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Sample records for enzymatically inactive recombinant

  1. Non-Enzymatic Template-Directed Recombination of RNAs

    PubMed Central

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

    2009-01-01

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

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

    PubMed Central

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

    1992-01-01

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

  3. 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

  4. 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.

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

    PubMed

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

    2015-07-01

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

  6. 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

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

    PubMed

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

    2015-12-01

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

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

    PubMed Central

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

    1991-01-01

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

  9. 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

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

    PubMed

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

    2011-09-01

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

  11. 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.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2014-12-02

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

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

    PubMed

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

    2007-06-01

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

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

    PubMed

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

    2015-05-01

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

  17. 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

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

    PubMed

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

    2014-02-01

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

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

    PubMed

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

    2010-01-01

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

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

    PubMed

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

    2013-08-01

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

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

    PubMed

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

    1998-10-01

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

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

    PubMed

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

    2013-12-01

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

  3. 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.

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

    DOE PAGES

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

    2014-06-27

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

  5. 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

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

    PubMed

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

    2015-08-20

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

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

    PubMed

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

    2012-03-01

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

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

    PubMed Central

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

    2009-01-01

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

  9. 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.

  10. 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.

  11. Inactive and abandoned noncoal mines

    SciTech Connect

    Not Available

    1991-08-01

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

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

    PubMed

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

    2013-03-01

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

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

    PubMed

    Janse, B J; Pretorius, I S

    1995-03-01

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

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

    PubMed

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

    2013-08-01

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

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

    PubMed

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

    2015-07-21

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

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

    PubMed Central

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

    2015-01-01

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

  18. Enzymatic vitreous disruption.

    PubMed

    Gandorfer, A

    2008-10-01

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

  19. Inactive sites and the evolution of cooperation

    NASA Astrophysics Data System (ADS)

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

    2016-10-01

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

  20. 21 CFR 312.45 - Inactive status.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  1. 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…

  2. 21 CFR 201.117 - Inactive ingredients.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

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

  3. 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...

  4. Obesity and Physical Inactivity in Rural America

    ERIC Educational Resources Information Center

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

    2004-01-01

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

  5. 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...

  6. 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.

  7. 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.

  8. 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

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

    PubMed

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

    2016-11-17

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

  10. 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.

  11. 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.

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

    ERIC Educational Resources Information Center

    Piggin, Joe; Bairner, Alan

    2016-01-01

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

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

    PubMed

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

    2016-07-08

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

  14. Enzymatic glycosylation of multivalent scaffolds.

    PubMed

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

    2013-06-07

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

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

    PubMed

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

    2015-09-01

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

  16. Photoelectrochemical enzymatic biosensors.

    PubMed

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

    2017-06-15

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

  17. 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.

  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. Recombinant microorganisms for increased production of organic acids

    DOEpatents

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

    2012-02-21

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

  20. 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...

  1. 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.

  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. 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.

  5. 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.

  6. 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)

  7. Enzymatic synthesis of isotopically labeled isoprenoid diphosphates.

    PubMed

    Christensen, D J; Poulter, C D

    1994-07-01

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

  8. Physical inactivity and arterial stiffness in COPD

    PubMed Central

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

    2015-01-01

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

  9. DNA recombination: the replication connection.

    PubMed

    Haber, J E

    1999-07-01

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

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

    PubMed

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

    2015-01-01

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

  11. 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...

  12. 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...

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

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  14. 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...

  15. 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...

  16. 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

  17. Enzymatic vitreous surgery.

    PubMed

    Trese, M T

    2000-06-01

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

  18. 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.

  19. 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. ...

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

    PubMed

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

    2014-06-01

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

  1. 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)

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

    PubMed

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

    2017-02-07

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

  3. Recombinant allergens

    PubMed Central

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

    2012-01-01

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

  4. Recombinant gonadotropins.

    PubMed

    Lathi, R B; Milki, A A

    2001-10-01

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

  5. 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)

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2011-01-01

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

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

    PubMed

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

    2011-03-11

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

  9. 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.

  10. 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.

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

    PubMed

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

    2017-02-02

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

  12. 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.

  13. 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

  14. [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.

  15. Fluorometric enzymatic assay of L-arginine

    NASA Astrophysics Data System (ADS)

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

    2017-01-01

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

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

    PubMed

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

    2007-09-01

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

  17. Enzymatic Glycosylation by Transferases

    NASA Astrophysics Data System (ADS)

    Blixt, Ola; Razi, Nahid

    Glycosyltransferases are important biological catalysts in cellular systems generating complex cell surface glycans involved in adhesion and signaling processes. Recent advances in glycoscience have increased the demands to access significant amount of glycans representing the glycome. Glycosyltransferases are now playing a key role for in vitro synthesis of oligosaccharides and the bacterial genome are increasingly utilized for cloning and over expression of active transferases in glycosylation reactions. This chapter highlights the recent progress towards preparative synthesis of oligosaccharides representing terminal sequences of glycoproteins and glycolipids using recombinant transferases. Transferases are also being explored in the context of solid-phase synthesis, immobilized on resins and over expression in vivo by engineered bacteria.

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

    ERIC Educational Resources Information Center

    New Hampshire Health Careers Council, Concord.

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

  19. A New Direction for Wisconsin Inactive Health Personnel.

    ERIC Educational Resources Information Center

    Wisconsin Univ., Madison. Univ. Extension.

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  2. 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...

  3. 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...

  4. 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...

  5. 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...

  6. 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...

  7. Identification of inactive medications in narrative medical text.

    PubMed

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

    2008-11-06

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

  8. 30 CFR 57.5045 - Posting of inactive workings.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

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

  9. 30 CFR 57.5045 - Posting of inactive workings.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

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

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

    PubMed

    Long, D M

    1997-05-01

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

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  13. 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

  14. 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.

  15. Enzymatic hydrolysis of spent coffee ground.

    PubMed

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

    2013-04-01

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

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

    PubMed

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

    2001-02-23

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

  17. 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.

  18. Enzymatic degradation of endomorphins.

    PubMed

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

    2008-11-01

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

  19. 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.

  20. 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)

  1. Enzymatic reactions in confined environments.

    PubMed

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

    2016-05-05

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

  2. 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.

  3. Homologous recombination using bacterial artificial chromosomes.

    PubMed

    Lai, Cary; Fischer, Tobias; Munroe, Elizabeth

    2015-02-02

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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. Improving baculovirus recombination

    PubMed Central

    Zhao, Yuguang; Chapman, David A. G.; Jones, Ian M.

    2003-01-01

    Recombinant baculoviruses have established themselves as a favoured technology for the high-level expression of recombinant proteins. The construction of recombinant viruses, however, is a time consuming step that restricts consideration of the technology for high throughput developments. Here we use a targeted gene knockout technology to inactivate an essential viral gene that lies adjacent to the locus used for recombination. Viral DNA prepared from the knockout fails to initiate an infection unless rescued by recombination with a baculovirus transfer vector. Modified viral DNA allows 100% recombinant virus formation, obviates the need for further virus purification and offers an efficient means of mass parallel recombinant formation. PMID:12527795

  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. 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...

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

    PubMed

    Wang, Jincheng; Guo, Chunlei

    2006-12-15

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

  14. 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.

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

    PubMed

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

    2009-08-01

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

  16. 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...

  17. Bioluminescence methods for enzymatic determinations

    DOEpatents

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

    1982-01-01

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

  18. 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.

  19. Enzymatic reactions on immobilised substrates.

    PubMed

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

    2013-08-07

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

  20. Multifractality in intracellular enzymatic reactions.

    PubMed

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

    2006-05-21

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

  1. 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...

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2008-03-01

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

  4. 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...

  5. 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…

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

    PubMed

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

    2016-10-01

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

  7. 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.

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

    PubMed

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

    2010-08-01

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

  9. 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.

  10. Acid mine drainage from inactive eastern coal operations.

    PubMed

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

    1985-03-01

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

  11. 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

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

    PubMed

    Mavromoustakos, T; Papahatjis, D; Laggner, P

    2001-06-06

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

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

    PubMed

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

    2010-05-01

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

  14. Enzymatic synthesis of prebiotic oligosaccharides.

    PubMed

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

    2006-04-01

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

  15. 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.

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

    PubMed

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

    2012-03-01

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

  17. 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

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

    SciTech Connect

    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. 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.

  20. 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

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

    PubMed Central

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

    1984-01-01

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

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

    PubMed

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

    2007-12-15

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

  3. 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.

  4. Mice with Catalytically Inactive Cathepsin A Display Neurobehavioral Alterations

    PubMed Central

    Calhan, O. Y.

    2017-01-01

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

  5. 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

  6. Resurrecting inactive antimicrobial peptides from the lipopolysaccharide trap.

    PubMed

    Mohanram, Harini; Bhattacharjya, Surajit

    2014-01-01

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

  7. EPA Facility Registry Service (FRS): RCRA_INACTIVE

    EPA Pesticide Factsheets

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

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

    PubMed

    Legh-Jones, Hannah; Moore, Spencer

    2012-05-01

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

  9. 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

  10. 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.

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

    PubMed

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

    2014-02-01

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

  12. Biofuel cells: enhanced enzymatic bioelectrocatalysis.

    PubMed

    Meredith, Matthew T; Minteer, Shelley D

    2012-01-01

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

  13. 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

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

    PubMed

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

    2015-08-01

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

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

    PubMed Central

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

    2016-01-01

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

  16. 37 CFR 11.19 - Disciplinary jurisdiction; Jurisdiction to transfer to disability inactive status.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Office following the resignation. (b) Grounds for discipline; Grounds for transfer to disability inactive... pending before the Office, constitute grounds for discipline or grounds for transfer to disability inactive status. (1) Grounds for discipline include: (i) Conviction of a serious crime; (ii) Discipline...

  17. 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...

  18. 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...

  19. 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...

  20. 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...

  1. 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...

  2. 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...

  3. 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...

  4. Enzymatic biotransformation of synthetic dyes.

    PubMed

    Rodríguez-Couto, S

    2009-11-01

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

  5. 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

  6. 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

  7. 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

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

    PubMed Central

    Xiang, Xiaoling; Yang, Yan; Yan, Hai

    2016-01-01

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

  9. 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.

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

    PubMed

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

    2015-10-01

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

  11. Recombinant Baculovirus Isolation.

    PubMed

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

    2016-01-01

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

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

    PubMed

    Albarracin, Dolores; Hart, William

    2011-08-01

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

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

    PubMed Central

    Albarracin, Dolores; Hart, William

    2013-01-01

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

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

    PubMed

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

    2000-09-01

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

  15. Monitoring enzymatic ATP hydrolysis by EPR spectroscopy.

    PubMed

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

    2014-07-14

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

  16. 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.

  17. Enzymatic disease of the podocyte

    PubMed Central

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

    2014-01-01

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

  18. Physical Inactivity Differentially Alters Dietary Oleate and Palmitate Trafficking

    PubMed Central

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

    2009-01-01

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

  19. 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...

  20. 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 ...

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

    PubMed Central

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

    2016-01-01

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

  2. 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

  3. The landscape of recombination in African Americans.

    PubMed

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

    2011-07-20

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

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

    PubMed

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

    2010-04-02

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2017-02-14

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

  7. Dissociative recombination in aeronomy

    NASA Technical Reports Server (NTRS)

    Fox, J. L.

    1989-01-01

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

  8. 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.

  9. 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.

  10. 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.

  11. Multiphoton Assisted Recombination

    NASA Astrophysics Data System (ADS)

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

    2008-12-01

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

  12. 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.

  13. 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.

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

    PubMed

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

    2008-09-01

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

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

    PubMed

    Albarracin, Dolores; Hepler, Justin; Tannenbaum, Melanie

    2011-04-01

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

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

    PubMed Central

    Albarracin, Dolores; Hepler, Justin; Tannenbaum, Melanie

    2013-01-01

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

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

    PubMed Central

    Hepler, Justin; Albarracin, Dolores; McCulloch, Kathleen C.; Noguchi, Kenji

    2013-01-01

    Although self-control often requires behavioral inaction (i.e., not eating a piece of cake), the process of inhibiting impulsive behavior is commonly characterized as cognitively active (i.e., actively exerting self-control). Two experiments examined whether motivation for action or inaction facilitates self-control behavior in the presence of tempting stimuli. Experiment 1 used a delay discounting task to assess the ability to delay gratification with respect to money. Experiment 2 used a Go/No-Go task to assess the ability to inhibit a dominant but incorrect motor response to the words “condom” and “sex”. The results demonstrate that goals for inaction promote self-control, whereas goals for action promote impulsive behavior. These findings are discussed in light of recent evidence suggesting that goals for action and inaction modulate physiological resources that promote behavioral execution. PMID:23766548

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

    PubMed

    Hepler, Justin; Albarracin, Dolores; McCulloch, Kathleen C; Noguchi, Kenji

    2012-12-01

    Although self-control often requires behavioral inaction (i.e., not eating a piece of cake), the process of inhibiting impulsive behavior is commonly characterized as cognitively active (i.e., actively exerting self-control). Two experiments examined whether motivation for action or inaction facilitates self-control behavior in the presence of tempting stimuli. Experiment 1 used a delay discounting task to assess the ability to delay gratification with respect to money. Experiment 2 used a Go/No-Go task to assess the ability to inhibit a dominant but incorrect motor response to the words "condom" and "sex". The results demonstrate that goals for inaction promote self-control, whereas goals for action promote impulsive behavior. These findings are discussed in light of recent evidence suggesting that goals for action and inaction modulate physiological resources that promote behavioral execution.

  19. 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

  20. 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...

  1. 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...

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

    PubMed

    Aladjem, Mirit I; Fu, Haiqing

    2014-06-01

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

  3. 2014 consensus statement from the first Economics of Physical Inactivity Consensus (EPIC) conference (Vancouver).

    PubMed

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

    2014-06-01

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

  4. 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

  5. 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

  6. Light saturation response of inactive photosystem II reaction centers in spinach.

    PubMed

    Chylla, R A; Whitmarsh, J

    1990-07-01

    The effective absorption cross section of inactive photosystem II (PS II) centers, which is the product of the effective antenna size and the quantum yield for photochemistry, was investigated by comparing the light saturation curves of inactive PS II and active reaction centers in intact chloroplasts and thylakoid membranes of spinach (Spinacia oleracea). Inactive PS II centers are defined as the impaired PS II reaction centers that require greater than 50 ms for the reoxidation of QA (-) subsequent to a single turnover flash. Active reaction centers are defined as the rapidly turning over PS II centers (recovery time less than 50 ms) and all of the PS I centers. The electrochromic shift, measured by the flash-induced absorbance increase at 518 nm, was used to probe the activity of the reaction centers. Light saturation curves were generated for inactive PS II centers and active reaction centers by measuring the extent of the absorbance increase at 518 nm induced by red actinic flashes of variable energy. The light saturation curves show that inactive PS II centers required over twice as many photons as active reaction centers to achieve the same yield. The ratio of the flash energy required for 50% saturation for active reaction centers (PS II active + PS I) compared to inactive PS II centers was 0.45±0.04 in intact chloroplasts, and 0.54±0.11 in thylakoid membranes. Analysis of the light saturation curves using a Poisson statistical model in which the ratio of the antenna size of active PS II centers to that of PS I is considered to range from 1 to 1.5, indicates that the effective absorption cross section of inactive PS II centers was 0.54-0.37 times that of active PS II centers. If the quantum yield for photochemistry is assumed to be one, we estimate that the antenna system serving the inactive PS II centers contains approx. 110 chlorophyll molecules.

  7. 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.

  8. Enzymatic Reactions in Microfluidic Devices

    NASA Astrophysics Data System (ADS)

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

    2008-11-01

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

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

    PubMed Central

    Chaligné, Ronan; Popova, Tatiana; Mendoza-Parra, Marco-Antonio; Saleem, Mohamed-Ashick M.; Gentien, David; Ban, Kristen; Piolot, Tristan; Leroy, Olivier; Mariani, Odette

    2015-01-01

    Disappearance of the Barr body is considered a hallmark of cancer, although whether this corresponds to genetic loss or to epigenetic instability and transcriptional reactivation is unclear. Here we show that breast tumors and cell lines frequently display major epigenetic instability of the inactive X chromosome, with highly abnormal 3D nuclear organization and global perturbations of heterochromatin, including gain of euchromatic marks and aberrant distributions of repressive marks such as H3K27me3 and promoter DNA methylation. Genome-wide profiling of chromatin and transcription reveal modified epigenomic landscapes in cancer cells and a significant degree of aberrant gene activity from the inactive X chromosome, including several genes involved in cancer promotion. We demonstrate that many of these genes are aberrantly reactivated in primary breast tumors, and we further demonstrate that epigenetic instability of the inactive X can lead to perturbed dosage of X-linked factors. Taken together, our study provides the first integrated analysis of the inactive X chromosome in the context of breast cancer and establishes that epigenetic erosion of the inactive X can lead to the disappearance of the Barr body in breast cancer cells. This work offers new insights and opens up the possibility of exploiting the inactive X chromosome as an epigenetic biomarker at the molecular and cytological levels in cancer. PMID:25653311

  10. 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...

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-09-02

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

  13. Accelerated progression of chronic lymphocytic leukemia in Eμ-TCL1 mice expressing catalytically inactive RAG1.

    PubMed

    Nganga, Vincent K; Palmer, Victoria L; Naushad, Hina; Kassmeier, Michele D; Anderson, Dirk K; Perry, Greg A; Schabla, Nathan M; Swanson, Patrick C

    2013-05-09

    Chronic lymphocytic leukemia (CLL) is a prevalent B-cell neoplasia that is often preceded by a more benign monoclonal CD5(+) B-cell lymphocytosis. We previously generated transgenic mice expressing catalytically inactive RAG1 (dominant-negative recombination activating gene 1 [dnRAG1] mice) that develop an early-onset indolent CD5(+) B-cell lymphocytosis attributed to a defect in secondary V(D)J rearrangements initiated to edit autoreactive B-cell receptor (BCR) specificity. Hypothesizing that CD5(+) B cells in these animals represent potential CLL precursors, we crossed dnRAG1 mice with CLL-prone Eμ-TCL1 mice to determine whether dnRAG1 expression in Eμ-TCL1 mice accelerates CLL onset. Consistent with this hypothesis, CD5(+) B-cell expansion and CLL progression occurred more rapidly in double-transgenic mice compared with Eμ-TCL1 mice. Nevertheless, CD5(+) B cells in the 2 mouse strains exhibited close similarities in phenotype, immunoglobulin gene usage, and mutation status, and expression of genes associated with immune tolerance and BCR signaling. Gene expression profiling further revealed a potential role for prolactin signaling in regulating BCR editing. These results suggest a model in which benign accumulation of CD5(+) B cells can be initiated through a failure to successfully edit autoreactive BCR specificity and may, in turn, progress to CLL upon introduction of additional genetic mutations.

  14. Accelerated progression of chronic lymphocytic leukemia in Eμ-TCL1 mice expressing catalytically inactive RAG1

    PubMed Central

    Nganga, Vincent K.; Palmer, Victoria L.; Naushad, Hina; Kassmeier, Michele D.; Anderson, Dirk K.; Perry, Greg A.; Schabla, Nathan M.

    2013-01-01

    Chronic lymphocytic leukemia (CLL) is a prevalent B-cell neoplasia that is often preceded by a more benign monoclonal CD5+ B-cell lymphocytosis. We previously generated transgenic mice expressing catalytically inactive RAG1 (dominant-negative recombination activating gene 1 [dnRAG1] mice) that develop an early-onset indolent CD5+ B-cell lymphocytosis attributed to a defect in secondary V(D)J rearrangements initiated to edit autoreactive B-cell receptor (BCR) specificity. Hypothesizing that CD5+ B cells in these animals represent potential CLL precursors, we crossed dnRAG1 mice with CLL-prone Eμ-TCL1 mice to determine whether dnRAG1 expression in Eμ-TCL1 mice accelerates CLL onset. Consistent with this hypothesis, CD5+ B-cell expansion and CLL progression occurred more rapidly in double-transgenic mice compared with Eμ-TCL1 mice. Nevertheless, CD5+ B cells in the 2 mouse strains exhibited close similarities in phenotype, immunoglobulin gene usage, and mutation status, and expression of genes associated with immune tolerance and BCR signaling. Gene expression profiling further revealed a potential role for prolactin signaling in regulating BCR editing. These results suggest a model in which benign accumulation of CD5+ B cells can be initiated through a failure to successfully edit autoreactive BCR specificity and may, in turn, progress to CLL upon introduction of additional genetic mutations. PMID:23502221

  15. 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

  16. Atom Recombination on Surface

    NASA Astrophysics Data System (ADS)

    Kim, Young Chai

    Upon high speed re-entry of the Space Shuttle Orbiter (SSO) through the earth's atmosphere, oxygen and nitrogen atoms produced in the shock wave in front of the SSO recombine on the surface of the SSO, releasing heat. To minimize the rise of surface temperature due to the reaction, surface material of the SSO should have a low recombination probability, gamma, of atoms impinging on it. To design such material, it is necessary to understand the mechanism of atom recombination. With this in mind, gamma values were measured for recombination of O, N, and H atoms in a diffusion tube reactor between 700 and 1250 K (HT), 300 and 700 K (MT), and at 194 K (LT) on silica. The rate of recombination was first order with respect to the atom concentration from LT to HT. The Arrhenius plots, gamma vs. 1/T, were very complex. All observations are explained by assuming a surface with a small fraction of active sites that irreversibly bind chemisorbed atoms. Everything happens as if the active sites were surrounded by collection zones within which all atoms striking the surface are adsorbed reversibly with an assumed sticking probability of unity. These atoms then diffuse on the surface. Some of them reach the active sites where they can recombine with the chemisorbed atoms. At LT, all atoms striking the surface reach the active sites. As a result of desorption at MT, the collection zones shrink with increasing temperature. At HT, only atoms striking active sites directly from the gas phase lead to recombination. An analytical solution of the diffusion-reaction problem obtained for a model where the active sites are distributed uniformly fits with the experimental data from LT to HT. The two novel features of this work are the identification of the active sites on silica for recombination of H on silica at HT as surface OH groups and the suggestion that another kind of active site is responsible for recombination of O and N atoms at HT as well as for H atoms at LT and MT. Although

  17. 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.

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

    PubMed

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

    2012-12-01

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

  19. Adult physical inactivity prevalence in the Muslim world: Analysis of 38 countries

    PubMed Central

    Kahan, David

    2015-01-01

    Objective Physical inactivity surveillance informs policy and treatment options toward meeting the World Health Organization's (WHO) goal of a 10% reduction in its prevalence by 2025. We currently do not know the aggregate prevalence for Muslim-majority countries, many of which have extremely high rates of comorbidities associated with physical inactivity. Method Based on data for 163, 556 persons in 38 Muslim countries that were collected by the Global Physical Activity Questionnaire and the International Physical Activity Questionnaire, unweighted and weighted physical inactivity prevalence estimates were calculated. I used two-proportion Z tests to determine gender and ethnic differences within the sample and between the sample and 94 non-Muslim countries and odds ratios to determine the magnitude of significant differences. Results Total physical inactivity prevalence was 32.3% (95% CI: 31.9, 32.7). Prevalence among males and females was 28.8% and 35.5%, respectively. Prevalence among non-Arabs and Arabs was 28.6% and 43.7%, respectively. Females and Arabs were more likely physically inactive than their respective counterparts [OR = 1.36 (1.33, 1.39) and OR = 1.94 (1.90, 1.98)]. Muslim countries were more likely physically inactive [OR = 1.23 (1.22, 1.25)] than non-Muslim ones, which was primarily due to the influence of Arabs [OR = 2.01 (1.97, 2.04)], and in particular female Arabs [OR = 2.22 (2.17, 2.27)]. Conclusion Physical inactivity prevalence in the Muslim world is higher than non-Muslim countries and the difference is primarily due to higher rates among Arabs. PMID:26844051

  20. Exploring the Linkage between Activity-Friendly Zoning, Inactivity, and Cancer Incidence in the United States.

    PubMed

    Nicholson, Lisa M; Leider, Julien; Chriqui, Jamie F

    2017-03-07

    Background: Physical activity (PA) protects against cancer and enhances cancer survivorship. Given high inactivity rates nationwide, population-level physical activity facilitators are needed. Several authoritative bodies have recognized that zoning and planning helps create activity-friendly environments. This study examined the association between activity-friendly zoning, inactivity, and cancer in 478 of the most populous U.S. counties.Methods: County geocodes linked county-level data: cancer incidence and smoking (State Cancer Profiles), inactivity (Behavioral Risk Factor Surveillance System), 11 zoning measures (compiled by the study team), and covariates (from the American Community Survey and NAVTEQ). For each zoning measure, single mediation regression models and Sobel tests examined whether activity-friendly zoning was associated with reduced cancer incidence, and whether inactivity mediated those associations. All models were clustered on state with robust SEs and significance at the P < 0.05 level.Results: Zoning for crosswalks, bike-pedestrian connectivity, and bike-pedestrian trails/paths were associated with reduced cancer incidence (β between -0.71 and -1.27, P < 0.05), about 1 case per 100,000 for each 10 percentage-point increase in county population exposure to zoning. Except for crosswalks, each association was mediated by inactivity. However, county smoking attenuated these results, with only crosswalks remaining significant. Results were similar for males (with zoning for bike-pedestrian connectivity, street connectivity, and bike-pedestrian trails/paths), but not females, alone.Conclusions: Zoning can help to create activity-friendly environments that support decreased inactivity, and possibly reduced cancer incidence.Impact: Given low physical activity levels nationwide, cross-sectoral collaborations with urban planning can inform cancer prevention and public health efforts to decrease inactivity and cancer. Cancer Epidemiol Biomarkers Prev

  1. Active-site mutants of beta-lactamase: use of an inactive double mutant to study requirements for catalysis.

    PubMed

    Dalbadie-McFarland, G; Neitzel, J J; Richards, J H

    1986-01-28

    We have studied the catalytic activity and some other properties of mutants of Escherichia coli plasmid-encoded RTEM beta-lactamase (EC 3.5.2.6) with all combinations of serine and threonine residues at the active-site positions 70 and 71. (All natural beta-lactamases have conserved serine-70 and threonine-71.) From the inactive double mutant Ser-70----Thr, Thr-71----Ser [Dalbadie-McFarland, G., Cohen, L. W., Riggs, A. D., Morin, C., Itakura, K., & Richards, J. H. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6409-6413], an active revertant, Thr-71----Ser (i.e., residue 70 in the double mutant had changed from threonine to the serine conserved at position 70 in the wild-type enzyme), was isolated by an approach that allows identification of active revertants in the absence of a background of wild-type enzyme. This mutant (Thr-71----Ser) has about 15% of the catalytic activity of wild-type beta-lactamase. The other possible mutant involving serine and threonine residues at positions 70 and 71 (Ser-70----Thr) shows no catalytic activity. The primary nucleophiles of a serine or a cysteine residue [Sigal, I. S., Harwood, B. G., & Arentzen, R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7157-7160] at position 70 thus seem essential for enzymatic activity. Compared to wild-type enzyme, all three mutants show significantly reduced resistance to proteolysis; for the active revertant (Thr-71----Ser), we have also observed reduced thermal stability and reduced resistance to denaturation by urea.

  2. 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.

  3. 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

  4. A theory for fluidelastic instability of tube-support-plate inactive modes

    SciTech Connect

    Cai, Y.; Chen, S.S. ); Chandra, S. )

    1990-01-01

    Fluidelastic instability of loosely supported tubes, vibrating in a tube support plate (TSP)-inactive mode, is suspected to be one of the main causes of tube failures in some operating steam generators and heat exchangers. A mathematical model, which incorporates all motion-dependent fluid-forces based on the unsteady flow theory, is presented here for fluidelastic instability of loosely supported tubes exposed to nonuniform crossflow. In the unstable region associated with a TSP-inactive mode, the tube motion can be described by two linear models: TSP-inactive mode when tubes do not contact with the TSP and TSP-active mode when tubes contact the TSP. A bilinear model, consisting of these linear models, is presented in this paper to simulate the characteristics of fluidelastic instability of loosely supported tubes in the stable and unstable region associated with TSP-inactive modes. The analytical results are compared with published experimental data; they agree reasonably well. The prediction procedure presented for fluidelastic instability response of loosely supported tubes is applicable in the stable regions of TSP-inactive mode. 25 refs., 10 figs., 2 tabs.

  5. Association between Natural Resources for Outdoor Activities and Physical Inactivity: Results from the Contiguous United States.

    PubMed

    Jiang, Yan; Yuan, Yongping; Neale, Anne; Jackson, Laura; Mehaffey, Megan

    2016-08-17

    Protected areas including national/state parks and recreational waters are excellent natural resources that promote physical activity and interaction with Nature, which can relieve stress and reduce disease risk. Despite their importance, however, their contribution to human health has not been properly quantified. This paper seeks to evaluate quantitatively how national/state parks and recreational waters are associated with human health and well-being, taking into account of the spatial dependence of environmental variables for the contiguous U.S., at the county level. First, we describe available natural resources for outdoor activities (ANROA), using national databases that include features from the Protected Areas Database, NAVSTREETS, and ATTAINSGEO 305(b) Waters. We then use spatial regression techniques to explore the association of ANROA and socioeconomic status factors on physical inactivity rates. Finally, we use variance analysis to analyze ANROA's influence on income-related health inequality. We found a significantly negative association between ANROA and the rate of physical inactivity: ANROA and the spatial effect explained 69%, nationwide, of the variation in physical inactivity. Physical inactivity rate showed a strong spatial dependence-influenced not only by its own in-county ANROA, but also by that of its neighbors ANROA. Furthermore, community groups at the same income level and with the highest ANROA, always had the lowest physical inactivity rate. This finding may help to guide future land use planning and community development that will benefit human health and well-being.

  6. 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

  7. Recombinant renewable polyclonal antibodies.

    PubMed

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

    2015-01-01

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

  8. The dissociative recombination of ?

    NASA Astrophysics Data System (ADS)

    Laubé, S.; Lehfaoui, L.; Rowe, B. R.; Mitchell, J. B. A.

    1998-09-01

    The dissociative recombination rate coefficient for 0953-4075/31/18/016/img2 has been measured at 300 K using a flowing afterglow Langmuir probe-mass spectrometer apparatus. A value of 0953-4075/31/18/016/img3 has been found.

  9. Introduction to dissociative recombination

    NASA Technical Reports Server (NTRS)

    Guberman, Steven L.; Mitchell, J. Brian A.

    1989-01-01

    Dissociative recombination (DR) of molecular ions with electrons has important consequences in many areas of physical science. Ab-initio calculations coupled with resonant scattering theory and multichannel quantum defect studies have produced detailed results illuminating the role of ion vibrational excitation, the quantum yields of the DR products, and the role of Rydberg states. The theoretical and experimental results are discussed.

  10. Recombineering linear BACs.

    PubMed

    Chen, Qingwen; Narayanan, Kumaran

    2015-01-01

    Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.

  11. 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…

  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. 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.

  14. Mental health, places and people: a multilevel analysis of economic inactivity and social deprivation.

    PubMed

    Fone, David L; Dunstan, Frank

    2006-09-01

    Using data on 24,975 respondents to the Welsh Health Survey 1998 aged 17-74 years, we investigated associations between individual mental health status measured using the SF-36 instrument, social class, economic inactivity and the electoral division Townsend deprivation score. In a multilevel modelling analysis, we found mental health was significantly associated with the Townsend score after adjusting for composition, and this effect was strongest in respondents who were economically inactive. Further contextual effects were shown by significant random variability in the slopes of the relation between mental health and economic inactivity at the electoral division level. Our results suggest that the places in which people live affect their mental health, supporting NHS policy that multi-agency planning to reduce inequalities in mental health status should address the wider determinants of health, as well as services for individual patients.

  15. Light saturation curves show competence of the water splitting complex in inactive Photosystem II reaction centers.

    PubMed

    Nedbal, L; Gibas, C; Whitmarsh, J

    1991-12-01

    Photosystem II complexes of higher plants are structurally and functionally heterogeneous. While the only clearly defined structural difference is that Photosystem II reaction centers are served by two distinct antenna sizes, several types of functional heterogeneity have been demonstrated. Among these is the observation that in dark-adapted leaves of spinach and pea, over 30% of the Photosystem II reaction centers are unable to reduce plastoquinone to plastoquinol at physiologically meaningful rates. Several lines of evidence show that the impaired reaction centers are effectively inactive, because the rate of oxidation of the primary quinone acceptor, QA, is 1000 times slower than in normally active reaction centers. However, there are conflicting opinions and data over whether inactive Photosystem II complexes are capable of oxidizing water in the presence of certain artificial electron acceptors. In the present study we investigated whether inactive Photosystem II complexes have a functional water oxidizing system in spinach thylakoid membranes by measuring the flash yield of water oxidation products as a function of flash intensity. At low flash energies (less that 10% saturation), selected to minimize double turnovers of reaction centers, we found that in the presence of the artificial quinone acceptor, dichlorobenzoquinone (DCBQ), the yield of proton release was enhanced 20±2% over that observed in the presence of dimethylbenzoquinone (DMBQ). We argue that the extra proton release is from the normally inactive Photosystem II reaction centers that have been activated in the presence of DCBQ, demonstrating their capacity to oxidize water in repetitive flashes, as concluded by Graan and Ort (Biochim Biophys Acta (1986) 852: 320-330). The light saturation curves indicate that the effective antenna size of inactive reaction centers is 55±12% the size of active Photosystem II centers. Comparison of the light saturation dependence of steady state oxygen evolution

  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.

  17. Changes in diagnosed diabetes, obesity, and physical inactivity prevalence in US counties, 2004-2012

    PubMed Central

    Kirtland, Karen; Lin, Ji; Shrestha, Sundar; Thompson, Ted; Albright, Ann; Gregg, Edward W.

    2017-01-01

    Recent studies suggest that prevalence of diagnosed diabetes in the United States reached a plateau or slowed around 2008, and that this change coincided with obesity plateaus and increases in physical activity. However, national estimates can obscure important variations in geographic subgroups. We examine whether a slowing or leveling off in diagnosed diabetes, obesity, and leisure time physical inactivity prevalence is also evident across the 3143 counties of the United States. We used publicly available county estimates of the age-adjusted prevalence of diagnosed diabetes, obesity, and leisure-time physical inactivity, which were generated by the Centers for Disease Control and Prevention (CDC). Using a Bayesian multilevel regression that included random effects by county and year and applied cubic splines to smooth these estimates over time, we estimated the average annual percentage point change (APPC) from 2004 to 2008 and from 2008 to 2012 for diabetes, obesity, and physical inactivity prevalence in each county. Compared to 2004–2008, the median APPCs for diabetes, obesity, and physical inactivity were lower in 2008–2012 (diabetes APPC difference = 0.16, 95%CI 0.14, 0.18; obesity APPC difference = 0.65, 95%CI 0.59, 0.70; physical inactivity APPC difference = 0.43, 95%CI 0.37, 0.48). APPCs and APPC differences between time periods varied among counties and U.S. regions. Despite improvements, levels of these risk factors remained high with most counties merely slowing rather than reversing, which suggests that all counties would likely benefit from reductions in these risk factors. The diversity of trajectories in the prevalence of these risk factors across counties underscores the continued need to identify high risk areas and populations for preventive interventions. Awareness of how these factors are changing might assist local policy makers in targeting and tracking the impact of efforts to reduce diabetes, obesity and physical inactivity. PMID

  18. Changes in diagnosed diabetes, obesity, and physical inactivity prevalence in US counties, 2004-2012.

    PubMed

    Geiss, Linda S; Kirtland, Karen; Lin, Ji; Shrestha, Sundar; Thompson, Ted; Albright, Ann; Gregg, Edward W

    2017-01-01

    Recent studies suggest that prevalence of diagnosed diabetes in the United States reached a plateau or slowed around 2008, and that this change coincided with obesity plateaus and increases in physical activity. However, national estimates can obscure important variations in geographic subgroups. We examine whether a slowing or leveling off in diagnosed diabetes, obesity, and leisure time physical inactivity prevalence is also evident across the 3143 counties of the United States. We used publicly available county estimates of the age-adjusted prevalence of diagnosed diabetes, obesity, and leisure-time physical inactivity, which were generated by the Centers for Disease Control and Prevention (CDC). Using a Bayesian multilevel regression that included random effects by county and year and applied cubic splines to smooth these estimates over time, we estimated the average annual percentage point change (APPC) from 2004 to 2008 and from 2008 to 2012 for diabetes, obesity, and physical inactivity prevalence in each county. Compared to 2004-2008, the median APPCs for diabetes, obesity, and physical inactivity were lower in 2008-2012 (diabetes APPC difference = 0.16, 95%CI 0.14, 0.18; obesity APPC difference = 0.65, 95%CI 0.59, 0.70; physical inactivity APPC difference = 0.43, 95%CI 0.37, 0.48). APPCs and APPC differences between time periods varied among counties and U.S. regions. Despite improvements, levels of these risk factors remained high with most counties merely slowing rather than reversing, which suggests that all counties would likely benefit from reductions in these risk factors. The diversity of trajectories in the prevalence of these risk factors across counties underscores the continued need to identify high risk areas and populations for preventive interventions. Awareness of how these factors are changing might assist local policy makers in targeting and tracking the impact of efforts to reduce diabetes, obesity and physical inactivity.

  19. A simplified mathematical model of directional DNA site-specific recombination by serine integrases

    PubMed Central

    Zhao, Jia; Stark, W. Marshall; Colloms, Sean D.; Ebenhöh, Oliver

    2017-01-01

    Serine integrases catalyse site-specific recombination to integrate and excise bacteriophage genomes into and out of their host's genome. These enzymes exhibit remarkable directionality; in the presence of the integrase alone, recombination between attP and attB DNA sites is efficient and irreversible, giving attL and attR products which do not recombine further. However, in the presence of the bacteriophage-encoded recombination directionality factor (RDF), integrase efficiently promotes recombination between attL and attR to re-form attP and attB. The DNA substrates and products of both reactions are approximately isoenergetic, and no cofactors (such as adenosine triphosphate) are required for recombination. The thermodynamic driving force for directionality of these reactions is thus enigmatic. Here, we present a minimal mathematical model which can explain the directionality and regulation of both ‘forward’ and ‘reverse’ reactions. In this model, the substrates of the ‘forbidden’ reactions (between attL and attR in the absence of RDF, attP and attB in the presence of RDF) are trapped as inactive protein–DNA complexes, ensuring that these ‘forbidden’ reactions are extremely slow. The model is in good agreement with the observed in vitro kinetics of recombination by ϕC31 integrase, and defines core features of the system necessary and sufficient for directionality. PMID:28077763

  20. 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

  1. Associations between physical inactivity and sedentary behaviors among adolescents in 10 cities in China

    PubMed Central

    2014-01-01

    Background Studies in western countries have revealed that excessive sedentary behavior is a major risk factor for physical inactivity in adolescents. This study was performed to investigate the association between sedentary behavior and physical inactivity in Chinese adolescents using a large-scale cross-sectional survey design. Methods This study was part of the 2011 Chinese Youth Risk Behavior Survey. Between March and September 2011, 10,214 11–18-year-olds were recruited for survey participation in 18 schools in 10 cities in China. Demographic and socioeconomic characteristics, and the prevalences of physical inactivity and sedentary behaviors, were examined. Correlations between sedentary behavior and physical inactivity were analyzed using baseline logistic regression. Results Among the final 9,901 students, physical inactivity (~80%) and sedentary behaviors (television viewing, 43%; computer use, 30.2%) were prevalent. More male than female students reported sedentary behaviors (television viewing > 2 h: 5.5% vs. 3.9%; computer use > 2 h: 7.2% vs. 3.5%; both p < 0.05), but more males were physically active than females (25.1% vs.14.6%; p < 0.05). Television viewing was associated with lower odds of no physical activity (No PA) in males [0–2 h: adjusted odds ratio (AOR) = 0.81, 95% confidence interval (CI) = 0.68–0.96; >4 h: OR = 0.34, 95% CI = 0.18–0.64], but not in females. A similar pattern between insufficient physical activity and >4 h TV viewing (AOR = 0.42, 95% CI = 0.23–0.76) and >4 h computer use (AOR = 0.49, 95% CI = 0.30–0.78) was observed in males. In females, 0–2 h daily computer use was associated with higher odds of physical inactivity (No PA: AOR = 1.42, 95% CI = 1.10–1.82; Insufficient PA: AOR = 1.58, 95% CI = 1.24–2.01), while TV viewing was not associated with No PA or Insufficient PA. The probability of physical inactivity significantly increased with

  2. "Inactive" ingredients in pharmaceutical products: update (subject review). American Academy of Pediatrics Committee on Drugs.

    PubMed

    1997-02-01

    Because of an increasing number of reports of adverse reactions associated with pharmaceutical excipients, in 1985 the Committee on Drugs issued a position statement recommending that the Food and Drug Administration mandate labeling of over-the-counter and prescription formulations to include a qualitative list of inactive ingredients. However, labeling of inactive ingredients remains voluntary. Adverse reactions continue to be reported, although some are no longer considered clinically significant, and other new reactions have emerged. The original statement, therefore, has been updated and its information expanded.

  3. Systemic endothelial function is preserved in men with both active and inactive variant angina pectoris.

    PubMed

    Ito, K; Akita, H; Kanazawa, K; Yamada, S; Shiga, N; Terashima, M; Matsuda, Y; Takai, E; Iwai, C; Takaoka, H; Yokoyama, M

    1999-12-01

    To test the hypothesis that coronary spasm could be a coronary manifestation of systemic endothelial dysfunction and that the activity of coronary spasm could influence systemic endothelial function, we examined brachial flow-mediated, endothelium-dependent vasodilation and nitroglycerin-induced endothelium-independent vasodilation with high-resolution ultrasound in 11 men with variant angina pectoris (6 active and 5 inactive) without established coronary atherosclerosis. Endothelium-dependent vasodilation in peripheral circulation was preserved in men with active and inactive variant angina pectoris, suggesting that systemic endothelial dysfunction is not involved in either the pathogenesis or the activity of coronary spasm.

  4. Flavocytochrome b2-Based Enzymatic Method of L-Lactate Assay in Food Products

    PubMed Central

    Smutok, Halyna

    2013-01-01

    L-lactate, a key metabolite of the anaerobic glycolytic pathway, plays an important role as a biomarker in medicine, in the nutritional sector and food quality control. For these reasons, there is a need for very specific, sensitive, and simple analytical methods for the accurate L-lactate measuring. A new highly selective enzymatic method for L-lactate determination based on the use of flavocytochrome b2 (EC 1.1.2.3; FC b2) isolated from the recombinant strain of the yeast Hansenula polymorpha has been developed. A proposed enzymatic method exploits an enzymatic oxidation of L-lactate to pyruvate coupled with nitrotetrazolium blue (NTZB) reduction to a colored product, formazan. The maximal absorption peak of the colored product is near λ = 525 nm and the linear range is observed in the interval 0.005–0.14 mM of L-lactate. The main advantages of the proposed method when compared to the LDH-based routine approaches are a higher sensitivity (2.0 μM of L-lactate), simple procedure of analysis, usage of inexpensive, nontoxic reagents, and small amount of the enzyme. Enzymatic oxidation of L-lactate catalyzed by flavocytochrome b2 and coupled with formazan production from nitrotetrazolium blue was shown to be used for L-lactate assay in food samples. A high correlation between results of the proposed method and reference ones proves the possibility to use flavocytochrome b2-catalysed reaction for enzymatic measurement of L-lactate in biotechnology and food chemistry. PMID:24223505

  5. Preparation of chain-end clickable recombinant protein and its bio-orthogonal modification.

    PubMed

    Wang, Lin; Jiang, Rui; Wang, Lin; Liu, Yang; Sun, Xue-Long

    2016-04-01

    Introducing unique functional group into protein is an attractive approach for site-selective protein modification applications. In this report, we systemically investigated four site-selective strategies to introduce azide functionality into recombinant thrombomodulin (TM456), via direct recombinant expression with unnatural amino acid, chemical, and enzymatic modification for its bio-orthogonal modification application. First, a straightforward recombinant method to express TM456 with azide functionality near C-terminus by replacing methionine with azidohomoanlanine from methionine auxotroph Escherichia coli cell was investigated. Next, a sortase-mediated ligation (SML) method to incorporate azide functionality into the C-terminus of recombinant TM456 was demonstrated. The third is to add azide functionality to the N-terminal amine of recombinant TM456via amidation chemistry, and the fourth is tyrosine selective three-component Mannich reaction to introduce azide functionality to recombinant TM456. Overall, SML of recombinant protein affords the highest overall yield for incorporating azide functionality into the C-terminus recombinant TM456 since the key protein expression step uses natural amino acids. Also, single site modification facilitates the highest TM456 activity.

  6. Revocation of Office of Generic Drug's interim policy statement on inactive ingredients. Food and Drug Administration, HHS. Notice.

    PubMed

    1999-04-30

    The Food and Drug Administration (FDA) is revoking an interim policy statement on inactive ingredients in parenteral, ophthalmic, otic, and topical generic drug products (Interim Inactive Ingredient Policy). These generic drug products are the subjects of abbreviated new drug applications (ANDA's). The Interim Inactive Ingredient Policy was issued as a memorandum from the Acting Director of the Center for Drug Evaluation and Research's (CDER's) Office of Generic Drugs, FDA, to CDER's Associate Director for Science and Medical Affairs, FDA. FDA is taking this action because the Interim Inactive Ingredient Policy no longer represents current agency policy.

  7. 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.

  8. Strand invasion promoted by recombination protein of coliphage

    NASA Astrophysics Data System (ADS)

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

    2004-12-01

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

  9. Production and physicochemical properties of recombinant Lactobacillus plantarum tannase.

    PubMed

    Curiel, José Antonio; Rodríguez, Héctor; Acebrón, Iván; Mancheño, José Miguel; De Las Rivas, Blanca; Muñoz, Rosario

    2009-07-22

    Tannase is an enzyme with important biotechnological applications in the food industry. Previous studies have identified the tannase encoding gene in Lactobacillus plantarum and also have reported the description of the purification of recombinant L. plantarum tannase through a protocol involving several chromatographic steps. Here, we describe the high-yield production of pure recombinant tannase (17 mg/L) by a one-step affinity procedure. The purified recombinant tannase exhibits optimal activity at pH 7 and 40 degrees C. Addition of Ca(2+) to the reaction mixture greatly increased tannase activity. The enzymatic activity of tannase was assayed against 18 simple phenolic acid esters. Only esters derived from gallic acid and protocatechuic acid were hydrolyzed. In addition, tannase activity was also assayed against the tannins tannic acid, gallocatechin gallate, and epigallocatechin gallate. Despite L. plantarum tannase representing a novel family of tannases, which shows no significant similarity to tannases from fungal sources, both families of enzymes shared similar substrate specificity range. The physicochemical characteristics exhibited by L. plantarum recombinant tannase make it an adequate alternative to the currently used fungal tannases.

  10. The recombination epoch revisited

    NASA Technical Reports Server (NTRS)

    Krolik, Julian H.

    1989-01-01

    Previous studies of cosmological recombination have shown that this process produces as a by-product a highly superthermal population of Ly-alpha photons which retard completion of recombination. Cosmological redshifting was thought to determine the frequency distribution of the photons, while two-photon decay of hydrogen's 2s state was thought to control their numbers. It is shown here that frequency diffusion due to photon scattering dominate the cosmological redshift in the frequency range near line center which fixes the ratio of ground state to excited state population, while incoherent scattering into the far-red damping wing effectively destroys Ly-alpha photons as a rate which is competitive with two-photon decay. The former effect tends to hold back recombination, while the latter tends to accelerate it; the net results depends on cosmological parameters, particularly the combination Omega(b) h/sq rt (2q0), where Omega(b) is the fraction of the critical density provided by baryons.

  11. Variables associated with active and inactive behavior during the after-school period.

    PubMed

    Wickel, Eric

    2013-05-01

    This study analyzed time-use interviews to report levels of active and inactive behavior during the after-school period (3-6 pm). Interviews were conducted on random days from three separate seasons during third and fourth grade. Youth with at least two interviews during third (356 completed 2 interviews; 506 completed 3 interviews [9 yrs; 50% boys]) and fourth (186 completed 2 interviews; 768 completed 3 interviews [10 yrs; 50% boys]) grade were included to report levels of moderate-to-vigorous physical activity (MVPA), light physical activity, inactive screen time, inactive nonscreen time, and travel by location and who the activity was undertaken with. Reporting time outside the home and with peers (single or group) was related to higher levels of MVPA. While inside the home, screen and nonscreen proportions were comparable (38% and 40%, respectively), despite unique patterns (screen: boys > girls; nonscreen: girls > boys). Reporting time with both parents was associated with more nonscreen time; whereas reporting time with peer groups was associated with lower screen time. Understanding active and inactive patterns of children's behavior outside of school hours can be very important in contributing toward the development of innovative interventions for increasing physical activity.

  12. 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…

  13. 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…

  14. 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…

  15. Inactive tanks remediation program strategy and plans for Oak Ridge National Laboratory, Oak Ridge, Tennessee

    SciTech Connect

    1997-03-01

    This report presents plans and strategies for remediation of the liquid low-level waste (LLLW) tanks that have been removed from service (also known as inactive tanks) at Oak Ridge National Laboratory (ORNL) in Oak Ridge, Tennessee. These plans and strategies will be carried out by the Environmental Restoration Program`s Inactive LLLW Tank Program at ORNL. These tanks are defined as Category D tanks because they are existing tank systems without secondary containment that are removed from service. The approach to remediation of each tank or tank farm must be adapted in response to the specific circumstances of individual tank sites. The approach will be tailored to accommodate feedback on lessons learned from previous tank remediation activities and will not be a rigid step-by-step approach that must be conducted identically for every tank system. However, the approach will follow a multistep decision process. The overall objective of the Inactive Tank Program is to remediate all LLLW tanks that have been removed from service to the extent practicable in accordance with the FFA requirements. The Inactive Tank Program will focus on the remediation of the tank residues (i.e., contents after tank has been emptied) and tank shell. This strategy is discussed in detail in this report.

  16. Inactive tanks remediation program strategy and plans for Oak Ridge National Laboratory, Oak Ridge, Tennessee

    SciTech Connect

    1997-11-01

    This report presents plans and strategies for remediation of the liquid low-level waste (LLLW) tanks that have been removed from service (also known as inactive tanks) at Oak Ridge National Laboratory (ORNL) in Oak Ridge, Tennessee. These plans and strategies will be carried out by the Environmental Restoration Program`s Inactive LLLW Tank Program at ORNL. The approach to remediation of each tank or tank farm must be adapted in response to the specific circumstances of individual tank sites. The approach will be tailored to accommodate feedback on lessons learned from previous tank remediation activities and will not be a rigid step-by-step approach that must be conducted identically for every tank system. However, the approach will follow a multistep decision process. The overall objective of the Inactive Tank Program is to remediate all LLLW tanks that have been removed from service to the extent practicable in accordance with the FFA requirements. The Inactive Tank Program will focus on the remediation of the tank residues and tank shell. This strategy is discussed in detail in this report.

  17. 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…

  18. 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...

  19. 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...

  20. 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...

  1. 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...

  2. 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…

  3. 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.

  4. 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,...

  5. 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...

  6. 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...

  7. 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,...

  8. 38 CFR 4.89 - Ratings for inactive nonpulmonary tuberculosis in effect on August 19, 1968.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

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

  9. 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...

  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 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,...

  12. 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,...

  13. 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...

  14. "Learning-in-Action" and "Learning Inaction": Advancing the Theory and Practice of Critical Action Learning

    ERIC Educational Resources Information Center

    Vince, Russ

    2008-01-01

    This paper seeks to improve our understanding of the emotional and political dynamics that are generated (and too often avoided) in action learning. The idea at the centre of the paper is a distinction between "learning-in-action" and "learning inaction". The phrase "learning-in-action" represents the value of action…

  15. 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...

  16. 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...

  17. Teacher Educators' In-Action Mental Models in Different Teaching Situations

    ERIC Educational Resources Information Center

    Mevorach, Miriam; Strauss, Sidney

    2012-01-01

    In previous studies on teachers' cognition, we discovered that teachers' teaching can be described via a general in-action mental model (IAMM) concerning the structure of the mind and the roles of teaching in fostering children's learning. The purpose of our study was to examine teacher educators' IAMM regarding student teachers' minds and…

  18. Expression, purification and characterization of inactive and active forms of ERK2 from insect expression system.

    PubMed

    Yan, Kelly; Merritt, Hanne; Crawford, Kenneth; Pardee, Gwynn; Cheng, Jan Marie; Widger, Stephania; Hekmat-Nejad, Mohammad; Zaror, Isabel; Sim, Janet

    2015-06-01

    Extracellular signal-regulated kinase 2 (ERK2) is a serine/threonine protein kinase involved in many cellular programs, such as cell proliferation, differentiation, motility and programed cell-death. It is therefore considered an important target in the treatment of cancer. In an effort to support biochemical screening and small molecule drug discovery, we established a robust system to generate both inactive and active forms of ERK2 using insect expression system. We report here, for the first time, that inactive ERK2 can be expressed and purified with 100% homogeneity in the unphosphorylated form using insect system. This resulted in a significant 20-fold yield improvement compared to that previously reported using bacterial expression system. We also report a newly developed system to generate active ERK2 in insect cells through in vivo co-expression with a constitutively active MEK1 (S218D S222D). Isolated active ERK2 was confirmed to be doubly phosphorylated at the correct sites, T185 and Y187, in the activation loop of ERK2. Both ERK2 forms, inactive and active, were well characterized by biochemical activity assay for their kinase function. Inactive and active ERK2 were the two key reagents that enabled successful high through-put biochemical assay screen and structural drug discovery studies.

  19. 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…

  20. Cell biology of mitotic recombination.

    PubMed

    Lisby, Michael; Rothstein, Rodney

    2015-03-02

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination.

  1. Cell Biology of Mitotic Recombination

    PubMed Central

    Lisby, Michael; Rothstein, Rodney

    2015-01-01

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination. PMID:25731763

  2. Engineering of vault nanocapsules with enzymatic and fluorescent properties.

    PubMed

    Kickhoefer, Valerie A; Garcia, Yvette; Mikyas, Yeshi; Johansson, Erik; Zhou, Jing C; Raval-Fernandes, Sujna; Minoofar, Payam; Zink, Jeffrey I; Dunn, Bruce; Stewart, Phoebe L; Rome, Leonard H

    2005-03-22

    One of the central issues facing the emerging field of nanotechnology is cellular compatibility. Nanoparticles have been proposed for diagnostic and therapeutic applications, including drug delivery, gene therapy, biological sensors, and controlled catalysis. Viruses, liposomes, peptides, and synthetic and natural polymers have been engineered for these applications, yet significant limitations continue to prevent their use. Avoidance of the body's natural immune system, lack of targeting specificity, and the inability to control packaging and release are remaining obstacles. We have explored the use of a naturally occurring cellular nanoparticle known as the vault, which is named for its morphology with multiple arches reminiscent of cathedral ceilings. Vaults are 13-MDa ribonucleoprotein particles with an internal cavity large enough to sequester hundreds of proteins. Here, we report a strategy to target and sequester biologically active materials within the vault cavity. Attachment of a vault-targeting peptide to two proteins, luciferase and a variant of GFP, resulted in their sequestration within the vault cavity. The targeted proteins confer enzymatic and fluorescent properties on the recombinant vaults, both of which can be detected by their emission of light. The modified vaults are compatible with living cells. The ability to engineer vault particles with designed properties and functionalities represents an important step toward development of a biocompatible nanocapsule.

  3. 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.

  4. 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…

  5. 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...

  6. Enhanced enzymatic hydrolysis of cellulose in microgels.

    PubMed

    Chang, Aiping; Wu, Qingshi; Xu, Wenting; Xie, Jianda; Wu, Weitai

    2015-07-04

    A cellulose-based microgel, where an individual microgel contains approximately one cellulose chain on average, is synthesized via free radical polymerization of a difunctional small-molecule N,N'-methylenebisacrylamide in cellulose solution. This microgelation leads to a low-ordered cellulose, favoring enzymatic hydrolysis of cellulose to generate glucose.

  7. 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…

  8. 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

  9. Lactate and H+ uptake in inactive muscles during intense exercise in man.

    PubMed Central

    Bangsbo, J; Aagaard, T; Olsen, M; Kiens, B; Turcotte, L P; Richter, E A

    1995-01-01

    1. The present study examined how uptake of lactate and H+ in resting muscle is affected by blood flow, arterial lactate concentration and muscle metabolism. 2. Six males subjects performed intermittent arm exercise in two separate 32 min periods (Part I and Part II) and in one subsequent 20 min period in which one leg knee-extensor exercise was also performed (Part III). The exercise was performed at various intensities in order to obtain different steady-state arterial blood lactate concentrations. In the inactive leg, femoral venous blood flow (draining about 7.7 kg of muscles) was measured and femoral arterial and venous blood was collected frequently. Biopsies were taken from m. vastus lateralis of the inactive leg at rest and 10 and 30 min into both Part I and Part II as well as 10 min into recovery from Part II. 3. The arterial plasma lactate concentrations were 7, 9 and 16 mmol l-1 after 10 min of Parts I, II and III, respectively, and the corresponding arterial-venous difference (a-vdiff) for lactate in the resting leg was 1.3, 1.4 and 2.0 mmol l-1. The muscle lactate concentration was 2.8 mmol (kg wet wt)-1 after 10 min of Part I and remained constant throughout the experiment. During Parts I and II, a-vdiff lactate decreased although the arterial lactate concentration and plasma-muscle lactate gradient were unaltered throughout each period. Thus, membrane transport of lactate decreased during each period. 4. Blood flow in the inactive leg was about 2-fold higher during arm exercise compared to the rest periods, resulting in a 2-fold higher lactate uptake. Thus, lactate uptake by inactive muscles was closely related to blood flow. 5. Throughout the experiment a-vdiff for actual base excess and for lactate were of similar magnitude. Thus, in inactive muscles lactate uptake appears to be coupled to the transport of H+. PMID:8568658

  10. Enzymatic biotransformation of terpenes as bioactive agents.

    PubMed

    Sultana, Nighat; Saify, Zafar Saeed

    2013-12-01

    The plant-derived terpenoids are considered to be the most potent anticancer, anti-inflammatory and anticarcinogenic compounds known. Enzymatic biotransformation is a very useful approach to expand the chemical diversity of natural products. Recent enzymatic biotransformation studies on terpenoids have resulted in the isolation of novel compounds. 14-hydroxy methyl caryophyllene oxide produced from caryophyllene oxide showed a potent inhibitory activity against the butyryl cholinesterase enzyme, and was found to be more potent than parent caryophyllene oxide. The metabolites 3β,7β-dihydroxy-11-oxo-olean-12-en-30-oic acid, betulin, betulonic acid, argentatin A, incanilin, 18β glycyrrhetinic acid, 3,11-dioxo-olean-12-en-30-oic acid produced from 18β glycyrrhetinic acid were screened against the enzyme lipoxygenase. 3,11-Dioxo-olean-12-en-30-oic acid, was found to be more active than the parent compound. The metabolites 3β-hydroxy sclareol 18α-hydroxy sclareol, 6α,18α-dihydroxy sclareol, 11S,18α-dihydroxy sclareol, and 1β-hydroxy sclareol and 11S,18α-dihydroxy sclareol produced from sclareol were screened for antibacterial activity. 1β-Hydroxy sclareol was found to be more active than parent sclareol. There are several reports on natural product enzymatic biotransformation, but few have been conducted on terpenes. This review summarizes the classification, advantages and agents of enzymatic transformation and examines the potential role of new enzymatically transformed terpenoids and their derivatives in the chemoprevention and treatment of other diseases.

  11. Synthesis of the milk oligosaccharide 2'-fucosyllactose using recombinant bacterial enzymes.

    PubMed

    Albermann, C; Piepersberg, W; Wehmeier, U F

    2001-08-23

    The enzymatic synthesis of GDP-beta-L-fucose and its enzymatic transfer reaction using recombinant enzymes from bacterial sources was examined. The GDP-D-mannose 4,6-dehydratase and the GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase from Escherichia coli K-12, respectively, were used to catalyse the conversion of GDP-alpha-D-mannose to GDP-beta-L-fucose with 78% yield. For the transfer of the L-fucose to an acceptor, we cloned and overproduced the alpha-(1-->2)-fucosyltransferase (FucT2) protein from Helicobacter pylori. We were able to synthesise 2'-fucosyllactose using the overproduced FucT2 enzyme, enzymatically synthesised GDP-L-fucose and lactose. The isolation of 2'-fucosyllactose was accomplished by anion-exchange chromatography and gel filtration to give 65% yield.

  12. Recombineering: genetic engineering in bacteria using homologous recombination.

    PubMed

    Thomason, Lynn C; Sawitzke, James A; Li, Xintian; Costantino, Nina; Court, Donald L

    2014-04-14

    The bacterial chromosome and bacterial plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage-encoded recombination proteins efficiently recombine sequences with homologies as short as 35 to 50 bases. Recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with dsDNA or ssDNA. It then presents support protocols that describe several two-step selection/counter-selection methods of making genetic alterations without leaving any unwanted changes in the targeted DNA, and a method for retrieving onto a plasmid a genetic marker (cloning by retrieval) from the Escherichia coli chromosome or a co-electroporated DNA fragment. Additional protocols describe methods to screen for unselected mutations, removal of the defective prophage from recombineering strains, and other useful techniques.

  13. 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.

  14. Orientation Dependence in Homologous Recombination

    PubMed Central

    Yamamoto, K.; Takahashi, N.; Fujitani, Y.; Yoshikura, H.; Kobayashi, I.

    1996-01-01

    Homologous recombination was investigated in Escherichia coli with two plasmids, each carrying the homologous region (two defective neo genes, one with an amino-end deletion and the other with a carboxyl-end deletion) in either direct or inverted orientation. Recombination efficiency was measured in recBC sbcBC and recBC sbcA strains in three ways. First, we measured the frequency of cells carrying neo(+) recombinant plasmids in stationary phase. Recombination between direct repeats was much more frequent than between inverted repeats in the recBC sbcBC strain but was equally frequent in the two substrates in the recBC sbcA strain. Second, the fluctuation test was used to exclude bias by a rate difference between the recombinant and parental plasmids and led to the same conclusion. Third, direct selection for recombinants just after transformation with or without substrate double-strand breaks yielded essentially the same results. Double-strand breaks elevated recombination in both the strains and in both substrates. These results are consistant with our previous findings that the major route of recombination in recBC sbcBC strains generates only one recombinant DNA from two DNAs and in recBC sbcA strains generates two recombinant DNAs from two DNAs. PMID:8722759

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

    ERIC Educational Resources Information Center

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

    2008-01-01

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

  16. 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...

  17. 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.

  18. Enzymic properties of recombinant BACE2.

    PubMed

    Kim, Yong-Tae; Downs, Deborah; Wu, Shili; Dashti, Azar; Pan, Yujun; Zhai, Peng; Wang, Xinjuan; Zhang, Xuejun C; Lin, Xinli

    2002-11-01

    BACE2 (Memapsin 1) is a membrane-bound aspartic protease that is highly homologous with BACE1 (Memapsin 2). While BACE1 processes the amyloid precursor protein (APP) at a key step in generating the beta-amyloid peptide and presumably causes Alzheimer's disease (AD), BACE2 has not been demonstrated to be directly involved in APP processing, and its physiological functions remain to be determined. In vivo, BACE2 is expressed as a precursor protein containing pre-, pro-, protease, transmembrane, and cytosolic domains/peptides. To determine the enzymatic properties of BACE2, two variants of its pro-protease domain, pro-BACE2-T1 (PB2-T1) and pro-BACE2-T2 (PB2-T2), were constructed. They have been expressed in Escherichia coli as inclusion bodies, refolded and purified. These two recombinant proteins have the same N terminus but differ at their C-terminal ends: PB2-T1 ends at Pro466, on the boundary of the postulated transmembrane domain, and PB2-T2 ends at Ser431, close to the homologous ends of other aspartic proteases such as pepsin. While PB2-T1 shares similar substrate specificities with BACE1 and other 'general' aspartic proteases, the specificity of PB2-T2 is more constrained, apparently preferring to cleave at the NH2-terminal side of paired basic residues. Unlike other 'typical' aspartic proteases, which are active only under acidic conditions, the recombinant BACE2, PB2-T1, was active at a broad pH range. In addition, pro-BACE2 can be processed at its in vivo maturation site by BACE1.

  19. 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..

  20. 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

  1. Germline methylation patterns determine the distribution of recombination events in the dog genome.

    PubMed

    Berglund, Jonas; Quilez, Javier; Arndt, Peter F; Webster, Matthew T

    2014-12-19

    The positive-regulatory domain containing nine gene, PRDM9, which strongly associates with the location of recombination events in several vertebrates, is inferred to be inactive in the dog genome. Here, we address several questions regarding the control of recombination and its influence on genome evolution in dogs. First, we address whether the association between CpG islands (CGIs) and recombination hotspots is generated by lack of methylation, GC-biased gene conversion (gBGC), or both. Using a genome-wide dog single nucleotide polymorphism data set and comparisons of the dog genome with related species, we show that recombination-associated CGIs have low CpG mutation rates, and that CpG mutation rate is negatively correlated with recombination rate genome wide, indicating that nonmethylation attracts the recombination machinery. We next use a neighbor-dependent model of nucleotide substitution to disentangle the effects of CpG mutability and gBGC and analyze the effects that loss of PRDM9 has on these rates. We infer that methylation patterns have been stable during canid genome evolution, but that dog CGIs have experienced a drastic increase in substitution rate due to gBGC, consistent with increased levels of recombination in these regions. We also show that gBGC is likely to have generated many new CGIs in the dog genome, but these mostly occur away from genes, whereas the number of CGIs in gene promoter regions has not increased greatly in recent evolutionary history. Recombination has a major impact on the distribution of CGIs that are detected in the dog genome due to the interaction between methylation and gBGC. The results indicate that germline methylation patterns are the main determinant of recombination rates in the absence of PRDM9.

  2. 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

  3. Making recombinant extracellular matrix proteins.

    PubMed

    Ruggiero, Florence; Koch, Manuel

    2008-05-01

    A variety of approaches to understand extracellular matrix protein structure and function require production of recombinant proteins. Moreover, the expression of heterologous extracellular matrix proteins, in particular collagens, using the recombinant technology is of major interest to the biomedical industry. Although extracellular matrix proteins are large, modular and often multimeric, most of them have been successfully produced in various expression systems. This review provides important factors, including the design of the construct, the cloning strategies, the expression vectors, the transfection method and the host cell systems, to consider in choosing a reliable and cost-effective way to make recombinant extracellular matrix proteins. Advantages and drawbacks of each system have been appraised. Protocols that may ease efficient recombinant production of extracellular matrix are described. Emphasis is placed on the recombinant collagen production. Members of the collagen superfamily exhibit specific structural features and generally require complex post-translational modifications to retain full biological activity that make more arduous their recombinant production.

  4. Expression of recombinant antibodies.

    PubMed

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with "human-like" post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  5. Expression of Recombinant Antibodies

    PubMed Central

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications. PMID:23908655

  6. 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.

  7. Graphene based enzymatic bioelectrodes and biofuel cells.

    PubMed

    Karimi, Anahita; Othman, Ali; Uzunoglu, Aytekin; Stanciu, Lia; Andreescu, Silvana

    2015-04-28

    The excellent electrical conductivity and ease of functionalization make graphene a promising material for use in enzymatic bioelectrodes and biofuel cells. Enzyme based biofuel cells have attracted substantial interest due to their potential to harvest energy from organic materials. This review provides an overview of the functional properties and applications of graphene in the construction of biofuel cells as alternative power sources. The review covers the current state-of-the-art research in graphene based nanomaterials (physicochemical properties and surface functionalities), the role of these parameters in enhancing electron transfer, the stability and activity of immobilized enzymes, and how enhanced power density can be achieved. Specific examples of enzyme immobilization methods, enzyme loading, stability and function on graphene, functionalized graphene and graphene based nanocomposite materials are discussed along with their advantages and limitations. Finally, a critical evaluation of the performance of graphene based enzymatic biofuel cells, the current status, challenges and future research needs are provided.

  8. 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.

  9. Production of MAG via enzymatic glycerolysis

    NASA Astrophysics Data System (ADS)

    Jamlus, Norul Naziraa Ahmad; Derawi, Darfizzi; Salimon, Jumat

    2015-09-01

    Enzymatic glycerolysis of a medium chain methyl ester, methyl laurate was performed using lipase Candida antarctica (Novozyme 435) for 6 hours at 55°C. The percentage of components mixture of product were determined by using gas chromatography technique. The enzymatic reaction was successfully produced monolaurin (45.9 %), dilaurin (47.1 %) and trilaurin (7.0 %) respectively. Thin layer chromatography (TLC) plate also showed a good separation of component spots. Fourier transformation infra-red (FTIR) spectrum showed the presence of ester carbonyl at wavenumber 1739.99 cm-1 and hydrogen bonded O-H at 3512.03 cm-1. The product is potentially to be used as emulsifier and additive in food industry, pharmaceutical, as well as antibacterial.

  10. Enzymatic hydrolysis of poly(ethylene furanoate).

    PubMed

    Pellis, Alessandro; Haernvall, Karolina; Pichler, Christian M; Ghazaryan, Gagik; Breinbauer, Rolf; Guebitz, Georg M

    2016-10-10

    The urgency of producing new environmentally-friendly polyesters strongly enhanced the development of bio-based poly(ethylene furanoate) (PEF) as an alternative to plastics like poly(ethylene terephthalate) (PET) for applications that include food packaging, personal and home care containers and thermoforming equipment. In this study, PEF powders of various molecular weights (6, 10 and 40kDa) were synthetized and their susceptibility to enzymatic hydrolysis was investigated for the first time. According to LC/TOF-MS analysis, cutinase 1 from Thermobifida cellulosilytica liberated both 2,5-furandicarboxylic acid and oligomers of up to DP4. The enzyme preferentially hydrolyzed PEF with higher molecular weights but was active on all tested substrates. Mild enzymatic hydrolysis of PEF has a potential both for surface functionalization and monomers recycling.

  11. A Networks Approach to Modeling Enzymatic Reactions.

    PubMed

    Imhof, P

    2016-01-01

    Modeling enzymatic reactions is a demanding task due to the complexity of the system, the many degrees of freedom involved and the complex, chemical, and conformational transitions associated with the reaction. Consequently, enzymatic reactions are not determined by precisely one reaction pathway. Hence, it is beneficial to obtain a comprehensive picture of possible reaction paths and competing mechanisms. By combining individually generated intermediate states and chemical transition steps a network of such pathways can be constructed. Transition networks are a discretized representation of a potential energy landscape consisting of a multitude of reaction pathways connecting the end states of the reaction. The graph structure of the network allows an easy identification of the energetically most favorable pathways as well as a number of alternative routes.

  12. 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

  13. Transport dynamics of molecular motors that switch between an active and inactive state.

    PubMed

    Pinkoviezky, I; Gov, N S

    2013-08-01

    Molecular motors are involved in key transport processes in the cell. Many of these motors can switch from an active to a nonactive state, either spontaneously or depending on their interaction with other molecules. When active, the motors move processively along the filaments, while when inactive they are stationary. We treat here the simple case of spontaneously switching motors, between the active and inactive states, along an open linear track. We use our recent analogy with vehicular traffic, where we go beyond the mean-field description. We map the phase diagram of this system, and find that it clearly breaks the symmetry between the different phases, as compared to the standard total asymmetric exclusion process. We make several predictions that may be testable using molecular motors in vitro and in living cells.

  14. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

  16. 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.

  17. 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.

  18. 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.

  19. Heterogeneous reallocation of presynaptic efficacy in recurrent excitatory circuits adapting to inactivity.

    PubMed

    Mitra, Ananya; Mitra, Siddhartha S; Tsien, Richard W

    2011-12-18

    Recurrent excitatory circuits face extreme challenges in balancing efficacy and stability. We recorded from CA3 pyramidal neuron pairs in rat hippocampal slice cultures to characterize synaptic and circuit-level changes in recurrent synapses resulting from long-term inactivity. Chronic tetrodotoxin treatment greatly reduced the percentage of connected CA3-CA3 neurons, but enhanced the strength of the remaining connections; presynaptic release probability sharply increased, whereas quantal size was unaltered. Connectivity was decreased in activity-deprived circuits by functional silencing of synapses, whereas three-dimensional anatomical analysis revealed no change in spine or bouton density or aggregate dendrite length. The silencing arose from enhanced Cdk5 activity and could be reverted by acute Cdk5 inhibition with roscovitine. Our results suggest that recurrent circuits adapt to chronic inactivity by reallocating presynaptic weights heterogeneously, strengthening certain connections while silencing others. This restricts synaptic output and input, preserving signaling efficacy among a subset of neuronal ensembles while protecting network stability.

  20. 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.

  1. Extracellular enzymatic activity of Microsporum canis isolates.

    PubMed

    Papini, R; Mancianti, F

    The enzymatic activity of 70 feline and canine Microsporum canis isolates was determined by the Api-Zym test. The liquid phase of cultures, inoculated into Tryptic Soy Broth, was used to examine 19 enzymes. Considerable differences were observed among the extracellular enzymatic patterns. All the isolates produced alkaline phosphatase and beta-glucosidase, while lipase (C14), trypsin, chymotrypsin, beta-glucuronidase, and alpha-fucosidase activity was never revealed. Esterase (C4) activity was present in 57 samples (81%), esterase lipase (C8) in 31 (44%), leucine arylamidase in 35 (50%), valine arylamidase and cystine arylamidase in 7 (10%), acid phosphatase in 64 (91%), naphthol-AS-BI-phosphohydrolase in 60 (86%), alpha-galactosidase in 5 (7%), beta-galactosidase in 6 (8%), alpha-glucosidase in 25 (36%), N-acetyl-beta-glucosaminidase in 41 (58%), and alpha-mannosidase in 51 (73%). The beta-galactosidase activity of M. canis has not been reported previously. Remarkable variations of intensity for each enzymatic activity were also detected. It is believed that these results could provide basic data for further investigations on the pathogenic role of enzymes secreted by M. canis.

  2. Enzymatic transformation of nonfood biomass to starch

    PubMed Central

    You, Chun; Chen, Hongge; Myung, Suwan; Sathitsuksanoh, Noppadon; Ma, Hui; Zhang, Xiao-Zhou; Li, Jianyong; Zhang, Y.-H. Percival

    2013-01-01

    The global demand for food could double in another 40 y owing to growth in the population and food consumption per capita. To meet the world’s future food and sustainability needs for biofuels and renewable materials, the production of starch-rich cereals and cellulose-rich bioenergy plants must grow substantially while minimizing agriculture’s environmental footprint and conserving biodiversity. Here we demonstrate one-pot enzymatic conversion of pretreated biomass to starch through a nonnatural synthetic enzymatic pathway composed of endoglucanase, cellobiohydrolyase, cellobiose phosphorylase, and alpha-glucan phosphorylase originating from bacterial, fungal, and plant sources. A special polypeptide cap in potato alpha-glucan phosphorylase was essential to push a partially hydrolyzed intermediate of cellulose forward to the synthesis of amylose. Up to 30% of the anhydroglucose units in cellulose were converted to starch; the remaining cellulose was hydrolyzed to glucose suitable for ethanol production by yeast in the same bioreactor. Next-generation biorefineries based on simultaneous enzymatic biotransformation and microbial fermentation could address the food, biofuels, and environment trilemma. PMID:23589840

  3. Enzymatic transformation of nonfood biomass to starch.

    PubMed

    You, Chun; Chen, Hongge; Myung, Suwan; Sathitsuksanoh, Noppadon; Ma, Hui; Zhang, Xiao-Zhou; Li, Jianyong; Zhang, Y-H Percival

    2013-04-30

    The global demand for food could double in another 40 y owing to growth in the population and food consumption per capita. To meet the world's future food and sustainability needs for biofuels and renewable materials, the production of starch-rich cereals and cellulose-rich bioenergy plants must grow substantially while minimizing agriculture's environmental footprint and conserving biodiversity. Here we demonstrate one-pot enzymatic conversion of pretreated biomass to starch through a nonnatural synthetic enzymatic pathway composed of endoglucanase, cellobiohydrolyase, cellobiose phosphorylase, and alpha-glucan phosphorylase originating from bacterial, fungal, and plant sources. A special polypeptide cap in potato alpha-glucan phosphorylase was essential to push a partially hydrolyzed intermediate of cellulose forward to the synthesis of amylose. Up to 30% of the anhydroglucose units in cellulose were converted to starch; the remaining cellulose was hydrolyzed to glucose suitable for ethanol production by yeast in the same bioreactor. Next-generation biorefineries based on simultaneous enzymatic biotransformation and microbial fermentation could address the food, biofuels, and environment trilemma.

  4. 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.

  5. At Least I Tried: The Relationship between Regulatory Focus and Regret Following Action vs. Inaction

    PubMed Central

    Itzkin, Adi; Van Dijk, Dina; Azar, Ofer H.

    2016-01-01

    Regret is an unpleasant feeling that may arise following decisions that ended poorly, and may affect the decision-maker's well-being and future decision making. Some studies show that a decision to act leads to greater regret than a decision not to act when both resulted in failure, because the latter is usually the norm. In some cases, when the norm is to act, this pattern is reversed. We suggest that the decision maker's regulatory focus, affects regret after action or inaction. Specifically, promotion-focused individuals, who tend to be more proactive, view action as more normal than prevention-focused individuals, and therefore experience regulatory fit when an action decision is made. Hence, we hypothesized that promotion-focused individuals will feel less regret than prevention-focused individuals when a decision to act ended poorly. In addition, we hypothesized that a trigger for change implied in the situation, decreases the level of regret following action. We tested our hypotheses on a sample of 330 participants enrolled in an online survey. The participants received six decision scenarios, in which they were asked to evaluate the level of regret following action and inaction. Individual regulatory focus was measured by two different scales. Promotion-focused individuals attributed less regret than prevention-focused individuals to action decisions. Regret following inaction was not affected by regulatory focus. In addition, a trigger for change decreases regret following action. Orthodox people tend to attribute more regret than non-orthodox to a person who made an action decision. The results contribute to the literature by showing that not only the situation but also the decision maker's orientation affects the regret after action vs. inaction. PMID:27833581

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

    PubMed

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

    2016-01-19

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

  7. Comparison of Vitamin D Levels in Naive, Treated, and Inactive Carriers with Chronic Hepatitis B Virus

    PubMed Central

    Sali, Shahnaz; Tavakolpour, Soheil; Farkhondemehr, Baharan

    2016-01-01

    Abstract Background and Aims: During recent years, the relationship between vitamin D levels and chronic hepatitis B (CHB) infection has attracted many researchers’ attention. However, the results relating to the association of vitamin D levels and HBV infection have been conflicting and there remains a lack of knowledge about the effects of antiviral treatments on vitamin D level. Methods: Eighty-four patients with CHB were assessed and divided into three groups: inactive carriers (n = 28), treated (n = 34), and new (treatment-naïve) cases (n = 22). Thirty-two healthy controls (HCs) were included to enable comparison with the CHB groups. The levels of vitamin D3 were measured and statistically compared among the various groups. Results: Male subjects had higher levels of vitamin D3 (41.25 vs 28.85, p < 0.01). No association was found among any of the groups when compared with the HC group. Despite the significant association, the HCs demonstrated a higher level of vitamin D3, which was lower in the treated group, the inactive carrier group, and the new cases group (new case [29.82] < inactive carrier [32.91] < treated [39.56] < control [44.88]). The HBV DNA levels were not associated with vitamin D3 levels in the inactive carriers (p = 0.171), the treated groups (p = 0.192), and the new cases (p = 0.369). Moreover, the alanine transaminase and aspartate transaminase levels were not associated with vitamin D3 levels for any of the HBV-infected groups. Conclusions: Vitamin D3 contributes to the clinical statues of CHB patients. There is also a possible correlation between clinically healthy CHB patients and vitamin D3 level. PMID:28097099

  8. 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.

  9. 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.

  10. Inactive DNMT3B splice variants modulate de novo DNA methylation.

    PubMed

    Gordon, Catherine A; Hartono, Stella R; Chédin, Frédéric

    2013-01-01

    Inactive DNA methyltransferase (DNMT) 3B splice isoforms are associated with changes in DNA methylation, yet the mechanisms by which they act remain largely unknown. Using biochemical and cell culture assays, we show here that the inactive DNMT3B3 and DNMT3B4 isoforms bind to and regulate the activity of catalytically competent DNMT3A or DNMT3B molecules. DNMT3B3 modestly stimulated the de novo methylation activity of DNMT3A and also counteracted the stimulatory effects of DNMT3L, therefore leading to subtle and contrasting effects on activity. DNMT3B4, by contrast, significantly inhibited de novo DNA methylation by active DNMT3 molecules, most likely due to its ability to reduce the DNA binding affinity of co-complexes, thereby sequestering them away from their substrate. Immunocytochemistry experiments revealed that in addition to their effects on the intrinsic catalytic function of active DNMT3 enzymes, DNMT3B3 and DNMT34 drive distinct types of chromatin compaction and patterns of histone 3 lysine 9 tri-methylation (H3K9me3) deposition. Our findings suggest that regulation of active DNMT3 members through the formation of co-complexes with inactive DNMT3 variants is a general mechanism by which DNMT3 variants function. This may account for some of the changes in DNA methylation patterns observed during development and disease.

  11. Health culture and the clinical encounter: Vietnamese refugees' responses to preventive drug treatment of inactive tuberculosis.

    PubMed

    Ito, K L

    1999-09-01

    A majority of Vietnamese refugees entering the United States test positive for inactive tuberculosis (TB). In asymptomatic conditions like inactive TB, it is often difficult to obtain compliance with medical treatment. The clinical encounter has been analyzed as a form of symbolic action between doctor and patient critical to patient trust and compliance. However, it is equally, if not more, important to understand the health culture of patients, that is, the broader sociocultural context of the patient within which his or her illness is interpreted and understood. In this article I look at health culture elements that influence compliance and noncompliance by Vietnamese American clients with courses of preventive drug therapy for inactive TB. Key factors in compliance are: (1) cultural interpretations of the therapy's side effects as "hot"; (2) the role of family members and peers; and (3) community perceptions of the drug treatment. Culturally incongruent elements of the clinical encounter and the funding of community-based organizations for health education also are examined.

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

    PubMed Central

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

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Convertino, Victor A.

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

  14. Redox-Inactive Metals Modulate the Reduction Potential in Heterometallic Manganese-Oxido Clusters

    PubMed Central

    Tsui, Emily Y.; Tran, Rosalie; Yano, Junko; Agapie, Theodor

    2013-01-01

    Redox-inactive metals are found in biological and heterogeneous water oxidation catalysts, but their roles in catalysis are currently not well understood. A series of high oxidation state tetranuclear-dioxido clusters comprised of three manganese centers and a redox-inactive metal (M) of various charge is reported. Crystallographic studies show an unprecedented Mn3M(μ4-O)(μ2-O) core that remains intact upon changing M or the manganese oxidation state. Electrochemical studies reveal that the reduction potentials span a window of 700 mV, dependent upon the Lewis acidity of the second metal. With the pKa of the redox-inactive metal-aqua complex as a measure of Lewis acidity, these compounds display a linear dependence between reduction potential and acidity with a slope of ca. 100 mV per pKa unit. The Sr2+ and Ca2+ compounds show similar potentials, an observation that correlates with the behavior of the OEC, which is active only in the presence of one of these two metals. PMID:23511417

  15. 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.

  16. Cold enzymatic bleaching of fluid whey.

    PubMed

    Campbell, R E; Drake, M A

    2013-01-01

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

  17. 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.

  18. Putrescine metabolism: enzymatic formation and non-enzymatic isotope exchange of delta1-pyrroline.

    PubMed

    Callery, P S; Nayar, M S; Geelhaar, L A

    1984-03-01

    The deamination of putrescine catalysed by diamine oxidase was carried out in deuterium oxide and deuterated buffers. Enamine and alpha, beta-unsaturated intermediates were excluded, based on the observation that deuterium was not incorporated into delta 1-pyrroline during its enzymatic formation in deuterium oxide. When the reaction mixture was buffered with phosphate, isolated delta 1-pyrroline contained two deuterium atoms at C-3, indicating that a phosphate-promoted, non-enzymatic isotope exchange had occurred. Using 5,5-dimethyl-delta 1-pyrroline as a model compound, the nature of the non-enzymatic deuterium exchange was studied and a bifunctional catalysis mechanism proposed. The results suggest that the choice of buffer could alter the conclusions drawn from enzyme mechanism studies involving imine-enamine tautomerism .

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

    PubMed

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

    2016-05-01

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

  20. Auger recombination in sodium iodide

    NASA Astrophysics Data System (ADS)

    McAllister, Andrew; Kioupakis, Emmanouil; Åberg, Daniel; Schleife, André

    2014-03-01

    Scintillators are an important tool used to detect high energy radiation - both in the interest of national security and in medicine. However, scintillator detectors currently suffer from lower energy resolutions than expected from basic counting statistics. This has been attributed to non-proportional light yield compared to incoming radiation, but the specific mechanism for this non-proportionality has not been identified. Auger recombination is a non-radiative process that could be contributing to the non-proportionality of scintillating materials. Auger recombination comes in two types - direct and phonon-assisted. We have used first-principles calculations to study Auger recombination in sodium iodide, a well characterized scintillating material. Our findings indicate that phonon-assisted Auger recombination is stronger in sodium iodide than direct Auger recombination. Computational resources provided by LLNL and NERSC. Funding provided by NA-22.

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

    PubMed

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

    2015-08-01

    Effects of substrate-selective inoculum prepared by growing on glucose, xylose, arabinose, GXA (glucose, xylose, arabinose, 1:1:1) and corn stover hydrolyzate (dilute acid pretreated and enzymatically hydrolyzed, CSH) on ethanol production from CSH by a mixed sugar utilizing recombinant Escherichia coli (strain FBR5) were investigated. The initial ethanol productivity was faster for the seed grown on xylose followed by GXA, CSH, glucose and arabinose. Arabinose grown seed took the longest time to complete the fermentation. Delayed saccharifying enzyme addition in simultaneous saccharification and fermentation of dilute acid pretreated CS by the recombinant E. coli strain FBR5 allowed the fermentation to finish in a shorter time than adding the enzyme simultaneously with xylose grown inoculum. Use of substrate selective inoculum and fermenting pentose sugars first under glucose limited condition helped to alleviate the catabolite repression of the recombinant bacterium on ethanol production from lignocellulosic hydrolyzate.

  2. Recombinant protein to analyze autoantibodies to proteinase 3 in systemic vasculitis.

    PubMed

    Rarok, Agnieszka A; Huitema, Minke G; van der Leij, Marcel J; van der Geld, Ymke M; Berthold, Heike; Schmitt, Jacky; Stegeman, Coen A; Limburg, Pieter C; Kallenberg, Cees G

    2003-10-01

    The presence of antineutrophil cytoplasmic autoantibodies with specificity for proteinase 3 (PR3-ANCA) usually is detected by enzyme-linked immunosorbent assay (ELISA) with purified PR3 as a substrate. We studied the technical performance of direct and capture ELISA using a recombinant proteolytically inactive form of PR3 produced in the baculovirus expression system for the detection of PR3-ANCA in 114 patients with systemic vasculitis at diagnosis. We found that ELISA using recombinant PR3 produced in insect cells is a promising alternative for ELISA with native PR3. We found a correlation between tests using recombinant or native PR3, as well as correlation of the ELISA results with ANCA titers measured by the indirect immunofluorescence technique. However, the specificity for ANCA-associated vasculitis of ELISA with recombinant PR3 was lower than ELISA using native PR3. Compared with the direct assay, capture ELISA is a more sensitive method for PR3-ANCA detection, with both native and recombinant PR3, and its results depend on the monoclonal antibody used to capture the antigen.

  3. Nonrandom distribution of interhomolog recombination events induced by breakage of a dicentric chromosome in Saccharomyces cerevisiae.

    PubMed

    Song, Wei; Gawel, Malgorzata; Dominska, Margaret; Greenwell, Patricia W; Hazkani-Covo, Einat; Bloom, Kerry; Petes, Thomas D

    2013-05-01

    Dicentric chromosomes undergo breakage in mitosis, resulting in chromosome deletions, duplications, and translocations. In this study, we map chromosome break sites of dicentrics in Saccharomyces cerevisiae by a mitotic recombination assay. The assay uses a diploid strain in which one homolog has a conditional centromere in addition to a wild-type centromere, and the other homolog has only the wild-type centromere; the conditional centromere is inactive when cells are grown in galactose and is activated when the cells are switched to glucose. In addition, the two homologs are distinguishable by multiple single-nucleotide polymorphisms (SNPs). Under conditions in which the conditional centromere is activated, the functionally dicentric chromosome undergoes double-stranded DNA breaks (DSBs) that can be repaired by mitotic recombination with the homolog. Such recombination events often lead to loss of heterozygosity (LOH) of SNPs that are centromere distal to the crossover. Using a PCR-based assay, we determined the position of LOH in multiple independent recombination events to a resolution of ∼4 kb. This analysis shows that dicentric chromosomes have recombination breakpoints that are broadly distributed between the two centromeres, although there is a clustering of breakpoints within 10 kb of the conditional centromere.

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

    PubMed

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

    2014-12-01

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

  5. Continuous enzymatic liquefaction of starch for saccharification.

    PubMed

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

    1982-11-01

    A process was explored for continuous enzymatic liquefaction of corn starch at high concentration and subsequently 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 variable such as starch concentration (40-70 degrees ), 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 min). 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 process conditions were achieved by using 50-60% starch concentration, at 95 degrees C, with 0.4% alpha-amylase, and a 6-min residence period in the mixture. Under these conditions, rate and extents of glucose obtained after glucoamylase treatment approached those obtained in longer laboratory batch liquefactions. The amount of glucose formed in 24h 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).

  6. 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).

  7. Mutations affecting enzymatic activity in liver arginase

    SciTech Connect

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

    1994-09-01

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

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

    PubMed

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

    2015-09-01

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

  9. 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.

  10. Cryptococcus gattii urease as a virulence factor and the relevance of enzymatic activity in cryptococcosis pathogenesis.

    PubMed

    Feder, Vanessa; Kmetzsch, Lívia; Staats, Charley Christian; Vidal-Figueiredo, Natalia; Ligabue-Braun, Rodrigo; Carlini, Célia Regina; Vainstein, Marilene Henning

    2015-04-01

    Ureases (EC 3.5.1.5) are Ni(2+) -dependent metalloenzymes produced by plants, fungi and bacteria that hydrolyze urea to produce ammonia and CO2 . The insertion of nickel atoms into the apo-urease is better characterized in bacteria, and requires at least three accessory proteins: UreD, UreF, and UreG. Our group has demonstrated that ureases possess ureolytic activity-independent biological properties that could contribute to the pathogenicity of urease-producing microorganisms. The presence of urease in pathogenic bacteria strongly correlates with pathogenesis in some human diseases. Some medically important fungi also produce urease, including Cryptococcus neoformans and Cryptococcus gattii. C. gattii is an etiological agent of cryptococcosis, most often affecting immunocompetent individuals. The cryptococcal urease might play an important role in pathogenesis. It has been proposed that ammonia produced via urease action might damage the host endothelium, which would enable yeast transmigration towards the central nervous system. To analyze the role of urease as a virulence factor in C. gattii, we constructed knockout mutants for the structural urease-coding gene URE1 and for genes that code the accessory proteins Ure4 and Ure6. All knockout mutants showed reduced multiplication within macrophages. In intranasally infected mice, the ure1Δ (lacking urease protein) and ure4Δ (enzymatically inactive apo-urease) mutants caused reduced blood burdens and a delayed time of death, whereas the ure6Δ (enzymatically inactive apo-urease) mutant showed time and dose dependency with regard to fungal burden. Our results suggest that C. gattii urease plays an important role in virulence, in part possibly through enzyme activity-independent mechanism(s).

  11. [Enzymatic utilization of cotton soap stock].

    PubMed

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

    2000-01-01

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

  12. 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.

  13. 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...

  14. 38 CFR 4.88c - Ratings for inactive nonpulmonary tuberculosis initially entitled after August 19, 1968.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

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

  15. 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...

  16. 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...

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

    Code of Federal Regulations, 2012 CFR

    2012-07-01

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

  18. Testing for recombinant erythropoietin.

    PubMed

    Delanghe, Joris R; Bollen, Mathieu; Beullens, Monique

    2008-03-01

    Erythropoietin (Epo) is a glycoprotein hormone that promotes the production of red blood cells. Recombinant human Epo (rhEpo) is illicitly used to improve performance in endurance sports. Doping in sports is discouraged by the screening of athletes for rhEpo. Both direct tests (indicating the presence of exogeneous Epo isoforms) and indirect tests (indicating hematological changes induced by exogenous Epo administration) can be used for Epo detection. At present, the test adopted by the World Anti Doping Agency is based on a combination of isoelectric focusing and double immunoblotting, and distinguishes between endogenous and rhEpo. However, the adopted monoclonal anti-Epo antibodies are not monospecific. Therefore, the test can occasionally lead to the false-positive detection of rhEpo (epoetin-beta) in post-exercise, protein-rich urine, or in case of contamination of the sample with microorganisms. An improved preanalytical care may counteract a lot of these problems. Adaptation of the criteria may be helpful to further refine direct Epo testing. Indirect tests have the disadvantage that they require blood instead of urine samples, but they can be applied to detect a broader range of performance improving techniques which are illicitly used in sports.

  19. Activities of wildtype and mutant p53 in suppression of homologous recombination as measured by a retroviral vector system.

    PubMed

    Lu, Xiongbin; Lozano, Guillermina; Donehower, Lawrence A

    2003-01-28

    DNA repair of double strand breaks, interstrand DNA cross-links, and other types of DNA damage utilizes the processes of homologous recombination and non-homologous end joining to repair the damage. Aberrant homologous recombination is likely to be responsible for a significant fraction of chromosomal deletions, duplications, and translocations that are observed in cancer cells. To facilitate measurement of homologous recombination frequencies in normal cells, mutant cells, and cancer cells, we have developed a high titer retroviral vector containing tandem repeats of mutant versions of a GFP-Zeocin resistance fusion gene and an intact neomycin resistance marker. Recombination between the tandem repeats regenerates a functional GFP-Zeo(R) marker that can be easily scored. This retroviral vector was used to assess homologous recombination frequencies in human cancer cells and rodent fibroblasts with differing dosages of wild type or mutant p53. Absence of wild type p53 stimulated spontaneous and ionizing radiation-induced homologous recombination, confirming previous studies. Moreover, p53(+/-) mouse fibroblasts show elevated levels of homologous recombination compared to their p53(+/+) counterparts following retroviral vector infection, indicating that p53 is haploinsufficient for suppression of homologous recombination. Transfection of vector-containing p53 null Saos-2 cells with various human cancer-associated p53 mutants revealed that these altered p53 proteins retain some recombination suppression function despite being totally inactive for transcriptional transactivation. The retroviral vector utilized in these studies may be useful in performing recombination assays on a wide array of cell types, including those not readily transfected by normal vectors.

  20. Comparative Effectiveness Research Using Observational Data: Active Comparators to Emulate Target Trials with Inactive Comparators

    PubMed Central

    Huitfeldt, Anders; Hernan, Miguel A.; Kalager, Mette; Robins, James M.

    2016-01-01

    Introduction: Because a comparison of noninitiators and initiators of treatment may be hopelessly confounded, guidelines for the conduct of observational research often recommend using an “active” comparator group consisting of people who initiate a treatment other than the medication of interest. In this paper, we discuss the conditions under which this approach is valid if the goal is to emulate a trial with an inactive comparator. Identification of Effects: We provide conditions under which a target trial in a subpopulation can be validly emulated from observational data, using an active comparator that is known or believed to be inactive for the outcome of interest. The average treatment effect in the population as a whole is not identified, but under certain conditions this approach can be used to emulate a trial in the subset of individuals who were treated with the treatment of interest, in the subset of individuals who were treated with the treatment of interest but not with the comparator, or in the subset of individuals who were treated with both the treatment of interest and the active comparator. The Plausibility of the Comparability Conditions: We discuss whether the required conditions can be expected to hold in pharmacoepidemiologic research, with a particular focus on whether the conditions are plausible in situations where the standard analysis fails due to unmeasured confounding by access to health care or health seeking behaviors. Discussion: The conditions discussed in this paper may at best be approximately true. Investigators using active comparator designs to emulate trials with inactive comparators should exercise caution. PMID:27891526

  1. Chemically Linked Vemurafenib Inhibitors Promote an Inactive BRAFV600E Conformation

    PubMed Central

    Grasso, Michael; Estrada, Michelle A.; Ventocilla, Christian; Samanta, Minu; Maksimoska, Jasna; Villanueva, Jessie; Winkler, Jeffrey D.; Marmorstein, Ronen

    2016-01-01

    The BRAF kinase, within the mitogen activated protein kinase (MAPK) signaling pathway, harbors activating mutations in about half of melanomas and to a significant extent in many other cancers. A single valine to glutamic acid substitution at residue 600 (BRAFV600E) accounts for about 90% of these activating mutations. While BRAFV600E-selective small molecule inhibitors, such as debrafenib and vemurafenib, have shown therapeutic benefit, almost all patients develop resistance. Resistance often arises through reactivation of the MAPK pathway, typically through mutation of upstream RAS, downstream MEK or splicing variants. RAF kinases signal as homo- and hetero-dimers, and another complication associated with small molecule BRAFV600E inhibition is drug-induced allosteric activation of a wild-type RAF subunit (BRAF or CRAF) of the kinase dimer, a process called ‘transactivation’ or ‘paradoxical activation.’ Here we used BRAFV600E and vemurafenib as a model system to develop chemically linked kinase inhibitors to lock RAF dimers in an inactive conformation that cannot undergo transactivation. This structure-based design effort resulted in the development of Vem-BisAmide-2, a compound containing two vemurafenib molecules connected by a bis amide linker. We show that Vem-BisAmide-2 has comparable inhibitory potency as vemurafenib to BRAFV600E both in vitro and in cells, but promotes an inactive dimeric BRAFV600E conformation unable to undergo transactivation. The crystal structure of a BRAFV600E/Vem-BisAmide-2 complex and associated biochemical studies reveal the molecular basis for how Vem-BisAmide-2 mediates selectivity for an inactive over active dimeric BRAFV600E conformation. These studies have implications for targeting BRAFV600E/RAF heterodimers and other kinase dimers for therapy. PMID:27571413

  2. 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

  3. Same origins of DNA replication function on the active and inactive human X chromosomes.

    PubMed

    Cohen, Stephanie M; Brylawski, Bruna P; Cordeiro-Stone, Marila; Kaufman, David G

    2003-04-01

    We previously characterized a functional origin of DNA replication at the transcriptional promoter of the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene (Cohen et al. [2002] J. Cell. Biochem. 85:346-356). This origin was mapped using a quantitative PCR assay to evaluate the relative abundance of HPRT markers in short nascent DNA strands isolated from asynchronous cultures of male fibroblasts. The HPRT gene on the X chromosome is transcriptionally active in male human fibroblasts. It is known that on the heterochromatic X chromosome in female cells the HPRT gene is transcriptionally silenced and its replication timing changes from early to late in S phase. This change in replication timing could indicate that replication of the HPRT gene is under the control of different origins of DNA replication in the active (euchromatic, early replicating) and the inactive (heterochromatic, late replicating) X chromosomes. In the present study, we identified the location of the origin of replication of a second X chromosome gene, glucose-6-phosphate dehydrogenase (G6PD), which we mapped to its transcriptional promoter, in normal male human fibroblasts. Then, we determined the activity of the previously identified HPRT and the G6PD human origins in hybrid hamster cells carrying either the active or the inactive human X chromosome. The results of these studies clearly demonstrated that the human HPRT and G6PD origins of replication were utilized to the same extent in the active and the inactive X chromosomes. Therefore, transcription activity at the HPRT and G6PD genes is not necessary for initiation of DNA replication at the origins mapped to these chromosomal loci.

  4. 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

  5. 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

  6. 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.

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

    PubMed

    Wang, Hui; Kobayashi, Shinichi; Mochidzuki, Kazuhiro

    2015-08-01

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

  8. 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

  9. Enzymatic activities in coniferous leaf litter

    SciTech Connect

    Spalding, B.P.

    1980-07-01

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

  10. Enzymatic processing of municipal solid waste.

    PubMed

    Jensen, Jacob Wagner; Felby, Claus; Jørgensen, Henning; Rønsch, Georg Ørnskov; Nørholm, Nanna Dreyer

    2010-12-01

    The focus of this work was to investigate an enzymatic liquefaction of MSW organics, paper and cardboard. Liquefaction trials were conducted in different trial volumes: 50 g lab-scale trials and 5 0kg vessel-tests and evaluated based on particle size and viscosity. The viscosity results showed that Celluclast 1.5L had the singular significant effect on liquefaction of model MSW. No effect of α-amylase, protease and interaction in between and with cellulases on viscosity and particle size distribution was found in this study. Degradable material with a particle size above 1mm after treatment was evaluated using SEM microscopy. These results showed that paper particles were the main obstacles needing additional treatment in order to become fully liquefied. In a pilot scale test treating authentic MSW; more than 90% of initial organic and paper dry matter (DM) was recovered as liquid slurry after sieving through a 5-mm sieve. These tests were performed at up to 35% DM, showing that this process can easily manage high DM loadings. MSW enzymatic liquefaction promotes the separation of organics and paper from solids, which facilitate the use of these degradable fractions, with minimal loss, capable to enter a biogas plant through existing pipes.

  11. Enzymatically Controlled Vacancies in Nanoparticle Crystals

    SciTech Connect

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

    2016-08-01

    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.

  12. 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.

  13. Allergenic potential and enzymatic resistance of buckwheat

    PubMed Central

    Lee, Sujin; Han, Youngshin; Do, Jeong-Ryong

    2013-01-01

    Buckwheat is known as a health food but is one of the major food allergens triggering potentially fatal anaphylaxis in Asia, especially in Japan and Korea. This study was conducted to investigate the characteristic of enzymatic resistance of buckwheat protein and allergenic potential. Enzymatic resistance of buckwheat protein was performed with in vitro digestibility test in simulated gastric fluid (SGF), pH 1.2, using pepsin and simulated intestinal fluid (SIF) using chymotrypsin. Reactivity of buckwheat proteins to human IgE was performed using six allergic patients sensitized to buckwheat. Buckwheat's IgE levels were measured using the Phadia UniCAP-system. Buckwheat protein, 16 kDa, still remained after 30 min treatment of pepsin on SDS-PAGE. Even though 16 kDa almost disappeared after 60 min treatment, two out of the six buckwheat patients' sera showed reactivity to hydrolysate after 60 min treatment, indicating that allergenicity still remained. In simulated intestinal fluid (SIF) using chymotrypsin, buckwheat protein, 24 kDa, showed resistance to hydrolysis with chymotrypsin on SDS-PAGE, and still had allergenicity based on the result of ELISA. Our results suggest that buckwheat proteins have strong resistance to enzyme degradation. This may be attributed in part to the allergenic potential of buckwheat. Further study should be continued regarding buckwheat allergy. PMID:23423876

  14. Enzymatic labeling of proteins: techniques and approaches.

    PubMed

    Rashidian, Mohammad; Dozier, Jonathan K; Distefano, Mark D

    2013-08-21

    Site-specific modification of proteins is a major challenge in modern chemical biology due to the large number of reactive functional groups typically present in polypeptides. Because of its importance in biology and medicine, the development of methods for site-specific modification of proteins is an area of intense research. Selective protein modification procedures have been useful for oriented protein immobilization, for studies of naturally occurring post-translational modifications, for creating antibody–drug conjugates, for the introduction of fluorophores and other small molecules on to proteins, for examining protein structure, folding, dynamics, and protein–protein interactions, and for the preparation of protein–polymer conjugates. One of the most important approaches for protein labeling is to incorporate bioorthogonal functionalities into proteins at specific sites via enzymatic reactions. The incorporated tags then enable reactions that are chemoselective, whose functional groups not only are inert in biological media, but also do not occur natively in proteins or other macromolecules. This review article summarizes the enzymatic strategies, which enable site-specific functionalization of proteins with a variety of different functional groups. The enzymes covered in this review include formylglycine generating enzyme, sialyltransferases, phosphopantetheinyltransferases, O-GlcNAc post-translational modification, sortagging, transglutaminase, farnesyltransferase, biotin ligase, lipoic acid ligase, and N-myristoyltransferase.

  15. 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

  16. 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)

  17. 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

  18. 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.

  19. 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.

  20. Perovskite photovoltaics: Slow recombination unveiled

    NASA Astrophysics Data System (ADS)

    Moser, Jacques-E.

    2017-01-01

    One of the most salient features of hybrid lead halide perovskites is the extended lifetime of their photogenerated charge carriers. This property has now been shown experimentally to originate from a slow, thermally activated recombination process.

  1. Controlled release from recombinant polymers.

    PubMed

    Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

    2014-09-28

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

  2. 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.

  3. Influenza Vaccine, Inactivated or Recombinant

    MedlinePlus

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

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

    PubMed

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

    2015-03-01

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

  5. 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.

  6. Recombinant protein expression in Nicotiana.

    PubMed

    Matoba, Nobuyuki; Davis, Keith R; Palmer, Kenneth E

    2011-01-01

    Recombinant protein pharmaceuticals are now widely used in treatment of chronic diseases, and several recombinant protein subunit vaccines are approved for human and veterinary use. With growing demand for complex protein pharmaceuticals, such as monoclonal antibodies, manufacturing capacity is becoming limited. There is increasing need for safe, scalable, and economical alternatives to mammalian cell culture-based manufacturing systems, which require substantial capital investment for new manufacturing facilities. Since a seminal paper reporting immunoglobulin expression in transgenic plants was published in 1989, there have been many technological advances in plant expression systems to the present time where production of proteins in leaf tissues of nonfood crops such as Nicotiana species is considered a viable alternative. In particular, transient expression systems derived from recombinant plant viral vectors offer opportunities for rapid expression screening, construct optimization, and expression scale-up. Extraction of recombinant proteins from Nicotiana leaf tissues can be achieved by collection of secreted protein fractions, or from a total protein extract after grinding the leaves with buffer. After separation from solids, the major purification challenge is contamination with elements of the photosynthetic complex, which can be solved by application of a variety of facile and proven strategies. In conclusion, the technologies required for safe, efficient, scalable manufacture of recombinant proteins in Nicotiana leaf tissues have matured to the point where several products have already been tested in phase I clinical trials and will soon be followed by a rich pipeline of recombinant vaccines, microbicides, and therapeutic proteins.

  7. 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.

  8. 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.

  9. 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.

  10. Inactive corrinoid-compound significantly decreases in Spirulina platensis grown in a cobalt-deficient medium.

    PubMed

    Watanabe, F; Miyamoto, E; Nakano, Y

    2001-11-01

    Spirulina platensis NIES-39 was grown under open culture system in the presence or absence of CoSO(4) (12 microg/L) and/or vitamin B(12) (10 microg/L) to confirm whether CoSO(4) and/or vitamin B(12) stimulate or are essential for growth of the algal cells and for accumulation of vitamin B(12). The addition of CoSO(4) and/or vitamin B(12) could not affect both cell growth and cell yield of the alga. The amount of corrinoid-compound was increased significantly by the addition of CoSO(4) but not by vitamin B(12). A C18 reversed-phase HPLC pattern of the Spirulina corrinoid-compound increased by the addition of CoSO(4) was identical to that of authentic pseudovitamin B(12), which is inactive for human. These results indicate that the algal cells grown in the absence of CoSO(4) are suitable for use of human health foods because the inactive corrinoid-compound can be reduced significantly.

  11. Physical (in)activity and endothelium-derived constricting factors: overlooked adaptations.

    PubMed

    Thijssen, D H J; Rongen, G A; Smits, P; Hopman, M T E

    2008-01-15

    The inner surrounding of arterial vessels, the endothelium, is optimally located to detect changes in blood characteristics or blood flow that may result from changes in physical activity or from diseases. In response to physical stimuli, the endothelium varies its release of circulating vasoactive substances and serves as a source of local and systemic endothelium-derived dilator and vasoconstrictor factors. Endothelial dysfunction is one of the earliest markers of vascular abnormalities observed in cardiovascular disease and ageing. Exercise training is an efficient therapeutic strategy to improve endothelial function. Traditionally, studies on endothelial dysfunction and physical (in)activity-related effects on vascular adaptations are primarily focused on vasodilator substances (i.e. nitric oxide). One may suggest that augmentation of vasoconstrictor pathways (such as endothelin-1 and angiotensin II) contributes to the endothelial dysfunction observed after physical inactivity. Moreover, these pathways may also explain the exercise-induced beneficial cardiovascular adaptations. This review summarizes the current knowledge on the effects of physical (in)activity on several endothelium-derived vasoconstrictor substances.

  12. Detection of (in)activity periods in human body motion using inertial sensors: a comparative study.

    PubMed

    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.

  13. 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

  14. Reduction potentials of heterometallic manganese-oxido cubane complexes modulated by redox-inactive metals.

    PubMed

    Tsui, Emily Y; Agapie, Theodor

    2013-06-18

    Understanding the effect of redox-inactive metals on the properties of biological and heterogeneous water oxidation catalysts is important both fundamentally and for improvement of future catalyst designs. In this work, heterometallic manganese-oxido cubane clusters [MMn3O4] (M = Sr(2+), Zn(2+), Sc(3+), Y(3+)) structurally relevant to the oxygen-evolving complex (OEC) of photosystem II were prepared and characterized. The reduction potentials of these clusters and other related mixed metal manganese-tetraoxido complexes are correlated with the Lewis acidity of the apical redox-inactive metal in a manner similar to a related series of heterometallic manganese-dioxido clusters. The redox potentials of the [SrMn3O4] and [CaMn3O4] clusters are close, which is consistent with the observation that the OEC is functional only with one of these two metals. Considering our previous studies of [MMn3O2] moieties, the present results with more structurally accurate models of the OEC ([MMn3O4]) suggest a general relationship between the reduction potentials of heterometallic oxido clusters and the Lewis acidities of incorporated cations that applies to diverse structural motifs. These findings support proposals that one function of calcium in the OEC is to modulate the reduction potential of the cluster to allow electron transfer.

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

    PubMed

    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

    2016-01-29

    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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. Intestinal pseudo-obstruction in inactive systemic lupus erythematosus: An unusual finding

    PubMed Central

    Leonardi, Giulia; de Bortoli, Nicola; Bellini, Massimo; Mumolo, Maria Gloria; Costa, Francesco; Ricchiuti, Angelo; Bombardieri, Stefano; Marchi, Santino

    2010-01-01

    Chronic intestinal pseudo-obstruction (CIP) is an infrequent complication of an active systemic lupus erythematosus (SLE). We illustrate a case of SLE inactive-related CIP. A 51-year old female with inactive SLE (ECLAM score 2) was hospitalized with postprandial fullness, vomiting, abdominal bloating and abdominal pain. She had had no bowel movements for five days. Plain abdominal X-ray revealed multiple fluid levels and dilated small and large bowel loops with air-fluid levels. Intestinal contrast radiology detected dilated loops. CIP was diagnosed. The patient was treated with prokinetics, octreotide, claritromycin, rifaximin, azathioprine and tegaserod without any clinical improvement. Then methylprednisolone (500 mg iv daily) was started. After the first administration, the patient showed peristaltic movements. A bowel movement was reported after the second administration. A plain abdominal X-ray revealed no air-fluid levels. Steroid therapy was slowly reduced with complete resolution of the symptoms. The patient is still in a good clinical condition. SLE-related CIP is generally reported as a complication of an active disease. In our case, CIP was the only clinical demonstration of the SLE. PMID:21577309

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

    PubMed

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

    2014-07-15

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

  3. Engineered Mononuclear Variants in Bacillus cereus Metallo-beta-lactamase BcII Are Inactive

    SciTech Connect

    Abriata,L.; Gonzalez, L.; Llarrull, L.; Tomatis, P.; Myers, W.; Costello, A.; Tierney, D.; Vila, A.

    2008-01-01

    Metallo-{beta}-lactamases (M{beta}Ls) are zinc enzymes able to hydrolyze almost all {beta}-lactam antibiotics, rendering them inactive, at the same time endowing bacteria high levels of resistance. The design of inhibitors active against all classes of M{beta}Ls has been hampered by their structural diversity and by the heterogeneity in metal content in enzymes from different sources. BcII is the metallo-{beta}-lactamase from Bacillus cereus, which is found in both the mononuclear and dinuclear forms. Despite extensive studies, there is still controversy about the nature of the active BcII species. Here we have designed two mutant enzymes in which each one of the metal binding sites was selectively removed. Both mutants were almost inactive, despite preserving most of the structural features of each metal site. These results reveal that neither site isolated in the M{beta}L scaffold is sufficient to render a fully active enzyme. This suggests that only the dinuclear species is active or that the mononuclear variants can be active only if aided by other residues that would be metal ligands in the dinuclear species.

  4. 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.

  5. "Spatial Energetics": Integrating Data From GPS, Accelerometry, and GIS to Address Obesity and Inactivity.

    PubMed

    James, Peter; Jankowska, Marta; Marx, Christine; Hart, Jaime E; Berrigan, David; Kerr, Jacqueline; Hurvitz, Philip M; Hipp, J Aaron; Laden, Francine

    2016-11-01

    To address the current obesity and inactivity epidemics, public health researchers have attempted to identify spatial factors that influence physical inactivity and obesity. Technologic and methodologic developments have led to a revolutionary ability to examine dynamic, high-resolution measures of temporally matched location and behavior data through GPS, accelerometry, and GIS. These advances allow the investigation of spatial energetics, high-spatiotemporal resolution data on location and time-matched energetics, to examine how environmental characteristics, space, and time are linked to activity-related health behaviors with far more robust and detailed data than in previous work. Although the transdisciplinary field of spatial energetics demonstrates promise to provide novel insights on how individuals and populations interact with their environment, there remain significant conceptual, technical, analytical, and ethical challenges stemming from the complex data streams that spatial energetics research generates. First, it is essential to better understand what spatial energetics data represent, the relevant spatial context of analysis for these data, and if spatial energetics can establish causality for development of spatially relevant interventions. Second, there are significant technical problems for analysis of voluminous and complex data that may require development of spatially aware scalable computational infrastructures. Third, the field must come to agreement on appropriate statistical methodologies to account for multiple observations per person. Finally, these challenges must be considered within the context of maintaining participant privacy and security. This article describes gaps in current practice and understanding and suggests solutions to move this promising area of research forward.

  6. 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.

  7. Pretreatment and enzymatic hydrolysis of lignocellulosic biomass

    NASA Astrophysics Data System (ADS)

    Corredor, Deisy Y.

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

  8. Recombination and Population Mosaic of a Multifunctional Viral Gene, Adeno-Associated Virus cap

    PubMed Central

    Takeuchi, Yasuhiro; Myers, Richard; Danos, Olivier

    2008-01-01

    Homologous recombination is a dominant force in evolution and results in genetic mosaics. To detect evidence of recombination events and assess the biological significance of genetic mosaics, genome sequences for various viral populations of reasonably large size are now available in the GenBank. We studied a multi-functional viral gene, the adeno-associated virus (AAV) cap gene, which codes for three capsid proteins, VP1, VP2 and VP3. VP1-3 share a common C-terminal domain corresponding to VP3, which forms the viral core structure, while the VP1 unique N-terminal part contains an enzymatic domain with phospholipase A2 activity. Our recombinant detection program (RecI) revealed five novel recombination events, four of which have their cross-over points in the N-terminal, VP1 and VP2 unique region. Comparison of phylogenetic trees for different cap gene regions confirmed discordant phylogenies for the recombinant sequences. Furthermore, differences in the phylogenetic tree structures for the VP1 unique (VP1u) region and the rest of cap highlighted the mosaic nature of cap gene in the AAV population: two dominant forms of VP1u sequences were identified and these forms are linked to diverse sequences in the rest of cap gene. This observation together with the finding of frequent recombination in the VP1 and 2 unique regions suggests that this region is a recombination hot spot. Recombination events in this region preserve protein blocks of distinctive functions and contribute to convergence in VP1u and divergence of the rest of cap. Additionally the possible biological significance of two dominant VP1u forms is inferred. PMID:18286191

  9. 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

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

    PubMed Central

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

    2016-01-01

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

  11. 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.

  12. Efficient production of lactulose from whey powder by cellobiose 2-epimerase in an enzymatic membrane reactor.

    PubMed

    Wu, Lingtian; Xu, Cen; Li, Sha; Liang, Jinfeng; Xu, Hong; Xu, Zheng

    2017-06-01

    In this study, the gene encoding cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE) was successfully expressed in Bacillus subtilis WB800. After the fermentation medium optimization, the activity of recombinant strain was 4.5-fold higher than the original medium in a 7.5L fermentor. The optimal catalytic pH and temperature of crude CsCE were 7.0 and 80°C, respectively. An enzymatic synthesis of lactulose was developed using cheese-whey lactose as its substrate. The maximum conversion rate of whey powder obtained was 58.5% using 7.5 U/mL CsCE. The enzymatic membrane reactor system exhibited a great operational stability, confirmed with the higher lactose conversion (42.4%) after 10 batches. To our best knowledge, this is the first report of lactulose synthesis in food grade strain, which improve the food safety, and we not only realize the biological production of lactulose, but also make good use of industrial waste, which have positive impact on environment.

  13. In silico screening for agonists and blockers of the β2 adrenergic receptor: implications of inactive and activated state structures

    PubMed Central

    Costanzi, Stefano; Vilar, Santiago

    2011-01-01

    Ten crystal structures of the β2 adrenergic receptor (β2AR) have been published, reflecting different signaling states. Here, through controlled docking experiments, we examined the implications of using inactive or activated structures on the in silico screening for agonists and blockers of the receptor. Specifically, we targeted the crystal structures solved in complex with carazolol (2RH1), the neutral antagonist alprenalol (3NYA), the irreversible agonist FAUC50 (3PDS) and the full agonist BI-167017 (3P0G). Our results indicate that activated structures favor agonists over blockers while inactive structures favor blockers over agonists. This tendency is more marked for activated than for inactive structures. Additionally, agonists tend to receive more favorable docking scores when docked at activated rather than inactive structures, while blockers do the opposite. Hence, the difference between the docking scores attained with an activated and an inactive structure is an excellent means for the classification of ligands into agonists and blockers, as we determined through receiver operating characteristic (ROC) curves and linear discriminant analysis (LDA). With respect to virtual screening, all structures prioritized well agonists and blockers over non-binders. However, inactive structures worked better for blockers and activated structures worked better for agonists. Notably, the combination of individual docking experiments through receptor ensemble docking (RED) resulted in an excellent performance in the retrieval of both agonists and blockers. Finally, we demonstrated that the induced fit docking of agonists is a viable way of modifying an inactive crystal structure and bias it towards the in silico recognition of agonists rather than blockers. PMID:22170280

  14. 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...

  15. 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.

  16. Thermal stability of bioactive enzymatic papers.

    PubMed

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

    2010-01-01

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

  17. Enzymatic degradation of multiwalled carbon nanotubes.

    PubMed

    Zhao, Yong; Allen, Brett L; Star, Alexander

    2011-09-01

    Because of their unique properties, carbon nanotubes and, in particular, multiwalled carbon nanotubes (MWNTs) have been used for the development of advanced composite and catalyst materials. Despite their growing commercial applications and increased production, the potential environmental and toxicological impacts of MWNTs are not fully understood; however, many reports suggest that they may be toxic. Therefore, a need exists to develop protocols for effective and safe degradation of MWNTs. In this article, we investigated the effect of chemical functionalization of MWNTs on their enzymatic degradation with horseradish peroxidase (HRP) and hydrogen peroxide (H(2)O(2)). We investigated HRP/H(2)O(2) degradation of purified, oxidized, and nitrogen-doped MWNTs and proposed a layer-by-layer degradation mechanism of nanotubes facilitated by side wall defects. These results provide a better understanding of the interaction between HRP and carbon nanotubes and suggest an eco-friendly way of mitigating the environmental impact of nanotubes.

  18. 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

  19. 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.

  20. Enzymatic synthesis of lipid II and analogues.

    PubMed

    Huang, Lin-Ya; Huang, Shih-Hsien; Chang, Ya-Chih; Cheng, Wei-Chieh; Cheng, Ting-Jen R; Wong, Chi-Huey

    2014-07-28

    The emergence of antibiotic resistance has prompted active research in the development of antibiotics with new modes of action. Among all essential bacterial proteins, transglycosylase polymerizes lipid II into peptidoglycan and is one of the most favorable targets because of its vital role in peptidoglycan synthesis. Described in this study is a practical enzymatic method for the synthesis of lipid II, coupled with cofactor regeneration, to give the product in a 50-70% yield. This development depends on two key steps: the overexpression of MraY for the synthesis of lipid I and the use of undecaprenol kinase for the preparation of polyprenol phosphates. This method was further applied to the synthesis of lipid II analogues. It was found that MraY and undecaprenol kinase can accept a wide range of lipids containing various lengths and configurations. The activity of lipid II analogues for bacterial transglycolase was also evaluated.

  1. Recent development of miniatured enzymatic biofuel cell

    NASA Astrophysics Data System (ADS)

    Song, Yin; Penmatsa, Varun; Wang, Chunlei

    2011-06-01

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

  2. Heavy atom isotope effects on enzymatic reactions

    NASA Astrophysics Data System (ADS)

    Paneth, Piotr

    1994-05-01

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

  3. Proteinuria: an enzymatic disease of the podocyte?

    PubMed Central

    Mundel, Peter; Reiser, Jochen

    2014-01-01

    Proteinuria is a major health-care problem that affects several hundred million people worldwide. Proteinuria is a cardinal sign and a prognostic marker of kidney disease, and also an independent risk factor for cardiovascular morbidity and mortality. Microalbuminuria is the earliest cue of renal complications of diabetes, obesity, and the metabolic syndrome. It can often progress to overt proteinuria that in 10–50% of patients is associated with the development of chronic kidney disease, ultimately requiring dialysis or transplantation. Therefore, reduction or prevention of proteinuria is highly desirable. Here we review recent novel insights into the pathogenesis and treatment of proteinuria, with a special emphasis on the emerging concept that proteinuria can result from enzymatic cleavage of essential regulators of podocyte actin dynamics by cytosolic cathepsin L (CatL), resulting in a motile podocyte phenotype. Finally, we describe signaling pathways controlling the podocyte actin cytoskeleton and motility and how these pathways can be manipulated for therapeutic benefit. PMID:19924101

  4. Enzymatic Hydrogen Production from Starch and Water

    SciTech Connect

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

    2007-01-01

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

  5. Isothermal Titration Calorimetry to Characterize Enzymatic Reactions.

    PubMed

    Mazzei, Luca; Ciurli, Stefano; Zambelli, Barbara

    2016-01-01

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

  6. 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.

  7. Enzymatic production of glycerol acetate from glycerol.

    PubMed

    Oh, Seokhyeon; Park, Chulhwan

    2015-02-01

    In this study, we report the enzymatic production of glycerol acetate from glycerol and methyl acetate. Lipases are essential for the catalysis of this reaction. To find the optimum conditions for glycerol acetate production, sequential experiments were designed. Type of lipase, lipase concentration, molar ratio of reactants, reaction temperature and solvents were investigated for the optimum conversion of glycerol to glycerol acetate. As the result of lipase screening, Novozym 435 (Immobilized Candida antarctica lipase B) was turned out to be the optimal lipase for the reaction. Under the optimal conditions (2.5 g/L of Novozym 435, 1:40 molar ratio of glycerol to methyl acetate, 40 °C and tert-butanol as the solvent), glycerol acetate production was achieved in 95.00% conversion.

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

    PubMed

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

    2016-02-01

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

  9. 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)

  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. Characterizing Enzymatic Deposition for Microelectrode Neurotransmitter Detection

    SciTech Connect

    Hosein, W. K.; Yorita, A. M.; Tolosa, V. M.

    2016-08-12

    The enzyme immobilization process, one step in creating an enzymatic biosensor, was characterized and analyzed as a function of its physical properties. The neural glutamic biosensor is a flexible device, effectively minimizing trauma to the area of implantation. The Multielectrode Array (MEA) is composed primarily of a proprietary polymer which has been successfully implanted into human subjects in recent years. This polymer allows the device the pliability that other devices normally lack, though this poses some challenges to implantation. The electrodes are made of Platinum (Pt), and can range in number from eight to thirty two electrodes per device. These electrodes are electroplated with a semipermeable polymer layer to improve selectivity of the electrode to the neurotransmitter of interest, in this case glutamate. A signal is created from the interaction of glutamate in the brain with the glutamate oxidase (GluOx) which is immobilized on the surface of the electrode by using crosslinking chemistry in conjunction with glutaraldehyde and Bovine Serum Albumin (BSA). The glutamate is oxidized by glutamate oxidase, producing α-ketoglutarate and hydrogen peroxide (H2O2) as a by-product. The production of H2O2 is crucial for detection of the presence of the glutamate within the enzymatic coating, as it diffuses through the enzyme layer and oxidizes at the surface of the electrode. This oxidation is detectable by measurable change in the current using amperometry. Hence, the MEA allows for in vivo monitoring of neurotransmitter activity in real time. The sensitivity of the sensor to these neurotransmitters is dependent on the thickness of the layer, which is investigated in these experiments in order to optimize the efficacy of the device to detecting the substrate, once implanted.

  12. The enzymatic oxidation of graphene oxide.

    PubMed

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

    2011-03-22

    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, ultraviolet-visible, electron paramagnetic resonance, Fourier transform infrared spectroscopy, transmission electron microscopy, atomic force microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gas chromatography-mass spectrometry. Computational docking studies indicated that HRP was preferentially bound to the basal plane rather than the edge for both graphene oxide and RGO. Owing 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.

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

    PubMed

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

    2016-03-01

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

  14. DNA recombination. Recombination initiation maps of individual human genomes.

    PubMed

    Pratto, Florencia; Brick, Kevin; Khil, Pavel; Smagulova, Fatima; Petukhova, Galina V; Camerini-Otero, R Daniel

    2014-11-14

    DNA double-strand breaks (DSBs) are introduced in meiosis to initiate recombination and generate crossovers, the reciprocal exchanges of genetic material between parental chromosomes. Here, we present high-resolution maps of meiotic DSBs in individual human genomes. Comparing DSB maps between individuals shows that along with DNA binding by PRDM9, additional factors may dictate the efficiency of DSB formation. We find evidence for both GC-biased gene conversion and mutagenesis around meiotic DSB hotspots, while frequent colocalization of DSB hotspots with chromosome rearrangement breakpoints implicates the aberrant repair of meiotic DSBs in genomic disorders. Furthermore, our data indicate that DSB frequency is a major determinant of crossover rate. These maps provide new insights into the regulation of meiotic recombination and the impact of meiotic recombination on genome function.

  15. Enzymatic and Non-Enzymatic Virulence Activities of Dermatophytes on Solid Media

    PubMed Central

    Elavarashi, Elangovan; Rangarajan, Sudha

    2017-01-01

    Introduction Dermatophytes are keratinophilic fungi causing superficial cutaneous infections that account 20-25% of the global population. As per literature search, there is a dearth in the study on virulence factors of dermatophytes from the Indian sub-continent and moreover the association of the virulence factors and the host tissue in vitro helps in understanding the host-pathogen interaction. Aim To analyse the enzymatic and non-enzymatic virulence activities of dermatophytes on solid media. Materials and Methods A total of 11 isolates, three standard American Type Culture Collection (ATCC) strains- Trichophyton rubrum- 28188, Trichophyton mentagrophytes- 9533, Trichophyton tonsurans- 28942, one CBS KNAW Fungal Biodiversity Centre strain- Arthroderma grubyi- 243.66, five clinical isolates- T. rubrum, T. mentagrophytes, Trichophyton rubrum var. raubitschekii, Trichophyton interdigitale, Epidermophyton floccosum, and two laboratory isolates - Microsporum gypseum and Microsporum canis were screened for the production of virulence enzymes such as phospholipase, lipase, protease, gelatinase and non-enzyme virulence factors (haemolytic activity) of dermatophytes. The clinical isolates were identified from a tertiary care hospital, Chennai. These dermatophytes were tested upon specific substrates on solid media such as egg yolk, tween 80, bovine serum albumin, gelatin powder and sheep blood respectively. Results The virulence activity of phospholipase, lipase, protease and gelatinase was observed from all the dermatophyte species. T. rubrum, T. rubrum ATCC strain, T. rubrum var. raubitschekii, T. mentagrophytes, T. mentagrophytes ATCC strain, T. interdigitale and A. grubyi CBS strain produced complete haemolysis, whereas other dermatophytes showed no haemolytic activity. Conclusion Phospholipase, lipase, protease and gelatinase act as enzymatic virulence marker and the T. rubrum complex, T. mentagrophytes complex and A. grubyi showed complete haemolysis and hence

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

    NASA Astrophysics Data System (ADS)

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

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

  17. Physical inactivity prevalence and trends among Mexican adults: results from the National Health and Nutrition Survey (ENSANUT) 2006 and 2012

    PubMed Central

    2013-01-01

    Background Lifestyles such as unhealthy diets and the lack of physical activity have been contributed to the increased prevalence of obesity. In 2012, the world health organization published the first global recommendation for physical activity and health. People who do not meet at least 150 minutes of moderate-to-vigorous physical activity are considered to be physically inactive. The prevalence of physical inactivity worldwide is 31%, however there is insufficient data from prevalence and trends of physical inactivity in Mexican population. The purposes of this study are to describe the physical inactivity prevalence and recent trends in Mexican adults and to examine the association between physical inactivity with biologic and sociodemographic characteristics. Methods Representative samples of 17,183 and 10,729 adults (aged 20 to 69 years) who participated in the National Health and Nutrition Survey (ENSANUT) in 2006 and 2012, respectively. Moderate-to-vigorous physical activity (MVPA) was assessed using the short form version of the International Physical Activity Questionnaire (IPAQ), which was administered in face-to-face interviews. Self-reported IPAQ MVPA levels were adjusted using an equation derived from a previous validation study. Participants were considered inactive if they engaged in <150-minutes/week of moderate physical activity or <75 minutes/week of vigorous physical activity according to WHO classification criteria. Results The prevalence of physical inactivity was significantly higher in 2012 (19.4%, 95% CI: 18.1, 20.7) than in 2006 (13.4%, 95% CI: 12.5, 14.5). Adults in the obese category, 60–69 age group, and those in the highest socioeconomic status tertile were more likely to be physically inactive. Conclusions The proportion of the Mexican adult population who do not meet the minimum WHO physical activity criteria has increased by 6% points between 2006 and 2012. Given the increasing prevalence of obesity, the aging of the population

  18. 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.

  19. [Inactive dumps in Santa Catarina's carboniferous area: analysis of risks to the public health and the environment].

    PubMed

    Possamai, Fernando Pagani; Viana, Ednilson; Schulz, Harry Edmar; de Costa, Marcel Madeira; Casagrande, Everson

    2007-01-01

    The existence of inactive dumps considerably increases the so-called "environmental liability"; so much so that the final destination of urban solid residues takes a prominent place on the list of environmental issues of societies(1). In the state of Santa Catarina, it can be said that, currently, the real conditions of the "final destination deposits", or simply the inactive dumps, is officially unknown. This is maybe most evident in the carboniferous area of the state that already suffers from the environmental impact of coal mining. This study attempts to make a survey of the inactive dumps in the carboniferous area of Santa Catarina, analysing the risks they represent to public health and to the environment. The results gathered show that, of the eleven municipal districts in the carboniferous area, nine have inactive dumps. In these nine districts, there are eleven inactive dumps that, according to the this evaluation, represent a large risk to public health and the environment when the parameters analysed are taken into acount.

  20. The Proximal J Kappa Germline-Transcript Promoter Facilitates Receptor Editing through Control of Ordered Recombination

    PubMed Central

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

    2015-01-01

    V(D)J recombination creates antibody light chain diversity by joining a Vκ gene segment with one of four Jκ segments. Two Jκ germline-transcript (GT) promoters control Vκ-Jκ joining, but the mechanisms that govern Jκ choice are unclear. Here, we show in gene-targeted mice that the proximal GT promoter helps targeting rearrangements to Jκ1 by preventing premature DNA breaks at Jκ2. Consequently, cells lacking the proximal GT promoter show a biased utilization of downstream Jκ segments, resulting in a diminished potential for receptor editing. Surprisingly, the proximal—in contrast to the distal—GT promoter is transcriptionally inactive prior to Igκ recombination, indicating that its role in Jκ choice is independent of classical promoter function. Removal of the proximal GT promoter increases H3K4me3 levels at Jκ segments, suggesting that this promoter could act as a suppressor of recombination by limiting chromatin accessibility to RAG. Our findings identify the first cis-element critical for Jκ choice and demonstrate that ordered Igκ recombination facilitates receptor editing. PMID:25559567

  1. High-level secretory production of recombinant bovine enterokinase light chain by Pichia pastoris.

    PubMed

    Peng, Lisheng; Zhong, Xiaofen; Ou, Jingxing; Zheng, Suilan; Liao, Jian; Wang, Lei; Xu, Anlong

    2004-03-04

    Enterokinase (EC 3.4.21.9) is a serine proteinase with a specific digest sequence (Asp)4-Lys in the duodenum. Its high specificity for the recognition site makes enterokinase (EK) a useful tool for an in vitro cleavage of fusion proteins. In this work, an active bovine enterokinase light chain (EK(L)) was produced in secretory form by a recombinant strain of the methylotrophic yeast Pichia pastoris. The influences of methanol utilization phenotype of the host strain, induction pH, and carbon source on the recombinant production were studied. The production of recombinant EK(L) by Mut(s) strain was much higher than that by Mut+ strain. When inducted at pH 6.0, on a glycerol/methanol medium, the concentration of recombinant EK(L) (rEK(L)) reached 350 mg l(-1), which was 20-fold higher than that reported previously. The recombinant EK(L) was purified in a simple procedure on the anion exchange chromatography and 15 mg pure active EK(L) were obtained from 100 ml culture broth supernatant. The specific activity of purified rEK(L) was approximately 9000 u mg(-1). To facilitate purification and removal of rEKL after cleavage of fusion protein, the C-terminal His-tagged EK(L) (EK(L)/His) was also expressed in P. pastoris, and this His-tagged EK(L) exhibited a similar enzymatic activity to the untagged EK(L).

  2. 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.

  3. 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

  4. Cleanup of inactive Uranium Mill Tailings Sites in the Navajo Nation

    SciTech Connect

    Martin, B.

    1994-12-31

    The U.S. Congress passed the Uranium Mill Tailings Radiation Control Act (UMTRCA) in 1978 to address potential and significant radiation health hazards to the public from active and inactive mill operations. Title I to the UMTRCA identified sites to be designated for remedial action. These include four uranium mill tailings remedial action (UMTRA) sites in the Navajo Nation. These sites are located in Shiprock, New Mexico; Tuba City, Arizona; Cane Valley, Arizona; and Halchita, Utah. The U.S. Department of Energy (DOE) was directed to select and execute a plan of remedial action that provides long-term stabilization and control of radioactive materials and satisfies the U.S. Environmental Protection Agency standards and other applicable laws and regulations.

  5. Groundwater contamination from an inactive uranium mill tailings pile: 1. Application of a chemical mixing model

    NASA Astrophysics Data System (ADS)

    White, A. F.; Delany, J. M.; Narasimhan, T. N.; Smith, A.

    1984-11-01

    Low-pH process waters contained in a number of inactive and abandoned uranium mill tailings in the United States represent potential sources of radionuclide and trace metal contamination of groundwater. Detailed investigations at a typical site at Riverton, Wyoming, indicate that chemical transport occurs from initial dewatering of the tailings, downward infiltration due to precipitation, and groundwater intrusion into the base of the tailings pile. Except for elevated uranium and molybdenum concentrations, current radionuclide and trace metal transport is limited by the near-neutral pH conditions of the groundwater. Significant reactions include the dissolution of calcite, production of CO2, and precipitation of gypsum and the hydroxides of iron and aluminum. A geochemical mixing model employing the PHREEQE computer code is used to estimate current rates of the groundwater contamination by tailings water. A maximum mixing of 1.7% of pore water is a factor of 2 less than steady state estimates based on hydraulic parameters.

  6. Revegetation/rock cover for stabilization of inactive uranium mill tailings disposal sites

    SciTech Connect

    Beedlow, P.A.; McShane, M.C.; Cadwell, L.L.

    1982-07-01

    Pacific Northwest Laboratory is developing design and performance guidelines for surface stabilization of inactive uranium mill tailings. In this work, vegetation and rock covers are being evaluated for maintaining long-term integrity of impoundment systems. Methods are being developed to estimate erosion rates associated with rock and/or vegetation covers, and to determine the effects of surface treatments on soil moisture. Interactions between surface treatments and barriers (radon and biological) are being studied as well. The product will be a set of guidelines to aid in designing surface covers. This report presents the status of this program and a discussion of considerations pertinent to the application of surface covers to tailings. Test plots located in Grand Junction, Colorado and Waterflow, New Mexico are being used to study: (1) the interactions between vegetation and radon and biological barriers, (2) the effects of surface covers on soil moisture, and (3) the effects of rock covers on vegetation.

  7. Plant antimutagenic agents, 4. Isolation and structure elucidation of maesol, an inactive constituent of Maesa spp.

    PubMed

    Wall, M E; Wani, M C; Gaetano, K; Manikumar, G; Taylor, H; McGivney, R

    1988-01-01

    Maesol, a novel dimeric phenol, was isolated from seeds of Maesa montana and Maesa indica. Maesol was shown to have the formula C28H42O4 with structure 1, a dimeric, symmetrical 1,12-bis(3,3'-dihydroxy-4,4'-dimethyl-5,5'-dimethoxyphenyl)dodecane. It is the first compound with such structure to be isolated from plant material. Structure elucidation was based largely on 1H- and 13C-nmr techniques and comparison with a known synthetic isomeric dimer 3. Although crude extracts showed strong inhibition of 2-aminoanthracene activity against Salmonella typhimurium (T-98), the pure compound was inactive when tested for inhibition of the mutagenic activity of several mutagens.

  8. YY1 tethers Xist RNA to the inactive X nucleation center

    PubMed Central

    Jeon, Yesu; Lee, Jeannie T.

    2011-01-01

    SUMMARY The long noncoding Xist RNA inactivates one X-chromosome in the female mammal. Current models posit that Xist induces silencing as it spreads along X and recruits Polycomb complexes. However, the mechanisms for Xist loading and spreading are currently unknown. Here, we define the nucleation center for Xist RNA and show that YY1 docks Xist particles onto the X chromosome. YY1 is a ‘bivalent’ protein, capable of binding both RNA and DNA through different sequence motifs. Xist’s exclusive attachment to the inactive X is determined by an epigenetically regulated trio of YY1 sites as well as allelic origin. Specific YY1-to-RNA and YY1-to-DNA contacts are required to load Xist particles onto X. YY1 interacts with Xist RNA through Repeat C. We propose that YY1 acts as adaptor between regulatory RNA and chromatin targets. PMID:21729784

  9. Radiological survey of the inactive uranium-mill tailings at Naturita, Colorado

    SciTech Connect

    Haywood, F.F.; Jacobs, D.J.; Hubbard, H.M. Jr.; Ellis, B.S.; Shinpaugh, W.H.

    1980-03-01

    Results of a radiological survey of the inactive uranium-mill site a Naturita, Colorado, conducted in May 1976, are presented. The spread of tailings was detected in the area surrounding the site by means of direct above ground gamma measurements and analysis of surface and subsurface soil samples. Radiochemical analyses of water samples in the vicinity of the tailings pile indicate local surface water contamination immediately downstream from the pile, although the /sup 226/Ra concentration in the water at that point as well below the concentration guide for drinking water. The calculated subsoil distribution of /sup 226/Ra in onsite holes is presented graphically. The tailings at this site were removed and reprocessed at another location. This operation was completed and reclamation of the site was conducted in 1978. Consequently the information in this report documents radiological conditions that no longer exist.

  10. Radiological survey of the inactive uranium-mill tailings at Durango, Colorado

    SciTech Connect

    Haywood, F.F.; Perdue, P.T.; Shinpaugh, W.H.; Ellis, B.S.; Chou, K.D.

    1980-03-01

    Results of a radiological survey of the inactive uranium-mill site at Durango, Colorado, conducted in April 1976, in cooperation with a team from Ford, Bacon and Davis Utah Inc., are presented together with descriptions of the instruments and techniques used to obtain the data. Direct above-ground gamma measurements and analysis of surface soil and sediment samples indicate movement of tailings from the piles toward Lightner Creek on the north and the Animas River on the east side of the piles. The concentration of /sup 226/Ra in the former raffinate pond area is only slightly above the background level. Two structures in Durango were found to contain high concentrations of airborne radon daughters, where tailings are known to have been utilized in construction. Near-background concentrations of radon daughters were found in a well-ventilated building close to the tailings.

  11. Radiological survey of the inactive uranium-mill tailings at Gunnison, Colorado

    SciTech Connect

    Haywood, F.F.; Jacobs, D.G.; Hubbard, H.M. Jr.; Ellis, B.S.; Shinpaugh, W.H.

    1980-03-01

    The findings of a radiological survey of the inactive uranium-mill site at Gunnison, Colorado, conducted in May 1976, are presented. Results of surface soil sample analyses and direct gamma radiation measurements indicate limited spread of tailings off the site. The only significant above background measurements off the site were obtained in an area previously covered by the tailings pile. There was little evidence of contamination of the surface or of unconfined groundwater in the vicinity of the tailings pile; however, the hydrologic conditions at the site indicate a potential for such contamination. The concentration of /sup 226/Ra in all water samples except one from the tailings pile was well below the concentration guide for drinking water. The subsurface distribution of /sup 226/Ra in 14 bore holes located on and around the tailings pile was calculated from gamma ray monitoring data obtained jointly with Ford, Bacon and Davis Utah Inc.

  12. Radiological survey of the inactive uranium-mill tailings at Maybell, Colorado

    SciTech Connect

    Haywood, F.F.; Perdue, P.T.; Ellis, B.S.

    1980-03-01

    Results of a radiological survey of the inactive uranium-mill tailings near Maybell, Colorado are presented. Measurements of external gamma exposure rate at 1 m above the tailings ranged 16 to 340 ..mu..R/hr with an average value of 65 ..mu..R/hr. Radionuclide analysis of offsite soil and sediment samples, as well as above-ground gamma exposure rate measurements defined the spread of contamination around the tailings pile. This spread is greatest toward the east, in the direction of surface water runoff. Calculated concentrations of /sup 226/Ra in all of the holes drilled in the tailngs, based on gamma monitoring data, showed maximum concentrations in the range 100 to 800 pCi/g.

  13. Assessment of the radiological impact of the inactive uranium-mill tailings at Mexican Hat, Utah

    SciTech Connect

    Haywood, F.F.; Goldsmith, W.A.; Ellis, B.S.; Hubbard, H.M. Jr.; Fox, W.F.; Shinpaugh, W.H.

    1980-03-01

    High surface soil concentrations of /sup 226/Ra and high above-ground measurements of gamma-ray intensity in the vicinity of the inactive uranium-mill tailings at Mexican Hat show both wind and water erosion of the tailings. The former mill area, occupied by a trade school at the time of this survey, shows a comparatively high level of contamination, probably from unprocessed ore on the surface of the ore storage area near the location of the former mill buildings. However, the estimated health effect of exposure to gamma rays during a 2000-hr work year in the area represents an increase of 0.1% in the risk of death from cancer. Exposure of less than 600 persons within 1.6 km of the tailings to radon daughters results in an estimated 0.2%/year increase in risk of lung cancer.

  14. Sampling and Analysis Plan for Flammable Gases in Inactive Miscellaneous Underground Storage Tanks

    SciTech Connect

    NGUYEN, D.M.

    2000-02-01

    This sampling and analysis plan (SAP) identifies the field measurements for a screening of flammable gases in the vapor space of the inactive miscellaneous underground storage tanks (IMUSTs) currently assigned to the River Protection Project (RPP). If a measurement exceeds 25% of the lower flammability limit (LFL), vapor grab samples will be collected for laboratory analysis. This SAP also specifies the sample collection, laboratory analysis, quality assurance/quality control (QA/QC), and reporting objectives for grab sampling. Technical bases for the sampling objectives are provided in the Tank Safety Screening Data Quality Objectives (Dukelow et al 1995). The screening data will be used to determine if additional data are needed to support closure of a flammable gas unreviewed safety question for these facilities.

  15. The inactive-active phase transition in the noisy additive (exclusive-or) probabilistic cellular automaton

    NASA Astrophysics Data System (ADS)

    Mendonça, J. Ricardo G.

    2016-07-01

    We investigate the inactive-active phase transition in an array of additive (exclusive-or) cellular automata (CA) under noise. The model is closely related with the Domany-Kinzel (DK) probabilistic cellular automaton (PCA), for which there are rigorous as well as numerical estimates on the transition probabilities. Here, we characterize the critical behavior of the noisy additive cellular automaton by mean field analysis and finite-size scaling and show that its phase transition belongs to the directed percolation universality class of critical behavior. As a by-product of our analysis, we argue that the critical behavior of the noisy elementary CA 90 and 102 (in Wolfram’s enumeration scheme) must be the same. We also perform an empirical investigation of the mean field equations to assess their quality and find that away from the critical point (but not necessarily very far away) the mean field approximations provide a reasonably good description of the dynamics of the PCA.

  16. Physiology of Fluid and Electrolyte Responses During Inactivity: Water Immersion and Bed Rest

    NASA Technical Reports Server (NTRS)

    Greenleaf, John E.

    1984-01-01

    This manuscript emphasizes the physiology of fluid-electrolyte-hormonal responses during the prolonged inactivity of bed rest and water immersion. An understanding of the total mechanism of adaptation (deconditioning) should provide more insight into the conditioning process. Findings that need to be confirmed during bed rest and immersion are: (1) the volume and tissues of origin of fluid shifted to the thorax and head; (2) interstitial fluid pressure changes in muscle and subcutaneous tissue, particularly during immersion; and (3) the composition of the incoming presumably interstitial fluid that contributes to the early hypervolemia. Better resolution of the time course and source of the diuretic fluid is needed. Important data will be forthcoming when hypotheses are tested involving the probable action of the emerging diuretic and natriuretic hormones, between themselves and among vasopressin and aldosterone, on diuresis and blood pressure control.

  17. Physical inactivity as a risk factor for diabetic retinopathy? A review.

    PubMed

    Dirani, Mohamed; Crowston, Jonathan G; van Wijngaarden, Peter

    2014-08-01

    Physical inactivity and sedentary behaviour have been identified as modifiable risk factors for diabetes. However, little is known of the associations between physical activity, sedentary behaviour and diabetic retinopathy. The development of diabetic retinopathy is associated with longer duration of diabetes, elevated blood pressure and poor glycaemic control. However these factors only explain a proportion of the risk of retinopathy in individuals with diabetes. Several studies have suggested a protective role for physical activity in diabetic retinopathy. Other work has shown that the time spent watching television is independently associated with abnormal retinal vascular signs. Limitations of the existing studies, such as the absence of objective measures of physical activity, a lack of sedentary behaviour measures, the inclusion of only those with type 1 diabetes and a lack of longitudinal data, make it difficult to draw firm conclusions about the strength of these associations.

  18. Radiological survey of the inactive uranium-mill tailings at Rifle, Colorado

    SciTech Connect

    Haywood, F.F.; Jacobs, D.J.; Ellis, B.S.; Hubbard, H.M. Jr.; Shinpaugh, W.H.

    1980-06-01

    Results of radiological surveys of two inactive uranium-mill sites near Rifle, Colorado, in May 1976 are presented. These sites are referred to as Old Rifle and New Rifle. The calculated /sup 226/Ra inventory of the latter site is much higher than at the older mill location. Data on above-ground measurements of gamma exposure rates, surface and near-surface concentration of /sup 226/Ra in soil and sediment samples, concentration of /sup 226/Ra in water, calculated subsurface distribution of /sup 226/Ra, and particulate radionuclide concentrations in air samples are given. The data serve to define the extent of contamination in the vicinity of the mill sites and their immediate surrounding areas with tailings particles. Results of these measurements were utilized as technical input for an engineering assessment of these two sites.

  19. Hazardous waste site assessment: Inactive landfill, Site 300, Lawrence Livermore National Laboratory

    SciTech Connect

    Not Available

    1985-01-01

    This report presents the results of an investigation of an inactive landfill (Pit 6) at Lawrence Livermore National Laboratory's (LLNL) Site 300. The primary objectives were to: collect and review background information pertaining to past waste disposal practices and previous environmental characterization studies; conduct a geophysical survey of the landfill area to locate the buried wastes; conduct a hydrogeologic investigation to provide additional data on the rate and direction of groundwater flow, the extent of any groundwater contamination, and to investigate the connection, if any, of the shallow groundwater beneath the landfill with the local drinking water supply; conduct a risk assessment to identify the degree of threat posed by the landfill to the public health and environment; compile a preliminary list of feasible long-term remedial action alternatives for the landfill; and develop a list of recommendations for any interim measures necessary at the landfill should the long-term remedial action plan be needed.

  20. 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.

  1. 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.

  2. 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.

  3. Accessibility of chromosomal recombination breaks in nuclei of wild-type and DNA-PKcs-deficient cells.

    PubMed

    Franco, Daniel; Chang, Yung

    2009-07-04

    V(D)J recombination is a highly regulated process, proceeding from a site-specific cleavage to an imprecise end joining. After the DNA excision catalyzed by the recombinase encoded by recombination activating genes 1 and 2 (RAG1/2), newly generated recombination ends are believed held by a post-cleavage complex (PC) consisting of RAG1/2 proteins, and are subsequently resolved by non-homologous end joining (NHEJ) machinery. The relay of these ends from PC to NHEJ remains elusive. It has been speculated that NHEJ factors modify the RAG1/2-PC to gain access to the ends or act on free ends after the disassembly of the PC. Thus, recombination ends may either be retained in a complex throughout the recombination process or left as unprotected free ends after cleavage, a condition that may permit an alternative, non-classical NHEJ end joining pathway. To directly test these scenarios on recombination induced chromosomal breaks, we have developed a recombination end protection assay to monitor the accessibility of recombination ends to exonuclease-V in intact nuclei. We demonstrate that these ends are well protected in the nuclei of wild-type cells, suggesting a seamless cleavage-joining reaction. However, divergent end protection of coding versus signal ends was found in cells derived from severe combined immunodeficient (scid) mice that are defective in the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). While signal ends are resistant, opened coding ends are susceptible to enzymatic modification. Our data suggests a role of DNA-PKcs in protecting chromosomal coding ends. Furthermore, using recombination inducible scid cell lines, we demonstrate that conditional protection of coding ends is inversely correlated with the level of their resolution, i.e., the greater the accessibility of the coding ends, the higher level of coding joints formed. Taken together, our findings provide important insights into the resolution of recombination ends by error

  4. The generation and quality of streamflow on inactive uranium tailings near Elliot Lake, Ontario

    NASA Astrophysics Data System (ADS)

    Blowes, David W.; Gillham, Robert W.

    1988-01-01

    This study was undertaken to investigate the physical and chemical interactions between groundwater and surface water in an area of inactive uranium mill tailings near Elliot Lake, Ontario. The study area had a shallow water table and was drained by a single channel. Streamflow chemistry and discharge and the hydraulic response of the groundwater system were monitored for several runoff events during the period of September 1981 through May 1982. The hydrographs measured for each event were separated into groundwater and surface-water components using the mass-balance technique with Cl - and 18O as conservative tracers. The mechanisms of streamflow generation and their effect on runoff quality were evaluated on the basis of the hydrograph separations, the observed groundwater response, and the characteristics of the precipitation events. The results indicate that groundwater constitutes a significant amount of the stormflow generated in the study area. The magnitude of this contribution was dependent upon the intensity and duration of rainfall and on the initial depth to the water table. The capillary fringe played an important role in the processes of streamflow generation, both in causing a large water-table rise which increased groundwater flow to the stream, and in forming an expanding seepage face which enhanced overland flow. During runoff events the stream contained high concentrations of several dissolved inorganic species. The groundwater contribution was a major source of these constituents. The findings of this study indicate that groundwater can be a major component of streamflow during runoff events in inactive tailings areas, and as a result, can have a major influence on runoff quality. The groundwater contribution to streamflow represents an important pathway of contaminant migration to the biosphere which can be anticipated to persist for long periods of time.

  5. Short term aerobic exercise alters the reinforcing value of food in inactive adults.

    PubMed

    Panek, Leah M; Jones, Kelly R; Temple, Jennifer L

    2014-10-01

    Motivation to eat, or the reinforcing value of food, may be influenced by a number of factors, including physical activity. The purpose of these studies was to test the hypothesis that short-term moderate-vigorous intensity aerobic exercise would alter the reinforcing value of high (HED) and low (LED) energy density foods in inactive adults. The reinforcing value of LED and HED food was measured at baseline and again after two weeks of aerobic exercise. In Experiment 1, 41 participants were randomized to a no exercise condition or aerobic exercise for 3 days per week for two weeks. In Experiment 2, 76 participants were randomized to one of four aerobic exercise frequencies, 0, 1, 3, or 5 days per week for two weeks. In both experiments, exercise reduced the reinforcing value of HED food compared to baseline and to non-exercise controls. In Experiment 2, the 5 day group also showed a significant increase in the reinforcing value of LED food compared to baseline and other exercise frequencies. Liking of HED and LED foods and consumption of HED food were not affected by exercise treatment. Finally, in Experiment 2, the 5 day group reported consuming more energy outside of the laboratory than the other groups. Taken together, these data suggest, in inactive individuals, motivation to obtain HED and LED foods can be altered with a short-term moderate-vigorous intensity exercise intervention. Further research is needed to understand the cognitive and physiological processes involved in food choices paired with exercise.

  6. Differences in Allosteric Communication Pipelines in the Inactive and Active States of a GPCR

    PubMed Central

    Bhattacharya, Supriyo; Vaidehi, Nagarajan

    2014-01-01

    G-protein-coupled receptors (GPCRs) are membrane proteins that allosterically transduce the signal of ligand binding in the extracellular (EC) domain to couple to proteins in the intracellular (IC) domain. However, the complete pathway of allosteric communication from the EC to the IC domain, including the role of individual amino acids in the pathway is not known. Using the correlation in torsion angle movements calculated from microseconds-long molecular-dynamics simulations, we elucidated the allosteric pathways in three different conformational states of β2-adrenergic receptor (β2AR): 1), the inverse-agonist-bound inactive state; 2), the agonist-bound intermediate state; and (3), the agonist- and G-protein-bound fully active state. The inactive state is less dynamic compared with the intermediate and active states, showing dense clusters of allosteric pathways (allosteric pipelines) connecting the EC with the IC domain. The allosteric pipelines from the EC domain to the IC domain are weakened in the intermediate state, thus decoupling the EC domain from the IC domain and making the receptor more dynamic compared with the other states. Also, the orthosteric ligand-binding site becomes the initiator region for allosteric communication in the intermediate state. This finding agrees with the paradigm that the nature of the agonist governs the specific signaling state of the receptor. These results provide an understanding of the mechanism of allosteric communication in class A GPCRs. In addition, our analysis shows that mutations that affect the ligand efficacy, but not the binding affinity, are located in the allosteric pipelines. This clarifies the role of such mutations, which has hitherto been unexplained. PMID:25028884

  7. The Prevalence and Incidence of Epiretinal Membranes in Eyes With Inactive Extramacular CMV Retinitis

    PubMed Central

    Kozak, Igor; Vaidya, Vijay; Van Natta, Mark L.; Pak, Jeong W.; May, K. Patrick; Thorne, Jennifer E.

    2014-01-01

    Purpose. To determine the prevalence and incidence of epiretinal membranes (ERM) in eyes with inactive extramacular cytomegalovirus (CMV) retinitis in patients with acquired immune deficiency syndrome (AIDS). Methods. A case–control report from a longitudinal multicenter observational study by the Studies of the Ocular Complications of AIDS (SOCA) Research Group. A total of 357 eyes of 270 patients with inactive CMV retinitis and 1084 eyes of 552 patients with no ocular opportunistic infection (OOI) were studied. Stereoscopic views of the posterior pole from fundus photographs were assessed at baseline and year 5 visits for the presence of macular ERM. Generalized estimating equations (GEE) logistic regression was used to compare the prevalence and 5-year incidence of ERM in eyes with and without CMV retinitis at enrollment. Crude and adjusted logistic regression was performed adjusting for possible confounders. Main outcome measures included the prevalence, incidence, estimated prevalence, and incidence odds ratios. Results. The prevalence of ERM at enrollment was 14.8% (53/357) in eyes with CMV retinitis versus 1.8% (19/1084) in eyes with no OOI. The incidence of ERM at 5 years was 18.6% (16/86) in eyes with CMV retinitis versus 2.4% (6/253) in eyes with no OOI. The crude odds ratio (OR) (95% confidence interval, CI) for prevalence was 9.8 (5.5–17.5) (P < 0.01). The crude OR (95% CI) for incidence was 9.4 (3.2–27.9) (P < 0.01). Conclusions. A history of extramacular CMV retinitis is associated with increased prevalence and incidence of ERM formation compared to what is seen in eyes without ocular opportunistic infections in AIDS patients. PMID:24925880

  8. Colistin Methanesulfonate Is an Inactive Prodrug of Colistin against Pseudomonas aeruginosa

    PubMed Central

    Bergen, Phillip J.; Li, Jian; Rayner, Craig R.; Nation, Roger L.

    2006-01-01

    There is a dearth of information on the pharmacodynamics of “colistin,” despite its increasing use as a last line of defense for treatment of infections caused by multidrug-resistant gram-negative organisms. The antimicrobial activities of colistin and colistin methanesulfonate (CMS) were investigated by studying the time-kill kinetics of each against a type culture of Pseudomonas aeruginosa in cation-adjusted Mueller-Hinton broth. The appearance of colistin from CMS spiked at 8.0 and 32 mg/liter was measured by high-performance liquid chromatography, which generated colistin concentration-time profiles. These concentration-time profiles were subsequently mimicked in other incubations, independent of CMS, by incrementally spiking colistin. When the cultures were spiked with CMS at either concentration, there was a substantial delay in the onset of the killing effect which was not evident until the concentrations of colistin generated from the hydrolysis of CMS had reached approximately 0.5 to 1 mg/liter (i.e., ∼0.5 to 1 times the MIC for colistin). The time course of the killing effect was similar when colistin was added incrementally to achieve the same colistin concentration-time course observed from the hydrolysis of CMS. Given that the killing kinetics of CMS can be accounted for by the appearance of colistin, CMS is an inactive prodrug of colistin with activity against P. aeruginosa. This is the first study to demonstrate the formation of colistin in microbiological media containing CMS and to demonstrate that CMS is an inactive prodrug of colistin. These findings have important implications for susceptibility testing involving “colistin,” in particular, for MIC measurement and for microbiological assays and pharmacokinetic and pharmacodynamic studies. PMID:16723551

  9. Metabolic rates and biochemical compositions of Apostichopus japonicus (Selenka) tissue during periods of inactivity

    NASA Astrophysics Data System (ADS)

    Bao, Jie; Dong, Shuanglin; Tian, Xiangli; Wang, Fang; Gao, Qinfeng; Dong, Yunwei

    2010-03-01

    Estivation, hibernation, and starvation are indispensable inactive states of sea cucumbers Apostichopus japonicus in nature and in culture ponds. Generally, temperature is the principal factor that induces estivation or hibernation in the sea cucumber. The present study provided insight into the physiological adaptations of A. japonicus during the three types of inactivity (hibernation, estivation, and starvation) by measuring the oxygen consumption rates ( Vo2) and biochemical compositions under laboratory conditions of low (3°C), normal (17°C) and high (24°C) temperature. The results show that the characteristics of A. japonicus in dormancy (hibernation and estivation) states were quite different from higher animals, such as fishes, amphibians, reptiles, and mammals, but more closely resembled a semi-dormant state. It was observed that the shift in the A. japonicus physiological state from normal to dormancy was a chronic rather than acute process, indicated by the gradual depression of metabolic rate. While metabolic rates declined 44.9% for the estivation group and 71.7% for the hibernation group, relative to initial rates, during the 36 d culture period, metabolic rates were not maintained at constant levels during these states. The metabolic depression processes for sea cucumbers in hibernation and estivation appeared to be a passive and an active metabolic suppression, respectively. In contrast, the metabolic rates (128.90±11.70 μg/g h) of estivating sea cucumbers were notably higher (107.85±6.31 μg/g h) than in starving sea cucumbers at 17°C, which indicated that the dormancy mechanism here, as a physiological inhibition, was not as efficient as in higher animals. Finally, the principle metabolic substrate or energy source of sea cucumbers in hibernation was lipid, whereas in estivation they mainly consumed protein in the early times and both protein and lipid thereafter.

  10. Unusual chromatin status and organization of the inactive X chromosome in murine trophoblast giant cells.

    PubMed

    Corbel, Catherine; Diabangouaya, Patricia; Gendrel, Anne-Valerie; Chow, Jennifer C; Heard, Edith

    2013-02-01

    Mammalian X-chromosome inactivation (XCI) enables dosage compensation between XX females and XY males. It is an essential process and its absence in XX individuals results in early lethality due primarily to extra-embryonic defects. This sensitivity to X-linked gene dosage in extra-embryonic tissues is difficult to reconcile with the reported tendency of escape from XCI in these tissues. The precise transcriptional status of the inactive X chromosome in different lineages has mainly been examined using transgenes or in in vitro differentiated stem cells and the degree to which endogenous X-linked genes are silenced in embryonic and extra-embryonic lineages during early postimplantation stages is unclear. Here we investigate the precise temporal and lineage-specific X-inactivation status of several genes in postimplantation mouse embryos. We find stable gene silencing in most lineages, with significant levels of escape from XCI mainly in one extra-embryonic cell type: trophoblast giant cells (TGCs). To investigate the basis of this epigenetic instability, we examined the chromatin structure and organization of the inactive X chromosome in TGCs obtained from ectoplacental cone explants. We find that the Xist RNA-coated X chromosome has a highly unusual chromatin content in TGCs, presenting both heterochromatic marks such as H3K27me3 and euchromatic marks such as histone H4 acetylation and H3K4 methylation. Strikingly, Xist RNA does not form an overt silent nuclear compartment or Cot1 hole in these cells. This unusual combination of silent and active features is likely to reflect, and might underlie, the partial activity of the X chromosome in TGCs.

  11. Maternal inflammation during late pregnancy is lower in physically active compared with inactive obese women.

    PubMed

    Tinius, Rachel A; Cahill, Alison G; Strand, Eric A; Cade, W Todd

    2016-02-01

    The primary purpose of this study was to compare maternal plasma inflammation between physically active and inactive obese women during late pregnancy. The secondary purpose was to examine the relationships between maternal plasma inflammation and lipid metabolism and maternal and neonatal metabolic health in these women. A cross-sectional, observational study design was performed in 16 obese-inactive (OBI; means ± SD; age, 25.0 ± 4.8 years; prepregnancy body mass index (BMI), 36.3 ± 4.3 kg/m(2); body fat percentage in late gestation, 37.7% ± 3.5%) and 16 obese-active (OBA; age, 28.9 ± 4.8 years; prepregnancy BMI, 34.0 ± 3.7 kg/m(2); body fat in late gestation, 36.6% ± 3.8%) women during the third trimester of pregnancy. Maternal plasma inflammation (C -reactive protein (CRP)) and insulin resistance (Homeostatic Model Assessment-Insulin Resistance) were measured at rest. Plasma lipid concentration and metabolism (lipid oxidation and lipolysis) were measured at rest, during a 30-min bout of low-intensity (40% peak oxygen uptake) exercise, and during a resting recovery period using indirect calorimetry. Umbilical cord blood was collected for measurement of neonatal plasma insulin resistance, inflammation, and lipid concentration. Neonatal body composition was measured via air displacement plethysmography. Maternal plasma CRP concentration was significantly higher in OBI compared with OBA women (9.1 ± 4.0 mg/L vs. 6.3 ± 2.5 mg/L, p = 0.02). Maternal plasma CRP concentration was significantly associated with maternal lipolysis (r = 0.43, p = 0.02), baseline lipid oxidation rate (r = 0.39, p = 0.03), and baseline plasma free fatty acid concentration (r = 0.36, p = 0.04). In conclusion, maternal physical activity may reduce inflammation during pregnancy in obese women. Maternal lipid metabolism is related to systemic inflammation.

  12. Variation in Recombination Rate: Adaptive or Not?

    PubMed

    Ritz, Kathryn R; Noor, Mohamed A F; Singh, Nadia D

    2017-03-27

    Rates of meiotic recombination are widely variable both within and among species. However, the functional significance of this variation remains largely unknown. Is the observed within-species variation in recombination rate adaptive? Recent work has revealed new insight into the scale and scope of population-level variation in recombination rate. These data indicate that the magnitude of within-population variation in recombination is similar among taxa. The apparent similarity of the variance in recombination rate among individuals between distantly related species suggests that the relative costs and benefits of recombination that establish the upper and lower bounds may be similar across species. Here we review the current data on intraspecific variation in recombination rate and discuss the molecular and evolutionary costs and benefits of recombination frequency. We place this variation in the context of adaptation and highlight the need for more empirical studies focused on the adaptive value of variation in recombination rate.

  13. Efficient enzymatic degradation process for hydrolysis activity of the Carrageenan from red algae in marine biomass.

    PubMed

    Kang, Dae Hee; Hyeon, Jeong Eun; You, Seung Kyou; Kim, Seung Wook; Han, Sung Ok

    2014-12-20

    Carrageenan is a generic name for a family of polysaccharides obtained from certain species of red algae. New methods to produce useful cost-efficiently materials from red algae are needed to convert enzymatic processes into fermentable sugars. In this study, we constructed chimeric genes cCgkA and cCglA containing the catalytic domain of κ-carrageenase CgkA and λ-carrageenase CglA from Pseudoalteromonas carrageenovora fused with a dockerin domain. Recombinant strains expressing the chimeric carrageenase resulted in a halo formation on the carrageenan plate by alcian blue staining. The recombinant cCgkA and cCglA were assembled with scaffoldin miniCbpA via cohesin and dockerin interaction. Carbohydrate binding module (CBM) in scaffoldin was used as a tag for cellulose affinity purification using cellulose as a support. The hydrolysis process was monitored by the amount of reducing sugar released from carrageenan. Interestingly, these results indicated that miniCbpA, cCgkA and cCglA assembled into a complex and that the dockerin-fused enzymes on the scaffoldin had synergistic activity in the degradation of carrageenan. The observed enhancement of activity by carrageenolytic complex was 3.1-fold-higher compared with the corresponding enzymes alone. Thus, the assemblies of advancement of active enzyme complexes will facilitate the commercial production of useful products from red algae biomass which represents inexpensive and sustainable feed-stocks.

  14. 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.

  15. 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.

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

    PubMed

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

    2003-10-10

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

  17. Sex differences in the physical inactivity and health-related quality of life relationship among rural adults.

    PubMed

    Hart, Peter D

    2016-01-01

    Background: The purpose of this study was to investigate the relationship between physical inactivity (PIA) and health-related quality of life (HRQOL) in rural adults and examine the extent to which sex differences exist in this relationship. Methods: A total of 5617 adults 18 years of age and older who indicated residing in a rural county was included in this analysis. PIA status was assessed by questions regarding recreational physical activity during the previous month. Five HRQOL measures (physical health, mental health, inactivity health, general health, & unhealthy days) were used as primary outcome variables. PIA and HRQOL prevalence estimates were computed with 95% CIs. Multiple logistic regression was used to obtain odds ratios (ORs) and 95% CIs adjusted for age, ethnicity, and income. Results: Physically inactive rural adults were significantly more likely to report poor HRQOL in all overall crude models with ORs ranging from 1.59 to 2.16. Additionally, sex-by-PIA interactions were significant across all crude HRQOL models with ORs ranging from 2.27 to 3.08 and 1.56 to 2.42 for women and men, respectively. Sex differences were maintained in fully adjusted models, except for mental health and inactivity health with ORs ranging from 1.80 to 2.58 and 1.41 to 1.79 for women and men, respectively. Conclusion: Results from this study show that PIA is a strong predictor of poor HRQOL even after controlling for confounding variables. Furthermore, physically inactive rural women appear more likely to report poor levels of HRQOL than physically inactive rural men.

  18. Metabolic profiles using (1)H-nuclear magnetic resonance spectroscopy in postpartum dairy cows with ovarian inactivity.

    PubMed

    Xu, Chuchu; Xia, Cheng; Sun, Yuhang; Xiao, Xinhuan; Wang, Gang; Fan, Ziling; Shu, Shi; Zhang, Hongyou; Xu, Chuang; Yang, Wei

    2016-10-01

    To understand the differences in metabolic changes between cows with ovarian inactivity and estrus cows, we selected cows at 60-90 days postpartum from an intensive dairy farm. According to clinical manifestations, B-ultrasound scan, rectal examination, 10 cows were assigned to the estrus group (A) and 10 to the ovarian inactivity group (B). All plasma samples were analyzed by (1)H-nuclear magnetic resonance spectroscopy to compare plasma metabolomic profiles between the groups. We used multivariate pattern recognition to screen for different metabolites in plasma of anestrus cows. Compared with normal estrous cows, there were abnormalities in 12 kinds of metabolites in postpartum cows with ovarian inactivity (|r|> 0.602), including an increase in acetic acid (r = -0.817), citric acid (r = -0.767), and tyrosine (r = -0.714), and a decrease in low-density lipoprotein (r = 0.820), very low-density lipoprotein (r = 0.828), lipids (r = 0.769), alanine (r = 0.816), pyruvate (r = 0.721), creatine (r = 0.801), choline (r = 0.639), phosphorylcholine (r = 0.741), and glycerophosphorylcholine (r = 0.881). These metabolites were closely related to abnormality of glucose, amino acid, lipoprotein and choline metabolism, which may disturb the normal estrus. The decrease in plasma creatine and the increase in tyrosine were new changes for ovarian inactivity of postpartum cows. The decrease in plasma creatine and choline and the increase in tyrosine and p-hydroxyphenylalanine in cows with ovarian inactivity provide new directions for research on the mechanism of ovarian inactivity in cows.

  19. Life and death of deep-sea vents: bacterial diversity and ecosystem succession on inactive hydrothermal sulfides.

    PubMed

    Sylvan, Jason B; Toner, Brandy M; Edwards, Katrina J

    2012-01-01

    Hydrothermal chimneys are a globally dispersed habitat on the seafloor associated with mid-ocean ridge (MOR) spreading centers. Active, hot, venting sulfide structures from MORs have been examined for microbial diversity and ecology since their discovery in the mid-1970s, and recent work has also begun to explore the microbiology of inactive sulfides--structures that persist for decades to millennia and form moderate to massive deposits at and below the seafloor. Here we used tag pyrosequencing of the V6 region of the 16S rRNA and full-length 16S rRNA sequencing on inactive hydrothermal sulfide chimney samples from 9°N on the East Pacific Rise to learn their bacterial composition, metabolic potential, and succession from venting to nonventing (inactive) regimes. Alpha-, beta-, delta-, and gammaproteobacteria and members of the phylum Bacteroidetes dominate all inactive sulfides. Greater than 26% of the V6 tags obtained are closely related to lineages involved in sulfur, nitrogen, iron, and methane cycling. Epsilonproteobacteria represent <4% of the V6 tags recovered from inactive sulfides and 15% of the full-length clones, despite their high abundance in active chimneys. Members of the phylum Aquificae, which are common in active vents, were absent from both the V6 tags and full-length 16S rRNA data sets. In both analyses, the proportions of alphaproteobacteria, betaproteobacteria, and members of the phylum Bacteroidetes were greater than those found on active hydrothermal sulfides. These shifts in bacterial population structure on inactive chimneys reveal ecological succession following cessation of venting and also imply a potential shift in microbial activity and metabolic guilds on hydrothermal sulfides, the dominant biome that results from seafloor venting.

  20. 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.