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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. Construction and characterization of recombinant Vibrio cholerae strains producing inactive cholera toxin analogs.

    PubMed

    Häse, C C; Thai, L S; Boesman-Finkelstein, M; Mar, V L; Burnette, W N; Kaslow, H R; Stevens, L A; Moss, J; Finkelstein, R A

    1994-08-01

    The catalytic A subunit of cholera toxin (CT-A) is capable of ADP-ribosylating the guanine nucleotide-binding protein, which regulates cell adenylyl cyclase, leading to the life-threatening diarrhea of cholera. Amino acids involved in the enzymatic activity of CT-A have previously been identified. By means of site-directed mutagenesis, an analog of the CT-A subunit gene was created with codon substitutions for both Arg-7 and Glu-112, each of which has been shown to produce subunits lacking ADP-ribosyltransferase activity. The mutated gene fragment was exchanged for the wild-type copy in the previously cloned ctxAB operon from El Tor biotype, Ogawa serotype Vibrio cholerae strain 3083, which produces CT-2. Further, the zonula occludens toxin gene, zot, was inactivated by an insertional mutation to create the new plasmid construct pCT-2*. Additionally, a DNA fragment encoding the B subunit of CT-1 (CT produced by classical biotype, Inaba serotype V. cholerae strain 569B) was exchanged for the homologous part in pCT-2*, resulting in the creation of pCT-1*. These plasmid constructs were introduced into the CT-negative V. cholerae mutant strain JBK70 (E1 Tor biotype, Inaba serotype); CT-A-B+ derivatives CVD101 and CVD103 of classical biotype Ogawa and Inaba serotype strains 395 and 569B, respectively; El Tor biotype Inaba and Ogawa serotype strains C6706 and C7258, respectively, recently isolated in Peru; and O139 (synonym Bengal) strain SG25-1 from the current epidemic in India. Recombinant toxins (CT-1* and CT-2*), partially purified from culture supernatants of transformed JBK70, were shown to be inactive on mouse Y1 adrenal tumor cells and in an in vitro ADP-ribosyltransferase assay. CT-1* and CT-2* reacted with polyclonal and monoclonal antibodies against both A and B subunits of CT. The toxin analogs reacted with antibodies against CT-A and CT-B on cellulose acetate strips and in a GM1 enzyme-linked immunosorbent assay; they reacted appropriately with B

  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. 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

  5. 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

  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. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. Recombinant human proteinase 3, the Wegener's autoantigen, expressed in HMC-1 cells is enzymatically active and recognized by c-ANCA.

    PubMed

    Specks, U; Fass, D N; Fautsch, M P; Hummel, A M; Viss, M A

    1996-07-29

    We developed a stable expression system for conformationally intact recombinant human PR3 (rPR3) using the human mast cell line HMC-1. Like in U937 cells, the rPR3 is processed from a 34 kDa precursor to the 29 kDa mature form, primarily as the result of oligosaccharide trimming. The rPR3 binds [3H]DFP and hydrolyzes the substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-pNA. The enzymatic activity is inhibited by greater than 95% by alpha 1-PI. The rPR3 and the enzymatically inactive mutant rPR3-S176A are both packaged in granules. Thus, proteolytic autoprocessing is not required for PR3's targeting to granules. This rPR3 is the first to be recognized by most c-ANCA from WG patients and all anti-PR3 ANCA that were detected by standard anti-PR3 specific ELISA. This expression system for rPR3 represents a versatile tool for the analysis of its intracellular processing, structure-function relationships and interaction with autoantibodies. PMID:8706874

  13. Highly efficient recombinant production and purification of streptococcal cysteine protease streptopain with increased enzymatic activity.

    PubMed

    Lane, Michael D; Seelig, Burckhard

    2016-05-01

    Streptococcus pyogenes produces the cysteine protease streptopain (SpeB) as a critical virulence factor for pathogenesis. Despite having first been described seventy years ago, this protease still holds mysteries which are being investigated today. Streptopain can cleave a wide range of human proteins, including immunoglobulins, the complement activation system, chemokines, and structural proteins. Due to the broad activity of streptopain, it has been challenging to elucidate the functional results of its action and precise mechanisms for its contribution to S. pyogenes pathogenesis. To better study streptopain, several expression and purification schemes have been developed. These methods originally involved isolation from S. pyogenes culture but were more recently expanded to include recombinant Escherichia coli expression systems. While substantially easier to implement, the latter recombinant approach can prove challenging to reproduce, often resulting in mostly insoluble protein and poor purification yields. After extensive optimization of a wide range of expression and purification conditions, we applied the autoinduction method of protein expression and developed a two-step column purification scheme that reliably produces large amounts of purified soluble and highly active streptopain. This method reproducibly yielded 3 mg of streptopain from 50 mL of expression culture at >95% purity, with an activity of 5306 ± 315 U/mg, and no remaining affinity tags or artifacts from recombinant expression. This improved method therefore enables the facile production of the important virulence factor streptopain at higher yields, with no purification scars that might bias functional studies, and with an 8.1-fold increased enzymatic activity compared to previously described procedures. PMID:26773742

  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. PMID:27199917

  15. 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

  16. Enzymatic Properties of Populus α- and β-NAD-ME Recombinant Proteins

    PubMed Central

    Liu, Jinwen; Yu, Qiguo; Elsheery, Nabil I.; Cheng, Yuxiang

    2013-01-01

    Plant mitochondrial NAD-malic enzyme (NAD-ME), which is composed of α- and β-subunits in many species, participates in many plant biosynthetic pathways and in plant respiratory metabolism. However, little is known about the properties of woody plant NAD-MEs. In this study, we analyzed four NAD-ME genes (PtNAD-ME1 through PtNAD-ME4) in the genome of Populus trichocarpa. PtNAD-ME1 and -2 encode putative α-subunits, while PtNAD-ME3 and -4 encode putative β-subunits. The Populus NAD-MEs were expressed in Escherichia coli cells as GST-tagged fusion proteins. Each recombinant GST-PtNAD-ME protein was purified to near homogeneity by glutathione-Sepharose 4B affinity chromatography. Milligram quantities of each native protein were obtained from 1 L bacterial cultures after cleavage of the GST tag. Analysis of the enzymatic properties of these proteins in vitro indicated that α-NAD-MEs are more active than β-NAD-MEs and that α- and β-NAD-MEs presented different kinetic properties (Vmax, kcat and kcat/Km). The effect of different amounts of metabolites on the activities of Populus α- and β-NAD-MEs was assessed in vitro. While none of the metabolites evaluated in our assays activated Populus NAD-ME, oxalacetate and citrate inhibited all α- and β-NAD-MEs and glucose-6-P and fructose inhibited only the α-NAD-MEs. PMID:23797660

  17. 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.

  18. Enzymatic degradation of aromatic hydrocarbon intermediates using a recombinant dioxygenase immobilized onto surfactant-activated carbon nanotube.

    PubMed

    Suma, Yanasinee; Lim, Heejun; Kwean, Oh Sung; Cho, Suyeon; Yang, Junwon; Kim, Yohan; Kang, Christina S; Kim, Han S

    2016-06-01

    This study examined the enzymatic decomposition of aromatic hydrocarbon intermediates (catechol, 4-chlorocatechol, and 3-methylcatechol) using a dioxygenase immobilized onto single-walled carbon nanotube (SWCNT). The surfaces of SWCNTs were activated with surfactants. The dioxygenase was obtained by recombinant technique: the corresponding gene was cloned from Arthrobacter chlorophenolicus A6, and the enzyme was overexpressed and purified subsequently. The enzyme immobilization yield was 62%, and the high level of enzyme activity was preserved (60-79%) after enzyme immobilization. Kinetic analyses showed that the substrate utilization rates and the catalytic efficiencies of the immobilized enzyme for all substrates (target aromatic hydrocarbon intermediates) tested were similar to those of the free enzyme, indicating that the loss of enzyme activity was minimal during enzyme immobilization. The immobilized enzyme was more stable than the free enzyme against abrupt changes in pH, temperature, and ionic strength. Moreover, it retained high enzyme activity even after repetitive use. PMID:26810145

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

    PubMed Central

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

    2014-01-01

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

  20. 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

  1. 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

  2. Generation of recombinant, enzymatically active human thyroid peroxidase and its recognition by antibodies in the sera of patients with Hashimoto's thyroiditis.

    PubMed Central

    Kaufman, K D; Rapoport, B; Seto, P; Chazenbalk, G D; Magnusson, R P

    1989-01-01

    A full-length cDNA clone for human thyroid peroxidase (TPO) inserted into the mammalian cell expression vector pECE was stably transfected into Chinese hamster ovary (CHO) cells. Clones were assayed for human TPO mRNA, TPO protein, and TPO enzymatic activity. One subclone, expressing the highest TPO enzymatic activity, was used in further studies. FACS analysis of these cells preincubated in Hashimoto's serum revealed approximately 100-fold greater fluorescence compared with controls, indicating that recombinant TPO is expressed on the cell surface. Particulate antigen was extracted from these cells and studied by Western blot analysis using a panel of Hashimoto's sera of known antimicrosomal antibody (anti-MSA) titer. Under nonreducing conditions a broad, immunoreactive band of approximately 200 kD was observed, as well as a doublet of approximately 110 kD. All of the 36 Hashimoto's sera tested reacted with these bands, most in proportion to their anti-MSA titer. Six normal sera tested against this antigen(s) were nonreactive, as were the Hashimoto's sera tested against nontransfected CHO cells. Western blots under reducing conditions revealed a considerably diminished signal, with some of the sera of lower anti-MSA titer becoming negative, the loss of the 200-kD broad band, and the apparent conversion of the 110-kD doublet into a single band. Preincubation of cells in tunicamycin revealed no decrease in TPO immunoreactivity. In conclusion, we expressed enzymatically active human TPO in nonthyroidal eukaryotic cells. Our data prove that functionally active TPO is a major component of the thyroid microsomal antigen. Images PMID:2474568

  3. An evaluation of different enzymatic cleavage methods for recombinant fusion proteins, applied on des(1-3)insulin-like growth factor I.

    PubMed

    Forsberg, G; Baastrup, B; Rondahl, H; Holmgren, E; Pohl, G; Hartmanis, M; Lake, M

    1992-04-01

    Different enzymatic methods for cleavage of recombinant fusion proteins were compared. To find an efficient cleavage method, five different fusion proteins were produced. The fusion proteins differed only in the linker region between the fusion partner and the desired product, human des(1-3)insulin-like growth factor I. A cleavage study was performed with enterokinase, plasmin, thrombin, urokinase, and recombinant H64A subtilisin. Significant cleavage was obtained using thrombin, H64A subtilisin, and enterokinase. Thrombin cleavage was studied on a larger scale and des(1-3)IGF-I was recovered at a final yield of 3 mg/L growth medium. Thrombin and enterokinase were also studied as immobilized proteases and they cleaved the fusion proteins with retained activity. To further improve thrombin cleavage, a continuous reactor was constructed, consisting of a closed system with a thrombin column and an ion exchange column in series. Here, the fusion protein circulated while free des(1-3)IGF-I was bound to the ion exchange column after release from the fusion protein. In the reactor, thrombin was as efficient as the free enzyme but gave a diminished rate of product degradation. PMID:1388667

  4. Recombinant production of enzymatically active male contraceptive drug target hTSSK2 - Localization of the TSKS domain phosphorylated by TSSK2.

    PubMed

    Shetty, Jagathpala; Sinville, Rondedrick; Shumilin, Igor A; Minor, Wladek; Zhang, Jianhai; Hawkinson, Jon E; Georg, Gunda I; Flickinger, Charles J; Herr, John C

    2016-05-01

    The testis-specific serine/threonine kinase 2 (TSSK2) has been proposed as a candidate male contraceptive target. Development of a selective inhibitor for this kinase first necessitates the production of highly purified, soluble human TSSK2 and its substrate, TSKS, with high yields and retention of biological activity for crystallography and compound screening. Strategies to produce full-length, soluble, biologically active hTSSK2 in baculovirus expression systems were tested and refined. Soluble preparations of TSSK2 were purified by immobilized-metal affinity chromatography (IMAC) followed by gel filtration chromatography. The biological activities of rec.hTSSK2 were verified by in vitro kinase and mobility shift assays using bacterially produced hTSKS (isoform 2), casein, glycogen synthase peptide (GS peptide) and various TSKS peptides as target substrates. Purified recombinant hTSSK2 showed robust kinase activity in the in vitro kinase assay by phosphorylating hTSKS isoform 2 and casein. The ATP Km values were similar for highly and partially purified fractions of hTSSK2 (2.2 and 2.7 μM, respectively). The broad spectrum kinase inhibitor staurosporine was a potent inhibitor of rec.hTSSK2 (IC50 = 20 nM). In vitro phosphorylation experiments carried out with TSKS (isoform 1) fragments revealed particularly strong phosphorylation of a recombinant N-terminal region representing aa 1-150 of TSKS, indicating that the N-terminus of human TSKS is phosphorylated by human TSSK2. Production of full-length enzymatically active recombinant TSSK2 kinase represents the achievement of a key benchmark for future discovery of TSSK inhibitors as male contraceptive agents. PMID:26777341

  5. 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

  6. 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

  7. 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. PMID:24972714

  8. 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.

  9. 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

  10. 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

  11. 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

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

    PubMed

    Cheng, Gang; Wang, Lu; Lan, Haiyan

    2016-02-01

    Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of C4 photosynthetic pathway and plays an important biochemical role in higher plants and micro organisms. To gain understanding of the role of PEPC in stress adaptation in plant, we cloned PEPC gene from Suaeda aralocaspica, a C4 species without Kranz anatomy, and performed a series of experiments with PEPC gene expressed in Escherichia coli under various abiotic stresses. Results showed that, based on the homology cloning and 5'-RACE technique, the full-length cDNA sequence of PEPC (2901 bp) from S. aralocaspica was obtained, which shares the typical conserved domains to documented PEPCs and was identified as PEPC-1 in accord to the reported partial sequence (ppc-1) in S. aralocaspica. qRT-PCR analysis revealed the expression patterns of PEPC-1 and PEPC-2 (known as ppc-2, another plant type of PEPC) in S. aralocaspica, suggesting that PEPC-1 was up-regulated during seed germination and under NaCl stress, and presented higher level in chlorenchyma than other tissues, which were significantly different with PEPC-2. Afterwards, PEPC-1 was recombinant in E. coli (pET-28a-PEPC) and expressed as an approximate 110 kDa protein. Under various abiotic stresses, the recombinant E. coli strain harboring with PEPC-1 showed significant advantage in growth at 400-800 mmol L(-1) NaCl, 10-20% PEG6000, 25 and 30 °C lower temperature, 50-200 μmol L(-1) methyl viologen, and pH 5.0 and 9.0 condition, compared to control. Further analysis of the enzymatic characteristics of the recombinant PEPC-1 suggests that it was the higher enzyme activity of PEPC-1 which might confer the stress tolerance to E. coli. We speculate that over expression of PEPC-1 is probably related to regulation of oxaloacetate (OAA) in tricarboxylic acid (TCA) cycle in E. coli, which may contribute to further understanding of the physiological function of PEPC in S. aralocaspica. PMID:26777251

  13. 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.

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

    PubMed Central

    Tudyka, T.; Skerra, A.

    1997-01-01

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

  15. 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

  16. Enzymatic characterization of recombinant α-amylase in the Drosophila melanogaster species subgroup: is there an effect of specialization on digestive enzyme?

    PubMed

    Commin, Céline; Aumont-Nicaise, Magali; Claisse, Gaëlle; Feller, Georges; Da Lage, Jean-Luc

    2013-01-01

    We performed a comparative study on the enzymological features of purified recombinant α-amylase of three species belonging to the Drosophila melanogaster species subgroup: D. melanogaster, D. erecta and D. sechellia. D. erecta and D. sechellia are specialist species, with host plant Pandanus candelabrum (Pandanaceae) and Morinda citrifolia (Rubiaceae), respectively. The temperature optima were around 57-60℃ for the three species. The pH optima were 7.2 for D. melanogaster, 8.2 for D. erecta and 8.5 for D. sechellia. The kcat and Km were also estimated for each species with different substrates. The specialist species D. erecta and D. sechellia display a higher affinity for starch than D. melanogaster. α-Amylase activity is higher on starch than on glycogen in all species. α-Amylases of D. erecta and D. sechellia have a higher activity on maltooligosaccharides (G6 and G7) than on starch, contrary to D. melanogaster. Such differences in the enzymological features between the species might reflect adaptation to different ecological niches and feeding habits. PMID:24463528

  17. Enzymatic cyclization of 22,23-dihydro-2,3-oxidosqualene into euph-7-en-3beta-ol and bacchar-12-en-3beta-ol by recombinant beta-amyrin synthase.

    PubMed

    Abe, Ikuro; Sakano, Yuichi; Tanaka, Hideya; Lou, Weiwei; Noguchi, Hiroshi; Shibuya, Masaaki; Ebizuka, Yutaka

    2004-03-24

    Recombinant beta-amyrin synthase from Pisum sativum converted 22,23-dihydro-2,3-oxidosqualene, a substrate analogue lacking the terminal double bond of 2,3-oxidosqualene, into a 4:1 mixture of euph-7-en-3beta-ol and bacchar-12-en-3beta-ol. This is the first demonstration of the enzymatic formation of the baccharene skeleton with a six-membered D-ring. In the absence of the terminal double bond, the proton-initiated cyclization first generated the tetracyclic dammarenyl cation, followed by a backbone rearrangement with loss of H-7alpha leading to the formation of euph-7-en-3beta-ol, while D-ring expansion to the baccharenyl cation and subsequent 1,2-hydride shifts with H-12alpha elimination yielded bacchar-12-en-3beta-ol. It is remarkable that the formation of the anti-Markovnikov six-membered D-ring did not depend on the participation of the terminal pi-electrons. PMID:15025461

  18. [Health hazards of physical inactivity].

    PubMed

    Helajärvi, Harri; Lindholm, Harri; Vasankari, Tommi; Heinonen, Olli J

    2015-01-01

    Obesity and non-communicable diseases and related costs increase with physical inactivity. In addition to the lack of recreational exercise, a sedentary lifestyle also seems to have a negative effect of health, independently of other lifestyle and risks. New means, as well as multidisciplinary and multiprofessional collaboration, are required in order to improve health and well-being on the population level and to reduce health-related costs. New, more effective operational models are also needed in health communication in order to achieve the desired and more permanent results. PMID:26591649

  19. 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...

  20. 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: GENERAL LABELING Exemptions From Adequate Directions for Use § 201.117 Inactive ingredients. A...

  1. 21 CFR 201.117 - Inactive ingredients.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  2. 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…

  3. 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…

  4. 29 CFR 1404.6 - Inactive status.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 4 2010-07-01 2010-07-01 false Inactive status. 1404.6 Section 1404.6 Labor Regulations Relating to Labor (Continued) FEDERAL MEDIATION AND CONCILIATION SERVICE ARBITRATION SERVICES Roster of Arbitrators; Admission and Retention § 1404.6 Inactive status. A member of the Roster who continues to...

  5. λ Recombination and Recombineering.

    PubMed

    Murphy, Kenan C

    2016-05-01

    The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics. PMID:27223821

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

    NASA Astrophysics Data System (ADS)

    Borjigin, Jimo; Nathans, Jeremy

    1993-01-01

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

  7. 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. PMID:25292019

  8. 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

  9. Enzymatic intracrine regulation of white adipose tissue.

    PubMed

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

    2014-07-01

    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

  10. 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

  11. Temperature-Compensating Inactive Strain Gauge

    NASA Technical Reports Server (NTRS)

    Moore, Thomas C., Sr.

    1993-01-01

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

  12. Neuromuscular adaptations to respiratory muscle inactivity

    PubMed Central

    Mantilla, Carlos B.; Sieck, Gary C.

    2009-01-01

    Cervical spinal cord injury results in significant functional impairment. It is important to understand the neuroplasticity in response to inactivity of respiratory muscles in order to prevent any associated effects that limit functional recovery. Recent studies have examined the mechanisms involved in inactivity-induced neuroplasticity of diaphragm motor units. Both spinal hemisection at C2 (C2HS) and tetrodotoxin (TTX)-induced phrenic nerve blockade result in diaphragm paralysis and inactivity of axon terminals. However, phrenic motoneurons are inactive with C2HS but remain active after TTX. Diaphragm muscle fibers ipsilateral to C2HS display minimal changes post-injury. Neuromuscular transmission is enhanced following C2HS but impaired following TTX. Synaptic vesicle pool size at diaphragm neuromuscular junctions increases after C2HS, but decreases after TTX. Thus, inactivity-induced neuromuscular plasticity reflects specific adaptations that depend on inactivity at the motoneuron rather than at axon terminals or muscle fibers. Theses results indicate that neuromuscular transmission and functional properties of DIAm fibers can be maintained after spinal cord injury, providing a substrate for functional recovery and/or specific therapeutic approaches such as phrenic pacing. PMID:19744580

  13. 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

  14. 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. PMID:17902621

  15. 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

  16. 24 CFR 214.200 - Inactive status.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 24 Housing and Urban Development 2 2010-04-01 2010-04-01 false Inactive status. 214.200 Section 214.200 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF ASSISTANT SECRETARY FOR HOUSING-FEDERAL HOUSING COMMISSIONER, DEPARTMENT OF HOUSING AND...

  17. 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.

  18. 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.

  19. Enzymatic modification of schizophyllan

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  20. Pretreatment and Enzymatic Hydrolysis

    SciTech Connect

    2006-06-01

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

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

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

    PubMed

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

    2007-12-01

    A thiamine biosynthesis gene, thi3, from maize Zea mays has been identified through cloning and sequencing of cDNA and heterologous overexpression of the encoded protein, THI3, in Escherichia coli. The recombinant THI3 protein was purified to homogeneity and shown to possess two essentially different enzymatic activities of HMP(-P) [4-amino-5-hydroxymethyl-2-methylpyrimidine (phosphate)] kinase and TMP (thiamine monophosphate) synthase. Both activities were characterized in terms of basic kinetic constants, with interesting findings that TMP synthase is uncompetitively inhibited by excess of one of the substrates [HMP-PP (HMP diphosphate)] and ATP. A bioinformatic analysis of the THI3 sequence suggested that these activities were located in two distinct, N-terminal kinase and C-terminal synthase, domains. Models of the overall folds of THI3 domains and the arrangements of active centre residues were obtained with the SWISS-MODEL protein modelling server, on the basis of the known three-dimensional structures of Salmonella enterica serotype Typhimurium HMP(-P) kinase and Bacillus subtilis TMP synthase. The essential roles of Gln98 and Met134 residues for HMP kinase activity and of Ser444 for TMP synthase activity were experimentally confirmed by site-directed mutagenesis. PMID:17696876

  7. 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. PMID:26044846

  8. 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)

  9. 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…

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

    PubMed

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

    2007-01-01

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

  11. 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.

  12. Enzymatic DNA molecules

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  13. Enzymatic Modifications of Polysaccharides

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    2016-04-20

    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. PMID:26762578

  15. Cosmological Recombination

    NASA Astrophysics Data System (ADS)

    Wong, Wan Yan

    2008-11-01

    In this thesis we focus on studying the physics of cosmological recombination and how the details of recombination affect the Cosmic Microwave Background (CMB) anisotropies. We present a detailed calculation of the spectral line distortions on the CMB spectrum arising from the Lyman-alpha and the lowest two-photon transitions in the recombination of hydrogen (H), and the corresponding lines from helium (He). The peak of these distortions mainly comes from the Lyman-alpha transition and occurs at about 170 microns, which is the Wien part of the CMB. The major theoretical limitation for extracting cosmological parameters from the CMB sky lies in the precision with which we can calculate the cosmological recombination process. With this motivation, we perform a multi-level calculation of the recombination of H and He with the addition of the spin-forbidden transition for neutral helium (He I), plus the higher order two-photon transitions for H and among singlet states of He I. We find that the inclusion of the spin-forbidden transition results in more than a percent change in the ionization fraction, while the other transitions give much smaller effects. Last we modify RECFAST by introducing one more parameter to reproduce recent numerical results for the speed-up of helium recombination. Together with the existing hydrogen `fudge factor', we vary these two parameters to account for the remaining dominant uncertainties in cosmological recombination. By using a Markov Chain Monte Carlo method with Planck forecast data, we find that we need to determine the parameters to better than 10% for He I and 1% for H, in order to obtain negligible effects on the cosmological parameters.

  16. AN INACTIVE PRECURSOR OF STREPTOCOCCAL PROTEINASE

    PubMed Central

    Elliott, Stuart D.; Dole, Vincent P.

    1947-01-01

    1. Streptococcal proteinase is derived from an inactive precursor found in culture filtrates of proteinase-producing streptococci. 2. The precursor can be converted into the proteinase by low concentrations of trypsin but not by chymotrypsin. 3. In cultures grown in suitable media the conversion of precursor to proteinase is effected autocatalytically. This reaction occurs under reducing conditions and is initiated by active proteinase present in low concentrations with the precursor. 4. The autocatalytic reaction is suppressed or retarded by conditions which decrease the activity of the proteinase, e.g. by growing cultures at 22°C. instead of at 37°C. or by growing them under markedly aerobic conditions. It is also retarded in the presence of casein. PMID:19871616

  17. 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)

  18. Enzymatic production of cyclodextrins.

    PubMed

    Biwer, A; Antranikian, G; Heinzle, E

    2002-09-01

    Cyclodextrins (CD) are enzymatically modified starches with a wide range of applications in food, pharmaceutical and chemical industries, agriculture and environmental engineering. They are produced from starch via enzymatic conversion using cyclodextrin glycosyl transferases (CGTases) and partly alpha-amylases. Due to its low solubility in water, separation and purification of beta-CD is relatively easy compared to alpha- and gamma-CD. In recent years more economic processes for gamma-CD and especially alpha-CD production have been developed using improved CGTases and downstream processing. New purification steps, e.g. affinity adsorption, may reduce the use of complexing agents. The implementation of thermostable CGTases can simplify the production process and increase the selectivity of the reaction. A tabular overview of alpha-CD production processes is presented. PMID:12226716

  19. 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)

  20. 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

  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. 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

  3. 25 CFR 23.63 - Appeals from inaction of official.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

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

  4. 25 CFR 23.63 - Appeals from inaction of official.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  5. 25 CFR 23.63 - Appeals from inaction of official.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

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

  6. 25 CFR 23.63 - Appeals from inaction of official.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

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

  7. 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

  8. 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.

  9. 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.

  10. Pretreatment and enzymatic hydrolysis of corn fiber

    SciTech Connect

    Grohmann, K.; Bothast, R.J.

    1996-10-01

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

  11. 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

  12. [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. PMID:15989270

  13. Molecular abnormality of an inactive aldehyde dehydrogenase variant commonly found in Orientals.

    PubMed Central

    Yoshida, A; Huang, I Y; Ikawa, M

    1984-01-01

    Usual human livers contain two major aldehyde dehydrogenase [(ALDH) aldehyde:NAD+ oxidoreductase] isozymes--i.e., a cytosolic ALDH1 component and a mitochondrial ALDH2 component--whereas approximately equal to 50% of Orientals are "atypical" and have only the ALDH1 isozyme and are missing the ALDH2 isozyme. We previously demonstrated that atypical livers contain an enzymatically inactive but immunologically crossreactive material (CRM) corresponding to the ALDH2 component. The enzymatically active ALDH2 obtained from a usual liver and the CRM obtained from an atypical liver were reduced, S-carboxymethylated, and digested by trypsin. Separation of their digests by high-performance reverse-phase chromatography and by two-dimensional paper chromatography and electrophoresis revealed that ALDH2 contained a peptide sequence of -Glu-Leu-Gly-Glu-Ala-Gly-Leu-Gln-Ala-Asn-Val-Gln-Val-Lys- and that the glutamine adjacent to lysine was substituted by lysine in CRM. All other tryptic peptides, including eight peptides containing S-carboxymethylcysteine, were common in ALDH2 and CRM. It is concluded that a point mutation in the human ALDH2 locus produced the glutamine leads to lysine substitution and enzyme inactivation. Images PMID:6582480

  14. 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

  15. Fluorometric enzymatic assay of l-arginine.

    PubMed

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

    2017-01-01

    The enzymes of l-arginine (further - Arg) metabolism are promising tools for elaboration of selective methods for quantitative Arg analysis. In our study we propose an enzymatic method for Arg assay based on fluorometric monitoring of ammonia, a final product of Arg splitting by human liver arginase I (further - arginase), isolated from the recombinant yeast strain, and commercial urease. The selective analysis of ammonia (at 415nm under excitation at 360nm) is based on reaction with o-phthalaldehyde (OPA) in the presence of sulfite in alkali medium: these conditions permit to avoid the reaction of OPA with any amino acid. A linearity range of the fluorometric arginase-urease-OPA method is from 100nM to 6μМ with a limit of detection of 34nM Arg. The method was used for the quantitative determination of Arg in the pooled sample of blood serum. The obtained results proved to be in a good correlation with the reference enzymatic method and literature data. The proposed arginase-urease-OPA method being sensitive, economical, selective and suitable for both routine and micro-volume formats, can be used in clinical diagnostics for the simultaneous determination of Arg as well as urea and ammonia in serum samples. PMID:27450117

  16. Enzymatic modification of schizophyllan.

    PubMed

    Leathers, Timothy D; Sutivisedsak, Nongnuch; Nunnally, Melinda S; Price, Neil P J; Stanley, April M

    2015-03-01

    An enzymatic method was developed for the progressive modification of the polysaccharide schizophyllan. Fungal strains Hypocrea nigricans NRRL 62555, Penicillium crustosum NRRL 62558, and Penicillium simplicissimum NRRL 62550 were previously identified as novel sources of β-endoglucanase with specificity towards schizophyllan. Concentrated enzyme preparations from these strains showed specific activities of 1.7-4.3 U β-glucanase/mg protein. Using dilutions of these enzymes in time course digestions, schizophyllan was progressively modified to reduced molecular weight species. Glucose and oligosaccharides were found only in the more complete digestions, and thus modified schizophyllan can be produced quantitatively, without loss, to small molecules. Permethylation analysis confirmed that modified schizophyllan retains the fundamental linkage structure of native schizophyllan. Modified schizophyllan species showed progressively reduced viscosity profiles, and all exhibited pseudoplasticity in response to shear thinning. PMID:25335747

  17. Inactive Women May Face Higher Risk for Cervical Cancer

    MedlinePlus

    ... html Inactive Women May Face Higher Risk for Cervical Cancer But study found just 30 minutes of exercise ... who are sedentary appear more likely to develop cervical cancer, but just 30 minutes of exercise each week ...

  18. 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

  19. 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

  20. Enzymatically active ultrathin pepsin membranes.

    PubMed

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

    2015-05-11

    Enzymatically active proteins enable efficient and specific cleavage reactions of peptide bonds. Covalent coupling of the enzymes permits immobilization, which in turn reduces autolysis-induced deactivation. Ultrathin pepsin membranes were prepared by facile interfacial polycondensation of pepsin and trimesoyl chloride. The pepsin membrane allows for simultaneous enzymatic conversion and selective removal of digestion products. The large water fluxes through the membrane expedite the transport of large molecules through the pepsin layers. The presented method enables the large-scale production of ultrathin, cross-linked, enzymatically active membranes. PMID:25779668

  1. Association between Physical Inactivity and Academic Record in Korean Adolescents

    PubMed Central

    Shin, Yi-Soo; So, Wi-Young

    2012-01-01

    Background: The purpose of this study was to investigate the association between physical inactivity and academic record in Korean adolescents. Methods: Adolescent students from the first grade of middle school to the third grade of high school (n=75,066) participated in the 5th Korea Youth Risk Behavior Web-based Survey project in 2009. The association between physical inactivity and academic record was assessed using multivariate logistic regression analysis after adjusting for gender, age, body mass index, family’s socioeconomic status, parents’ education level, and frequency of vigorous or moderate physical activity (PA) as well as muscular strength exercises. Results: During weekdays, the odds ratios (ORs) (95% confidence interval [CI]) for reporting a higher than average academic record, as compared with <1 hour of physical inactivity per day, was 0.796 (0.761–0.832, for ≥1 to <2 hours, 0.632 (0.603–0.663, for ≥2 to <3 hours, 0.567 (0.535–0.601, for ≥3 to <4 hours, and 0.494 (0.468–0.522, P < 0.001 for all cases) for ≥4 hours of physical inactivity per day. During the weekends, the ORs (95% CI) for reporting a higher than average academic record, as compared with <1 hour of physical inactivity per day, were 0.901 (0.848–0.957, P = 0.001) for ≥3 to <4 hours and 0.785 (0.743–0.830, P < 0.001) for ≥4 hours of physical inactivity per day. Conclusion: Korean adolescents who spend more time engaged in physical inactivity are predisposed to a below-average academic record. PMID:23308350

  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. 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.

  4. 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)

  5. Fluorous enzymatic synthesis of phosphatidylinositides.

    PubMed

    Huang, Weigang; Proctor, Angela; Sims, Christopher E; Allbritton, Nancy L; Zhang, Qisheng

    2014-03-18

    A fluorous tagging strategy coupled with enzymatic synthesis is introduced to efficiently synthesize multiple phosphatidylinositides, which are then directly immobilized on a fluorous polytetrafluoroethylene (PTFE) membrane to probe protein-lipid interactions. PMID:24496473

  6. Enzymatic reactions in confined environments.

    PubMed

    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. PMID:27146955

  7. Recombination hotspots: Models and tools for detection.

    PubMed

    Paul, Prosenjit; Nag, Debjyoti; Chakraborty, Supriyo

    2016-04-01

    Recombination hotspots are the regions within the genome where the rate, and the frequency of recombination are optimum with a size varying from 1 to 2kb. The recombination event is mediated by the double-stranded break formation, guided by the combined enzymatic action of DNA topoisomerase and Spo 11 endonuclease. These regions are distributed non-uniformly throughout the human genome and cause distortions in the genetic map. Numerous lines of evidence suggest that the number of hotspots known in humans has increased manifold in recent years. A few facts about the hotspot evolutions were also put forward, indicating the differences in the hotspot position between chimpanzees and humans. In mice, recombination hot spots were found to be clustered within the major histocompatibility complex (MHC) region. Several models, that help explain meiotic recombination has been proposed. Moreover, scientists also developed some computational tools to locate the hotspot position and estimate their recombination rate in humans is of great interest to population and medical geneticists. Here we reviewed the molecular mechanisms, models and in silico prediction techniques of hot spot residues. PMID:26991854

  8. The Costs of Inaction with Respect to Climate Change

    SciTech Connect

    Cline, William R.

    2005-04-14

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

  9. 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

  10. 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

  11. 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.

  12. Directed Ig class switch recombination in activated murine B cells.

    PubMed Central

    Winter, E; Krawinkel, U; Radbruch, A

    1987-01-01

    Immunoglobulin class switch recombination occurs at frequencies of up to 10%/cell/generation in activated murine B-lymphocytes. We analysed cH gene rearrangements and switch recombinations from active and inactive IgH loci of B-cells activated in various ways and immortalized by cell fusion. Although about half of the IgM+ cells show rearrangement of c mu genes, the deletion of c mu is a rare event. Half of the IgG3+ and IgG1+ cells show rearrangement of c mu genes on the inactive IgH locus and the other half of the IgG+ cells have deleted c mu from both IgH loci by switch recombination. This recombination is directed to the same switch regions on both IgH loci in 60-80% of all cases. Interleukin 4 may play a critical role in programming murine B-lymphocytes for specific switch recombination. Images Fig. 1. Fig. 2. Fig. 6. PMID:3038529

  13. Identification of inactive medications in narrative medical text.

    PubMed

    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 patients medication list in more than one in ten notes. Documentation of inactive medications is common in narrative provider notes and can be computationally extracted. This technology could be employed in real-time patient care as well as for research and quality of care monitoring. PMID:18999079

  14. 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...

  15. 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...

  16. 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 Waste Disposal Programs § 256.25 Recommendation...

  17. 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...

  18. 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...

  19. 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...

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

    NASA Technical Reports Server (NTRS)

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

    1975-01-01

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

  1. Phospholipases a2 from Viperidae snakes: Differences in membranotropic activity between enzymatically active toxin and its inactive isoforms.

    PubMed

    Ghazaryan, Narine A; Ghulikyan, Lusine; Kishmiryan, Arsen; Andreeva, Tatyana V; Utkin, Yuri N; Tsetlin, Victor I; Lomonte, Bruno; Ayvazyan, Naira M

    2015-02-01

    We describe the interaction of various phospholipases A2 (PLA2) from snake venoms of the family Viperidae (Macrovipera lebetina obtusa, Vipera ursinii renardi, Bothrops asper) with giant unilamellar vesicles (GUVs) composed of natural brain phospholipids mixture, visualized through fluorescence microscopy. The membrane fluorescent probes 8-anilino-1-naphthalenesulfonicacid (ANS), LAUDRAN and PRODAN were used to assess the state of the membrane and specifically mark the lipid packing and membrane fluidity. Our results have shown that the three PLA2s which contain either of aspartic acid, serine, or lysine residues at position 49 in the catalytic center, have different effects on the vesicles. The PLA2 with aspartic acid at this position causes the oval deformation of the vesicles, while serine and lysine-containing enzymes lead to an appreciable increase of fluorescence intensity in the vesicles membrane, wherein the shape and dimensions of GUVs have not changed, but in this case GUV aggregation occurs. LAURDAN and PRODAN detect the extent of water penetration into the bilayer surface. We calculated generalized polarization function (GP), showing that for all cases (D49 PLA2, S49 PLA2 and K49 PLA2) both LAUDRAN and PRODAN GP values decrease. A higher LAURDAN GP is indicative of low water penetration in the lipid bilayer in case of K49 PLA2 compared with D49 PLA2, whereas the PRODAN mainly gives information when lipid is in liquid crystalline phase. PMID:25450350

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

    PubMed

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

    2015-12-01

    Macrophage migration inhibitory factor (MIF) is an important player in the regulation of the inflammatory response. Elevated plasma MIF is found in sepsis, arthritis, cystic fibrosis and atherosclerosis. Immunomodulatory activities of MIF include the ability to promote survival and recruitment of inflammatory cells and to amplify pro-inflammatory cytokine production. MIF has an unusual nucleophilic N-terminal proline with catalytic tautomerase activity. It remains unclear whether tautomerase activity is required for MIF function, but small molecules that inhibit tautomerase activity also inhibit the pro-inflammatory activities of MIF. A prominent feature of the acute inflammatory response is neutrophil activation and production of reactive oxygen species, including myeloperoxidase (MPO)-derived hypochlorous acid and hypothiocyanous acid. We hypothesized that MPO-derived oxidants would oxidize the N-terminal proline of MIF and alter its biological activity. MIF was exposed to hypochlorous acid and hypothiocyanous acid and the oxidative modifications on MIF were examined by LC-MS/MS. Imine formation and carbamylation was observed on the N-terminal proline in response to MPO-dependent generation of hypochlorous and hypothiocyanous acid, respectively. These modifications led to a complete loss of tautomerase activity. However, modified MIF still increased CXCL-8/IL-8 production by peripheral blood mononuclear cells (PBMCs) and blocked neutrophil apoptosis, indicating that tautomerase activity is not essential for these biological functions. Pre-treatment of MIF with hypochlorous acid protected the protein from covalent modification by the MIF inhibitor 4-iodo-6-phenylpyrimidine (4-IPP). Therefore, oxidant generation at inflammatory sites may protect MIF from inactivation by more disruptive electrophiles, including drugs designed to target the tautomerase activity of MIF. PMID:26453918

  3. 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

  4. Enzymatic Activity of Xyloglucan Xylosyltransferase 5.

    PubMed

    Culbertson, Alan T; Chou, Yi-Hsiang; Smith, Adrienne L; Young, Zachary T; Tietze, Alesia A; Cottaz, Sylvain; Fauré, Régis; Zabotina, Olga A

    2016-07-01

    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

  5. Enzymatic hydrolysis of organic phosphorus

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. 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.

  7. Bioluminescence methods for enzymatic determinations

    SciTech Connect

    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.

  8. Enzymatic conversion of carbon dioxide.

    PubMed

    Shi, Jiafu; Jiang, Yanjun; Jiang, Zhongyi; Wang, Xueyan; Wang, Xiaoli; Zhang, Shaohua; Han, Pingping; Yang, Chen

    2015-10-01

    With the continuous increase in fossil fuels consumption and the rapid growth of atmospheric CO2 concentration, the harmonious state between human and nature faces severe challenges. Exploring green and sustainable energy resources and devising efficient methods for CO2 capture, sequestration and utilization are urgently required. Converting CO2 into fuels/chemicals/materials as an indispensable element for CO2 capture, sequestration and utilization may offer a win-win strategy to both decrease the CO2 concentration and achieve the efficient exploitation of carbon resources. Among the current major methods (including chemical, photochemical, electrochemical and enzymatic methods), the enzymatic method, which is inspired by the CO2 metabolic process in cells, offers a green and potent alternative for efficient CO2 conversion due to its superior stereo-specificity and region/chemo-selectivity. Thus, in this tutorial review, we firstly provide a brief background about enzymatic conversion for CO2 capture, sequestration and utilization. Next, we depict six major routes of the CO2 metabolic process in cells, which are taken as the inspiration source for the construction of enzymatic systems in vitro. Next, we focus on the state-of-the-art routes for the catalytic conversion of CO2 by a single enzyme system and by a multienzyme system. Some emerging approaches and materials utilized for constructing single-enzyme/multienzyme systems to enhance the catalytic activity/stability will be highlighted. Finally, a summary about the current advances and the future perspectives of the enzymatic conversion of CO2 will be presented. PMID:26055659

  9. Recombinant laccase: I. Enzyme cloning and characterization.

    PubMed

    Nicolini, Claudio; Bruzzese, Debora; Cambria, Maria Teresa; Bragazzi, Nicola Luigi; Pechkova, Eugenia

    2013-03-01

    We obtained structural and functional characterization of a recombinant Laccase from Rigidoporus lignosus (formerly Rigidoporus microporus), a white-rot basidiomycete, by means of circular dichroism (CD) spectra, cyclic voltammetry (CV) and biochemical assays. Here we report the optimization of expression and purification procedures of a recombinant Laccase expressed in supercompetent Escherichia coli cells. We amplified the coding sequence of Laccase using PCR from cDNA and cloned into a bacterial expression system. The resulting expression plasmid, pET-28b, was under a strong T7/Lac promoter induced by IPTG (isopropyl-β-d-thiogalactoipyranoside). We obtained purification by fast protein liquid chromatography (FPLC) method. We recorded the variation of the current of a solution containing purified Laccase with increasing Syringaldazine (SGZ) concentration using a potentiometer as proof of principle, showing its compatibility with the development of a new enzymatic biosensor for medical purposes, as described in Part II. PMID:22991171

  10. 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...

  11. 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...

  12. 200-Area plateau inactive miscellaneous underground storage tanks locations

    SciTech Connect

    Brevick, C.H.

    1997-12-01

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

  13. 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…

  14. 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.

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

    PubMed

    Bull, Fiona C; Bauman, Adrian E

    2011-08-01

    There is strong evidence demonstrating the direct and indirect pathways by which physical activity prevents many of the major noncommunicable diseases (NCD) responsible for premature death and disability. Physical inactivity was identified as the 4th leading risk factor for the prevention of NCD, preceded only by tobacco use, hypertension, and high blood glucose levels, and accounting for more than 3 million preventable deaths globally in 2010. Physical inactivity is a global public health priority but, in most countries, this has not yet resulted in widespread recognition nor specific physical activity-related policy action at the necessary scale. Instead, physical inactivity could be described as the Cinderella of NCD risk factors, defined as "poverty of policy attention and resourcing proportionate to its importance." The pressing question is "Why is this so?" The authors identify and discuss 8 possible explanations and the need for more effective communication on the importance of physical activity in the NCD prevention context. Although not all of the issues identified will be relevant for any 1 country, it is likely that at different times and in different combinations these 8 problems continue to delay national-level progress on addressing physical inactivity in many countries. The authors confirm that there is sufficient evidence to act, and that much better use of well-planned, coherent communication strategies are needed in most countries and at the international level. Significant opportunities exist. The Toronto Charter on Physical Activity and the Seven Investments that Work are 2 useful tools to support increased advocacy on physical activity within and beyond the context of the crucial 2011 UN High-Level Meeting on NCDs. PMID:21916710

  16. Isolation, purification, and study of properties of recombinant hepsin from Escherichia coli.

    PubMed

    Raevskaya, A A; Kuznetsova, E M; Savvateeva, M V; Severin, S E

    2010-07-01

    A recombinant hepsin-producing strain of Escherichia coli was obtained and the conditions for hepsin expression in a bacterial system were optimized. To study the physicochemical properties of the enzyme, a procedure for purification of active recombinant hepsin using metal-chelate affinity chromatography and ion-exchange chromatography was developed. The interaction of recombinant hepsin with various peptide substrates is characterized. The dose-dependent inhibition of the recombinant hepsin enzyme activity by anthralin in vitro and an increase in the hepsin enzymatic activity in the presence of resveratrol were revealed. PMID:20673210

  17. Recombineering homologous recombination constructs in Drosophila.

    PubMed

    Carreira-Rosario, Arnaldo; Scoggin, Shane; Shalaby, Nevine A; Williams, Nathan David; Hiesinger, P Robin; Buszczak, Michael

    2013-01-01

    The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner. PMID:23893070

  18. 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.

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

    PubMed

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

  20. 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

  1. 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.

  2. 37 CFR 11.19 - Disciplinary jurisdiction; Jurisdiction to transfer to disability inactive status.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...; Jurisdiction to transfer to disability inactive status. 11.19 Section 11.19 Patents, Trademarks, and Copyrights... disability inactive status. (a) All practitioners engaged in practice before the Office; all practitioners... transferred to disability inactive status, reprimanded, suspended, or excluded from the practice of law by...

  3. 37 CFR 11.29 - Reciprocal transfer or initial transfer to disability inactive status.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... initial transfer to disability inactive status. 11.29 Section 11.29 Patents, Trademarks, and Copyrights..., Sanctions, Investigations, and Proceedings § 11.29 Reciprocal transfer or initial transfer to disability inactive status. (a) Notification of OED Director. (1) Transfer to disability inactive status in...

  4. 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...

  5. Physical inactivity in COPD and increased patient perception of dyspnea

    PubMed Central

    Katajisto, Milla; Kupiainen, Henna; Rantanen, Piritta; Lindqvist, Ari; Kilpeläinen, Maritta; Tikkanen, Heikki; Laitinen, Tarja

    2012-01-01

    Objective: To study patients’ levels of exercise activity and the clinical characteristics that relate to physical activity and inactivity among patients with chronic obstructive pulmonary disease (COPD). Methods: A postal questionnaire was administered to 719 patients with COPD in 2010; patients were recruited from the Helsinki and Turku University Central Hospitals in Finland and have been followed since 2005. The questionnaire asked participants about their exercise routines and other daily activities, potential restrictions to exercise, health-related quality of life, and subjective sensations of dyspnea upon exertion. Results: A total of 50% of the participants reported exercising > 2 times a week throughout the year. The proportion of the exercise inactive patients increased in parallel with disease progression, but the participants exhibited great variation in the degree of activity as well as in sport choices. Year-round activity was better maintained among patients who exercised both indoors and outdoors. Training activity was significantly correlated with patients’ reported subjective dyspnea (r = 0.32, P < 0.001), health-related quality of life (r = 0.25, P < 0.001), mobility score (r = 0.37, P < 0.001), and bronchial obstruction (r = 0.18, P < 0.001). Active patients did not differ from inactive patients in terms of sex, age, smoking status, somatic comorbidities, or body mass index. Irrespective of the level of severity of patients’ COPD, the most significant barrier to exercising was the subjective sensation of dyspnea. Conclusion: When a patient with COPD suffers from dyspnea and does not have regular exercise routines, the patient will most likely benefit from an exercise program tailored to his or her physical capabilities. PMID:23152679

  6. 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.

  7. 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

  8. Recombination in electron coolers

    NASA Astrophysics Data System (ADS)

    Wolf, A.; Gwinner, G.; Linkemann, J.; Saghiri, A. A.; Schmitt, M.; Schwalm, D.; Grieser, M.; Beutelspacher, M.; Bartsch, T.; Brandau, C.; Hoffknecht, A.; Müller, A.; Schippers, S.; Uwira, O.; Savin, D. W.

    2000-02-01

    An introduction to electron-ion recombination processes is given and recent measurements are described as examples, focusing on low collision energies. Discussed in particular are fine-structure-mediated dielectronic recombination of fluorine-like ions, the moderate recombination enhancement by factors of typically 1.5-4 found for most ion species at relative electron-ion energies below about 10 meV, and the much larger enhancement occurring for specific highly charged ions of complex electronic structure, apparently caused by low-energy dielectronic recombination resonances. Recent experiments revealing dielectronic resonances with very large natural width are also described.

  9. 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

  10. 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

  11. Alterations in protein metabolism during space flight and inactivity.

    PubMed

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

    2002-10-01

    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. PMID:12361775

  12. 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

  13. 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.

  14. 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

  15. 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

  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. 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

  18. Economics of enzymatic hydrolysis processes

    SciTech Connect

    Wright, J.D.

    1988-02-01

    Enzymatic hydrolysis processes have the ability to produce high yields of sugars for fermentation to fuel ethanol from lignocellulosic biomass. However, these systems have been plagued with yields, product concentrations, and reactions rates far below those that are theoretically possible. Engineering and economic analyses are presented on several fungal enzyme hydrolysis processes to illustrate the effects of the important process parameters, to quantify the progress that has been made to date, and to estimate the cost reductions that can be made through research improvements. All enzymatic hydrolysis processes require pretreatment, hydrolysis, fermentation, and enzyme production. The key effect of pretreatment is to allow access of the enzymes to the substrate. Pretreatments have been devised that make the biomass completely digestible that increase the xylose yield and concentration, and that integrate pretreatment with lignin utilization. Major improvements in enzyme activity and use of simultaneous saccharification and fermentation (SSF) have greatly reduced the inhibition of the enzymes. It now appears that ethanol inhibition of the yeast is the limiting factor. Enzyme production costs have been dramatically reduced because use of SSF has reduced enzyme loading. However, further improvements may be possible by using soluble carbon sources for production. Over the past decade, the predicted cost of ethanol from such processes has dropped from more than $4.00/gallon to approximately $1.60. Research is currently under way in the United States and has the potential to reduce the projected cost to less than $1.00/gallon. 65 refs., 16 figs., 1 tab.

  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. PMID:25854425

  20. 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.

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

    PubMed Central

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

    2010-01-01

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

  3. 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

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

    PubMed

    Atkinson, Kaitlin; Lowe, Samantha; Moore, Spencer

    2016-06-01

    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

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

    SciTech Connect

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

    1986-01-01

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

  6. Validation of doubled haploid plants by enzymatic mismatch cleavage

    PubMed Central

    2013-01-01

    Background Doubled haploidy is a fundamental tool in plant breeding as it provides the fastest way to generate populations of meiotic recombinants in a genetically fixed state. A wide range of methods has been developed to produce doubled haploid (DH) plants and recent advances promise efficient DH production in otherwise recalcitrant species. Since the cellular origin of the plants produced is not always certain, rapid screening techniques are needed to validate that the produced individuals are indeed homozygous and genetically distinct from each other. Ideal methods are easily implemented across species and in crops where whole genome sequence and marker resources are limited. Results We have adapted enzymatic mismatch cleavage techniques commonly used for TILLING (Targeting Induced Local Lesions IN Genomes) for the evaluation of heterozygosity in parental, F1 and putative DH plants. We used barley as a model crop and tested 26 amplicons previously developed for TILLING. Experiments were performed using self-extracted single-strand-specific nuclease and standard native agarose gels. Eleven of the twenty-six tested primers allowed unambiguous assignment of heterozygosity in material from F1 crosses and loss of heterozygosity in the DH plants. Through parallel testing of previously developed Simple Sequence Repeat (SSR) markers, we show that 3/32 SSR markers were suitable for screening. This suggests that enzymatic mismatch cleavage approaches can be more efficient than SSR based screening, even in species with well-developed markers. Conclusions Enzymatic mismatch cleavage has been applied for mutation discovery in many plant species, including those with little or no available genomic DNA sequence information. Here, we show that the same methods provide an efficient system to screen for the production of DH material without the need of specialized equipment. This gene target based approach further allows discovery of novel nucleotide polymorphisms in candidate

  7. Continuous ethanol production from wheat straw hydrolysate by recombinant ethanologenic Escherichia coli strain FBR5

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Continuous production of ethanol from alkaline peroxide pretreated and enzymatically saccharified wheat straw hydrolyzate by ethanologenic recombinant Escherichia coli strain FBR5 was investigated under various conditions at controlled pH 6.5 and 35 deg C. The strain FBR5 was chosen because of its a...

  8. 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 ...

  9. 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...

  10. Ethanol production from wheat straw by recombinant Escherichia coli strain FBR5 at high solid loading

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ethanol production by a recombinant bacterium from wheat straw (WS) at high solid loading by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) was studied. The yield of total sugars from dilute acid pretreated WS (150 g/L) after enzymatic saccharific...

  11. 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.

  12. 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.

  13. Prevalence of atherosclerosis in patients with inactive rheumatoid arthritis.

    PubMed

    Dehghan, Pooneh; Rajaei, Alireza; Moeineddin, Reza; Alizadeh, Afshin Mohammad

    2015-08-01

    Patients with rheumatoid arthritis (RA) exhibit higher cardiovascular morbidity and mortality than the general population. The common carotid artery intima-media thickness (CCA-IMT) measured by ultrasound is a validated surrogate marker of atherosclerosis. We studied the prevalence of subclinical atherosclerosis in patients with inactive RA. CCA-IMT was measured at the level of the carotid bifurcation in 35 patients with RA and 35 age- and sex-matched controls. CCA-IMT measurements more than two standard deviations above the mean measurement of the control group were defined as abnormal. Patients and controls with known cardiovascular risk factors were excluded from the study. The following data were taken into consideration for each patient: age, sex, duration of RA, and medication received by the patient. These variables were adjusted in data analysis. The patients were required to have had normal C-reactive protein levels for at least 1 year and no clinical signs or symptoms of active disease. An independent t test and chi-squared test were used for statistical analysis. Nine patients with RA (25.7 %) and two controls (5.7 %) had an abnormal IMT. Patients showed more carotid atherosclerotic plaques and a higher mean CCA-IMT than did controls (48.6 vs. 14.3 % and 0.705 ± 0.140 vs. 0.580 ± 0.125, respectively). An abnormal CCA-IMT was more prevalent in patients >50 years of age with >5 years of involvement (P = 0.001). These results support the theory that chronic inflammation in patients with inactive RA is associated with atherosclerosis and suggest the use of carotid ultrasonography as a useful tool with which to establish the risk of cardiovascular disease. PMID:26122966

  14. 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.

  15. 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

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

    PubMed Central

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

    2011-01-01

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

  17. 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.

  18. 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.

  19. 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

    2011-08-11

    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. PMID:21775986

  20. 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

  1. [Recombinant antibodies against bioweapons].

    PubMed

    Thullier, Philippe; Pelat, Thibaut; Vidal, Dominique

    2009-12-01

    The threat posed by bioweapons (BW) could lead to the re-emergence of such deadly diseases as plague or smallpox, now eradicated from industrialized countries. The development of recombinant antibodies allows tackling this risk because these recombinant molecules are generally well tolerated in human medicine, may be utilized for prophylaxis and treatment, and because antibodies neutralize many BW. Recombinant antibodies neutralizing the lethal toxin of anthrax, botulinum toxins and the smallpox virus have in particular been isolated recently, with different technologies. Our approach, which uses phage-displayed immune libraries built from non-human primates (M. fascicularis) to obtain recombinant antibodies, which may later be super-humanized (germlinized), has allowed us to obtain such BWs-neutralizing antibodies. PMID:20035695

  2. Modeling the minimum enzymatic requirements for optimal cellulose conversion

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

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

  3. Activated recombinant adenovirus proteinases

    DOEpatents

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

    1999-08-10

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

  4. 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.

  5. 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

  6. 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

  7. 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

  8. Accumulation of B1-like B cells in transgenic mice over-expressing catalytically inactive RAG1 in the periphery

    PubMed Central

    Hassaballa, Ashraf E; Palmer, Victoria L; Anderson, Dirk K; Kassmeier, Michele D; Nganga, Vincent K; Parks, Kevin W; Volkmer, Dustin L; Perry, Greg A; Swanson, Patrick C

    2011-01-01

    During their development, B lymphocytes undergo V(D)J recombination events and selection processes that, if successfully completed, produce mature B cells expressing a non-self-reactive B-cell receptor (BCR). Primary V(D)J rearrangements yield self-reactive B cells at high frequency, triggering attempts to remove, silence, or reprogramme them through deletion, anergy induction, or secondary V(D)J recombination (receptor editing), respectively. In principle, expressing a catalytically inactive V(D)J recombinase during a developmental stage in which V(D)J rearrangement is initiated may impair this process. To test this idea, we generated transgenic mice expressing a RAG1 active site mutant (dnRAG1 mice); RAG1 transcript was elevated in splenic, but not bone marrow, B cells in dnRAG1 mice relative to wild-type mice. The dnRAG1 mice accumulate splenic B cells with a B1-like phenotype that exhibit defects in B-cell activation, and are clonally diverse, yet repertoire restricted with a bias toward Jκ1 gene segment usage. The dnRAG1 mice show evidence of impaired B-cell development at the immature-to-mature transition, immunoglobulin deficiency, and poorer immune responses to thymus-independent antigens. Interestingly, dnRAG1 mice expressing the anti-dsDNA 3H9H56R heavy chain fail to accumulate splenic B1-like cells, yet retain peritoneal B1 cells. Instead, these mice show an expanded marginal zone compartment, but no difference is detected in the frequency of heavy chain gene replacement. Taken together, these data suggest a model in which dnRAG1 expression impairs secondary V(D)J recombination. As a result, selection and/or differentiation processes are altered in a way that promotes expansion of B1-like B cells in the spleen. PMID:22044391

  9. 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

  10. 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

  11. Enzymatic Vitrectomy and Pharmacologic Vitreodynamics.

    PubMed

    Shah, Ankoor R; Trese, Michael T

    2016-01-01

    The field of vitreoretinal surgery has evolved substantially over the last several decades. Scientific advances have improved our understanding of disease pathophysiology, and new surgical adjuncts and techniques have decreased surgical time and improved patient outcomes. Pharmacologic agents have recently been developed for intraocular use in order to enhance vitreous removal and even as a nonsurgical treatment for pathology due to an abnormal vitreoretinal interface. Plasmin can successfully cause vitreous liquefaction and induce a posterior vitreous detachment. Additionally, ocriplasmin has been approved for symptomatic vitreomacular adhesion and others appear to be promising for pharmacologic manipulation of the vitreous. The ability to induce vitreous liquefaction and a complete posterior vitreous detachment (PVD) with a single intravitreal injection has potential implications for the management of multiple vitreoretinopathies. Enzymatic vitrectomy may help to reduce vitreous viscosity, thereby facilitating removal during vitrectomy and reducing surgical time, especially when using smaller-gauge vitrectomy instruments. The induction of a PVD also has the potential to reduce intraoperative complications. As we improve our understanding of the molecular flux in the vitreous cavity, pharmacologic vitreodynamics will likely become more important as it may allow for improved manipulation of intravitreal molecules. PMID:26501959

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

    PubMed

    Utomo, Iwu Dwisetyani; McDonald, Peter

    2009-06-01

    This study examines the changing social and political context of adolescent sexual and reproductive health policy in Indonesia. We describe how, in 2001, Indonesia was on the brink of implementing an adolescent reproductive health policy that was consistent with international agreements to which the Indonesian government was a party. Although the health of young Indonesians was known to be at risk, the opportunity for reform passed quickly with the emergence of a new competing force, Middle Eastern fundamentalist Islam. Faced with the risk of regional separatism and competing politico-religious influences, the Indonesian government retreated to the safety of inaction in this area of policy. In the absence of a supportive and committed political environment that reinforces policy specifically targeted to young people's reproductive health, extremist approaches that involve considerable health risk prevailed. The sexual and reproductive values and behaviors that are emerging among single young people in contemporary Indonesia are conditioned by a political context that allows the conflicting forces of traditional Indonesian values, Westernization, and the strong emerging force of fundamentalist Islam to compete for the allegiance of young people. PMID:19662805

  13. Genetic and pharmacological reactivation of the mammalian inactive X chromosome

    PubMed Central

    Bhatnagar, Sanchita; Zhu, Xiaochun; Ou, Jianhong; Lin, Ling; Chamberlain, Lynn; Zhu, Lihua J.; Wajapeyee, Narendra; Green, Michael R.

    2014-01-01

    X-chromosome inactivation (XCI), the random transcriptional silencing of one X chromosome in somatic cells of female mammals, is a mechanism that ensures equal expression of X-linked genes in both sexes. XCI is initiated in cis by the noncoding Xist RNA, which coats the inactive X chromosome (Xi) from which it is produced. However, trans-acting factors that mediate XCI remain largely unknown. Here, we perform a large-scale RNA interference screen to identify trans-acting XCI factors (XCIFs) that comprise regulators of cell signaling and transcription, including the DNA methyltransferase, DNMT1. The expression pattern of the XCIFs explains the selective onset of XCI following differentiation. The XCIFs function, at least in part, by promoting expression and/or localization of Xist to the Xi. Surprisingly, we find that DNMT1, which is generally a transcriptional repressor, is an activator of Xist transcription. Small-molecule inhibitors of two of the XCIFs can reversibly reactivate the Xi, which has implications for treatment of Rett syndrome and other dominant X-linked diseases. A homozygous mouse knockout of one of the XCIFs, stanniocalcin 1 (STC1), has an expected XCI defect but surprisingly is phenotypically normal. Remarkably, X-linked genes are not overexpressed in female Stc1−/− mice, revealing the existence of a mechanism(s) that can compensate for a persistent XCI deficiency to regulate X-linked gene expression. PMID:25136103

  14. 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

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

    Code of Federal Regulations, 2014 CFR

    2014-07-01

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

  16. 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...

  17. 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...

  18. 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...

  19. 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...

  20. 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

  1. 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...

  2. 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...

  3. 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...

  4. 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...

  5. In Search of Lost Springs: A Protocol for Locating Active and Inactive Springs.

    PubMed

    Fensham, R J; Silcock, J L; Powell, O; Habermehl, M A

    2016-05-01

    Groundwater springs are significant landscape features for humans and the biota that occupies their habitat. Many springs become inactive where groundwater exploitation by humans has lowered the water table or artesian pressure. In order to assess this impact, it is important to identify and locate active, and with more difficulty, inactive springs. Using a variety of archival, environmental and field-based data, this study presents a protocol for the determination of the location and status of springs across the Great Artesian Basin of Australia. This protocol underpins a database of springs, which is not only important for the assessment of spring ecosystems, but also contributes to understand groundwater extraction impacts and hydrogeological processes. The database indicates that 30.0% of discharge (artesian) springs in the Great Artesian Basin are entirely inactive and another 11.8% are partially inactive. For the outcrop (gravity) springs of the Basin, only 1.9% are entirely inactive and 7.4% partially inactive, and for the outcrop springs in the Tertiary sandstone overlying the Basin 30.9% are inactive and 18.2% are partially inactive. PMID:26436535

  6. 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...

  7. 37 CFR 11.20 - Disciplinary sanctions; Transfer to disability inactive status.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

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

  8. 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...

  9. 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

  10. 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

  11. 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

  12. 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. PMID:25829338

  13. The subunit composition of human extracellular superoxide dismutase (EC-SOD) regulate enzymatic activity

    PubMed Central

    Petersen, Steen V; Valnickova, Zuzana; Oury, Tim D; Crapo, James D; Chr Nielsen, Niels; Enghild, Jan J

    2007-01-01

    Background Human extracellular superoxide dismutase (EC-SOD) is a tetrameric metalloenzyme responsible for the removal of superoxide anions from the extracellular space. We have previously shown that the EC-SOD subunit exists in two distinct folding variants based on differences in the disulfide bridge pattern (Petersen SV, Oury TD, Valnickova Z, Thøgersen IB, Højrup P, Crapo JD, Enghild JJ. Proc Natl Acad Sci USA. 2003;100(24):13875–80). One variant is enzymatically active (aEC-SOD) while the other is inactive (iEC-SOD). The EC-SOD subunits are associated into covalently linked dimers through an inter-subunit disulfide bridge creating the theoretical possibility of 3 dimers (aa, ai or ii) with different antioxidant potentials. We have analyzed the quaternary structure of the endogenous EC-SOD disulfide-linked dimer to investigate if these dimers in fact exist. Results The analyses of EC-SOD purified from human tissue show that all three dimer combinations exist including two homo-dimers (aa and ii) and a hetero-dimer (ai). Because EC-SOD is a tetramer the dimers may combine to generate 5 different mature EC-SOD molecules where the specific activity of each molecule is determined by the ratio of aEC-SOD and iEC-SOD subunits. Conclusion This finding shows that the aEC-SOD and iEC-SOD subunits combine in all 3 possible ways supporting the presence of tetrameric enzymes with variable enzymatic activity. This variation in enzymatic potency may regulate the antioxidant level in the extracellular space and represent a novel way of modulating enzymatic activity. PMID:17937792

  14. 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

  15. Simple enzymatic procedure for L-carnosine synthesis: whole-cell biocatalysis and efficient biocatalyst recycling.

    PubMed

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

    2010-01-01

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

  16. [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. PMID:27183789

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

    PubMed Central

    Motl, R. W.; Goldman, M.

    2011-01-01

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

  18. 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

  19. 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

  20. Comprehensive Management Strategies for Physical Inactivity in Youth

    PubMed Central

    MYER, GREGORY D.; FAIGENBAUM, AVERY D.; STRACCIOLINI, ANDREA; HEWETT, TIMOTHY E.; MICHELI, LYLE J.; BEST, THOMAS M.

    2013-01-01

    Despite the widely recognized benefits of daily play, recreation, sports, and physical education on the physical and psychosocial well-being of children and adolescents, many contemporary children and adolescents worldwide do not meet the recommendations for daily physical activity. The decline in physical activity seems to start early in life which leads to conditions characterized by reduced levels of physical activity in the pediatric population that are inconsistent with current public health recommendations. Unlike many other diseases and disorders in pediatrics, physical inactivity in youth is unique in that it currently lacks a clinical gold standard for diagnosis. This makes the diagnosis and treatment medically challenging, though no less important, as the resultant ramifications of a missed diagnosis are of significant detriment. Exercise deficient children need to be identified early in life and treated with developmentally appropriate exercise programs designed to target movement deficiencies and physical weaknesses in a supportive environment. Without such interventions early in life, children are more likely to become resistant to our interventions later in life and consequently suffer from adverse health consequences. Integrative approaches that link health care professionals, pediatric exercise specialists, school administrators, community leaders, and policy makers, may provide the best opportunity to promote daily physical activity, reinforce desirable behaviors, and educate parents about the exercise-health link. If health care providers miss the window of opportunity to identify exercise deficit disorder in youth and promote healthy lifestyle choices, the eventual decline and disinterest in physical activity will begin to take shape and new health care concerns will continue to emerge. PMID:23851413

  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. Biologically inactive leptin and early-onset extreme obesity.

    PubMed

    Wabitsch, Martin; Funcke, Jan-Bernd; Lennerz, Belinda; Kuhnle-Krahl, Ursula; Lahr, Georgia; Debatin, Klaus-Michael; Vatter, Petra; Gierschik, Peter; Moepps, Barbara; Fischer-Posovszky, Pamela

    2015-01-01

    Mutations in the gene encoding leptin (LEP) typically lead to an absence of circulating leptin and to extreme obesity. We describe a 2-year-old boy with early-onset extreme obesity due to a novel homozygous transversion (c.298G→T) in LEP, leading to a change from aspartic acid to tyrosine at amino acid position 100 (p.D100Y) and high immunoreactive levels of leptin. Overexpression studies confirmed that the mutant protein is secreted but neither binds to nor activates the leptin receptor. The mutant protein failed to reduce food intake and body weight in leptin-deficient ob/ob mice. Treatment of the patient with recombinant human leptin (metreleptin) rapidly normalized eating behavior and resulted in weight loss. PMID:25551525

  3. Structural and functional characterization of mutants of recombinant single-chain urokinase-type plasminogen activator obtained by site-specific mutagenesis of Lys158, Ile159 and Ile160.

    PubMed

    Lijnen, H R; Nelles, L; Van Hoef, B; Demarsin, E; Collen, D

    1988-11-15

    Single-chain urokinase-type plasminogen activator (scu-PA) is converted to urokinase by hydrolysis of the Lys158-Ile159 peptide bond. Site-directed mutagenesis of Lys158 to Gly or Glu yields plasmin-resistant mutants with a 10-20-fold reduced catalytic efficiency for the activation of plasminogen [Nelles et al. (1987) J. Biol. Chem. 262, 5682-5689]. In the present study, we have further evaluated the enzymatic properties of derivatives of recombinant scu-PA (rscu-PA), produced by site-directed mutagenesis of Lys158, Ile159 or Ile160, in order to obtain additional information on the structure/function relations underlying the enzymatic properties of the single- and two-chain u-PA moieties. [Arg158]rscu-PA (rscu-PA with Lys158 substituted with Arg) appeared to be indistinguishable from wild-type rscu-PA with respect to plasminogen-activating potential (catalytic efficiency k2/Km = 0.21 mM-1 s-1 versus 0.64 mM-1 s-1), conversion to active two-chain urokinase by plasmin (k2/Km = 0.13 microM-1 s-1 versus 0.28 microM-1 s-1), as well as its specific activity (48,000 IU/mg as compared to 60,000 IU/mg) and its fibrinolytic potential in a plasma medium (50% lysis in 2 h with 2.8 micrograms/ml versus 2.1 micrograms/ml). [Pro159]rscu-PA (Ile159 substituted with Pro) and [Gly159]rscu-PA (Ile159 converted to Gly) are virtually inactive towards plasminogen (k2/Km less than 0.004 mM-1 s-1). They are however converted to inactive two-chain derivatives by plasmin following cleavage of the Arg156-Phe157 peptide bond in [Pro159]rscu-PA and of the Lys158-Gly159 peptide bond in [Gly159]rscu-PA. [Gly158,Lys160]rscu-PA (with Lys158 converted to Gly and Ile160 to Lys) has a low catalytic efficiency towards plasminogen both as a single-chain form (k2/Km = 0.012 mM-1 s-1) and as the two-chain derivative (k2/Km = 0.13 mM-1 s-1) generated by cleavage of both the Arg156-Phe157 and/or the Lys160-Gly161 peptide bonds by plasmin. These findings suggest that the enzymatic properties of rscu-PA are

  4. A novel computational method to simulate non-enzymatic self-replication. [Abstract only

    NASA Technical Reports Server (NTRS)

    Navarro-Gonzalez, Rafael; Reggia, James A.; Wu, Jayoung; Chou, Hui-Hsien

    1994-01-01

    Non-enzymatic, template-directed synthesis of oligonucleotides has been extensively studied in the laboratory as a model to understand the kind of chemical processes that might have contributed to the origin of life on Earth. Several oligonucleotides have been shown to catalyze the synthesis of their complements from activated mononucleotides; however, a restricted number of them have been found to self-replicate. Recently we developed an efficient modified cellular automata method that supports the study of self-replicating oligonucleotides. With this method the oligonucleotide molecules are represented as active cells imbedded in a two-dimensional array of inactive cells symbolizing the environment. Random movements and probability-governed chemical reactions occurring in a cellular space can effectively simulate the experimental behavior observed in self-directed replication of oligonucleotides.

  5. Enzymatic reduction of levulinic acid by engineering the substrate specificity of 3-hydroxybutyrate dehydrogenase.

    PubMed

    Yeon, Young Joo; Park, Hyung-Yeon; Yoo, Young Je

    2013-04-01

    Enzymatic reduction of levulinic acid (LA) was performed for the synthesis of 4-hydroxyvaleric acid (4HV)--a monomer of bio-polyester and a precursor of bio-fuels--using 3-hydroxybutyrate dehydrogenase (3HBDH) from Alcaligenes faecalis. Due to the catalytic inactivity of the wild-type enzyme toward LA, engineering of the substrate specificity of the enzyme was performed. A rational design approach with molecular docking simulation was applied, and a double mutant, His144Leu/Trp187Phe, which has catalytic activity (kcat/Km=578.0 min(-1) M(-1)) toward LA was generated. Approximately 57% conversion of LA to 4HV was achieved with this double mutant in 24 h, while no conversion was achieved with the wild-type enzyme. PMID:23489571

  6. 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

  7. Recombinant renewable polyclonal antibodies

    PubMed Central

    Ferrara, Fortunato; D’Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew RM

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products. PMID:25530082

  8. 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…

  9. 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...

  10. Oligonucleotide recombination in bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Today, there are more than 1,500 completed or draft bacterial genome sequences available for public access. To functionally analyze these genomes and to test the hypotheses that are generated from the sequence information we require new and generically useful tools. Recombineering (genetic engineer...

  11. Enzymatic synthesis of 2'-ara and 2'-deoxy analogues of c-di-GMP.

    PubMed

    Shchokolova, Anastasia S; Rymko, Alexander N; Kvach, Sergey V; Shabunya, Polina S; Fatykhava, Svetlana A; Zinchenko, Anatoly I

    2015-01-01

    The substrate specificity of recombinant full-length diguanylate cyclase (DGC) of Thermotoga maritima with mutant allosteric site was investigated. It has been originally shown that the enzyme could use GTP closest analogues - 2'-deoxyguanosine-5'-triphosphate (dGTP) and 9-β-D-arabinofuranosyl-guanine-5'-triphosphate (araGTP) as the substrates. The first demonstrations of an enzymatic synthesis of bis-(3'-5')-cyclic dimeric deoxyguanosine monophosphate (c-di-dGMP) and the previously unknown bis-(3'-5')-cyclic dimeric araguanosine monophosphate (c-di-araGMP) using DGC of T. maritima in the form of inclusion bodies have been provided. PMID:25965330

  12. 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.

  13. 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

  14. 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

  15. Enzymatic Enantioselective Decarboxylative Protonation of Heteroaryl Malonates

    PubMed Central

    Lewin, Ross; Goodall, Mark; Thompson, Mark L; Leigh, James; Breuer, Michael; Baldenius, Kai; Micklefield, Jason

    2015-01-01

    The enzyme aryl/alkenyl malonate decarboxylase (AMDase) catalyses the enantioselective decarboxylative protonation (EDP) of a range of disubstituted malonic acids to give homochiral carboxylic acids that are valuable synthetic intermediates. AMDase exhibits a number of advantages over the non-enzymatic EDP methods developed to date including higher enantioselectivity and more environmentally benign reaction conditions. In this report, AMDase and engineered variants have been used to produce a range of enantioenriched heteroaromatic α-hydroxycarboxylic acids, including pharmaceutical precursors, from readily accessible α-hydroxymalonates. The enzymatic method described here represents an improvement upon existing synthetic chemistry methods that have been used to produce similar compounds. The relationship between the structural features of these new substrates and the kinetics associated with their enzymatic decarboxylation is explored, which offers further insight into the mechanism of AMDase. PMID:25766433

  16. Spinal TNF is necessary for inactivity-induced phrenic motor facilitation.

    PubMed

    Broytman, Oleg; Baertsch, Nathan A; Baker-Herman, Tracy L

    2013-11-15

    A prolonged reduction in central neural respiratory activity elicits a form of plasticity known as inactivity-induced phrenic motor facilitation (iPMF), a 'rebound' increase in phrenic burst amplitude apparent once respiratory neural activity is restored. iPMF requires atypical protein kinase C (aPKC) activity within spinal segments containing the phrenic motor nucleus to stabilize an early transient increase in phrenic burst amplitude and to form long-lasting iPMF following reduced respiratory neural activity. Upstream signal(s) leading to spinal aPKC activation are unknown. We tested the hypothesis that spinal tumour necrosis factor-α (TNFα) is necessary for iPMF via an aPKC-dependent mechanism. Anaesthetized, ventilated rats were exposed to a 30 min neural apnoea; upon resumption of respiratory neural activity, a prolonged increase in phrenic burst amplitude (42 ± 9% baseline; P < 0.05) was apparent, indicating long-lasting iPMF. Pretreatment with recombinant human soluble TNF receptor 1 (sTNFR1) in the intrathecal space at the level of the phrenic motor nucleus prior to neural apnoea blocked long-lasting iPMF (2 ± 8% baseline; P > 0.05). Intrathecal TNFα without neural apnoea was sufficient to elicit long-lasting phrenic motor facilitation (pMF; 62 ± 7% baseline; P < 0.05). Similar to iPMF, TNFα-induced pMF required spinal aPKC activity, as intrathecal delivery of a ζ-pseudosubstrate inhibitory peptide (PKCζ-PS) 35 min following intrathecal TNFα arrested TNFα-induced pMF (28 ± 8% baseline; P < 0.05). These data demonstrate that: (1) spinal TNFα is necessary for iPMF; and (2) spinal TNFα is sufficient to elicit pMF via a similar aPKC-dependent mechanism. These data are consistent with the hypothesis that reduced respiratory neural activity elicits iPMF via a TNFα-dependent increase in spinal aPKC activity. PMID:23878370

  17. Spinal TNFα is necessary for inactivity-induced phrenic motor facilitation

    PubMed Central

    Broytman, Oleg; Baertsch, Nathan A; Baker-Herman, Tracy L

    2013-01-01

    A prolonged reduction in central neural respiratory activity elicits a form of plasticity known as inactivity-induced phrenic motor facilitation (iPMF), a ‘rebound’ increase in phrenic burst amplitude apparent once respiratory neural activity is restored. iPMF requires atypical protein kinase C (aPKC) activity within spinal segments containing the phrenic motor nucleus to stabilize an early transient increase in phrenic burst amplitude and to form long-lasting iPMF following reduced respiratory neural activity. Upstream signal(s) leading to spinal aPKC activation are unknown. We tested the hypothesis that spinal tumour necrosis factor-α (TNFα) is necessary for iPMF via an aPKC-dependent mechanism. Anaesthetized, ventilated rats were exposed to a 30 min neural apnoea; upon resumption of respiratory neural activity, a prolonged increase in phrenic burst amplitude (42 ± 9% baseline; P < 0.05) was apparent, indicating long-lasting iPMF. Pretreatment with recombinant human soluble TNF receptor 1 (sTNFR1) in the intrathecal space at the level of the phrenic motor nucleus prior to neural apnoea blocked long-lasting iPMF (2 ± 8% baseline; P > 0.05). Intrathecal TNFα without neural apnoea was sufficient to elicit long-lasting phrenic motor facilitation (pMF; 62 ± 7% baseline; P < 0.05). Similar to iPMF, TNFα-induced pMF required spinal aPKC activity, as intrathecal delivery of a ζ-pseudosubstrate inhibitory peptide (PKCζ-PS) 35 min following intrathecal TNFα arrested TNFα-induced pMF (28 ± 8% baseline; P < 0.05). These data demonstrate that: (1) spinal TNFα is necessary for iPMF; and (2) spinal TNFα is sufficient to elicit pMF via a similar aPKC-dependent mechanism. These data are consistent with the hypothesis that reduced respiratory neural activity elicits iPMF via a TNFα-dependent increase in spinal aPKC activity. PMID:23878370

  18. 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.

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

    PubMed Central

    Aladjem, Mirit I.; Fu, Haiqing

    2014-01-01

    While large portions of the mammalian genome are known to replicate sequentially in a distinct, tissue-specific order, recent studies suggest that the inactive X chromosome is duplicated rapidly via random, synchronous DNA synthesis at numerous adjacent regions. The rapid duplication of the inactive X chromosome was observed in high-resolution studies visualizing DNA replication patterns in the nucleus, and by allele-specific DNA sequencing studies measuring the extent of DNA synthesis. These studies conclude that inactive X chromosomes complete replication earlier than previously thought and suggest that the strict order of DNA replication detected in the majority of genomic regions is not preserved in non-transcribed, “silent” chromatin. These observations alter current concepts about the regulation of DNA replication in non-transcribed portions of the genome in general and in the inactive X-chromosome in particular. PMID:24706495

  20. 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

  1. 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.

  2. 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.

  3. 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...

  4. 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...

  5. Enzymatic desulfurization of coal: Third quarterly report

    SciTech Connect

    Marquis, J.K.; Kitchell, J.P.

    1989-03-14

    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. In this quarter we obtained important results both with the development of our understanding of the enzyme reaction systems and also with the microbial work at Woods Hole. 12 figs., 11 tabs.

  6. 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

  7. Population attributable fraction of type 2 diabetes due to physical inactivity in adults: a systematic review

    PubMed Central

    2014-01-01

    Background Physical inactivity is a global pandemic. The population attributable fraction (PAF) of type 2 diabetes mellitus (T2DM) associated with physical inactivity ranges from 3% to 40%. The purpose of this systematic review was to determine the best estimate of PAF for T2DM attributable to physical inactivity and absence of sport participation or exercise for men and women. Methods We conducted a systematic review that included a comprehensive search of MEDLINE, EMBASE, SportDiscus, and CINAHL (1946 to April 30 2013) limited by the terms adults and English. Two reviewers screened studies, extracted PAF related data and assessed the quality of the selected studies. We reconstructed 95% CIs for studies missing these data using a substitution method. Results Of the eight studies reporting PAF in T2DM, two studies included prospective cohort studies (3 total) and six were reviews. There were distinct variations in quality of defining and measuring physical inactivity, T2DM and adjusting for confounders. In the US, PAFs for absence of playing sport ranged from 13% (95% CI: 3, 22) in men and 29% (95% CI: 17, 41) in women. In Finland, PAFs for absence of exercise ranged from 3% (95% CI: -11, 16) in men to 7% (95% CI: -9, 20) in women. Conclusions The PAF of physical inactivity due to T2DM is substantial. Physical inactivity is a modifiable risk factor for T2DM. The contribution of physical inactivity to T2DM differs by sex; PAF also differs if physical inactivity is defined as the absence of ‘sport’ or absence of ‘exercise’. PMID:24885278

  8. Recombinant vaccines against leptospirosis.

    PubMed

    Dellagostin, Odir A; Grassmann, André A; Hartwig, Daiane D; Félix, Samuel R; da Silva, Éverton F; McBride, Alan J A

    2011-11-01

    Leptospirosis is an important neglected infectious disease that occurs in urban environments, as well as in rural regions worldwide. Rodents, the principal reservoir hosts of pathogenic Leptospira spp., and other infected animals shed the bacteria in their urine. During occupational or even recreational activities, humans that come into direct contact with infected animals or with a contaminated environment, particularly water, are at risk of infection. Prevention of urban leptospirosis is largely dependent on sanitation measures that are often difficult to implement, especially in developing countries. Vaccination with inactivated whole-cell preparations (bacterins) has limited efficacy due to the wide antigenic variation of the pathogen. Intensive efforts towards developing improved recombinant vaccines are ongoing. During the last decade, many reports on the evaluation of recombinant vaccines have been published. Partial success has been obtained with some surface-exposed protein antigens. The combination of protective antigens and new adjuvants or delivery systems may result in the much-needed effective vaccine. PMID:22048111

  9. Recombinant influenza vaccines.

    PubMed

    Sedova, E S; Shcherbinin, D N; Migunov, A I; Smirnov, Iu A; Logunov, D Iu; Shmarov, M M; Tsybalova, L M; Naroditskiĭ, B S; Kiselev, O I; Gintsburg, A L

    2012-10-01

    This review covers the problems encountered in the construction and production of new recombinant influenza vaccines. New approaches to the development of influenza vaccines are investigated; they include reverse genetics methods, production of virus-like particles, and DNA- and viral vector-based vaccines. Such approaches as the delivery of foreign genes by DNA- and viral vector-based vaccines can preserve the native structure of antigens. Adenoviral vectors are a promising gene-delivery platform for a variety of genetic vaccines. Adenoviruses can efficiently penetrate the human organism through mucosal epithelium, thus providing long-term antigen persistence and induction of the innate immune response. This review provides an overview of the practicability of the production of new recombinant influenza cross-protective vaccines on the basis of adenoviral vectors expressing hemagglutinin genes of different influenza strains. PMID:23346377

  10. 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.

  11. Strand invasion promoted by recombination protein of coliphage

    NASA Astrophysics Data System (ADS)

    Rybalchenko, Nataliya; Golub, Efim I.; Bi, Baoyuan; Radding, Charles M.

    2004-12-01

    Studies of phage in vivo have indicated that its own recombination enzymes, protein and exonuclease, are capable of catalyzing two dissimilar pathways of homologous recombination that are widely distributed in nature: single-strand annealing and strand invasion. The former is an enzymatic splicing of overlapping ends of broken homologous DNA molecules, whereas the latter is characterized by the formation of a three-stranded synaptic intermediate and subsequent strand exchange. Previous studies in vitro have shown that protein has annealing activity, and that exonuclease, acting on branched substrates, can produce a perfect splice that requires only ligation for completion. The present study shows that protein can initiate strand invasion in vitro, as evidenced both by the formation of displacement loops (D-loops) in superhelical DNA and by strand exchange between colinear single-stranded and double-stranded molecules. Thus, protein can catalyze steps that are central to both strand annealing and strand invasion pathways of recombination. These observations add protein to a set of diverse proteins that appear to promote recognition of homology by a unitary mechanism governed by the intrinsic dynamic properties of base pairs in DNA. genetic recombination | phage λ

  12. Motives for physical activity among active and inactive persons in their mid-30s.

    PubMed

    Aaltonen, S; Rottensteiner, M; Kaprio, J; Kujala, U M

    2014-08-01

    The purpose of this study was to examine the motives for leisure-time physical activity among active and inactive men and women in their mid-30s. We used both cross-sectional and longitudinal designs. Altogether, 2308 participants (mean age 33.9 years, 53.4% women) were identified from the population-based FinnTwin16 Cohort. Physically active and inactive individuals were identified on the basis of their leisure-time MET h/day. We evaluated participants' physical activity motivation with a modified version of the Recreational Exercise Motivation Measure. Comparisons between active and inactive individuals were analysed using the Wald test for equality of means, and effect sizes were calculated as Cohen's d. Motives related to mastery, physical fitness, social aspect of physical activity, psychological state, enjoyment, willingness to be fitter/look better than others, and appearance were significantly more important for the active than inactive participants. Conforming to others' expectations was the only item on which the inactive persons scored higher than active persons. The longitudinal results for physical activity were parallel to the cross-sectional results. This study supports the view that motivation factors differ between active and inactive persons, and that intrinsic motives are associated with consistent leisure-time physical activity. PMID:23331765

  13. The inactive X chromosome is epigenetically unstable and transcriptionally labile in breast cancer.

    PubMed

    Chaligné, Ronan; Popova, Tatiana; Mendoza-Parra, Marco-Antonio; Saleem, Mohamed-Ashick M; Gentien, David; Ban, Kristen; Piolot, Tristan; Leroy, Olivier; Mariani, Odette; Gronemeyer, Hinrich; Vincent-Salomon, Anne; Stern, Marc-Henri; Heard, Edith

    2015-04-01

    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

  14. 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.

  15. 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

  16. [Recombinant expression and antibacterial activity of i-type lysozyme from sea cucumber Stichopus japonicus].

    PubMed

    Wang, Xiuxia; Cong, Lina; Wang, Dan; Yang, Xijian; Zhu, Beiwei

    2009-02-01

    The cDNA of an i type lysozyme was cloned from Stichopus japonicus (named as SjLys). The DNA fragment of the mature SjLys was subcloned into expression vector of pET-32a (+) to construct the recombinant plasmid of pET32a (+)-SjLys. The recombinant plasmid was then transformed into Escherichia coli BL21 (DE3) pLysS and induced by isopropylthio-beta-D-galactoside (IPTG). The recombinant protein expressed as inclusion bodies was denatured, partially purified and refolded to be an active form. The bacteriolytic activity of recombinant protein purified by the metal-chelating was 19.2 U/mg. The antibacterial activity of the purified recombinant SjLys (rSjLys) was analyzed. The rSjLys protein displayed inhibitive effect on the growth of the tested Gram-positive and Gram-negative bacteria. In particular, rSjLys had a strong inhibitive activity on Vibrio parahaemolyticus and Pseudomonas aeruginosa, both the most common pathogenic bacteria in the marine animals. The heat-treated rSjLys exhibited more potent activities against all tested bacteria. These results indicated that the S. japonicus lysozyme was the enzyme with combined enzymatic (glycosidase) and non-enzymatic antibacterial action, and it had a wide antibacterial spectrum. Therefore, it is suggested that the S. japonicus lysozyme should be one of the important molecules against pathogens in the innate immunity of sea cucumbers. PMID:19459322

  17. 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

  18. 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

  19. Preparation of chitooligosaccharides from fungal waste mycelium by recombinant chitinase.

    PubMed

    Lv, Mengyuan; Hu, Ying; Gänzle, Michael G; Lin, Jianguo; Wang, Changgao; Cai, Jun

    2016-07-22

    This study aimed to develop an enzymatic method for conversion of chitin from fungal waste mycelia to chitooligosaccharides. The recombinant chitinase LlChi18A from Lactococcus lactis was over-expressed by Escherichia coli BL21 (DE3) and purified by affinity chromatography. The enzymatic properties of the purified enzyme were studied by chitin oligosaccharides. Waste mycelium was pre-treated by alkaline. The optimal conditions for hydrolysis of fungal chitin by recombinant chitinase were determined by Schales method. HPLC/ESI-MS was used to determine the content of N-acetylglucosamine and chitooligosaccharides after hydrolysis. The level of reducing sugar released from pretreated mycelium by chitinase increased with the reaction time during 6 days. The main product in the hydrolysates was N,N'-diacetylchitobiose. After hydrolysis by chitinase for 5 d, the yield of N,N'-diacetylchitobiose from waste mycelium was around 10% with estimated purity of around 70%. Combination of chitinase and snailase remarkably increased the yield to 24% with purity of 78%. Fungal mycelium which contains chitin is a new potential source for obtaining food grade chitooligosaccharides. PMID:27153004

  20. 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...

  1. 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

  2. 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…

  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. Comparison of enzymatic and non-enzymatic nitroethane anion formation: thermodynamics and contribution of tunneling.

    PubMed

    Valley, Michael P; Fitzpatrick, Paul F

    2004-05-26

    In the reaction of nitroalkane oxidase (NAO), the oxidation of nitroalkanes to the corresponding aldehydes or ketones is initiated by the deprotonation of the neutral nitroalkane. The energetics of nitroethane ionization for both the enzymatic and non-enzymatic reactions have been determined by measuring rate constants as a function of temperature. At 25 degrees C, the rate constant for the acetate-catalyzed reaction is a billionfold smaller than the kcat/Km value for NAO. This corresponds to a difference of 12.3 kcal/mol in the free energy of activation that is largely due to a difference in the activation enthalpy. Analysis of the temperature dependence of the deuterium kinetic isotope effects on the reactions yields similar DeltaEa and AH/AD values for the acetate, phosphate, and NAO-catalyzed reactions that fall within the semiclassical limits, consistent with similar contributions of tunneling to the enzymatic and non-enzymatic reactions. PMID:15149217

  6. Bacterial Recombineering: Genome Engineering via Phage-Based Homologous Recombination.

    PubMed

    Pines, Gur; Freed, Emily F; Winkler, James D; Gill, Ryan T

    2015-11-20

    The ability to specifically modify bacterial genomes in a precise and efficient manner is highly desired in various fields, ranging from molecular genetics to metabolic engineering and synthetic biology. Much has changed from the initial realization that phage-derived genes may be employed for such tasks to today, where recombineering enables complex genetic edits within a genome or a population. Here, we review the major developments leading to recombineering becoming the method of choice for in situ bacterial genome editing while highlighting the various applications of recombineering in pushing the boundaries of synthetic biology. We also present the current understanding of the mechanism of recombineering. Finally, we discuss in detail issues surrounding recombineering efficiency and future directions for recombineering-based genome editing. PMID:25856528

  7. Preparation and comparative characterization of immobilized Aspergillus oryzae expressing Fusarium heterosporum lipase for enzymatic biodiesel production.

    PubMed

    Hama, Shinji; Tamalampudi, Sriappareddy; Suzuki, Yuya; Yoshida, Ayumi; Fukuda, Hideki; Kondo, Akihiko

    2008-12-01

    In this paper, we provide the first report of utilizing recombinant fungal whole cells in enzymatic biodiesel production. Aspergillus oryzae, transformed with a heterologous lipase-encoding gene from Fusarium heterosporum, produced fully processed and active forms of recombinant F. heterosporum lipase (FHL). Cell immobilization within porous biomass support particles enabled the convenient usage of FHL-producing A. oryzae as a whole-cell biocatalyst for lipase-catalyzed methanolysis. The addition of 5% water to the reaction mixture was effective in both preventing the lipase inactivation by methanol and facilitating the acyl migration in partial glycerides, resulting in the final methyl ester content of 94% even in the tenth batch cycle. A comparative study showed that FHL-producing A. oryzae attained a higher final methyl ester content and higher lipase stability than Rhizopus oryzae, the previously developed whole-cell biocatalyst. Although both FHL and R. oryzae lipase exhibit 1,3-regiospecificity towards triglyceride, R. oryzae accumulated a much higher amount of sn-2 isomers of partial glycerides, whereas FHL-producing A. oryzae maintained a low level of the sn-2 isomers. This is probably because FHL efficiently facilitates the acyl migration from the sn-2 to the sn-1(3) position in partial glycerides. These findings indicate that the newly developed FHL-producing A. oryzae is an effective whole-cell biocatalyst for enzymatic biodiesel production. PMID:18795281

  8. 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.

  9. 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.

  10. 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..

  11. Did the universe recombine?

    NASA Technical Reports Server (NTRS)

    Bartlett, James G.; Stebbins, Albert

    1991-01-01

    The Zel'dovich-Sunyaev model-independent arguments for the existence of a neutral hydrogen phase is reviewed in light of new limits on the Compton y parameter from COBE. It is concluded that with baryon densities compatible with standard cosmological nucleosynthesis, the universe could have remained fully ionized throughout its history without producing a detectable spectral distortion. It is argued that it is unlikely that spectral observations of the cosmic microwave background will ever require the universe to have recombined for flat cosmologies.

  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. 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

  14. 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

  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

    2016-01-01

    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

  16. 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

  17. Economic burden of physical inactivity: healthcare costs associated with cardiovascular disease.

    PubMed

    Oldridge, Neil B

    2008-04-01

    Increasingly important objectives for developed and especially for developing countries include increasing the numbers of individuals who do not smoke, who eat healthy diets and who are physically active at levels that are health enhancing. In developing countries, deaths from chronic disease are projected to increase from 56% of all deaths in 2005 to 65% by 2030 (driven largely driven by deaths due to cardiovascular and coronary heart disease); in developed countries, however, the increase is only from 87.5 to 88.5%. The data on physical inactivity presented in this review were derived primarily from World Health Organization (WHO) publications and data warehouses. The prevalence of physical inactivity at less than the levels recommended for enhancing health is high; from 17 to 91% in developing countries and from 4 to 84% in developed countries. In developed countries, physical inactivity is associated with considerable economic burden, with 1.5-3.0% of total direct healthcare costs being accounted for by physical inactivity. Other than on some exciting work in Brazil, there is little information on the effectiveness and cost-effectiveness of physical activity-enhancement strategies in developing countries. The WHO has signaled a shift from the treatment of illness to promotion of health, with an emphasis on changing modifiable health-risk factors, including smoking, unhealthy diets and physical inactivity: the real question, especially for developing countries, is 'what is the future healthcare cost of not encouraging healthier lifestyles today?' PMID:18391637

  18. 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

  19. Recombinant factor VIIa.

    PubMed

    Aitken, Michael G

    2004-01-01

    Human coagulation factor (F) VII is a single chain protease that circulates in the blood as a weakly active zymogen at concentrations of approximately 10 nmol/L. When converted to the active 2 chain form (FVIIa), it is a powerful initiator of haemostasis. Recombinant factor VIIa (rFVIIa, eptacog alfa, NovoSeven) is a genetically engineered product that was first introduced in 1988 for the treatment of patients with haemophilia A and B with high inhibitory antibody titres to factors VIII and IX. Recent reports in the form of case studies and series, and early trial data, have suggested a role for rFVIIa across a diverse range of indications including bleeding associated with trauma, surgery, thrombocytopaenia, liver disease and oral anticoagulant toxicity. This review describes the physiology of the coagulation pathway and in particular the role of recombinant factor VIIa. It will also focus on the emerging role of rFVIIa in both trauma and non-trauma bleeding and its potential use in the ED. PMID:15537408

  20. 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

  1. 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

  2. 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.

  3. 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.

  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. PMID:27497170

  5. Enzymatic degradation of polycaprolactone-gelatin blend

    NASA Astrophysics Data System (ADS)

    Banerjee, Aditi; Chatterjee, Kaushik; Madras, Giridhar

    2015-04-01

    Blends of polycaprolactone (PCL), a synthetic polymer and gelatin, natural polymer offer a optimal combination of strength, water wettability and cytocompatibility for use as a resorbable biomaterial. The enzymatic degradation of PCL, gelatin and PCL-gelatin blended films was studied in the presence of lipase (Novozym 435, immobilized) and lysozyme. Novozym 435 degraded the PCL films whereas lysozyme degraded the gelatin. Though Novozym 435 and lysozyme individually could degrade PCL-gelatin blended films, the combination of these enzymes showed the highest degradation of these blended films. Moreover, the enzymatic degradation was much faster when fresh enzymes were added at regular intervals. The changes in physico-chemical properties of polymer films due to degradation were studied by scanning electron microscopy, Fourier transform infrared spectroscopy and differential scanning calorimetry. These results have important implications for designing resorbable biomedical implants.

  6. Identification of a region in the S1 subunit of pertussis toxin that is required for enzymatic activity and that contributes to the formation of a neutralizing antigenic determinant.

    PubMed Central

    Cieplak, W; Burnette, W N; Mar, V L; Kaljot, K T; Morris, C F; Chen, K K; Sato, H; Keith, J M

    1988-01-01

    The S1 subunit of pertussis toxin possesses two regions (homology boxes), each spanning 8 residues, that are nearly identical in sequence to similarly located regions in the enzymatically active A fragments of two other ADP-ribosylating toxins: cholera toxin and Escherichia coli heat-labile toxin. This observation suggests a functional role for one or both of these regions in enzymatic activity. We have examined the role of one of these regions, located near the amino terminus of the S1 subunit, by using a high-level recombinant expression system and progressive truncation of the gene sequence encoding the amino terminus of the molecule. A series of six truncated, recombinant proteins were produced at high levels in E. coli and examined for their enzymatic and antigenic properties. The three molecules that lacked most or all of the homology box delimited by amino acid residues 8 and 15 lacked detectable enzymatic activity. All of the three molecules in which the box was retained exhibited detectable activity. Only those recombinant molecules that possessed the homology box reacted with a neutralizing and passively protective monoclonal anti-S1 antibody. These findings identify the region of homology located near the amino terminus of S1 as an apparent enzymatic subsite and a potentially important antigenic determinant. Images PMID:2455296

  7. Uveitic angle closure glaucoma in a patient with inactive cytomegalovirus retinitis and immune recovery uveitis.

    PubMed

    Goldberg, Daniel E; Freeman, William R

    2002-01-01

    We report a case of uveitic acute angle closure glaucoma in a patient with acquired immunodeficiency syndrome (AIDS) associated with inactive cytomegalovirus retinitis and immune recovery vitritis. We conducted a long-term, follow-up examination of a 47-year-old male with AIDS and inactive cytomegalovirus retinitis caused by immune recovery on highly active antiretroviral therapy (HAART). We found vitritis and ultimate development of uveitic glaucoma in the postoperative periods following repair of retinal detachment and extracapsular cataract extraction with intraocular lens implant. An episode of acute angle closure secondary to posterior synechiae and iris bombé subsequently developed, requiring peripheral laser iridotomy. Immune recovery in the setting of inactive cytomegalovirus retinitis can result in intraocular inflammation severe enough to cause angle closure glaucoma and profound ocular morbidity. PMID:12358297

  8. Effect of acute exercise on some haematological parameters and neutrophil functions in active and inactive subjects.

    PubMed

    Benoni, G; Bellavite, P; Adami, A; Chirumbolo, S; Lippi, G; Brocco, G; Cuzzolin, L

    1995-01-01

    In this work we studied the possible effects of acute exercise on some haematological parameters and on some functions of neutrophils in seven active and six inactive subjects. Physical exercise (10 min on a cycle ergometer at a heart rate of 150 beats.min-1) induced a significant increase in total leucocyte, lymphocyte and neutrophil concentrations in active subjects; serum iron and ferritin concentrations were lower in active compared to inactive subjects. Cellular adhesion, bactericidal activity and superoxide anion production did not change after exercise, while we also observed some differences between active and inactive subjects before exercise. In particular, the neutrophils from active subjects showed a significantly higher percentage of adhesion, higher bactericidal activity and lower superoxide anion production. In conclusion, the training induced changes in some neutrophil functions, while acute exercise influenced, overall, leucocyte concentrations. PMID:7768243

  9. 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.

  10. 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

  11. 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

  12. Algebraic theory of recombination spaces.

    PubMed

    Stadler, P F; Wagner, G P

    1997-01-01

    A new mathematical representation is proposed for the configuration space structure induced by recombination, which we call "P-structure." It consists of a mapping of pairs of objects to the power set of all objects in the search space. The mapping assigns to each pair of parental "genotypes" the set of all recombinant genotypes obtainable from the parental ones. It is shown that this construction allows a Fourier decomposition of fitness landscapes into a superposition of "elementary landscapes." This decomposition is analogous to the Fourier decomposition of fitness landscapes on mutation spaces. The elementary landscapes are obtained as eigenfunctions of a Laplacian operator defined for P-structures. For binary string recombination, the elementary landscapes are exactly the p-spin functions (Walsh functions), that is, the same as the elementary landscapes of the string point mutation spaces (i.e., the hypercube). This supports the notion of a strong homomorphism between string mutation and recombination spaces. However, the effective nearest neighbor correlations on these elementary landscapes differ between mutation and recombination and among different recombination operators. On average, the nearest neighbor correlation is higher for one-point recombination than for uniform recombination. For one-point recombination, the correlations are higher for elementary landscapes with fewer interacting sites as well as for sites that have closer linkage, confirming the qualitative predictions of the Schema Theorem. We conclude that the algebraic approach to fitness landscape analysis can be extended to recombination spaces and provides an effective way to analyze the relative hardness of a landscape for a given recombination operator. PMID:10021760

  13. 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

  14. 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

  15. Enzymatic induction of supramolecular order and bioactivity.

    PubMed

    Yang, Chengbiao; Ren, Xinrui; Ding, Dan; Wang, Ling; Yang, Zhimou

    2016-05-19

    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. PMID:27161242

  16. 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. PMID:24475066

  17. 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

  18. 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

  19. Production, purification, and functional analysis of recombinant human and mouse 17beta-hydroxysteroid dehydrogenase type 7.

    PubMed

    Törn, Svea; Nokelainen, Pasi; Kurkela, Riitta; Pulkka, Anitta; Menjivar, Marta; Ghosh, Sikha; Coca-Prados, Miguel; Peltoketo, Hellevi; Isomaa, Veli; Vihko, Pirkko

    2003-05-23

    17beta-Hydroxysteroid dehydrogenases (17HSDs) have a central role in the regulation of the biological activity of sex steroid hormones. There is increasing evidence that in addition to their importance in gonads, these hormones also have substantial metabolic roles in a variety of peripheral tissues. In the present study, the cDNA of human 17HSD type 7 was cloned. In silico, the gene corresponding to the cDNA was localized on chromosome 1q23, close to the locus of hereditary prostate cancer 1 (HPC1) (1q24-25) and primary open-angle glaucoma (GLC1A) (1q23-25). Further, a pseudogene was found on chromosome 1q44, close to the locus of predisposing for early-onset prostate cancer (PCaP) (1q42.2-43). Both human (h17HSD7) and mouse 17HSD type 7 (m17HSD7) were for the first time produced as recombinant proteins and purified for functional analyses. Further, kinetic parameters and specific activities were described. h17HSD7 converted estrone (E1) to a more potent estrogen, estradiol (E2), and dihydrotestosterone (DHT), a potent androgen, to an estrogenic metabolite 5alpha-androstane-3beta, 17beta-diol (3betaA-diol) equally, thereby catalyzing the reduction of the keto group in either 17- or 3-position of the substrate. Minor 3betaHSD-like activity towards progesterone (P) and 20-hydroxyprogesterone (20-OH-P), leading to the inactivation of P by h17HSD7, was also detected. m17HSD7 efficiently catalyzed the reaction from E1 to E2 and moderately converted DHT to an inactive metabolite 5alpha-androstane-3alpha,17beta-diol (3alphaA-diol) and to an even lesser degree 3betaA-diol. The mouse enzyme did not metabolize P or 20-OH-P. The expression of 17HSD type 7 was observed widely in human tissues, most distinctly in adrenal gland, liver, lung, and thymus. Based on the enzymatic characteristics and tissue distribution, we conclude that h17HSD7 might be an intracrine regulator of steroid metabolism, fortifying the estrogenic milieu in peripheral tissues. PMID:12732193

  20. Enzymatic glycoprotein synthesis: Preparation of ribonuclease glycoforms via enzymatic glycopeptide condensation and glycosylation

    SciTech Connect

    Witte, K.; Sears, P.; Martin, R.; Wong, C.H.

    1997-03-05

    In order to study the effects carbohydrates have on glycoprotein structure and funciton, it is imperative to be able to synthesize the appropriate natural and non-natural glycoprotein variants in a single form. Because the available in vivo techniques provide only heterogeneous mixtures of different glycoforms, enzymatic in vitro methodologies have been pursued. Using the N-glycoprotein RNase B as a model system, the oligosaccharide was removed leaving only the N-acetylglucosamine as a `tag` to the site of glycosylation. Glycosyltransferases were then used to build a unique carbohydrate moiety. A new RNase glycoform containing the branched oligosccharide, sialyl Lewis X or the Hg derivative, was synthesized enzymatically to demonstrate the feasibility of the method. In addition, the monoglycosylated protein was digested into several smaller pieces by subtilisin BPN`. These fragments were religated by subtilisin 8397 to the full length RNase by addition glycerol; this method points to a new chemical-enzymatic process for the synthesis of glycoproteins using synthetic peptides and glycopeptides as substrates for enzymatic ligation followed by further enzymatic glycosylations. 29 refs., 6 figs.

  1. 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

  2. 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.

  3. 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.

  4. Microbial antigenic variation mediated by homologous DNA recombination.

    PubMed

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

    2012-09-01

    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 the modification of the amino acid sequence of a particular antigen or to alterations in the expression of biosynthesis genes that induce changes in the expression of a variant antigen. Here, we will review antigenic variation systems that rely on homologous DNA recombination and that are found in a wide range of cellular, human pathogens, including bacteria (such as Neisseria spp., Borrelia spp., Treponema pallidum, and Mycoplasma spp.), fungi (such as 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

  5. Coalescent Simulation of Intracodon Recombination

    PubMed Central

    Arenas, Miguel; Posada, David

    2010-01-01

    The coalescent with recombination is a very useful tool in molecular population genetics. Under this framework, genealogies often represent the evolution of the substitution unit, and because of this, the few coalescent algorithms implemented for the simulation of coding sequences force recombination to occur only between codons. However, it is clear that recombination is expected to occur most often within codons. Here we have developed an algorithm that can evolve coding sequences under an ancestral recombination graph that represents the genealogies at each nucleotide site, thereby allowing for intracodon recombination. The algorithm is a modification of Hudson's coalescent in which, in addition to keeping track of events occurring in the ancestral material that reaches the sample, we need to keep track of events occurring in ancestral material that does not reach the sample but that is produced by intracodon recombination. We are able to show that at typical substitution rates the number of nonsynonymous changes induced by intracodon recombination is small and that intracodon recombination does not generally result in inflated estimates of the overall nonsynonymous/synonymous substitution ratio (ω). On the other hand, recombination can bias the estimation of ω at particular codons, resulting in apparent rate variation among sites and in the spurious identification of positively selected sites. Importantly, in this case, allowing for variable synonymous rates across sites greatly reduces the false-positive rate and recovers statistical power. Finally, coalescent simulations with intracodon recombination could be used to better represent the evolution of nuclear coding genes or fast-evolving pathogens such as HIV-1.We have implemented this algorithm in a computer program called NetRecodon, freely available at http://darwin.uvigo.es. PMID:19933876

  6. 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

  7. 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

  8. 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…

  9. 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…

  10. 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...

  11. 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...

  12. 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,...

  13. 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,...

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

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 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,...

  15. 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...

  16. 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...

  17. 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...

  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. When Physical Activity Participation Promotes Inactivity: Negative Experiences of Spanish Adolescents in Physical Education and Sport

    ERIC Educational Resources Information Center

    Beltran-Carrillo, Vicente J.; Devis-Devis, Jose; Peiro-Velert, Carmen; Brown, David H. K.

    2012-01-01

    This article analyses negative experiences in physical education and sport reported during qualitative interviews of a group of inactive adolescent Spanish boys and girls. The purpose of this analysis is twofold. First and most important, it seeks to give voice to these young people reporting negative experiences and connect them to contexts of…

  20. 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...

  1. 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…

  2. 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…

  3. 17 CFR 210.3-11 - Financial statements of an inactive registrant.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 17 Commodity and Securities Exchanges 2 2010-04-01 2010-04-01 false Financial statements of an... COMMISSION FORM AND CONTENT OF AND REQUIREMENTS FOR FINANCIAL STATEMENTS, SECURITIES ACT OF 1933, SECURITIES... Financial Statements § 210.3-11 Financial statements of an inactive registrant. If a registrant is...

  4. Past-Year Sexual Inactivity among Older Married Persons and Their Partners

    ERIC Educational Resources Information Center

    Karraker, Amelia; DeLamater, John

    2013-01-01

    Family scholars have focused on the onset of sexual activity early in the life course, but little is known about the cessation of sexual activity in relationships in later life. We use event-history analysis techniques and logistic regression to identify the correlates of sexual inactivity among older married men and women. We analyze data for…

  5. 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...

  6. Evaluating Equity: A Framework for Understanding Action and Inaction on Social Justice Issues

    ERIC Educational Resources Information Center

    Opfer, V. Darleen

    2006-01-01

    This article relies on a case study of a policy evaluation to illustrate how issues of social justice arise for action or inaction in a political environment. The article uses the case study to show that social justice issue formation is shaped by the personal beliefs of the actors, the prevailing political culture, the evolutionary path of the…

  7. 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...

  8. 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...

  9. 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...

  10. 9 CFR 105.4 - Termination of licenses and permits for inactivity.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Termination of licenses and permits for inactivity. 105.4 Section 105.4 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND...

  11. 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.

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

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2012-07-01 2012-07-01 false Ratings for inactive nonpulmonary tuberculosis in effect on August 19, 1968. 4.89 Section 4.89 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS SCHEDULE FOR RATING DISABILITIES Disability Ratings Infectious Diseases, Immune Disorders and...

  13. 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.

  14. Enzymatic and nonenzymatic functions of viral RNA-dependent RNA polymerases within oligomeric arrays

    PubMed Central

    Spagnolo, Jeannie F.; Rossignol, Evan; Bullitt, Esther; Kirkegaard, Karla

    2010-01-01

    Few antivirals are effective against positive-strand RNA viruses, primarily because the high error rate during replication of these viruses leads to the rapid development of drug resistance. One of the favored current targets for the development of antiviral compounds is the active site of viral RNA-dependent RNA polymerases. However, like many subcellular processes, replication of the genomes of all positive-strand RNA viruses occurs in highly oligomeric complexes on the cytosolic surfaces of the intracellular membranes of infected host cells. In this study, catalytically inactive polymerases were shown to participate productively in functional oligomer formation and catalysis, as assayed by RNA template elongation. Direct protein transduction to introduce either active or inactive polymerases into cells infected with mutant virus confirmed the structural role for polymerase molecules during infection. Therefore, we suggest that targeting the active sites of polymerase molecules is not likely to be the best antiviral strategy, as inactivated polymerases do not inhibit replication of other viruses in the same cell and can, in fact, be useful in RNA replication complexes. On the other hand, polymerases that could not participate in functional RNA replication complexes were those that contained mutations in the amino terminus, leading to altered contacts in the folded polymerase and mutations in a known polymerase–polymerase interaction in the two-dimensional protein lattice. Thus, the functional nature of multimeric arrays of RNA-dependent RNA polymerase supplies a novel target for antiviral compounds and provides a new appreciation for enzymatic catalysis on membranous surfaces within cells. PMID:20051491

  15. 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.

  16. 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).

  17. The Impact of Smoking on Airflow Limitation in Subjects with History of Asthma and Inactive Tuberculosis

    PubMed Central

    Kim, Hyun Jung; Baek, Seunghee; Kim, Hee Jin; Lee, Jae Seung; Oh, Yeon-Mok; Lee, Sang-Do; Lee, Sei Won

    2015-01-01

    Background Although smoking is the most important and modifiable cause of chronic obstructive pulmonary disease (COPD), other risk factors including asthma and tuberculosis (TB) are also associated. It is common for COPD patients to have more than one of these risk factors. The aims of this study were to determine the prevalence of airflow limitation (FEV1/FVC<0.7) according to the risk factors and to investigate their impact and interaction in airflow limitation. Methods From the Korean National Health and Nutrition Examination Survey between 2008 and 2012, we analyzed participants over 40 years of age by spirometry, chest radiograph and questionnaire about asthma and smoking history. Results Of 12,631 participants, 1,548 (12.3%) had airflow limitation. The prevalence of airflow limitation in smokers (≥10 pack-year), asthmatics, and those with inactive TB was 23.9%, 32.1%, and 33.6%. The prevalence increased with the number of risk factors: 86.1% had airflow limitation if they had all three risk factors. Impacts of inactive TB and asthma on airflow limitation were equivalent to 47 and 69 pack-years of smoking, respectively. Airflow limitation resulted from lower levels of smoking in those with inactive TB and asthma. A potential interaction between smoking and inactive tuberculosis in the development of airflow limitation was identified (p = 0.054). Conclusions Asthma and inactive TB lesions increase susceptibility to smoking in the development of airflow limitation. People with these risk factors should be seen as a major target population for anti-smoking campaigns to prevent COPD. PMID:25915938

  18. The association of current and sustained area-based adverse socioeconomic environment with physical inactivity.

    PubMed

    Pascual, Cruz; Regidor, Enrique; Astasio, Paloma; Ortega, Paloma; Navarro, Pedro; Domínguez, Vicente

    2007-08-01

    This paper evaluates the association between socioeconomic environment in the province of residence and physical inactivity, using measures of current and sustained area-based adverse socioeconomic environment. The analysis included 19,324 individuals representative of the Spanish non-institutionalised population aged 16-74 years. The measure of association estimated was the prevalence odds ratio for physical inactivity by current gross domestic product per capita (GDPpc) and current Gini coefficient, and by number of times each province has had a low GDPpc and number of times each province has had a high Gini coefficient in the last two decades. After adjusting for age, individual socioeconomic characteristics, and number of sports facilities per 1,000 population, the odds ratio for physical inactivity in residents of provinces with the lowest current GDPpc versus those with the highest was 1.64 in men and 2.01 in women. The odds ratio in residents of provinces that had always been among those with the lowest GDPpc versus residents in provinces that had never been among those with lowest GDPpc was 1.54 in men and 1.91 in women. Neither the current Gini coefficient nor the indicator that reflects sustained high Gini coefficient were associated with physical inactivity. These findings show that physical inactivity is associated with current socioeconomic context and with the duration of exposure of the area of residence to adverse socioeconomic circumstances when the indicators of socioeconomic environment are based on GDPpc, but not on income inequality. Also, this association is not explained by individual socioeconomic characteristics or the number of sports facilities. PMID:17466424

  19. 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

  20. 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

  1. Enzymatic processing of protein-based fibers.

    PubMed

    Fu, Jiajia; Su, Jing; Wang, Ping; Yu, Yuanyuan; Wang, Qiang; Cavaco-Paulo, Artur

    2015-12-01

    Wool and silk are major protein fiber materials used by the textile industry. Fiber protein structure-function relationships are briefly described here, and the major enzymatic processing routes for textiles and other novel applications are deeply reviewed. Fiber biomodification is described here with various classes of enzymes such as protease, transglutaminase, tyrosinase, and laccase. It is expected that the reader will get a perspective on the research done as a basis for new applications in other areas such as cosmetics and pharma. PMID:26428240

  2. 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

  3. 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

  4. Enzymatic surface modification of acrylonitrile fibers

    NASA Astrophysics Data System (ADS)

    Battistel, Ezio; Morra, Marco; Marinetti, Massimo

    2001-06-01

    The surface of polyacrylonitrile polymer (containing 10% acetate groups) as fibers and finely ground powder have been modified by enzymatic treatment. The enzyme used was a nitrile hydratase, member of the class of nitrile converting enzymes, present in the microorganisms Brevibacterium imperiale and Corynebacterium nitrilophilus. The pendant nitrile groups were selectively converted into the corresponding amides as assessed by XPS analysis. As indicated by the increase of the O/C atomic ratio, the fiber surface showed a significant increase in hydrophilicity. The newly formed amide groups were then able to react with the acid dyes typically used to stain natural fibers, conferring the coloring properties to the otherwise inert polymer surface.

  5. 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.

  6. Ribonuclease activity and RNA binding of recombinant human Dicer

    PubMed Central

    Provost, Patrick; Dishart, David; Doucet, Johanne; Frendewey, David; Samuelsson, Bengt; Rådmark, Olof

    2002-01-01

    RNA silencing phenomena, known as post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference (RNAi) in animals, are mediated by double-stranded RNA (dsRNA) and mechanistically intersect at the ribonuclease Dicer. Here, we report cloning and expression of the 218 kDa human Dicer, and characterization of its ribonuclease activity and dsRNA-binding properties. The recombinant enzyme generated ∼21–23 nucleotide products from dsRNA. Processing of the microRNA let-7 precursor by Dicer produced an apparently mature let-7 RNA. Mg2+ was required for dsRNase activity, but not for dsRNA binding, thereby uncoupling these reaction steps. ATP was dispensable for dsRNase activity in vitro. The Dicer·dsRNA complex formed at high KCl concentrations was catalytically inactive, suggesting that ionic interactions are involved in dsRNA cleavage. The putative dsRNA-binding domain located at the C-terminus of Dicer was demonstrated to bind dsRNA in vitro. Human Dicer expressed in mammalian cells colocalized with calreticulin, a resident protein of the endoplasmic reticulum. Availability of the recombinant Dicer protein will help improve our understanding of RNA silencing and other Dicer-related processes. PMID:12411504

  7. 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

    ... Government misrepresentation, inaction, or error. 407.32 Section 407.32 Public Health CENTERS FOR MEDICARE... enrollment rights because of Federal Government misrepresentation, inaction, or error. If an individual's enrollment or nonenrollment in SMI is unintentional, inadvertent, or erroneous because of the...

  8. 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...

  9. 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…

  10. Production of recombinant peptides as fusions with SUMO.

    PubMed

    Satakarni, Makkapati; Curtis, Robin

    2011-08-01

    Recombinant production of non-native peptides requires using protein fusion technology to prevent peptide degradation by host-cell proteases. In this work, we have used SUMO protein as a fusion partner for the production of difficult-to-express, antimicrobial, self-assembling and amyloidogenic peptides using Escherichia coli. SUMO-peptide fusions were expressed as intracellular products by utilizing pET based expression vectors constructed by Life Sensors Inc., USA. Histidine tagged SUMO-peptide fusions were purified using Ni-NTA affinity chromatography. Complete (100%) cleavage of the SUMO-peptide fusion was achieved using SUMO protease-1. Our findings demonstrate that SUMO fusion technology is a promising alternative for production of peptides in E. coli. The key advantage of this technology is that the enzymatic activity of SUMO protease-1 is specific and efficient leading to inexpensive costs for cleaving the peptide fusion when compared with other fusion systems. PMID:21586326

  11. 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)

  12. 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

  13. 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.

  14. Stable recombination hotspots in birds.

    PubMed

    Singhal, Sonal; Leffler, Ellen M; Sannareddy, Keerthi; Turner, Isaac; Venn, Oliver; Hooper, Daniel M; Strand, Alva I; Li, Qiye; Raney, Brian; Balakrishnan, Christopher N; Griffith, Simon C; McVean, Gil; Przeworski, Molly

    2015-11-20

    The DNA-binding protein PRDM9 has a critical role in specifying meiotic recombination hotspots in mice and apes, but it appears to be absent from other vertebrate species, including birds. To study the evolution and determinants of recombination in species lacking the gene that encodes PRDM9, we inferred fine-scale genetic maps from population resequencing data for two bird species: the zebra finch, Taeniopygia guttata, and the long-tailed finch, Poephila acuticauda. We found that both species have recombination hotspots, which are enriched near functional genomic elements. Unlike in mice and apes, most hotspots are shared between the two species, and their conservation seems to extend over tens of millions of years. These observations suggest that in the absence of PRDM9, recombination targets functional features that both enable access to the genome and constrain its evolution. PMID:26586757

  15. 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.

  16. 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.

  17. 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. PMID:22161212

  18. 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.

  19. 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

  20. 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

  1. Palm Date Fibers: Analysis and Enzymatic Hydrolysis

    PubMed Central

    Shafiei, Marzieh; Karimi, Keikhosro; Taherzadeh, Mohammad J.

    2010-01-01

    Waste palm dates were subjected to analysis for composition and enzymatic hydrolysis of their flesh fibers. The fruit contained 32% glucose and 30% fructose, while the water-insoluble fibers of its flesh consisted of 49.9% lignin and 20.9% polysaccharides. Water-insoluble fibers were settled to 55% of its initial volume in 12 h. The presence of skin and flesh colloidal fibers results in high viscosity and clogging problems during industrial processes. The settling velocity of the fibers was improved by enzymatic hydrolysis. Hydrolysis resulted in 84.3% conversion of the cellulosic part of the fibers as well as reducing the settling time to 10 minutes and the final settled volume to 4% of the initial volume. It implies easier separation of the fibers and facilitates fermentation processes in the corresponding industries. Two kinds of high- and low-lignin fibers were identified from the water-insoluble fibers. The high-lignin fibers (75% lignin) settled easily, while the low-lignin fibers (41.4% lignin) formed a slurry suspension which settled very slowly. The hydrophilicity of these low-lignin fibers is the major challenge of the industrial processes. PMID:21151438

  2. 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

  3. Enzymatic deconstruction of xylan for biofuel production

    PubMed Central

    DODD, DYLAN; CANN, ISAAC K. O.

    2010-01-01

    The combustion of fossil-derived fuels has a significant impact on atmospheric carbon dioxide (CO2) levels and correspondingly is an important contributor to anthropogenic global climate change. Plants have evolved photosynthetic mechanisms in which solar energy is used to fix CO2 into carbohydrates. Thus, combustion of biofuels, derived from plant biomass, can be considered a potentially carbon neutral process. One of the major limitations for efficient conversion of plant biomass to biofuels is the recalcitrant nature of the plant cell wall, which is composed mostly of lignocellulosic materials (lignin, cellulose, and hemicellulose). The heteropolymer xylan represents the most abundant hemicellulosic polysaccharide and is composed primarily of xylose, arabinose, and glucuronic acid. Microbes have evolved a plethora of enzymatic strategies for hydrolyzing xylan into its constituent sugars for subsequent fermentation to biofuels. Therefore, microorganisms are considered an important source of biocatalysts in the emerging biofuel industry. To produce an optimized enzymatic cocktail for xylan deconstruction, it will be valuable to gain insight at the molecular level of the chemical linkages and the mechanisms by which these enzymes recognize their substrates and catalyze their reactions. Recent advances in genomics, proteomics, and structural biology have revolutionized our understanding of the microbial xylanolytic enzymes. This review focuses on current understanding of the molecular basis for substrate specificity and catalysis by enzymes involved in xylan deconstruction. PMID:20431716

  4. 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. PMID:27428463

  5. [Antithrombotic recombinant antibodies].

    PubMed

    Muzard, Julien; Loyau, Stéphane; Ajzenberg, Nadine; Billiald, Philippe; Jandrot-Perrus, Martine

    2006-01-01

    Coronary syndromes, stroke and other ischaemic arterial diseases are the leading cause of death in the world and will probably remain it at least until 2020. Cardiovascular diseases kill 17 million people each year with an expected increase to 20 million in 2020 and 24 million in 2030. The global impact of recurrence and death during the 6 months following an acute coronary syndrome remains at 8-15% in the present state of medical practice. Acute ischaemic syndromes have a common aetiology that is the formation of a platelet-rich clot at the site of severe coronary stenosis and of eroded atherosclerotic plaques. Therapy consists of medical treatments associating thrombolysis, antiplatelet drugs, and the re-opening of the coronary artery by angioplasty. But these treatments do not prevent morbidity and mortality reaching 15% at 6 months. Finally the treatment of stroke is very limited. There is thus a real clinical need to improve existing treatments and to discover new molecules. Platelet activation is a critical step in ischaemic cardiovascular diseases. This is the reason why antiplatelet drugs are most often prescribed in these cases. Currently, only one recombinant antithrombotic antibody is used in therapy. This is a chimeric Fab, c7E3 or abciximab, which inhibits the final phase of platelet aggregation. Abciximab is prescribed in acute coronary syndromes treated by angioplasty. However, treatment by abciximab can induce severe complications, principally, hemorrages and thrombopenia. Other platelet receptors involved in the earlier steps of platelet activation, such as the phases of contact with and of activation by the subendothelium matrix, have been identified as potential targets for the development of antithrombotic antibodies and are described in this revue. PMID:17652972

  6. Genetic recombination in Streptomyces griseus.

    PubMed Central

    Parag, Y

    1978-01-01

    Low-frequency (10(-6)) genetic recombination was observed in a cephamycin-producing strain of Streptomyces griseus. The recombinants were predominantly heteroclones. Heteroclone analysis was performed involving four heteroclones of one cross. In 100 mutants correlation was found between the type of auxotrophy and the level of antibiotic activity. A cross of this strain with a streptomycin-producing strain of S. griesus is described. PMID:415037

  7. [Vaccine application of recombinant herpesviruses].

    PubMed

    Yokoyama, N; Xuan, X; Mikami, T

    2000-04-01

    Recently, genetic engineering using recombinant DNA techniques has been applied to design new viral vaccines in order to reduce some problems which the present viral vaccines have. Up to now, many viruses have been investigated for development of recombinant attenuated vaccines or live viral vectors for delivery of foreign genes coding immunogenic antigens. In this article, we introduced the new vaccine strategy using genetically engineered herpesviruses. PMID:10774221

  8. Combinatorics in Recombinational Population Genomics

    NASA Astrophysics Data System (ADS)

    Parida, Laxmi

    The work that I will discuss is motivated by the need for understanding, and processing, the manifestations of recombination events in chromosome sequences. In this talk, we focus on two related problems. First, we explore the very general problem of reconstructability of pedigree history. How plausible is it to unravel the history of a complete unit (chromosome) of inheritance? The second problem deals with reconstructing the recombinational history of a collection of chromosomes.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. DNA shuffling method for generating highly recombined genes and evolved enzymes.

    PubMed

    Coco, W M; Levinson, W E; Crist, M J; Hektor, H J; Darzins, A; Pienkos, P T; Squires, C H; Monticello, D J

    2001-04-01

    We introduce a method of in vitro recombination or "DNA shuffling" to generate libraries of evolved enzymes. The approach relies on the ordering, trimming, and joining of randomly cleaved parental DNA fragments annealed to a transient polynucleotide scaffold. We generated chimeric libraries averaging 14.0 crossovers per gene, a several-fold higher level of recombination than observed for other methods. We also observed an unprecedented four crossovers per gene in regions of 10 or fewer bases of sequence identity. These properties allow generation of chimeras unavailable by other methods. We detected no unshuffled parental clones or duplicated "sibling" chimeras, and relatively few inactive clones. We demonstrated the method by molecular breeding of a monooxygenase for increased rate and extent of biodesulfurization on complex substrates, as well as for 20-fold faster conversion of a nonnatural substrate. This method represents a conceptually distinct and improved alternative to sexual PCR for gene family shuffling. PMID:11283594

  14. Application of Ni(II)-Assisted Peptide Bond Hydrolysis to Non-Enzymatic Affinity Tag Removal

    PubMed Central

    Kopera, Edyta; Belczyk-Ciesielska, Agnieszka; Bal, Wojciech

    2012-01-01

    In this study, we demonstrate a non-enzymatic method for hydrolytic peptide bond cleavage, applied to the removal of an affinity tag from a recombinant fusion protein, SPI2-SRHWAP-His6. This method is based on a highly specific Ni(II) reaction with (S/T)XHZ peptide sequences. It can be applied for the protein attached to an affinity column or to the unbound protein in solution. We studied the effect of pH, temperature and Ni(II) concentration on the efficacy of cleavage and developed an analytical protocol, which provides active protein with a 90% yield and ∼100% purity. The method works well in the presence of non-ionic detergents, DTT and GuHCl, therefore providing a viable alternative for currently used techniques. PMID:22574150

  15. Characterization of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties.

    PubMed

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

    2016-07-01

    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 CSASs 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 characterized 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 Zn(2+), Fe(2+), Co(2+) and Mn(2+), whereas the activity with Mg(2+) 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 co-ordinating 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

  16. 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

  17. Cloning, expression and enzymatic properties analysis of dihydrofolate reductase gene from the silkworm, Bombyx mori.

    PubMed

    Wang, Wenjing; Gao, Junshan; Wang, Jing; Liu, Chaoliang; Meng, Yan

    2012-12-01

    Tetrahydrobiopterin (BH4) is an essential cofactor for aromatic acid hydroxylases, which control the levels of monoamine neurotransmitters. BH4 deficiency has been associated with many neuropsychological disorders. Dihydrofolate reductase (DHFR) can catalyze 7,8-dihydrobiopterin to 5,6,7,8-tetrahydrobiopterin (BH4) in the salvage pathway of BH4 synthesis from sepiapterin (SP), a major pigment component contained in the integument of silkworm Bombyx mori mutant lemon (lem) in high concentration. In this study, we report the cloning of DHFR gene from the silkworm B. mori (BmDhfr) and identification of enzymatic properties of BmDHFR. BmDhfr is located on scaffold Bm_199 with a predicted gene model BGIBMGA013340, which encodes a 185-aa polypeptide with a predicted molecular mass of about 21 kDa. Biochemical analyses showed that the recombinant BmDHFR protein exhibited high enzymatic activity and suitable parameters to substrate. Together with our previous studies on SP reductase of B. mori (BmSPR) and the lem mutant, it may be an effective way to industrially extract SP from the lem silkworms in large scale to produce BH4 in vitro by co-expressing BmSPR and BmDHFR and using the extracted SP as a substrate in the future. PMID:23065260

  18. Hypermutation of the Inactive X Chromosome Is a Frequent Event in Cancer

    PubMed Central

    Jäger, Natalie; Schlesner, Matthias; Jones, David T.W.; Raffel, Simon; Mallm, Jan-Philipp; Junge, Kristin M.; Weichenhan, Dieter; Bauer, Tobias; Ishaque, Naveed; Kool, Marcel; Northcott, Paul A.; Korshunov, Andrey; Drews, Ruben M.; Koster, Jan; Versteeg, Rogier; Richter, Julia; Hummel, Michael; Mack, Stephen C.; Taylor, Michael D.; Witt, Hendrik; Swartman, Benedict; Schulte-Bockholt, Dietrich; Sultan, Marc; Yaspo, Marie-Laure; Lehrach, Hans; Hutter, Barbara; Brors, Benedikt; Wolf, Stephan; Plass, Christoph; Siebert, Reiner; Trumpp, Andreas; Rippe, Karsten; Lehmann, Irina; Lichter, Peter; Pfister, Stefan M.; Eils, Roland

    2013-01-01

    Summary Mutation is a fundamental process in tumorigenesis. However, the degree to which the rate of somatic mutation varies across the human genome and the mechanistic basis underlying this variation remain to be fully elucidated. Here, we performed a cross-cancer comparison of 402 whole genomes comprising a diverse set of childhood and adult tumors, including both solid and hematopoietic malignancies. Surprisingly, we found that the inactive X chromosome of many female cancer genomes accumulates on average twice and up to four times as many somatic mutations per megabase, as compared to the individual autosomes. Whole-genome sequencing of clonally expanded hematopoietic stem/progenitor cells (HSPCs) from healthy individuals and a premalignant myelodysplastic syndrome (MDS) sample revealed no X chromosome hypermutation. Our data suggest that hypermutation of the inactive X chromosome is an early and frequent feature of tumorigenesis resulting from DNA replication stress in aberrantly proliferating cells. PMID:24139898

  19. 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. PMID:21541146

  20. [Relationships between temperature change and microbial amount in inactive ice wedges in Yitulihe, Northeast China].

    PubMed

    Yang, Si-Zhong; Jin, Hui-Jun; Wen, Xi; Luo, Dong-Liang; Yu, Shao-Peng

    2009-11-01

    Ice-wedge is an indicator of paleoclimate change. The delta18 O concentration in different layers could reflect the change of paleotemperature during ice-wedge growth. In the late 1980s, inactive ice wedges were found in Yitulihe, Northeast China, which were the south-most ones so far and were important in climatic and environmental research. In this paper, the delta18 O concentration and microbial number in the inactive ice-wedges were analyzed by using stable isotope, fluorescence microscopy counting, and flow cytometer (FCM). During the ice-wedge growth in Yitulihe area, there were three short-term paleotemperature fluctuation, and three times of fluctuation in microbial amount in different ice-wedge layer. Correlation analysis indicated that there was a converging relationship between the temperature change and microbial amount in the ice-wedges. The lower the temperature when ice-wedge layer formed, the less the microbes survived in the layer. PMID:20136017

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

    PubMed Central

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

    2013-01-01

    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. PMID:24416051

  2. Dynamic characteristics of heat exchanger tubes vibrating in a tube support plate inactive mode

    SciTech Connect

    Jendrzejczyk, J.A.

    1984-06-01

    Tubes in shell-and-tube heat exchangers, including nuclear plant steam generators, derive their support from longitudinally positioned tube support plates (TSPs). Typically there is a clearance between the tube and TSP hole. Depending on design and fabrication tolerances, the tube may or may not contract all of the TSPs. Noncontact results in an inactive TSP which can lead to detrimental flow induced tube vibrations under certain conditions dependent on the resulting tube-TSP interaction dynamics and the fluid excitation forces. The purpose of this study is to investigate the tube-TSP interaction dynamics. Results of an experimental study of damping and natural frequency as functions of tube-TSP diametral clearance and TSP thickness are reported. Calculated values of damping ratio and frequency of a tube vibrating within an inactive TSP are also presented together with a comparison of calculated and experimental quantities.

  3. Dynamic characteristics of heat exchanger tubes vibrating in a tube support plate inactive mode

    SciTech Connect

    Jendrzejczyk, J.A.

    1985-01-01

    Tubes in shell-and-tube heat exchangers, including nuclear plant steam generators, derive their support from longitudinally positioned tube support plates (TSPs). Typically there is a clearance between the tube and TSP hole. Depending on design and fabrication tolerances, the tube may or may not contact all of the TSPs. Noncontact results in an inactive TSP which can lead to detrimental flow induced tube vibrations under certain conditions dependent on the resulting tube-TSP interaction dynamics and the fluid excitation forces. The purpose of this study is to investigate the tube-TSP interaction dynamics. Results of an experimental study of damping and natural frequency as functions of tube-TSP diametral clearance and TSP thickness are reported. Calculated values of damping ratio and frequency of a tube vibrating within an inactive TSP are also presented together with a comparison of calculated and experimetnal quantities.

  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. 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

  6. Artificial cytoskeletal structures within enzymatically active bio-inorganic protocells.

    PubMed

    Kumar, Ravinash Krishna; Li, Mei; Olof, Sam N; Patil, Avinash J; Mann, Stephen

    2013-02-11

    The fabrication of enzymatically active, semi-permeable bio-inorganic protocells capable of self-assembling a cytoskeletal-like interior and undergoing small-molecule dephosphorylation reactions is described. Reversible disassembly of an amino acid-derived supramolecular hydrogel within the internalized reaction space is used to tune the enzymatic activity of the nanoparticle-bounded inorganic compartments. PMID:23027575

  7. 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...

  8. Sizing up single-molecule enzymatic conformational dynamics.

    PubMed

    Lu, H Peter

    2014-02-21

    Enzymatic reactions and related protein conformational dynamics are complex and inhomogeneous, playing crucial roles in biological functions. The relationship between protein conformational dynamics and enzymatic reactions has been a fundamental focus in modern enzymology. It is extremely difficult to characterize and analyze such complex dynamics in an ensemble-averaged measurement, especially when the enzymes are associated with multiple-step, multiple-conformation complex chemical interactions and transformations. Beyond the conventional ensemble-averaged studies, real-time single-molecule approaches have been demonstrated to be powerful in dissecting the complex enzymatic reaction dynamics and related conformational dynamics. Single-molecule enzymology has come a long way since the early demonstrations of the single-molecule spectroscopy studies of enzymatic dynamics about two decades ago. The rapid development of this fundamental protein dynamics field is hand-in-hand with the new development of single-molecule imaging and spectroscopic technology and methodology, theoretical model analyses, and correlations with biological preparation and characterization of the enzyme protein systems. The complex enzymatic reactions can now be studied one molecule at a time under physiological conditions. Most exciting developments include active manipulation of enzymatic conformational changes and energy landscape to regulate and manipulate the enzymatic reactivity and associated conformational dynamics, and the new advancements have established a new stage for studying complex protein dynamics beyond by simply observing but by actively manipulating and observing the enzymatic dynamics at the single-molecule sensitivity temporally and spatially. PMID:24306450

  9. 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...

  10. Recombination Drives Vertebrate Genome Contraction

    PubMed Central

    Nam, Kiwoong; Ellegren, Hans

    2012-01-01

    Selective and/or neutral processes may govern variation in DNA content and, ultimately, genome size. The observation in several organisms of a negative correlation between recombination rate and intron size could be compatible with a neutral model in which recombination is mutagenic for length changes. We used whole-genome data on small insertions and deletions within transposable elements from chicken and zebra finch to demonstrate clear links between recombination rate and a number of attributes of reduced DNA content. Recombination rate was negatively correlated with the length of introns, transposable elements, and intergenic spacer and with the rate of short insertions. Importantly, it was positively correlated with gene density, the rate of short deletions, the deletion bias, and the net change in sequence length. All these observations point at a pattern of more condensed genome structure in regions of high recombination. Based on the observed rates of small insertions and deletions and assuming that these rates are representative for the whole genome, we estimate that the genome of the most recent common ancestor of birds and lizards has lost nearly 20% of its DNA content up until the present. Expansion of transposable elements can counteract the effect of deletions in an equilibrium mutation model; however, since the activity of transposable elements has been low in the avian lineage, the deletion bias is likely to have had a significant effect on genome size evolution in dinosaurs and birds, contributing to the maintenance of a small genome. We also demonstrate that most of the observed correlations between recombination rate and genome contraction parameters are seen in the human genome, including for segregating indel polymorphisms. Our data are compatible with a neutral model in which recombination drives vertebrate genome size evolution and gives no direct support for a role of natural selection in this process. PMID:22570634

  11. Recombination processes in ionised plasmas

    NASA Astrophysics Data System (ADS)

    Bastin, Robert

    The observational analysis of astrophysical plasmas relies on accurate calculations of the atomic processes involved. The recombination spectra of singly ionised oxygen (O il) and carbon (C il) present excellent tools for investigating regions such as planetary nebulae and H II regions. In this thesis, detailed treatments of the recombination processes of both O II and C II are presented. Using the R-matrix solution to the close coupling equations, I present the results of accurate photoionisation calculations. Bound state energy levels are determined and oscillator strengths calculated for both species. Recombination coefficients were evalu ated for low n and 1, for C II in LS-coupling, and 0 II in intermediate coupling, taking particular care to treat resonances effectively. Sample photoionisation cross-sections are presented for both species, and compared to previous work. A complete radiative-cascade model is treated for both species, in order to determine line emissivities under nebular conditions at a wide range of temperatures and densities. Collisional effects are treated for C II, along with, for the first time, the effects of high temperature dielectronic recombination, allowing the modelling of regions of much higher electron temperature than previous work. The O II calculations were performed under intermediate coupling for the first time, allowing the effects of non-statistical popula tions of the parent ion fine-structure levels and dielectronic recombination onto bound states within this fine-structure to be taken into account in line emissivities. Detailed comparison with previous theoretical work was made for both species. The application of the C II and 0 n recombination spectra to determining tempera ture and densities from the observed spectra of a number of ionised nebulae is considered. The potential for using the new recombination spectra as diagnostic tools to solve some of the key problems in the study of ionised nebulae is demonstrated.

  12. 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.

  13. 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.

  14. 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. PMID:27072508

  15. 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

  16. School Social Fragmentation, Economic Deprivation and Social Cohesion and Adolescent Physical Inactivity: A Longitudinal Study

    PubMed Central

    Pabayo, Roman; Janosz, Michel; Bisset, Sherri; Kawachi, Ichiro

    2014-01-01

    Objectives To examine the independent influence of school economic deprivation, social fragmentation, and social cohesion on the likelihood of participating in no physical activity among students. Methods Data are from a large-scale longitudinal study of schools based in disadvantaged communities in Quebec, Canada. Questionnaires were administered every year between 2002 and 2008 among n = 14,924 students aged 12 to 18 from a sample of 70 schools. Cross-sectional and longitudinal analyses were conducted. Multilevel modeling was utilized to account for the clustering of students within schools. Schools were categorized as being low, moderate or high economic deprivation, social fragmentation and social cohesion. Those who indicated that they do no participate in any physical activity during the week were identified as being physically inactive. Results In baseline multilevel cross-sectional analyses, adolescents attending schools in the highest (compared to the lowest) levels of socioeconomic deprivation and social fragmentation were more likely to be physically inactive (OR = 1.33, 95% CI = 1.03, 1.72; and OR = 1.24, 95% CI = 0.98, 1.56, respectively). Conversely, students attending schools with the highest cohesion were less likely to be physically inactive (OR = 0.78, 95% CI = 0.61, 0.99). In longitudinal analysis, physically active students who attended schools with the highest social fragmentation were more likely to become physically inactive over two years (OR = 1.65, 95% CI = 1.09, 2.51). Conclusion The school socioeconomic environment appears to be an important contextual influence on participation in no physical activity among adolescents. Following adolescents beyond two years is necessary to determine if these environments have a lasting effect on physical activity behavior. PMID:24932679

  17. 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)

  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. PMID:25542368

  19. 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

  20. 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. PMID:26120796

  1. 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.

  2. 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.

  3. Enzymatic bioremediation: from enzyme discovery to applications.

    PubMed

    Sutherland, T D; Horne, I; Weir, K M; Coppin, C W; Williams, M R; Selleck, M; Russell, R J; Oakeshott, J G

    2004-11-01

    1. Enzymatic bioremediation is potentially a rapid method of removing environmental pesticide residues. Applications include the treatment of residues resulting from agricultural production and processing industries, such as the treatment of irrigation waters, surface-contaminated fruit and vegetables and spent dip liquors. 2. A specific application for some organophosphate-degrading enzymes involves detoxification of nerve agent stockpiles. Effective and affordable remediation requires highly specialized enzymes, so protein engineering techniques are being used to improve properties of various source enzymes to enhance catalytic rates, stability and substrate range. 3. Trials with an optimized organophosphate-degrading enzyme have shown the feasibility of such technology in various applications. 4. The enzymes developed for environmental remediation for specific pesticide classes also have applications as antidotes for high-dose pesticide poisonings and as prophylaxis for people at risk of high pesticide doses. PMID:15566400

  4. 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. PMID:25708541

  5. 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

  6. Effective enzymatic caffeoylation of natural glucopyranosides.

    PubMed

    Chyba, Andrej; Mastihuba, Vladimír; Mastihubová, Mária

    2016-03-15

    Reaction system was developed for enzymatic caffeoylation of model saccharidic acceptor methyl β-d-glucopyranoside to obtain exclusively methyl 6-O-caffeoyl-β-D-glucopyranoside. Reaction with starting concentration of acceptor 0.2 M provided 73% yield of purified product within 17 days. Reactions with low acceptor concentrations (0.04 and 0.08 M) run to the completion within 7 days. Such highly effective and regioselective reaction was promoted by Lipozyme TL IM in tert-butanol, using vinyl caffeate as acylation donor. The optimized reaction conditions were used in preparative caffeoylation of natural substances-arbutin and salidroside, giving 75% of 6-O-caffeoylated arbutin (robustaside B) and 74% of 6-O-caffeoylated salidroside as the only products after 12 and 16 days, respectively. PMID:26896186

  7. Enzymatic pretreatment for preparing starch nanocrystals.

    PubMed

    LeCorre, Déborah; Vahanian, Elina; Dufresne, Alain; Bras, Julien

    2012-01-01

    Starch nanocrystals (SNCs) are crystalline platelets resulting from the acid hydrolysis of starch. A limiting factor for their more widespread use is their preparation duration. Therefore, this study investigates the possibility of developing an enzymatic pretreatment of starch to reduce the acid hydrolysis duration. A screening of three types of enzymes, namely, α-amylase, β-amylase, and glucoamylase, is proposed, and the latter was selected for a pretreatment. Compared with the regular kinetics of hydrolysis for preparing SNC, that of pretreated starch was much faster. The extent of hydrolysis normally reached in 24 h was obtained after only 6 h, and the regular final yield (15% after 5 days) was reached in 45 h. AFM and X-ray diffraction measurements confirmed that the obtained nanoparticles were indeed SNC. PMID:22133316

  8. Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

    PubMed Central

    Parvin, Jeffrey; Chiba, Natsuko; Ransburgh, Derek

    2011-01-01

    The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was

  9. 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

  10. How the rice weevil breaks down the pectin network: Enzymatic synergism and sub-functionalization.

    PubMed

    Kirsch, Roy; Heckel, David G; Pauchet, Yannick

    2016-04-01

    Pectin is the most complex polysaccharide in nature and highly abundant in plant cell walls and middle lamellae, where it functions in plant growth and development. Phytopathogens utilize plant pectin as an energy source through enzyme-mediated degradation. These pectolytic enzymes include polygalacturonases (PGs) of the GH28 family and pectin methylesterases (PMEs) of the CE8 family. Recently, PGs were also identified in herbivorous insects of the distantly related plant bug, stick insect and Phytophaga beetle lineages. Unlike all other insects, weevils possess PMEs in addition to PGs. To investigate pectin digestion in insects and the role of PMEs in weevils, all PME and PG family members of the rice weevil Sitophilus oryzae were heterologously expressed and functionally characterized. Enzymatically active and inactive PG and PME family members were identified. The loss of activity can be explained by a lack of substrate binding correlating with substitutions of functionally important amino acid residues. We found subfunctionalization in both enzyme families, supported by expression pattern and substrate specificities as well as evidence for synergistic pectin breakdown. Our data suggest that the rice weevil might be able to use pectin as an energy source, and illustrates the potential of both PG and PME enzyme families to functionally diversify after horizontal gene transfer. PMID:26899322

  11. 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

  12. 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.

  13. Recombinant allergens for specific immunotherapy.

    PubMed

    Cromwell, Oliver; Häfner, Dietrich; Nandy, Andreas

    2011-04-01

    Recombinant DNA technology provides the means for producing allergens that are equivalent to their natural counterparts and also genetically engineered variants with reduced IgE-binding activity. The proteins are produced as chemically defined molecules with consistent structural and immunologic properties. Several hundred allergens have been cloned and expressed as recombinant proteins, and these provide the means for making a very detailed diagnosis of a patient's sensitization profile. Clinical development programs are now in progress to assess the suitability of recombinant allergens for both subcutaneous and sublingual immunotherapy. Recombinant hypoallergenic variants, which are developed with the aim of increasing the doses that can be administered while at the same time reducing the risks for therapy-associated side effects, are also in clinical trials for subcutaneous immunotherapy. Grass and birch pollen preparations have been shown to be clinically effective, and studies with various other allergens are in progress. Personalized or patient-tailored immunotherapy is still a very distant prospect, but the first recombinant products based on single allergens or defined mixtures could reach the market within the next 5 years. PMID:21377719

  14. Three-dimensional Structure and Enzymatic Function of Proapoptotic Human p53-inducible Quinone Oxidoreductase PIG3*

    PubMed Central

    Porté, Sergio; Valencia, Eva; Yakovtseva, Evgenia A.; Borràs, Emma; Shafqat, Naeem; Debreczeny, Judit É.; Pike, Ashley C. W.; Oppermann, Udo; Farrés, Jaume; Fita, Ignacio; Parés, Xavier

    2009-01-01

    Tumor suppressor p53 regulates the expression of p53-induced genes (PIG) that trigger apoptosis. PIG3 or TP53I3 is the only known member of the medium chain dehydrogenase/reductase superfamily induced by p53 and is used as a proapoptotic marker. Although the participation of PIG3 in the apoptotic pathway is proven, the protein and its mechanism of action were never characterized. We analyzed human PIG3 enzymatic function and found NADPH-dependent reductase activity with ortho-quinones, which is consistent with the classification of PIG3 in the quinone oxidoreductase family. However, the activity is much lower than that of ζ-crystallin, a better known quinone oxidoreductase. In addition, we report the crystallographic structure of PIG3, which allowed the identification of substrate- and cofactor-binding sites, with residues fully conserved from bacteria to human. Tyr-59 in ζ-crystallin (Tyr-51 in PIG3) was suggested to participate in the catalysis of quinone reduction. However, kinetics of Tyr/Phe and Tyr/Ala mutants of both enzymes demonstrated that the active site Tyr is not catalytic but may participate in substrate binding, consistent with a mechanism based on propinquity effects. It has been proposed that PIG3 contribution to apoptosis would be through oxidative stress generation. We found that in vitro activity and in vivo overexpression of PIG3 accumulate reactive oxygen species. Accordingly, an inactive PIG3 mutant (S151V) did not produce reactive oxygen species in cells, indicating that enzymatically active protein is necessary for this function. This supports that PIG3 action is through oxidative stress produced by its enzymatic activity and provides essential knowledge for eventual control of apoptosis. PMID:19349281

  15. 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

  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. 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

  19. 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.

  20. Stable recombination hotspots in birds

    PubMed Central

    Singhal, Sonal; Leffler, Ellen M.; Sannareddy, Keerthi; Turner, Isaac; Venn, Oliver; Hooper, Daniel M.; Strand, Alva I.; Li, Qiye; Raney, Brian; Balakrishnan, Christopher N.; Griffith, Simon C.; McVean, Gil; Przeworski, Molly

    2016-01-01

    The DNA-binding protein PRDM9 has a critical role in specifying meiotic recombination hotspots in mice and apes, but appears to be absent from other vertebrate species, including birds. To study the evolution and determinants of recombination in species lacking PRDM9, we inferred fine-scale genetic maps from population resequencing data for two bird species, the zebra finch Taeniopygia guttata and the long-tailed finch Poephila acuticauda. We find that both species have hotspots, which are enriched near functional genomic elements. Unlike in mice and apes, the two species share most hotspots, with conservation seemingly extending over tens of millions of years. These observations suggest that in the absence of PRDM9, recombination targets functional features that both enable access to the genome and constrain its evolution. PMID:26586757

  1. 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

  2. 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.

  3. 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.

  4. 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. PMID:23334017

  5. 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

  6. A Redundant Role of Human Thyroid Peroxidase Propeptide for Cellular, Enzymatic, and Immunological Activity

    PubMed Central

    Góra, Monika; Buckle, Ashley M.; Porebski, Benjamin T.; Kemp, E. Helen; Sutton, Brian J.; Czarnocka, Barbara; Banga, J. Paul

    2014-01-01

    Background: Thyroid peroxidase (TPO) is a dimeric membrane-bound enzyme of thyroid follicular cells, responsible for thyroid hormone biosynthesis. TPO is also a common target antigen in autoimmune thyroid disease (AITD). With two active sites, TPO is an unusual enzyme, and thus there is much interest in understanding its structure and role in AITD. Homology modeling has shown TPO to be composed of different structural modules, as well as a propeptide sequence. During the course of studies to obtain homogeneous preparations of recombinant TPO for structural studies, we investigated the role of the large propeptide sequence in TPO. Methods: An engineered recombinant human TPO preparation expressed in Chinese hamster ovary (CHO) cells lacking the propeptide (TPOΔpro; amino acid residues 21–108) was characterized and its properties compared to wild-type TPO. Plasma membrane localization was determined by cell surface protein biotinylation, and biochemical studies were performed to evaluate enzymatic activity and the effect of deglycosylation. Immunological investigations using autoantibodies from AITD patients and other epitope-specific antibodies that recognize conformational determinants on TPO were evaluated for binding to TPOΔpro by flow cytometry, immunocytochemistry, and capture enzyme-linked immunosorbent assay. Molecular modeling and dynamics simulation of TPOΔpro comprising a dimer of myeloperoxidase-like domains was performed in order to investigate the impact of propeptide removal and the role of glycosylation. Results: The TPOΔpro was expressed on the cell surface at comparable levels to wild-type TPO. The TPOΔpro was enzymatically active and recognized by patients' autoantibodies and a panel of epitope-specific antibodies, confirming structural integrity of the two major conformational determinants recognized by autoantibodies. Faithful intracellular trafficking and N-glycosylation of TPOΔpro was also maintained. Molecular modeling and dynamics

  7. Overexpression of Cytochrome c by a Recombinant Rabies Virus Attenuates Pathogenicity and Enhances Antiviral Immunity

    PubMed Central

    Pulmanausahakul, Rojjanaporn; Faber, Milosz; Morimoto, Kinjiro; Spitsin, Sergei; Weihe, Eberhard; Hooper, D. Craig; Schnell, Matthias J.; Dietzschold, Bernhard

    2001-01-01

    The pathogenicity of individual rabies virus strains appears to correlate inversely with the extent of apoptotic cell death they induce and with the expression of rabies virus glycoprotein, a major inducer of an antiviral immune response. To determine whether the induction of apoptosis by rabies virus contributes to a decreased pathogenicity by stimulating antiviral immunity, we have analyzed these parameters in tissue cultures and in mice infected with a recombinant rabies virus construct that expresses the proapoptotic protein cytochrome c. The extent of apoptosis was strongly increased in primary neuron cultures infected with the recombinant virus carrying the active cytochrome c gene [SPBN-Cyto c(+)], compared with cells infected with the recombinant virus containing the inactive cytochrome c gene [SPBN-Cyto c(−)]. Mortality in mice infected intranasally with SPBN-Cyto c(+) was substantially lower than in SPBN-Cyto c(−)-infected mice. Furthermore, virus-neutralizing antibody (VNA) titers were significantly higher in mice immunized with SPBN-Cyto c(+) at the same dose. The VNA titers induced by these recombinant viruses paralleled their protective activities against a lethal rabies virus challenge infection, with SPBN-Cyto c(+) revealing an effective dose 20 times lower than that of SPBN-Cyto c(−). The strong increase in immunogenicity, coupled with the marked reduction in pathogenicity, identifies the SPBN-Cyto c(+) construct as a candidate for a live rabies virus vaccine. PMID:11602721

  8. 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.

  9. Molecular expression and enzymatic characterization of thioredoxin from the carcinogenic human liver fluke Opisthorchis viverrini.

    PubMed

    Suttiprapa, Sutas; Matchimakul, Pitchaya; Loukas, Alex; Laha, Thewarach; Wongkham, Sopit; Kaewkes, Sasithorn; Brindley, Paul J; Sripa, Banchob

    2012-03-01

    The human liver fluke, Opisthorchis viverrini, induces inflammation of the hepatobiliary system. Despite being constantly exposed to inimical oxygen radicals released from inflammatory cells, the parasite survives for years. Defense against oxidative damage can be mediated through glutathione and/or thioredoxin utilizing systems. Here, we report the molecular expression and biochemical characterization of a thioredoxin (Trx) from O. viverrini. O. viverrini Trx cDNA encoded a polypeptide of 105 amino acid residues, of molecular mass 11.63 kDa. The predicted protein has similarity to previously characterized thioredoxins with 26-51% identity. Recombinant O. viverrini Trx (Ov-Trx-1) was expressed as soluble protein in E. coli. The recombinant protein showed insulin reduction activity and supported the enzymatic function of O. viverrini thioredoxin peroxidase. Expression of Ov-Trx-1 at mRNA and protein levels was observed in all obtainable developmental stages of the liver fluke. Ov-Trx-1 was also detected in excretory-secretory products released by adult O. viverrini. Immunohistochemistry, Ov-Trx-1 was expressed in nearly all parasite tissue excepted ovary and mature sperms. Interestingly, Ov-Trx-1 was observed in the infected biliary epithelium but not in normal bile ducts. These results suggest that Ov-Trx-1 is essential for the parasite throughout the life cycle. In the host-parasite interaction aspect, Ov-Trx-1 may support thioredoxin peroxidase in protecting the parasite against damage induced by reactive oxygen species from inflammation. PMID:21740981

  10. A functional analysis of the spacer of V(D)J recombination signal sequences.

    PubMed

    Lee, Alfred Ian; Fugmann, Sebastian D; Cowell, Lindsay G; Ptaszek, Leon M; Kelsoe, Garnett; Schatz, David G

    2003-10-01

    During lymphocyte development, V(D)J recombination assembles antigen receptor genes from component V, D, and J gene segments. These gene segments are flanked by a recombination signal sequence (RSS), which serves as the binding site for the recombination machinery. The murine Jbeta2.6 gene segment is a recombinationally inactive pseudogene, but examination of its RSS reveals no obvious reason for its failure to recombine. Mutagenesis of the Jbeta2.6 RSS demonstrates that the sequences of the heptamer, nonamer, and spacer are all important. Strikingly, changes solely in the spacer sequence can result in dramatic differences in the level of recombination. The subsequent analysis of a library of more than 4,000 spacer variants revealed that spacer residues of particular functional importance are correlated with their degree of conservation. Biochemical assays indicate distinct cooperation between the spacer and heptamer/nonamer along each step of the reaction pathway. The results suggest that the spacer serves not only to ensure the appropriate distance between the heptamer and nonamer but also regulates RSS activity by providing additional RAG:RSS interaction surfaces. We conclude that while RSSs are defined by a "digital" requirement for absolutely conserved nucleotides, the quality of RSS function is determined in an "analog" manner by numerous complex interactions between the RAG proteins and the less-well conserved nucleotides in the heptamer, the nonamer, and, importantly, the spacer. Those modulatory effects are accurately predicted by a new computational algorithm for "RSS information content." The interplay between such binary and multiplicative modes of interactions provides a general model for analyzing protein-DNA interactions in various biological systems. PMID:14551903

  11. 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. PMID:25184300

  12. 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

  13. 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

  14. 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...

  15. 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...

  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, 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...

  18. 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

  19. Physical inactivity amplifies the sensitivity of skeletal muscle to the lipid-induced downregulation of lipoprotein lipase activity.

    PubMed

    Zderic, Theodore W; Hamilton, Marc T

    2006-01-01

    Physical inactivity is a risk factor for lipoprotein disorders and the metabolic syndrome. Physical inactivity has a powerful effect on suppressing lipoprotein lipase (LPL) activity in skeletal muscle, the rate-limiting enzyme for hydrolysis of triglyceride (TG)-rich lipoproteins. We tested the ability of several compounds to prevent the decrease in LPL. The present study minimized standing and ordinary light nonexercise movements in rats to compare the effects of inactivity and nonexercise activity thermogenesis (NEAT) on LPL activity. The key new insight was that the typically quick decrease in LPL activity of oxidative muscle caused by physical inactivity was prevented by nicotinic acid (NA), whereas inhibitors of TNF-alpha, inducible nitric oxide synthase, and NF-kappaB had no such effect. NA was administered at a dose known to acutely impede the appearance of plasma TG from the liver and free fatty acids from adipose tissue, and it was effective at intentionally lowering plasma lipid concentrations to the same level in active and inactive groups. As measured from heparin-releasable LPL activity, LPL in the microvasculature of the most oxidative muscles was approximately 90% lower in the inactive group compared with controls, and this suppression was completely blocked by NA. In contrast to inactivity, NA did not raise muscle LPL in ambulatory controls, whereas a large exogenous fat delivery did decrease LPL activity. In vitro control studies revealed that NA did not have a direct effect on skeletal muscle LPL activity. In conclusion, physical inactivity amplifies the ability of plasma lipids to suppress muscle LPL activity. The light ambulatory contractions responsible for NEAT are sufficient for mitigating these deleterious effects. PMID:16195388

  20. Improving recombinant protein purification yield

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Production of adequate amounts of recombinant proteins is essential for antibody production, biochemical activity study, and structural determination during the post-genomic era. It’s technologically challenging and a limiting factor for tung oil research because analytical reagents such as high qua...

  1. CRMAGE: CRISPR Optimized MAGE Recombineering

    PubMed Central

    Ronda, Carlotta; Pedersen, Lasse Ebdrup; Sommer, Morten O. A.; Nielsen, Alex Toftgaard

    2016-01-01

    A bottleneck in metabolic engineering and systems biology approaches is the lack of efficient genome engineering technologies. Here, we combine CRISPR/Cas9 and λ Red recombineering based MAGE technology (CRMAGE) to create a highly efficient and fast method for genome engineering of Escherichia coli. Using CRMAGE, the recombineering efficiency was between 96.5% and 99.7% for gene recoding of three genomic targets, compared to between 0.68% and 5.4% using traditional recombineering. For modulation of protein synthesis (small insertion/RBS substitution) the efficiency was increased from 6% to 70%. CRMAGE can be multiplexed and enables introduction of at least two mutations in a single round of recombineering with similar efficiencies. PAM-independent loci were targeted using degenerate codons, thereby making it possible to modify any site in the genome. CRMAGE is based on two plasmids that are assembled by a USER-cloning approach enabling quick and cost efficient gRNA replacement. CRMAGE furthermore utilizes CRISPR/Cas9 for efficient plasmid curing, thereby enabling multiple engineering rounds per day. To facilitate the design process, a web-based tool was developed to predict both the λ Red oligos and the gRNAs. The CRMAGE platform enables highly efficient and fast genome editing and may open up promising prospective for automation of genome-scale engineering. PMID:26797514

  2. Inhomogeneous recombinations during cosmic reionization

    NASA Astrophysics Data System (ADS)

    Sobacchi, Emanuele; Mesinger, Andrei

    2014-05-01

    By depleting the ionizing photon budget available to expand cosmic H II regions, recombining systems (or Lyman limit systems) can have a large impact during (and following) cosmic reionization. Unfortunately, directly resolving such structures in large-scale reionization simulations is computationally impractical. Instead, here we implement a subgrid prescription for tracking inhomogeneous recombinations in the intergalactic medium. Building on previous work parametrizing photoheating feedback on star formation, we present large-scale, seminumeric reionization simulations which self-consistently track the local (subgrid) evolution of both sources and sinks of ionizing photons. Our simple, single-parameter model naturally results in both an extended reionization and a modest, slowly evolving emissivity, consistent with observations. Recombinations are instrumental in slowing the growth of large H II regions, and damping the rapid rise of the ionizing background in the late stages of (and following) reionization. As a result, typical H II regions are smaller by factors of ˜2 to 3 throughout reionization. The large-scale (k ≲ 0.2 Mpc-1) ionization power spectrum is suppressed by factors of ≳2-3 in the second half of reionization. Therefore properly modelling recombinations is important in interpreting virtually all reionization observables, including upcoming interferometry with the redshifted 21cm line. Consistent with previous works, we find the clumping factor of ionized gas to be C H II ˜ 4 at the end of reionization.

  3. Consecutive enzymatic modification of ornithine generates the hydroxamate moieties of the siderophore erythrochelin.

    PubMed

    Robbel, Lars; Helmetag, Verena; Knappe, Thomas A; Marahiel, Mohamed A

    2011-07-12

    Biosynthesis of the hydroxamate-type siderophore erythrochelin requires the generation of δ-N-acetyl-δ-N-hydroxy-L-ornithine (L-haOrn), which is incorporated into the tetrapeptide at positions 1 and 4. Bioinformatic analysis revealed the FAD-dependent monooxygenase EtcB and the bifunctional malonyl-CoA decarboxylase/acetyltransferase Mcd to be putatively involved in the generation of L-haOrn. To investigate if EtcB and Mcd constitute a two-enzyme pathway for the biosynthesis of L-haOrn, they were produced in a recombinant manner and subjected to biochemical studies in vitro. Hydroxylation assays employing recombinant EtcB gave rise to δ-N-hydroxy-L-ornithine (L-hOrn) and confirmed the enzyme to be involved in building block assembly. Acetylation assays were carried out by incubating L-hOrn with recombinant Mcd and malonyl-CoA as the acetyl group donor. Substrate turnover was increased by substituting malonyl-CoA with acetyl-CoA, bypassing the decarboxylation reaction which represents the rate-limiting step. Consecutive enzymatic synthesis of L-haOrn was accomplished in coupled assays employing both the L-ornithine hydroxylase and Mcd. In summary, a biosynthetic route for the generation of δ-N-acetyl-δ-N-hydroxy-L-ornithine starting from L-ornithine has been established in vitro by tandem action of the FAD-dependent monooxygenase EtcB and the bifunctional malonyl-CoA decarboxylase/acetyltransferase Mcd. PMID:21650455

  4. Predictors of hazardous drinking, tobacco smoking and physical inactivity in vocational school students

    PubMed Central

    2013-01-01

    Background Tobacco smoking, hazardous drinking and physical inactivity during adolescence are risk factors that are associated with poorer health in adulthood. The identification of subgroups of young people with a high prevalence of one or more of these risk factors allows an optimised allocation of preventive measures. This study aimed at investigating hazardous drinking, tobacco smoking and physical inactivity as well as their associations and demographic predictors in vocational school students. Methods Out of 57 contacted vocational schools in Switzerland, a total of 24 schools participated in a survey assessing gender, age, immigrant background, educational attainment and vocational field as well as the above mentioned health risk factors. Out of the 2659 students present in 177 included vocational school classes, 2647 (99.5%) completed the survey. Binary logistic regression analyses were conducted to investigate the demographic predictors of each health risk factor and a multinomial logistic regression analysis was conducted to investigate predictors of different risk factor combinations. Results Of the surveyed students, 79.4% showed at least one risk factor, 43.6% showed two or more and 9.6% showed all three health risk factors. Hazardous drinking was more prevalent in male, physical inactivity was more prevalent in female vocational school students. The proportion of students with low physical activity and tobacco smoking increased with increasing age. While the combination of hazardous drinking and tobacco smoking was higher in males, the other risk factor combinations were observed particularly among females. Conclusions Multiple risk factors were ascertained in a significant proportion of vocational school students. Specifically, tobacco smoking and hazardous drinking were coexistent. The study underlines the need for preventive measures in specific subpopulations of adolescents and young adults with lower educational level. PMID:23672294

  5. Previously unrecognized now-inactive strand of the North Anatolian fault in the Thrace basin

    SciTech Connect

    Perincek, D. )

    1988-08-01

    The North Anatolian fault is a major 1,200 km-long transform fault bounding the Anatolian plate to the north. It formed in late middle Miocene time as a broad shear zone with a number of strands splaying westward in a horsetail fashion. Later, movement became localized along the stem, and the southerly and northerly splays became inactive. One such right-lateral, now-inactive splay is the west-northwest-striking Thrace strike-slip fault system, consisting of three subparallel strike-slip faults. From north to south these are the Kirklareli, Lueleburgaz, and Babaeski fault zones, extending {plus minus} 130 km along the strike. The Thrace fault zone probably connected with the presently active northern strand of the North Anatolian fault in the Sea of Marmara in the southeast and may have joined the Plovdiv graben zone in Bulgaria in the northwest. The Thrace basin in which the Thrace fault system is located, is Cenozoic with a sedimentary basin fill from middle Eocene to Pliocene. The Thrace fault system formed in pre-Pliocene time and had become inactive by the Pliocene. Strike-slip fault zones with normal and reverse separation are detected by seismic reflection profiles and subsurface data. Releasing bend extensional structures (e.g., near the town of Lueleburgaz) and restraining bend compressional structures (near Vakiflar-1 well) are abundant on the fault zones. Umurca and Hamitabad fields are en echelon structures on the Lueleburgaz fault zone. The Thrace strike-slip fault system has itself a horsetail shape, the various strands of which become younger southward. The entire system died before the Pliocene, and motion on the North Anatolian fault zone began to be accommodated in the Sea of Marmara region. Thus the Thrace fault system represents the oldest strand of the North Anatolian fault in the west.

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

    PubMed Central

    Strey, K.A.; Nichols, N.L.; Baertsch, N.A.; Broytman, O.; Baker-Herman, T.L.

    2012-01-01

    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/p62 in spinal regions associated with the phrenic motor pool. Although PKCζ/Ι activity is necessary for iPMF, spinal aPKC 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 where prolonged inactivity ends life. PMID:23152633

  7. 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

  8. 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

  9. Enzymatic hydrolysis of biomass from wood.

    PubMed

    Álvarez, Consolación; Reyes-Sosa, Francisco Manuel; Díez, Bruno

    2016-03-01

    Current research and development in cellulosic ethanol production has been focused mainly on agricultural residues and dedicated energy crops such as corn stover and switchgrass; however, woody biomass remains a very important feedstock for ethanol production. The precise composition of hemicellulose in the wood is strongly dependent on the plant species, therefore different types of enzymes are needed based on hemicellulose complexity and type of pretreatment. In general, hardwood species have much lower recalcitrance to enzymes than softwood. For hardwood, xylanases, beta-xylosidases and xyloglucanases are the main hemicellulases involved in degradation of the hemicellulose backbone, while for softwood the effect of mannanases and beta-mannosidases is more relevant. Furthermore, there are different key accessory enzymes involved in removing the hemicellulosic fraction and increasing accessibility of cellulases to the cellulose fibres improving the hydrolysis process. A diversity of enzymatic cocktails has been tested using from low to high densities of biomass (2-20% total solids) and a broad range of results has been obtained. The performance of recently developed commercial cocktails on hardwoods and softwoods will enable a further step for the commercialization of fuel ethanol from wood. PMID:26833542

  10. Computational Investigations on Enzymatic Catalysis and Inhibition

    NASA Astrophysics Data System (ADS)

    Simard, Daniel

    Enzymes are the bimolecular "workhorses" of the cell due to their range of functions and their requirement for cellular success. The atomistic details of how they function can provide key insights into the fundamentals of catalysis and in turn, provide a blueprint for biotechnological advances. A wide range of contemporary computational techniques has been applied with the aim to characterize recently discovered intermediates or to provide insights into enzymatic mechanisms and inhibition. More specifically, an assessment of methods was conducted to evaluate the presence of the growing number 3-- and 4--coordinated sulfur intermediates in proteins/enzymes. Furthermore, two mechanisms have been investigated, the mu-OH mechanism of the hydrolysis of dimethylphosphate in Glycerophosphodiesterase (GpdQ) using five different homonuclear metal combinations Zn(II)/Zn(II), Co(II)/Co(II), Mn(II)/Mn(II), Cd(II)/Cd(II) and Ca(II)/Ca(II) as well as a preliminary study into the effectivness of boron as an inhibitor in the serine protease reaction of class A TEM-1 beta-lactamases.

  11. Non-eluting Enzymatic Antibiofilm Coatings

    PubMed Central

    Pavlukhina, Svetlana V.; Kaplan, Jeffrey B.; Xu, Li; Chang, Wei; Yu, Xiaojun; Madhyastha, Srinivasa; Yakandawala, Nandadeva; Mentbayeva, Almagul; Khan, Babar; Sukhishvili, Svetlana A.

    2012-01-01

    We developed a highly efficient, biocompatible surface coating that disperses bacterial biofilms through enzymatic cleavage of the extracellular biofilm matrix. The coating was fabricated by binding the naturally existing enzyme dispersin B (DspB) to surface-attached polymer matrices constructed via a layer-by-layer (LbL) deposition technique. LbL matrices were assembled through electrostatic interactions of poly(allylamine hydrochloride) (PAH) and poly(methacrylic acid) (PMAA), followed by chemical crosslinking with glutaraldehyde and pH triggered removal of PMAA, producing a stable PAH hydrogel matrix used for DspB loading. The amount of DspB loaded increased linearly with the number of PAH layers in surface hydrogels. DspB was retained within these coatings in the pH range from 4 to 7.5. DspB-loaded coatings inhibited biofilm formation by two clinical strains of Staphylococcus epidermidis. Biofilm inhibition was ≥ 98% compared to mock-loaded coatings as determined by CFU enumeration. In addition, DspB-loaded coatings did not inhibit attachment or growth of cultured human osteoblast cells. We suggest that the use of DspB-loaded multilayer coatings presents a promising method for creating biocompatible surfaces with high antibiofilm efficiency, especially when combined with conventional antimicrobial treatment of dispersed bacteria. PMID:22909396

  12. Natural Product Sugar Biosynthesis and Enzymatic Glycodiversification**

    PubMed Central

    Thibodeaux, Christopher J.; Melançon, Charles E.; Liu, Hung-wen

    2009-01-01

    Many biologically active small molecule natural products produced by microorganisms derive their activities from sugar substituents. Changing the structures of these sugars can have a profound impact on the biological properties of the parent compounds. This realization has inspired attempts to derivatize the sugar moieties of these natural products through exploitation of the sugar biosynthetic machinery. This approach requires an understanding of the biosynthetic pathway of each target sugar and detailed mechanistic knowledge of the key enzymes. Scientists have begun to unravel the biosynthetic logic behind the assembly of many glycosylated natural products, and have found that a core set of enzyme activities is mixed and matched to synthesize the diverse sugar structures observed in nature. Remarkably, many of these sugar biosynthetic enzymes and glycosyltransferases also exhibit relaxed substrate specificity. The promiscuity of these enzymes has prompted efforts to modify the sugar structures and/or alter the glycosylation patterns of natural products via metabolic pathway engineering and/or enzymatic glycodiversification. In applied biomedical research, these studies will enable the development of new glycosylation tools and generate novel glycoforms of secondary metabolites with useful biological activity. PMID:19058170

  13. Aqueous enzymatic extraction of Moringa oleifera oil.

    PubMed

    Mat Yusoff, Masni; Gordon, Michael H; Ezeh, Onyinye; Niranjan, Keshavan

    2016-11-15

    This paper reports on the extraction of Moringa oleifera (MO) oil by using aqueous enzymatic extraction (AEE) method. The effect of different process parameters on the oil recovery was discovered by using statistical optimization, besides the effect of selected parameters on the formation of its oil-in-water cream emulsions. Within the pre-determined ranges, the use of pH 4.5, moisture/kernel ratio of 8:1 (w/w), and 300stroke/min shaking speed at 40°C for 1h incubation time resulted in highest oil recovery of approximately 70% (goil/g solvent-extracted oil). These optimized parameters also result in a very thin emulsion layer, indicating minute amount of emulsion formed. Zero oil recovery with thick emulsion were observed when the used aqueous phase was re-utilized for another AEE process. The findings suggest that the critical selection of AEE parameters is key to high oil recovery with minimum emulsion formation thereby lowering the load on the de-emulsification step. PMID:27283648

  14. The enzymatic synthesis of rubber polymer

    SciTech Connect

    Venkatachalam, K.V.; Wooten, L.; Benedict, C.R. )

    1990-05-01

    Washed rubber particles (WRP) isolated from stem homogenates of Parthenium argentatum by ultracentrifugation and gel filtration on columns of LKB Ultrogel AcA34 contain a tightly bound rubber transferase which catalyzes the polymerization of IPP into rubber polymer. The polymerization reaction requires Mg{sup 2+}, IPP and an allylic-PP. The Km values for Mg{sup 2+}, IPP and DMAPP are 5.2{times}10{sup {minus}4}M, 8.3{times}10{sup {minus}5} M and 9.6{times}10{sup {minus}5}M respectively. Gel permeation chromatography of the enzymatic polymer product on 3 linear columns of 1{times}10{sup 6} to 500 {angstrom} Ultrastyragel shows that the in vitro formed polymer has a similar mol wt to natural rubber. Over 90% of the in vitro formation of the rubber polymer was a de novo polymerization reaction from DMAPP initiator and IPP monomers. The bound rubber polymerase substantially differs from cytosolic rubber transferase which catalyzes only chain lengthening reactions. Treatment of the WRP with Chaps solubilized the bound rubber transferase which was further purified by DEAE-cellulose chromatography. The purified preparation primarily consists of a 52 kD polypeptide which binds to a photolabile substrate analog. The soluble rubber transferase catalyzes the synthesis of a 1{times}10{sup 5} mol wt rubber polymer from Mg{sup 2+}, DMAPP, IPP and detergent.

  15. Method for the enzymatic production of hydrogen

    DOEpatents

    Woodward, Jonathan; Mattingly, Susan M.

    1999-01-01

    The present invention is an enzymatic method for producing hydrogen comprising the steps of: a) forming a reaction mixture within a reaction vessel comprising a substrate capable of undergoing oxidation within a catabolic reaction, such as glucose, galactose, xylose, mannose, sucrose, lactose, cellulose, xylan and starch. The reaction mixture further comprises an amount of glucose dehydrogenase in an amount sufficient to catalyze the oxidation of the substrate, an amount of hydrogenase sufficient to catalyze an electron-requiring reaction wherein a stoichiometric yield of hydrogen is produced, an amount of pH buffer in an amount sufficient to provide an environment that allows the hydrogenase and the glucose dehydrogenase to retain sufficient activity for the production of hydrogen to occur and also comprising an amount of nicotinamide adenine dinucleotide phosphate sufficient to transfer electrons from the catabolic reaction to the electron-requiring reaction; b) heating the reaction mixture at a temperature sufficient for glucose dehydrogenase and the hydrogenase to retain sufficient activity and sufficient for the production of hydrogen to occur, and heating for a period of time that continues until the hydrogen is no longer produced by the reaction mixture, wherein the catabolic reaction and the electron-requiring reactions have rates of reaction dependent upon the temperature; and c) detecting the hydrogen produced from the reaction mixture.

  16. Method for the enzymatic production of hydrogen

    DOEpatents

    Woodward, J.; Mattingly, S.M.

    1999-08-24

    The present invention is an enzymatic method for producing hydrogen comprising the steps of: (a) forming a reaction mixture within a reaction vessel comprising a substrate capable of undergoing oxidation within a catabolic reaction, such as glucose, galactose, xylose, mannose, sucrose, lactose, cellulose, xylan and starch; the reaction mixture also comprising an amount of glucose dehydrogenase in an amount sufficient to catalyze the oxidation of the substrate, an amount of hydrogenase sufficient to catalyze an electron-requiring reaction wherein a stoichiometric yield of hydrogen is produced, an amount of pH buffer in an amount sufficient to provide an environment that allows the hydrogenase and the glucose dehydrogenase to retain sufficient activity for the production of hydrogen to occur and also comprising an amount of nicotinamide adenine dinucleotide phosphate sufficient to transfer electrons from the catabolic reaction to the electron-requiring reaction; (b) heating the reaction mixture at a temperature sufficient for glucose dehydrogenase and the hydrogenase to retain sufficient activity and sufficient for the production of hydrogen to occur, and heating for a period of time that continues until the hydrogen is no longer produced by the reaction mixture, wherein the catabolic reaction and the electron-requiring reactions have rates of reaction dependent upon the temperature; and (c) detecting the hydrogen produced from the reaction mixture. 8 figs.

  17. Enzymatic production of hydrogen from glucose

    SciTech Connect

    Woodward, J.; Mattingly, S.M.

    1995-06-01

    The objective of this research is to optimize conditions for the enzymatic production of hydrogen gas from biomass-derived glucose. This new project is funded at 0.5 PY level of effort for FY 1995. The rationale for the work is that cellulose is, potentially, a vast source of hydrogen and that enzymes offer a specific and efficient method for its extraction with minimal environmental impact. This work is related to the overall hydrogen program goal of technology development and validation. The approach is based on knowledge that glucose is oxidized by the NADP{sup +} requiring enzyme glucose dehydrogenase (GDH) and that the resulting NADPH can donate its electrons to hydrogenase (H{sub 2}ase) which catalyzes the evolution of H{sub 2}. Thus hydrogen production from glucose was achieved using calf liver GDH and Pyrococcus furiosus H{sub 2}ase yielding 17% of theoretical maximum expected. The cofactor NADP{sup +} for this reaction was regenerated and recycled. Current and future work includes understanding the rate limiting steps of this process and the stabilization/immobilization of the enzymes for long term hydrogen production. Cooperative interactions with the Universities of Georgia and Bath for obtaining thermally stable enzymes are underway.

  18. Enzymatic production of hydrogen from glucose

    NASA Astrophysics Data System (ADS)

    Woodward, J.; Mattingly, S. M.

    The objective of this research is to optimize conditions for the enzymatic production of hydrogen gas from biomass-derived glucose. This new project is funded at 0.5 PY level of effort for FY 1995. The rationale for the work is that cellulose is, potentially, a vast source of hydrogen and that enzymes offer a specific and efficient method for its extraction with minimal environmental impact. This work is related to the overall hydrogen program goal of technology development and validation. The approach is based on knowledge that glucose is oxidized by the NADP(sup +) requiring enzyme glucose dehydrogenase (GDH) and that the resulting NADPH can donate its electrons to hydrogenase (H2ase) which catalyzes the evolution of H2. Thus hydrogen production from glucose was achieved using calf liver GDH and Pyrococcus furiosus H2ase yielding 17% of theoretical maximum expected. The cofactor NADP(sup +) for this reaction was regenerated and recycled. Current and future work includes understanding the rate limiting steps of this process and the stabilization/immobilization of the enzymes for long term hydrogen production. Cooperative interactions with the Universities of Georgia and Bath for obtaining thermally sta

  19. Nanoparticle Mediated Remote Control of Enzymatic Activity

    PubMed Central

    Knecht, Leslie D.; Ali, Nur; Wei, Yinan; Hilt, J. Zach; Daunert, Sylvia

    2012-01-01

    Nanomaterials have found numerous applications as tunable, remotely controlled platforms for drug delivery, hyperthermia cancer treatment, and various other biomedical applications. The basis for the interest lies in their unique properties achieved at the nanoscale that can be accessed via remote stimuli. These properties could then be exploited to simultaneously activate secondary systems that are not remotely actuatable. In this work, iron oxide nanoparticles are encapsulated in a bisacrylamide-crosslinked polyacrylamide hydrogel network along with a model dehalogenase enzyme, L-2-HADST. This thermophilic enzyme is activated at elevated temperatures and has been shown to have optimal activity at 70 °C. By exposing the Fe3O4 nanoparticles to a remote stimulus, an alternating magnetic field (AMF), enhanced system heating can be achieved, thus remotely activating the enzyme. The internal heating of the nanocomposite hydrogel network in the AMF results in a 2-fold increase in enzymatic activity as compared to the same hydrogel heated externally in a water bath, suggesting that the internal heating of the nanoparticles is more efficient than the diffusion limited heating of the water bath. This system may prove useful for remote actuation of biomedical and environmentally relevant enzymes and find applications in a variety of fields. PMID:22989219

  20. 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.

  1. 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

  2. Methylation of the mouse hprt gene differs on the active and inactive X chromosomes.

    PubMed Central

    Lock, L F; Melton, D W; Caskey, C T; Martin, G R

    1986-01-01

    It has been proposed that DNA methylation is involved in the mechanism of X inactivation, the process by which equivalence of levels of X-linked gene products is achieved in female (XX) and male (XY) mammals. In this study, Southern blots of female and male DNA digested with methylation-sensitive restriction endonucleases and hybridized to various portions of the cloned mouse hprt gene were compared, and sites within the mouse hprt gene were identified that are differentially methylated in female and male cells. The extent to which these sites are methylated when carried on the active and inactive X chromosomes was directly determined in a similar analysis of DNA from clonal cell lines established from a female embryo derived from a mating of two species of mouse, Mus musculus and Mus caroli. The results revealed two regions of differential methylation in the mouse hprt gene. One region, in the first intron of the gene, includes four sites that are completely unmethylated when carried on the active X and extensively methylated when carried on the inactive X. These same sites are extensively demethylated in hprt genes reactivated either spontaneously or after 5-azacytidine treatment. The second region includes several sites in the 3' 20kilobases of the gene extending from exon 3 to exon 9 that show the converse pattern; i.e., they are completely methylated when carried on the active X and completely unmethylated when carried on the inactive X. At least one of these sites does not become methylated after reactivation of the gene. The results of this study, together with the results of previous studies by others of the human hprt gene, indicate that these regions of differential methylation on the active and inactive X are conserved between mammalian species. Furthermore, the data described here are consistent with the idea that at least the sites in the 5' region of the gene play a role in the X inactivation phenomenon and regulation of expression of the mouse hprt

  3. Identification and characterization of a bacterial hyaluronidase and its production in recombinant form.

    PubMed

    Messina, Luciano; Gavira, Jose A; Pernagallo, Salvatore; Unciti-Broceta, Juan D; Sanchez Martin, Rosario M; Diaz-Mochon, Juan J; Vaccaro, Susanna; Conejero-Muriel, Mayte; Pineda-Molina, Estela; Caruso, Salvatore; Musumeci, Luca; Di Pasquale, Roberta; Pontillo, Angela; Sincinelli, Francesca; Pavan, Mauro; Secchieri, Cynthia

    2016-07-01

    Hyaluronidases (Hyals) are broadly used in medical applications to facilitate the dispersion and/or absorption of fluids or medications. This study reports the isolation, cloning, and industrial-scale recombinant production, purification and full characterization, including X-ray structure determination at 1.45 Å, of an extracellular Hyal from the nonpathogenic bacterium Streptomyces koganeiensis. The recombinant S. koganeiensis Hyal (rHyal_Sk) has a novel bacterial catalytic domain with high enzymatic activity, compared with commercially available Hyals, and is more thermostable and presents higher proteolytic resistance, with activity over a broad pH range. Moreover, rHyal_Sk exhibits remarkable substrate specificity for hyaluronic acid (HA) and poses no risk of animal cross-infection. PMID:27311405

  4. A catalytically-inactive snake venom Lys49 phospholipase A₂ homolog induces expression of cyclooxygenase-2 and production of prostaglandins through selected signaling pathways in macrophages.

    PubMed

    Moreira, Vanessa; de Castro Souto, Pollyana Cristina Maggio; Ramirez Vinolo, Marco Aurélio; Lomonte, Bruno; María Gutiérrez, José; Curi, Rui; Teixeira, Catarina

    2013-05-15

    The effects of a snake venom Lys-49 phospholipase A2 (PLA2) homolog named MT-II, devoid of enzymatic activity, on the biosynthesis of prostaglandins and protein expression of cyclooxygenase-2 (COX-2) and signaling pathways involved were evaluated in mouse macrophages in culture and in peritoneal cells ex vivo. Stimulation of macrophages with MT-II leads to production of prostaglandin D2 (PGD2) and prostaglandin E2 (PGE2) and protein expression of COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1). Inhibition of cytosolic PLA2 (cPLA2), but not Ca(2+) independent PLA2 (iPLA2) reduced release of PGD2 and PGE2 and expression of COX-2 induced by MT-II. Inhibition of nuclear factor κB (NF-κB) significantly reduced MT-II-induced PGE2, but not PGD2 production and COX-2 expression. Inhibitors of either protein kinase C (PKC), protein tyrosine kinase (PTK), or extracellular signal-regulated kinase (ERK) pathways abrogated MT-II-induced NF-κB activation and reduced COX-2 expression and PGE2 release, whereas the p38 mitogen-activated protein kinase (MAPK) inhibitor reduced MT-II-induced COX-2 expression and PGD2 production. Inhibition of phosphatidylinositol-3-kinase (PI3K) pathway abrogated MT-II-induced NF-κB activation, but affected neither prostaglandins production nor COX-2 expression. MT-II-induced production of PGD2 and PGE2 and COX-2 expression were also observed in vivo after intraperitoneal injection into mice. Collectively, our data demonstrate that a catalytically-inactive PLA2 homolog is capable of inducing prostaglandins biosynthesis and COX-2 expression in macrophages in both in vitro and in vivo models, indicating that the enzymatic activity of PLA2 is not necessary to trigger these effects. MT-II-activated NF-κB, cPLA2 and distinct protein kinases are the principal steps involved in these cellular events. PMID:23416211

  5. 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)

  6. Antibacterial activity of recombinant murine beta interferon.

    PubMed Central

    Fujiki, T; Tanaka, A

    1988-01-01

    Recombinant murine beta interferon was protective and therapeutic for mice against Listeria monocytogenes infection in vivo. The recombinant murine beta interferon caused enhanced H2O2 release by macrophages in vivo, but not in vitro. PMID:3343048

  7. Enzymatic production of specifically distributed hyaluronan oligosaccharides.

    PubMed

    Yuan, Panhong; Lv, Mengxian; Jin, Peng; Wang, Miao; Du, Guocheng; Chen, Jian; Kang, Zhen

    2015-09-20

    High-molecular-mass hyaluronan (HA) was controllably depolymerized in pure aqueous solution with recombinant leech hyaluronidase (HAase). The HAase concentration per unit HA and hydrolysis time played important roles in molecular mass distribution. By modulating the concentrations of HAase and controlling the hydrolysis time, any molar-mass-defined HA oligomers could be efficiently and specifically produced on a large scale (40 g/L), such as HA oligosaccharides with weight-average molar mass of 4000, 10,000, and 30,000Da and end hydrolysates containing only HA6 and HA4. High performance liquid chromatography-size exclusion chromatography, polyacrylamide gel electrophoresis, capillary zone electrophoresis, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry confirmed low polydispersity of the produced molar-mass-defined HA oligosaccharides. Therefore, large-scale production of defined HA oligosaccharides with narrow molecular mass distribution will significantly promote progress in related research and its potential applications. PMID:26050905

  8. 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

  9. Recent insights in enzymatic synthesis of fructooligosaccharides from inulin.

    PubMed

    Singh, Ram Sarup; Singh, Rupinder Pal; Kennedy, John F

    2016-04-01

    In the past few years, people are paying more attention to their dietary habits, and functional foods are playing a key role in maintaining the health of man. Prebiotics are considered as a main component of the functional foods which are usually composed of short chains of carbohydrates. Fructooligosaccharides (FOSs) are considered as one of the main group of prebiotics which have recognisable bifidogenic properties. FOSs are obtained either by extraction from various plant materials or by enzymatic synthesis from different substrates. Enzymatically, these can be obtained either from sucrose using fructosyltransferase or from inulin by endoinulinase. Inulin is a potent substrate for the enzymatic production of FOSs. This review article will provide an overview on the inulin as potent substrate, microbial sources of endoinulinases, enzymatic synthesis of FOSs from inulin, commercial status of FOSs, and their future perspectives. PMID:26791586

  10. Enzymatic hydrolysis and recrystallization behavior of initially amorphous cellulose.

    PubMed

    Bertran, M S; Dale, B E

    1985-02-01

    Cellulose samples from cotton and wood pulps with varying low degrees of crystallinity (mechanically decrystallized) were studied. The influence of initial cellulose crystallinity on sugar yield after enzymatic hydrolysis was determined by two different methods. As expected, samples with low crystallinity were much more accessible to enzymatic attack and glucose yields were higher than were samples of high initial crystallinity. Hydrolysis of cellulose seems more dependent on cellulose crystallinity than on the source of cellulose. It is known that decrystallized or amorphous cellulose can recrystallize under proper conditions, e.g., during acid hydrolysis. The data reported here also reveal some recrystallization during enzymatic hydrolysis which probably occurs simulataneously with a selective enzymatic attack on the amorphous regions of cellulose. In all cases, the amorphous celluloses recrystallized in the original lattice form, that of native cellulose. PMID:18553653

  11. [Recombination in Drosophila in space flight].

    PubMed

    Filatova, L P; Vaulina, E N; Lapteva, N Sh; Grozdova, T Ia

    1988-04-01

    An experiment with Drosophila melanogaster males was performed aboard the Artificial Satellite "Kosmos-1667". Mutagenic effects of a 7-day space flight on intergene recombination in chromosome 2 were studied. The space flight factors decreased the frequency of recombination. A model experiment on a laboratory centrifuge demonstrated insignificant increase in recombination frequency caused by acceleration. PMID:3135244

  12. Enzymatically induced motion at nano- and micro-scales.

    PubMed

    Gáspár, Szilveszter

    2014-07-21

    In contrast to adenosine triphosphate (ATP)-dependent motor enzymes, other enzymes are little-known as "motors" or "pumps", that is, for their ability to induce motion. The enhanced diffusive movement of enzyme molecules, the self-propulsion of enzyme-based nanomotors, and liquid pumping with enzymatic micropumps were indeed only recently reported. Enzymatically induced motion can be achieved in mild conditions and without the use of external fields. It is thus better suited for use in living systems (from single-cell to whole-body) than most other ways to achieve motion at small scales. Enzymatically induced motion is thus not only new but also important. Therefore, the present work reviews the most significant discoveries in enzymatically induced motion. As we will learn, freely diffusing enzymes enhance their diffusive movement by nonreciprocal conformational changes which parallel their catalytic cycles. Meanwhile, enzyme-modified nano- and micro-objects turn chemical energy into kinetic energy through mechanisms such as bubble recoil propulsion, self-electrophoresis, and self-diffusiophoresis. Enzymatically induced motion of small objects ranges from enhanced diffusive movement to directed motion at speeds as high as 1 cm s(-1). In spite of the progress made in understanding how the energy of enzyme reactions is turned into motion, most enzymatically powered devices remain inefficient and need improvements before we will witness their application in real world environments. PMID:24931666

  13. Enzymatically induced motion at nano- and micro-scales

    NASA Astrophysics Data System (ADS)

    Gáspár, Szilveszter

    2014-06-01

    In contrast to adenosine triphosphate (ATP)-dependent motor enzymes, other enzymes are little-known as ``motors'' or ``pumps'', that is, for their ability to induce motion. The enhanced diffusive movement of enzyme molecules, the self-propulsion of enzyme-based nanomotors, and liquid pumping with enzymatic micropumps were indeed only recently reported. Enzymatically induced motion can be achieved in mild conditions and without the use of external fields. It is thus better suited for use in living systems (from single-cell to whole-body) than most other ways to achieve motion at small scales. Enzymatically induced motion is thus not only new but also important. Therefore, the present work reviews the most significant discoveries in enzymatically induced motion. As we will learn, freely diffusing enzymes enhance their diffusive movement by nonreciprocal conformational changes which parallel their catalytic cycles. Meanwhile, enzyme-modified nano- and micro-objects turn chemical energy into kinetic energy through mechanisms such as bubble recoil propulsion, self-electrophoresis, and self-diffusiophoresis. Enzymatically induced motion of small objects ranges from enhanced diffusive movement to directed motion at speeds as high as 1 cm s-1. In spite of the progress made in understanding how the energy of enzyme reactions is turned into motion, most enzymatically powered devices remain inefficient and need improvements before we will witness their application in real world environments.

  14. Purification and refolding of recombinant human proMMP-7 (pro-matrilysin) expressed in Escherichia coli and its characterization.

    PubMed

    Itoh, M; Masuda, K; Ito, Y; Akizawa, T; Yoshioka, M; Imai, K; Okada, Y; Sato, H; Seiki, M

    1996-04-01

    Human matrix metalloproteinase-7 (MMP-7 = matrilysin) was overproduced in Escherichia coli as a recombinant zymogen (31 kDa), the C-terminus of which bears artificial hexa-histidines. Most of the enzyme was isolated from the insoluble fraction of the cell lysate and purified by a single step using Ni-NTA resin after solubilization of the precipitates with 8 M urea solution. The resin-bound recombinant protein was refolded into a form that is activatable by p-amino-phenylmercuric acetate in an autocatalytic manner. The activated enzyme cleaved a synthetic peptide substrate at the reported site for MMP-7. Digestion of carboxymethylated transferrin (a natural substrate of MMP-7) by the recombinant proteinase generated fragments with the same peptide map as in the case of native purified MMP-7. The autocatalytic activation and enzyme reaction were entirely dependent on the presence of calcium and zinc ions. The enzyme activity to cleave carboxymethylated transferrin was inhibited by tissue inhibitors of metalloproteinases-1 and -2, MMP-specific inhibitors. The activity of the recombinant MMP-7 was also inhibited by a synthetic peptide derived from a part of the cysteine switch that maintains the zymogen in an inactive state. Thus, we report here a simple means of preparing a large quantity of recombinant proMMP-7 that can be used to study the activation mechanism and to screen synthetic inhibitors. PMID:8743567

  15. Balancing the Expression and Production of a Heterodimeric Protein: Recombinant Agkisacutacin as a Novel Antithrombotic Drug Candidate.

    PubMed

    Guo, Yugang; Wu, Jing; Jia, Hao; Chen, Wei; Shao, Changsheng; Zhao, Lei; Ma, Jiajia; Li, Rui; Zhong, Yongjun; Fang, Fang; Wang, Dong; Sun, Jie; Qian, Fang; Dai, Xiangrong; Zhang, Guohui; Tian, Zhigang; Xiaoyi Li, Benjamin; Xiao, Weihua

    2015-01-01

    Agkisacucetin extracted from the venom of Agkistrodon acutus has been demonstrated to be a promising antithrombotic drug candidate in clinical studies due to its function as a novel platelet membrane glycoprotein (GP) Ib inhibitor. Agkisacucetin is a heterodimeric protein composed of α- and β-subunits with seven disulphide bonds. Both subunits form inactive homodimeric products, which cause difficulties for recombinant production. In this study, Agkisacucetin α- and β-subunits were inserted sequentially into the chromosome of Pichia pastoris at the mutant histidinol dehydrogenase gene and ribosomal DNA repeat sites, respectively. By optimizing the gene copies and productivity of each subunit by drug screening, we successfully obtained a recombinant strain with balanced expression of the two subunits. Using this strain, a yield greater than 100 mg/L recombinant Agkisacucetin in fed-batch fermentation was reached. The recombinant Agkisacucetin possessed extremely similar binding affinity to recombinant GPIb and human platelets in in vitro assays, and its ristocetin-induced platelet aggregation activity ex vivo was identical to that of the extracted native Agkisacucetin, demonstrating that the yeast-derived Agkisacucetin could be an effective alternative to native Agkisacucetin. Moreover, this study provides an effective strategy for balancing the expression and production of heterodimeric proteins in P. pastoris. PMID:26144864

  16. Detection of (In)activity Periods in Human Body Motion Using Inertial Sensors: A Comparative Study

    PubMed Central

    Olivares, Alberto; Ramírez, Javier; Górriz, Juan M.; Olivares, Gonzalo; Damas, Miguel

    2012-01-01

    Determination of (in)activity periods when monitoring human body motion is a mandatory preprocessing step in all human inertial navigation and position analysis applications. Distinction of (in)activity needs to be established in order to allow the system to recompute the calibration parameters of the inertial sensors as well as the Zero Velocity Updates (ZUPT) of inertial navigation. The periodical recomputation of these parameters allows the application to maintain a constant degree of precision. This work presents a comparative study among different well known inertial magnitude-based detectors and proposes a new approach by applying spectrum-based detectors and memory-based detectors. A robust statistical comparison is carried out by the use of an accelerometer and angular rate signal synthesizer that mimics the output of accelerometers and gyroscopes when subjects are performing basic activities of daily life. Theoretical results are verified by testing the algorithms over signals gathered using an Inertial Measurement Unit (IMU). Detection accuracy rates of up to 97% are achieved. PMID:22778613

  17. 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. PMID:25111015

  18. Active and Inactive Metabolic Pathways in Tumor Spheroids: Determination by GC-MS

    PubMed Central

    Hunnewell, Michael; Forbes, Neil S.

    2016-01-01

    Active metabolic pathways in three-dimensional cancer-cell cultures are potential chemotherapeutic targets that would be effective throughout tumors. Chaotic vasculature creates cellular regions in tumors with distinct metabolic behavior that are only present in aggregate cell masses. To quantify cancer cell metabolism, transformed mouse fibroblasts were grown as spheroids and fed isotopically labeled culture medium. Metabolite uptake and production rates were measured as functions of time. Gas chromatography - mass spectrometry was used quantify the extent of labeling on amino acids present in cytoplasmic extracts. The labeling pattern identified several active and inactive metabolic pathways: glutaminolysis was found to be active, and malic enzyme and gluconeogenesis were inactive. Transformed cells in spheroids were also found to actively synthesize serine, cysteine, alanine, aspartate, glutamate, and proline; and not synthesize glutamine. The activities of these pathways suggest that cancer cells consume glutamine for biosynthesis and not to provide cellular energy. Determining active metabolic pathways indicates how cells direct carbon flow and may lead to the discovery of novel molecular targets for anti-cancer therapy. PMID:20014107

  19. 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

  20. 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

  1. Imprinting control regions (ICRs) are marked by mono-allelic bivalent chromatin when transcriptionally inactive

    PubMed Central

    Maupetit-Méhouas, Stéphanie; Montibus, Bertille; Nury, David; Tayama, Chiharu; Wassef, Michel; Kota, Satya K.; Fogli, Anne; Cerqueira Campos, Fabiana; Hata, Kenichiro; Feil, Robert; Margueron, Raphael; Nakabayashi, Kazuhiko; Court, Franck; Arnaud, Philippe

    2016-01-01

    Parental allele-specific expression of imprinted genes is mediated by imprinting control regions (ICRs) that are constitutively marked by DNA methylation imprints on the maternal or paternal allele. Mono-allelic DNA methylation is strictly required for the process of imprinting and has to be faithfully maintained during the entire life-span. While the regulation of DNA methylation itself is well understood, the mechanisms whereby the opposite allele remains unmethylated are unclear. Here, we show that in the mouse, at maternally methylated ICRs, the paternal allele, which is constitutively associated with H3K4me2/3, is marked by default by H3K27me3 when these ICRs are transcriptionally inactive, leading to the formation of a bivalent chromatin signature. Our data suggest that at ICRs, chromatin bivalency has a protective role by ensuring that DNA on the paternal allele remains unmethylated and protected against spurious and unscheduled gene expression. Moreover, they provide the proof of concept that, beside pluripotent cells, chromatin bivalency is the default state of transcriptionally inactive CpG island promoters, regardless of the developmental stage, thereby contributing to protect cell identity. PMID:26400168

  2. Interaction of anammox bacteria and inactive methanogenic granules under high nitrogen selective pressure.

    PubMed

    Ni, Shou-Qing; Fessehaie, Anania; Lee, Po-Heng; Gao, Bao-Yu; Xu, Xing; Sung, Shihwu

    2010-09-01

    Granular anammox reactors usually adopted anaerobic/aerobic granules as source sludge, in which the washout of other species and enrichment of anammox biomass were very slow because of the competition of the coexisting bacteria. In this study, inactive methanogenic granules were proved to be suitable for rapid anammox granulation under high nitrogen concentrations by investigating their interaction with anammox bacteria. The start-up nitrite concentration was significantly higher than the published toxic level for anammox bacteria and other lab-scale studies. The nitrogen loading rate increased from 141 to 480 mg/L/d in 120 days operation with a total nitrogen removal efficiency of 96.0+/-0.6%. Anammox granules with a diameter of 1.3+/-0.4mm were observed over the course of three months. Molecular analysis showed that over 67% of the cells in the anammox granules were anammox bacteria after 90 days. The accommodations and proliferations of anammox bacteria in the inactive methanogenic granules might be the main reason for the high anammox purity in a short period. The important role of the extracellular polymer in the granule structure was observed via morphological observation. PMID:20444597

  3. Dominant lethality by expression of a catalytically inactive class I tRNA synthetase.

    PubMed Central

    Schmidt, E; Schimmel, P

    1993-01-01

    Alignment-guided mutagenesis was used to create an inactive, but toxic, aminoacyl-tRNA synthetase. An Asp-96-->Ala (D96A) replacement in the nucleotide binding fold of the class I Escherichia coli isoleucyl-tRNA synthetase inactivates the enzyme without disrupting its competence for binding isoleucine tRNA. Expression of plasmid-encoded mutant enzyme in a cell with a wild-type ileS chromosomal allele resulted in cell death. Introduction of a second K732T substitution previously shown to weaken tRNA binding gives an inactive D96A/K732T double mutant. Expression of the double mutant is not lethal to E. coli. D96A but not the double mutant significantly inhibited in vitro charging of isoleucine tRNA by the wild-type enzyme. The results suggest a dominant tRNA binding-dependent arrest of cell growth caused by a reduction in the pool of a specific tRNA. Specific tRNA binding drugs may have therapeutic applications for treatment of microbial pathogens. Images Fig. 1 Fig. 3 PMID:8346197

  4. 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

  5. Overweight, obesity, and inactivity and urban design in rapidly growing Chinese cities.

    PubMed

    Day, Kristen; Alfonzo, Mariela; Chen, Yufei; Guo, Zhan; Lee, Karen K

    2013-05-01

    China faces rising rates of overweight, obesity, and physical inactivity among its citizens. Risk is highest in China's rapidly growing cities and urban populations. Current urban development practices and policies in China heighten this risk. These include policies that support decentralization in land use planning; practices of neighborhood gating; and policies and practices tied to motor vehicle travel, transit planning, and bicycle and pedestrian infrastructure. In this paper, we review cultural, political, and economic issues that influence overweight, obesity, and inactivity in China. We examine key urban planning features and policies that shape urban environments that may compromise physical activity as part of everyday life, including walking and bicycling. We review the empirical research to identify planning and design strategies that support physical activity in other high-density cities in developing and developed countries. Finally, we identify successful strategies to increase physical activity in another growing, high-density city - New York City - to suggest strategies that may have relevance for rapidly urbanizing Chinese cities. PMID:23416231

  6. 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

  7. 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.

  8. Heterometallic Triiron-Oxo/Hydroxo Clusters: Effect of Redox-Inactive Metals

    PubMed Central

    Herbert, David E.; Lionetti, Davide; Rittle, Jonathan; Agapie, Theodor

    2014-01-01

    A series of tetranuclear oxo/hydroxo clusters comprised of three Fe centers and a redox-inactive metal (M) of various charge is reported. Crystallographic studies show an unprecedented Fe3M(μ4-O)(μ2-OH) core that remains intact upon changing M or the oxidation state of iron. Electrochemical studies reveal that the reduction potentials (E1/2) span a window of 500 mV and depend upon the Lewis acidity of M. Using the pKa of the redox-inactive metal aqua complex as a measure of Lewis acidity, these compounds display a linear dependence between E1/2 and acidity with a slope of ca. 70 mV per pKa unit. The current study of [Fe3MO(OH)] and previous ones of [Mn3MOn] (n = 2, 4) moieties support the generality of the above relationship between the reduction potentials of heterometallic oxido clusters and the Lewis acidity of incorporated cations, as applied to clusters of different redox-active metals. PMID:24304416

  9. Enzymatic reduction of azo and indigoid compounds.

    PubMed

    Pricelius, S; Held, C; Murkovic, M; Bozic, M; Kokol, V; Cavaco-Paulo, A; Guebitz, G M

    2007-11-01

    A customer- and environment-friendly method for the decolorization azo dyes was developed. Azoreductases could be used both to bleach hair dyed with azo dyes and to reduce dyes in vat dyeing of textiles. A new reduced nicotinamide adenine dinucleotide-dependent azoreductase of Bacillus cereus, which showed high potential for reduction of these dyes, was purified using a combination of ammonium sulfate precipitation and chromatography and had a molecular mass of 21.5 kDa. The optimum pH of the azoreductase depended on the substrate and was within the range of pH 6 to 7, while the maximum temperature was reached at 40 degrees C. Oxygen was shown to be an alternative electron acceptor to azo compounds and must therefore be excluded during enzymatic dye reduction. Biotransformation of the azo dyes Flame Orange and Ruby Red was studied in more detail using UV-visible spectroscopy, high-performance liquid chromatography, and mass spectrometry (MS). Reduction of the azo bonds leads to cleavage of the dyes resulting in the cleavage product 2-amino-1,3 dimethylimidazolium and N approximately 1 approximately ,N approximately 1 approximately -dimethyl-1,4-benzenediamine for Ruby Red, while only the first was detected for Flame Orange because of MS instability of the expected 1,4-benzenediamine. The azoreductase was also found to reduce vat dyes like Indigo Carmine (C.I. Acid Blue 74). Hydrogen peroxide (H(2)O(2)) as an oxidizing agent was used to reoxidize the dye into the initial form. The reduction and oxidation mechanism of Indigo Carmine was studied using UV-visible spectroscopy. PMID:17891390

  10. Nanocrystal Bioassembly: Asymmetry, Proximity, and Enzymatic Manipulation

    SciTech Connect

    Claridge, Shelley A.

    2008-05-01

    Research at the interface between biomolecules and inorganic nanocrystals has resulted in a great number of new discoveries. In part this arises from the synergistic duality of the system: biomolecules may act as self-assembly agents for organizing inorganic nanocrystals into functional materials; alternatively, nanocrystals may act as microscopic or spectroscopic labels for elucidating the behavior of complex biomolecular systems. However, success in either of these functions relies heavily uponthe ability to control the conjugation and assembly processes.In the work presented here, we first design a branched DNA scaffold which allows hybridization of DNA-nanocrystal monoconjugates to form discrete assemblies. Importantly, the asymmetry of the branched scaffold allows the formation of asymmetric2assemblies of nanocrystals. In the context of a self-assembled device, this can be considered a step toward the ability to engineer functionally distinct inputs and outputs.Next we develop an anion-exchange high performance liquid chromatography purification method which allows large gold nanocrystals attached to single strands of very short DNA to be purified. When two such complementary conjugates are hybridized, the large nanocrystals are brought into close proximity, allowing their plasmon resonances to couple. Such plasmon-coupled constructs are of interest both as optical interconnects for nanoscale devices and as `plasmon ruler? biomolecular probes.We then present an enzymatic ligation strategy for creating multi-nanoparticle building blocks for self-assembly. In constructing a nanoscale device, such a strategy would allow pre-assembly and purification of components; these constructs can also act as multi-label probes of single-stranded DNA conformational dynamics. Finally we demonstrate a simple proof-of-concept of a nanoparticle analog of the polymerase chain reaction.

  11. 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

  12. Selection of Recombinant Human Antibodies.

    PubMed

    Tomszak, Florian; Weber, Susanne; Zantow, Jonas; Schirrmann, Thomas; Hust, Michael; Frenzel, André

    2016-01-01

    Since the development of therapeutic antibodies the demand of recombinant human antibodies is steadily increasing. Traditionally, therapeutic antibodies were generated by immunization of rat or mice, the generation of hybridoma clones, cloning of the antibody genes and subsequent humanization and engineering of the lead candidates. In the last few years, techniques were developed that use transgenic animals with a human antibody gene repertoire. Here, modern recombinant DNA technologies can be combined with well established immunization and hybridoma technologies to generate already affinity maturated human antibodies. An alternative are in vitro technologies which enabled the generation of fully human antibodies from antibody gene libraries that even exceed the human antibody repertoire. Specific antibodies can be isolated from these libraries in a very short time and therefore reduce the development time of an antibody drug at a very early stage.In this review, we describe different technologies that are currently used for the in vitro and in vivo generation of human antibodies. PMID:27236551

  13. Chemical kinetics of geminal recombination

    SciTech Connect

    Levin, P.P.; Khudyakov, I.V.; Brin, E.F.; Kuz'min, V.A.

    1988-09-01

    The kinetics of geminal recombination of triplet radical pairs formed in photoreduction of benzophenone by p-cresol in glycerin solution was studied by pulsed laser photolysis. The experiments were conducted at several temperatures and in a constant magnetic field of H = 0.34 T. The parameters in six kinetic equations describing geminal recombination were determined with a computer. The values of the sums of the squares of the residual deviations of the approximation were obtained. It was found that the kinetics are best described by the functions proposed by Noyes and Shushin. It was shown that it is necessary to use the mutual diffusion coefficient of the radicals, which is significantly smaller than the sum of the estimations of the experimental values of the radical diffusion coefficients, for describing the kinetics due to the correlations of the molecular motions of the radicals in the cage.

  14. Recombination Catalysts for Hypersonic Fuels

    NASA Technical Reports Server (NTRS)

    Chinitz, W.

    1998-01-01

    The goal of commercially-viable access to space will require technologies that reduce propulsion system weight and complexity, while extracting maximum energy from the products of combustion. This work is directed toward developing effective nozzle recombination catalysts for the supersonic and hypersonic aeropropulsion engines used to provide such access to space. Effective nozzle recombination will significantly reduce rk=le length (hence, propulsion system weight) and reduce fuel requirements, further decreasing the vehicle's gross lift-off weight. Two such catalysts have been identified in this work, barium and antimony compounds, by developing chemical kinetic reaction mechanisms for these materials and determining the engine performance enhancement for a typical flight trajectory. Significant performance improvements are indicated, using only 2% (mole or mass) of these compounds in the combustor product gas.

  15. Surface recombination statistics at traps

    NASA Astrophysics Data System (ADS)

    Landsberg, P. T.; Abrahams, M. S.

    1983-09-01

    The Shockley-Read-Hall recombination statistics was recently generalised by Dhariwal, Kothari and Jain to include the effect of a finite time of relaxation before the captured carrier settles into its ground state, and by Landsberg to allow for Auger effects and so-called "extra" carriers supplied to the semiconductor from the outside. The combined result of these effects is studied here theoretically, together with the consideration of a simple distribution of trap states. It is found that the surface recombination velocity s has the usual minimum in the near intrinsic state and that s passes through a maximum as a function of excess electron concentration. Both extrema are enhanced if the trap states are distributed over an energy range. Experimental plots of s as a function of excess electron and hole concentrations should yield insight concerning the numerical importance of (a) Auger effects with the participation of traps and (b) relaxation times.

  16. Non-redundancy of cytidine deaminases in class switch recombination.

    PubMed

    Fugmann, Sebastian D; Rush, James S; Schatz, David G

    2004-03-01

    Class switch recombination (CSR), somatic hypermutation, and gene conversion are immunoglobulin diversification mechanisms that are strictly dependent on the activity of the activation-induced cytidine deaminase (AID). The precise role and substrate(s) of AID in these processes remain to be well defined. The closest homologue of AID is APOBEC-1, a bona fide mRNA-editing enzyme, which shares with AID the ability to deaminate cytidines within single-stranded DNA in vitro and in prokaryotic cells. To determine whether APOBEC-1 can therefore substitute for AID in activated B cells, we expressed human AID, a catalytic mutant thereof, and rat APOBEC-1 in AID-deficient murine B cells. Whereas AID rescued CSR, neither the inactive mutant nor APOBEC-1 could complement AID deficiency. This indicates that cytidine deaminase activity is necessary but not sufficient to initiate CSR, and suggests that AID is specifically targeted to its cognate substrate, the immunoglobulin genes or a distinct mRNA, by an as-yet-unknown mechanism. PMID:14991614

  17. Expression and characterization of a recombinant endoglucanase from western corn rootworm, in Pichia pastoris.

    PubMed

    Valencia Jiménez, Arnubio; Wang, Haichuan; Siegfried, Blair D

    2014-01-01

    The endoglucanase cDNA, Dvv-ENGase I, from western corn rootworm, Diabrotica virgifera virgifera LeConte was expressed using the GS115 methylotrophic strain of Pichia pastoris. The Dvv-ENGase I gene was cloned into the integrative plasmid pPICZαA under the control of AOX1, which is a methanol-inducible promoter. Positive clones were selected for their ability to produce the recombinant endoglucanase upon continuous methanol induction. The secreted recombinant insect endoglucanase Dvv-ENGase I has an apparent molecular mass of 29 kDa. The recombinant endo-1,4-β-glucanase (ENGase) was able to digest the substrates: hydroxyethyl cellulose (HEC), carboxymethyl cellulose (CMC), and Whatman No. 1 filter paper. A higher accumulation of reducing sugar was evident when the P. pastoris expression medium contained HEC (1%) instead of CMC (1%). An enzymatic activity band was detected after performing electrophoretic separation under nondenaturing conditions. The biological activity of the recombinant Dvv-ENGase I was influenced by the presence of protease inhibitors in the culture medium. PMID:25434035

  18. Recombinant Toxins for Cancer Treatment

    NASA Astrophysics Data System (ADS)

    Pastan, Ira; Fitzgerald, David

    1991-11-01

    Recombinant toxins target cell surface receptors and antigens on tumor cells. They kill by mechanisms different from conventional chemotherapy, so that cross resistance to conventional chemotherapeutic agents should not be a problem. Furthermore, they are not mutagens and should not induce secondary malignancies or accelerate progression of benign malignancies. They can be mass-produced cheaply in bacteria as homogeneous proteins. Either growth factor-toxin fusions or antibody-toxin fusions can be chosen, depending on the cellular target.

  19. Introductory experiments in recombinant DNA.

    PubMed

    Tait, R C

    2000-07-01

    Nine practical exercises demonstrate the basic principles in recombinant DNA. The exercises explain the principles that DNA equals genes and that changes in DNA cause changes in genetic properties. The aim is to provide a teaching resource that can be used to illustrate the theory and applications of molecular biology to highschool students, undergraduate students, medics, dentists, doctors, nurses, life scientists, and anyone learning the basics of DNA technology. PMID:11471559

  20. Recombinant vector and eukaryotic host transformed thereby

    SciTech Connect

    Sugden, W.M.

    1987-08-11

    A recombinant plasmid is described comprising: a segment from a first plasmid which is not a lymphotrophic herpes virus segment and which facilitates the replication of the recombinant plasmid in a prokaryotic host; a segment from a lymphotrophic herpes virus which is linked to the first plasmid segment such that is a capable of assisting in maintaining the recombinant plasmid as a plasmid if the recombinant plasmid is inserted into a eukaryotic host that has been transformed by the lymphotrophic herpes virus; and a foreign eukaryotic gene component linked as part of the recombinant plasmid.