WorldWideScience

Sample records for acid residues required

  1. Amino acid residues of heparin cofactor II required for stimulation of thrombin inhibition by sulphated polyanions.

    Colwell, N S; Grupe, M J; Tollefsen, D M

    1999-04-12

    A variety of sulphated polyanions in addition to heparin and dermatan sulphate stimulate the inhibition of thrombin by heparin cofactor II (HCII). Previous investigations indicated that the binding sites on HCII for heparin and dermatan sulphate overlap but are not identical. In this study we determined the concentrations (IC50) of various polyanions required to stimulate thrombin inhibition by native recombinant HCII in comparison with three recombinant HCII variants having decreased affinity for heparin (Lys-173-->Gln), dermatan sulphate (Arg-189-->His), or both heparin and dermatan sulphate (Lys-185-->Asn). Pentosan polysulphate, sulphated bis-lactobionic acid amide, and sulphated bis-maltobionic acid amide resembled dermatan sulphate, since their IC50 values were increased to a much greater degree (>/=8-fold) by the mutations Arg-189-->His and Lys-185-->Asn than by Lys-173-->Gln (Gln and Lys-185-->Asn (>/=6-fold) than by Arg-189-->His (requiring the N-terminal acidic domain. PMID:10209287

  2. Identification of an amino acid residue required for differential recognition of a viral movement protein by the Tomato mosaic virus resistance gene Tm-2(2).

    Kobayashi, Michie; Yamamoto-Katou, Ayako; Katou, Shinpei; Hirai, Katsuyuki; Meshi, Tetsuo; Ohashi, Yuko; Mitsuhara, Ichiro

    2011-07-01

    The Tm-2 gene of tomato and its allelic gene, Tm-2(2), confer resistance to Tomato mosaic virus (ToMV) and encode a member of the coiled-coil/nucleotide binding-ARC/leucine-rich repeat (LRR) protein class of plant resistance (R) genes. Despite exhibiting only four amino acid differences between the products of Tm-2 and Tm-2(2), Tm-2(2) confers resistance to ToMV mutant B7, whereas Tm-2 is broken by ToMV-B7. An Agrobacterium-mediated transient expression system was used to study the mechanism of differential recognition of the movement proteins (MPs), an avirulence factor for ToMV resistance, of ToMV-B7 by Tm-2 and Tm-2(2). Although resistance induced by Tm-2 and Tm-2(2) is not usually accompanied by hypersensitive response (HR), Tm-2 and Tm-2(2) induced HR-like cell death by co-expression with MP of a wild-type ToMV, a strain that causes resistance for these R genes, and Tm-2(2) but not Tm-2 induced cell death with B7-MP in this system. Site-directed amino acid mutagenesis revealed that Tyr-767 in the LRR of Tm-2(2) is required for the specific recognition of the B7-MP. These results suggest that the Tyr residue in LRR contributes to the recognition of B7-MP, and that Tm-2 and Tm-2(2) are involved in HR cell death. PMID:21310506

  3. Identification of Amino Acid Residues in Fibroblast Growth Factor 14 (FGF14) Required for Structure-Function Interactions with Voltage-gated Sodium Channel Nav1.6.

    Ali, Syed R; Singh, Aditya K; Laezza, Fernanda

    2016-05-20

    The voltage-gated Na(+) (Nav) channel provides the basis for electrical excitability in the brain. This channel is regulated by a number of accessory proteins including fibroblast growth factor 14 (FGF14), a member of the intracellular FGF family. In addition to forming homodimers, FGF14 binds directly to the Nav1.6 channel C-tail, regulating channel gating and expression, properties that are required for intrinsic excitability in neurons. Seeking amino acid residues with unique roles at the protein-protein interaction interface (PPI) of FGF14·Nav1.6, we engineered model-guided mutations of FGF14 and validated their impact on the FGF14·Nav1.6 complex and the FGF14:FGF14 dimer formation using a luciferase assay. Divergence was found in the β-9 sheet of FGF14 where an alanine (Ala) mutation of Val-160 impaired binding to Nav1.6 but had no effect on FGF14:FGF14 dimer formation. Additional analysis revealed also a key role of residues Lys-74/Ile-76 at the N-terminal of FGF14 in the FGF14·Nav1.6 complex and FGF14:FGF14 dimer formation. Using whole-cell patch clamp electrophysiology, we demonstrated that either the FGF14(V160A) or the FGF14(K74A/I76A) mutation was sufficient to abolish the FGF14-dependent regulation of peak transient Na(+) currents and the voltage-dependent activation and steady-state inactivation of Nav1.6; but only V160A with a concomitant alanine mutation at Tyr-158 could impede FGF14-dependent modulation of the channel fast inactivation. Intrinsic fluorescence spectroscopy of purified proteins confirmed a stronger binding reduction of FGF14(V160A) to the Nav1.6 C-tail compared with FGF14(K74A/I76A) Altogether these studies indicate that the β-9 sheet and the N terminus of FGF14 are well positioned targets for drug development of PPI-based allosteric modulators of Nav channels. PMID:26994141

  4. Amino Acid Sequence Requirements at Residues 69 and 238 for the SME-1 β-Lactamase To Confer Resistance to β-Lactam Antibiotics

    Majiduddin, Fahd K.; Palzkill, Timothy

    2003-01-01

    Carbapenem antibiotics have been used to counteract resistant strains of bacteria harboring β-lactamases and extended-spectrum β-lactamases. Four enzymes from the class A group of β-lactamases, NMC-A, IMI-1, SME-1, and KPC-1, efficiently hydrolyze carbapenem antibiotics. Sequence comparisons and structural information indicate that cysteines at amino acid residues 69 and 238, which are conserved in all four of these enzymes, form a disulfide bond that is unique to these β-lactamases. To test ...

  5. Efficient autophosphorylation and phosphorylation of the beta-subunit by casein kinase-2 require the integrity of an acidic cluster 50 residues downstream from the phosphoacceptor site

    Boldyreff, B; Meggio, F; Pinna, L A; Issinger, O G

    1994-01-01

    Various beta-mutants were investigated either as subunits or as substrates for casein kinase 2 (CK-2), in the absence of presence of polylysine. A total of 21 beta-mutants were characterized for their susceptibility to autophosphorylation, by combining them in equimolar amounts with the recombinant...... alpha-subunit. Six mutants, i.e. beta A5,6, beta A59-61,63,64, beta A55,57, beta 55-57, beta delta 171-215, and beta delta 150-215 exhibited a > 70% reduction in autophosphorylation. This strongly suggests that in addition to amino acid residues 5,6, distant amino acid residues within the sequence 55...... autophosphorylation of beta wt (86% inhibition) inducing a parallel increase of the alpha-subunit autophosphorylation. The autophosphorylation of all mutants, with the exception of beta A55-57 and beta A59-61,63,64, is also inhibited by polylysine (>64%). The alpha-subunit autophosphorylation is increased with all...

  6. 40 CFR 161.240 - Residue chemistry data requirements.

    2010-07-01

    ... requirement if their residues are regulated by the Food and Drug Administration at 21 CFR 178.1010. (11... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Residue chemistry data requirements... § 161.240 Residue chemistry data requirements. (a) Table. Sections 161.100 through 161.102 describe...

  7. Identification of amino acid residues in protein SRP72 required for binding to a kinked 5e motif of the human signal recognition particle RNA

    Zwieb Christian

    2010-11-01

    Full Text Available Abstract Background Human cells depend critically on the signal recognition particle (SRP for the sorting and delivery of their proteins. The SRP is a ribonucleoprotein complex which binds to signal sequences of secretory polypeptides as they emerge from the ribosome. Among the six proteins of the eukaryotic SRP, the largest protein, SRP72, is essential for protein targeting and possesses a poorly characterized RNA binding domain. Results We delineated the minimal region of SRP72 capable of forming a stable complex with an SRP RNA fragment. The region encompassed residues 545 to 585 of the full-length human SRP72 and contained a lysine-rich cluster (KKKKKKKKGK at postions 552 to 561 as well as a conserved Pfam motif with the sequence PDPXRWLPXXER at positions 572 to 583. We demonstrated by site-directed mutagenesis that both regions participated in the formation of a complex with the RNA. In agreement with biochemical data and results from chymotryptic digestion experiments, molecular modeling of SRP72 implied that the invariant W577 was located inside the predicted structure of an RNA binding domain. The 11-nucleotide 5e motif contained within the SRP RNA fragment was shown by comparative electrophoresis on native polyacrylamide gels to conform to an RNA kink-turn. The model of the complex suggested that the conserved A240 of the K-turn, previously identified as being essential for the binding to SRP72, could protrude into a groove of the SRP72 RNA binding domain, similar but not identical to how other K-turn recognizing proteins interact with RNA. Conclusions The results from the presented experiments provided insights into the molecular details of a functionally important and structurally interesting RNA-protein interaction. A model for how a ligand binding pocket of SRP72 can accommodate a new RNA K-turn in the 5e region of the eukaryotic SRP RNA is proposed.

  8. 40 CFR 158.1410 - Residue chemistry data requirements table.

    2010-07-01

    ... table list the data requirements for residue chemistry related to food uses. The table notes are shown in paragraph (e) of this section. Table—Residue Chemistry Data Requirements for Food Uses Guideline... feed. 2. Material safety data sheets must accompany standards as specified by OSHA in 29 CFR...

  9. Recovery of mercury from acid waste residues

    Greenhalgh, Wilbur O.

    1989-01-01

    Mercury can be recovered from nitric acid-containing fluids by reacting the fluid with aluminum metal to produce mercury metal, and then quenching the reactivity of the nitric acid prior to nitration of the mercury metal.

  10. Engineering Dehydrated Amino Acid Residues in the Antimicrobial Peptide Nisin

    Kuipers, Oscar P.; Rollema, Harry S.; Yap, Wyanda M.G.J.; Boot, Hein J.; Siezen, Roland J.; Vos, Willem M. de

    1992-01-01

    The small antimicrobial peptide nisin, produced by Lactococcus lactis, contains the uncommon amino acid residues dehydroalanine and dehydrobutyrine and five thio ether bridges. Since these structures are posttranslationally formed from Ser, Thr, and Cys residues, it is feasible to study their role i

  11. Fatty Acid Structure and Degradation Analysis in Fingerprint Residues.

    Pleik, Stefanie; Spengler, Bernhard; Schäfer, Thomas; Urbach, Dieter; Luhn, Steven; Kirsch, Dieter

    2016-09-01

    GC-MS investigations were carried out to elucidate the aging behavior of unsaturated fatty acids in fingerprint residues and to identify their degradation products in aged samples. For this purpose, a new sample preparation technique for fingerprint residues was developed that allows producing N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) derivatives of the analyzed unsaturated fatty acids and their degradation products. MSTFA derivatization catalyzed by iodotrimethylsilane enables the reliable identification of aldehydes and oxoacids as characteristic MSTFA derivatives in GCMS. The obtained results elucidate the degradation pathway of unsaturated fatty acids. Our study of aged fingerprint residues reveals that decanal is the main degradation product of the observed unsaturated fatty acids. Furthermore, oxoacids with different chain lengths are detected as specific degradation products of the unsaturated fatty acids. The detection of the degradation products and their chain length is a simple and effective method to determine the double bond position in unsaturated compounds. We can show that the hexadecenoic and octadecenoic acids found in fingerprint residues are not the pervasive fatty acids Δ9-hexadecenoic (palmitoleic acid) and Δ9-octadecenoic (oleic acid) acid but Δ6-hexadecenoic acid (sapienic acid) and Δ8-octadecenoic acid. The present study focuses on the structure identification of human sebum-specific unsaturated fatty acids in fingerprint residues based on the identification of their degradation products. These results are discussed for further investigations and method developments for age determination of fingerprints, which is still a tremendous challenge because of several factors affecting the aging behavior of individual compounds in fingerprints. Graphical Abstract ᅟ. PMID:27324649

  12. Topological features of proteins from amino acid residue networks

    Alves, N A; Alves, Nelson Augusto; Martinez, Alexandre Souto

    2006-01-01

    Topological properties of native folds are obtained from statistical analysis of 160 low homology proteins covering the four structural classes. This is done analysing one, two and three-vertex joint distribution of quantities related to the corresponding network of amino acid residues. Emphasis on the amino acid residue hydrophobicity leads to the definition of their center of mass as vertices in this contact network model with interactions represented by edges. The network analysis helps us to interpret experimental results such as hydrophobic scales and fraction of buried accessible surface area in terms of the network connectivity. To explore the vertex type dependent correlations, we build a network of hydrophobic and polar vertices. This procedure presents the wiring diagram of the topological structure of globular proteins leading to the following attachment probabilities between hydrophobic-hydrophobic 0.424(5), hydrophobic-polar 0.419(2) and polar-polar 0.157(3) residues.

  13. Amendment of Acid Soils with Crop Residues and Biochars

    YUAN Jin-Hua; XU Ren-Kou; WANG Ning; LI Jiu-Yu

    2011-01-01

    The liming potential of some crop residues and their biochars on an acid Ultisol was investigated using incubation experiments. Rice hulls showed greater liming potential than rice hull biochar, while soybean and pea straws had less liming potential than their biochars. Due to their higher alkalinity, biochars from legume materials increased soil pH much compared to biochars from non-legume materials. The alkalinity of biochars was a key factor affecting their liming potential,and the greater alkalinity of biochars led to greater reductions in soil acidity. The incorporation of biochars decreased soil exchangeable acidity and increased soil exchangeable base cations and base saturation, thus improving soil fertility.

  14. Optimization of acid hydrolysis from the hemicellulosic fraction of Eucalyptus grandis residue using response surface methodology.

    Canettieri, Eliana Vieira; de Moraes Rocha, George Jackson; de Carvalho, João Andrade; de Almeida e Silva, João Batista

    2007-01-01

    Biotechnological conversion of biomass into fuels and chemicals requires hydrolysis of the polysaccharide fraction into monomeric sugars. Hydrolysis can be performed enzymatically and with dilute or concentrate mineral acids. The present study used dilute sulfuric acid as a catalyst for hydrolysis of Eucalyptus grandis residue. The purpose of this paper was to optimize the hydrolysis process in a 1.4 l pilot-scale reactor and investigate the effects of the acid concentration, temperature and residue/acid solution ratio on the hemicellulose removal and consequently on the production of sugars (xylose, glucose and arabinose) as well as on the formation of by-products (furfural, 5-hydroxymethylfurfural and acetic acid). This study was based on a model composition corresponding to a 2(3) orthogonal factorial design and employed the response surface methodology (RSM) to optimize the hydrolysis conditions, aiming to attain maximum xylose extraction from hemicellulose of residue. The considered optimum conditions were: H(2)SO(4) concentration of 0.65%, temperature of 157 degrees C and residue/acid solution ratio of 1/8.6 with a reaction time of 20 min. Under these conditions, 79.6% of the total xylose was removed and the hydrolysate contained 1.65 g/l glucose, 13.65 g/l xylose, 1.55 g/l arabinose, 3.10 g/l acetic acid, 1.23 g/l furfural and 0.20 g/l 5-hydroxymethylfurfural. PMID:16473004

  15. Lactic Acid and Biosurfactants Production from Residual Cellulose Films.

    Portilla Rivera, Oscar Manuel; Arzate Martínez, Guillermo; Jarquín Enríquez, Lorenzo; Vázquez Landaverde, Pedro Alberto; Domínguez González, José Manuel

    2015-11-01

    The increasing amounts of residual cellulose films generated as wastes all over the world represent a big scale problem for the meat industry regarding to environmental and economic issues. The use of residual cellulose films as a feedstock of glucose-containing solutions by acid hydrolysis and further fermentation into lactic acid and biosurfactants was evaluated as a method to diminish and revalorize these wastes. Under a treatment consisting in sulfuric acid 6% (v/v); reaction time 2 h; solid liquid ratio 9 g of film/100 mL of acid solution, and temperature 130 °C, 35 g/L of glucose and 49% of solubilized film was obtained. From five lactic acid strains, Lactobacillus plantarum was the most suitable for metabolizing the glucose generated. The process was scaled up under optimized conditions in a 2-L bioreactor, producing 3.4 g/L of biomass, 18 g/L of lactic acid, and 15 units of surface tension reduction of a buffer phosphate solution. Around 50% of the cellulose was degraded by the treatment applied, and the liqueurs generated were useful for an efficient production of lactic acid and biosurfactants using L. plantarum. Lactobacillus bacteria can efficiently utilize glucose from cellulose films hydrolysis without the need of clarification of the liqueurs. PMID:26293409

  16. EHD3 Protein Is Required for Tubular Recycling Endosome Stabilization, and an Asparagine-Glutamic Acid Residue Pair within Its Eps15 Homology (EH) Domain Dictates Its Selective Binding to NPF Peptides.

    Bahl, Kriti; Xie, Shuwei; Spagnol, Gaelle; Sorgen, Paul; Naslavsky, Naava; Caplan, Steve

    2016-06-24

    An elaborate network of dynamic lipid membranes, termed tubular recycling endosomes (TRE), coordinates the process of endocytic recycling in mammalian cells. The C-terminal Eps15 homology domain (EHD)-containing proteins have been implicated in the bending and fission of TRE, thus regulating endocytic recycling. EHD proteins have an EH domain that interacts with proteins containing an NPF motif. We found that NPF-containing EHD1 interaction partners such as molecules interacting with CasL-like1 (MICAL-L1) and Syndapin2 are essential for TRE biogenesis. Also crucial for TRE biogenesis is the generation of phosphatidic acid, an essential lipid component of TRE that serves as a docking point for MICAL-L1 and Syndapin2. EHD1 and EHD3 have 86% amino acid identity; they homo- and heterodimerize and partially co-localize to TRE. Despite their remarkable identity, they have distinct mechanistic functions. EHD1 induces membrane vesiculation, whereas EHD3 supports TRE biogenesis and/or stabilization by an unknown mechanism. While using phospholipase D inhibitors (which block the conversion of glycerophospholipids to phosphatidic acid) to deplete cellular TRE, we observed that, upon inhibitor washout, there was a rapid and dramatic regeneration of MICAL-L1-marked TRE. Using this "synchronized" TRE biogenesis system, we determined that EHD3 is involved in the stabilization of TRE rather than in their biogenesis. Moreover, we identify the residues Ala-519/Asp-520 of EHD1 and Asn-519/Glu-520 of EHD3 as defining the selectivity of these two paralogs for NPF-containing binding partners, and we present a model to explain the atomic mechanism and provide new insight for their differential roles in vesiculation and tubulation, respectively. PMID:27189942

  17. 40 CFR 158.270 - Experimental use permit data requirements for residue chemistry.

    2010-07-01

    ... requirements for residue chemistry. 158.270 Section 158.270 Protection of Environment ENVIRONMENTAL PROTECTION... Experimental use permit data requirements for residue chemistry. All residue chemistry data, as described in... section 408(r) is sought. Residue chemistry data are not required for an experimental use permit issued...

  18. Methodology for detecting residual phosphoric acid in polybenzoxazole fibers.

    Park, Eun Su; Sieber, John; Guttman, Charles; Rice, Kirk; Flynn, Kathleen; Watson, Stephanie; Holmes, Gale

    2009-12-01

    Because of the premature failure of in-service soft-body armor containing the ballistic fiber poly[(benzo-[1,2-d:5,4-d']-benzoxazole-2,6-diyl)-1,4-phenylene] (PBO), the Office of Law Enforcement Standards (OLES) at the National Institute of Standards and Technology (NIST) initiated a research program to investigate the reasons for this failure and to develop testing methodologies and protocols to ensure that these types of failures do not reoccur. In a report that focused on the stability of the benzoxazole ring that is characteristic of PBO fibers, Holmes, G. A.; Rice, K.; Snyder, C. R. J. Mater. Sci. 2006, 41, 4105-4116, showed that the benzoxazole ring was susceptible to hydrolytic degradation under acid conditions. Because of the processing conditions for the fibers, it is suspected by many researchers that residual phosphoric acid may cause degradation of the benzoxazole ring resulting in a reduction of ballistic performance. Prior to this work, no definitive data have indicated the presence of phosphoric acid since the residual phosphorus is not easily extracted and the processed fibers are known to incorporate phosphorus containing processing aids. Methods to efficiently extract phosphorus from PBO are described in this article. Further, characterization determined that the majority of the extractable phosphorus in PBO was attributed to the octyldecyl phosphate processing aid with some phosphoric acid being detected. Analysis by matrix assisted laser desorption ionization of model PBO oligomers indicates that the nonextractable phosphorus is attached to the PBO polymer chain as a monoaryl phosphate ester. The response of model aryl phosphates to NaOH exposure indicates that monoaryl phosphate ester is stable to NaOH washes used in the manufacturing process to neutralize the phosphoric acid reaction medium and to extract residual phosphorus impurities. PMID:19899783

  19. Residual load, renewable surplus generation and storage requirements in Germany

    I examine the effects of increasing amounts of fluctuating renewable energy on residual load, which is defined as the difference between actual power demand and the feed-in of non-dispatchable and inflexible generators. I draw on policy-relevant scenarios for Germany and make use of extensive sensitivity analyses. Whereas yearly renewable surplus energy is low in most scenarios analyzed, peak surplus power can become very high. Decreasing thermal must-run requirements and increasing biomass flexibility substantially reduce surpluses. I use an optimization model to determine the storage capacities required for taking up renewable surpluses. Allowing curtailment of 1% of the yearly feed-in of non-dispatchable renewables would render storage investments largely obsolete until 2032 under the assumption of a flexible power system. Further restrictions of curtailment as well as lower system flexibility strongly increase storage requirements. By 2050, at least 10 GW of storage are required for surplus integration, of which a sizeable share is seasonal storage. Results suggest that policy makers should work toward avoiding surplus generation, in particular by decreasing the must-run of thermal generators. Concerns about surpluses should not be regarded as an obstacle to further renewable expansion. The findings are also relevant for other countries that shift toward fluctuating renewables. - Highlights: • I examine the effects of fluctuating renewable energy on residual load. • Surplus energies are generally low, but there are high surplus power peaks. • Increasing the flexibility of thermal generators substantially reduces surpluses. • Allowing curtailment of 1% renders storage investments largely obsolete by 2032. • Both storage requirements and the share of seasonal storage increase by 2050

  20. Human Protein and Amino Acid Requirements.

    Hoffer, L John

    2016-05-01

    Human protein and amino acid nutrition encompasses a wide, complex, frequently misunderstood, and often contentious area of clinical research and practice. This tutorial explains the basic biochemical and physiologic principles that underlie our current understanding of protein and amino acid nutrition. The following topics are discussed: (1) the identity, measurement, and essentiality of nutritional proteins; (2) the definition and determination of minimum requirements; (3) nutrition adaptation; (4) obligatory nitrogen excretion and the minimum protein requirement; (5) minimum versus optimum protein intakes; (6) metabolic responses to surfeit and deficient protein intakes; (7) body composition and protein requirements; (8) labile protein; (9) N balance; (10) the principles of protein and amino acid turnover, including an analysis of the controversial indicator amino acid oxidation technique; (11) general guidelines for evaluating protein turnover articles; (12) amino acid turnover versus clearance; (13) the protein content of hydrated amino acid solutions; (14) protein requirements in special situations, including protein-catabolic critical illness; (15) amino acid supplements and additives, including monosodium glutamate and glutamine; and (16) a perspective on the future of protein and amino acid nutrition research. In addition to providing practical information, this tutorial aims to demonstrate the importance of rigorous physiologic reasoning, stimulate intellectual curiosity, and encourage fresh ideas in this dynamic area of human nutrition. In general, references are provided only for topics that are not well covered in modern textbooks. PMID:26796095

  1. Linoleic acid intake and vitamin E requirement

    Jager, F.C.

    1973-01-01

    In experiments with rats and Peking ducklings it has been investigated to what extent the linoleic acid content of the diet is of influence on the requirement of vitamin E. This requirement was determined by adding D-α-tocopheryl acetate in increasing doses to vitamin E-free diets and to determine h

  2. The effect of delignification process with alkaline peroxide on lactic acid production from furfural residues

    Yong Tang; Lingxi Bu; Lihong Deng; Liwei Zhu; Jianxin Jiang

    2012-01-01

    Furfural residues produced from the furfural industry were investigated as a substrate for lactic acid production by simultaneous saccharification and fermentation (SSF). Alkaline peroxide was used for delignification of furfural residues to improve the final lactic acid concentration. The residue was treated with 1.3% to 1.7% hydrogen peroxide at 80 °C for 1 h with a substrate concentration of 3.33%. SSF of furfural residues with different delignification degrees were carried out to eval...

  3. Nitrous acid damage to duplex deoxyribonucleic acid: distinction between deamination of cytosine residues and a novel mutational lesion.

    Frankel, A D; Duncan, B K; Hartman, P E

    1980-01-01

    The rate of nitrous acid deamination of labeled cytosine residues in native Escherichia coli deoxyribonucleic acid was monitored in vitro by release of acid-soluble counts after treatment with uracil deoxyribonucleic acid glycosylase. The reaction exhibited a lag and was not stimulate by several agents previously shown to enhance base substitution mutagenesis during nitrous acid treatment of duplex deoxyribonucleic acid. We conclude that a significant proportion of nitrous acid induced mutage...

  4. Acid leaching of uranium present in a residue from mining industry

    Braulio, Walace S.; Ladeira, Ana C.Q. [Center for Development of Nuclear Technology (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil). Dept. of Mineral Technology

    2011-07-01

    The acid mine drainage is one of the most important environmental problems associated with mining of ores containing sulfides. The treatment of these acid effluents, which contains high concentrations of dissolved metals and anions, is generally by liming. The wastes generated in the liming process may present significant toxicity and their storage in inappropriate places waiting for treatment is a common issue that requires solution. Osamu Utsumi Mine located in the city of Caldas, Minas Gerais, has been facing this problem. The residue of this mine consists of an alkaline sludge generated from the neutralization of the pH of acid mine drainage and is rich in various metals, including uranium. The main concern is the long term stability of this residue, which is in permanent contact with the acid water in the open pit. The recovery of uranium by hydrometallurgical techniques, such as acid leaching, can be a viable alternative on the reuse of this material. This study aimed at establishing a specific leaching process for the recovery of uranium present in the sludge from Caldas uranium mine. Some parameters such as solid/liquid ratio (0.09 to 0.17), time of leaching (1 to 24 hours) and concentration of sulfuric acid (pH from 0 to 3.0) were assessed. The results showed that it is possible to extract 100% of uranium present in the sludge. The concentration of U{sub 3}O{sub 8} in the residue was 0.25%, similar to the content of the vein ores which is around 0.20% to 1.0%. The best experimental leaching condition is solid/liquid ratio of 0.17, pH 1.0 and 2 hours of reaction at room temperature (25 deg C). The content of uranium in the liquor is around 440 mgL{sup -1}. The recovery of the uranium from the liquor is under investigation by ionic exchange. (author)

  5. Acid leaching of uranium present in a residue from mining industry

    The acid mine drainage is one of the most important environmental problems associated with mining of ores containing sulfides. The treatment of these acid effluents, which contains high concentrations of dissolved metals and anions, is generally by liming. The wastes generated in the liming process may present significant toxicity and their storage in inappropriate places waiting for treatment is a common issue that requires solution. Osamu Utsumi Mine located in the city of Caldas, Minas Gerais, has been facing this problem. The residue of this mine consists of an alkaline sludge generated from the neutralization of the pH of acid mine drainage and is rich in various metals, including uranium. The main concern is the long term stability of this residue, which is in permanent contact with the acid water in the open pit. The recovery of uranium by hydrometallurgical techniques, such as acid leaching, can be a viable alternative on the reuse of this material. This study aimed at establishing a specific leaching process for the recovery of uranium present in the sludge from Caldas uranium mine. Some parameters such as solid/liquid ratio (0.09 to 0.17), time of leaching (1 to 24 hours) and concentration of sulfuric acid (pH from 0 to 3.0) were assessed. The results showed that it is possible to extract 100% of uranium present in the sludge. The concentration of U3O8 in the residue was 0.25%, similar to the content of the vein ores which is around 0.20% to 1.0%. The best experimental leaching condition is solid/liquid ratio of 0.17, pH 1.0 and 2 hours of reaction at room temperature (25 deg C). The content of uranium in the liquor is around 440 mgL-1. The recovery of the uranium from the liquor is under investigation by ionic exchange. (author)

  6. 40 CFR 158.2082 - Experimental use permit biochemical pesticides residue data requirements table.

    2010-07-01

    ... pesticides residue data requirements table. 158.2082 Section 158.2082 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS DATA REQUIREMENTS FOR PESTICIDES Biochemical Pesticides § 158.2082 Experimental use permit biochemical pesticides residue data requirements table. (a)...

  7. 40 CFR 158.2172 - Experimental use permit microbial pesticides residue data requirements table.

    2010-07-01

    ... pesticides residue data requirements table. 158.2172 Section 158.2172 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS DATA REQUIREMENTS FOR PESTICIDES Microbial Pesticides § 158.2172 Experimental use permit microbial pesticides residue data requirements table. (a)...

  8. Protein and Amino Acid Requirements during Pregnancy.

    Elango, Rajavel; Ball, Ronald O

    2016-07-01

    Protein forms an essential component of a healthy diet in humans to support both growth and maintenance. During pregnancy, an exceptional stage of life defined by rapid growth and development, adequate dietary protein is crucial to ensure a healthy outcome. Protein deposition in maternal and fetal tissues increases throughout pregnancy, with most occurring during the third trimester. Dietary protein intake recommendations are based on factorial estimates because the traditional method of determining protein requirements, nitrogen balance, is invasive and undesirable during pregnancy. The current Estimated Average Requirement and RDA recommendations of 0.88 and 1.1 g · kg(-1) · d(-1), respectively, are for all stages of pregnancy. The single recommendation does not take into account the changing needs during different stages of pregnancy. Recently, with the use of the minimally invasive indicator amino acid oxidation method, we defined the requirements to be, on average, 1.2 and 1.52 g · kg(-1) · d(-1) during early (∼16 wk) and late (∼36 wk) stages of pregnancy, respectively. Although the requirements are substantially higher than current recommendations, our values are ∼14-18% of total energy and fit within the Acceptable Macronutrient Distribution Range. Using swine as an animal model we showed that the requirements for several indispensable amino acids increase dramatically during late gestation compared with early gestation. Additional studies should be conducted during pregnancy to confirm the newly determined protein requirements and to determine the indispensable amino acid requirements during pregnancy in humans. PMID:27422521

  9. Oxidation in Acidic Medium of Lignins from Agricultural Residues

    Labat, Gisele Aparecida Amaral; Gonçalves, Adilson Roberto

    Agricultural residues as sugarcane straw and bagasse are burned in boilers for generation of energy in sugar and alcohol industries. However, excess of those by-products could be used to obtain products with higher value. Pulping process generates cellulosic pulps and lignin. The lignin could be oxidized and applied in effluent treatments for heavy metal removal. Oxidized lignin presents very strong chelating properties. Lignins from sugarcane straw and bagasse were obtained by ethanol-water pulping. Oxidation of lignins was carried out using acetic acid and Co/Mn/Br catalytical system at 50, 80, and 115 °C for 5 h. Kinetics of the reaction was accomplished by measuring the UV-visible region. Activation energy was calculated for lignins from sugarcane straw and bagasse (34.2 and 23.4 kJ mol-1, respectively). The first value indicates higher cross-linked formation. Fourier-transformed infrared spectroscopy data of samples collected during oxidation are very similar. Principal component analysis applied to spectra shows only slight structure modifications in lignins after oxidation reaction.

  10. Properties of nanocellulose isolated from corncob residue using sulfuric acid, formic acid, oxidative and mechanical methods.

    Liu, Chao; Li, Bin; Du, Haishun; Lv, Dong; Zhang, Yuedong; Yu, Guang; Mu, Xindong; Peng, Hui

    2016-10-20

    In this work, nanocellulose was extracted from bleached corncob residue (CCR), an underutilized lignocellulose waste from furfural industry, using four different methods (i.e. sulfuric acid hydrolysis, formic acid (FA) hydrolysis, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxidation, and pulp refining, respectively). The self-assembled structure, morphology, dimension, crystallinity, chemical structure and thermal stability of prepared nanocellulose were investigated. FA hydrolysis produced longer cellulose nanocrystals (CNCs) than the one obtained by sulfuric acid hydrolysis, and resulted in high crystallinity and thermal stability due to its preferential degradation of amorphous cellulose and lignin. The cellulose nanofibrils (CNFs) with fine and individualized structure could be isolated by TEMPO-mediated oxidation. In comparison with other nanocellulose products, the intensive pulp refining led to the CNFs with the longest length and the thickest diameter. This comparative study can help to provide an insight into the utilization of CCR as a potential source for nanocellulose production. PMID:27474618

  11. Plant residues: short term effect on sulphate, borate, zinc and copper adsorption by an acid oxisol

    2003-01-01

    Laboratory experiments were carried out to examine the effects of plant residues on Cu, Zn, B and S adsorption by an acidic oxisol. The plant residues were: black oats (Avena strigosa), oil seed radish(Raphanus sativus), velvet beans (Stizolobium cinereum), and pigeon pea (Cajanus cajan) collected at flowering stage. Plant residues increased Cu and Zn adsorptions and decreased B and S adsorptions. The results indicated that for short term effect plant residues decreased the availabilities of ...

  12. 40 CFR 158.2040 - Biochemical pesticides residue data requirements table.

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Biochemical pesticides residue data... (CONTINUED) PESTICIDE PROGRAMS DATA REQUIREMENTS FOR PESTICIDES Biochemical Pesticides § 158.2040 Biochemical pesticides residue data requirements table. (a) General. Sections 158.100 through 158.130 describe how to...

  13. 40 CFR 158.2130 - Microbial pesticides residue data requirements table.

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Microbial pesticides residue data... (CONTINUED) PESTICIDE PROGRAMS DATA REQUIREMENTS FOR PESTICIDES Microbial Pesticides § 158.2130 Microbial pesticides residue data requirements table. (a) General. Sections 158.100 through 158.130 describe how to...

  14. Residue Geometry Networks: A Rigidity-Based Approach to the Amino Acid Network and Evolutionary Rate Analysis.

    Fokas, Alexander S; Cole, Daniel J; Ahnert, Sebastian E; Chin, Alex W

    2016-01-01

    Amino acid networks (AANs) abstract the protein structure by recording the amino acid contacts and can provide insight into protein function. Herein, we describe a novel AAN construction technique that employs the rigidity analysis tool, FIRST, to build the AAN, which we refer to as the residue geometry network (RGN). We show that this new construction can be combined with network theory methods to include the effects of allowed conformal motions and local chemical environments. Importantly, this is done without costly molecular dynamics simulations required by other AAN-related methods, which allows us to analyse large proteins and/or data sets. We have calculated the centrality of the residues belonging to 795 proteins. The results display a strong, negative correlation between residue centrality and the evolutionary rate. Furthermore, among residues with high closeness, those with low degree were particularly strongly conserved. Random walk simulations using the RGN were also successful in identifying allosteric residues in proteins involved in GPCR signalling. The dynamic function of these residues largely remain hidden in the traditional distance-cutoff construction technique. Despite being constructed from only the crystal structure, the results in this paper suggests that the RGN can identify residues that fulfil a dynamical function. PMID:27623708

  15. Mutation of aspartic acid residues in the fructosyltransferase of Streptococcus salivarius ATCC 25975.

    Song, D D; Jacques, N A

    1999-01-01

    The site-directed mutated fructosyltransferases (Ftfs) of Streptococcus salivarius ATCC 25975, D312E, D312S, D312N and D312K were all active at 37 degrees C, indicating that Asp-312 present in the 'sucrose box' was not the nucleophilic Asp residue responsible for the formation of a covalent fructosyl-enzyme intermediate required for enzyme activity. Analysis of the kinetic constants of the purified mutated forms of the enzyme showed that Asp-312 was most likely an essential amino acid involved in determining acceptor recognition and/or stabilizing a beta-turn in the protein. In contrast, when the Asp-397 of the Ftf present in the conserved triplet RDP motif of all 60 bacterial and plant family-32 glycosylhydrolases was mutated to a Ser residue, both sucrose hydrolysis and polymerization ceased. Tryptophan emission spectra confirmed that this mutation did not alter protein structure. Comparison of published data from other site-directed mutated enzymes implicated the Asp residue in the RDP motif as the one that may form a transient covalent fructosyl intermediate during the catalysis of sucrose by the Ftf of S. salivarius. PMID:10548559

  16. The effect of delignification process with alkaline peroxide on lactic acid production from furfural residues

    Yong Tang

    2012-11-01

    Full Text Available Furfural residues produced from the furfural industry were investigated as a substrate for lactic acid production by simultaneous saccharification and fermentation (SSF. Alkaline peroxide was used for delignification of furfural residues to improve the final lactic acid concentration. The residue was treated with 1.3% to 1.7% hydrogen peroxide at 80 °C for 1 h with a substrate concentration of 3.33%. SSF of furfural residues with different delignification degrees were carried out to evaluate the effect of delignification degree on lactic acid production. Using corn hydrolysates/ furfural residues as substrates, SSF with different media were carried out to investigate the effect of lignin on the interaction between enzymes and lactic acid bacteria. Lactic acid bacteria had a negative effect on cellulase, thus resulting in the reduction of enzyme activity. Lignin and nutrients slowed down the decreasing trend of enzyme activity. A higher delignification resulted in a slower fermentation rate and lower yield due to degradation products of lignin and the effect of lignin on the interaction between enzymes and lactic acid bacteria. For the purpose of lactic acid production, a moderate delignification (furfural residues with the lignin content of 14.8% was optimum.

  17. Teichuronic acid reducing terminal N-acetylglucosamine residue linked by phosphodiester to peptidoglycan of Micrococcus luteus

    Teichuronic acid-peptidoglycan complex isolated from Micrococcus luteus cells by lysozyme digestion in osmotically stabilized medium was treated with mild acid to cleave the linkage joining teichuronic acid to peptidoglycan. This labile linkage was shown to be the phosphodiester which joins N-acetylglucosamine, the residue located at the reducing end of the teichuronic acid, through its anomeric hydroxyl group to a 6-phosphomuramic acid, a residue of the glycan strand of peptidoglycan. 31P nuclear magnetic resonance spectroscopy of the lysozyme digest of cell walls demonstrated the presence of a phosphodiester which was converted to a phosphomonoester by the conditions which released teichuronic acid from cell walls. Reduction of acid-liberated reducing end groups by NaB3H4 followed by complete acid hydrolysis yielded [3H] glucosaminitol from the true reducing end residue of teichuronic acid and [3H]glucitol from the sites of fragmentation of teichuronic acid. The amount of N-acetylglucosamine detected was approximately stoichiometric with the amount of phosphate in the complex. Partial fragmentation of teichuronic acid provides an explanation of the previous erroneous identification of the reducing end residue

  18. On the distribution of amino acid residues in transmembrane alpha-helix bundles.

    Samatey, F A; Xu, C.; Popot, J L

    1995-01-01

    The periodic distribution of residues in the sequence of 469 putative transmembrane alpha-helices from eukaryotic plasma membrane polytopic proteins has been analyzed with correlation matrices. The method does not involve any a priori assumption about the secondary structure of the segments or about the physicochemical properties of individual amino acid residues. Maximal correlation is observed at 3.6 residues per period, characteristic of alpha-helices. A scale extracted from the data descr...

  19. Finding coevolving amino acid residues using row and column weighting of mutual information and multi-dimensional amino acid representation

    Pedersen Anders G

    2007-10-01

    Full Text Available Abstract Background Some amino acid residues functionally interact with each other. This interaction will result in an evolutionary co-variation between these residues – coevolution. Our goal is to find these coevolving residues. Results We present six new methods for detecting coevolving residues. Among other things, we suggest measures that are variants of Mutual Information, and measures that use a multidimensional representation of each residue in order to capture the physico-chemical similarities between amino acids. We created a benchmarking system, in silico, able to evaluate these methods through a wide range of realistic conditions. Finally, we use the combination of different methods as a way of improving performance. Conclusion Our best method (Row and Column Weighed Mutual Information has an estimated accuracy increase of 63% over Mutual Information. Furthermore, we show that the combination of different methods is efficient, and that the methods are quite sensitive to the different conditions tested.

  20. Generation of organic acids and monosaccharides by hydrolytic and oxidative transformation of food processing residues.

    Fischer, Klaus; Bipp, Hans-Peter

    2005-05-01

    Carbohydrate-rich biomass residues, i.e. sugar beet molasses, whey powder, wine yeast, potato peel sludge, spent hops, malt dust and apple marc, were tested as starting materials for the generation of marketable chemicals, e.g. aliphatic acids, sugar acids and mono-/disaccharides. Residues were oxidized or hydrolyzed under acidic or alkaline conditions applying conventional laboratory digestion methods and microwave assisted techniques. Yields and compositions of the oxidation products differed according to the oxidizing agent used. Main products of oxidation by 30% HNO(3) were acetic, glucaric, oxalic and glycolic acids. Applying H(2)O(2)/CuO in alkaline solution, the organic acid yields were remarkably lower with formic, acetic and threonic acids as main products. Gluconic acid was formed instead of glucaric acid throughout. Reaction of a 10% H(2)O(2) solution with sugar beet molasses generated formic and lactic acids mainly. Na(2)S(2)O(8) solutions were very inefficient at oxidizing the residues. Glucose, arabinose and galactose were formed during acidic hydrolysis of malt dust and apple marc. The glucose content reached 0.35 g per gram of residue. Important advantages of the microwave application were lower reaction times and reduced reagent demands. PMID:15607197

  1. The Evolving Profile of the Signature Amino Acid Residues in HIV-1 Subtype C Tat.

    Aralaguppe, Shambhu Prasad G; Sharma, Shilpee; Menon, Malini; Prasad, Vinayaka R; Saravanan, Shanmugam; Murugavel, Kailapuri G; Solomon, Suniti; Ranga, Udaykumar

    2016-05-01

    Using several HIV-1 tat exon 1 amino acid sequences available from public databases and additional sequences derived from a southern Indian clinical cohort, we compared the profile of the signature amino acid residues (SAR) between two different time periods, 1986-2004 and 2005-2014. The analysis identified eight positions as signature residues in subtype C Tat and demonstrated a changing pattern at four of these positions between the two periods. At three locations (histidine 29, serine 57, and proline 60), there appears to be a nonuniform negative selection against the SAR. The negative selection appears to be severe, especially against histidine 29 (p < .0001) and moderate against proline 60 (p < .0001). The negative selection against serine 57 is statistically insignificant and appears to have begun recently. At position 63, the frequency of signature residue glutamic acid increased over the past decade, although the difference was not significant. Importantly, at the three locations where the negative selection is in progress, the substitute amino acids are the generic residues present in most of the other HIV-1 subtypes. Our data demonstrate that viral evolution can subject specific amino acid residues to subtle and progressive selection pressures without affecting the prevalence of other amino acid residues. PMID:26678403

  2. 40 CFR 180.1023 - Propanoic acid; exemptions from the requirement of a tolerance.

    2010-07-01

    ...) Propanoic acid is exempt from the requirement of a tolerance for residues in or on cattle, meat; cattle, meat byproducts; goat, meat; goat, meat byproducts; hog, meat; hog meat byproducts; horse, meat; horse, meat byproducts; sheep, meat; sheep meat byproducts; and, poultry, fat; poultry meat; poultry......

  3. Plant residues: short term effect on sulphate, borate, zinc and copper adsorption by an acid oxisol

    Dias Ana Cristi Basile

    2003-01-01

    Full Text Available Laboratory experiments were carried out to examine the effects of plant residues on Cu, Zn, B and S adsorption by an acidic oxisol. The plant residues were: black oats (Avena strigosa, oil seed radish(Raphanus sativus, velvet beans (Stizolobium cinereum, and pigeon pea (Cajanus cajan collected at flowering stage. Plant residues increased Cu and Zn adsorptions and decreased B and S adsorptions. The results indicated that for short term effect plant residues decreased the availabilities of Cu and Zn through metal organic complex reactions and increased availabilities of S and B through competition with organic anions by the adsorption sites on soil.

  4. Coevolution of amino acid residues in the key photosynthetic enzyme Rubisco

    Kapralov Maxim V

    2011-09-01

    Full Text Available Abstract Background One of the key forces shaping proteins is coevolution of amino acid residues. Knowing which residues coevolve in a particular protein may facilitate our understanding of protein evolution, structure and function, and help to identify substitutions that may lead to desired changes in enzyme kinetics. Rubisco, the most abundant enzyme in biosphere, plays an essential role in the process of carbon fixation through photosynthesis, thus facilitating life on Earth. This makes Rubisco an important model system for studying the dynamics of protein fitness optimization on the evolutionary landscape. In this study we investigated the selective and coevolutionary forces acting on large subunit of land plants Rubisco using Markov models of codon substitution and clustering approaches applied to amino acid substitution histories. Results We found that both selection and coevolution shape Rubisco, and that positively selected and coevolving residues have their specifically favored amino acid composition and pairing preference. The mapping of these residues on the known Rubisco tertiary structures showed that the coevolving residues tend to be in closer proximity with each other compared to the background, while positively selected residues tend to be further away from each other. This study also reveals that the residues under positive selection or coevolutionary force are located within functionally important regions and that some residues are targets of both positive selection and coevolution at the same time. Conclusion Our results demonstrate that coevolution of residues is common in Rubisco of land plants and that there is an overlap between coevolving and positively selected residues. Knowledge of which Rubisco residues are coevolving and positively selected could be used for further work on structural modeling and identification of substitutions that may be changed in order to improve efficiency of this important enzyme in crops.

  5. Protonation of Individual Histidine Residues Is Not Required for the pH-Dependent Entry of West Nile Virus: Evaluation of the “Histidine Switch” Hypothesis▿

    Nelson, Steevenson; Poddar, Subhajit; Lin, Tsai-Yu; Pierson, Theodore C.

    2009-01-01

    Histidine residues have been hypothesized to function as sensors of environmental pH that can trigger the activity of viral fusion proteins. We investigated a requirement for histidine residues in the envelope (E) protein of West Nile virus during pH-dependent entry into cells. Each histidine was individually replaced with a nonionizable amino acid and tested functionally. In each instance, mutants capable of orchestrating pH-dependent infection were identified. These results do not support a...

  6. 75 FR 1773 - Notice of Receipt of a Pesticide Petition Filed for Residues of Polymeric Polyhydroxy Acid in or...

    2010-01-13

    ... AGENCY Notice of Receipt of a Pesticide Petition Filed for Residues of Polymeric Polyhydroxy Acid in or... establishment of a regulation for residues of the plant growth regulator, polymeric polyhydroxy acid, in or on... (FFDCA), 21 U.S.C. 346a, proposing the establishment of a regulation in 40 CFR part 180 for residues...

  7. Effect of water and ethanol radicals on the protein part of human hemoglobin. Pt. 2. Damage to amino acid residues

    This work is devoted to the effect of interactions of water and ethanol radicals with human hemoglobin (Hb) amino acid residues. The OH radicals caused the greatest destruction of human hemoglobin amino acid residues. Among them the most sensitive to irradiation were cysteine tryptophan and histidine residues. (author). 11 refs, 2 tabs

  8. Studies on distribution and residue of sulfur in simulated acid rain in vegetable and soil by using 35S

    Distribution and residue of sulfur in simulated acid rain in two kinds of vegetables (lettuce and Chinese cabbage) and three types of soils (acid yellow earth, acid and neutral purple soils) were studied by using 35S tracer method. The results showed that the higher concentration of acid rain was sprayed, the more residue of sulfur in vegetable there would be. The residue of sulfur in vegetable varied with the different physical and chemical properties of soils, the order of sulfur residue in vegetable was: acid purple soil>acid yellow earth>neutral purple soil. In the same soil, the residue of sulfur in lettuce was higher than that in Chinese cabbage, for the same vegetable, the residue of sulfur in leaves were higher than that in stems. The order of sulfur residue in different soils was acid purple soil>acid yellow earth>neutral purple soil. The higher concentration of acid rain was sprayed, the more residue of sulfur in soil surface there would be. The sulfur residue varied with the depth of soil and the pH value of acid rain. With the increase of soil depth, a slight increase of sulfur residue with rain of ph 6 and a slight decrease with rain of pH 4.0 and 2.5 were found

  9. Amino acid residues in the CDC25 guanine nucleotide exchange factor critical for interaction with Ras.

    Park, W.; Mosteller, R D; Broek, D.

    1994-01-01

    Previously we found that negatively charged residues at positions 62, 63, and 69 of H-Ras are involved in binding to the CDC25 guanine nucleotide exchange factor (GEF). Using site-directed mutagenesis, we have changed conserved, positively charged residues of CDC25GEF to glutamic acid. We find the nonfunctional CDC25R1374E mutant and the nonfunctional H-RasE63K mutant cooperate in suppression of the loss of CDC25 function in Saccharomyces cerevisiae. Also, peptides corresponding to residues 1...

  10. Identification of amino acid residues in Streptococcus mutans glucosyltransferases influencing the structure of the glucan product.

    Shimamura, A; Nakano, Y J; Mukasa, H; Kuramitsu, H K

    1994-01-01

    The glucosyltransferases (GTFs) of mutans streptococci are important virulence factors in the sucrose-dependent colonization of tooth surfaces by these organisms. To investigate the structure-function relationship of the GTFs, an approach was initiated to identify amino acid residues of the GTFs which affect the incorporation of glucose residues into the glucan polymer. Conserved amino acid residues were identified in the GTF-S and GTF-I enzymes of the mutans streptococci and were selected for site-directed mutagenesis in the corresponding enzymes from Streptococcus mutans GS5. Conversion of six amino acid residues of the GTF-I enzyme to those present at the corresponding positions in GTF-S, either singly or in multiple combinations, resulted in enzymes synthesizing increased levels of soluble glucans. The enzyme containing six alterations synthesized 73% water-soluble glucan in the absence of acceptor dextran T10, while parental enzyme GTF-I synthesized no such glucan product. Conversely, when residue 589 of the GTF-S enzyme was converted from Thr to either Asp or Glu, the resulting enzyme synthesized primarily water-insoluble glucan in the absence of the acceptor. Therefore, this approach has identified several amino acid positions which influence the nature of the glucan product synthesized by GTFs. PMID:8050997

  11. Functional analyses of carnivorous plant-specific amino acid residues in S-like ribonucleases.

    Arai, Naoki; Nishimura, Emi; Kikuchi, Yo; Ohyama, Takashi

    2015-09-11

    Unlike plants with no carnivory, carnivorous plants seem to use S-like ribonucleases (RNases) as an enzyme for carnivory. Carnivorous plant-specific conserved amino acid residues are present at four positions around the conserved active site (CAS). The roles of these conserved amino acid residues in the enzymatic function were explored in the current study by preparing five recombinant variants of DA-I, the S-like RNase of Drosera adelae. The kcat and kcat/Km values of the enzymes revealed that among the four variants with a single mutation, the serine to glycine mutation at position 111 most negatively influenced the enzymatic activity. The change in the bulkiness of the amino acid residue side-chain seemed to be the major cause of the above effect. Modeling of the three dimensional (3D) structures strongly suggested that the S to G mutation at 111 greatly altered the overall enzyme conformation. The conserved four amino acid residues are likely to function in keeping the two histidine residues, which are essential for the cleavage of RNA strands, and the CAS in the most functional enzymatic conformation. PMID:26235877

  12. Residual learning rates in lead-acid batteries: Effects on emerging technologies

    The low price of lead-acid, the most popular battery, is often used in setting cost targets for emerging energy storage technologies. Future cost reductions in lead acid batteries could increase investment and time scales needed for emerging storage technologies to reach cost-parity. In this paper the first documented model of cost reductions for lead-acid batteries is developed. Regression to a standard experience curve using 1989–2012 data yield a poor fit, with R2 values of 0.17 for small batteries and 0.05 for larger systems. To address this problem, battery costs are separated into material and residual costs, and experience curves developed for residual costs. Depending on the year, residual costs account for 41–86% of total battery cost. Using running-time averages to address volatility in material costs, a 4-year time average experience curve for residual costs yield much higher R2, 0.78 for small and 0.74 for large lead-acid batteries. The learning rate for residual costs in lead-acid batteries is 20%, a discovery with policy implications. Neglecting to consider cost reductions in lead-acid batteries could result in failure of energy storage start-ups and public policy programs. Generalizing this result, learning in incumbent technologies must be understood to assess the potential of emerging ones. -- Highlights: •We analyze potential cost reductions in lead-acid batteries. •Modified experience curve for non-material costs gives good empirical fit. •Historical learning rate for non-material costs from 1985–2012 is 19–24%. •Progress in incumbent technology raises barrier to new entrants

  13. Involvement of phylogenetically conserved acidic amino acid residues in catalysis by an oxidative DNA damage enzyme formamidopyrimidine glycosylase.

    Lavrukhin, O V; Lloyd, R S

    2000-12-12

    Formamidopyrimidine glycosylase (Fpg) is an important bacterial base excision repair enzyme, which initiates removal of damaged purines such as the highly mutagenic 8-oxoguanine. Similar to other glycosylase/AP lyases, catalysis by Fpg is known to proceed by a nucleophilic attack by an amino group (the secondary amine of its N-terminal proline) on C1' of the deoxyribose sugar at a damaged base, which results in the departure of the base from the DNA and removal of the sugar ring by beta/delta-elimination. However, in contrast to other enzymes in this class, in which acidic amino acids have been shown to be essential for glycosyl and phosphodiester bond scission, the catalytically essential acidic residues have not been documented for Fpg. Multiple sequence alignments of conserved acidic residues in all known bacterial Fpg-like proteins revealed six conserved glutamic and aspartic acid residues. Site-directed mutagenesis was used to change glutamic and aspartic acid residues to glutamines and asparagines, respectively. While the Asp to Asn mutants had no effect on the incision activity on 8-oxoguanine-containing DNA, several of the substitutions at glutamates reduced Fpg activity on the 8-oxoguanosine DNA, with the E3Q and E174Q mutants being essentially devoid of activity. The AP lyase activity of all of the glutamic acid mutants was slightly reduced as compared to the wild-type enzyme. Sodium borohydride trapping of wild-type Fpg and its E3Q and E174Q mutants on 8-oxoguanosine or AP site containing DNA correlated with the relative activity of the mutants on either of these substrates. PMID:11106507

  14. Synthesis of an Ursolic Acid Saponin with N-Acetylglucosamine-containing Trisaccharide Residue

    WANG Peng; LI Chun-Xia; WANG Guang-Fa; LI Ying-Xia

    2006-01-01

    The focus of this work is the synthesis of an ursolic acid saponin with an N-acetylglucosamine-containing trisaccharide residue. Therefore, ursolic acid 3-yl α-L-arabinopyranosyl-(1→2)-α-L-arabinopyranosyl-(1→6)-2-acetamido-2-deoxy-β-D-glucopyranoside (1) was concisely synthesized in convergent synthesis with 48.0% overall yield. The structure of saponin 1 was confirmed by 1H NMR, 13C NMR and mass spectra.

  15. Does the autoantibody immunodominant region on thyroid peroxidase include amino acid residues 742-771?

    Xiong, Z; Farilla, L; Guo, J; McLachlan, S; Rapoport, B

    2001-03-01

    Identification of the thyroid peroxidase (TPO) amino acid residues that comprise the autoantibody immunodominant region is an important goal that has proven difficult because of the conformational nature of the epitopes involved. Recent data suggest that the immunodominant region has been located. Thus, by autoantibody recognition of tryptic fragments of native TPO, as well as of conformational portions of TPO expressed as cell-free translates, the autoantibody immunodominant region appears to include amino acid residues 742-771, near the C terminus of the ectodomain. To evaluate this deduction, we expressed as cell-free translates the full TPO ectodomain, as well as TPO truncated after residues 741 and 771. The epitopic integrity of these molecules was first confirmed by immunoprecipitation by patient sera containing TPO autoantibodies. However, autoantibody recognition could involve a minority of TPO autoantibodies with the individual sera, not fulfilling the strict criteria for immunodominance. In order to obtain definitive data, we performed immunoprecipitations on these TPO variants with four recombinant human monoclonal autoantibodies that define the immunodominant region. All four monoclonal autoantibodies immunoprecipitated TPO 1-741 to the same extent as they did TPO 1-771 and the full TPO ectodomain, indicating that the immunodominant region comprises (at least in large part) amino acid residues upstream of residue 741. PMID:11327613

  16. Search for conserved amino acid residues of the [Formula: see text]-crystallin proteins of vertebrates.

    Shiliaev, Nikita G; Selivanova, Olga M; Galzitskaya, Oxana V

    2016-04-01

    [Formula: see text]-crystallin is the major eye lens protein and a member of the small heat-shock protein (sHsp) family. [Formula: see text]-crystallins have been shown to support lens clarity by preventing the aggregation of lens proteins. We performed the bioinformatics analysis of [Formula: see text]-crystallin sequences from vertebrates to find conserved amino acid residues as the three-dimensional (3D) structure of [Formula: see text]-crystallin is not identified yet. We are the first who demonstrated that the N-terminal region is conservative along with the central domain for vertebrate organisms. We have found that there is correlation between the conserved and structured regions. Moreover, amyloidogenic regions also correspond to the structured regions. We analyzed the amino acid composition of [Formula: see text]-crystallin A and B chains. Analyzing the occurrence of each individual amino acid residue, we have found that such amino acid residues as leucine, serine, lysine, proline, phenylalanine, histidine, isoleucine, glutamic acid, and valine change their content simultaneously in A and B chains in different classes of vertebrates. Aromatic amino acids occur more often in [Formula: see text]-crystallins from vertebrates than on the average in proteins among 17 animal proteomes. We obtained that the identity between A and B chains in the mammalian group is 0.35, which is lower than the published 0.60. PMID:26972563

  17. Protein reactivity with singlet oxygen: Influence of the solvent exposure of the reactive amino acid residues.

    Sjöberg, Béatrice; Foley, Sarah; Staicu, Angela; Pascu, Alexandru; Pascu, Mihail; Enescu, Mironel

    2016-06-01

    The singlet oxygen quenching rate constants were measured for three model proteins, bovine serum albumin, β-lactoglobulin and lysozyme. The results were analyzed by comparing them with the corresponding singlet oxygen quenching rate constants for a series of tripeptides with the basic formula GlyAAGly where the central amino acid (AA) was the oxidizable amino acid, tryptophan, tyrosine, methionine and histidine. It was found that the reaction rate constant in proteins can be satisfactorily modelled by the sum of the individual contributions of the oxidizable AA residues corrected for the solvent accessible surface area (SASA) effects. The best results were obtained when the SASA of the AA residues were determined by averaging over molecular dynamics simulated trajectories of the proteins. The limits of this geometrical correction of the AA residue reactivity are also discussed. PMID:27045278

  18. Isoelectric Point, Electric Charge, and Nomenclature of the Acid-Base Residues of Proteins

    Maldonado, Andres A.; Ribeiro, Joao M.; Sillero, Antonio

    2010-01-01

    The main object of this work is to present the pedagogical usefulness of the theoretical methods, developed in this laboratory, for the determination of the isoelectric point (pI) and the net electric charge of proteins together with some comments on the naming of the acid-base residues of proteins. (Contains 8 figures and 4 tables.)

  19. Residual mitochondrial transmembrane potential decreases unsaturated fatty acid level in sake yeast during alcoholic fermentation.

    Sawada, Kazutaka; Kitagaki, Hiroshi

    2016-01-01

    Oxygen, a key nutrient in alcoholic fermentation, is rapidly depleted during this process. Several pathways of oxygen utilization have been reported in the yeast Saccharomyces cerevisiae during alcoholic fermentation, namely synthesis of unsaturated fatty acid, sterols and heme, and the mitochondrial electron transport chain. However, the interaction between these pathways has not been investigated. In this study, we showed that the major proportion of unsaturated fatty acids of ester-linked lipids in sake fermentation mash is derived from the sake yeast rather than from rice or koji (rice fermented with Aspergillus). Additionally, during alcoholic fermentation, inhibition of the residual mitochondrial activity of sake yeast increases the levels of unsaturated fatty acids of ester-linked lipids. These findings indicate that the residual activity of the mitochondrial electron transport chain reduces molecular oxygen levels and decreases the synthesis of unsaturated fatty acids, thereby increasing the synthesis of estery flavors by sake yeast. This is the first report of a novel link between residual mitochondrial transmembrane potential and the synthesis of unsaturated fatty acids by the brewery yeast during alcoholic fermentation. PMID:26839744

  20. Process optimization of reaction of acid leaching residue of asbestos tailing and sodium hydroxide aqueous solution

    2009-01-01

    Silica is the major component of the acid leaching residue of asbestos tailing. The waterglass solution can be prepared by the reaction of the residue with sodium hydroxide aqueous solution. Compared to the high temperature reaction method, this process is environmental friendly and low cost. In this paper, the reaction process of the residue and the sodium hydroxide aqueous solution is optimized. The optimum reaction process parameters are as follows: the usage of sodium hydroxide is 26.4 g/100 g acid leaching residue, the reaction temperature is 90℃, the reaction time is 1 h, and the ratio of the liquid/solid is 2.0. The significance sequence of the process parameters to the alkali leaching reaction effect is the usage of sodium hydroxide > the ratio of the liquid/solid > the reaction time > the reaction temperature. The significance sequence to the leaching ratio of SiO2 is the ratio of the liquid/solid > the usage of sodium hydroxide > the reaction time > the reaction temperature. The significance sequence to the modulus of the sodium silicate is the ratio of the liquid/solid > the usage of sodium hydroxide > the reaction time > the reaction temperature. Under the optimum conditions, the leaching ratio of the SiO2 is 77.5%, and the modulus of the sodium silicate is 3.15. The XRD analysis result indicates that the major components of the alkali leaching residue are serpentine, talc, quartz and some albite.

  1. Characteristics of lactic acid bacteria isolates and their effect on silage fermentation of fruit residues.

    Yang, Jinsong; Tan, Haisheng; Cai, Yimin

    2016-07-01

    The natural lactic acid bacteria (LAB) population, chemical composition, and silage fermentation of fruit residues were studied. Eighty-two strains of LAB were isolated from fruit residues such as banana leaf and stem, pineapple peel, and papaya peel. All strains were gram-positive and catalase-negative bacteria, and they were divided into 7 groups (A-G) according to morphological and biochemical characters. Strains in groups A to F were rods, and group G was cocci. Group F produced gas from glucose; other groups did not. Groups A to C and F formed dl-lactic acid, whereas groups D, E, and G formed l-lactic acid. Based on the 16S rRNA gene sequence and DNA-DNA hybridization analysis, groups A to G strains were identified as Lactobacillus plantarum (54.9% of the total isolates), Lactobacillus paraplantarum (3.6%), Lactobacillus nagelii (8.5%), Lactobacillus perolens (4.9%), Lactobacillus casei (11.0%), Lactobacillus fermentum (9.8%), and Enterococcus gallinarum (7.3%), respectively. Lactobacillus plantarum and Lactobacillus casei are the most frequently isolated from fruit residues as a dominant species, and they could grow at a lower pH conditions and produce more lactic acid than other isolates. Pineapple and papaya peels contained higher crude protein (11.5-13.8%) and water-soluble carbohydrate (16.8-22.4%), but lower acid detergent fiber contents (21.2 to 26.4%) than banana stems and leaves (8.2% crude protein, 42.8% acid detergent fiber, and 5.1% water-soluble carbohydrate). Compared with banana stem and leaf silages, the pineapple and papaya peel silages were well preserved with a lower pH and higher lactate content. The study suggests that the fruit residues contain excellent LAB species and abundant feed nutrients, and that they can be preserved as silage to be potential food resources for livestock. PMID:27108171

  2. Fermentation quality and nutritive value of rice crop residue based silage ensiled with addition of epiphytic lactic acid bacteria

    2011-01-01

    Silage is the feedstuff resulted from the preservation of forages through lactic acid fermentation. The aim of this study was to evaluate nutritive value, fermentation characteristics and nutrients digestibility of rice crop residue based silage ensiled with epiphytic lactic acid bacteria (LAB). The mixture of rice crop residue (RC), soybean curd residue (SC) and cassava waste (CW) in a 90: 5: 5 (on dry matter basis) ratio was used as silage material. Three treatments silage were (A) RC + SC...

  3. Intramolecular cyclization of aspartic acid residues assisted by three water molecules: a density functional theory study

    Aspartic acid (Asp) residues in peptides and proteins (l-Asp) are known to undergo spontaneous nonenzymatic reactions to form l-β-Asp, d-Asp, and d-β-Asp residues. The formation of these abnormal Asp residues in proteins may affect their three-dimensional structures and hence their properties and functions. Indeed, the reactions have been thought to contribute to aging and pathologies. Most of the above reactions of the l-Asp residues proceed via a cyclic succinimide intermediate. In this paper, a novel three-water-assisted mechanism is proposed for cyclization of an Asp residue (forming a gem-diol precursor of the succinimide) by the B3LYP/6-31 + G(d,p) density functional theory calculations carried out for an Asp-containing model compound (Ace−Asp−Nme, where Ace = acetyl and Nme = NHCH3). The three water molecules act as catalysts by mediating ‘long-range’ proton transfers. In the proposed mechanism, the amide group on the C-terminal side of the Asp residue is first converted to the tautomeric iminol form (iminolization). Then, reorientation of a water molecule and a conformational change occur successively, followed by the nucleophilic attack of the iminol nitrogen on the carboxyl carbon of the Asp side chain to form the gem-diol species. A satisfactory agreement was obtained between the calculated and experimental energetics.

  4. Effect of Dodecylbenzene Sulfonic Acid Used as Additive on Residue Hydrotreating

    Sun Yudong; Yang Chaohe

    2015-01-01

    The effect of additive—dodecylbenzene sulfonic acid (DBSA)—on residue hydrotreating was studied in the au-toclave. The results showed that the additive improved stabilization of the colloid system of residue, which could delay the aggregation and coke formation from asphaltenes on the catalyst, and make heavy components transformed into light oil. The residue conversion in the presence of this additive increased by 1.94%, and the yield of light oil increased by 1.53% when the reaction time was 90 min. The surface properties of the catalyst in the presence of this additive were better than that of the blank test within a very short time (30 min) and deteriorated rapidly after a longer reaction time due to higher conversion and coke deposition. Compared with the blank test, the case using the said additive had shown that the structure of hydrotreated asphaltene units was smaller and the condensation degrees were higher. The test results indicated that the additive could improve the hydrotreating reactivity of residue via permeation and depolymerization, the heavier components could be transformed into light oil more easily, and the light oil yield and residue conversion were higher for the case using the said additive in residue hydrotreating process.

  5. A novel sono-assisted acid pretreatment of chili post harvest residue for bioethanol production.

    Sindhu, Raveendran; Binod, Parameswaran; Pandey, Ashok

    2016-08-01

    The objective of the present study was to develop a sono-assisted acid pretreatment strategy for the effective removal of lignin and hemicelluloses and to improve the sugar yield from chili post harvest residue. Operational parameters that affect the pretreatment efficiency were studied and optimized. Inhibitor analysis of the hydrolyzate revealed that major fermentation inhibitors like furfural, hydroxymethyl furfural and organic acids like citric acid, succinic acid and propionic acid were absent. Changes in structural properties of the biomass were studied in relation to the pretreatment process using scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier Transform Infrared (FTIR) analysis, and the changes in chemical composition was also monitored. The biomass pretreated with the optimized novel method yielded 0.465g/g of reducing sugars on enzymatic hydrolysis. Fermentation of the non-detoxified hydrolysate yielded 2.14% of bioethanol with a fermentation efficiency of 71.03%. PMID:26949055

  6. Effects of particle size and acid addition on the remediation of chromite ore processing residue using ferrous sulfate.

    Jagupilla, Santhi Chandra; Moon, Deok Hyun; Wazne, Mahmoud; Christodoulatos, Christos; Kim, Min Gyu

    2009-08-30

    A bench-scale treatability study was conducted to assess the effects of particle size and acid addition on the remediation of chromite ore processing residue (COPR) using ferrous sulfate. The remediation scheme entailed the chemical reduction of hexavalent chromium [Cr(VI)] and the mitigation of swell potential. Leaching tests and the EQ3/6 geochemical model were used to estimate the acid dosage required to destabilize Cr(VI)-bearing and swell-causing minerals. The model predicted greater acid dosage than that estimated from the batch leaching tests. This indicated that mass transfer limitation may be playing a significant role in impeding the dissolution of COPR minerals following acid addition and hence hindering the remediation of COPR. Cr(VI) concentrations determined by alkaline digestion for the treated samples were less than the current NJDEP standard. However, Cr(VI) concentrations measured by X-ray absorption near edge structure (XANES) were greater than those measured by alkaline digestion. Greater Cr(VI) percentages were reduced for acid pretreated and also for smaller particle size COPR samples. Upon treatment, brownmillerite content was greatly reduced for the acid pretreated samples. Conversely, ettringite, a swell-causing mineral, was not observed in the treated COPR. PMID:19272700

  7. Extension of microwave-accelerated residue-specific acid cleavage to proteins with carbohydrate side chains and disulfide linkages

    Li, Jinxi; Shefcheck, Kevin; Callahan, John; Fenselau, Catherine

    2008-12-01

    This laboratory has introduced a chemical method for residue-specific protein cleavage and has provided a preliminary assessment of the suitability of microwave-accelerated acid cleavage as a proteomic tool. This report is a continuing assessment of the fate of common protein modifications in microwave-accelerated acid cleavage. We have examined the cleavage of ribonuclease A and the related N-linked glycoprotein ribonuclease B, and the O-linked glycoprotein alpha crystallin A chain, using MALDI-TOF and LC-ESI-MS to identify the peptide products. RNase A and B each contains four disulfide bonds, and the addition of a reducing reagent, such as dithiothreitol, was found to be required to achieve efficient acidic proteolysis. The linkage of the glycosidic group to the asparagine side chain in ribonuclease B was found not to be cleaved by brief microwave treatment in 12.5% acetic acid. The distribution of the heterogeneous carbohydrate side chain in the glycopeptide products of acid cleavage was compared to that of the glycopeptide products of tryptic digestion. Hydrolysis within the carbohydrate chain itself is minimal under the conditions used. The O-linked side chain on alpha crystalline A was found to be cleaved during acid cleavage of the protein.

  8. A method for computing the inter-residue interaction potentials for reduced amino acid alphabet

    Abhinav Luthra; Anupam Nath Jha; G K Ananthasuresh; Saraswathi Vishveswara

    2007-08-01

    Inter-residue potentials are extensively used in the design and evaluation of protein structures. However, dealing with all (20×20) interactions becomes computationally difficult in extensive investigations. Hence, it is desirable to reduce the alphabet of 20 amino acids to a smaller number. Currently, several methods of reducing the residue types exist; however a critical assessment of these methods is not available. Towards this goal, here we review and evaluate different methods by comparing with the complete (20×20) matrix of Miyazawa-Jernigan potential, including a method of grouping adopted by us, based on multi dimensional scaling (MDS). The second goal of this paper is the computation of inter-residue interaction energies for the reduced amino acid alphabet, which has not been explicitly addressed in the literature until now. By using a least squares technique, we present a systematic method of obtaining the interaction energy values for any type of grouping scheme that reduces the amino acid alphabet. This can be valuable in designing the protein structures.

  9. Acidic Residues C-Terminal to the A2 Domain Facilitate Thrombin-Catalyzed Activation of Factor VIII

    Newell, Jennifer L.; Fay, Philip J.

    2008-01-01

    Factor VIII is activated by thrombin through proteolysis at Arg740, Arg372, and Arg1689. One region implicated in this exosite-dependent interaction is the factor VIII a2 segment (residues 711-740) separating the A2 and B domains. Residues 717-725 (DYYEDSYED) within this region consist of five acidic residues and three sulfo-Tyr residues, thus representing a high density of negative charge potential. The contributions of these residues to thrombin-catalyzed activation of factor VIII were asse...

  10. Process optimization of reaction of acid leaching residue of asbestos tailing and sodium hydroxide aqueous solution

    DU GaoXiang; ZHENG ShuiLin; DING Hao

    2009-01-01

    Silica is the major component of the acid leaching residue of asbestos tailing. The waterglass solution can be prepared by the reaction of the residue with sodium hydroxide aqueous solution. Compared to the high temperature reaction method, this process is environmental friendly and low cost. In this paper, the reaction process of the residue and the sodium hydroxide aqueous solution is optimized. The op-timum reaction process parameters are as follows: the usage of sodium hydroxide is 26.4 g/100 g acid leaching residue, the reaction temperature is 90℃, the reaction time is 1 h, and the ratio of the liq-uid/solid is 2.0. The significance sequence of the process parameters to the alkali leaching reaction effect is the usage of sodium hydroxide > the ratio of the liquid/solid > the reaction time > the reaction temperature. The significance sequence to the leaching ratio of SiO2 is the ratio of the liquid/solid > the usage of sodium hydroxide > the reaction time > the reaction temperature. The significance sequence to the modulus of the sodium silicate is the ratio of the liquid/solid > the usage of sodium hydroxide > the reaction time > the reaction temperature. Under the optimum conditions, the leaching ratio of the SiO2 is 77.5%, and the modulus of the sodium silicate is 3.15. The XRD analysis result indicates that the major components of the alkali leaching residue are serpentine, talc, quartz and some albite.

  11. Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family.

    Broussard, Tyler C; Miller, Darcie J; Jackson, Pamela; Nourse, Amanda; White, Stephen W; Rock, Charles O

    2016-03-18

    Fatty acid kinase (Fak) is a ubiquitous Gram-positive bacterial enzyme consisting of an ATP-binding protein (FakA) that phosphorylates the fatty acid bound to FakB. In Staphylococcus aureus, Fak is a global regulator of virulence factor transcription and is essential for the activation of exogenous fatty acids for incorporation into phospholipids. The 1.2-Å x-ray structure of S. aureus FakB2, activity assays, solution studies, site-directed mutagenesis, and in vivo complementation were used to define the functions of the five conserved residues that define the FakB protein family (Pfam02645). The fatty acid tail is buried within the protein, and the exposed carboxyl group is bound by a Ser-93-fatty acid carboxyl-Thr-61-His-266 hydrogen bond network. The guanidinium of the invariant Arg-170 is positioned to potentially interact with a bound acylphosphate. The reduced thermal denaturation temperatures of the T61A, S93A, and H266A FakB2 mutants illustrate the importance of the hydrogen bond network in protein stability. The FakB2 T61A, S93A, and H266A mutants are 1000-fold less active in the Fak assay, and the R170A mutant is completely inactive. All FakB2 mutants form FakA(FakB2)2 complexes except FakB2(R202A), which is deficient in FakA binding. Allelic replacement shows that strains expressing FakB2 mutants are defective in fatty acid incorporation into phospholipids and virulence gene transcription. These conserved residues are likely to perform the same critical functions in all bacterial fatty acid-binding proteins. PMID:26774272

  12. Composition, texture and methane potential of cellulosic residues from Lewis acids organosolv pulping of wheat straw.

    Constant, Sandra; Barakat, Abdellatif; Robitzer, Mike; Di Renzo, Francesco; Dumas, Claire; Quignard, Françoise

    2016-09-01

    Cellulosic pulps have been successfully isolated from wheat straw through a Lewis acids organosolv treatment. The use of Lewis acids with different hardness produced pulps with different delignification degrees. The cellulosic residue was characterised by chemical composition, X-ray diffraction, FT-IR spectroscopy, N2 physisorption, scanning electron microscopy and potential for anaerobic digestibility. Surface area and pore volume increased with the hardness of the Lewis acid, in correspondence with the decrease of the amount of lignin and hemicellulose in the pulp. The non linearity of the correlation between porosity and composition suggests that an agglomeration of cellulose fibrils occurs in the early stages of pulping. All organosolv pulps presented a significantly higher methane potential than the parent straw. A methane evolution of 295Ncm(3)/g OM was reached by a moderate improvement of the accessibility of the native straw. PMID:27295251

  13. Radionuclide Leaching from Residual Solids Remaining after Acid Dissolution of Composite K East Canister Sludge

    Laboratory tests were performed to examine mixed nitric/hydrofluoric acid leach treatments for decontaminating dissolver residual solids (KECDVSR24H-2) produced during a 20- to 24-hr dissolution of a composite K East (KE) Basin canister sludge in 95 C 6 M nitric acid (HNO3). The scope of this testing has been described in Section 4.5 of ''Testing Strategy to Support the Development of K Basin Sludge Treatment Process'' (Flament 1998). Radionuclides sorbed or associated with the residual solids generated in the K Basin sludge treatment process can restrict disposal of this solid to the Environmental Restoration Disposal Facility (ERDF). The starting dissolver residual solid for this testing, KECDVSR24H-2, contains radionuclides at concentrations which exceed the ERDF Waste Acceptance Criteria for TRU by about a factor of 70, for 239Pu by a factor of 200, and for 241Am by a factor of 50. The solids also exceed the ERDF criterion for 137Cs by a factor of 2 and uranium by a factor of 5. Therefore, the radionuclides of greatest interest in this leaching study are first 239Pu and 241Am (both components of TRU) and then uranium and 137Cs

  14. Leaching of lead from zinc leach residue in acidic calcium chloride aqueous solution

    Wang, Le; Mu, Wen-ning; Shen, Hong-tao; Liu, Shao-ming; Zhai, Yu-chun

    2015-05-01

    A process with potentially reduced environmental impacts and occupational hazards of lead-bearing zinc plant residue was studied to achieve a higher recovery of lead via a cost-effective and environmentally friendly process. This paper describes an optimization study on the leaching of lead from zinc leach residue using acidic calcium chloride aqueous solution. Six main process conditions, i.e., the solution pH value, stirring rate, concentration of CaCl2 aqueous solution, liquid-to-solid (L/S) ratio, leaching temperature, and leaching time, were investigated. The microstructure and components of the residue and tailing were characterized using scanning electron microscopy (SEM) and X-ray diffraction (XRD). On the basis of experimental results, the optimum reaction conditions were determined to be a solution pH value of 1, a stirring rate of 500 r·min-1, a CaCl2 aqueous solution concentration of 400 g·L-1, a liquid-to-solid mass ratio of 7:1, a leaching temperature of 80°C, and a leaching time of 45 min. The leaching rate of lead under these conditions reached 93.79%, with an iron dissolution rate of 19.28%. Silica did not take part in the chemical reaction during the leaching process and was accumulated in the residue.

  15. Dimerization of the DYT6 dystonia protein, THAP1, requires residues within the coiled-coil domain.

    Sengel, Cem; Gavarini, Sophie; Sharma, Nutan; Ozelius, Laurie J; Bragg, D Cristopher

    2011-09-01

    Thanatos-associated [THAP] domain-containing apoptosis-associated protein 1 (THAP1) is a DNA-binding protein that has been recently associated with DYT6 dystonia, a hereditary movement disorder involving sustained, involuntary muscle contractions. A large number of dystonia-related mutations have been identified in THAP1 in diverse patient populations worldwide. Previous reports have suggested that THAP1 oligomerizes with itself via a C-terminal coiled-coil domain, raising the possibility that DYT6 mutations in this region might affect this interaction. In this study, we examined the ability of wild-type THAP1 to bind itself and the effects on this interaction of the following disease mutations: C54Y, F81L, ΔF132, T142A, I149T, Q154fs180X, and A166T. The results confirmed that wild-type THAP1 associated with itself and most of the DYT6 mutants tested, except for the Q154fs180X variant, which loses most of the coiled-coil domain because of a frameshift at position 154. However, deletion of C-terminal residues after position 166 produced a truncated variant of THAP1 that was able to bind the wild-type protein. The interaction of THAP1 with itself therefore required residues within a 13-amino acid region (aa 154-166) of the coiled-coil domain. Further inspection of this sequence revealed elements highly consistent with previous descriptions of leucine zippers, which serve as dimerization domains in other transcription factor families. Based on this similarity, a structural model was generated to predict how hydrophobic residues in this region may mediate dimerization. These observations offer additional insight into the role of the coiled-coil domain in THAP1, which may facilitate future analyses of DYT6 mutations in this region. PMID:21752024

  16. Critical lysine residues of Klf4 required for protein stabilization and degradation

    Highlights: • Klf4 undergoes the 26S proteasomal degradation by ubiquitination on its multiple lysine residues. • Essential Klf4 ubiquitination sites are accumulated between 190–263 amino acids. • A mutation of lysine at 232 on Klf4 elongates protein turnover. • Klf4 mutants dramatically suppress p53 expression both under normal and UV irradiated conditions. - Abstract: The transcription factor, Krüppel-like factor 4 (Klf4) plays a crucial role in generating induced pluripotent stem cells (iPSCs). As the ubiquitination and degradation of the Klf4 protein have been suggested to play an important role in its function, the identification of specific lysine sites that are responsible for protein degradation is of prime interest to improve protein stability and function. However, the molecular mechanism regulating proteasomal degradation of the Klf4 is poorly understood. In this study, both the analysis of Klf4 ubiquitination sites using several Klf4 deletion fragments and bioinformatics predictions showed that the lysine sites which are signaling for Klf4 protein degradation lie in its N-terminal domain (aa 1–296). The results also showed that Lys32, 52, 232, and 252 of Klf4 are responsible for the proteolysis of the Klf4 protein. These results suggest that Klf4 undergoes proteasomal degradation and that these lysine residues are critical for Klf4 ubiquitination

  17. Critical lysine residues of Klf4 required for protein stabilization and degradation

    Lim, Key-Hwan; Kim, So-Ra; Ramakrishna, Suresh; Baek, Kwang-Hyun, E-mail: baek@cha.ac.kr

    2014-01-24

    Highlights: • Klf4 undergoes the 26S proteasomal degradation by ubiquitination on its multiple lysine residues. • Essential Klf4 ubiquitination sites are accumulated between 190–263 amino acids. • A mutation of lysine at 232 on Klf4 elongates protein turnover. • Klf4 mutants dramatically suppress p53 expression both under normal and UV irradiated conditions. - Abstract: The transcription factor, Krüppel-like factor 4 (Klf4) plays a crucial role in generating induced pluripotent stem cells (iPSCs). As the ubiquitination and degradation of the Klf4 protein have been suggested to play an important role in its function, the identification of specific lysine sites that are responsible for protein degradation is of prime interest to improve protein stability and function. However, the molecular mechanism regulating proteasomal degradation of the Klf4 is poorly understood. In this study, both the analysis of Klf4 ubiquitination sites using several Klf4 deletion fragments and bioinformatics predictions showed that the lysine sites which are signaling for Klf4 protein degradation lie in its N-terminal domain (aa 1–296). The results also showed that Lys32, 52, 232, and 252 of Klf4 are responsible for the proteolysis of the Klf4 protein. These results suggest that Klf4 undergoes proteasomal degradation and that these lysine residues are critical for Klf4 ubiquitination.

  18. Rubber recovery from centrifuged natural rubber latex residue using sulfuric acid

    Wirach Taweepreda

    2013-04-01

    Full Text Available Waste latex sludge from centrifuged residue, which is a null by-product of concentrated latex manufacturing, wasdigested to retrieve the rubber by using sulfuric acid. It was found that the acid concentration and digestion time have aneffect on the amount and purity of the retrieved rubber. Sulfuric acid at concentrations of more than 10% by weight with adigestion time of 48 hours completely digested waste latex sludge and gave rubber 10% by weight. The quality of the retrievedrubber was examined for Mooney viscosity (MV, plasticity retention index, nitrogen content, and ash content. The averagemolecular weight of the retrieved rubber, using gel permeation chromatography, was lower than that of normal natural rubber(NR which corresponds with the MV and initial plasticity (Po. The molecular structure from Fourier transform infraredspectroscopy (FT-IR indicated that the retrieved rubber surface is wet composed with hydroxyl functional ended group.The residue solution was evaporated and crystallized. The structure of crystals was determined using power X-ray diffractometer.

  19. Lead Isotope Compositions of Acid Residues from Olivine-Phyric Shergottite Tissint: Implications for Heterogeneous Shergottite Source Reservoirs

    Moriwaki, R.; Usui, T.; Yokoyama, T.; Simon, J. I.; Jones, J. H.

    2015-01-01

    Geochemical studies of shergottites suggest that their parental magmas reflect mixtures between at least two distinct geochemical source reservoirs, producing correlations between radiogenic isotope compositions and trace element abundances. These correlations have been interpreted as indicating the presence of a reduced, incompatible element- depleted reservoir and an oxidized, incompatible- element-enriched reservoir. The former is clearly a depleted mantle source, but there is ongoing debate regarding the origin of the enriched reservoir. Two contrasting models have been proposed regarding the location and mixing process of the two geochemical source reservoirs: (1) assimilation of oxidized crust by mantle derived, reduced magmas, or (2) mixing of two distinct mantle reservoirs during melting. The former requires the ancient Martian crust to be the enriched source (crustal assimilation), whereas the latter requires isolation of a long-lived enriched mantle domain that probably originated from residual melts formed during solidification of a magma ocean (heterogeneous mantle model). This study conducts Pb isotope and trace element concentration analyses of sequential acid-leaching fractions (leachates and the final residues) from the geochemically depleted olivine-phyric shergottite Tissint. The results suggest that the Tissint magma is not isotopically uniform and sampled at least two geochemical source reservoirs, implying that either crustal assimilation or magma mixing would have played a role in the Tissint petrogenesis.

  20. DNA Three Way Junction Core Decorated with Amino Acids-Like Residues-Synthesis and Characterization.

    Addamiano, Claudia; Gerland, Béatrice; Payrastre, Corinne; Escudier, Jean-Marc

    2016-01-01

    Construction and physico-chemical behavior of DNA three way junction (3WJ) functionalized by protein-like residues (imidazole, alcohol and carboxylic acid) at unpaired positions at the core is described. One 5'-C(S)-propargyl-thymidine nucleotide was specifically incorporated on each strand to react through a post synthetic CuACC reaction with either protected imidazolyl-, hydroxyl- or carboxyl-azide. Structural impacts of 5'-C(S)-functionalization were investigated to evaluate how 3WJ flexibility/stability is affected. PMID:27563857

  1. Collision-induced dissociation of protonated tetrapeptides containing beta-alanine, gamma-aminobutyric acid, epsilon-aminocaproic acid or 4-aminomethylbenzoic acid residues.

    Talaty, Erach R; Cooper, Travis J; Osburn, Sandra; Van Stipdonk, Michael J

    2006-01-01

    The influence of the presence and position of a single beta-alanine, gamma-aminobutyric acid, epsilon-aminocaproic acid or 4-aminomethylbenzoic acid residue on the tendency to form b(n)+ -and y(n)+ -type product ions was determined using a group of protonated tetrapeptides with general sequence XAAG, AXAG and AAXG (where X refers to the position of amino acid substitution). The hypothesis tested was that the 'alternative' amino acids would influence product ion signal intensities by inhibiting or suppressing either the nucleophilic attack or key proton transfer steps by forcing the adoption of large cyclic intermediates or blocking cyclization altogether. We found that specific b ions are diminished or eliminated completely when betaA, gammaAbu, Cap or 4AMBz residues are positioned such that they should interfere with the intramolecular nucleophilic attack step. In addition, differences in the relative proton affinities of the alternative amino acids influence the competition between complementary b(n) and y(n) ions. For both the AXAG and the XAAG series of peptides, collision-induced dissociation (CID) generated prominent b ions despite potential inhibition or suppression of intramolecular proton migration by the betaA, gammaAbu, Cap or 4AMBz residues. The prominent appearance of b ions from the AXAG and XAAG peptide is noteworthy, and suggests either that proton migration occurs through larger, 'whole' peptide cyclic intermediates or that fragmentation proceeds through a population of [M+H]+ isomers that are initially protonated at amide O atoms. PMID:17066369

  2. Amino acid residues involved in ligand preference of the Snf3 transporter-like sensor in Saccharomyces cerevisiae

    Dietvorst, J.; Karhumaa, Kaisa; Kielland-Brandt, Morten; Brandt, Anders Bøving

    2010-01-01

    Snf3 is a plasma membrane protein in Saccharomyces cerevisiae cerevisiae able to sense the presence of glucose. Although the Snf3 protein does not transport sugars, it shares sequence similarity with various glucose transporters from other organisms. we investigated the sugar specificity....../preferences of Snf3. The ability of cells to sense sugars in vivo was monitored by following the degradation of the Mth1 protein, :ill earl., event ill the signal pathway. Our study reveals that Snf3. ill addition to glucose. also senses fructose and mannose, as well as the glucose analogues 2-deoxyglucose, 3-O......-methylglucoside and 6-deoxyglucose. The signalling proficiency of a non-phosphorylatable analogue strongly supports the notion that sensing through Snf3 does not require sugar phosphorylation. Sequence comparisons of Snf3 to glucose transporters indicated amino acid residues possibly involved in sensing of sugars...

  3. Identification of key amino acid residues modulating intracellular and in vitro microcin E492 amyloid formation

    Paulina eAguilera

    2016-01-01

    Full Text Available Microcin E492 (MccE492 is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492. Besides its antibacterial activity, excreted MccE492 can form amyloid fibrils in vivo as well as in vitro. It has been proposed that bacterial amyloids can be functional playing a biological role, and in the particular case of MccE492 it would control the antibacterial activity. MccE492 amyloid fibril’s morphology and formation kinetics in vitro have been well characterized, however it is not known which amino acid residues determine its amyloidogenic propensity, nor if it forms intracellular amyloid inclusions as has been reported for other bacterial amyloids. In this work we found the conditions in which MccE492 forms intracellular amyloids in E. coli cells, that were visualized as round-shaped inclusion bodies recognized by two amyloidophillic probes, 2-4´-methylaminophenyl benzothiazole and thioflavin-S. We used this property to perform a flow cytometry-based assay to evaluate the aggregation propensity of MccE492 mutants, that were designed using an in silico prediction of putative aggregation hotspots. We established that the predicted amino acid residues 54-63, effectively act as a pro-amyloidogenic stretch. As in the case of other amyloidogenic proteins, this region presented two gatekeeper residues (P57 and P59, which disfavor both intracellular and in vitro MccE492 amyloid formation, preventing an uncontrolled aggregation. Mutants in each of these gatekeeper residues showed faster in vitro aggregation and bactericidal inactivation kinetics, and the two mutants were accumulated as dense amyloid inclusions in more than 80% of E. coli cells expressing these variants. In contrast, the MccE492 mutant lacking residues 54-63 showed a significantly lower intracellular aggregation propensity and slower in vitro polymerization kinetics. Electron microscopy analysis of the amyloids formed in vitro by these mutants revealed that, although

  4. Studies on the riboflavin, pantothenic acid, nicotinic acid and choline requirements of young Embden geese

    Serafin, J.A.

    1981-01-01

    Four experiments were conducted to examine the riboflavin, pantothenic acid, nicotinic acid, and choline requirements of young Embden geese fed purified diets. Goslings fed diets deficient in either riboflavin, pantothenic acid, nicotinic acid, or choline grew poorly. Feeding a pantothenic acid-deficient diet resulted in 100% mortality. Goslings fed diets containing 530 mg/kg of choline or less developed perosis. Under the conditions of these experiments it was found that: 1) goslings require no more than 3.84 mg/kg of riboflavin and 31.2 mg/kg of nicotinic acid in the diet for rapid growth and normal development, 2) the pantothenic acid requirement of goslings is no more than 12.6 mg/kg of diet, and 3) a dietary choline level of 1530 mg/kg is adequate for both the prevention of perosis and rapid growth of goslings. The levels of vitamins found to support normal growth and development of goslings appear to be similar to requirements of other species that have been examined.

  5. Studies on the riboflavin, pantothenic acid, nicotinic acid, and choline requirements of young Embden geese.

    Serafin, J A

    1981-08-01

    Four experiments were conducted to examine the riboflavin, pantothenic acid, nicotinic acid, and choline requirements of young Embden geese fed purified diets. Goslings fed diets deficient in either riboflavin, pantothenic acid, nicotinic acid, or choline grew poorly. Feeding a pantothenic acid-deficient diet resulted in 100% mortality. Goslings fed diets containing 530 mg/kg of choline or less developed perosis. Under the conditions of these experiments it was found that: 1) goslings require no more than 3.84 mg/kg of riboflavin and 31.2 mg/kg of nicotinic acid in the diet for rapid growth and normal development, 2) the pantothenic acid requirement of goslings is no more than 12.6 mg/kg of diet, and 3) a dietary choline level of 1530 mg/kg is adequate for both the prevention of perosis and rapid growth of goslings. The levels of vitamins found to support normal growth and development of goslings appear to be similar to requirements of other species that have been examined. PMID:7322986

  6. Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System.

    Worst, Emanuel G; Exner, Matthias P; De Simone, Alessandro; Schenkelberger, Marc; Noireaux, Vincent; Budisa, Nediljko; Ott, Albrecht

    2016-01-01

    The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any

  7. Quantitative solid state NMR analysis of residues from acid hydrolysis of loblolly pine wood.

    Sievers, Carsten; Marzialetti, Teresita; Hoskins, Travis J C; Valenzuela Olarte, Mariefel B; Agrawal, Pradeep K; Jones, Christopher W

    2009-10-01

    The composition of solid residues from hydrolysis reactions of loblolly pine wood with dilute mineral acids is analyzed by (13)C Cross Polarization Magic Angle Spinning (CP MAS) NMR spectroscopy. Using this method, the carbohydrate and lignin fractions are quantified in less than 3h as compared to over a day using wet chemical methods. In addition to the quantitative information, (13)C CP MAS NMR spectroscopy provides information on the formation of additional extractives and pseudo lignin from the carbohydrates. Being a non-destructive technique, NMR spectroscopy provides unambiguous evidence of the presence of side reactions and products, which is a clear advantage over the wet chemical analytical methods. Quantitative results from NMR spectroscopy and proximate analysis are compared for the residues from hydrolysis of loblolly pine wood under 13 different conditions; samples were treated either at 150 degrees C or 200 degrees C in the presence of various acids (HCl, H(2)SO(4), H(3)PO(4), HNO(3) and TFA) or water. The lignin content determined by both methods differed on averaged by 2.9 wt% resulting in a standard deviation of 3.5 wt%. It is shown that solid degradation products are formed from saccharide precursors under harsh reaction conditions. These degradation reactions limit the total possible yield of monosaccharides from any subsequent reaction. PMID:19477123

  8. Accurate prediction of hot spot residues through physicochemical characteristics of amino acid sequences

    Chen, Peng

    2013-07-23

    Hot spot residues of proteins are fundamental interface residues that help proteins perform their functions. Detecting hot spots by experimental methods is costly and time-consuming. Sequential and structural information has been widely used in the computational prediction of hot spots. However, structural information is not always available. In this article, we investigated the problem of identifying hot spots using only physicochemical characteristics extracted from amino acid sequences. We first extracted 132 relatively independent physicochemical features from a set of the 544 properties in AAindex1, an amino acid index database. Each feature was utilized to train a classification model with a novel encoding schema for hot spot prediction by the IBk algorithm, an extension of the K-nearest neighbor algorithm. The combinations of the individual classifiers were explored and the classifiers that appeared frequently in the top performing combinations were selected. The hot spot predictor was built based on an ensemble of these classifiers and to work in a voting manner. Experimental results demonstrated that our method effectively exploited the feature space and allowed flexible weights of features for different queries. On the commonly used hot spot benchmark sets, our method significantly outperformed other machine learning algorithms and state-of-the-art hot spot predictors. The program is available at http://sfb.kaust.edu.sa/pages/software.aspx. © 2013 Wiley Periodicals, Inc.

  9. Amino acid residues Tyr-67, Asn-72, and Asp-73 of the TVB receptor are important for subgroup E avian sarcoma and leukosis virus interaction

    The chicken TVBS1 protein serves as the cellular receptor for the cytopathic subgroups B and D avian sarcoma and leukosis viruses (ASLVs) as well as for the non-cytopathic subgroup E ASLV. Previous studies had mapped the subgroup B viral interaction determinants to a region that was located between residues 32 and 46 of TVBS1 [J. Virol. 76 (2002) 5404]. To gain a greater insight into ASLV Env-receptor interactions and the possible role of these interactions in viral cytopathic effects, we employed a homolog-scanning mutagenesis approach to identify amino acid residues important for subgroup E viral receptor function by exchanging amino acid residues between TVBS1 and its human homolog, DR5. These studies identified residues Tyr-67, Asn-72, and Asp-73 of TVBS1 as important subgroup E viral interaction determinants. Intriguingly, these three residues are conserved between TVBS1 and DR5, demonstrating that the human protein contains critical subgroup E viral interaction determinants, but in this context, they cannot support viral entry. These data confirm that the molecular determinants of the TVB receptor required for subgroup E viral entry are completely distinct from those used by subgroup B viruses

  10. Kinetics of sulfuric acid leaching of cadmium from Cd-Ni zinc plant residues.

    Safarzadeh, Mohammad Sadegh; Moradkhani, Davood; Ojaghi-Ilkhchi, Mehdi

    2009-04-30

    Cd-Ni filtercakes are produced continuously at the third purification step in the electrolytic production of zinc in the National Iranian Lead and Zinc Company (NILZ) in northwestern Iran. In this research, the dissolution kinetics of cadmium from Cd-Ni residues produced in NILZ plant has been investigated. Hence, the effects of temperature, sulfuric acid concentration, particle size and stirring speed on the kinetics of cadmium dissolution in sulfuric acid were studied. The dissolution kinetics at 25-55 degrees C and tshrinking core model, with inter-diffusion of cadmium and sulfate ions through the porous region of alloying layer (Cd(5)Ni, Cd(2)Ni(1.9) and Cd(10)Cu(3)) as the rate determining step. This finding is in accordance with the apparent activation energy (E(a)) of 13.363 kJ/mol and a linear relationship between the rate constant and the reciprocal of squared particle size. Arrhenius constant was calculated as 6.3942 min(-1). The order of reaction with respect to sulfuric acid concentration, solid/liquid ratio and particle size were also achieved. The rate of reaction at first 5 min based on diffusion-controlled process can be expressed by a semi-empirical equation as:It was determined that the dissolution rate increased with increasing sulfuric acid concentration and decreasing particle size. PMID:18755541

  11. Structural requirements for the procoagulant activity of nucleic acids.

    Julia Gansler

    Full Text Available Nucleic acids, especially extracellular RNA, are exposed following tissue- or vessel damage and have previously been shown to activate the intrinsic blood coagulation pathway in vitro and in vivo. Yet, no information on structural requirements for the procoagulant activity of nucleic acids is available. A comparison of linear and hairpin-forming RNA- and DNA-oligomers revealed that all tested oligomers forming a stable hairpin structure were protected from degradation in human plasma. In contrast to linear nucleic acids, hairpin forming compounds demonstrated highest procoagulant activities based on the analysis of clotting time in human plasma and in a prekallikrein activation assay. Moreover, the procoagulant activities of the DNA-oligomers correlated well with their binding affinity to high molecular weight kininogen, whereas the binding affinity of all tested oligomers to prekallikrein was low. Furthermore, four DNA-aptamers directed against thrombin, activated protein C, vascular endothelial growth factor and nucleolin as well as the naturally occurring small nucleolar RNA U6snRNA were identified as effective cofactors for prekallikrein auto-activation. Together, we conclude that hairpin-forming nucleic acids are most effective in promoting procoagulant activities, largely mediated by their specific binding to kininogen. Thus, in vivo application of therapeutic nucleic acids like aptamers might have undesired prothrombotic or proinflammatory side effects.

  12. Requirement of tyrosine residues 333 and 338 of the growth hormone (GH) receptor for selected GH-stimulated function

    Lobie, P E; Allevato, G; Norstedt, G;

    1995-01-01

    We have examined the involvement of tyrosine residues 333 and 338 of the growth hormone (GH) receptor in the cellular response to GH. Stable Chinese hamster ovary (CHO) cell clones expressing a receptor with tyrosine residues at position 333 and 338 of the receptor substituted for phenylalanine (...... acetyltransferase cDNA expression driven by the GH-responsive region of the SPI 2.1 gene) was not affected by Y333F,Y338F substitution. Thus we provide the first experimental evidence that specific tyrosine residues of the GH receptor are required for selected cellular responses to GH....

  13. Amphoteric surfactants containing ?-hydroxy ester group and an amino acid residue

    Eissa, A. M. F.

    2006-09-01

    Full Text Available A series of amphoteric surfactants containing α-hydroxy ester group and an amino acid residue were prepared with the addition of epoxy derivatives (which were prepared from epoxidation of alkyl methacrylate to different types of amino acids (glycine, alanine, valine, isoleucine, phenylalanine, tyrosine, serine, threonine, aspartic and anthranilic acid.The structures of the prepared compounds were confirmed by infrared spectra, proton magnetic resonance spectra, Mass spectra and elementary analysis. Surface tension, Kraft point, foaming power, critical micelle concentration emulsion and Ca++ stabilities were determined. Antimicrobial activity and biodegradability were also screened.Se prepararon una serie de tensioactivos anfóteros conteniendo un grupo alfa hidroxi éster y un residuo de aminoácido por adición de derivados epoxy (obtenidos mediante epoxidación de metacrilato de alquilo a diferentes tipos de aminoácidos (glicina, alanina, valina, isoleucina, fenilalanina, tirosina, serina, treonina y ácidos aspártico y antranílico. Las estructuras de los compuestos preparados se confirmaron por los espectros de infrarrojo, de masa, resonancia magnética nuclear de protones y análisis elemental. Se determinaron la tensión superficial, el punto de Kraft, el poder espumante, la concentración micelar crítica en emulsión y las estabilidades de Ca++. También se estudiaron la actividad antimicrobiana y la biodegradabilidad.

  14. Differential isotope-labeling for Leu and Val residues in a protein by E. coli cellular expression using stereo-specifically methyl labeled amino acids

    Miyanoiri, Yohei; Takeda, Mitsuhiro [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan); Okuma, Kosuke; Ono, Akira M.; Terauchi, Tsutomu [Tokyo Metropolitan University, Center for Priority Areas (Japan); Kainosho, Masatsune, E-mail: kainosho@tmu.ac.jp [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan)

    2013-09-21

    The {sup 1}H–{sup 13}C HMQC signals of the {sup 13}CH{sub 3} moieties of Ile, Leu, and Val residues, in an otherwise deuterated background, exhibit narrow line-widths, and thus are useful for investigating the structures and dynamics of larger proteins. This approach, named methyl TROSY, is economical as compared to laborious methods using chemically synthesized site- and stereo-specifically isotope-labeled amino acids, such as stereo-array isotope labeling amino acids, since moderately priced, commercially available isotope-labeled α-keto acid precursors can be used to prepare the necessary protein samples. The Ile δ{sub 1}-methyls can be selectively labeled, using isotope-labeled α-ketobutyrates as precursors. However, it is still difficult to prepare a residue-selectively Leu and Val labeled protein, since these residues share a common biosynthetic intermediate, α-ketoisovalerate. Another hindering drawback in using the α-ketoisovalerate precursor is the lack of stereo-selectivity for Leu and Val methyls. Here we present a differential labeling method for Leu and Val residues, using four kinds of stereo-specifically {sup 13}CH{sub 3}-labeled [U–{sup 2}H;{sup 15}N]-leucine and -valine, which can be efficiently incorporated into a protein using Escherichia coli cellular expression. The method allows the differential labeling of Leu and Val residues with any combination of stereo-specifically isotope-labeled prochiral methyls. Since relatively small amounts of labeled leucine and valine are required to prepare the NMR samples; i.e., 2 and 10 mg/100 mL of culture for leucine and valine, respectively, with sufficient isotope incorporation efficiency, this approach will be a good alternative to the precursor methods. The feasibility of the method is demonstrated for 82 kDa malate synthase G.

  15. Extension of microwave-accelerated residue-specific acid cleavage to proteins with carbohydrate side chains and disulfide linkages

    Li, Jinxi; Shefcheck, Kevin; Callahan, John; Fenselau, Catherine

    2008-01-01

    This laboratory has introduced a chemical method for residue-specific protein cleavage and has provided a preliminary assessment of the suitability of microwave accelerated acid cleavage as a proteomic tool. This report is a continuing assessment of the fate of common protein modifications in microwave-accelerated acid cleavage. We have examined the cleavage of ribonuclease A and the related N-linked glycoprotein ribonuclease B, and the O-linked glycoprotein alpha crystallin A chain, using MA...

  16. Complex formation of cadmium with sugar residues, nucleobases, phosphates, nucleotides, and nucleic acids.

    Sigel, Roland K O; Skilandat, Miriam; Sigel, Astrid; Operschall, Bert P; Sigel, Helmut

    2013-01-01

    Cadmium(II), commonly classified as a relatively soft metal ion, prefers indeed aromatic-nitrogen sites (e.g., N7 of purines) over oxygen sites (like sugar-hydroxyl groups). However, matters are not that simple, though it is true that the affinity of Cd(2+) towards ribose-hydroxyl groups is very small; yet, a correct orientation brought about by a suitable primary binding site and a reduced solvent polarity, as it is expected to occur in a folded nucleic acid, may facilitate metal ion-hydroxyl group binding very effectively. Cd(2+) prefers the guanine(N7) over the adenine(N7), mainly because of the steric hindrance of the (C6)NH(2) group in the adenine residue. This Cd(2+)-(N7) interaction in a guanine moiety leads to a significant acidification of the (N1)H meaning that the deprotonation reaction occurs now in the physiological pH range. N3 of the cytosine residue, together with the neighboring (C2)O, is also a remarkable Cd(2+) binding site, though replacement of (C2)O by (C2)S enhances the affinity towards Cd(2+) dramatically, giving in addition rise to the deprotonation of the (C4)NH(2) group. The phosphodiester bridge is only a weak binding site but the affinity increases further from the mono- to the di- and the triphosphate. The same also holds for the corresponding nucleotides. Complex stability of the pyrimidine-nucleotides is solely determined by the coordination tendency of the phosphate group(s), whereas in the case of purine-nucleotides macrochelate formation takes place by the interaction of the phosphate-coordinated Cd(2+) with N7. The extents of the formation degrees of these chelates are summarized and the effect of a non-bridging sulfur atom in a thiophosphate group (versus a normal phosphate group) is considered. Mixed ligand complexes containing a nucleotide and a further mono- or bidentate ligand are covered and it is concluded that in these species N7 is released from the coordination sphere of Cd(2+). In the case that the other ligand

  17. Influence of sodium chloride and weak organic acids (flux residues) on electrochemical migration of tin on surface mount chip components

    Verdingovas, Vadimas; Jellesen, Morten Stendahl; Ambat, Rajan

    2013-01-01

    contamination. The probability of migration becomes less dependent on the amount of contamination for sodium chloride at high concentrations. However, for organic acids from flux residues the migration probability shows an abrupt decrease with increasing concentration, which is attributed to a pH change in the...

  18. Sequence and structural features of binding site residues in protein-protein complexes: comparison with protein-nucleic acid complexes

    Selvaraj S

    2011-10-01

    Full Text Available Abstract Background Protein-protein interactions are important for several cellular processes. Understanding the mechanism of protein-protein recognition and predicting the binding sites in protein-protein complexes are long standing goals in molecular and computational biology. Methods We have developed an energy based approach for identifying the binding site residues in protein–protein complexes. The binding site residues have been analyzed with sequence and structure based parameters such as binding propensity, neighboring residues in the vicinity of binding sites, conservation score and conformational switching. Results We observed that the binding propensities of amino acid residues are specific for protein-protein complexes. Further, typical dipeptides and tripeptides showed high preference for binding, which is unique to protein-protein complexes. Most of the binding site residues are highly conserved among homologous sequences. Our analysis showed that 7% of residues changed their conformations upon protein-protein complex formation and it is 9.2% and 6.6% in the binding and non-binding sites, respectively. Specifically, the residues Glu, Lys, Leu and Ser changed their conformation from coil to helix/strand and from helix to coil/strand. Leu, Ser, Thr and Val prefer to change their conformation from strand to coil/helix. Conclusions The results obtained in this study will be helpful for understanding and predicting the binding sites in protein-protein complexes.

  19. Conserved charged amino acid residues in the extracellular region of sodium/iodide symporter are critical for iodide transport activity

    Liang Ji-An

    2010-11-01

    Full Text Available Abstract Background Sodium/iodide symporter (NIS mediates the active transport and accumulation of iodide from the blood into the thyroid gland. His-226 located in the extracellular region of NIS has been demonstrated to be critical for iodide transport in our previous study. The conserved charged amino acid residues in the extracellular region of NIS were therefore characterized in this study. Methods Fourteen charged residues (Arg-9, Glu-79, Arg-82, Lys-86, Asp-163, His-226, Arg-228, Asp-233, Asp-237, Arg-239, Arg-241, Asp-311, Asp-322, and Asp-331 were replaced by alanine. Iodide uptake abilities of mutants were evaluated by steady-state and kinetic analysis. The three-dimensional comparative protein structure of NIS was further modeled using sodium/glucose transporter as the reference protein. Results All the NIS mutants were expressed normally in the cells and targeted correctly to the plasma membrane. However, these mutants, except R9A, displayed severe defects on the iodide uptake. Further kinetic analysis revealed that mutations at conserved positively charged amino acid residues in the extracellular region of NIS led to decrease NIS-mediated iodide uptake activity by reducing the maximal rate of iodide transport, while mutations at conserved negatively charged residues led to decrease iodide transport by increasing dissociation between NIS mutants and iodide. Conclusions This is the first report characterizing thoroughly the functional significance of conserved charged amino acid residues in the extracellular region of NIS. Our data suggested that conserved charged amino acid residues, except Arg-9, in the extracellular region of NIS were critical for iodide transport.

  20. D-amino acid residue in a defensin-like peptide from platypus venom: effect on structure and chromatographic properties.

    Torres, Allan M; Tsampazi, Chryssanthi; Geraghty, Dominic P; Bansal, Paramjit S; Alewood, Paul F; Kuchel, Philip W

    2005-10-15

    The recent discovery that the natriuretic peptide OvCNPb (Ornithorhynchus venom C-type natriuretic peptide B) from platypus (Ornithorynchus anatinus) venom contains a D-amino acid residue suggested that other D-amino-acid-containing peptides might be present in the venom. In the present study, we show that DLP-2 (defensin-like peptide-2), a 42-amino-acid residue polypeptide in the platypus venom, also contains a D-amino acid residue, D-methionine, at position 2, while DLP-4, which has an identical amino acid sequence, has all amino acids in the L-form. These findings were supported further by the detection of isomerase activity in the platypus gland venom extract that converts DLP-4 into DLP-2. In the light of this new information, the tertiary structure of DLP-2 was recalculated using a new structural template with D-Met2. The structure of DLP-4 was also determined in order to evaluate the effect of a D-amino acid at position 2 on the structure and possibly to explain the large retention time difference observed for the two molecules in reverse-phase HPLC. The solution structures of the DLP-2 and DLP-4 are very similar to each other and to the earlier reported structure of DLP-2, which assumed that all amino acids were in the L-form. Our results suggest that the incorporation of the D-amino acid at position 2 has minimal effect on the overall fold in solution. PMID:16033333

  1. 40 CFR 180.1090 - Lactic acid; exemption from the requirement of a tolerance.

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Lactic acid; exemption from the... Exemptions From Tolerances § 180.1090 Lactic acid; exemption from the requirement of a tolerance. Lactic acid (2-hydroxypropanoic acid) is exempted from the requirement of a tolerance when used as a plant...

  2. 40 CFR 72.31 - Information requirements for Acid Rain permit applications.

    2010-07-01

    ... 40 Protection of Environment 16 2010-07-01 2010-07-01 false Information requirements for Acid Rain... (CONTINUED) AIR PROGRAMS (CONTINUED) PERMITS REGULATION Acid Rain Permit Applications § 72.31 Information requirements for Acid Rain permit applications. A complete Acid Rain permit application shall include...

  3. Influence of shear force on floc properties and residual aluminum in humic acid treatment by nano-Al13

    Graphical abstract: Effect of shear forces on total residual Al and dissolved residual Al in humic acid coagulations by PACl and Al13 at dosages of 5.5 mg/L. Fig. a Variations of total residual Al concentrations with shear force. Fig. b Variations of dissolved residual Al concentrations with shear force. - Highlights: • Increased shear reduced floc size, strength and re-growth but improved floc Df. • HA-Al13 flocs were smaller but showed higher Sf, Rf and Df than HA-PACl flocs. • Higher shear resulted in higher residual Al in finished water. • Residual Al increased less with enhanced shear rate for Al13 than that for PACl. • Higher Sf and Rf mitigated residual Al increment but Df had little effect on it. - Abstract: The impacts of various shear forces on floc sizes and structures in humic acid coagulations by polyaluminum chloride (PACl) and nano-Al13 were comparatively studied in this paper. The dynamic floc size was monitored by use of a laser diffraction particle sizing device. The floc structure was evaluated in terms of fractal dimension, analyzed by small-angle laser light scattering (SALLS). The effect of increased shear rate on residual Al of the coagulation effluents was then analyzed on the basis of different floc characteristics generated under various shear conditions. The results showed that floc size decreased with the increasing shear rate for both Al13 and PACl. Besides, floc strength and re-formation ability were also weakened by the enhanced shear force. Al13 resulted in small, strong and better recoverable flocs than PACl and moreover, in the shear range of 100–300 revolution per minute (rpm) (G = 40.7–178.3 s−1), the characteristics of HA-Al13 flocs displayed smaller scale changes than those of HA-PACl flocs. The results of residual Al measurements proved that with shear increased, the residual Al increased continuously but Al13 presented less sensitivity to the varying shear forces. PACl contributed higher residual Al than Al13

  4. Influence of shear force on floc properties and residual aluminum in humic acid treatment by nano-Al{sub 13}

    Xu, Weiying, E-mail: stu_xuwy@ujn.edu.cn [School of Resources and Environmental Sciences, University of Jinan, Ji’nan 250022, Shandong (China); Gao, Baoyu [Shandong Key Laboratory of Water Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Shandong University, Ji’nan 250100, Shandong (China); Du, Bin; Xu, Zhenghe; Zhang, Yongfang; Wei, Dong [School of Resources and Environmental Sciences, University of Jinan, Ji’nan 250022, Shandong (China)

    2014-04-01

    Graphical abstract: Effect of shear forces on total residual Al and dissolved residual Al in humic acid coagulations by PACl and Al{sub 13} at dosages of 5.5 mg/L. Fig. a Variations of total residual Al concentrations with shear force. Fig. b Variations of dissolved residual Al concentrations with shear force. - Highlights: • Increased shear reduced floc size, strength and re-growth but improved floc D{sub f}. • HA-Al{sub 13} flocs were smaller but showed higher S{sub f}, R{sub f} and D{sub f} than HA-PACl flocs. • Higher shear resulted in higher residual Al in finished water. • Residual Al increased less with enhanced shear rate for Al{sub 13} than that for PACl. • Higher S{sub f} and R{sub f} mitigated residual Al increment but D{sub f} had little effect on it. - Abstract: The impacts of various shear forces on floc sizes and structures in humic acid coagulations by polyaluminum chloride (PACl) and nano-Al{sub 13} were comparatively studied in this paper. The dynamic floc size was monitored by use of a laser diffraction particle sizing device. The floc structure was evaluated in terms of fractal dimension, analyzed by small-angle laser light scattering (SALLS). The effect of increased shear rate on residual Al of the coagulation effluents was then analyzed on the basis of different floc characteristics generated under various shear conditions. The results showed that floc size decreased with the increasing shear rate for both Al{sub 13} and PACl. Besides, floc strength and re-formation ability were also weakened by the enhanced shear force. Al{sub 13} resulted in small, strong and better recoverable flocs than PACl and moreover, in the shear range of 100–300 revolution per minute (rpm) (G = 40.7–178.3 s{sup −1}), the characteristics of HA-Al{sub 13} flocs displayed smaller scale changes than those of HA-PACl flocs. The results of residual Al measurements proved that with shear increased, the residual Al increased continuously but Al{sub 13

  5. A kinetic model for the competitive reactions of ozone with amino acid residues in proteins in reverse micelles

    Pryor, W.A.; Uppu, R.M. (Louisiana State Univ., Baton Rouge (United States))

    1993-02-15

    Lysozyme and 10 other proteins are solubilized in reverse micelles formed by 0.1 M sodium di-2-ethyl-hexylsulfosuccinate and 2.0-2.5 M water (pH 7.4) in isooctane solvent. Exposure of the protein-containing reverse micellar solutions to ozone causes oxidative damage to the proteins, as assessed by the oxidation of tryptophan residues. The oxidation product of the protein-bound tryptophan has a molar absorption coefficient of 3275 +/- 81 M-1 cm-1 (mean +/- S.D., n = 6) at 320 nm. The product is suggested to be a Criegee ozonide or a tautomer of the Criegee ozonide and not N-formylkynurenine. Ozonation of lysozyme in reverse micelles results in the formation of hydrogen peroxide in yields of only approximately 0.07 mol/mol of tryptophan residues oxidized. The recovery of hydrogen peroxide added as an internal standard to the lysozyme-containing reverse micellar solutions ranges from 84 to 88%, whether or not the samples are subjected to ozonation. This suggests that hydrogen peroxide is neither destroyed during the process of ozonation nor consumed by the protein to a significant extent in an adventitious reaction. A kinetic model for the overall reaction of ozone with the proteins is developed, taking into account the concentrations and the reactivities of individual amino acid residues toward ozone. The model predicts the fractional reaction of ozone with tryptophan residues in the proteins, despite differences in amino acid composition, molecular weight, and tertiary structures. The lack of influence of protein structure is confirmed further by the observation that the native lysozyme (with and without external S-carboxymethylcysteine) and S-carboxymethylated lysozyme give identical values of the fractional reaction of ozone with tryptophan residues. The kinetic equations for the competitive reactions of ozone with amino acid residues in proteins, with some minor modification, are applicable to ozonations on complex mixtures of lipids, proteins, and antioxidants.

  6. Identification of amino acid residues that determine the substrate specificity of mammalian membrane-bound front-end fatty acid desaturases.

    Watanabe, Kenshi; Ohno, Makoto; Taguchi, Masahiro; Kawamoto, Seiji; Ono, Kazuhisa; Aki, Tsunehiro

    2016-01-01

    Membrane-bound desaturases are physiologically and industrially important enzymes that are involved in the production of diverse fatty acids such as polyunsaturated fatty acids and their derivatives. Here, we identified amino acid residues that determine the substrate specificity of rat Δ6 desaturase (D6d) acting on linoleoyl-CoA by comparing its amino acid sequence with that of Δ5 desaturase (D5d), which converts dihomo-γ-linolenoyl-CoA. The N-terminal cytochrome b5-like domain was excluded as a determinant by domain swapping analysis. Substitution of eight amino acid residues (Ser209, Asn211, Arg216, Ser235, Leu236, Trp244, Gln245, and Val344) of D6d with the corresponding residues of D5d by site-directed mutagenesis switched the substrate specificity from linoleoyl-CoA to dihomo-γ-linolenoyl-CoA. In addition, replacement of Leu323 of D6d with Phe323 on the basis of the amino acid sequence of zebra fish Δ5/6 bifunctional desaturase was found to render D6d bifunctional. Homology modeling of D6d using recent crystal structure data of human stearoyl-CoA (Δ9) desaturase revealed that Arg216, Trp244, Gln245, and Leu323 are located near the substrate-binding pocket. To our knowledge, this is the first report on the structural basis of the substrate specificity of a mammalian front-end fatty acid desaturase, which will aid in efficient production of value-added fatty acids. PMID:26590171

  7. Retinoic acid activates two pathways required for meiosis in mice.

    Jana Koubova

    2014-08-01

    Full Text Available In all sexually reproducing organisms, cells of the germ line must transition from mitosis to meiosis. In mice, retinoic acid (RA, the extrinsic signal for meiotic initiation, activates transcription of Stra8, which is required for meiotic DNA replication and the subsequent processes of meiotic prophase. Here we report that RA also activates transcription of Rec8, which encodes a component of the cohesin complex that accumulates during meiotic S phase, and which is essential for chromosome synapsis and segregation. This RA induction of Rec8 occurs in parallel with the induction of Stra8, and independently of Stra8 function, and it is conserved between the sexes. Further, RA induction of Rec8, like that of Stra8, requires the germ-cell-intrinsic competence factor Dazl. Our findings strengthen the importance of RA and Dazl in the meiotic transition, provide important details about the Stra8 pathway, and open avenues to investigate early meiosis through analysis of Rec8 induction and function.

  8. Evaluation of methods to estimate the essential amino acids requirements of fish from the muscle amino acid profile

    Álvaro José de Almeida Bicudo

    2014-03-01

    Full Text Available Many methods to estimate amino acid requirement based on amino acid profile of fish have been proposed. This study evaluates the methodology proposed by Meyer & Fracalossi (2005 and by Tacon (1989 to estimate amino acids requirement of fish, which do exempt knowledge on previous nutritional requirement of reference amino acid. Data on amino acid requirement of pacu, Piaractus mesopotamicus, were used to validate de accuracy of those methods. Meyer & Fracalossi's and Tacon's methodology estimated the lysine requirement of pacu, respectively, at 13 and 23% above requirement determined using dose-response method. The values estimated by both methods lie within the range of requirements determined for other omnivorous fish species, the Meyer & Fracalossi (2005 method showing better accuracy.

  9. Sample Preparation for Headspace GC Analysis of Residual Solvents in Hyaluronic Acid Derivative Fiber

    Kim, Hoon Joo; Kim, Dong Min; Yang, Jeong Soo [LG life Sciences, Ltd./R and D Park, Daejeon (Korea, Republic of); Kim, Chan Wha [Korea University, Seoul (Korea, Republic of)

    2006-02-15

    The aim of this study is to develop efficient sample preparation method for HS-GC analysis of residual solvents in HA derivative fiber. Compared to direct extraction of residual solvents from HA derivative fiber, the extraction through the hydrolysis of HA derivative fiber by HAse gave more complete and higher reproducible quantification of residual solvent. To validate HS-GC analysis method of residual solvents, specificity, limits of detection and quantification, linearity, accuracy and precision are investigated in the study. HA derivative fiber was hydrolyzed using HAse for headspace gas chromatographic analysis of residual solvents of ethanol, acetone and isopropanol in HA derivative fiber. This study showed that the developed method had specificity, linearity, accuracy and precision. In addition, it demonstrated that HS-GC coupled with matrix-breaking method such as hydrolysis was available for the determination of residual solvents in a matrix like HA derivative fiber.

  10. Sample Preparation for Headspace GC Analysis of Residual Solvents in Hyaluronic Acid Derivative Fiber

    The aim of this study is to develop efficient sample preparation method for HS-GC analysis of residual solvents in HA derivative fiber. Compared to direct extraction of residual solvents from HA derivative fiber, the extraction through the hydrolysis of HA derivative fiber by HAse gave more complete and higher reproducible quantification of residual solvent. To validate HS-GC analysis method of residual solvents, specificity, limits of detection and quantification, linearity, accuracy and precision are investigated in the study. HA derivative fiber was hydrolyzed using HAse for headspace gas chromatographic analysis of residual solvents of ethanol, acetone and isopropanol in HA derivative fiber. This study showed that the developed method had specificity, linearity, accuracy and precision. In addition, it demonstrated that HS-GC coupled with matrix-breaking method such as hydrolysis was available for the determination of residual solvents in a matrix like HA derivative fiber

  11. Reuse of acid coagulant-recovered drinking waterworks sludge residual to remove phosphorus from wastewater

    Yang, Lan; Wei, Jie; Zhang, Yumei; Wang, Jianli; Wang, Dongtian

    2014-06-01

    Acid coagulant-recovered drinking waterworks sludge residual (DWSR) is a waste product from drinking waterworks sludge (DWS) treatment with acid for coagulant recovery. In this study, we evaluated DWSR as a potential phosphorus (P) removing material in wastewater treatment by conducting a series of batch and semi-continuous tests. Batch tests were carried out to study the effects of pH, initial concentration, and sludge dose on P removal. Batch test results showed that the P removal efficiency of DWSR was highly dependent on pH. Calcinated DWSR (C-DWSR) performed better in P removal than DWSR due to its higher pH. At an optimum initial pH value of 5-6 and a sludge dose of 10 g/L, the P removal rates of DWSR and DWS decreased from 99% and 93% to 84% and 14%, respectively, and the specific P uptake of DWSR and DWS increased from 0.19 and 0.19 mg P/g to 33.60 and 5.72 mg P/g, respectively, when the initial concentration was increased from 2 to 400 mg/L. The effective minimum sludge doses of DWSR and DWS were 0.5 g/L and 10 g/L, respectively, when the P removal rates of 90% were obtained at an initial concentration of 10 mg/L. Results from semi-continuous test indicated that P removal rates over 99% were quickly achieved for both synthetic and actual wastewater (lake water and domestic sewage). These rates could be maintained over a certain time under a certain operational conditions including sludge dose, feed flow, and initial concentration. The physicochemical properties analysis results showed that the contents of aluminum (Al) and iron (Fe) in DWSR were reduced by 50% and 70%, respectively, compared with DWS. The insoluble Al and Fe hydroxide in DWS converted into soluble Al and Fe in DWSR. Metal leaching test results revealed that little soluble Al and Fe remained in effluent when DWSR was used for P removal. We deduced that chemical precipitation might be the major action for P removal by DWSR and that adsorption played only a marginal role.

  12. Formation of [b3 - 1 + cat]+ ions from metal-cationized tetrapeptides containing beta-alanine, gamma-aminobutyric acid or epsilon-aminocaproic acid residues.

    Osburn, Sandra M; Ochola, Sila O; Talaty, Erach R; Van Stipdonk, Michael J

    2008-11-01

    The presence and position of a single beta-alanine (betaA), gamma-aminobutyric acid (gammaABu) or epsilon-aminocaproic acid (Cap) residue has been shown to have a significant influence on the formation of b(n)+ and y(n)+ product ions from a series of model, protonated peptides. In this study, we examined the effect of the same residues on the formation of analogous [b3 - 1 + cat]+ products from metal (Li+, Na+ and Ag+)-cationized peptides. The larger amino acids suppress formation of b3+ from protonated peptides with general sequence AAXG (where X = beta-alanine, gamma-aminobutyric acid or epsilon-aminocaproic acid), presumably because of the prohibitive effect of larger cyclic intermediates in the 'oxazolone' pathway. However, abundant [b3 - 1 + cat]+ products are generated from metal-cationized versions of AAXG. Using a group of deuterium-labeled and exchanged peptides, we found that formation of [b3 - 1 + cat]+ involves transfer of either amide or alpha-carbon position H atoms, and the tendency to transfer the atom from the alpha-carbon position increases with the size of the amino acid in position X. To account for the transfer of the H atom, a mechanism involving formation of a ketene product as [b3 - 1 + cat]+ is proposed. PMID:18449851

  13. Determination of residues in honey after treatments with formic and oxalic acid under field conditions

    Bogdanov, Stefan; Charrière, Jean-Daniel; IMDORF, Anton; KILCHENMANN, Verena; Fluri, Peter

    2002-01-01

    International audience Formic acid and oxalic acid field trials for control of Varroa destructor were carried out in autumn according to the Swiss prescriptions during three successive years in different apiaries in Switzerland. The following parameters were determined in honey that was harvested the year after treatment: formic acid, oxalic acid and free acidity. The following range of values were found in honeys of untreated colonies: formic acid, from 17 to 284 mg/kg, n = 34; oxalic aci...

  14. Treatment of air pollution control residues with iron rich waste sulfuric acid: does it work for antimony (Sb)?

    Okkenhaug, Gudny; Breedveld, Gijs D; Kirkeng, Terje; Lægreid, Marit; Mæhlum, Trond; Mulder, Jan

    2013-03-15

    Antimony (Sb) in air pollution control (APC) residues from municipal solid waste incineration has gained increased focus due to strict Sb leaching limits set by the EU landfill directive. Here we study the chemical speciation and solubility of Sb at the APC treatment facility NOAH Langøya (Norway), where iron (Fe)-rich sulfuric acid (∼3.6M, 2.3% Fe(II)), a waste product from the industrial extraction of ilmenite, is used for neutralization. Antimony in water extracts of untreated APC residues occurred exclusively as pentavalent antimonate, even at low pH and Eh values. The Sb solubility increased substantially at pHettringite (at alkaline pH) or calcium (Ca)-antimonate. Treated APC residues, stored anoxically in the laboratory, simulating the conditions at the NOAH Langøya landfill, gave rise to decreasing concentrations of Sb in porewater, occurring exclusively as Sb(V). Concentrations of Sb decreased from 87-918μgL(-1) (day 3) to 18-69μgL(-1) (day 600). We hypothesize that an initial sorption of Sb to Fe(II)-Fe(III) hydroxides (green rust) and eventually precipitation of Ca- and Fe-antimonates (tripuhyite; FeSbO4) occurred. We conclude that Fe-rich, sulfuric acid waste is efficient to immobilize Sb in APC residues from waste incineration. PMID:23465722

  15. CHOICE FEEDING AND AMINO ACID REQUIREMENTS FOR BROILERS

    B. Indarsih

    2011-12-01

    Full Text Available The study was conducted as a completely randomized design, with a factorial arrangement to determine the response of commercial broilers to choice feeding and limiting amino acids on growth and carcass performance. A total of 432 male birds were weighed at one-d-old and randomly distributed to 48 wire-floored brooder cage each 1.0 m2. There were 2 sexes and 4 dietary treatments with 6 replicates each of 9 birds. Birds were given one of three dietary regimens with dietary change every 7 days. All groups were fed free choice of summit and dilution diets. The estimated dietary level of crude protein at day-old was 240 g/kg and the level at 42 d was either 120, 150 or 180 g/kg for females or 130, 160 and 190 g/kg for males. At 43 d of age, all birds from each dietary treatment were slaughtered for measurement of body composition. Results reveal that lysine requirement for maximum gain in this study was higher than NRC recommendation. The free choice-fed bird was significantly higher, in terms of growth and body composition than that obtained on the low dietary protein regimen.Keyword

  16. CHOICE FEEDING AND AMINO ACID REQUIREMENTS FOR BROILERS

    B. Indarsih

    2014-10-01

    Full Text Available The study was conducted as a completely randomized design, with a factorial arrangement todetermine the response of commercial broilers to choice feeding and limiting amino acids on growth andcarcass performance. A total of 432 male birds were weighed at one-d-old and randomly distributed to48 wire-floored brooder cage each 1.0 m2. There were 2 sexes and 4 dietary treatments with 6 replicateseach of 9 birds. Birds were given one of three dietary regimens with dietary change every 7 days. Allgroups were fed free choice of summit and dilution diets. The estimated dietary level of crude protein atday-old was 240 g/kg and the level at 42 d was either 120, 150 or 180 g/kg for females or 130, 160 and190 g/kg for males. At 43 d of age, all birds from each dietary treatment were slaughtered formeasurement of body composition. Results reveal that lysine requirement for maximum gain in thisstudy was higher than NRC recommendation. The free choice-fed bird was significantly higher, in termsof growth and body composition than that obtained on the low dietary protein regimen.

  17. Specific RNA residue interactions required for enzymatic functions of Tetrahymena telomerase.

    Gilley, D; Blackburn, E H

    1996-01-01

    The ribonucleoprotein enzyme telomerase is a specialized reverse transcriptase that synthesizes telomeric DNA by copying a template sequence within the telomerase RNA. Here we analyze the actions of telomerase from Tetrahymena thermophila assembled in vivo with mutated or wild-type telomerase RNA to define further the roles of particular telomerase RNA residues involved in essential enzymatic functions: templating, substrate alignment, and promotion of polymerization. Position 49 of the telom...

  18. The influence of the residual copper on the pipes steel hot plasticity according to environmental requirements

    Rusănescu C.O.; Rusănescu M.

    2013-01-01

    Considering the importance of gaseous and/or liquid fuels impact on the environment, the resistance of pipelines at hot plastic deformation is important. Therefore, in order to avoid or reduce any adverse impact on the environment, the influence of residual copper on hot deformability of steel pipes was investigated in this paper. The negative copper influence was experimentally proved using torsion deformation at temperatures above 1000o, under the air and...

  19. Frequencies of amino acid strings in globular protein sequences indicate suppression of blocks of consecutive hydrophobic residues

    Schwartz, Russell; Istrail, Sorin; King, Jonathan

    2001-01-01

    Patterns of hydrophobic and hydrophilic residues play a major role in protein folding and function. Long, predominantly hydrophobic strings of 20–22 amino acids each are associated with transmembrane helices and have been used to identify such sequences. Much less attention has been paid to hydrophobic sequences within globular proteins. In prior work on computer simulations of the competition between on-pathway folding and off-pathway aggregate formation, we found that long sequences of cons...

  20. Effect of Drying Medium on Residual Moisture Content and Viability of Freeze-Dried Lactic Acid Bacteria

    Graciela F. Valdez; de Giori, Graciela S.; Aida P de Ruiz Holgado; Oliver, Guillermo

    1985-01-01

    The effect of various substances on the relationship between residual moisture content and the viability of freeze-dried lactic acid bacteria has been studied. Compounds such as polymers, which display considerable ability in displacing water, showed no protective action during freeze-drying. Adonitol, on the other hand, produced the smallest change in water content at various times during drying and allowed the highest rate of survival.

  1. Synthesis of two hyaluronic-acid-related oligosaccharide 4-methoxyphenyl glycosides having a beta-D-glucuronic acid residue at the reducing end

    Halkes, K.M.; Slaghek, T.M.; Hypponen, T.K.; Kamerling, J.P.; Vliegenthart, J.F.G.

    1999-01-01

    Synthesis of two hyaluronic-acid-related oligosaccharides, the 4-methoxyphenyl β-glycosides of β-D-GlcpA-(1→3)-β-D-GlcpNAc-(1→4)-D-GlcpA and β-D-GlcpA-(1→3)-β-D-GlcpNAc-(1→4)-β-D-GlcpA-(1→3)- β-D-GJcpNAc-(1→4)-D-GlcpA, is described. D-Glucopyranosyluronic acid residues were obtained by selective oxi

  2. Nature of Soil Acidity in Relation to Properties and Lime Requirement of Some Inceptisols

    A. K. DOLUI; S. BHATTACHARJEE

    2003-01-01

    Some Inceptisols representing the Singla catchment area in Karimgaunge district of Assam, India, were studied for lime requirement as influenced by the nature of soil acidity. The electrostatically bonded (EB)-H+ and EB-Al3+ acidities constituted 33 and 67 percent of exchangeable acidity while EB-H+, EB-Al3+,exchangeable and pH-dependent acidities comprised 6, 14, 20 and 80 percent of total potential acidity. The pH-dependent acidity made a major contribution towards the total potential acidity (67%~84%). Grand mean of lime requirement determined by the laboratory incubation method and estimated by the methods of New Woodruff, Woodruff and Peech as expressed in MgCaCO3 ha-1 was in the order: Woodruff (15.6) > New Woodruff (14.9) > Peech (5.1) > incubation (5.0). Correlations analysis among different forms of acidity and lime requirement methods with selected soil properties showed that pH in three media, namely water, 1 mol L-1 KCl and 0.01 mol L-1 CaCl2, had a significant negative correlation with different forms of acidity and lime requirement methods. Exchangeable Fe and Al showed significant positive correlations with EB-Al3+ acidity, exchangeable acidity, pH-dependent acidity and total potential acidity, and also lime requirement methods. Extractable Al showed positive correlations with different forms of acidity except EB-H+ and EB-Al3+ acidities. The lime requirement by different methods depended upon the extractable aluminium.Significant positive correlations existed between lime requirements and different forms of acidity of the soils except EB-H+ acidity and incubation method. The nature of soil acidity was mostly pH-dependent. Statistically, the Woodruff method did slightly better than the New Woodruff, incubation and Peech methods at estimating lime requirement and hence the Woodruff procedure may be recommended for routine soil testing because of its speed and simplicity.

  3. The influence of the residual copper on the pipes steel hot plasticity according to environmental requirements

    Rusănescu C.O.

    2013-01-01

    Full Text Available Considering the importance of gaseous and/or liquid fuels impact on the environment, the resistance of pipelines at hot plastic deformation is important. Therefore, in order to avoid or reduce any adverse impact on the environment, the influence of residual copper on hot deformability of steel pipes was investigated in this paper. The negative copper influence was experimentally proved using torsion deformation at temperatures above 1000o, under the air and argon atmosphere. The samples were heated and then deformed at different temperatures with constant deformation rate. Also, structural analysis of investigated materials was done, using metallographic and SEM analysis.

  4. Chapter 4. Uranium extraction technology from wastes of uranium industry. 4.1. Sulfuric acid leaching kinetics of residues from anthropogenic deposit Map 1-9

    Present article is devoted to sulfuric acid leaching kinetics of residues from anthropogenic deposit Map 1-9. Since residues from anthropogenic deposit Map 1-9 basically contain quartz (69%), it went through acid leaching. Results of laboratory investigations on the opening degree and uranium conversion to solution were summarized. The influence of sulfuric acid consumption on uranium extraction during leaching (U = 0,018%, t =20 deg C, t = 10 hours) was considered. The kinetic and energetic parameters of residues leaching of anthropogenic deposit Map 1-9 was discussed. The basic process flow diagram of uranium industry wastes reprocessing was proposed.

  5. Catalytic mechanism of S-type phycobiliprotein lyase: chaperone-like action and functional amino acid residues.

    Kupka, Michaela; Zhang, Juan; Fu, Wei-Lei; Tu, Jun-Ming; Böhm, Stephan; Su, Ping; Chen, Yu; Zhou, Ming; Scheer, Hugo; Zhao, Kai-Hong

    2009-12-25

    The phycobilin:cysteine 84-phycobiliprotein lyase, CpcS1, catalyzes phycocyanobilin (PCB) and phycoerythrobilin (PEB) attachment at nearly all cysteine 82 binding sites (consensus numbering) of phycoerythrin, phycoerythrocyanin, phycocyanin, and allophycocyanin (Zhao, K. H., Su, P., Tu, J. M., Wang, X., Liu, H., Plöscher, M., Eichacker, L., Yang, B., Zhou, M., and Scheer, H. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 14300-14305). We now show that CpcS1 binds PCB and PEB rapidly with bi-exponential kinetics (38/119 and 12/8300 ms, respectively). Chromophore binding to the lyase is reversible and much faster than the spontaneous, but low fidelity chromophore addition to the apo-protein in the absence of the lyase. This indicates kinetic control by the enzyme, which then transfers the chromophore to the apo-protein in a slow (tens of minutes) but stereo- and regioselectively corrects the reaction. This mode of action is reminiscent of chaperones but does not require ATP. The amino acid residues Arg-18 and Arg-149 of the lyase are essential for chromophore attachment in vitro and in Escherichia coli, mutations of His-21, His-22, Trp-75, Trp-140, and Arg-147 result in reduced activity (<30% of wild type in vitro). Mutants R147Q and W69M were active but had reduced capacity for PCB binding; additionally, with W69M there was loss of fidelity in chromophore attachment. Imidazole is a non-competitive inhibitor, supporting a bilin-binding function of histidine. Evidence was obtained that CpcS1 also catalyzes exchange of C-beta84-bound PCB in biliproteins by PEB. PMID:19864423

  6. Basic amino acid residues located in the N-terminal region of BEND3 are essential for its nuclear localization

    Shiheido, Hirokazu, E-mail: shiheido@ak.med.kyoto-u.ac.jp; Shimizu, Jun

    2015-02-20

    BEN domain-containing protein 3 (BEND3) has recently been reported to function as a heterochromatin-associated protein in transcriptional repression in the nucleus. BEND3 should have nuclear localization signals (NLSs) to localize to the nucleus in light of its molecular weight, which is higher than that allowed to pass through nuclear pore complexes. We here analyzed the subcellular localization of deletion/site-directed mutants of human BEND3 by an immunofluorescence assay in an attempt to identify the amino acids essential for its nuclear localization. We found that three basic amino acid residues located in the N-terminal region of BEND3 (BEND3{sub 56–58}, KRK) are essential, suggesting that these residues play a role as a functional NLS. These results provide valuable information for progressing research on BEND3. - Highlights: • BEND3 localizes to the nucleus. • The N-terminal 60 amino acids region of BEND3 contains NLS. • Amino acids located between 56 and 58 of BEND3 (KRK) are part of NLS. • KRK motif is highly conserved among BEND3 homologs.

  7. Basic amino acid residues located in the N-terminal region of BEND3 are essential for its nuclear localization

    BEN domain-containing protein 3 (BEND3) has recently been reported to function as a heterochromatin-associated protein in transcriptional repression in the nucleus. BEND3 should have nuclear localization signals (NLSs) to localize to the nucleus in light of its molecular weight, which is higher than that allowed to pass through nuclear pore complexes. We here analyzed the subcellular localization of deletion/site-directed mutants of human BEND3 by an immunofluorescence assay in an attempt to identify the amino acids essential for its nuclear localization. We found that three basic amino acid residues located in the N-terminal region of BEND3 (BEND356–58, KRK) are essential, suggesting that these residues play a role as a functional NLS. These results provide valuable information for progressing research on BEND3. - Highlights: • BEND3 localizes to the nucleus. • The N-terminal 60 amino acids region of BEND3 contains NLS. • Amino acids located between 56 and 58 of BEND3 (KRK) are part of NLS. • KRK motif is highly conserved among BEND3 homologs

  8. Impact of arterial occlusion during partial nephrectomy on residual renal function. An evaluation with 99mtechnetium-dimercaptosuccinic acid scintigraphy

    Partial nephrectomy (PNx) has been performed with temporary renal arterial occlusion and in situ renal hypothermia (conventional PNx). However, the impact of temporary renal arterial occlusion on residual renal function has not been well assessed. To address this question, we performed renal scintigraphy with 99mtechnetium-dimercaptosuccinic acid (DMSA) for the quantitative measurement of postoperative residual renal function after conventional PNx and partial nephrectomy without arterial occlusion (non-clamping PNx). Thirty-four patients underwent postoperative DMSA scintigraphy after PNx for renal cell carcinoma. No obvious difference in preoperative renal function between the diseased kidney and the contralateral kidney was found in any of the patients. Of these patients, 24 underwent conventional PNx, and 10 underwent non-clamping PNx. Residual renal function was evaluated using the relative DMSA uptake of the operated kidney. The relative DMSA uptake of the operated kidney was 39.9±7.3% (25.1-58.8) after conventional PNx compared to 34.8±8.9% (13.5-45.5) after non-clamping PNx. This difference was not statistically significant (P=0.15). Total ischemic time during conventional PNx had no adverse influence on the residual renal function. In the analysis of the other determinant factors influencing residual renal function, tumor size was the only significant factor that inversely correlated with the relative DMSA uptake. Our results showed that arterial clamping during PNx has no negative impact on the functional residual capacity as long as in situ renal hypothermia is adequately performed. (author)

  9. Absence of acetohydroxy acid synthetase in a clinical isolate of Neisseria gonorrhoeae requiring isoleucine and valine.

    Lerner, S A; Friedman, E L; Dudek, E J; Kominski, G; Bohnhoff, M.; Morello, J A

    1980-01-01

    A clinical isolate of Neisseria gonorrhoeae with an unusual growth requirement for isoleucine and valine lacked the activity of acetohydroxy acid synthetase, one of the enzymes required for the biosynthesis of these amino acids. A spontaneous mutant which no longer required isoleucine and valine had acquired this enzymatic activity.

  10. Multiple roles of the extracellular vestibule amino acid residues in the function of the rat P2X4 receptor.

    Milos B Rokic

    Full Text Available The binding of ATP to trimeric P2X receptors (P2XR causes an enlargement of the receptor extracellular vestibule, leading to opening of the cation-selective transmembrane pore, but specific roles of vestibule amino acid residues in receptor activation have not been evaluated systematically. In this study, alanine or cysteine scanning mutagenesis of V47-V61 and F324-N338 sequences of rat P2X4R revealed that V49, Y54, Q55, F324, and G325 mutants were poorly responsive to ATP and trafficking was only affected by the V49 mutation. The Y54F and Y54W mutations, but not the Y54L mutation, rescued receptor function, suggesting that an aromatic residue is important at this position. Furthermore, the Y54A and Y54C receptor function was partially rescued by ivermectin, a positive allosteric modulator of P2X4R, suggesting a rightward shift in the potency of ATP to activate P2X4R. The Q55T, Q55N, Q55E, and Q55K mutations resulted in non-responsive receptors and only the Q55E mutant was ivermectin-sensitive. The F324L, F324Y, and F324W mutations also rescued receptor function partially or completely, ivermectin action on channel gating was preserved in all mutants, and changes in ATP responsiveness correlated with the hydrophobicity and side chain volume of the substituent. The G325P mutant had a normal response to ATP, suggesting that G325 is a flexible hinge. A topological analysis revealed that the G325 and F324 residues disrupt a β-sheet upon ATP binding. These results indicate multiple roles of the extracellular vestibule amino acid residues in the P2X4R function: the V49 residue is important for receptor trafficking to plasma membrane, the Y54 and Q55 residues play a critical role in channel gating and the F324 and G325 residues are critical for vestibule widening.

  11. Investigation of a substrate-specifying residue within Papaver somniferum and Catharanthus roseus aromatic amino acid decarboxylases.

    Torrens-Spence, Michael P; Lazear, Michael; von Guggenberg, Renee; Ding, Haizhen; Li, Jianyong

    2014-10-01

    Plant aromatic amino acid decarboxylases (AAADs) catalyze the decarboxylation of aromatic amino acids with either benzene or indole rings. Because the substrate selectivity of AAADs is intimately related to their physiological functions, primary sequence data and their differentiation could provide significant physiological insights. However, due to general high sequence identity, plant AAAD substrate specificities have been difficult to identify through primary sequence comparison. In this study, bioinformatic approaches were utilized to identify several active site residues within plant AAAD enzymes that may impact substrate specificity. Next a Papaver somniferum tyrosine decarboxylase (TyDC) was selected as a model to verify our putative substrate-dictating residues through mutation. Results indicated that mutagenesis of serine 372 to glycine enables the P. somniferum TyDC to use 5-hydroxytryptophan as a substrate, and reduces the enzyme activity toward 3,4-dihydroxy-L-phenylalanine (dopa). Additionally, the reverse mutation in a Catharanthus roseus tryptophan decarboxylase (TDC) enables the mutant enzyme to utilize tyrosine and dopa as substrates with a reduced affinity toward tryptophan. Molecular modeling and molecular docking of the P. somniferum TyDC and the C. roseus TDC enzymes provided a structural basis to explain alterations in substrate specificity. Identification of an active site residue that impacts substrate selectivity produces a primary sequence identifier that may help differentiate the indolic and phenolic substrate specificities of individual plant AAADs. PMID:25107664

  12. A conserved acidic patch in the Myb domain is required for activation of an endogenous target gene and for chromatin binding

    Chen Carolyn

    2008-10-01

    Full Text Available Abstract The c-Myb protein is a transcriptional regulator initially identified by homology to the v-Myb oncoprotein, and has since been implicated in human cancer. The most highly conserved portion of the c-Myb protein is the DNA-binding domain which consists of three imperfect repeats. Many other proteins contain one or more Myb-related domains, including a number of proteins that do not bind directly to DNA. We performed a phylogenetic analysis of diverse classes of Myb-related domains and discovered a highly conserved patch of acidic residues common to all Myb-related domains. These acidic residues are positioned in the first of three alpha-helices within each of the three repeats that comprise the c-Myb DNA-binding domain. Interestingly, these conserved acidic residues are present on a surface of the protein which is distinct from that which binds to DNA. Alanine mutagenesis revealed that the acidic patch of the third c-Myb repeat is essential for transcriptional activity, but neither for nuclear localization nor DNA-binding. Instead, these acidic residues are required for efficient chromatin binding and interaction with the histone H4 N-terminal tail.

  13. Two Arginine Residues of Streptococcus gordonii Sialic Acid-Binding Adhesin Hsa Are Essential for Interaction to Host Cell Receptors

    Urano-Tashiro, Yumiko; Takahashi, Yukihiro; Oguchi, Riyo; Konishi, Kiyoshi

    2016-01-01

    Hsa is a large, serine-rich protein of Streptococcus gordonii DL1 that mediates binding to α2-3-linked sialic acid termini of glycoproteins, including platelet glycoprotein Ibα, and erythrocyte membrane protein glycophorin A, and band 3. The binding of Hsa to platelet glycoprotein Ibα contributes to the pathogenesis of infective endocarditis. This interaction appears to be mediated by a second non-repetitive region (NR2) of Hsa. However, the molecular details of the interaction between the Hsa NR2 region and these glycoproteins are not well understood. In the present study, we identified the amino acid residues of the Hsa NR2 region that are involved in sialic acid recognition. To identify the sialic acid-binding site of Hsa NR2 region, we prepared various mutants of Hsa NR2 fused with glutathione transferase. Fusion proteins harboring Arg340 to Asn (R340N) or Arg365 to Asn (R365N) substitutions in the NR2 domain exhibited significantly reduced binding to human erythrocytes and platelets. A sugar-binding assay showed that these mutant proteins abolished binding to α2-3-linked sialic acid. Furthermore, we established S. gordonii DL1 derivatives that encoded the corresponding Hsa mutant protein. In whole-cell assays, these mutant strains showed significant reductions in hemagglutination, in platelet aggregation, and in adhesion to human leukocytes. These results indicate that the Arg340 and Arg365 residues of Hsa play an important role in the binding of Hsa to α2-3-linked sialic acid-containing glycoproteins. PMID:27101147

  14. Interactions between acidic proteins and crystals: stereochemical requirements in biomineralization.

    Addadi, L; Weiner, S

    1985-01-01

    Acidic matrix macromolecules are intimately involved in biological crystal growth. In vitro experiments, in which crystals of calcium dicarboxylate salts were grown in the presence of aspartic acid-rich proteins, revealed a stereochemical property common to all the interacting faces. Calcite crystals are nucleated on stereochemically analogous faces when proteins are adsorbed onto a rigid substrate. The importance of this property in biomineralization is discussed.

  15. Identification of functionally important amino acid residues in the mitochondria targeting sequence of Hepatitis B virus X protein

    Chronic hepatitis B virus (HBV) infection has been strongly associated with hepatocellular carcinoma (HCC) and the X protein (HBx) is thought to mediate the cellular changes associated with carcinogenesis. Recently, isolation of the hepatitis B virus integrants from HCC tissue by others have established the fact that the X gene is often truncated at its C-terminus. Expression of the GFP fusion proteins of HBx and its truncation mutants with a GFP tag in human liver cell-lines in this study revealed that the C-terminus of HBx is indispensable for its specific localization in the mitochondria. A crucial region of seven amino acids at the C-terminus has been mapped out in which the cysteine residue at position 115 serves as the most important residue for the subcellular localization. When cysteine 115 of HBx is mutated to alanine the mitochondria targeting property of HBx is abrogated

  16. Discrimination between acid and alkali-labile phosphorylated residues on Immobilon: phosphorylation studies of nucleoside diphosphate kinase

    Biondi, R M; Walz, K; Issinger, O G;

    1996-01-01

    We have critically analyzed current methodologies for distinguishing histidine and serine phosphorylated residues in proteins and report a simple technique that assures a reliable discrimination. Electro-transfer of a phosphorylated enzyme to Immobilon membranes and its treatment at pH 1 and 14 in...... buffers containing 5% methanol allows unambiguous distinction between serine/threonine and histidine phosphorylation (O-phosphomonoesters and phosphoramide, respectively) since under these conditions only one type of residue is dephosphorylated. The addition of 5% methanol to all buffers was indispensable...... to deplete phosphate from membranes incubated successively under acid and basic conditions. The technique was applied to the study of nucleoside diphosphate kinase (NDP kinase) phosphorylation. In this enzyme, autophosphorylation of active site histidine is an accepted intermediate step in the...

  17. Key amino acid residues for the endo-processive activity of GH74 xyloglucanase.

    Matsuzawa, Tomohiko; Saito, Yuji; Yaoi, Katsuro

    2014-05-01

    Unlike endo-dissociative-xyloglucanases, Paenibacillus XEG74 is an endo-processive xyloglucanase that contains four unique tryptophan residues in the negative subsites (W61 and W64) and the positive subsites (W318 and W319), as indicated by three-dimensional homology modelling. Selective replacement of the positive subsite residues with alanine mutations reduced the degree of processive activity and resulted in the more endo-dissociative-activity. The results showed that W318 and W319, which are found in the positive subsites, are essential for processive degradation and are responsible for maintaining binding interactions with xyloglucan polysaccharide through a stacking effect. PMID:24657616

  18. Studies on Models,Patterns and Require-ments of Digestible Amino Acids for Layers by Nitrogen Metabolism

    1999-01-01

    The nitrogen (N) metabolic experiments were made to estimate separately amino acid requirements of 43~48 weeks old layers for maintenance, for protein accretion to estabolish models to estimate digestible amino acid requirements. The regression relationship of nitrogen retention vs amino acid intake was estimated for each amino acid by giving, at rate of N intake of 0.91, 0.52, 0.15 and 0.007g.kg-1 body-weight (W0.75) per d, the semi-synthetic diets was made specially deficient in one amino acid. From the regression coefficients, it was calculated that, for the accretion of 1 g protein, the dietary digestible amino acid requirements were (mg) Thr 63.1, Val 100.4, Met 39.9, Ile 88.6, Leu 114.3, Phe 63.2, Lys 87.0, His 20.5, Arg 87.9, Trp 21.4, Met+Cys 77.6, and Phe+Tyr 114.3. Daily amino acid requirements for N equilibrium were estimated to be (mg.kg-1W0.75 per day) Thr 50.6, Val 74.7, Met 30.3, ILe 66.7 Leu 81.4, Phe 44.8, Lys 60.5 His 14.7, Arg 73.9 ,Trp 17.3, Met+Cys 58.6, and Phe+Tyr 83.9 The dietary degestible amino acid patterns for protein accretion and N equilibrium were also proposed. The models of estimating digestible amino acid requirements for the different productions were developed.

  19. Evidence for a Proton Transfer Network and a Required Persulfide-Bond-Forming Cysteine Residue in Ni-Containing Carbon Monoxide Dehydrogenases

    Eun Jin Kim; Jian Feng; Matthew R. Bramlett; Paul A. Lindahl

    2004-05-18

    OAK-B135 Carbon monoxide dehydrogenase from Moorella thermoacetica catalyzes the reversible oxidation of CO to CO2 at a nickel-iron-sulfur active-site called the C-cluster. Mutants of a proposed proton transfer pathway and of a cysteine residue recently found to form a persulfide bond with the C-cluster were characterized. Four semi-conserved histidine residues were individually mutated to alanine. His116 and His122 were essential to catalysis, while His113 and His119 attenuated catalysis but were not essential. Significant activity was ''rescued'' by a double mutant where His116 was replaced by Ala and His was also introduced at position 115. Activity was also rescued in double mutants where His122 was replaced by Ala and His was simultaneously introduced at either position 121 or 123. Activity was also ''rescued'' by replacing His with Cys at position 116. Mutation of conserved Lys587 near the C-cluster attenuated activity but did not eliminate it. Activity was virtually abolished in a double mutant where Lys587 and His113 were both changed to Ala. Mutations of conserved Asn284 also attenuated activity. These effects suggest the presence of a network of amino acid residues responsible for proton transfer rather than a single linear pathway. The Ser mutant of the persulfide-forming Cys316 was essentially inactive and displayed no EPR signals originating from the C-cluster. Electronic absorption and metal analysis suggests that the C-cluster is absent in this mutant. The persulfide bond appears to be essential for either the assembly or stability of the C-cluster, and/or for eliciting the redox chemistry of the C-cluster required for catalytic activity.

  20. Evidence for a Proton Transfer Network and a Required Persulfide-Bond-Forming Cysteine Residue in Ni-Containing Carbon Monoxide Dehydrogenases

    OAK-B135 Carbon monoxide dehydrogenase from Moorella thermoacetica catalyzes the reversible oxidation of CO to CO2 at a nickel-iron-sulfur active-site called the C-cluster. Mutants of a proposed proton transfer pathway and of a cysteine residue recently found to form a persulfide bond with the C-cluster were characterized. Four semi-conserved histidine residues were individually mutated to alanine. His116 and His122 were essential to catalysis, while His113 and His119 attenuated catalysis but were not essential. Significant activity was ''rescued'' by a double mutant where His116 was replaced by Ala and His was also introduced at position 115. Activity was also rescued in double mutants where His122 was replaced by Ala and His was simultaneously introduced at either position 121 or 123. Activity was also ''rescued'' by replacing His with Cys at position 116. Mutation of conserved Lys587 near the C-cluster attenuated activity but did not eliminate it. Activity was virtually abolished in a double mutant where Lys587 and His113 were both changed to Ala. Mutations of conserved Asn284 also attenuated activity. These effects suggest the presence of a network of amino acid residues responsible for proton transfer rather than a single linear pathway. The Ser mutant of the persulfide-forming Cys316 was essentially inactive and displayed no EPR signals originating from the C-cluster. Electronic absorption and metal analysis suggests that the C-cluster is absent in this mutant. The persulfide bond appears to be essential for either the assembly or stability of the C-cluster, and/or for eliciting the redox chemistry of the C-cluster required for catalytic activity

  1. Effects of the substitution of amino acid residues, through chemical synthesis, on the conformation and activity of antimicrobial peptides

    Regina C. Adão

    2012-06-01

    Full Text Available Antimicrobial peptides make up an assorted group of molecules which contain from 12 to 50 amino acid residues and which may be produced by microorganisms, plants and animals. From the discovery that these biomolecules are lethal to bacteria, inhibiting the pathogenic organism’s growth, and are also related to innate and adapted defense mechanisms, the investigation of such molecules came to be an emergent research field, in which more than 1800 antimicrobial peptides have so far been discovered throughout the last three decades. These molecules are potential representatives of a new generation of antibiotic agents and the main motivation for such use is their activity against a wide variety of pathogens, including Gram-positive and Gram-negative bacteria as well as fungi and viruses. An important class of comprising some of these peptides may be found in anurans, from which it has been isolated, a considerable number of antimicrobial peptides with diverse sequences and structures, including linear and dimeric ones. In this work monomeric chains (CH1 e CH2 of the heterodimeric antimicrobial peptide distinctin (isolated in 1999 from Phyllomedusa distincta anurans, as well as its mutated monomers (CH1-S and CH2-S and the heterodimer itself were synthesized. The distinctin is the peptide with two chains of different sequences (Table 1 bound each other by disulfide bond from the cystein residues constituting the heterodimer. To investigate the effects on the biological activity by amino acids substitution at normal distinctin CH1 and CH2 chains, both were synthesized as well as their similar chains (CH1-S and CH2-S in which the cystein (Fig.1 a residues of each chain were changed by serin residues (Fig. 1 b. The new chains were named mutants. The synthesis was carried out in solid phase, using Fmoc strategy. The heterodimer distinctin was obtained from CH1 and CH2 chains coupling through cystein residues air oxidation. The results from HPLC

  2. Simulation of acid hydrolysis of lignocellulosic residues to fermentable sugars for bioethanol production

    Sidiras, Dimitris

    2012-12-01

    The dilute acid hydrolysis of fir sawdust with sulfuric acid was undertaken in a batch reactor system (autoclave). The experimental data and reaction kinetic analysis indicate that this is a potential process for cellulose and hemicelluloses hydrolysis, due to a rapid hydrolysis reaction for acid concentration 0.045 N at 160-180°C. It was found that significant sugar degradation occurred at these conditions. The optimum conditions gave a yield of 38% total fermentable sugars. The kinetics of dilute acid hydrolysis of cellulose and hemicelluloses (polysaccharides) were simulated using four pseudo-kinetic models. The reaction rate constants were calculated in each case.

  3. DNA Three Way Junction Core Decorated with Amino Acids-Like Residues-Synthesis and Characterization †

    Claudia Addamiano

    2016-08-01

    Full Text Available Construction and physico-chemical behavior of DNA three way junction (3WJ functionalized by protein-like residues (imidazole, alcohol and carboxylic acid at unpaired positions at the core is described. One 5′-C(S-propargyl-thymidine nucleotide was specifically incorporated on each strand to react through a post synthetic CuACC reaction with either protected imidazolyl-, hydroxyl- or carboxyl-azide. Structural impacts of 5′-C(S-functionalization were investigated to evaluate how 3WJ flexibility/stability is affected.

  4. Reasoned opinion on the review of the existing maximum residue levels (MRLs for indolylacetic acid according to Article 12 of Regulation (EC No 396/2005

    European Food Safety Authority

    2014-07-01

    Full Text Available According to Article 12 of Regulation (EC No 396/2005, the European Food Safety Authority (EFSA has reviewed the Maximum Residue Levels (MRLs currently established at European level for the pesticide active substance indolylacetic acid. Considering that this active substance is no longer authorised within the European Union, that no MRLs are established by the Codex Alimentarius Commission, and that no import tolerances were notified to EFSA, residues of indolylacetic acid are not expected to occur in any plant or animal commodity. Available data were also not sufficient to derive a residue definition or a limit of quantification (LOQ for enforcement against potential illegal uses.

  5. Reasoned opinion on the review of the existing maximum residue levels (MRLs for indolylbutyric acid according to Article 12 of Regulation (EC No 396/2005

    European Food Safety Authority

    2014-07-01

    Full Text Available According to Article 12 of Regulation (EC No 396/2005, the European Food Safety Authority (EFSA has reviewed the Maximum Residue Levels (MRLs currently established at European level for the pesticide active substance indolylbutyric acid. Considering that this active substance is not authorised for use on edible crops within the European Union, that no MRLs are established by the Codex Alimentarius Commission, and that no import tolerances were notified to EFSA, residues of indolylbutyric acid are not expected to occur in any plant or animal commodity. Available data were also not sufficient to derive a residue definition or a limit of quantification (LOQ for enforcement against potential illegal uses.

  6. Optimization of a Nucleic Acids united-RESidue 2-Point model (NARES-2P) with a maximum-likelihood approach

    He, Yi; Scheraga, Harold A., E-mail: has5@cornell.edu [Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853 (United States); Liwo, Adam [Faculty of Chemistry, University of Gdańsk, Wita Stwosza 63, 80-308 Gdańsk (Poland)

    2015-12-28

    Coarse-grained models are useful tools to investigate the structural and thermodynamic properties of biomolecules. They are obtained by merging several atoms into one interaction site. Such simplified models try to capture as much as possible information of the original biomolecular system in all-atom representation but the resulting parameters of these coarse-grained force fields still need further optimization. In this paper, a force field optimization method, which is based on maximum-likelihood fitting of the simulated to the experimental conformational ensembles and least-squares fitting of the simulated to the experimental heat-capacity curves, is applied to optimize the Nucleic Acid united-RESidue 2-point (NARES-2P) model for coarse-grained simulations of nucleic acids recently developed in our laboratory. The optimized NARES-2P force field reproduces the structural and thermodynamic data of small DNA molecules much better than the original force field.

  7. Optimization of a Nucleic Acids united-RESidue 2-Point model (NARES-2P) with a maximum-likelihood approach

    Coarse-grained models are useful tools to investigate the structural and thermodynamic properties of biomolecules. They are obtained by merging several atoms into one interaction site. Such simplified models try to capture as much as possible information of the original biomolecular system in all-atom representation but the resulting parameters of these coarse-grained force fields still need further optimization. In this paper, a force field optimization method, which is based on maximum-likelihood fitting of the simulated to the experimental conformational ensembles and least-squares fitting of the simulated to the experimental heat-capacity curves, is applied to optimize the Nucleic Acid united-RESidue 2-point (NARES-2P) model for coarse-grained simulations of nucleic acids recently developed in our laboratory. The optimized NARES-2P force field reproduces the structural and thermodynamic data of small DNA molecules much better than the original force field

  8. Optimization of a Nucleic Acids united-RESidue 2-Point model (NARES-2P) with a maximum-likelihood approach

    He, Yi; Liwo, Adam; Scheraga, Harold A.

    2015-12-01

    Coarse-grained models are useful tools to investigate the structural and thermodynamic properties of biomolecules. They are obtained by merging several atoms into one interaction site. Such simplified models try to capture as much as possible information of the original biomolecular system in all-atom representation but the resulting parameters of these coarse-grained force fields still need further optimization. In this paper, a force field optimization method, which is based on maximum-likelihood fitting of the simulated to the experimental conformational ensembles and least-squares fitting of the simulated to the experimental heat-capacity curves, is applied to optimize the Nucleic Acid united-RESidue 2-point (NARES-2P) model for coarse-grained simulations of nucleic acids recently developed in our laboratory. The optimized NARES-2P force field reproduces the structural and thermodynamic data of small DNA molecules much better than the original force field.

  9. An Acidic Thermostable Recombinant Aspergillus nidulans Endoglucanase Is Active towards Distinct Agriculture Residues

    Marcio Jose Poças-Fonseca; Ildinete Silva-Pereira; Cynthia Maria Kyaw; Edivaldo Ximenes Ferreira Filho; Fabrícia Paula de Faria; Gilvan Caetano Duarte; Marciano Regis Rubini; Thiago Machado Mello-de-Sousa; Eveline Queiroz de Pinho Tavares

    2013-01-01

    Aspergillus nidulans is poorly exploited as a source of enzymes for lignocellulosic residues degradation for biotechnological purposes. This work describes the A. nidulans Endoglucanase A heterologous expression in Pichia pastoris, the purification and biochemical characterization of the recombinant enzyme. Active recombinant endoglucanase A (rEG A) was efficiently secreted as a 35 kDa protein which was purified through a two-step chromatography procedure. The highest enzyme activity was dete...

  10. Stabile Chlorine Isotope Study of Martian Shergottites and Nakhlites; Whole Rock and Acid Leachates and Residues

    Nakamura, N.; Nyquist, L. E.; Reese, Y.; Shih, C-Y; Fujitani, T.; Okano, O.

    2011-01-01

    We have established a precise analytical technique for stable chlorine isotope measurements of tiny planetary materials by TIMS (Thermal Ionization Mass Spectrometry) [1], for which the results are basically consistent with the IRMS tech-nique (gas source mass spectrometry) [2,3,4]. We present here results for Martian shergottites and nakhlites; whole rocks, HNO3-leachates and residues, and discuss the chlorine isotope evolution of planetary Mars.

  11. Radionuclide Leaching from Residual Solids Remaining after Acid Dissolution of K East Area Sludge Composite

    Laboratory tests were performed to examine the efficacy of various leach treatments for decontaminating dissolver residual solids (KEACRESID1) produced during a 24-hour dissolution of K East Basin floor and Weasel Pit sludge composite in boiling 6 M HNO3. The scope of this testing has been described in Section 4.5 of ''Testing Strategy to Support the Development of K Basin Sludge Treatment Process'' (Flament 1998). Radionuclides sorbed or associated with the residual solids generated in the K Basin sludge treatment process can restrict disposal of this solid to the Environmental Restoration Disposal Facility (ERDF). The starting dissolver residual solid for this testing, KEACRESID1, is a visibly heterogeneous material. This material contains radionuclides at concentrations above the ERDF Waste Acceptance Criteria for transuranics (TRU) by about a factor of 3, for 239Pu by a factor of 10, and for 241Am by a factor of 1.6. It meets the ERDF criterion for 137Cs by a factor of 4 and for uranium by a factor of 10. Therefore, the radionuclides of greatest interest in this leaching study are first 239Pu, and then 241Am, 137Cs, and uranium

  12. Improving volatile fatty acids production by exploiting the residual substrates in post-fermented sludge: Protease catalysis of refractory protein.

    Yin, Bo; Liu, Hongbo; Wang, Yuanyuan; Bai, Jie; Liu, He; Fu, Bo

    2016-03-01

    The real cause to the low yield of volatile fatty acids (VFAs), from inhibition or low biodegradation, is uncertain in sludge anaerobic fermentation. In this study, poor biodegradability of proteins and fast decrease of the indigenous hydrolase activity in the residual post-fermented sludge were found to be the major reasons. With the addition of trypsin or alkaline protease in residual post-fermented sludge after primary alkaline fermentation, degradation efficiency of refractory protein increased by 33.6% and 34.8%, respectively. Accordingly, the VFAs yields were improved by 69.7% and 106.1%, respectively. Furthermore, the activities of added trypsin and alkaline protease could maintain at 13.52 U/mL and 19.11 U/mL in the alkaline fermentation process. This study demonstrated that exploiting the refractory proteins in residual post-fermented sludge by protease addition seems to be a very promising way for improving VFAs yield of conventional alkaline fermentations with waste activated sludge. PMID:26722812

  13. Amino acid residues important for substrate specificity of the amino acid permeases Can I p and Gnp I p in Saccharomyces cerevisiae

    Regenberg, Birgitte; Kielland-Brandt, M.C.

    2001-01-01

    mutations affecting six predicted domains (helices III and X, and loops 1. 2, 6 and 7) of the permeases. Helix III and loop 7 are candidates for domains in direct contact with the transported amino acid. Helix III was affected in both CAN1 (Y173H, Y173D) and GNP1 (W239C) mutants and has previously been...... found to be important for substrate preference in other members of the family. Furthermore, the mutations affecting loop 7 (residue T354, S355, Y356) are close to a glutamate side chain (E367) potentially interacting with the positively charged substrate, a notion supported by conservation of the side......Deletion of the general amino acid permease gene GAP1 abolishes uptake of L-citrulline in Saccharomyces cerevisiae, resulting in the inability to grow on L-citrulline as sole nitrogen source. Selection for suppressor mutants that restored growth on L-citrulline led to isolation of 21 mutations in...

  14. Identification of Two Tyrosine Residues Required for the Intramolecular Mechanism Implicated in GIT1 Activation

    Antonio Totaro; Veronica Astro; Diletta Tonoli; Ivan de Curtis

    2014-01-01

    GIT1 is an ArfGAP and scaffolding protein regulating cell adhesion and migration. The multidomain structure of GIT1 allows the interaction with several partners. Binding of GIT1 to some of its partners requires activation of the GIT1 polypeptide. Our previous studies indicated that binding of paxillin to GIT1 is enhanced by release of an intramolecular interaction between the amino-terminal and carboxy-terminal portions that keeps the protein in a binding-incompetent state. Here we have addre...

  15. Residual mitochondrial transmembrane potential decreases unsaturated fatty acid level in sake yeast during alcoholic fermentation

    Sawada, Kazutaka; Kitagaki, Hiroshi

    2016-01-01

    Oxygen, a key nutrient in alcoholic fermentation, is rapidly depleted during this process. Several pathways of oxygen utilization have been reported in the yeast Saccharomyces cerevisiae during alcoholic fermentation, namely synthesis of unsaturated fatty acid, sterols and heme, and the mitochondrial electron transport chain. However, the interaction between these pathways has not been investigated. In this study, we showed that the major proportion of unsaturated fatty acids of ester-linked ...

  16. Replacing 32 Proline Residues by a Noncanonical Amino Acid Results in a Highly Active DNA Polymerase

    Holzberger, Bastian; Marx, Andreas

    2010-01-01

    Protein engineering may be achieved by rational design, directed evolution-based methods, or computational protein design. Mostly these methods make recourse to the restricted pool of the 20 natural amino acids. With the ability to introduce different new kinds of functionalities into proteins, the use of noncanonical amino acids became a promising new method in protein engineering. Here, we report on the generation of a multifluorinated DNA polymerase. DNA polymerases are highly dynamic enzy...

  17. JAK2 V617F constitutive activation requires JH2 residue F595: a pseudokinase domain target for specific inhibitors.

    Alexandra Dusa

    Full Text Available The JAK2 V617F mutation present in over 95% of Polycythemia Vera patients and in 50% of Essential Thrombocythemia and Primary Myelofibrosis patients renders the kinase constitutively active. In the absence of a three-dimensional structure for the full-length protein, the mechanism of activation of JAK2 V617F has remained elusive. In this study, we used functional mutagenesis to investigate the involvement of the JH2 alphaC helix in the constitutive activation of JAK2 V617F. We show that residue F595, located in the middle of the alphaC helix of JH2, is indispensable for the constitutive activity of JAK2 V617F. Mutation of F595 to Ala, Lys, Val or Ile significantly decreases the constitutive activity of JAK2 V617F, but F595W and F595Y are able to restore it, implying an aromaticity requirement at position 595. Substitution of F595 to Ala was also able to decrease the constitutive activity of two other JAK2 mutants, T875N and R683G, as well as JAK2 K539L, albeit to a lower extent. In contrast, the F595 mutants are activated by erythropoietin-bound EpoR. We also explored the relationship between the dimeric conformation of EpoR and several JAK2 mutants. Since residue F595 is crucial to the constitutive activation of JAK2 V617F but not to initiation of JAK2 activation by cytokines, we suggest that small molecules that target the region around this residue might specifically block oncogenic JAK2 and spare JAK2 wild-type.

  18. CHOICE FEEDING AND AMINO ACID REQUIREMENTS FOR BROILERS

    B. Indarsih; R.A.E.Pym

    2011-01-01

    The study was conducted as a completely randomized design, with a factorial arrangement to determine the response of commercial broilers to choice feeding and limiting amino acids on growth and carcass performance. A total of 432 male birds were weighed at one-d-old and randomly distributed to 48 wire-floored brooder cage each 1.0 m2. There were 2 sexes and 4 dietary treatments with 6 replicates each of 9 birds. Birds were given one of three dietary regimens with dietary change every 7 days. ...

  19. Waste management from reprocessing: a stringent regulatory requirements for high quality conditioned residues

    Nuclear waste production and management in France is governed by safety requirements imposed to all operators. French nuclear safety relies on two basic principles: · Responsibility of the nuclear operator, which expands to waste generated, · Safety basic objectives issued by national Safety Authority. For a long time the regulatory framework for waste production and management has been satisfactorily applied and has benefited to each actor of the process. LLW/MLW and HLW nuclear waste are currently conditioned in safe matrices or packages either likely to be disposed in surface repositories or designed with the intention to be disposed underground according to their radioactive content. France is looking into the case of VLLW and has already carried out a design for future disposal, the design being in the pipe. Other types of waste (i. e. radium bearing waste, graphite, and tritium content waste) are also considered in the whole framework of French waste management. (author)

  20. Asthma induction in mice leads to appearance of alpha2-3- and alpha2-6-linked sialic acid residues in respiratory goblet-like cells

    Kirkeby, Svend; Jensen, Niels-Erik Viby; Mandel, Ulla;

    2008-01-01

    demonstrate binding of lectins and antibodies that detect alpha2-3- and alpha2-6-linked sialic acid residues. After sensitization and challenge, the histology of the lung changed markedly, and goblet-like cells appeared, most likely caused by Clara cell metaplasia. Normal Clara cells showed no reaction after...... incubation with the sialic acid detecting agents, while the goblet-like cells expressed both alpha2-3- and alpha2-6-linked sialic acid residues in the asthmatic animals. The lectins but not the antibodies reacted with intestinal goblet cells. Instead, an antibody recognizing a disialoganglioside, stained...

  1. The cysteine, total sulfur amino acid, tyrosine, phenylalanine + tyrosine, and non-essential amino acid maintenance requirements of broiler breeders.

    Ekmay, R D; Mei, S J; Sakomura, N K; Coon, C N

    2016-06-01

    Two hundred and fifty Cobb-Vantress broiler breeders were used to determine the maintenance requirement and efficiency of utilization of dietary Cys, Tyr, and non-essential amino acids (AA) in a 21-day experiment. The breeders were fed crystalline amino acid diets containing graded levels of Cys or Tyr representing 0, 10, 20, 30, and 40% of their suggested requirement level with all other amino acids maintained at 40% of their suggested requirement level. To determine the non-essential AA maintenance requirement, graded levels of non-essential AA were provided by glutamic acid to represent 12, 19, 26, 33, and 40% of the ideal level of glutamic acid with all other amino acids maintained at their maintenance requirement level. The total sulfur amino acid (TSAA) and Phe + Tyr requirements were calculated by combining Cys and Tyr results, respectively, with previously determined Met and Phe, respectively. The slope of Cys, Tyr, and non-essential AA accretion regression line indicated that 29% Cys, 24% TSAA, 21% Tyr, 20% Phe + Tyr, and 9% non-essential AA of crystalline amino acids were retained. The Cys requirement for zero protein accretion was calculated to be 30.48 mg/d or 17.006 mg/ kgBW(0.75)/d or 75.426 mg/kgCP/d. The TSAA requirement for zero accretion was calculated to be 132.25 mg/b/d, 71.48 mg/kgBW(0.75)/d, and 307.55 mg/kgCP/d. The Tyr requirement for zero protein accretion was calculated to be 65.907 mg/d or 37.233 mg/ kgBW(0.75)/d or 175.566 mg/kgCP/d. The Phe + Tyr requirement for zero protein accretion was calculated to be 352.18 mg/b/d, 190.37 mg/kgBW(0.75)/d, and 749.33 mg/kgCP/d. The non-essential AA requirement for zero protein accretion was calculated to be 3715.194 mg/d or 2003.155 mg/kgBW(0.75)/d or 9452.954 mg/kgCP/d. PMID:26994191

  2. Estimates of methionine and sulfur amino acid requirements for laying hens using different models

    AA Saki; R Naseri Harsini; MM Tabatabaei; Zamani, P.; M Haghight

    2012-01-01

    This experiment was conducted to evaluate the effects of dietary methionine (Met) content on the performance of white commercial laying hens and to determine Met and total sulfur amino acids requirements (TSAA). These requirements were estimated using three statistical models (broken-line regression, exponential and second order equations) to evaluate their abilit to determine amino acid requirements. A total of 216 laying hens (23 wks of age) was used in a completely randomized design (CRD) ...

  3. Lysophosphatidic Acid Signaling through the Lysophosphatidic Acid-1 Receptor Is Required for Alveolarization.

    Funke, Manuela; Knudsen, Lars; Lagares, David; Ebener, Simone; Probst, Clemens K; Fontaine, Benjamin A; Franklin, Alicia; Kellner, Manuela; Kühnel, Mark; Matthieu, Stephanie; Grothausmann, Roman; Chun, Jerold; Roberts, Jesse D; Ochs, Matthias; Tager, Andrew M

    2016-07-01

    Lysophosphatidic acid (LPA) signaling through one of its receptors, LPA1, contributes to both the development and the pathological remodeling after injury of many organs. Because we found previously that LPA-LPA1 signaling contributes to pulmonary fibrosis, here we investigated whether this pathway is also involved in lung development. Quantitative assessment of lung architecture of LPA1-deficient knock-out (KO) and wild-type (WT) mice at 3, 12, and 24 weeks of age using design-based stereology suggested the presence of an alveolarization defect in LPA1 KO mice at 3 weeks, which persisted as alveolar numbers increased in WT mice into adulthood. Across the ages examined, the lungs of LPA1 KO mice exhibited decreased alveolar numbers, septal tissue volumes, and surface areas, and increased volumes of the distal airspaces. Elastic fibers, critical to the development of alveolar septa, appeared less organized and condensed and more discontinuous in KO alveoli starting at P4. Tropoelastin messenger RNA expression was decreased in KO lungs, whereas expression of matrix metalloproteinases degrading elastic fibers was either decreased or unchanged. These results are consistent with the abnormal lung phenotype of LPA1 KO mice, being attributable to reduced alveolar septal formation during development, rather than to increased septal destruction as occurs in the emphysema of chronic obstructive pulmonary disease. Peripheral septal fibroblasts and myofibroblasts, which direct septation in late alveolarization, demonstrated reduced production of tropoelastin and matrix metalloproteinases, and diminished LPA-induced migration, when isolated from LPA1 KO mice. Taken together, our data suggest that LPA-LPA1 signaling is critically required for septation during alveolarization. PMID:27082727

  4. Models of protein and amino acid requirements for cattle

    Luis Orlindo Tedeschi

    2015-03-01

    Full Text Available Protein supply and requirements by ruminants have been studied for more than a century. These studies led to the accumulation of lots of scientific information about digestion and metabolism of protein by ruminants as well as the characterization of the dietary protein in order to maximize animal performance. During the 1980s and 1990s, when computers became more accessible and powerful, scientists began to conceptualize and develop mathematical nutrition models, and to program them into computers to assist with ration balancing and formulation for domesticated ruminants, specifically dairy and beef cattle. The most commonly known nutrition models developed during this period were the National Research Council (NRC in the United States, Agricultural Research Council (ARC in the United Kingdom, Institut National de la Recherche Agronomique (INRA in France, and the Commonwealth Scientific and Industrial Research Organization (CSIRO in Australia. Others were derivative works from these models with different degrees of modifications in the supply or requirement calculations, and the modeling nature (e.g., static or dynamic, mechanistic, or deterministic. Circa 1990s, most models adopted the metabolizable protein (MP system over the crude protein (CP and digestible CP systems to estimate supply of MP and the factorial system to calculate MP required by the animal. The MP system included two portions of protein (i.e., the rumen-undegraded dietary CP - RUP - and the contributions of microbial CP - MCP as the main sources of MP for the animal. Some models would explicitly account for the impact of dry matter intake (DMI on the MP required for maintenance (MPm; e.g., Cornell Net Carbohydrate and Protein System - CNCPS, the Dutch system - DVE/OEB, while others would simply account for scurf, urinary, metabolic fecal, and endogenous contributions independently of DMI. All models included milk yield and its components in estimating MP required for lactation

  5. Site-directed mutagenesis of the heterotrimeric killer toxin zymocin identifies residues required for early steps in toxin action.

    Wemhoff, Sabrina; Klassen, Roland; Meinhardt, Friedhelm

    2014-10-01

    Zymocin is a Kluyveromyces lactis protein toxin composed of αβγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αβ-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activity in vitro that might be important during γ import. Saccharomyces cerevisiae strains carrying k1-derived hybrid elements deficient in either αβ (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α's chitinase domain and the sole cysteine residue of β (Cys250). Since βγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of β C250 and γ C231 in zymocin assembly. To test the capability of αβ to carry alternative cargos, the heterologous ACNase from Pichia acaciae (P. acaciae Orf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αβ's capability to deliver other cargo proteins into target cells. PMID:25128337

  6. Models of protein and amino acid requirements for cattle

    Luis Orlindo Tedeschi; Danny Gene Fox; Mozart Alves Fonseca; Luigi Francis Lima Cavalcanti

    2015-01-01

    Protein supply and requirements by ruminants have been studied for more than a century. These studies led to the accumulation of lots of scientific information about digestion and metabolism of protein by ruminants as well as the characterization of the dietary protein in order to maximize animal performance. During the 1980s and 1990s, when computers became more accessible and powerful, scientists began to conceptualize and develop mathematical nutrition models, and to program them into comp...

  7. Identification of Ourmiavirus 30K movement protein amino acid residues involved in symptomatology, viral movement, subcellular localization and tubule formation.

    Margaria, Paolo; Anderson, Charles T; Turina, Massimo; Rosa, Cristina

    2016-09-01

    Several plant viruses encode movement proteins (MPs) classified in the 30K superfamily. Despite a great functional diversity, alignment analysis of MP sequences belonging to the 30K superfamily revealed the presence of a central core region, including amino acids potentially critical for MP structure and functionality. We performed alanine-scanning mutagenesis of the Ourmia melon virus (OuMV) MP, and studied the effects of amino acid substitutions on MP properties and virus infection. We identified five OuMV mutants that were impaired in systemic infection in Nicotiana benthamiana and Arabidopsis thaliana, and two mutants showing necrosis and pronounced mosaic symptoms, respectively, in N. benthamiana. Green fluorescent protein fusion constructs (GFP:MP) of movement-defective MP alleles failed to localize in distinct foci at the cell wall, whereas a GFP fusion with wild-type MP (GFP:MPwt) mainly co-localized with plasmodesmata and accumulated at the periphery of epidermal cells. The movement-defective mutants also failed to produce tubular protrusions in protoplasts isolated from infected leaves, suggesting a link between tubule formation and the ability of OuMV to move. In addition to providing data to support the importance of specific amino acids for OuMV MP functionality, we predict that these conserved residues might be critical for the correct folding and/or function of the MP of other viral species in the 30K superfamily. PMID:26637973

  8. Distinguishing of Ile/Leu amino acid residues in the PP3 protein by (hot) electron capture dissociation in Fourier transform ion cyclotron resonance mass spectrometry.

    Kjeldsen, Frank; Haselmann, Kim F; Sørensen, Esben S; Zubarev, Roman A

    2003-03-15

    In hot electron capture dissociation (HECD), multiply protonated polypeptides fragment upon capturing approximately 11-eV electrons. The excess of energy upon the primary c, z* cleavage induces secondary fragmentation in z* fragments. The resultant w ions allow one to distinguish between the isomeric Ile and Leu residues. The analytical utility of HECD is evaluated using tryptic peptides from the bovine milk protein PP3 containing totally 135 amino acid residues. Using a formal procedure for Ile/Leu (Xle) residue assignment, the identities of 20 out of 25 Xle residues (80%) were determined. The identity of an additional two residues could be correctly guessed from the absence of the alternative w ions, and only two residues, for which neither expected nor alternative w ions were observed, remained unassigned. Reinspection of conventional ECD spectra also revealed the presence of Xle w ions, although at lower abundances, with 44% of all Xle residues distinguished. Using a dispenser cathode as an electron source, identification of four out of five Xle residues in a 2.7-kDa peptide was possible with one acquisition 2 s long, with identification of all five residues by averaging of five such acquisitions. Unlike the case of high-energy collision-induced dissociation, no d ions were observed in the HECD of tryptic peptides. PMID:12659185

  9. Radiation-induced crosslinking between poly(deoxyadenylic-deoxythymidylic acid) and tripeptides containing aromatic residues

    OH radical-induced covalent peptide-nucleotide adducts have been isolated by reverse-phase chromatography from the enzymic hydrolyzates of gamma-ray irradiated solutions containing double-stranded poly(deoxyadenylic-deoxythymidylic acid) and one of the tripeptides, lysyl-tryptophyl-lysine or lysyl-tyrosyl-lysine. Numerous compounds were formed, resulting presumably from different modes of radical addition. All isomers appeared to have the same general structure peptide-d(ApTpA), based mostly on double-labelling experiments of bases and phosphate groups in DNA. The major adduct fraction obtained from Lys-Trp-Lys and poly(dA-dT) was purified to homogeneity by sequential reverse-phase and ion-exchange chromatography, and characterized spectrally. The pattern of acid and alkaline hydrolysis suggests that thymine is the site of peptide-nucleotide binding in this particular adduct fraction. (author)

  10. Enzymatic Conversion of Xylan Residues from Dilute Acid-Pretreated Corn Stover

    Shekiro, Joseph; Kuhn, Erik M; Selig, Michael J; Nagle, Nicholas J; Decker, Stephen R.; Elander, Richard T

    2012-01-01

    Enzymatic conversion of oligomeric xylose and insoluble xylan remaining after effective pretreatment offers significant potential to improve xylan-to-xylose yields while minimizing yields of degredation products and fermentation inhibitors. In this work, a commercial enzyme cocktail is demonstrated to convert up to 70 % of xylo-oligomers found in dilute acid-pretreated hydrolyzate liquor at varying levels of dilution when supplemented with accessory enzymes targeting common side chains. Comme...

  11. Required catalytic properties for alkane production from carboxylic acids: Hydrodeoxygenation of acetic acid

    Zhong; He; Xianqin; Wang

    2013-01-01

    The supported Pt catalysts(1 wt%)were prepared by the incipient impregnation method and analyzed using synchrotron-based X-ray diffraction,BET surface area,oxygen adsorption,CO pulse chemisorption,temperature-programmed desorption(TPD)of acetic acid,H2-TPD,NH3-TPD,O2-TPD,and H2-TPR.The reactivity of Pt-based catalysts was studied using a fixed bed reactor at 300 C and 4 MPa for hydrodeoxygenation of acetic acid,where Pt/TiO2 was very selective for ethane production.TPD experiments revealed that several conditions must be satisfied to achieve this high selectivity to ethane from acetic acid,such as Pt sites,moderate acidity,and medium metal-oxygen bond strength in the oxide support.This work provides insights in developing novel catalytic materials for hydrocarbon productions from various organics including bio-fuels.

  12. Purification, cloning, characterization and essential amino acid residues analysis of a new ι-carrageenase from Cellulophaga sp. QY3.

    Su Ma

    Full Text Available ι-Carrageenases belong to family 82 of glycoside hydrolases that degrade sulfated galactans in the red algae known as ι-carrageenans. The catalytic mechanism and some substrate-binding residues of family GH82 have been studied but the substrate recognition and binding mechanism of this family have not been fully elucidated. We report here the purification, cloning and characterization of a new ι-carrageenase CgiA_Ce from the marine bacterium Cellulophaga sp. QY3. CgiA_Ce was the most thermostable carrageenase described so far. It was most active at 50°C and pH 7.0 and retained more than 70% of the original activity after incubation at 50°C for 1 h at pH 7.0 or at pH 5.0-10.6 for 24 h. CgiA_Ce was an endo-type ι-carrageenase; it cleaved ι-carrageenan yielding neo-ι-carrabiose and neo-ι-carratetraose as the main end products, and neo-ι-carrahexaose was the minimum substrate. Sequence analysis and structure modeling showed that CgiA_Ce is indeed a new member of family GH82. Moreover, sequence analysis of ι-carrageenases revealed that the amino acid residues at subsites -1 and +1 were more conserved than those at other subsites. Site-directed mutagenesis followed by kinetic analysis identified three strictly conserved residues at subsites -1 and +1 of ι-carrageenases, G228, Y229 and R254 in CgiA_Ce, which played important roles for substrate binding. Furthermore, our results suggested that Y229 and R254 in CgiA_Ce interacted specifically with the sulfate groups of the sugar moieties located at subsites -1 and +1, shedding light on the mechanism of ι-carrageenan recognition in the family GH82.

  13. Branched chain amino acids requirements and metabolism in pigs

    Assadi Soumeh, Elham

    2015-01-01

    investigation. A non-targeted metabolomics study was thereafter conducted in order to screen the bio-fluids of pigs for discriminating metabolites and identify biomarkers of BCAA, when the pigs were fed the optimum level of BCAA for the highest growth performance. The results of the Ile dose-response study...... requirements at 0.93. Metabolomics, one of the last “-omics”, is a global analysis and interpretation of metabolome in specific health or nutritional status. Non-targeted metabolomics is used for screening the metabolic profile, and the metabolic signature could be used for hypothesis generation. The results...... of a non-targeted LC-MS metabolomics approach in the current study provided novel knowledge of the metabolic response of pigs to increasing dietary BCAA and enabled us to identify the biomarkers of BCAA in plasma and urine of pigs when fed the optimum dietary Ile, Val, and Leu for the highest growth...

  14. Identification of essential amino-acid residues in Azotobacter vinelandii isocitrate dehydrogenase by radical anions and H atoms

    Pure TPN+-specific isocitrate dehydrogenase from Azotobacter vinelandii was irradiated with H atoms generated in a γ-irradiated solution at pH 6.5. A G(-activity) = 0.12 +- 0.01 was found. At the same time no corresponding loss in free sulfhydryls was observed. These results confirmed the essentiality of methionine for the enzymatic activity as known from previous studies. Irradiation with the radical anions, (CNS)2- and Br2- generated in γ-irradiated solutions at pH 6.5, strongly inactivated isocitrate dehydrogenase with yields of G(-activity) of 2.1 and 3.9, respectively. Part of the inactivating effect, however, is due to oxidation of sulfhydryl groups. These results lead to the conclusion that tryptophan is an essential amino-acid residue to isocitrate dehydrogenase from A. vinelandii. The presence of tryptophan in the enzyme was demonstrated by pulse radiolysis

  15. Sialic Acids on Varicella-Zoster Virus Glycoprotein B Are Required for Cell-Cell Fusion.

    Suenaga, Tadahiro; Matsumoto, Maki; Arisawa, Fuminori; Kohyama, Masako; Hirayasu, Kouyuki; Mori, Yasuko; Arase, Hisashi

    2015-08-01

    Varicella-zoster virus (VZV) is a member of the human Herpesvirus family that causes varicella (chicken pox) and zoster (shingles). VZV latently infects sensory ganglia and is also responsible for encephalomyelitis. Myelin-associated glycoprotein (MAG), a member of the sialic acid (SA)-binding immunoglobulin-like lectin family, is mainly expressed in neural tissues. VZV glycoprotein B (gB) associates with MAG and mediates membrane fusion during VZV entry into host cells. The SA requirements of MAG when associating with its ligands vary depending on the specific ligand, but it is unclear whether the SAs on gB are involved in the association with MAG. In this study, we found that SAs on gB are essential for the association with MAG as well as for membrane fusion during VZV infection. MAG with a point mutation in the SA-binding site did not bind to gB and did not mediate cell-cell fusion or VZV entry. Cell-cell fusion and VZV entry mediated by the gB-MAG interaction were blocked by sialidase treatment. N-glycosylation or O-glycosylation inhibitors also inhibited the fusion and entry mediated by gB-MAG interaction. Furthermore, gB with mutations in N-glycosylation sites, i.e. asparagine residues 557 and 686, did not associate with MAG, and the cell-cell fusion efficiency was low. Fusion between the viral envelope and cellular membrane is essential for host cell entry by herpesviruses. Therefore, these results suggest that SAs on gB play important roles in MAG-mediated VZV infection. PMID:26105052

  16. Determination of cyanuric acid residues in catfish, trout, tilapia, salmon and shrimp by liquid chromatography-tandem mass spectrometry

    In May 2007, investigators discovered that waste material from the pet food manufacturing process contaminated with melamine (MEL) and/or cyanuric acid (CYA) had been added to hog and chicken feeds. At this time, investigators also learned that adulterated wheat gluten had been used in the manufacture of aquaculture feeds. Concern that the contaminated feed had been used in aquaculture and could enter the human food supply prompted the development of a method for the determination of CYA residues in the edible tissues of fish and shrimp. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed as a sensitive technique for the analysis of CYA in catfish, tilapia, salmon, trout and shrimp tissue. CYA was extracted from ground fish or shrimp with an acetic acid solution, defatted with hexane, and isolated with a graphitic carbon black solid-phase extraction column. Residues were separated from matrix components using a porous graphitic carbon LC column, and then analyzed with electrospray ionization in negative ion mode on a triple quadrupole mass spectrometer. Selective reaction monitoring was performed on the [M-H]-m/z 128 ion resulting in the product ions m/z 85 and 42. Recoveries from catfish, tilapia and trout fortified with 10-100 μg kg-1 of CYA averaged 67% with a relative standard deviation (R.S.D.) of 18% (n = 107). The average method detection limit (MDL) for catfish, tilapia and trout is 3.5 μg kg-1. An internal standard, 13C3-labeled CYA, was used in the salmon and shrimp extractions. Average recovery of CYA from salmon was 91% (R.S.D. = 15%, n = 18) with an MDL of 7.4 μg kg-1. Average recovery of CYA from shrimp was 85% (R.S.D. = 10%, n = 13) with an MDL of 3.5 μg kg-1

  17. 40 CFR 74.3 - Relationship to the Acid Rain program requirements.

    2010-07-01

    ... 40 Protection of Environment 16 2010-07-01 2010-07-01 false Relationship to the Acid Rain program requirements. 74.3 Section 74.3 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) SULFUR DIOXIDE OPT-INS Background and Summary § 74.3 Relationship to the Acid...

  18. Thyroid hormone requirement for retinoic acid induction of mouse mammary tumor virus expression.

    Bolander, F F; Blackstone, M E

    1990-01-01

    In normal mouse mammary epithelium, insulin, cortisol, and prolactin are absolute requirements for mouse mammary tumor virus expression. Retinoic acid further increased mouse mammary tumor virus expression two- to threefold but only when triiodothyronine was also present; neither retinoic acid nor triiodothyronine alone had any effect.

  19. 40 CFR 180.1178 - Formic acid; exemption from the requirement of a tolerance.

    2010-07-01

    ... pesticide formic acid is exempted from the requirement of a tolerance in or on honey and honeycomb when used to control tracheal mites and suppress varroa mites in bee colonies, and applied in accordance...

  20. Just three water molecules can trigger the undesired nonenzymatic reactions of aspartic acid residues: new insight from a quantum-chemical study

    Aspartic acid (Asp) residues in peptides and proteins (L-Asp) can undergo spontaneous, nonenzymatic reactions under physiological conditions by which abnormal L-β-Asp, D-Asp, and/or D-β-Asp residues are formed. These altered Asp residues may affect the three-dimensional structures of the peptides and proteins and hence their properties and functions. In fact, the altered Asp residues are relevant to age-related diseases such as cataract and Alzheimer's disease. Most of the above reactions of the L-Asp residue proceed via a cyclic succinimide intermediate. In this paper, I propose a detailed mechanism of cyclization of an Asp residue (forming a precursor of the succinimide) by the B3LYP/6-31+G(d,p) density functional theory calculations carried out for a small Asp-containing model compound complexed with three water molecules which act as general acid-base catalysts in proton transfers. In the proposed mechanism, the amide group on the C-terminal side of the Asp residue is first converted to the tautomeric iminol form. Then, successive reorientation of a water molecule and conformational change occur followed by the nucleophilic attack of the iminol nitrogen atom on the carboxyl carbon atom of the Asp side chain to form a five-membered ring. A satisfactory agreement was obtained between the calculated and experimental energetics

  1. Further evidence for the association of distinct amino acid residues with in vitro and in vivo growth of infectious bursal disease virus.

    Noor, M; Mahmud, M S; Ghose, P R; Roy, U; Nooruzzaman, M; Chowdhury, E H; Das, P M; Islam, M R; Müller, H

    2014-04-01

    A cell-culture-adapted reverse genetics strain of very virulent infectious bursal disease virus (IBDV) of chickens, designated as BD-3tcC, having four amino acid substitutions (Gln253His, Asp279Asn, Ala284Thr and Ser330Arg) in the capsid protein VP2 was tested for its genetic stability during serial passage in chickens and chicken embryo fibroblast (CEF) cell culture. Results of in vitro and in vivo experiments demonstrated that all four introduced mutations in BD-3tcC remained stable during serial passage in CEF cell culture, but during passage in chickens, amino acid residues at position 253 and 284 reverted from histidine to glutamine and threonine to alanine, respectively. In a parallel experiment, the same substitutions also occurred in a conventionally attenuated vaccine strain D-78 on serial passage in chickens. However, no reversion or substitution took place at positions 279 and 330 during in vivo passage of the mutant virus BD-3tcC or vaccine virus D-78. The findings provide conclusive evidence that while IBDV requires histidine and threonine at positions 253 and 284 for cell culture adaptation, glutamine and alanine at these positions are selected preferentially during in vivo replication. PMID:24136723

  2. Dilute Sulfuric Acid Pretreatment of Agricultural and Agro-Industrial Residues for Ethanol Production

    Martin, Carlos; Alriksson, Björn; Sjöde, Anders; Nilvebrant, Nils-Olof; Jönsson, Leif J.

    The potential of dilute-acid prehydrolysis as a pretreatment method for sugarcane bagasse, rice hulls, peanut shells, and cassava stalks was investigated. The prehydrolysis was performed at 122°C during 20, 40, or 60 min using 2% H2SO4 at a solid-to-liquid ratio of 1∶10. Sugar formation increased with increasing reaction time. Xylose, glucose, arabinose, and galactose were detected in all of the prehydrolysates, whereas mannose was found only in the prehydrolysates of peanut shells and cassava stalks. The hemicelluloses of bagasse were hydrolyzed to a high-extent yielding concentrations of xylose and arabinose of 19.1 and 2.2 g/L, respectively, and a xylan conversion of more than 80%. High-glucose concentrations (26-33.5 g/L) were found in the prehydrolysates of rice hulls, probably because of hydrolysis of starch of grain remains in the hulls. Peanut shells and cassava stalks rendered low amounts of sugars on prehydrolysis, indicating that the conditions were not severe enough to hydrolyze the hemicelluloses in these materials quantitatively. All prehydrolysates were readily fermentable by Saccharomyces cerevisiae. The dilute-acid prehydrolysis resulted in a 2.7-to 3.7-fold increase of the enzymatic convertibility of bagasse, but was not efficient for improving the enzymatic hydrolysis of peanut shells, cassava stalks, or rice hulls.

  3. ACID EVAPORATION OF ULTIMA GOLD TM AB LIQUID SCINTILLATION COCKTAIL RESIDUE

    Kyser, E.; Fondeur, F.; Crump, S.

    2011-12-21

    Prior analyses of samples from the F/H Lab solutions showed the presence of diisopropylnapthalene (DIN), a major component of Ultima Gold{trademark} AB liquid scintillation cocktail (LSC). These solutions are processed through H-Canyon Tank 10.5 and ultimately through the 17.8E evaporator. Similar solutions originated in SRNL streams sent to the same H Canyon tanks. This study examined whether the presence of these organics poses a process-significant hazard for the evaporator. Evaporation and calorimetry testing of surrogate samples containing 2000 ppm of Ultima Gold{trademark} AB LSC in 8 M nitric acid have been completed. These experiments showed that although reactions between nitric acid and the organic components do occur, they do not appear to pose a significant hazard for runaway reactions or generation of energetic compounds in canyon evaporators. The amount of off-gas generated was relatively modest and appeared to be well within the venting capacity of the H-Canyon evaporators. A significant fraction of the organic components likely survives the evaporation process primarily as non-volatile components that are not expected to represent any new process concerns during downstream operations such as neutralization. Laboratory Waste solutions containing minor amounts of DIN can be safely received, stored, transferred, and processed through the canyon waste evaporator.

  4. 113Cd NMR studies of a 1:1 Cd adduct with an 18-residue finger peptide from HIV-1 nucleic acid binding protein, p7

    The Zn2+ and Cd2+ adducts with the 18-residue peptide comprising the amino acid sequence of the first finger (residues 13 through 30) of retroviral nucleic acid binding proteins p7 from HIV-1 (the causative agent of AIDS) have been prepared. 1H NMR data indicate that the metal adducts are 1:1 compounds that are stable in aqueous solutions for at least a month. The 113Cd NMR spectral results for the adduct are presented and analyzed. 26 references, 3 figures

  5. Stainless steels with low contents in residual elements for nitric acid environments. Influence of melting processes

    Nitric acid solutions, as they are for instance employed in the chemical treatment of nuclear fuels, do not apparently pose any more corrosion problems and the safe use of the several steels is well documented. The most difficult corrosion problems are next those related to ''tunnel'' penetrations, in the hot-rolling direction (longitudinal). This phenomenon can be quite important such as to imperial complex structures. Up to the last few years, the only manner to eliminate such ''tunnel'' corrosions was to carry out special operations of remelting and refining under careful selected slag (Electro Slag Remelting or ESR). It turns out indeed that non metallic inclusions are the prime factors in this type of corrosion. Well adapted ladle refining processes made possible to obtain equivalent results at lower costs, while purity and cleanliness are improved to such a degree that a steel of type Cr 18 - Ni 10 thus produced exhibit a nearly absolute resistance to intergranular corrosion in the 14 N (65%) boiling nitric acid (the Huey test) whatever the ''sensitization'' treatment, between 10000C and 6000C. In view of its very high tolerance to varied thermal cycles and of the near complete disappearance of any ''tunnel'' corrosion, such a steel is of great interest when complex parts are produced, by machining forged or rolled metal, which is then assembled by welding techniques or procedures which would be forbidden in the case of usual Cr 18 -Ni 10 steels. Similar improvements are also obtained on the two other special steels used in nitric environments: Cr 25 - Ni 20 - C <= 0,020 (Nb) and Cr 17 - Ni 14 - Si 4 - C <= 0,015 (Nb)

  6. Probing the chemical mechanism and critical regulatory amino acid residues of Drosophila melanogaster arylalkylamine N-acyltransferase like 2.

    Dempsey, Daniel R; Carpenter, Anne-Marie; Ospina, Santiago Rodriguez; Merkler, David J

    2015-11-01

    Arylalkylamine N-acyltransferase like 2 (AANATL2) catalyzes the formation of N-acylarylalkylamides from the corresponding acyl-CoA and arylalkylamine. The N-acylation of biogenic amines in Drosophila melanogaster is a critical step for the inactivation of neurotransmitters, cuticle sclerotization, and melatonin biosynthesis. In addition, D. melanogaster has been used as a model system to evaluate the biosynthesis of fatty acid amides: a family of potent cell signaling lipids. We have previously showed that AANATL2 catalyzes the formation of N-acylarylakylamides, including long-chain N-acylserotonins and N-acyldopamines. Herein, we define the kinetic mechanism for AANATL2 as an ordered sequential mechanism with acetyl-CoA binding first followed by tyramine to generate the ternary complex prior to catalysis. Bell shaped kcat,app - acetyl-CoA and (kcat/Km)app - acetyl-CoA pH-rate profiles identified two apparent pKa,app values of ∼7.4 and ∼8.9 that are critical to catalysis, suggesting the AANATL2-catalyzed formation of N-acetyltyramine occurs through an acid/base chemical mechanism. Site-directed mutagenesis of a conserved glutamate that corresponds to the catalytic base for other D. melanogaster AANATL enzymes did not produce a substantial depression in the kcat,app value nor did it abolish the pKa,app value attributed to the general base in catalysis (pKa ∼7.4). These data suggest that AANATL2 catalyzes the formation of N-acylarylalkylamides using either different catalytic residues or a different chemical mechanism relative to other D. melanogaster AANATL enzymes. In addition, we constructed other site-directed mutants of AANATL2 to help define the role of targeted amino acids in substrate binding and/or enzyme catalysis. PMID:26476413

  7. Leaching and selective copper recovery from acidic leachates of Três Marias zinc plant (MG, Brazil) metallurgical purification residues.

    Sethurajan, Manivannan; Huguenot, David; Lens, Piet N L; Horn, Heinrich A; Figueiredo, Luiz H A; van Hullebusch, Eric D

    2016-07-15

    Zinc plant purification residue (ZPR), a typical Zn-hydrometallurgical waste, was collected from the Três Marias Zn plant (MG, Brazil). ZPR was characterized for its metal content and fractionation, mineralogy, toxicity and leachability. Toxicity characteristics leaching procedure (TCLP) and European Community Bureau of Reference (BCR) sequential extraction results revealed that this ZPR displays high percentages of metals (Cd, Cu, Zn and Pb) in the highly mobilizable fractions, increasing its hazardous potential. Bulk chemical analysis, pH dependent leaching and acid (H2SO4) leaching studies confirm that the ZPR is polymetallic, rich in Cd, Cu and Zn. The sulfuric acid concentration (1 M), agitation speed (450 rpm), temperature (40 °C) and pulp density (20 g L(-1)) were optimized to leach the maximum amount of heavy metals (Cd, Cu and Zn). Under optimum conditions, more than 50%, 70% and 60% of the total Cd, Cu and Zn present in the ZPR can be leached, respectively. The metals in the acid leachates were investigated for metal sulfide precipitation with an emphasis on selective Cu recovery. Metal sulfide precipitation process parameters such as initial pH and Cu to sulfide ratio were optimized as pH 1.5 and 1:0.5 (Cu:sulfide) mass ratio, respectively. Under optimum conditions, more than 95% of Cu can be selectively recovered from the polymetallic ZPR leachates. The Cu precipitates characterization studies reveal that they are approximately 0.1 μm in diameter and mainly consist of Cu and S. XRD analysis showed covellite (CuS), chalcanthite (CuSO4·5H2O) and natrochalcite (NaCu2(SO4)2(OH)·H2O) as the mineral phases. ZPRs can thus be considered as an alternative resource for copper production. PMID:27074201

  8. 46 CFR 153.556 - Special requirements for sulfuric acid and oleum.

    2010-10-01

    ... 46 Shipping 5 2010-10-01 2010-10-01 false Special requirements for sulfuric acid and oleum. 153.556 Section 153.556 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CERTAIN BULK DANGEROUS CARGOES SHIPS CARRYING BULK LIQUID, LIQUEFIED GAS, OR COMPRESSED GAS HAZARDOUS MATERIALS Design and Equipment Special Requirements §...

  9. Estimated quantitative amino acid requirements for Florida pompano reared in low-salinity

    As with most marine carnivores, Florida pompano require relatively high crude protein diets to obtain optimal growth. Precision formulations to match the dietary indispensable amino acid (IAA) pattern to a species’ requirements can be used to lower the overall dietary protein. However IAA requirem...

  10. Strains of Lactococcus lactis with a partial pyrimidine requirement show sensitivity toward aspartic acid

    Wadskov-Hansen, Steen Lyders Lerche; Martinussen, Jan

    2009-01-01

    that the partial pyrimidine requirement can be explained by a low specific activity of the pyrimidine biosynthetic enzymes. In conclusion, L. lactis LM0230 during the process of plasmid- and prophage-curing has acquired a partial pyrimidine requirement resulting in sensitivity toward aspartic acid....

  11. Studies on the riboflavin, niacin, pantothenic acid and choline requirements of young bobwhite quail

    Serafin, J.A.

    1974-01-01

    Four experiments were conducted to examine the riboflavin, niacin, pantothenic acid and choline requirements of young Bobwhite quail. Quail fed purified diets deficient in either riboflavin, niacin, pantothenic acid or choline grew poorly and high mortality occurred by 5 weeks of age. Under the conditions of these experiments, it was found that: (1) young quail require approximately 3.8 mg. riboflavin/kg. diet for satisfactory growth and survival; (2) no more than 31 mg. niacin/kg. diet are required for normal growth and survival of young quail; (3) the requirement for pantothenic acid is higher than has previously been reported, quail in these studies requiring 12.6 mg. pantothenic acid/kg. feed for growth and survival; and (4) the requirement for choline for reducing mortality is approximately 1000 mg./kg., while the amount necessary for normal growth of young quail is no greater than 1500 mg./kg. when the diet contains ample amounts of methionine. Quail fed a niacin-deficient diet developed stiff, shortened feathers and an erythema about the head; those receiving a riboflavin-deficient ration developed enlarged hocks and bowed legs, as did quail fed diets low or devoid of choline. Aside from slow growth, poor feathering was the only other indication that a deficient diet was being fed when quail were placed on a basal ration without pantothenic acid for five weeks.

  12. Amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by streptococcal IgA1 proteases.

    Batten, Margaret R; Senior, Bernard W; Kilian, Mogens; Woof, Jenny M

    2003-03-01

    The amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by IgA1 proteases of different species of Streptococcus were investigated. Recombinant IgA1 antibodies were generated with point mutations at proline 227 and threonine 228, the residues lying on either side of the peptide bond at which all streptococcal IgA1 proteases cleave wild-type human IgA1. The amino acid substitutions produced no major effect upon the structure of the mutant IgA1 antibodies or their functional ability to bind to Fcalpha receptors. However, the substitutions had a substantial effect upon sensitivity to cleavage with some streptococcal IgA1 proteases, with, in some cases, a single point mutation rendering the antibody resistant to a particular IgA1 protease. This effect was least marked with the IgA1 protease from Streptococcus pneumoniae, which showed no absolute requirement for either proline or threonine at residues 227 to 228. By contrast, the IgA1 proteases of Streptococcus oralis, Streptococcus sanguis, and Streptococcus mitis had an absolute requirement for proline at 227 but not for threonine at 228, which could be replaced by valine. There was evidence in S. mitis that proteases from different strains may have different amino acid requirements for cleavage. Remarkably, some streptococcal proteases appeared able to cleave the hinge at a distant alternative site if substitution prevented efficient cleavage of the original site. Hence, this study has identified key residues required for the recognition of the IgA1 hinge as a substrate by streptococcal IgA1 proteases, and it marks a preliminary step towards development of specific enzyme inhibitors. PMID:12595464

  13. The conserved glycine residues in the transmembrane domain of the Semliki Forest virus fusion protein are not required for assembly and fusion

    The alphavirus Semliki Forest virus (SFV) infects cells via a low pH-triggered fusion reaction mediated by the viral E1 protein. Both the E1 fusion peptide and transmembrane (TM) domain are essential for membrane fusion, but the functional requirements for the TM domain are poorly understood. Here we explored the role of the five TM domain glycine residues, including the highly conserved glycine pair at E1 residues 415/416. SFV mutants with alanine substitutions for individual or all five glycine residues (5G/A) showed growth kinetics and fusion pH dependence similar to those of wild-type SFV. Mutants with increasing substitution of glycine residues showed an increasingly more stringent requirement for cholesterol during fusion. The 5G/A mutant showed decreased fusion kinetics and extent in fluorescent lipid mixing assays. TM domain glycine residues thus are not required for efficient SFV fusion or assembly but can cause subtle effects on the properties of membrane fusion

  14. The omega-3 fatty acid eicosapentaenoic acid is required for normal alcohol response behaviors in C. elegans.

    Richard C Raabe

    Full Text Available Alcohol addiction is a widespread societal problem, for which there are few treatments. There are significant genetic and environmental influences on abuse liability, and understanding these factors will be important for the identification of susceptible individuals and the development of effective pharmacotherapies. In humans, the level of response to alcohol is strongly predictive of subsequent alcohol abuse. Level of response is a combination of counteracting responses to alcohol, the level of sensitivity to the drug and the degree to which tolerance develops during the drug exposure, called acute functional tolerance. We use the simple and well-characterized nervous system of Caenorhabditis elegans to model the acute behavioral effects of ethanol to identify genetic and environmental factors that influence level of response to ethanol. Given the strong molecular conservation between the neurobiological machinery of worms and humans, cellular-level effects of ethanol are likely to be conserved. Increasingly, variation in long-chain polyunsaturated fatty acid levels has been implicated in complex neurobiological phenotypes in humans, and we recently found that fatty acid levels modify ethanol responses in worms. Here, we report that 1 eicosapentaenoic acid, an omega-3 polyunsaturated fatty acid, is required for the development of acute functional tolerance, 2 dietary supplementation of eicosapentaenoic acid is sufficient for acute tolerance, and 3 dietary eicosapentaenoic acid can alter the wild-type response to ethanol. These results suggest that genetic variation influencing long-chain polyunsaturated fatty acid levels may be important abuse liability loci, and that dietary polyunsaturated fatty acids may be an important environmental modulator of the behavioral response to ethanol.

  15. Selective 'unlabeling' of amino acids in fractionally 13C labeled proteins: An approach for stereospecific NMR assignments of CH3 groups in Val and Leu residues

    A novel methodology for stereospecific NMR assignments of methyl (CH3) groups of Val and Leu residues in fractionally 13C-labeled proteins is presented. The approach is based on selective 'unlabeling' of specific amino acids in proteins while fractionally 13C-labeling the rest. A 2D [13C-1H] HSQC spectrum recorded on such a sample is devoid of peaks belonging to the 'unlabeled' amino acid residues. Such spectral simplification aids in unambiguous stereospecific assignment of diastereotopic CH3 groups in Val and Leu residues in large proteins. This methodology has been demonstrated on a 15 kDa calcium binding protein from Entamoeba histolytica (Eh-CaBP)

  16. Effect of Amino Acid Residue and Oligosaccharide Chain Chemical Modifications on Spectral and Hemagglutinating Activity of Millettia dielsiana Harms. ex Diels. Lectin

    Shun GAO; Jie AN; Chuan-Fang WU; Ying GU; Fang CHEN; Yuan YU; Qia-Qing WU; Jin-Ku BAO

    2005-01-01

    The effects of modifying the carbohydrate chain and amino acids on the conformation and activity of Millettia dielsiana Harms. ex Diels. lectin (MDL) were studied by hemagglutination, fluorescence and circular dichroism analysis. The modification of tryptophan residues led to a compete loss of hemagglutinating activity; however, the addition of mannose was able to prevent this loss of activity. The results indicate that two tryptophan residues are involved in the carbohydrate-binding site. Modifications of the carboxyl group residues produced an 80% loss of activity, but the presence of mannose protected against the modification. The results suggest that the carboxyl groups of aspartic and glutamic acids are involved in the carbohydrate-binding site of the lectin. However, oxidation of the carbohydrate chain and modification of the histidine and arginine residues did not affect the hemagglutinating activity of MDL. Fluorescence studies of MDL indicate that tryptophan residues are present in a relatively hydrophobic region, and the binding of mannose to MDL could quench tryptophan fluorescence without any change in λmax. The circular dichroism spectrum showed that all of these modifications affected the conformation of the MDL molecule to different extents, except the modification of arginine residues. Fluorescence quenching showed that acrylamide and iodoacetic acids are able to quench 77% and 98% of the fluorescence of tryptophan in MDL, respectively.However, KI produced a barely perceptible effect on the fluorescence of MDL, even when the concentration of I- was 0.15 M. This demonstrates that most of tryptophan residues are located in relatively hydrophobic or negatively charged areas near the surface of the MDL molecule.

  17. Evaluation of crop residues on potassium kinetics in an acid soil and potassium use efficiency in potato-garlic sequence using tracer 86Rb

    Greenhouse and laboratory studies were conducted on an acid soil in order to evaluate the role of two crop residues i.e. paddy and wheat along with farmyard manure on potassium kinetics and its availability in the potato-garlic sequence using tracer 86Rb. Under rapid equilibrium, application of crop residues of paddy, wheat straw and FYM were able to enhance soil pH and organic carbon content. In addition, their application helped in enhancing soil K availability indices like water soluble, available and non-exchangeable -K. This was further augmented by the Q/I studies using 86Rb where application of organic residues helped in lowering the potassium buffering capacity of the soil. Greenhouse study supplemented the results obtained from laboratory study where application of crop residues/FYM were able to improve the potato yield significantly and maintained higher concentration of K in potato leaf at early growth stages. A significant correlation was obtained between leaf K and haulms-K with that of 86Rb activities in potato leaf at 35 days and 86Rb absorbed in the haulms, respectively. Residues/ FYM and PK application to potato left sufficient residual effect on succeeding garlic crop. In potato-garlic sequence, K recovery was highest with FYM while N and P recoveries were higher with wheat residues. The nutrient recoveries with PK application followed law of diminishing returns. (author)

  18. Determination of cyanuric acid residues in catfish, trout, tilapia, salmon and shrimp by liquid chromatography-tandem mass spectrometry

    Karbiwnyk, Christine M. [Animal Drugs Research Center, U.S. Food and Drug Administration, P.O. Box 25087, Denver, CO 80225-0087 (United States)], E-mail: christine.karbiwnyk@fda.hhs.gov; Andersen, Wendy C.; Turnipseed, Sherri B. [Animal Drugs Research Center, U.S. Food and Drug Administration, P.O. Box 25087, Denver, CO 80225-0087 (United States); Storey, Joseph M.; Madson, Mark R. [Denver District Laboratory, U.S. Food and Drug Administration, P.O. Box 25087, Denver, CO 80225-0087 (United States); Miller, Keith E. [Center for Veterinary Medicine, U.S. Food and Drug Administration, 8401 Muirkirk Road, Laurel, MD 20708 (United States); Gieseker, Charles M.; Miller, Ron A.; Rummel, Nathan G.; Reimschuessel, Renate [University of Denver, Department of Chemistry and Biochemistry, Denver, CO 80208 (United States)

    2009-04-01

    In May 2007, investigators discovered that waste material from the pet food manufacturing process contaminated with melamine (MEL) and/or cyanuric acid (CYA) had been added to hog and chicken feeds. At this time, investigators also learned that adulterated wheat gluten had been used in the manufacture of aquaculture feeds. Concern that the contaminated feed had been used in aquaculture and could enter the human food supply prompted the development of a method for the determination of CYA residues in the edible tissues of fish and shrimp. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed as a sensitive technique for the analysis of CYA in catfish, tilapia, salmon, trout and shrimp tissue. CYA was extracted from ground fish or shrimp with an acetic acid solution, defatted with hexane, and isolated with a graphitic carbon black solid-phase extraction column. Residues were separated from matrix components using a porous graphitic carbon LC column, and then analyzed with electrospray ionization in negative ion mode on a triple quadrupole mass spectrometer. Selective reaction monitoring was performed on the [M-H]{sup -}m/z 128 ion resulting in the product ions m/z 85 and 42. Recoveries from catfish, tilapia and trout fortified with 10-100 {mu}g kg{sup -1} of CYA averaged 67% with a relative standard deviation (R.S.D.) of 18% (n = 107). The average method detection limit (MDL) for catfish, tilapia and trout is 3.5 {mu}g kg{sup -1}. An internal standard, {sup 13}C{sub 3}-labeled CYA, was used in the salmon and shrimp extractions. Average recovery of CYA from salmon was 91% (R.S.D. = 15%, n = 18) with an MDL of 7.4 {mu}g kg{sup -1}. Average recovery of CYA from shrimp was 85% (R.S.D. = 10%, n = 13) with an MDL of 3.5 {mu}g kg{sup -1}.

  19. Separating nano graphene oxide from the residual strong-acid filtrate of the modified Hummers method with alkaline solution

    Hu, Xuebing, E-mail: xuebinghu2010@gmail.com [Key Laboratory of Inorganic Membrane, Jingdezhen Ceramic Institute, Jingdezhen 333001 (China); Key Laboratory of Inorganic Coating Materials, Shanghai Institute of Ceramics, Chinese Academy of Science, Shanghai 201800 (China); Yu, Yun, E-mail: yunyush@mail.sic.ac.cn [Key Laboratory of Inorganic Coating Materials, Shanghai Institute of Ceramics, Chinese Academy of Science, Shanghai 201800 (China); Wang, Yongqing; Zhou, Jianer [Key Laboratory of Inorganic Membrane, Jingdezhen Ceramic Institute, Jingdezhen 333001 (China); Song, Lixin [Key Laboratory of Inorganic Coating Materials, Shanghai Institute of Ceramics, Chinese Academy of Science, Shanghai 201800 (China)

    2015-02-28

    Graphical abstract: By adding an alkaline (NaOH or KOH) solution, the unprecipitated nano graphene oxide undergoes fast aggregation from the residual strong-acid filtrate of the modified Hummers method and forms the stable floccules when the pH value of the filtrate is about 1.7. The acid–base interaction with the surface functional groups of the carbon layers plays a role in the aggregation of the unprecipitated nano graphene oxide. - Highlights: • The novel and high-efficient method for separating graphene oxide was showed. • Graphene oxide undergoes aggregation and forms the floccules when pH value is ∼1.7. • The acid–base interaction plays a role in the aggregation of graphene oxide. - Abstract: In the modified Hummers method for preparing graphene oxide, the yellow slurry can be obtained. After filtering through a quantitative filter paper, the strong-acid filtrate containing the unprecipitated nano graphene oxide was gained. The corresponding filtrate was added gradually with an alkaline (NaOH or KOH) solution at room temperature. The unprecipitated nano graphene oxide could undergo fast aggregation when the pH value of the filtrate was about 1.7 and formed the stable floccules. X-ray diffraction analysis shows the dominant peak of the floccules is about 11°, which accords to the peak of graphene oxide. Spectra of X-ray photoelectron spectroscopy confirm the presence in the floccules of an abundance of oxygen functional groups and the purified graphene oxide floccules can be obtained. Atomic force microscopy measurement shows the graphene oxide floccules consists of sheet-like objects, mostly containing only a few layers (about 5 layers). Zeta potential analysis demonstrates the surface charge of the graphene oxide is pH-sensitive and its isoelectric point is ∼1.7. The flocculation mechanism of graphene oxide ascribes to the acid–base interaction with the surface functional groups of the carbon layers.

  20. Separating nano graphene oxide from the residual strong-acid filtrate of the modified Hummers method with alkaline solution

    Graphical abstract: By adding an alkaline (NaOH or KOH) solution, the unprecipitated nano graphene oxide undergoes fast aggregation from the residual strong-acid filtrate of the modified Hummers method and forms the stable floccules when the pH value of the filtrate is about 1.7. The acid–base interaction with the surface functional groups of the carbon layers plays a role in the aggregation of the unprecipitated nano graphene oxide. - Highlights: • The novel and high-efficient method for separating graphene oxide was showed. • Graphene oxide undergoes aggregation and forms the floccules when pH value is ∼1.7. • The acid–base interaction plays a role in the aggregation of graphene oxide. - Abstract: In the modified Hummers method for preparing graphene oxide, the yellow slurry can be obtained. After filtering through a quantitative filter paper, the strong-acid filtrate containing the unprecipitated nano graphene oxide was gained. The corresponding filtrate was added gradually with an alkaline (NaOH or KOH) solution at room temperature. The unprecipitated nano graphene oxide could undergo fast aggregation when the pH value of the filtrate was about 1.7 and formed the stable floccules. X-ray diffraction analysis shows the dominant peak of the floccules is about 11°, which accords to the peak of graphene oxide. Spectra of X-ray photoelectron spectroscopy confirm the presence in the floccules of an abundance of oxygen functional groups and the purified graphene oxide floccules can be obtained. Atomic force microscopy measurement shows the graphene oxide floccules consists of sheet-like objects, mostly containing only a few layers (about 5 layers). Zeta potential analysis demonstrates the surface charge of the graphene oxide is pH-sensitive and its isoelectric point is ∼1.7. The flocculation mechanism of graphene oxide ascribes to the acid–base interaction with the surface functional groups of the carbon layers

  1. Cavity residue leucine 95 and channel residues glutamine 204, aspartic acid 211, and phenylalanine 269 of toluene o-xylene monooxygenase influence catalysis.

    Kurt, Cansu; Sönmez, Burcu; Vardar, Nurcan; Yanık-Yıldırım, K Cansu; Vardar-Schara, Gönül

    2016-09-01

    Structural analysis of toluene-o-xylene monooxygenase (ToMO) hydroxylase revealed the presence of three hydrophobic cavities, a channel, and a pore leading from the protein surface to the active site. Here, saturation mutagenesis was used to investigate the catalytic roles of alpha-subunit (TouA) second cavity residue L95 and TouA channel residues Q204, D211, and F269. By testing the substrates toluene, phenol, nitrobenzene, and/or naphthalene, these positions were found to influence the catalytic activity of ToMO. Several regiospecific variants were identified from TouA positions Q204, F269, and L95. For example, TouA variant Q204H had the regiospecificity of nitrobenzene changed significantly from 30 to 61 % p-nitrophenol. Interestingly, a combination of mutations at Q204H and A106V altered the regiospecificity of nitrobenzene back to 27 % p-nitrophenol. TouA variants F269Y, F269P, Q204E, and L95D improved the meta-hydroxylating capability of nitrobenzene by producing 87, 85, 82, and 77 % m-nitrophenol, respectively. For naphthalene oxidation, TouA variants F269V, Q204A, Q204S/S222N, and F269T had the regiospecificity changed from 16 to 9, 10, 23, and 25 % 2-naphthol, respectively. Here, two additional TouA residues, S222 and A106, were also identified that may have important roles in catalysis. Most of the isolated variants from D211 remained active, whereas having a hydrophobic residue at this position appeared to diminish the catalytic activity toward naphthalene. The mutational effects on the ToMO regiospecificity described here suggest that it is possible to further fine tune and engineer the reactivity of multicomponent diiron monooxygenases toward different substrates at positions that are relatively distant from the active site. PMID:27311562

  2. Antiretroviral intensification and valproic acid lack sustained effect on residual HIV-1 viremia or resting CD4+ cell infection.

    Nancie M Archin

    Full Text Available BACKGROUND: Human immunodeficiency virus (HIV infection that persists despite antiretroviral therapy (ART is a daunting problem. Given the limited evidence that resting CD4+ T cell infection (RCI is affected by the histone deacetylase (HDAC inhibitor valproic acid (VPA, we measured the stability of RCI and residual viremia in patients who added VPA with or without raltegravir (RAL, or enfuvirtide (ENF with or without VPA, to standard ART. METHODS: Patients with plasma HIV RNA50% was seen in six volunteers after the addition of RAL and VPA. In 4 of the 6 patients this lack of effect might be attributed to intermittent viremia, low VPA levels, or intermittent study therapy adherence. Overall, there was no effect of the addition of RAL or ENF on low-level viremia measured by SCA. CONCLUSIONS: The prospective addition of VPA and RAL, VPA and ENF, or ENF failed to progressively reduce the frequency of RCI, or ablate intermittent and low-level viremia. New approaches such as more potent HDAC inhibition, alone or in combination with intensified ART or other agents that may disrupt proviral latency must be pursued.

  3. Three amino acid residues bind corn odorants to McinOBP1 in the polyembryonic endoparasitoid of Macrocentrus cingulum Brischke.

    Tofael Ahmed

    Full Text Available Odorant binding proteins (OBPs play a central role in transporting odorant molecules from the sensillum lymph to olfactory receptors to initiate behavioral responses. In this study, the OBP of Macrocentrus cingulum McinOBP1 was expressed in Escherichia coli and purified by Ni ion affinity chromatography. Real-time PCR experiments indicate that the McinOBP1 is expressed mainly in adult antennae, with expression levels differing by sex. Ligand-binding experiments using N-phenyl-naphthylamine (1-NPN as a fluorescent probe demonstrated that the McinOBP1 can bind green-leaf volatiles, including aldehydes and terpenoids, but also can bind aliphatic alcohols with good affinity, in the order trans-2-nonenal>cis-3-hexen-1-ol>trans-caryophelle, suggesting a role of McinOBP1 in general odorant chemoreception. We chose those three odorants for further homology modeling and ligand docking based on their binding affinity. The Val58, Leu62 and Glu130 are the key amino acids in the binding pockets that bind with these three odorants. The three mutants, Val58, Leu62 and Glu130, where the valine, leucine and glutamic residues were replaced by alanine, proline and alanine, respectively; showed reduced affinity to these odorants. This information suggests, Val58, Leu62 and Glu130 are involved in the binding of these compounds, possibly through the specific recognition of ligands that forms hydrogen bonds with the ligands functional groups.

  4. RESIDUAL COMPRESSIVE STRENGTH OF FLY ASH BASED GLASS FIBRE REINFORCED HIGH PERFORMANCE CONCRETE SUBJECTED TO ACID ATTACK

    Dr.H.Sudarsana Rao

    2012-01-01

    Full Text Available In recent years, improvements in concrete properties have been achieved by the invention of High- Performance-Concrete (HPC. Improvements involving a combination of improved compaction, improved paste characteristics and aggregate-matrix bond, and reduced porosity are achieved through HPC. The ductility of HPC can be improved by altering its composition through the addition of glass fibers in the design mix. High- Performance-Concrete made with glass fibers inside is regarded as Glass Fiber Reinforced High Performance Concrete (GFRHPC. This paper presents the details of an experimental investigation planned to utilize fly ash in the production of Glass fibre reinforced High-Performance-Concrete (GFRHPC. The investigation examines the progressive deterioration of concrete mixtures containing various combinations of fly ash based GFRHPCmixes exposed to sulphate and chloride solutions. Acid attack tests have been conducted to measure the durability of GFRHPC. Cubes of 150X150X150 mm have been cast, cured and then kept immersed in 5%concentrated solutions of HCl, H2SO4 and MgSO4 for 30, 60 and 90 days and then tested to record the residual compressive strengths of GFRHPC produced with the fly ash mineral admixtures. The results have been analyzed and useful conclusions have been drawn.

  5. Stable nuclide tracer studies and human amino acid requirements. A summary

    The nutritional requirements for proteins have been estimated for various age groups. The current status of knowledge concerning the quantitative needs for specific indispensable amino acids was reviewed and it was concluded that, except for infants, current values for pre-school children, school age children and healthy adults are based on limited experimental data and/or on results from nitrogen balance determinations which are open to serious question regarding their nutritional significance. A review of 13C-labelled tracer studies carried out in MIT laboratories was undertaken to demonstrate the applicability of stable nuclide tracer studies for purposes of determining the amino acid requirements of humans. 5 refs

  6. Determination of small halogenated carboxylic acid residues in drug substances by high performance liquid chromatography-diode array detection following derivatization with nitro-substituted phenylhydrazines.

    Hou, Desheng; Fan, Jingjing; Han, Lingfei; Ruan, Xiaoling; Feng, Feng; Liu, Wenyuan; Zheng, Feng

    2016-03-18

    A method for the determination of small halogenated carboxylic acid (HCA) residues in drug substances is urgently needed because of the potential of HCAs for genotoxicity and carcinogenicity in humans. We have now developed a simple method, involving derivatization followed by high performance liquid chromatography-diode array detection (HPLC-DAD), for the determination of six likely residual HCAs (monochloroacetic acid, monobromoacetic acid, dichloroacetic acid, 2-chloropropionic acid, 2-bromopropionic acid and 3-chloropropionic acid) in drug substances. Different nitro-substituted phenylhydrazines (NPHs) derivatization reagents were systematically compared and evaluated. 2-Nitrophenylhydrazine hydrochloride (2-NPH·HCl) was selected as the most suitable choice since its derivatives absorb strongly at 392 nm, a region of the spectrum where most drug substances and impurities absorb very weakly. During the derivatization process, the commonly used catalyst, pyridine, caused rapid dechlorination or chlorine substitution of α-halogenated derivatives. To avoid these unwanted side reactions, a reliable derivatization method that did not use pyridine was developed. Reaction with 2-NPH·HCl using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride as coupling agent in acetonitrile-water (70:30) at room temperature for 2h gave complete reaction and avoided degradation products. The derivatives were analyzed, without any pretreatment, using gradient HPLC with detection in the near visible region. Organic acids commonly found in drug substances and other impurities did not interfere with the analysis. Good linearity (r>0.999) and low limits of quantitation (0.05-0.12 μg mL(-1)) were obtained. The mean recoveries were in the range of 80-115% with RSD <5.81% except for 3-CPA in ibuprofen which was 78.5%. The intra- and inter-day precisions were expressed as RSD <1.98% and <4.39%, respectively. Finally, the proposed method was successfully used for the residue

  7. Solid grape extract for protection against the sun consists of a residues based material containing gallic acid flavonol and e.g. procyanidine

    Torres, Josep Lluís; González, Pedro; Cascante, Marta; Comín, Begoña

    2001-01-01

    [EN]The solid grape extract for protection against the sun comprises a residues-based material containing gallic acid 1-5, flavonol and its glycosyl derivatives 1-20 and catechin and its oligomers or procyanidine 1-30 per cent respectively. The grape residue is extracted by aqueous ethanol with lyophilisation and suspension in water. The polyphenolic fraction is extracted in ethyl acetate for suspension in water, centrifugation and lyophilisation. [ES]Extracto de uva para protección solar. Se...

  8. D-Amino acid residue in the C-type natriuretic peptide from the venom of the mammal, Ornithorhynchus anatinus, the Australian platypus.

    Torres, Allan M; Menz, Ian; Alewood, Paul F; Bansal, Paramjit; Lahnstein, Jelle; Gallagher, Clifford H; Kuchel, Philip W

    2002-07-31

    The C-type natriuretic peptide from the platypus venom (OvCNP) exists in two forms, OvCNPa and OvCNPb, whose amino acid sequences are identical. Through the use of nuclear magnetic resonance, mass spectrometry, and peptidase digestion studies, we discovered that OvCNPb incorporates a D-amino acid at position 2 in the primary structure. Peptides containing a D-amino acid have been found in lower forms of organism, but this report is the first for a D-amino acid in a biologically active peptide from a mammal. The result implies the existence of a specific isomerase in the platypus that converts an L-amino acid residue in the protein to the D-configuration. PMID:12135762

  9. Reclamation of acidic mine residues by creation of technosoils with the addition of biochar and marble waste

    Moreno-Barriga, Fabián; Díaz, Vicente; Acosta, José; Faz, Ángel; Zornoza, Raul

    2016-04-01

    This study reports the short-term effect of biochar and marble waste addition for the reclamation of acidic mine residues. A lab incubation was carried out for 90 days. Biochars derived from pig manure (PM), crop residues (CR) and municipal solid waste (MSW) were added to the soil at a rate of 20 g kg-1. The marble waste (MW) was added at a rate of 200 g kg-1. Bochars and MW were applied independently and combined. A control soil was used without application of amendments. The evolution of different physical, chemical and biochemical properties and availability of heavy metals was periodically monitored. Results showed that original pH (2.8) was increased with all amendments, those samples containing MW being the ones with the highest pH (~8.0). The electrical conductivity (EC) decreased from 6.6 to 3.0-4.5 mS cm-1 in all the treatments receiving MW. Soil organic C (SOC) increased in all samples receiving biochar up to 18-20 g kg-1, with no shifts during the 90 d incubation, indicating the high stability of the C supplied. Recalcitrant organic C accounted for ~90-98% of the SOC. No significant effect of amendment addition was observed for carbohydrates, soluble C, microbial biomass C and β-glucosidase activity. However, arylesterase activity increased with amendments, highly related to pH. The availability of heavy metals decreased up to 90-95% owing to the addition of amendments, mainly in samples containing MW. The MW provided conditions to increase pH and decrease EC and metals mobility. Biochar was an effective strategy to increase SOC, recalcitrant C and AS, essential to create soil structure. However, a labile source of organic matter should be added together with the proposed amendments to promote the activation of microbial communities. Acknowledgement : This work has been funded by Fundación Séneca (Agency of Science and Technology of the Region of Murcia, Spain) by the project 18920/JLI/13

  10. An amino acid residue in the second extracellular loop determines the agonist-dependent tolerance property of the human D3 dopamine receptor.

    Gil-Mast, Sara; Kortagere, Sandhya; Kota, Kokila; Kuzhikandathil, Eldo V

    2013-06-19

    The D3 dopamine receptor is a therapeutic target for treating various nervous system disorders such as schizophrenia, Parkinson's disease, depression, and addictive behaviors. The crystal structure of the D3 receptor bound to an antagonist was recently described; however, the structural features that contribute to agonist-induced conformational changes and signaling properties are not well understood. We have previously described the conformation-dependent tolerance and slow response termination (SRT) signaling properties of the D3 receptor and identified the C147 residue in the second intracellular loop (IL2) of the D3 receptor as important for the tolerance property. Interestingly, while IL2 and the C147 residue, in particular, were important for dopamine- and quinpirole-induced tolerance, this residue did not affect the severe tolerance induced by the high affinity, D3 receptor-selective agonist, PD128907. Here, we used D2/D3 receptor chimeras and site-specific D3 receptor mutants to identify another residue, D187, in the second extracellular loop (EC2) of the human D3 receptor that mediates the tolerance property induced by PD128907, quinpirole, pramipexole, and dopamine. Molecular dynamics simulations confirmed the distinct conformation adopted by D3 receptor during tolerance and suggested that in the tolerant D3 receptor the D187 residue in EC2 forms a salt bridge with the H354 residue in EC3. Indeed, site-directed mutation of the H354 residue resulted in loss of PD1287907-induced tolerance. The mapping of specific amino acid residues that contribute to agonist-dependent conformation changes and D3 receptor signaling properties refines the agonist-bound D3 receptor pharmacophore model which will help develop novel D3 receptor agonists. PMID:23477444

  11. Effect of the inclusion of fish residue oils in diets on the fatty acid profile of muscles of males and females lambari (Astyanax altiparanae

    Ligia Uribe Gonçalves

    2012-09-01

    Full Text Available This study evaluated the effects of two lipids sources of fish residue (tilapia and salmon compared with a vegetable oil source (soybean oil on the fatty acid profiles of male and female lambari. This experiment was developed in a completely randomized experimental design in a 3 × 2 factorial arrangement, totaling 6 treatments resulting from the combination of the three experimental diets for both sexes, with four replications for each treatment. This study involved 120 male (2.58±0.13 g and 72 female lambari (4.00±0.09 g, fed the experimental diets twice a day until apparent satiation for a period of 60 days. Oleic, linoleic, palmitic and stearic fatty acids were found at higher concentrations in all experimental oils and diets, as well in the muscle of male and female lambari. The low amounts of arachidonic, eicosapentaenoic and docosahexaenoic acids in the experimental diets and subsequent greater concentrations in muscle tissue, suggested that lambari are able to desaturate and elongate the chain of fatty acids with 18 carbons. The fish of both sexes that received the diet with soybean oil showed high levels of n-6 fatty acids, especially of C18: 2n-6 and low levels of eicosapentaenoic and docosahexaenoic acids. The diet with salmon residue oil promoted higher levels of fatty acids of the n-3 series and resulted in the best n-3/n-6 ratio in the muscle of male and female lambari. The oils from fish residues can be a substitute for traditional fish oil and its use in the lambari diets does not impair its growth.

  12. Estimation of the dietary essential amino acid requirements of colliroja Astyanax fasciatus by using the ideal protein concept

    Wilson Massamitu Furuya; Mariana Michelato; Ana Lúcia Salaro; Thais Pereira da Cruz; Valéria Rossetto Barriviera-Furuya

    2015-01-01

    Colliroja, Astyanax fasciatus, is a new aquaculture species, and information on its dietary essential amino acid requirements is lacking. The whole body composition of 120 farmed fish (16.2 ± 8.8 g) was determined to estimate the dietary essential amino acid requirement based on the ideal protein concept ((each essential amino acid/lysine) x100), and the findings were correlated to the whole body essential amino acid content of Nile tilapia Oreochromis niloticus. The dietary essential amino a...

  13. Folic Acid and Vitamin B12 Fortification of Flour: A Global Basic Food Security Requirement

    Tulchinsky Theodore H

    2010-07-01

    Full Text Available Folic acid is an essential water soluble B vitamin which has been used for decades in the prevention of folate deficiency anemia of pregnancy. In 1991, folic acid taken prior to the start of pregnancy was shown unequivocally to prevent spina bifida and anencephaly—two of the most serious and common birth (neural tube defects. Soon governments recommended that women of reproductive age consume folic acid daily to prevent these birth defects. Because compliance was low and since more than half of pregnancies are unplanned, the United States Food and Drug Administration mandated in 1998 that all enriched flour be fortified with folic acid at a concentration estimated to give the average woman an intake of 100 micrograms of folic acid a day. Canada and Chile followed with similar requirements for folic acid fortification of wheat flour. Now there is mandatory fortification in more than 50 countries globally.Where fortification has been implemented and studied, it has led to dramatic increases in serum folate concentrations, reduction in neural tube defects, folate deficiency anemia, as well as the reduction in homocysteine concentrations and stroke mortality with no known risk. Australia implemented mandatory folic acid fortification in 2009. To date, no country in Europe has implemented mandatory folic acid fortification of flour, although it has been recommended by the UK Food Safety Authority. This review discusses the vital importance of mandatory flour fortification with folic acid and vitamin B12, for public health food security and as a challenge to the New Public Health in Europe and globally.

  14. The myeloperoxidase-derived oxidant hypothiocyanous acid inhibits protein tyrosine phosphatases via oxidation of key cysteine residues

    Cook, Naomi L.; Moeke, Cassidy H.; Fantoni, Luca I.;

    2016-01-01

    Phosphorylation of protein tyrosine residues is critical to cellular processes, and is regulated by kinases and phosphatases (PTPs). PTPs contain a redox-sensitive active site Cys residue, which is readily oxidized. Myeloperoxidase, released from activated leukocytes, catalyzes thiocyanate ion (S...

  15. Amino acid residues involved in the catalytic mechanism of NAD-dependent glutamate dehydrogenase from Halobacterium salinarum.

    Pérez-Pomares, F; Ferrer, J; Camacho, M; Pire, C; LLorca, F; Bonete, M J

    1999-02-01

    The pH dependence of kinetic parameters for a competitive inhibitor (glutarate) was determined in order to obtain information on the chemical mechanism for NAD-dependent glutamate dehydrogenase from Halobacterium salinarum. The maximum velocity is pH dependent, decreasing at low pHs giving a pK value of 7.19+/-0.13, while the V/K for l-glutamate at 30 degrees C decreases at low and high pHs, yielding pK values of 7.9+/-0.2 and 9.8+/-0.2, respectively. The glutarate pKis profile decreases at high pHs, yielding a pK of 9. 59+/-0.09 at 30 degrees C. The values of ionization heat calculated from the change in pK with temperature are: 1.19 x 10(4), 5.7 x 10(3), 7 x 10(3), 6.6 x 10(3) cal mol-1, for the residues involved. All these data suggest that the groups required for catalysis and/or binding are lysine, histidine and tyrosine. The enzyme shows a time-dependent loss in glutamate oxidation activity when incubated with diethyl pyrocarbonate (DEPC). Inactivation follows pseudo-first-order kinetics with a second-order rate constant of 53 M-1min-1. The pKa of the titratable group was pK1=6.6+/-0.6. Inactivation with ethyl acetimidate also shows pseudo-first-order kinetics as well as inactivation with TNM yielding second-order constants of 1.2 M-1min-1 and 2.8 M-1min-1, and pKas of 8.36 and 9.0, respectively. The proposed mechanism involves hydrogen binding of each of the two carboxylic groups to tyrosyl residues; histidine interacts with one of the N-hydrogens of the l-glutamate amino group. We also corroborate the presence of a conservative lysine that has a remarkable ability to coordinate a water molecule that would act as general base. PMID:10076069

  16. The role of a conserved acidic residue in calcium-dependent protein folding for a low density lipoprotein (LDL)-A module: implications in structure and function for the LDL receptor superfamily.

    Guo, Ying; Yu, Xuemei; Rihani, Kayla; Wang, Qing-Yin; Rong, Lijun

    2004-04-16

    One common feature of the more than 1,000 complement-type repeats (or low density lipoprotein (LDL)-A modules) found in LDL receptor and the other members of the LDL receptor superfamily is a cluster of five highly conserved acidic residues in the C-terminal region, DXXXDXXDXXDE. However, the role of the third conserved aspartate of these LDL-A modules in protein folding and ligand recognition has not been elucidated. In this report, using a model LDL-A module and several experimental approaches, we demonstrate that this acidic residue, like the other four conserved acidic residues, is involved in calcium-dependent protein folding. These results suggest an alternative calcium coordination conformation for the LDL-A modules. The proposed model provides a plausible explanation for the conservation of this acidic residue among the LDL-A modules. Furthermore, the model can explain why mutations of this residue in human LDL receptor cause familial hypercholesterolemia. PMID:14749324

  17. Evaluation of ozonation technique for pesticide residue removal and its effect on ascorbic acid, cyanidin-3-glucoside, and polyphenols in apple (Malus domesticus) fruits.

    Swami, Saurabh; Muzammil, Raunaq; Saha, Supradip; Shabeer, Ahammed; Oulkar, Dasharath; Banerjee, Kaushik; Singh, Shashi Bala

    2016-05-01

    Ozonated water dip technique was evaluated for the detoxification of six pesticides, i.e., chlorpyrifos, cypermethrin, azoxystrobin, hexaconazole, methyl parathion, and chlorothalonil from apple fruits. Results revealed that ozonation was better than washing alone. Ozonation for 15 min decreased residues of the test pesticides in the range of from 26.91 to 73.58%, while ozonation for 30 min could remove the pesticide residues by 39.39-95.14 % compared to 19.05-72.80 % by washing. Cypermethrin was the least removed pesticide by washing as well as by ozonation. Chlorothalonil, chlorpyrifos, and azoxystrobin were removed up to 71.45-95.14 % in a 30-min ozonation period. In case of methyl parathion removal, no extra advantage could be obtained by ozonation. The HPLC analysis indicated that ozonation also affected adversely the ascorbic acid and cyanidin-3-glucoside content of apples. However, 11 polyphenols studied showed a mixed trend. Gallic acid, 3,4-dihydroxybenzoic acid, catechin, epicatechin, p-coumaric acid, quercetin-3-O-glucoside, quercetin, and kaempferol were found to decrease while syringic acid, rutin, and resveratrol were found to increase in 30-min ozonation. PMID:27098519

  18. Functional analysis of three amino acid residues of purR re-pressor, Trp147, Gln-218 and Gln-292 in Salmonella typhi-murium

    2001-01-01

    The amber mutation sites of 6 purR(am) mutants were determined bycloning and DNA sequencing. The results showed that the mutations were distributed at three different sites in PurR coding region, G721(→A), C933(→T) and C1155(→T), which respectively turn Trp-147,Gln-218 and Gln-292 of PurR into TAG terminal codon. To determine the effect of the three amino acid residues on regulatory function of PurR protein 5 different kinds of tRNA suppressor genes, Su3, Su4, Su6, Su7 and Su9 were used for creating the PurR protein variants with single amino acid substitution. The results indicated that Cys, Glu, Gly, His and Arg which substituted Trp-147 respectively all could not recover the regulation function of PurR. It confirmed that Trp-147 is a critical amino acid for the PurR function. Gln-292 substituted respectively by the same amino acids also could not recover the PurR function, demonstrating that Gln-292 is also an important amino acid residue in PurR.

  19. Amino Acid Residues in the GIY-YIG Endonuclease II of Phage T4 Affecting Sequence Recognition and Binding as Well as Catalysis▿ †

    Lagerbäck, Pernilla; Carlson, Karin

    2008-01-01

    Phage T4 endonuclease II (EndoII), a GIY-YIG endonuclease lacking a carboxy-terminal DNA-binding domain, was subjected to site-directed mutagenesis to investigate roles of individual amino acids in substrate recognition, binding, and catalysis. The structure of EndoII was modeled on that of UvrC. We found catalytic roles for residues in the putative catalytic surface (G49, R57, E118, and N130) similar to those described for I-TevI and UvrC; in addition, these residues were found to be important for substrate recognition and binding. The conserved glycine (G49) and arginine (R57) were essential for normal sequence recognition. Our results are in agreement with a role for these residues in forming the DNA-binding surface and exposing the substrate scissile bond at the active site. The conserved asparagine (N130) and an adjacent proline (P127) likely contribute to positioning the catalytic domain correctly. Enzymes in the EndoII subfamily of GIY-YIG endonucleases share a strongly conserved middle region (MR, residues 72 to 93, likely helical and possibly substituting for heterologous helices in I-TevI and UvrC) and a less strongly conserved N-terminal region (residues 12 to 24). Most of the conserved residues in these two regions appeared to contribute to binding strength without affecting the mode of substrate binding at the catalytic surface. EndoII K76, part of a conserved NUMOD3 DNA-binding motif of homing endonucleases found to overlap the MR, affected both sequence recognition and catalysis, suggesting a more direct involvement in positioning the substrate. Our data thus suggest roles for the MR and residues conserved in GIY-YIG enzymes in recognizing and binding the substrate. PMID:18539732

  20. Amino acid residues in the GIY-YIG endonuclease II of phage T4 affecting sequence recognition and binding as well as catalysis.

    Lagerbäck, Pernilla; Carlson, Karin

    2008-08-01

    Phage T4 endonuclease II (EndoII), a GIY-YIG endonuclease lacking a carboxy-terminal DNA-binding domain, was subjected to site-directed mutagenesis to investigate roles of individual amino acids in substrate recognition, binding, and catalysis. The structure of EndoII was modeled on that of UvrC. We found catalytic roles for residues in the putative catalytic surface (G49, R57, E118, and N130) similar to those described for I-TevI and UvrC; in addition, these residues were found to be important for substrate recognition and binding. The conserved glycine (G49) and arginine (R57) were essential for normal sequence recognition. Our results are in agreement with a role for these residues in forming the DNA-binding surface and exposing the substrate scissile bond at the active site. The conserved asparagine (N130) and an adjacent proline (P127) likely contribute to positioning the catalytic domain correctly. Enzymes in the EndoII subfamily of GIY-YIG endonucleases share a strongly conserved middle region (MR, residues 72 to 93, likely helical and possibly substituting for heterologous helices in I-TevI and UvrC) and a less strongly conserved N-terminal region (residues 12 to 24). Most of the conserved residues in these two regions appeared to contribute to binding strength without affecting the mode of substrate binding at the catalytic surface. EndoII K76, part of a conserved NUMOD3 DNA-binding motif of homing endonucleases found to overlap the MR, affected both sequence recognition and catalysis, suggesting a more direct involvement in positioning the substrate. Our data thus suggest roles for the MR and residues conserved in GIY-YIG enzymes in recognizing and binding the substrate. PMID:18539732

  1. Practical starter pig amino acid requirements in relation to immunity, gut health and growth performance

    Bob Goodband; Mike Tokach; Steve Dritz; Joel DeRouchey; Jason Woodworth

    2014-01-01

    Immune system activation begins a host of physiological responses. Infectious agents are recognized by monocytes and macrophages which in turn stimulate cytokine production. It is the hormone-like factors called cytokines that orchestrate the immune response. The classic responses observed with immune system activation and cytokine production include:anorexia, fever, lethargy, recruitment of other immune cells, and phagocytosis. While production of immune system components is known to require some amino acids, increases in amino acid requirements are more than offset by the associated decrease in protein accretion and increased muscle protein degradation that also accompanies immune system activation. However, the biggest impact of cytokine production is a decrease in feed intake. Therefore, as feed intake decreases, the energy needed to drive protein synthesis is also decreased. This suggests that diets should still be formulated on a similar calorie:lysine ratio as those formulated for non-immune challenged pigs. The evidence is sparse or equivocal for increasing nutrient requirements during an immune chal enge. Nutritionists and swine producers should resist the pressure to alter the diet, limit feed, or add expensive feed additives during an immune chal enge. While immune stimulation does not necessitate changes in diet formulation, when pigs are challenged with non-pathogenic diarrhea there are potential advantages on gut health with the increased use of crystal ine amino acids rather than intact protein sources (i.e., soybean meal). This is because reducing crude protein decreases the quantity of fermentable protein entering the large intestine, which lowers post weaning diarrhea. It also lowers the requirement for expensive specialty protein sources or other protein sources such as soybean meal that present immunological chal enges to the gut. The objective of this review is two-fold. The first is to discuss immunity by nutrition interactions, or lack

  2. Amino acid residue Y196E substitution and C-terminal peptide synergistically alleviate the toxicity of Clostridium perfringens epsilon toxin.

    Yao, Wenwu; Kang, Lin; Gao, Shan; Zhuang, Xiangjin; Zhang, Tao; Yang, Hao; Ji, Bin; Xin, Wenwen; Wang, Jinglin

    2015-06-15

    Epsilon toxin (ETX) is produced by Clostridium perfringens type B and D strains, and is the causative agent of a lethal enterotoxemia in livestock animals and possibly in humans. However, many details of ETX structure and activity are not known. Therefore, it is important to clarify the relationship between ETX structure and activity. To explore the effect and mechanism of ETX amino acid residue Y196E substitution and C-terminal peptide on toxicity, four recombinant proteins, rETX (without 13 N-terminal peptides and 23 C-terminal peptides), rETX-C (rETX with 23 C-terminal peptides), rETX(Y196E) (rETX with an amino acid residue substitution at Y196) and rETX(Y196E)-C (rETX-C with a Y196E mutation), were constructed in this study. Both the amino acid residue Y196E substitution and the C-terminal peptide reduce ETX toxicity to a similar extent, and the two factors synergistically alleviate ETX toxicity. In addition, we demonstrated that the C-terminal peptides and Y196E amino acid mutation reduce the toxin toxicity in two different pathways: the C-terminal peptides inhibit the binding activity of toxins to target cells, and the Y196E amino acid mutation slightly inhibits the pore-forming or heptamer-forming process. Interaction between the two factors was not observed in pore-forming or binding assays but toxicity assays, which demonstrated that the relationship between domains of the toxin is more complicated than previously appreciated. However, the exact mechanism of synergistic action is not yet clarified. PMID:25912943

  3. Lysine residues K66, K109, and K110 in the bovine foamy virus transactivator protein are required for transactivation and viral replication.

    Zhang, Suzhen; Cui, Xiaoxu; Li, Jing; Liang, Zhibin; Qiao, Wentao; Tan, Juan

    2016-04-01

    Bovine foamy virus (BFV) is a complex retrovirus that infects cattle. Like all retroviruses, BFV encodes a transactivator Tas protein (BTas) that increases gene transcription from viral promoters. BFV encodes two promoters that can interact with BTas, a conserved promoter in the 5' long terminal repeat (LTR) and a unique internal promoter (IP). Our previous study showed that BTas is acetylated by p300 at residues K66, K109, and K110, which markedly enhanced the ability of BTas to bind to DNA. However, whether these residues are important for BFV replication was not determined. Therefore, in this study we provide direct evidence that BTas is required for BFV replication and demonstrate that residues K66, K109, and K110 are critical for BTas function and BFV replication. Full-length infectious clones were generated, which were BTas deficient or contained lysine to arginine (K→R) mutations at position 66, 109, and/or 110. In vivo data indicated that K→R mutations at positions 66, 109, and 110 in BTas impaired transactivation of both the LTR and IP promoters. In addition, the K→R mutations in full-length infectious clones reduced expression of viral proteins, and the triple mutant and BTas deletion completely abrogated viral replication. Taken together, these results indicate that lysine residues at positions 66, 109, and 110 in the BTas protein are crucial for BFV replication and suggest a potential role for BTas acetylation in regulating the viral life cycle. PMID:26980333

  4. Long-chain polyunsaturated fatty acid (LCPUFA requirement for brain development: A personal view

    Gibson Robert A

    2016-01-01

    Full Text Available Dietary docosahexaenoic acid (DHA is known to accumulate in the infant brain and clinical trials have established that dietary DHA is associated with improvements in visual and neural function in preterm infants. Thus, an elevated DHA status is considered to be important throughout infancy for brain development. While DHA can be added directly to infant foods, there have been important studies to show that infants can partially meet their own DHA requirements by consuming adequate levels of omega 3 alpha linolenic acid (ALA. A key requirement to allow for the conversion of ALA to DHA and to maximise its incorporation into tissues is a diet that is also low in omega 6 linoleic acid (LA. Such diets are hard to find commercially because dietary guidelines dictate that ~3% energy of infant diets should be in the form of LA. These estimates were based on early animal experiments in which basal diets were devoid of both LA and ALA. However, recent animal experiments have indicated that the level of LA required to avoid essential fatty acid deficiency is much lower when ALA is also present in the diet. When a wide range diets are evaluated in animal systems, it is possible to see that the level of DHA found in the blood of animals fed diets containing only LA and ALA can reach levels similar to that of animals fed diets rich in fish oil, but only when the ALA:LA ratio is high and the total amount of dietary polyunsaturated fatty acids (PUFA is low. Diets that are rich in either monounsaturates or saturates meet these requirements. Importantly, there are human infant studies that have tested such diets and demonstrated that human infants accumulate greater amounts of DHA than when diets are high in LA. It might be time to reconsider the dietary requirement of the two essential fatty acids LA and ALA in human infants in terms of their ability to enhance endogenous synthesis of DHA rather than more adult biomarkers like cholesterol levels.

  5. Sequence and structural features of binding site residues in protein-protein complexes: comparison with protein-nucleic acid complexes

    Selvaraj S; Jayaram B; Saranya N; Gromiha M; Fukui Kazuhiko

    2011-01-01

    Abstract Background Protein-protein interactions are important for several cellular processes. Understanding the mechanism of protein-protein recognition and predicting the binding sites in protein-protein complexes are long standing goals in molecular and computational biology. Methods We have developed an energy based approach for identifying the binding site residues in protein–protein complexes. The binding site residues have been analyzed with sequence and structure based parameters such...

  6. Current issues in determining dietary protein and amino-acid requirements

    Pencharz, P; Jahoor, F; Kurpad, A;

    2014-01-01

    Pregnancy and the first two years of life are periods of rapid growth and yet the knowledge of requirements for protein and dietary indispensable amino acids is very limited. The development of carbon oxidation methods opens the way to studies that should fill these important gaps in knowledge.Eu.......European Journal of Clinical Nutrition advance online publication, 15 January 2014; doi:10.1038/ejcn.2013.297....

  7. Effect of Extraction Conditions on the Saccharide (Neutral and Acidic) Composition of the Crude Pectic Extract from Various Agro-Industrial Residues.

    Babbar, Neha; Roy, Sandra Van; Wijnants, Marc; Dejonghe, Winnie; Caligiani, Augusta; Sforza, Stefano; Elst, Kathy

    2016-01-13

    The influence of different extraction methodologies was assessed on the composition of both neutral (arabinose, rhamnose, galactose) and acidic (galacturonic acid) pectic polysaccharides obtained from four agro-industrial residues, namely, berry pomace (BP), onion hulls (OH), pressed pumpkin (PP), and sugar beet pulp (SBP). For acidic pectic polysaccharides, the extraction efficiency was obtained as BP (nitric acid-assisted extraction, 2 h, 62.9%), PP (enzymatic-assisted extraction, 12 h, 75.0%), SBP (enzymatic-assisted extraction, 48 h, 89.8%; and nitric acid-assisted extraction, 4 h, 76.5%), and OH (sodium hexametaphosphate-assisted extraction, 0.5 h, 100%; and ammonium oxalate-assisted extraction, 0.5 h, 100%). For neutral pectic polysaccharides, the following results were achieved: BP (enzymatic-assisted extraction, 24 h, 85.9%), PP (nitric acid-assisted extraction, 6 h, 82.2%), and SBP (enzymatic assisted extraction, 48 h, 97.5%; and nitric acid-assisted extraction, 4 h, 83.2%). On the basis of the high recovery of pectic sugars, SBP and OH are interesting candidates for the further purification of pectin and production of pectin-derived products. PMID:26652767

  8. Studies on the protein and sulfur amino acid requirements of young bobwhite quail

    Serafin, J.A.

    1977-01-01

    Four experiments were conducted with purified diets to examine the influence of protein level and to estimate the sulfur amino acid (S.A.A.) requirement of young Bobwhite quail (Colinus virginianus). These studies demonstrated (I) that 26% protein was sufficient for rapid growth when the diet was supplemented with methionine; (2) that diets containing higher levels of protein (29.3% and 31.3%) failed to support satisfactory growth unless they contained supplemental methionine; and (3) that young Bobwhite quail require no more than 1.0% sulfur-containing amino acids for optimal growth and efficiency of feed utilization. A fifth experiment was conducted to examine the protein and S.A.A. requirements of young Bobwhite quail using practical rations and to compare results with those obtained with purified diets. Diets containing 24%, 26% and 28% protein were supplied with and without supplemental methionine in a five week study. Results showed significant growth responses to protein and supplemental methionine. Responses showed that Bobwhite quail require no more than 26% protein for maximum growth and efficiency of feed utilization when the S.A.A. level of the diet was approximately 1.0%. The results were in close agreement with those obtained with purified diets. These findings define more precisely than had been known the quantitative requirements of young Bobwhite quail for protein and for the S.A.A. necessary for optimal growth.

  9. An Aromatic Side Chain Is Required at Residue 8 of SU for Fusion of Ecotropic Murine Leukemia Virus

    Qian, Zhaohui; Albritton, Lorraine M.

    2004-01-01

    The surface glycoprotein (SU) of most gammaretroviruses contains a conserved histidine at its amino terminus. In ecotropic murine leukemia virus SU, replacement of histidine 8 with arginine (H8R) or deletion of H8 (H8del) abolishes infection and cell-cell fusion but has no effect on binding to the cellular receptor. We report here that an aromatic ring side chain is essential to the function of residue 8. The size of the aromatic ring appears to be important, as does its ability to form a hyd...

  10. A first estimate of the amino acid requirement for milk production of the high-producing female mink (Mustela vison)

    Fink, R; Tauson, A-H; Chwalibog, André;

    2006-01-01

    to estimate the amino acid requirement of the lactating mink. Twelve dams were held in an intensive care unit and subjected to balance experiments and the kits were injected with deuterium oxide to determine water kinetics and milk yield. Eighteen dams were kept under normal farm conditions but with feed...... costs for glucose production through less amino acids being used in gluconeogenesis. In conclusion, the improved performance of dams fed the LP diet suggested that their requirement of essential amino acids and non-specific N were covered, and the requirement of digestible amino acids of lactating mink...

  11. Three Basic Residues of Intracellular Loop 3 of the Beta-1 Adrenergic Receptor Are Required for Golgin-160-Dependent Trafficking

    Catherine E. Gilbert

    2014-02-01

    Full Text Available Golgin-160 is a member of the golgin family of proteins, which have been implicated in the maintenance of Golgi structure and in vesicle tethering. Golgin-160 is atypical; it promotes post-Golgi trafficking of specific cargo proteins, including the β-1 adrenergic receptor (β1AR, a G protein-coupled receptor. Here we show that golgin-160 binds directly to the third intracellular loop of β1AR and that this binding depends on three basic residues in this loop. Mutation of the basic residues does not affect trafficking of β1AR from the endoplasmic reticulum through the Golgi complex, but results in reduced steady-state levels at the plasma membrane. We hypothesize that golgin-160 promotes incorporation of β1AR into specific transport carriers at the trans-Golgi network to ensure efficient delivery to the cell surface. These results add to our understanding of the biogenesis of β1AR, and suggest a novel point of regulation for its delivery to the plasma membrane.

  12. Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach

    Mayer Kimberly M

    2007-01-01

    Full Text Available Abstract Background The large amount of available sequence information for the plant acyl-ACP thioesterases (TEs made it possible to use a bioinformatics-guided approach to identify amino acid residues involved in substrate specificity. The Conserved Property Difference Locator (CPDL program allowed the identification of putative specificity-determining residues that differ between the FatA and FatB TE classes. Six of the FatA residue differences identified by CPDL were incorporated into the FatB-like parent via site-directed mutagenesis and the effect of each on TE activity was determined. Variants were expressed in E. coli strain K27 that allows determination of enzyme activity by GCMS analysis of fatty acids released into the medium. Results Substitutions at four of the positions (74, 86, 141, and 174 changed substrate specificity to varying degrees while changes at the remaining two positions, 110 and 221, essentially inactivated the thioesterase. The effects of substitutions at positions 74, 141, and 174 (3-MUT or 74, 86, 141, 174 (4-MUT were not additive with respect to specificity. Conclusion Four of six putative specificity determining positions in plant TEs, identified with the use of CPDL, were validated experimentally; a novel colorimetric screen that discriminates between active and inactive TEs is also presented.

  13. Mapping of amino acid residues responsible for adhesion of cell culture-adapted foot-and-mouth disease SAT type viruses.

    Maree, Francois F; Blignaut, Belinda; de Beer, Tjaart A P; Visser, Nico; Rieder, Elizabeth A

    2010-10-01

    Foot-and-mouth disease virus (FMDV) infects host cells by adhering to the alpha(V) subgroup of the integrin family of cellular receptors in a Arg-Gly-Asp (RGD) dependent manner. FMD viruses, propagated in non-host cell cultures are reported to acquire the ability to enter cells via alternative cell surface molecules. Sequencing analysis of SAT1 and SAT2 cell culture-adapted variants showed acquisition of positively charged amino acid residues within surface-exposed loops of the outer capsid structural proteins. The fixation of positively charged residues at position 110-112 in the beta F-beta G loop of VP1 of SAT1 isolates is thought to correlate with the acquisition of the ability to utilise alternative glycosaminoglycan (GAG) molecules for cell entry. Similarly, two SAT2 viruses that adapted readily to BHK-21 cells accumulated positively charged residues at positions 83 and 85 of the beta D-beta E loop of VP1. Both regions surround the fivefold axis of the virion. Recombinant viruses containing positively charged residues at position 110 and 112 of VP1 were able to infect CHO-K1 cells (that expresses GAG) and demonstrated increased infectivity in BHK-21 cells. Therefore, recombinant SAT viruses engineered to express substitutions that induce GAG-binding could be exploited in the rational design of vaccine seed stocks with improved growth properties in cell cultures. PMID:20637812

  14. Does gasification and biochar amendment provide a viable solution to balance greenhouse gas emissions, energy requirements and orchard residue management?

    Pereira, Engil; Suddick, Emma; Six, Johan

    2015-04-01

    By converting biomass residue to biochar, we can generate power cleanly and sequester carbon resulting in overall greenhouse gas (GHG) savings when compared to typical fossil fuel burning and waste disposal. This on-farm research study provides a long-term and high frequency assessment of GHG emissions from biochar amended-soils in an organic walnut orchard in the Central Valley of California, USA. We also estimated the GHG offsets from the conversion of walnut residue into energy through gasification at the on-site walnut processing plant. Soil fluxes of carbon dioxide (CO2) and nitrous oxide (N2O) were monitored over 29 months in a 3.6 ha walnut orchard following management and precipitation events. We compared four treatments: control, biochar, compost, and biochar combined with compost. Events involving resource inputs such as fertilization or cover crop mowing induced the largest N2O peaks with average 0.13 kg N2O-N ha-1 day-1, while precipitation events produced the highest CO2 fluxes in average 0.124 Mg CO2-C ha-1 day-1. Biochar alone decreased N2O fluxes in two out of 23 measured events, however, not with enough significant magnitude to modify annual or seasonal totals. This indicates that biochar-induced decreases in N2O fluxes may occasionally occur without significant changes in total emissions. Additionally, biochar alone or in combination with compost did not alter annual or seasonal cumulative CO2 emissions. For this particular study, the conversion of orchard waste into energy and C sequestration through biochar amendment offset 100.3 Mg CO2-Ceq year-1. Thus, given that biochar did not alter cumulative GHG emissions from soils, we conclude that, in the scenario of this study, the use of biochar as a strategy to decrease farm-level GHG emissions is obtained through the gasification of orchard residue into energy and through biochar C sequestration, and not as a tool to decrease soil CO2 and N2O emissions.

  15. Genetic and functional analyses of the oeX174 DNA binding protein: the effects of substitutions for amino acid residues that spatially organize the two DNA binding domains

    The oeX174 DNA binding protein contains two DNA binding domains, containing a series of DNA binding basic amino acids, separated by a proline-rich linker region. Within each DNA binding domain, there is a conserved glycine residue. Glycine and proline residues were mutated and the effects on virion structure were examined. Substitutions for glycine residues yield particles with similar properties to previously characterized mutants with substitutions for DNA binding residues. Both sets of mutations share a common extragenic second-site suppressor, suggesting that the defects caused by the mutant proteins are mechanistically similar. Hence, glycine residues may optimize DNA-protein contacts. The defects conferred by substitutions for proline residues appear to be fundamentally different. The properties of the mutant particles along with the atomic structure of the virion suggest that the proline residues may act to guide the packaged DNA to the adjacent fivefold related asymmetric unit, thus preventing a chaotic packaging arrangement

  16. 常压渣油及其悬浮床加氢尾油中的酸性含氧化合物%Acid Oxygenated Compounds in Residue and Slurry-Bed Hydrocracking Cracked Residue

    文萍; 李传; 李庶峰; 邓文安

    2011-01-01

    以克拉玛依炼油厂常压渣油为原料,在重质油加氢实验室中试装置进行悬浮床加氢反应:反应时间为1h,催化荆质量分数为800 μg/g,反应温度分别为410,430,440℃.对反应后产物进行常减压蒸馏.采用柱色谱法对克炼常渣及其悬浮床加氢尾油进行四组分分离,测定了各组分的酸值,考察了常压渣油及其悬浮床加氢尾油中酸性含氧化合物的分布情况.研究结果表明,克炼常渣中的酸性含氧化合物主要分布在胶质中;克炼常渣悬浮床加氢尾油中的酸性含氧化合物主要分布在胶质和沥青质中.%Karamay atmospheric residue(KLAR) was hydrocracked in pilot experimental device of state key laboratory of heavy oil processing, with the presence of an oil - soluble catalyst. Reaction time was set to 1 h and reaction catalyst concentration at 800 μg/g, reaction temperature were 410,430 and 450 ℃. Then KLAR and its slurry-bed hydrocracking cracked residue were separated into four fractions. The acid number in each fraction was determined. The distribution of the acid oxygenated compounds in each material was obtained. The results show that the acid oxygenated compounds in KLAR are mainly distributed in resin, which in slurry-bed hydrocracking cracked residue of KLAR are mainly distributed in resin and asphaltene.

  17. Advanced treatment of residual nitrogen from biologically treated coke effluent by a microalga-mediated process using volatile fatty acids (VFAs) under stepwise mixotrophic conditions.

    Ryu, Byung-Gon; Kim, Woong; Heo, Sung-Woon; Kim, Donghyun; Choi, Gang-Guk; Yang, Ji-Won

    2015-09-01

    This work describes the development of a microalga-mediated process for simultaneous removal of residual ammonium nitrogen (NH4(+)-N) and production of lipids from biologically treated coke effluent. Four species of green algae were tested using a sequential mixotrophic process. In the first phase-CO2-supplied mixotrophic condition-all microalgae assimilated NH4(+)-N with no evident inhibition. In second phase-volatile fatty acids (VFAs)-supplied mixotrophic condition-removal rates of NH4(+)-N and biomass significantly increased. Among the microalgae used, Arctic Chlorella sp. ArM0029B had the highest rate of NH4(+)-N removal (0.97 mg/L/h) and fatty acid production (24.9 mg/L/d) which were 3.6- and 2.1-fold higher than those observed under the CO2-supplied mixotrophic condition. Redundancy analysis (RDA) indicated that acetate and butyrate were decisive factors for increasing NH4(+)-N removal and fatty acid production. These results demonstrate that microalgae can be used in a sequential process for treatment of residual nitrogen after initial treatment of activated sludge. PMID:25881553

  18. Direct fermentation of potato starch and potato residues to lactic acid by Geobacillus stearothermophilus under non-sterile conditions

    Smerilli, Marina; Neureiter, Markus; Wurz, Stefan; Haas, Cornelia; Frühauf, Sabine; Fuchs, Werner

    2015-01-01

    BACKGROUND Lactic acid is an important biorefinery platform chemical. The use of thermophilic amylolytic microorganisms to produce lactic acid by fermentation constitutes an efficient strategy to reduce operating costs, including raw materials and sterilization costs. RESULTS A process for the thermophilic production of lactic acid by Geobacillus stearothermophilus directly from potato starch was characterized and optimized. Geobacillus stearothermophilus DSM 494 was selected out of 12 strain...

  19. Evaluation of the Protein Requirement in Chinese Young Adults Using the Indicator Amino Acid Oxidation Technique

    LI Min; ZHANG Yu Hui; WANG Zhi Ling; GOU Ling Yan; LI Wei Dong; TIAN Yuan; HU Yi Chun; WANG Rui; PIAO Jian Hua; YANG Xiao Guang

    2013-01-01

    Objective To accurately calculate the protein requirements in Chinese young adults using the indicator amino acid oxidation technique. Methods Nine women and ten men received a restricted daily level of protein intake (0.75, 0.82, 0.89, 0.97, and 1.05 g/kg), along with L-[1-13C]-leucine. Subjects’ protein requirement was determined by a biphasic linear regression crossover analysis of F13CO2 data. In doing so, a breakpoint at the minimal rate of appearance of 13CO2 expiration specific to each level of dietary protein was identified. This trial was registered with the Chinese clinical trial registry as ChiCTR-ONC-11001407. Results The Estimated Average Requirement (EAR) and the Recommended Nutrient Intake (RNI) of protein for healthy Chinese young adults were determined to be 0.87 and 0.98 g/(kg·d), respectively, based on the indicator amino acid oxidation technique. Conclusion The EAR and RNI of mixed protein are 5% and 16% that are lower than the current proposed EAR and RNI (0.92 and 1.16 g/(kg·d), respectively), as determined by the nitrogen balance method. The respective EAR and RNI recommendations of 0.87 and 0.98 g/(kg·d) of mixed protein are estimated to be reasonable and suitable for Chinese young adults.

  20. Entry of Bluetongue Virus Capsid Requires the Late Endosome-specific Lipid Lysobisphosphatidic Acid.

    Patel, Avnish; Mohl, Bjorn-Patrick; Roy, Polly

    2016-06-01

    The entry of viruses into host cells is one of the key processes of infection. The mechanisms of cellular entry for enveloped virus have been well studied. The fusion proteins as well as the facilitating cellular lipid factors involved in the viral fusion entry process have been well characterized. The process of non-enveloped virus cell entry, in comparison, remains poorly defined, particularly for large complex capsid viruses of the family Reoviridae, which comprises a range of mammalian pathogens. These viruses enter cells without the aid of a limiting membrane and thus cannot fuse with host cell membranes to enter cells. Instead, these viruses are believed to penetrate membranes of the host cell during endocytosis. However, the molecular mechanism of this process is largely undefined. Here we show, utilizing an in vitro liposome penetration assay and cell biology, that bluetongue virus (BTV), an archetypal member of the Reoviridae, utilizes the late endosome-specific lipid lysobisphosphatidic acid for productive membrane penetration and viral entry. Further, we provide preliminary evidence that lipid lysobisphosphatidic acid facilitates pore expansion during membrane penetration, suggesting a mechanism for lipid factor requirement of BTV. This finding indicates that despite the lack of a membrane envelope, the entry process of BTV is similar in specific lipid requirements to enveloped viruses that enter cells through the late endosome. These results are the first, to our knowledge, to demonstrate that a large non-enveloped virus of the Reoviridae has specific lipid requirements for membrane penetration and host cell entry. PMID:27036941

  1. Lactobacillus gasseri requires peptides, not proteins or free amino acids, for growth in milk.

    Arakawa, K; Matsunaga, K; Takihiro, S; Moritoki, A; Ryuto, S; Kawai, Y; Masuda, T; Miyamoto, T

    2015-03-01

    Lactobacillus gasseri is a widespread commensal lactic acid bacterium inhabiting human mucosal niches and has many beneficial effects as a probiotic. However, L. gasseri is difficult to grow in milk, which hurts usability for the food industry. It had been previously reported that supplementation with yeast extract or proteose peptone, including peptides, enables L. gasseri to grow well in milk. In this study, our objective was to confirm peptide requirement of L. gasseri and evaluate efficacy of peptide release by enzymatic proteolysis on growth of L. gassei in milk. Three strains of L. gasseri did not grow well in modified DeMan, Rogosa, Sharpe broth without any nitrogen sources (MRS-N), but addition of a casein-derived peptide mixture, tryptone, promoted growth. In contrast, little effect was observed after adding casein or a casein-derived amino acid mixture, casamino acids. These results indicate that L. gasseri requires peptides, not proteins or free amino acids, among milk-derived nitrogen sources for growth. Lactobacillus gasseri JCM 1131T hardly had growth capacity in 6 kinds of milk-based media: bovine milk, human milk, skim milk, cheese whey, modified MRS-N (MRSL-N) supplemented with acid whey, and MRSL-N supplemented with casein. Moreover, treatment with digestive proteases, particularly pepsin, to release peptides made it grow well in each milk-based medium. The pepsin treatment was the most effective for growth of strain JCM 1131T in skim milk among the tested food-grade proteases such as trypsin, α-chymotrypsin, calf rennet, ficin, bromelain, and papain. As well as strain JCM 1131T, pepsinolysis of milk improved growth of other L. gasseri strains and some strains of enteric lactobacilli such as Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus johnsonii, and Lactobacillus reuteri. These results suggest that some relatives of L. gasseri also use peptides as desirable nitrogen sources, and that milk may be a good supplier of nutritious

  2. Estimates of methionine and sulfur amino acid requirements for laying hens using different models

    AA Saki

    2012-09-01

    Full Text Available This experiment was conducted to evaluate the effects of dietary methionine (Met content on the performance of white commercial laying hens and to determine Met and total sulfur amino acids requirements (TSAA. These requirements were estimated using three statistical models (broken-line regression, exponential and second order equations to evaluate their abilit to determine amino acid requirements. A total of 216 laying hens (23 wks of age was used in a completely randomized design (CRD with six treatments with four replicates of nine birds each. The basal diet contained 15.25% crude protein, 2830.16 kcal/kg ME and 0.24% Met. Synthetic DL-Met was added to the deficient (basal diet in 0.05% increments to make the other five experimental diets (0.29, 0.34, 0.39, 0.44 and 0.49% Met. Increasing Met level from 0.24 to 0.34% significantly increased egg production, egg weight, egg mass, egg content, and feed intake and decreased feed conversion ratio (p<0.05. However, further Met increases, from 0.34 to 0.49%, no longer influenced these parameters. Out of the three models, the broken-line regression model presented better estimates of AA requirements. Based on broken-line equations, average Met and TSAA requirements of the laying hens were 0.31 and 0.60% (245.50 and 469.25 mg/hen/day from 22 to 36 wks of age, respectively.

  3. TmiRUSite and TmiROSite scripts: searching for mRNA fragments with miRNA binding sites with encoded amino acid residues

    Berillo, Olga; Régnier, Mireille; Ivashchenko, Anatoly

    2014-01-01

    microRNAs are small RNA molecules that inhibit the translation of target genes. microRNA binding sites are located in the untranslated regions as well as in the coding domains. We describe TmiRUSite and TmiROSite scripts developed using python as tools for the extraction of nucleotide sequences for miRNA binding sites with their encoded amino acid residue sequences. The scripts allow for retrieving a set of additional sequences at left and at right from the binding site. The scripts presents ...

  4. The Role of Acidic Residues and of Sodium Ion Adduction on the Gas-Phase H/D Exchange of Peptides and Peptide Dimers

    Jurchen, John C.; Cooper, Russell E.; Williams, Evan R.

    2003-01-01

    Gas-phase H/D exchange is widely used for characterizing the structure of ions. However, many structural parameters that affect the rate of H/D exchange are poorly understood, which complicates the interpretation of experimental data. Here, the effects of sodium ion adduction on the rate of H/D exchange with D2O for a series of peptides and peptide dimers with varying numbers of acidic residues are described. The maximum number of sodium ion adducts that can be accommodated by the peptides an...

  5. Replacement of the N-terminal tyrosine residue in opioid peptides with 3-(2,6-dimethyl-4-carbamoylphenyl)propanoic acid (Dcp) results in novel opioid antagonists.

    Lu, Yixin; Lum, Tze Keong; Leow Augustine, Yoon Wui; Weltrowska, Grazyna; Nguyen, Thi M-D; Lemieux, Carole; Chung, Nga N; Schiller, Peter W

    2006-08-24

    3-(2,6-Dimethyl-4-carbamoylphenyl)propanoic acid (Dcp), a 2',6'-dimethyltyrosine analogue containing a carbamoyl group in place of the hydroxyl function and lacking the amino group, was synthesized. The replacement of Tyr1 in an enkephalin analogue and in dynorphin A(1-11)-NH2 with Dcp resulted in the first opioid peptide-derived antagonists that do not contain a phenolic hydroxyl group at the 1-position residue. The cyclic peptide Dcp-c[D-Cys-Gly-Phe(pNO2)-D-Cys]NH2 represents a novel, potent mu opioid antagonist. PMID:16913729

  6. Rapid determination of phenoxy acid residues in rice by modified QuEChERS extraction and liquid chromatography-tandem mass spectrometry

    A new method for the analysis of phenoxy acid herbicide residues in rice, based on the use of liquid extraction/partition and dispersive solid phase extraction (dispersive-SPE) followed by ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS), is reported. 5% (v/v) formic acid in acetonitrile as the extraction solvent and inclusion of citrate buffer helped partitioning of all the analytes into the acetonitrile phase. The extract was then cleaned up by dispersive-SPE using C18 and alumina neutral as selective sorbents. Further optimization of sample preparation and determination allowed recoveries of between 45 and 104% for all 13 phenoxy acid herbicides with RSD values lower than 13.3% at 5.0 μg kg-1 concentration level. Limit of detections (LODs) of 0.5 μg kg-1 or below were attained for all 13 phenoxy acids. Quantitative analysis was done in the multiple-reaction monitoring (MRM) mode using two combinations of selected precursor ion and product ion transition for each compound. This developed method produced relatively higher recoveries of the acid herbicides with a smaller range of variation and less susceptibility to matrix effects, than the original QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method

  7. Hereditary folate malabsorption: A positively charged amino acid at position 113 of the proton-coupled folate transporter (PCFT/SLC46A1) is required for folic acid binding

    The proton-coupled folate transporter (PCFT/SLC46A1) mediates intestinal folate uptake at acidic pH. Some loss of folic acid (FA) transport mutations in PCFT from hereditary folate malabsorption (HFM) patients cluster in R113, thereby suggesting a functional role for this residue. Herein, unlike non-conservative substitutions, an R113H mutant displayed 80-fold increase in the FA transport Km while retaining parental Vmax, hence indicating a major fall in folate substrate affinity. Furthermore, consistent with the preservation of 9% of parental transport activity, R113H transfectants displayed a substantial decrease in the FA growth requirement relative to mock transfectants. Homology modeling based on the crystal structures of the Escherichia coli transporter homologues EmrD and glycerol-3-phosphate transporter revealed that the R113H rotamer properly protrudes into the cytoplasmic face of the minor cleft normally occupied by R113. These findings constitute the first demonstration that a basic amino acid at position 113 is required for folate substrate binding.

  8. Hereditary folate malabsorption: A positively charged amino acid at position 113 of the proton-coupled folate transporter (PCFT/SLC46A1) is required for folic acid binding

    Lasry, Inbal; Berman, Bluma [The Fred Wyszkowski Cancer Research Laboratory, Dept. of Biology, Technion-Israel Institute of Technology, Haifa 32000 (Israel); Glaser, Fabian [Bioinformatics Knowledge Unit, The Lorry I. Lokey Interdisciplinary Center for Life Sciences and Engineering, Technion, Haifa 32000 (Israel); Jansen, Gerrit [Department of Rheumatology, VU University Medical Center, Amsterdam (Netherlands); Assaraf, Yehuda G., E-mail: assaraf@tx.technion.ac.il [The Fred Wyszkowski Cancer Research Laboratory, Dept. of Biology, Technion-Israel Institute of Technology, Haifa 32000 (Israel)

    2009-08-28

    The proton-coupled folate transporter (PCFT/SLC46A1) mediates intestinal folate uptake at acidic pH. Some loss of folic acid (FA) transport mutations in PCFT from hereditary folate malabsorption (HFM) patients cluster in R113, thereby suggesting a functional role for this residue. Herein, unlike non-conservative substitutions, an R113H mutant displayed 80-fold increase in the FA transport Km while retaining parental Vmax, hence indicating a major fall in folate substrate affinity. Furthermore, consistent with the preservation of 9% of parental transport activity, R113H transfectants displayed a substantial decrease in the FA growth requirement relative to mock transfectants. Homology modeling based on the crystal structures of the Escherichia coli transporter homologues EmrD and glycerol-3-phosphate transporter revealed that the R113H rotamer properly protrudes into the cytoplasmic face of the minor cleft normally occupied by R113. These findings constitute the first demonstration that a basic amino acid at position 113 is required for folate substrate binding.

  9. Use of radioactive glucosamine in the perfused rat liver to prepare α1-acid glycoprotein (orosomucoid) with 3H- or 14C-labelled sialic acid and N-acetylglucosamine residues

    A method was developed whereby [1-14C]glucosamine was used in a perfused rat liver system to prepare over 2 mg of α1-acid glycoprotein with highly radioactive sialic acid and glucosamine residues. The liver secreted radioactive α1-acid glycoprotein over a 4-6 h period, and this glycoprotein was purified from the perfusate by chromatography on DEAE-cellulose at pH3.6. The sialic acid on the isolated glycoprotein had a specific radioactivity of 3.1 Ci/mol, whereas the glucosamine-specific radioactivity was 4.3 Ci/mole. The latter amino-sugar residues on the isolated protein were only 13-fold less radioactive than the initially added [1-14C]glucosamine. Orosomucoid with a specific radioactivity of 31.3 μCi/mg of protein was obtainable by using [6-3H]glucosamine. Many other radioactive glycoproteins were found to be secreted into the perfusate by the liver. Thus this experimental system should prove useful for obtaining other serum glycoproteins with highly radioactive sugar moieties. (author)

  10. Bromopyruvate, an active site-directed inactivator of E. coli 2-keto-4-hydroxyglutarate(KHG) aldolase, modifies glutamic acid residue-45

    E. coli KHG-aldolase (2-keto-4-hydroxyglutarate ↔ pyruvate + glyoxylate), a novel trimeric Class I aldolase, requires one active-site lysine residue (Lys 133)/subunit for Schiff-base formation as well as one arginine residue (Arg 49)/subunit for catalytic activity. The substrate analog, 3-bromopyruvate (BRPY), causes a time- and concentration-dependent loss of KHG-aldolase activity. This inactivation is regarded as active site-directed since: (a) BRPY modification results in complete loss of enzymatic activity; (b) saturation kinetics are exhibited, suggesting that a reversible complex is formed between the aldolase and BRPY prior to the rate-limiting inactivation step; (c) over 90% of the initial aldolase activity is protected by either substrate, pyruvate or KHG; (d) 1.1 mol of 14C-BRPY is bound/enzyme subunit. Peptide isolation and sequencing show that the incorporated radioactivity is associated with residue Glu-45. Denaturation of the enzyme with guanidine x HCl following treatment with excess 14C-BRPY allows for the incorporation of carbon-14 at Cys-159 and Cys-180 as well. The presence of pyruvate protects Glu-45 from being esterified but does not prevent the alkylation of the two cysteine residues. These results suggest that Glu-45 is essential for the catalytic activity of E. coli KHG-aldolase, most likely functioning as the active-site amphoteric proton donor/acceptor moiety that is involved in the overall mechanism of the reaction catalyzed by this enzyme