herein suggest that obesity-associated maladies including skeletal muscle insulin resistance, hypothalamic leptin resistance, fatty acid oxidation impairments in skeletal muscle and adipose tissue, nonalcoholic fatty liver disease, and endothelial dysfunction are initiated in juvenile animals that are restrained from voluntary exercise via WL. The use of the juvenile rodent WL or other inactivity models will continue to provide a powerful clinical translational tool that can be used for primordial prevention of human childhood obesity.

  5. Maltodextrin-powered enzymatic fuel cell through a non-natural enzymatic pathway

    NASA Astrophysics Data System (ADS)

    Zhu, Zhiguang; Wang, Yiran; Minteer, Shelley D.; Percival Zhang, Y.-H.

    Enzymatic fuel cells (EFCs) use a variety of fuels to generate electricity through oxidoreductase enzymes, such as oxidases or dehydrogenases, as catalysts on electrodes. We have developed a novel synthetic enzymatic pathway containing two free enzymes (maltodextrin phosphorylase and phosphoglucomutase) and one immobilized glucose-6-phosphate dehydrogenase that can utilize an oligomeric substrate maltodextrin for producing electrons mediated via a diaphorase and vitamin K 3 electron shuttle system. Three different enzyme immobilization approaches were compared based on electrostatic force entrapment, chemical cross-linking, and cross-linking with the aid of carbon nanotubes. At 10 mM glucose-6-phosphate (G6P) as a substrate concentration, the maximum power density of 0.06 mW cm -2 and retaining 42% of power output after 11 days were obtained through the method of chemical cross-linking with carbon nanotubes, approximately 6-fold and 3.5-fold better than those of the electrostatic force-based method, respectively. When changed to maltodextrin (degree of polymerization = 19) as the substrate, the EFC achieved a maximum power density of 0.085 mW cm -2. With the advantages of stable, low cost, high energy density, non-inhibitor to enzymes, and environmental friendly, maltodextrin is suggested to be an ideal fuel to power enzymatic fuel cells.

  6. Classifications and comparisons of multilocus recombination distributions

    PubMed Central

    Karlin, Samuel; Liberman, Uri

    1978-01-01

    Various classifications and representations of multilocus recombination structures are delineated based on generalized notions of linkage values and recombination rates. An important class of recombination distributions (called the count-location chiasma process) is parameterized by a distribution of the number of crossover events and, for each such crossover count, by a conditional distribution of crossover locations. A number of properties of this recombination structure are developed. A multilocus definition of a “natural” recombination range is set forth. Orderings among recombination distributions in the multilocus setting are also discussed. Comparisons are made in terms of complete linkage, free assortment and noninterference schemes serving as standards. PMID:16592601

  7. The autolytic activity of the recombinant amidase of Staphylococcus saprophyticus is inhibited by its own recombinant GW repeats.

    PubMed

    Hell, Wolfgang; Reichl, Sylvia; Anders, Agnes; Gatermann, Sören

    2003-10-10

    The Aas (autolysin/adhesin of Staphylococcus saprophyticus) is a multifunctional surface protein containing two enzymatic domains an N-acetyl-muramyl-L-alanine amidase, an endo-beta-N-acetyl-D-glucosaminidase, and two different regions of repetitive sequences, an N-terminal and a C-terminal repetitive domain. The C-terminal repetitive domain is built up by the repeats R1, R2 and R3, which interconnect the putative active centers of the amidase and glucosaminidase. To investigate the influence of the C-terminal repeats and the N-terminal repeats on the amidase activity, the repetitive domains and fragments of them were cloned and expressed in Escherichia coli. The influence of the different fragments on the activity of the recombinant amidase of the Aas, consisting of the active center of the enzyme and repeat R1, was investigated in a turbidimetric microassay. The different fragments derived from the C-terminal repeats inhibited the amidase activity, while the N-terminal repeats did not influence the activity of the enzyme. The inhibiting activity increased with the number of GW repeats the recombinant fragment contained. Thus we conclude, that the C-terminal GW repeats and not the N-terminal repeats are necessary for the cell wall targeting and the autolytic function of the amidase.

  8. Risk assessment as a management tool for inactive hazardous materials disposal sites

    SciTech Connect

    Chrostowski, P.C.; Pearsall, L.J.; Shaw, C.

    1985-09-01

    A protocol for risk assessment at inactive hazardous material disposal sites has been developed by using mathematical modeling and estimation techniques. Compared with ranking models, the protocol yields quantitative results with a minimum of input data that may be used to select among management, scientific, or engineering alternatives for the site. This article reviews the background, presents the protocol, illustrates the application of the protocol, and demonstrates the use of the protocol in conjunction with the critical environmental area concept. Comparisons are also presented with other model systems such as the EPA HRS. 21 references, 2 figures, 2 tables.

  9. Living in a Box or Call of the Wild? Revisiting Lifetime Inactivity and Sarcopenia

    PubMed Central

    Hindle, Allyson

    2011-01-01

    Abstract Significance The accepted effects of aging in mammalian skeletal muscle are progressive atrophy and weakening, or sarcopenia. Canonical hallmarks of aging in skeletal muscle include a reduction in muscle fiber cross-sectional area, a loss in muscle fibers through apoptosis and denervation, and infiltration of connective tissue or fibrosis. Emerging thought suggests that pro-inflammatory signaling and oxidative stress may contribute to sarcopenia. Critical Issues Unfortunately, most of the mammalian models used to examine and understand sarcopenia are confounded by the pervasive influence of prolonged physical inactivity. Further, the potential for underlying metabolic disorder and chronic disease (e.g., type II diabetes and cardiovascular disease) may accelerate skeletal muscle wasting. Because physical inactivity may share elevated pro-inflammatory (tumor necrosis factor-alpha and inducible nitric oxide synthase) and insufficient stress response (insulin-like growth factor-1 [IGF-1], heat-shock protein 25 [HSP25], NAD-dependent deacetylase sirtuin-3 [SIRT-3], and peroxisome proliferator-activated receptor-gamma coactivator 1[PGC-1α]) signaling with aging and chronic disease, it is critical to distinguish true aging from chronic inactivity or underlying disease. Conversely, the efficacy of exercise and caloric restrictive interventions against sarcopenia in aging populations appears highly effective when (a) conducted across the lifespan, or (b) at higher intensities when commenced in middle age or later. Recent Advances While the prospective mechanisms by which exercise or daily activity provide have not been elucidated, upregulation of HSPs, PGC-1α, and IGF-1 may ameliorate inflammatory signaling, apoptosis, and sarcopenia. Limited data indicate that the aging phenotype exhibited by mammals living in their natural habitat (Weddell seal and shrews) express limited apoptosis and fiber atrophy, whereas significant collagen accumulation remains. In

  10. Remedial action plan for stabilization of the inactive uranium mill tailings site at Monument Valley, Arizona

    SciTech Connect

    1986-02-01

    This Remedial Action Plan (RAP) has been developed to serve a two-fold purpose. It presents the series of activities which are proposed by the U.S. Department of Energy (DOE) to accomplish long-term stabilization and control of radioactive materials at the inactive uranium processing site located near Monument Valley, Arizona It also serves to document the concurrence of both the Navajo Nation and the U.S. Nuclear Regulatory Commission (NRC) in the remedial action. This agreement, upon execution by DOE and the Navajo Nation and concurrence by NRC, becomes Appendix B of the Cooperative Agreement.

  11. Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

    SciTech Connect

    Marcondes, Marcelo F.; Torquato, Ricardo J.S.; Assis, Diego M.; Juliano, Maria A.; Hayashi, Mirian A.F.; Oliveira, Vitor

    2010-01-01

    In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, and enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.

  12. Toxicological evaluation of lactase derived from recombinant Pichia pastoris.

    PubMed

    Zou, Shiying; He, Xiaoyun; Liu, Yifei; Chen, Delong; Luo, Yunbo; Huang, Kunlun; Zhang, Wei; Xu, Wentao

    2014-01-01

    A recombinant lactase was expressed in Pichia pastoris, resulting in enzymatic activity of 3600 U/mL in a 5 L fermenter. The lactase product was subjected to a series of toxicological tests to determine its safety for use as an enzyme preparation in the dairy industry. This recombinant lactase had the highest activity of all recombinant strains reported thus far. Acute oral toxicity, mutagenicity, genotoxic, and subchronic toxicity tests performed in rats and mice showed no death in any groups. The lethal dose 50% (LD50) based on the acute oral toxicity study is greater than 30 mL/kg body weight, which is in accordance with the 1500 L milk consumption of a 50 kg human daily. The lactase showed no mutagenic activity in the Ames test or a mouse sperm abnormality test at levels of up to 5 mg/plate and 1250 mg/kg body weight, respectively. It also showed no genetic toxicology in a bone marrow cell micronucleus test at levels of up to 1250 mg/kg body weight. A 90-day subchronic repeated toxicity study via the diet with lactase levels up to 1646 mg/kg (1000-fold greater than the mean human exposure) did not show any treatment-related significant toxicological effects on body weight, food consumption, organ weights, hematological and clinical chemistry, or histopathology compared to the control groups. This toxicological evaluation system is comprehensive and can be used in the safety evaluation of other enzyme preparations. The lactase showed no acute, mutagenic, genetic, or subchronic toxicity under our evaluation system.

  13. Toxicological Evaluation of Lactase Derived from Recombinant Pichia pastoris

    PubMed Central

    Liu, Yifei; Chen, Delong; Luo, Yunbo; Huang, Kunlun; Zhang, Wei; Xu, Wentao

    2014-01-01

    A recombinant lactase was expressed in Pichia pastoris, resulting in enzymatic activity of 3600 U/mL in a 5 L fermenter. The lactase product was subjected to a series of toxicological tests to determine its safety for use as an enzyme preparation in the dairy industry. This recombinant lactase had the highest activity of all recombinant strains reported thus far. Acute oral toxicity, mutagenicity, genotoxic, and subchronic toxicity tests performed in rats and mice showed no death in any groups. The lethal dose 50% (LD50) based on the acute oral toxicity study is greater than 30 mL/kg body weight, which is in accordance with the 1500 L milk consumption of a 50 kg human daily. The lactase showed no mutagenic activity in the Ames test or a mouse sperm abnormality test at levels of up to 5 mg/plate and 1250 mg/kg body weight, respectively. It also showed no genetic toxicology in a bone marrow cell micronucleus test at levels of up to 1250 mg/kg body weight. A 90-day subchronic repeated toxicity study via the diet with lactase levels up to 1646 mg/kg (1000-fold greater than the mean human exposure) did not show any treatment-related significant toxicological effects on body weight, food consumption, organ weights, hematological and clinical chemistry, or histopathology compared to the control groups. This toxicological evaluation system is comprehensive and can be used in the safety evaluation of other enzyme preparations. The lactase showed no acute, mutagenic, genetic, or subchronic toxicity under our evaluation system. PMID:25184300

  14. Response surface optimization of corn stover pretreatment using dilute phosphoric acid for enzymatic hydrolysis and ethanol production.

    PubMed

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

    2013-02-01

    Dilute H(3)PO(4) (0.0-2.0%, v/v) was used to pretreat corn stover (10%, w/w) for conversion to ethanol. Pretreatment conditions were optimized for temperature, acid loading, and time using central composite design. Optimal pretreatment conditions were chosen to promote sugar yields following enzymatic digestion while minimizing formation of furans, which are potent inhibitors of fermentation. The maximum glucose yield (85%) was obtained after enzymatic hydrolysis of corn stover pretreated with 0.5% (v/v) acid at 180°C for 15min while highest yield for xylose (91.4%) was observed from corn stover pretreated with 1% (v/v) acid at 160°C for 10min. About 26.4±0.1g ethanol was produced per L by recombinant Escherichia coli strain FBR5 from 55.1±1.0g sugars generated from enzymatically hydrolyzed corn stover (10%, w/w) pretreated under a balanced optimized condition (161.81°C, 0.78% acid, 9.78min) where only 0.4±0.0g furfural and 0.1±0.0 hydroxylmethyl furfural were produced.

  15. Genetic recombination and molecular evolution.

    PubMed

    Charlesworth, B; Betancourt, A J; Kaiser, V B; Gordo, I

    2009-01-01

    Reduced rates of genetic recombination are often associated with reduced genetic variability and levels of adaptation. Several different evolutionary processes, collectively known as Hill-Robertson (HR) effects, have been proposed as causes of these correlates of recombination. Here, we use DNA sequence polymorphism and divergence data from the noncrossing over dot chromosome of Drosophila to discriminate between two of the major forms of HR effects: selective sweeps and background selection. This chromosome shows reduced levels of silent variability and reduced effectiveness of selection. We show that neither model fits the data on variability. We propose that, in large genomic regions with restricted recombination, HR effects among nonsynonymous mutations undermine the effective strength of selection, so that their background selection effects are weakened. This modified model fits the data on variability and also explains why variability in very large nonrecombining genomes is not completely wiped out. We also show that HR effects of this type can produce an individual selection advantage to recombination, as well as greatly reduce the mean fitness of nonrecombining genomes and genomic regions.

  16. Arabian Oryx (Oryx leucoryx) Respond to Increased Ambient Temperatures with a Seasonal Shift in the Timing of Their Daily Inactivity Patterns.

    PubMed

    Davimes, Joshua G; Alagaili, Abdulaziz N; Gravett, Nadine; Bertelsen, Mads F; Mohammed, Osama B; Ismail, Khairy; Bennett, Nigel C; Manger, Paul R

    2016-08-01

    The Arabian oryx inhabits an environment where summer ambient temperatures can exceed 40 °C for extended periods of time. While the oryx uses a suite of adaptations that aid survival, the effects of this extreme environment on inactivity are unknown. To determine how the oryx manages inactivity seasonally, we measured the daily rhythm of body temperature and used fine-grain actigraphy, in 10 animals, to reveal when the animals were inactive in relation to ambient temperature and photoperiod. We demonstrate that during the cooler winter months, the oryx was inactive during the cooler parts of the 24-h day (predawn hours), showing a nighttime (nocturnal) inactivity pattern. In contrast, in the warmer summer months, the oryx displayed a bimodal inactivity pattern, with major inactivity bouts (those greater than 1 h) occurring equally during both the coolest part of the night (predawn hours) and the warmest part of the day (afternoon hours). Of note, the timing of the daily rhythm of body temperature did not vary seasonally, although the amplitude did change, leading to a seasonal alteration in the phase relationship between inactivity and the body temperature rhythm. Because during periods of inactivity the oryx were presumably asleep for much of the time, we speculate that the daytime shift in inactivity may allow the oryx to take advantage of the thermoregulatory physiology of sleep, which likely occurs when the animal is inactive for more than 1 h, to mitigate environmentally induced increases in body temperature. PMID:27154303

  17. Expression of the heparinase gene from Flavobacterium heparinum in Escherichia coli and its enzymatic properties.

    PubMed

    Yu, Ping; Wu, Yan

    2012-09-01

    Heparinase has an important application in the preparation of low-molecular-weight heparins by heparin enzymolysis. A heparinase gene from Flavobacterium heparinum was cloned and expressed in Escherichia coli BL21 in order to enhance its activity. The expressed heparinase was purified to homogeneity by a metal chelating affinity column and its enzymatic properties were evaluated. A maximal heparinase activity of 1061 IU/L toward the substrate heparin was achieved when the recombinant strain was induced with 0.5 mM isopropyl-β-D-thiogalactoside at 28 °C for 9 h. The optimal temperature and pH of heparinase were 30 °C and 7.0, respectively. The recombinant heparinase was heat-unstable and had a higher stability at pHs from 7.0 to 10.0. Observed activities of heparinase were the highest in the presence of Ca(2+) and Cu(2+) and the lowest in the presence of Mn(2+) and Pb(2+). These results lay a good foundation for the preparation of LMWHs by heparin enzymolysis.

  18. Method to convert N-terminal glutamine to pyroglutamate for characterization of recombinant monoclonal antibodies.

    PubMed

    Xu, Wei; Peng, Yan; Wang, Fengqiang; Paporello, Brittany; Richardson, Daisy; Liu, Hongcheng

    2013-05-01

    Cyclization of N-terminal glutamine to pyroglutamate is a common modification of recombinant monoclonal antibodies that has often been identified by liquid chromatography mass spectrometry (LC-MS) analysis using separated fractions. An alternative approach of using glutaminyl-peptide cyclotransferase to convert the N-terminal glutamine to pyroglutamate was developed in the current study. Enzymatic conversion of the N-terminal glutamine to pyroglutamate not only provides an identification of the N-terminal amino acids without fraction collection but also can significantly simplify the chromatograms to assist fraction collections for the characterization of other antibody variants.

  19. Recombinant DNA: History of the Controversy.

    ERIC Educational Resources Information Center

    Vigue, Charles L.; Stanziale, William G.

    1979-01-01

    The hazards associated with recombinant DNA research are presented along with some social implications and the development of recombinant DNA research guidelines by the National Institutes of Health. (SA)

  20. Meiotic Recombination: The Essence of Heredity.

    PubMed

    Hunter, Neil

    2015-10-28

    The study of homologous recombination has its historical roots in meiosis. In this context, recombination occurs as a programmed event that culminates in the formation of crossovers, which are essential for accurate chromosome segregation and create new combinations of parental alleles. Thus, meiotic recombination underlies both the independent assortment of parental chromosomes and genetic linkage. This review highlights the features of meiotic recombination that distinguish it from recombinational repair in somatic cells, and how the molecular processes of meiotic recombination are embedded and interdependent with the chromosome structures that characterize meiotic prophase. A more in-depth review presents our understanding of how crossover and noncrossover pathways of meiotic recombination are differentiated and regulated. The final section of this review summarizes the studies that have defined defective recombination as a leading cause of pregnancy loss and congenital disease in humans.

  1. Epigenome changes in active and inactive Polycomb-group-controlled regions

    PubMed Central

    Breiling, Achim; O'Neill, Laura P; D'Eliseo, Donatella; Turner, Bryan M; Orlando, Valerio

    2004-01-01

    The Polycomb group (PcG) of proteins conveys epigenetic inheritance of repressed transcriptional states. In Drosophila, the Polycomb repressive complex 1 (PRC1) maintains the silent state by inhibiting the transcription machinery and chromatin remodelling at core promoters. Using immunoprecipitation of in vivo formaldehyde-fixed chromatin in phenotypically diverse cultured cell lines, we have mapped PRC1 components, the histone methyl transferase (HMT) Enhancer of zeste (E(z)) and histone H3 modifications in active and inactive PcG-controlled regions. We show that PRC1 components are present in both cases, but at different levels. In particular, active target promoters are nearly devoid of E(z) and Polycomb. Moreover, repressed regions are trimethylated at lysines 9 and 27, suggesting that these histone modifications represent a mark for inactive PcG-controlled regions. These PcG-specific repressive marks are maintained by the action of the E(z) HMT, an enzyme that has an important role not only in establishing but also in maintaining PcG repression. PMID:15448640

  2. Cold-active alkaline phosphatase is irreversibly transformed into an inactive dimer by low urea concentrations.

    PubMed

    Hjörleifsson, Jens Guðmundur; Ásgeirsson, Bjarni

    2016-07-01

    Alkaline phosphatase is a homodimeric metallo-hydrolase where both Zn(2+) and Mg(2+) are important for catalysis and stability. Cold-adapted alkaline phosphatase variants have high activity at low temperatures and lower thermal stability compared with variants from mesophilic hosts. The instability, and thus inactivation, could be due to loose association of the dimers and/or loosely bound Mg(2)(+) in the active site, but this has not been studied in detail for the cold-adapted variants. Here, we focus on using the intrinsic fluorescence of Trp in alkaline phosphatase from the marine bacterium Vibrio splendidus (VAP) to probe for dimerization. Trp→Phe substitutions showed that two out of the five native Trp residues contributed mostly to the fluorescence emission. One residue, 15Å away from the active site (W460) and highly solvent excluded, was phosphorescent and had a distant role in substrate binding. An additional Trp residue was introduced to the dimer interface to act as a possible probe for dimerization. Urea denaturation curves indicated that an inactive dimer intermediate, structurally equivalent to the native state, was formed before dimer dissociation took place. This is the first example of the transition of a native dimer to an inactive dimer intermediate for alkaline phosphatase without using mutagenesis, ligands, or competitive inhibition. PMID:27043172

  3. Cold-active alkaline phosphatase is irreversibly transformed into an inactive dimer by low urea concentrations.

    PubMed

    Hjörleifsson, Jens Guðmundur; Ásgeirsson, Bjarni

    2016-07-01

    Alkaline phosphatase is a homodimeric metallo-hydrolase where both Zn(2+) and Mg(2+) are important for catalysis and stability. Cold-adapted alkaline phosphatase variants have high activity at low temperatures and lower thermal stability compared with variants from mesophilic hosts. The instability, and thus inactivation, could be due to loose association of the dimers and/or loosely bound Mg(2)(+) in the active site, but this has not been studied in detail for the cold-adapted variants. Here, we focus on using the intrinsic fluorescence of Trp in alkaline phosphatase from the marine bacterium Vibrio splendidus (VAP) to probe for dimerization. Trp→Phe substitutions showed that two out of the five native Trp residues contributed mostly to the fluorescence emission. One residue, 15Å away from the active site (W460) and highly solvent excluded, was phosphorescent and had a distant role in substrate binding. An additional Trp residue was introduced to the dimer interface to act as a possible probe for dimerization. Urea denaturation curves indicated that an inactive dimer intermediate, structurally equivalent to the native state, was formed before dimer dissociation took place. This is the first example of the transition of a native dimer to an inactive dimer intermediate for alkaline phosphatase without using mutagenesis, ligands, or competitive inhibition.

  4. Any link between sexual inactivity and treadle pump performance characteristics: The Malawi case

    NASA Astrophysics Data System (ADS)

    Joseph, Chidanti-Malunga; Yamikani, Malunga

    In mitigating the effects of climate change in Malawi, government promotes the use of low cost irrigation technologies to small-scale farmers, especially in wetlands where water is available. The treadle pump is one such technology. The pump is a manual water lifting device operated by feet. Although the technology has been widely accepted by small-scale farmers, there are documented reports that some farmers abandon the technology, preferring other technologies such as river diversion. One theory for the abandonment is that female farmers claim that the technology makes their male counterparts sexually inactive. This research seeks to find an explanation to the misconception. The study analyzed the physical characteristics of the treadle pump and its users. The results show that the technology is male-dominated (30% were females out of a sample of 40). The results also show that the technology is labor-intensive with very small discharge rates (an average of 0.78 l/s) achieved regardless of the BMI of the operator. With such small discharge rates, in order to fulfill irrigation requirements of a crop, the operator has to pump for long hours. This exercise makes men naturally tired, perhaps making them sexually inactive as well.

  5. The use of periodization in exercise prescriptions for inactive adults: A systematic review

    PubMed Central

    Strohacker, Kelley; Fazzino, Daniel; Breslin, Whitney L.; Xu, Xiaomeng

    2015-01-01

    Background Periodization of exercise is a method typically used in sports training, but the impact of periodized exercise on health outcomes in untrained adults is unclear. Purpose This review aims to summarize existing research wherein aerobic or resistance exercise was prescribed to inactive adults using a recognized periodization method. Methods A search of relevant databases, conducted between January and February of 2014, yielded 21 studies published between 2000 and 2013 that assessed the impact of periodized exercise on health outcomes in untrained participants. Results Substantial heterogeneity existed between studies, even under the same periodization method. Compared to baseline values or non-training control groups, prescribing periodized resistance or aerobic exercise yielded significant improvements in health outcomes related to traditional and emerging risk factors for cardiovascular disease, low-back and neck/shoulder pain, disease severity, and quality of life, with mixed results for increasing bone mineral density. Conclusions Although it is premature to conclude that periodized exercise is superior to non-periodized exercise for improving health outcomes, periodization appears to be a feasible means of prescribing exercise to inactive adults within an intervention setting. Further research is necessary to understand the effectiveness of periodizing aerobic exercise, the psychological effects of periodization, and the feasibility of implementing flexible non-linear methods. PMID:26844095

  6. Flavonoid-induced conversion of catalase to its inactive form--Compound II.

    PubMed

    Krych, J; Gebicki, J L; Gebicka, L

    2014-11-01

    Flavonoids (FlaOHs), plant polyphenols, are ubiquitous components of human diet and are known as antioxidants. However, their prooxidant activity has also been reported. We have recently found that FlaOHs inhibit catalase, the heme enzyme which catalyzes the decomposition of hydrogen peroxide (H2O2) into water and molecular oxygen. The catalytic cycle proceeds with the formation of the intermediate, Compound I (Cpd I), an oxoferryl porphyrin π-cation radical, the two-electron oxidation product of a heme group. Under conditions of low H2O2 fluxes and in the presence of an appropriate substrate, Cpd I can undergo one-electron reduction to inactive Compound II (Cpd II), oxoferryl derivative without radical site. Here we show that in vitro, under low fluxes of H2O2, FlaOHs reduce Cpd I to inactive Cpd II. Measurable amounts of Cpd II can be formed even in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) at concentration comparable with the investigated FlaOHs. Possible mechanisms of electron transfer from FlaOH molecule to the heme are discussed.

  7. High prevalence of physical inactivity among adolescents living with HIV/AIDS

    PubMed Central

    Tanaka, Luana Fiengo; Latorre, Maria do Rosário Dias de Oliveira; da Silva, Aline Medeiros; Konstantyner, Thais Claudia Roma de Oliveira; Peres, Stela Verzinhasse; Marques, Heloisa Helena de Sousa

    2015-01-01

    Objective: To assess the prevalence of physical inactivity among adolescents with HIV/AIDS, as well as associated factors. Methods: Ninety-one adolescents (from 10 to 19 years old) with HIV/AIDS who are patients at a university follow-up service were interviewed. Anthropometric data (weight, height, and waist circumference) were measured twice; clinical information was obtained from medical records, and habitual physical activity was assessed by a questionnaire proposed by Florindo et al. The cutoff point for sedentariness was 300 minutes/week. Results: The prevalence of inadequate height for age, malnutrition, and overweight/obesity was 15.4%, 9.9% and 12.1%, respectively. The most common physical activities were soccer (44.4%), volleyball (14.4%) and cycling (7.8%). The median times spent with physical activity and walking/bicycling to school were 141 min and 39 min, respectively. Most adolescents (71.4%) were sedentary and this proportion was higher among girls (p=0.046). Conclusions: A high prevalence of physical inactivity among adolescents with HIV/AIDS was observed, similar to the general population. Promoting physical activity among adolescents, especially among girls with HIV/AIDS, as well as monitoring it should be part of the follow-up routine of these patients. PMID:25907024

  8. Astrometric and photometric data fusion for inactive space object mass and area estimation

    NASA Astrophysics Data System (ADS)

    Linares, Richard; Jah, Moriba K.; Crassidis, John L.; Leve, Fred A.; Kelecy, Tom

    2014-06-01

    This paper presents a new method to determine the mass of an inactive space object from the fusion of photometric and astrometric data. Typically, the effect of solar radiation pressure is used to determine area-to-mass ratio for space objects from angles observations. The area-to-mass ratio of a space object can greatly affect its orbital dynamics. As a consequence, angles data are sensitive to this quantity. On the other hand, photometric data is not sensitive to mass but is a strong function of the albedo-area and the rotational dynamics of the space object. The albedo-area can be used to determine the amount of energy reflected from solar radiation. Since these two data types are sensitive to albedo-area and area-to-mass, then through fusion of photometric data with angles data it is possible to determine the area and mass of a space object. This work employs an unscented Kalman filter to estimate rotational and translational states, area and mass of an inactive space object. Mass is not observable with only angles data or only photometric data alone, but it is shown in this work that with the two combined data types mass can be recovered. Recovery of space object characteristics and attitude and orbit trajectories with sufficient accuracy is demonstrated in this paper via simulation.

  9. Multimotor transport in a system of active and inactive kinesin-1 motors.

    PubMed

    Scharrel, Lara; Ma, Rui; Schneider, René; Jülicher, Frank; Diez, Stefan

    2014-07-15

    Long-range directional transport in cells is facilitated by microtubule-based motor proteins. One example is transport in a nerve cell, where small groups of motor proteins, such as kinesins and cytoplasmic dynein, work together to ensure the supply and clearance of cellular material along the axon. Defects in axonal transport have been linked to Alzheimer's and other neurodegenerative diseases. However, it is not known in detail how multimotor-based cargo transport is impaired if a fraction of the motors are defective. To mimic impaired multimotor transport in vitro, we performed gliding motility assays with varying fractions of active kinesin-1 motors and inactive kinesin-1 motor mutants. We found that impaired transport manifests in multiple motility regimes: 1), a fast-motility regime characterized by gliding at velocities close to the single-molecule velocity of the active motors; 2), a slow-motility regime characterized by gliding at close-to zero velocity or full stopping; and 3), a regime in which fast and slow motilities coexist. Notably, the transition from the fast to the slow regime occurred sharply at a threshold fraction of active motors. Based on single-motor parameters, we developed a stochastic model and a mean-field theoretical description that explain our experimental findings. Our results demonstrate that impaired multimotor transport mostly occurs in an either/or fashion: depending on the ratio of active to inactive motors, transport is either performed at close to full speed or is out of action.

  10. Major initiatives related to childhood obesity and physical inactivity in Canada: the year in review.

    PubMed

    Tremblay, Mark S

    2012-01-01

    This manuscript briefly reviews 15 significant initiatives related to childhood obesity and physical inactivity in Canada between September 2010 and September 2011. These include the: announcement of a Federal-Provincial-Territorial framework for action to promote healthy weights; implementation of the nutrition labeling initiative; launch of the CBC "Live Right Now" campaign; announcement of the Public Health Agency of Canada's innovation strategy funding related to obesity; publication of the Canadian Health Measures Survey physical activity findings; release of new Canadian physical activity and sedentary behaviour guidelines; launch of ParticipACTION's "Think Again" campaign; workshop on building trust to address the epidemic of obesity; start of the Canadian Pediatric Weight Management Registry; initiation of "Our Health Our Future: A National Dialogue on Healthy Weights"; release of the Active Healthy Kids Canada Report Card on Physical Activity for Children and Youth; National Obesity Summit; Nature Play Day and Sports Day in Canada; development of the Canadian Assessment of Physical Literacy; and the creation of Active Canada 20/20--A National Physical Activity Plan. The diversity and intensity of activity addressing the childhood obesity and physical inactivity "epidemic" in Canada is encouraging and must be maintained and enhanced.

  11. Major initiatives related to childhood obesity and physical inactivity in Canada: the year in review.

    PubMed

    Tremblay, Mark S

    2012-01-01

    This manuscript briefly reviews 15 significant initiatives related to childhood obesity and physical inactivity in Canada between September 2010 and September 2011. These include the: announcement of a Federal-Provincial-Territorial framework for action to promote healthy weights; implementation of the nutrition labeling initiative; launch of the CBC "Live Right Now" campaign; announcement of the Public Health Agency of Canada's innovation strategy funding related to obesity; publication of the Canadian Health Measures Survey physical activity findings; release of new Canadian physical activity and sedentary behaviour guidelines; launch of ParticipACTION's "Think Again" campaign; workshop on building trust to address the epidemic of obesity; start of the Canadian Pediatric Weight Management Registry; initiation of "Our Health Our Future: A National Dialogue on Healthy Weights"; release of the Active Healthy Kids Canada Report Card on Physical Activity for Children and Youth; National Obesity Summit; Nature Play Day and Sports Day in Canada; development of the Canadian Assessment of Physical Literacy; and the creation of Active Canada 20/20--A National Physical Activity Plan. The diversity and intensity of activity addressing the childhood obesity and physical inactivity "epidemic" in Canada is encouraging and must be maintained and enhanced. PMID:22905632

  12. [The economically active population in Verviers during the industrial revolution. Part 1. Inactivity and underemployment].

    PubMed

    Desama, C

    1979-01-01

    The industrial revolution during the 1st 1/2 of the 19th century in Wallonia had important social and demographic repercussions which are still being felt today. The author examines and compares the demographic structures of the working population of the city of Verviers in 1806 and 1846. 1/4 of the adult population was not working at that time. Married women constitute a manpower reserve which is drawn upon in line with the general line of activity; the more than proportional overall offer of employment with respect to demand is a cause for inactivity which is as restraining as the education of children or social pressures may be which force these women to perform household work. Underemployment is linked to age, affecting the youngest among potential workers; it is high between ages 12-19, decreases until age 30 and then becomes negligible, and increases again by age 65 in 1806 or age 70 in 1846. Underemployment is also linked to sex since it affects women, although less than inactivity does. Finally, it affects bachelors; the evident relation in 1806 becomes almost exclusive in 1846 when 90% of underemployed individuals were bachelors. The increase in the rate of underemployment between 1806-1846 is the result of the volume of immigration and its structure which brought about disequilibrium between supply and demand. (author's)

  13. Cloning and expression of pig kidney dopa decarboxylase: comparison of the naturally occurring and recombinant enzymes.

    PubMed Central

    Moore, P S; Dominici, P; Borri Voltattorni, C

    1996-01-01

    whereas the remaining PLP binding site is most probably occupied by an inactive covalently bound coenzyme derivative; some speculations are made about its origin. The coenzyme absorbing bands of recombinant DDC show a modest pH dependence at 335 and 425 nm. A putative working model is presented to explain this behaviour. PMID:8670114

  14. Two-Photon Small Molecule Enzymatic Probes.

    PubMed

    Qian, Linghui; Li, Lin; Yao, Shao Q

    2016-04-19

    Enzymes are essential for life, especially in the development of disease and on drug effects, but as we cannot yet directly observe the inside interactions and only partially observe biochemical outcomes, tools "translating" these processes into readable information are essential for better understanding of enzymes as well as for developing effective tools to fight against diseases. Therefore, sensitive small molecule probes suitable for direct in vivo monitoring of enzyme activities are ultimately desirable. For fulfilling this desire, two-photon small molecule enzymatic probes (TSMEPs) producing amplified fluorescent signals based on enzymatic conversion with better photophysical properties and deeper penetration in intact tissues and whole animals have been developed and demonstrated to be powerful in addressing the issues described above. Nonetheless, currently available TSMEPs only cover a small portion of enzymes despite the distinct advantages of two-photon fluorescence microscopy. In this Account, we would like to share design principles for TSMEPs as potential indicators of certain pathology-related biomarkers together with their applications in disease models to inspire more elegant work to be done in this area. Highlights will be addressed on how to equip two-photon fluorescent probes with features amenable for direct assessment of enzyme activities in complex pathological environments. We give three recent examples from our laboratory and collaborations in which TSMEPs are applied to visualize the distribution and activity of enzymes at cellular and organism levels. The first example shows that we could distinguish endogenous phosphatase activity in different organelles; the second illustrates that TSMEP is suitable for specific and sensitive detection of a potential Parkinson's disease marker (monoamine oxidase B) in a variety of biological systems from cells to patient samples, and the third identifies that TSMEPs can be applied to other enzyme

  15. DFGmodel: Predicting Protein Kinase Structures in Inactive States for Structure-Based Discovery of Type-II Inhibitors

    PubMed Central

    2015-01-01

    Protein kinases exist in equilibrium of active and inactive states, in which the aspartate-phenylalanine-glycine motif in the catalytic domain undergoes conformational changes that are required for function. Drugs targeting protein kinases typically bind the primary ATP-binding site of an active state (type-I inhibitors) or utilize an allosteric pocket adjacent to the ATP-binding site in the inactive state (type-II inhibitors). Limited crystallographic data of protein kinases in the inactive state hampers the application of rational drug discovery methods for developing type-II inhibitors. Here, we present a computational approach to generate structural models of protein kinases in the inactive conformation. We first perform a comprehensive analysis of all protein kinase structures deposited in the Protein Data Bank. We then develop DFGmodel, a method that takes either a known structure of a kinase in the active conformation or a sequence of a kinase without a structure, to generate kinase models in the inactive conformation. Evaluation of DFGmodel’s performance using various measures indicates that the inactive kinase models are accurate, exhibiting RMSD of 1.5 Å or lower. The kinase models also accurately distinguish type-II kinase inhibitors from likely nonbinders (AUC > 0.70), suggesting that they are useful for virtual screening. Finally, we demonstrate the applicability of our approach with three case studies. For example, the models are able to capture inhibitors with unintended off-target activity. Our computational approach provides a structural framework for chemical biologists to characterize kinases in the inactive state and to explore new chemical spaces with structure-based drug design. PMID:25420233

  16. Characteristics of the Activity-Affect Association in Inactive People: An Ambulatory Assessment Study in Daily Life

    PubMed Central

    von Haaren, Birte; Loeffler, Simone Nadine; Haertel, Sascha; Anastasopoulou, Panagiota; Stumpp, Juergen; Hey, Stefan; Boes, Klaus

    2013-01-01

    Acute and regular exercise as well as physical activity (PA) is related to well-being and positive affect. Recent studies have shown that even daily, unstructured physical activities increase positive affect. However, the attempt to achieve adherence to PA or exercise in inactive people through public health interventions has often been unsuccessful. Most studies analyzing the activity-affect association in daily life, did not report participants’ habitual activity behavior. Thus, samples included active and inactive people, but they did not necessarily exhibit the same affective reactions to PA in daily life. Therefore the present study investigated whether the association between PA and subsequent affective state in daily life can also be observed in inactive individuals. We conducted a pilot study with 29 inactive university students (mean age 21.3 ± 1.7 years) using the method of ambulatory assessment. Affect was assessed via electronic diary and PA was measured with accelerometers. Participants had to rate affect every 2 h on a six item bipolar scale reflecting the three basic mood dimensions energetic arousal, valence, and calmness. We calculated activity intensity level [mean Metabolic Equivalent (MET) value] and the amount of time spent in light activity over the last 15 min before every diary prompt and conducted within-subject correlations. We did not find significant associations between activity intensity and the three mood dimensions. Due to the high variability in within-subject correlations we conclude that not all inactive people show the same affective reactions to PA in daily life. Analyzing the PA-affect association of inactive people was difficult due to little variance and distribution of the assessed variables. Interactive assessment and randomized controlled trials might help solving these problems. Future studies should examine characteristics of affective responses of inactive people to PA in daily life. General assumptions

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

    PubMed Central

    Saha, Badal; Cotta, Michael A.

    2012-01-01

    Lignocellulosic biomass, upon pretreatment and enzymatic hydrolysis, generates a mixture of hexose and pentose sugars such as glucose, xylose, arabinose and galactose. While Escherichia coli utilizes all these sugars it lacks the ability to produce ethanol from them. Recombinant ethanologenic E. coli strains have been created with a goal to produce ethanol from both hexose and pentose sugars. Herein, we review the current state of the art on the production of ethanol from lignocellulosic hydrolyzates by an ethanologenic recombinant E. coli strain (FBR5). The bacterium is stable without antibiotics and can tolerate ethanol up to 50 gL-1. It produces up to 45 g ethanol per L and has the potential to be used for industrial production of ethanol from lignocellulosic hydrolyzates. PMID:22705843

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

    PubMed

    Saha, Badal; Cotta, Michael A

    2012-01-01

    Lignocellulosic biomass, upon pretreatment and enzymatic hydrolysis, generates a mixture of hexose and pentose sugars such as glucose, xylose, arabinose and galactose. While Escherichia coli utilizes all these sugars it lacks the ability to produce ethanol from them. Recombinant ethanologenic E. coli strains have been created with a goal to produce ethanol from both hexose and pentose sugars. Herein, we review the current state of the art on the production of ethanol from lignocellulosic hydrolyzates by an ethanologenic recombinant E. coli strain (FBR5). The bacterium is stable without antibiotics and can tolerate ethanol up to 50 gL(-1). It produces up to 45 g ethanol per L and has the potential to be used for industrial production of ethanol from lignocellulosic hydrolyzates.

  19. Effect of Ultrasonic Frequency on Enzymatic Hydrolysis of Cellulose

    NASA Astrophysics Data System (ADS)

    Yasuda, Keiji; Kato, Daiki; Xu, Zheng; Sakka, Makiko; Sakka, Kazuo

    2010-07-01

    The effect of ultrasonic frequency on the enzymatic hydrolysis of cellulose was examined. As the cellulose and enzyme, needle unbleached kraft pulp and cellulase were used. In the cases of the horn-type transducer at 20 kHz and the plate-type transducer at 28 kHz, the enzymatic hydrolysis was accelerated by ultrasonic irradiation. Total sugar concentration linearly increased with ultrasonic intensity. On the other hand, in the case of the plate-type transducer at 500 kHz, the enzymatic hydrolysis was inhibited. Total sugar concentration decreased with increasing ultrasonic intensity.

  20. The crystal structure of S. cerevisiae Sad1, a catalytically inactive deubiquitinase that is broadly required for pre-mRNA splicing

    PubMed Central

    Hadjivassiliou, Haralambos; Rosenberg, Oren S.; Guthrie, Christine

    2014-01-01

    Sad1 is an essential splicing factor initially identified in a genetic screen in Saccharomyces cerevisiae for snRNP assembly defects. Based on sequence homology, Sad1, or USP39 in humans, is predicted to comprise two domains: a zinc finger ubiquitin binding domain (ZnF-UBP) and an inactive ubiquitin-specific protease (iUSP) domain, both of which are well conserved. The role of these domains in splicing and their interaction with ubiquitin are unknown. We first used splicing microarrays to analyze Sad1 function in vivo and found that Sad1 is critical for the splicing of nearly all yeast intron-containing genes. By using in vitro assays, we then showed that it is required for the assembly of the active spliceosome. To gain structural insights into Sad1 function, we determined the crystal structure of the full-length protein at 1.8 Å resolution. In the structure, the iUSP domain forms the characteristic ubiquitin binding pocket, though with an amino acid substitution in the active site that results in complete inactivation of the enzymatic activity of the domain. The ZnF-UBP domain of Sad1 shares high structural similarly to other ZnF-UBPs; however, Sad1's ZnF-UBP does not possess the canonical ubiquitin binding motif. Given the precedents for ZnF-UBP domains to function as activators for their neighboring USP domains, we propose that Sad1's ZnF-UBP acts in a ubiquitin-independent capacity to recruit and/or activate Sad1's iUSP domain to interact with the spliceosome. PMID:24681967