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Sample records for acid residues previously

  1. Autumn vegetable response to residual herbicides applied the previous spring under low density polyethylene mulch

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Field studies were conducted in 2002 and 2005 to evaluate autumn vegetable tolerance to residual herbicides applied the previous spring under low density polyethylene (LDPE) mulch. Spring applications of 1.12 kg/ha S-metolachlor, 0.027 kg/ha halosulfuron, 0.28 kg/ha sulfentrazone, and 1.12 kg/ha S...

  2. Anaesthetic management for caesarean section in a case of previously operated with residual pituitary tumour

    PubMed Central

    Shah, Prerana N; Sonawane, Darshana; Appukutty, Jithesh

    2011-01-01

    Successful anaesthetic management for caesarean section in a case with previous pituitary tumour resection, with residual tumour, is reported. The pituitary gland undergoes global hyperplasia during pregnancy. Functional pituitary tumours may exhibit symptomatic enlargement during pregnancy. Growth hormone secreting tumour is associated with acromegaly which has associated anaesthetic implications of difficult airway, systemic hypertension, and diabetes and electrolyte imbalance. Intracranial space occupying lesions can increase intra cranial pressure and compromise cerebral perfusion or cause herniation. We report management of this case. PMID:22223910

  3. A previously undescribed organic residue sheds light on heat treatment in the Middle Stone Age.

    PubMed

    Schmidt, Patrick; Porraz, Guillaume; Bellot-Gurlet, Ludovic; February, Edmund; Ligouis, Bertrand; Paris, Céline; Texier, Pierre-Jean; Parkington, John E; Miller, Christopher E; Nickel, Klaus G; Conard, Nicholas J

    2015-08-01

    South Africa has in recent years gained increasing importance for our understanding of the evolution of 'modern human behaviour' during the Middle Stone Age (MSA). A key element in the suite of behaviours linked with modern humans is heat treatment of materials such as ochre for ritual purposes and stone prior to tool production. Until now, there has been no direct archaeological evidence for the exact procedure used in the heat treatment of silcrete. Through the analysis of heat-treated artefacts from the Howiesons Poort of Diepkloof Rock Shelter, we identified a hitherto unknown type of organic residue - a tempering-residue - that sheds light on the processes used for heat treatment in the MSA. This black film on the silcrete surface is an organic tar that contains microscopic fragments of charcoal and formed as a residue during the direct contact of the artefacts with hot embers of green wood. Our results suggest that heat treatment of silcrete was conducted directly using an open fire, similar to those likely used for cooking. These findings add to the discussion about the complexity of MSA behaviour and appear to contradict previous studies that had suggested that heat treatment of silcrete was a complex (i.e., requiring a large number of steps for its realization) and resource-consuming procedure. PMID:26073074

  4. Residual Impact of Previous Injury on Musculoskeletal Characteristics in Special Forces Operators

    PubMed Central

    Parr, Jeffrey J.; Clark, Nicholas C.; Abt, John P.; Kresta, Julie Y.; Keenan, Karen A.; Kane, Shawn F.; Lephart, Scott M.

    2015-01-01

    Background Musculoskeletal injuries are a significant burden to United States Army Special Operations Forces. The advanced tactical skill level and physical training required of Army Special Operators highlights the need to optimize musculoskeletal characteristics to reduce the likelihood of suffering a recurrent injury. Purpose To identify the residual impact of previous injury on musculoskeletal characteristics. Study Design Cross-sectional study; Level of evidence, 3. Methods Isokinetic strength of the knee, shoulder, and back and flexibility of the shoulder and hamstrings were assessed as part of a comprehensive human performance protocol, and self-reported musculoskeletal injury history was obtained. Subjects were stratified based on previous history of low back, knee, or shoulder injury, and within-group and between-group comparisons were made for musculoskeletal variables. Results Knee injury analysis showed no significant strength or flexibility differences. Shoulder injury analysis found internal rotation strength of the healthy subjects (H) was significantly higher compared with injured (I) and uninjured (U) limbs of the injured group (H, 60.8 ± 11.5 percent body weight [%BW]; I, 54.5 ± 10.5 %BW; U, 55.5 ± 11.3 %BW) (P = .014 [H vs I] and P = .05 [H vs U]). The external rotation/internal rotation strength ratio was significantly lower in the healthy subjects compared with injured and uninjured limbs of the injured group (H, 0.653 ± 0.122; I, 0.724 ± 0.121; U, 0.724 ± 0.124) (P = .026 [H vs I] and P = .018 [H vs U]). Posterior shoulder tightness was significantly different between the injured and uninjured limb of the injured group (I, 111.6° ± 9.4°; U, 114.4° ± 9.3°; P = .008). The back injury analysis found no significant strength differences between the healthy and injured groups. Conclusion Few physical differences existed between operators with prior knee or back injury. However, operators with a previous history of shoulder injury

  5. 40 CFR 180.180 - Orthoarsenic acid; tolerance for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... residues. 180.180 Section 180.180 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... § 180.180 Orthoarsenic acid; tolerance for residues. (a) General. A tolerance that expires on July 1... exemptions. (c) Tolerances with regional registrations. (d) Indirect or inadvertent residues....

  6. 40 CFR 180.180 - Orthoarsenic acid; tolerance for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... residues. 180.180 Section 180.180 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... § 180.180 Orthoarsenic acid; tolerance for residues. (a) General. A tolerance that expires on July 1... exemptions. (c) Tolerances with regional registrations. (d) Indirect or inadvertent residues....

  7. 40 CFR 180.180 - Orthoarsenic acid; tolerance for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... residues. 180.180 Section 180.180 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... § 180.180 Orthoarsenic acid; tolerance for residues. (a) General. A tolerance that expires on July 1... exemptions. (c) Tolerances with regional registrations. (d) Indirect or inadvertent residues....

  8. Recovery of mercury from acid waste residues

    DOEpatents

    Greenhalgh, W.O.

    1987-02-27

    Mercury can be recovered from nitric acid-containing fluids by reacting the fluid with aluminum metal to produce mercury metal, and thence quenching the reactivity of the nitric acid prior to nitration of the mercury metal. 1 fig.

  9. Recovery of mercury from acid waste residues

    DOEpatents

    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. Recovery of mercury from acid waste residues

    DOEpatents

    Greenhalgh, Wilbur O.

    1989-12-05

    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.

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

    PubMed

    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. Fatty Acid Structure and Degradation Analysis in Fingerprint Residues

    NASA Astrophysics Data System (ADS)

    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.

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

    SciTech Connect

    Gassner, G.T.; Dickie, J.P.; Hamerski, D.A.; Magnuson, J.K.; Anderson, J.S. )

    1990-05-01

    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. {sup 31}P 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 NaB{sup 3}H{sub 4} followed by complete acid hydrolysis yielded ({sup 3}H) glucosaminitol from the true reducing end residue of teichuronic acid and ({sup 3}H)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.

  14. Microscopic residues of bone from dissolving human remains in acids.

    PubMed

    Vermeij, Erwin; Zoon, Peter; van Wijk, Mayonne; Gerretsen, Reza

    2015-05-01

    Dissolving bodies is a current method of disposing of human remains and has been practiced throughout the years. During the last decade in the Netherlands, two cases have emerged in which human remains were treated with acid. In the first case, the remains of a cremated body were treated with hydrofluoric acid. In the second case, two complete bodies were dissolved in a mixture of hydrochloric and sulfuric acid. In both cases, a great variety of evidence was collected at the scene of crime, part of which was embedded in resin, polished, and investigated using SEM/EDX. Apart from macroscopic findings like residual bone and artificial teeth, in both cases, distinct microscopic residues of bone were found as follows: (partly) digested bone, thin-walled structures, and recrystallized calcium phosphate. Although some may believe it is possible to dissolve a body in acid completely, at least some of these microscopic residues will always be found. PMID:25677640

  15. 40 CFR 180.550 - Arsanilic acid [(4-aminophenyl) arsonic acid]; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Arsanilic acid ; tolerances for residues. 180.550 Section 180.550 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.550 Arsanilic acid ; tolerances...

  16. Electron microscopy of carbonaceous matter in Allende acid residues

    NASA Technical Reports Server (NTRS)

    Lumpkin, G. R.

    1982-01-01

    On the basis of characteristic diffuse ring diffraction patterns, much of the carbonaceous matter in a large suite of Allende acid residues has been identified as a variety of turbostratic carbon. Crystallites of this phase contain randomly stacked sp(2) hybridized carbon layers and diffraction patterns resemble those from carbon black and glassy carbon. Carbynes are probably absent, and are certainly restricted to less than 0.5% of these acid residues. The work of Ott et al. (1981) provides a basis for the possibility that turbostratic carbon is a carrier of noble gases, but an additional component - amorphous carbon - may be necessary to explain the high release temperatures of noble gases as well as the glassy character of many of the carbonaceous particles. Carbynes are considered to be questionable as important carriers of noble gases in the Allende acid residues.

  17. SO2 gas adsorption by modified kaolin clays: influence of previous heating and time acid treatments.

    PubMed

    Volzone, Cristina; Ortiga, Jose

    2011-10-01

    Modified kaolin clays were used as adsorbents for SO(2) gas adsorptions. The clays were heated up to 900 °C previous to acid treatments with 0.5 N sulfuric acid solutions at boiling temperature during different times up to 1440 min. Equilibrium adsorption at 25 °C and 0.1 MPa was carried out by using a volumetric apparatus. The samples were characterized by chemical analysis, X-ray diffraction and infrared analysis. The heating of the clays followed by acid treatment improved the adsorption capacity of the kaolin clays. The presence of amorphous silica and hydroxyl in the final products improved SO(2) adsorption capacity. Better properties for SO(2) adsorption were found in kaolin rich in not well ordered kaolinite clay mineral. PMID:21696883

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

    PubMed

    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

  19. Phosphate acceptor amino acid residues in structural proteins of rhabdoviruses.

    PubMed

    Sokol, F; Tan, K B; McFalls, M L; Madore, P

    1974-07-01

    Partial acid hydrolysates of the [(32)P]phosphate- or [(3)H]serine-labeled proteins of purified vesicular stomatitis, rabies, Lagos bat, Mokola, or spring viremia of carp virions and of purified intracellular nucleocapsids of these viruses have been analyzed by paper electrophoresis for the presence of phosphorylated amino acids. Both phosphoserine and phosphothreonine, with the former predominant, were present in virion and nucleocapsid preparations that contained phosphoproteins. An exception was the fish rhabdovirus, which contained only phosphoserine. When vesicular stomatitis or rabies virus proteins were phosphorylated in a cell-free system by the virion-associated protein kinase and analyzed for the presence of phosphorylated amino acid residues, phosphoserine was again found to be more abundant than phosphothreonine. After in vitro protein phosphorylation, another phospho-compound, possibly a third phosphoamino acid, was detected in the partial acid hydrolysates of these viruses. PMID:4365328

  20. Cancellous bone healing around strontium-doped hydroxyapatite in osteoporotic rats previously treated with zoledronic acid.

    PubMed

    Li, Yunfeng; Shui, Xueping; Zhang, Li; Hu, Jing

    2016-04-01

    Bisphosphonates (BPs) are potent anti-osteoporotic agents. Strontium-doped hydroxyapatite (HA) (SrHA) has been reported to increase bone density and improve trabecular microarchitecture in osteoporotic animals. But information about the effect of SrHA on the surrounding bone tissue in osteoporotic animals previously on BPs treatment is limited. We hypothesize that SrHA will induce increased bone density in the vicinity of the material when compared to HA, even in osteoporotic animals previously treated with BPs. HA and 10%SrHA (HA with 10 mol % calcium substituted by strontium) implants were prepared and characterized by scanning electronic microscopy (SEM), X-ray photoemission spectroscopy (XPS), and X-ray diffraction (XRD). Osteoporotic animal model was established by bilateral ovariectomy. Twelve weeks later, all OVX rats accepted subcutaneous injection of zoledronic acid (ZOL) at the dose of 1.5 μg/kg weekly for another twelve weeks. Subsequently, rod-shaped HA and SrHA implants were inserted in the distal femur of the OVX animals previously treated with ZOL. Eight weeks after implantation, specimens were harvested for histological and micro-computed tomography (micro-CT) analysis. Compared to HA, 10%SrHA raised the percent bone volume by 32.7%, the mean trabecular thickness by 36.5%, the mean trabecular number by 34.3%, the mean connectivity density by 38.4%, while the mean trabecular separation showed no significant difference. 10%SrHA also increased the bone area density by 36.3% in histological analysis. Results from this study indicated that 10%SrHA increased bone density and improved trabecular microarchitecture around implants in osteoporotic animals previously treated with ZOL when compared to HA. PMID:25891947

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

    PubMed

    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

  2. XPS and STEM studies of Allende acid insoluble residues

    NASA Technical Reports Server (NTRS)

    Housley, R. M.; Clarke, D. R.

    1980-01-01

    Data on Allende acid residues obtained both before and after etching with hot HNO3 are presented. X-ray photoelectron spectra show predominantly carbonaceous material plus Fe-deficient chromite in both cases. The HNO3 oxidizes the carbonaceous material to some extent. The small chromites in these residues have a wide range of compositions somewhat paralleling those observed in larger Allende chromites and in Murchison chromites, especially in the high Al contents; however, they are deficient in divalent cations, which makes them metastable and indicates that they must have formed at relatively low temperatures. It is suggested that they formed by precipitation of Cr(3+) and Fe(3+) from olivine at low temperature or during rapid cooling.

  3. Denosumab or Zoledronic Acid in Postmenopausal Women With Osteoporosis Previously Treated With Oral Bisphosphonates

    PubMed Central

    Pannacciulli, N.; Brown, J. P.; Czerwinski, E.; Nedergaard, B. S.; Bolognese, M. A.; Malouf, J.; Bone, H. G.; Reginster, J.-Y.; Singer, A.; Wang, C.; Wagman, R. B.; Cummings, S. R.

    2016-01-01

    Context: Denosumab and zoledronic acid (ZOL) are parenteral treatments for patients with osteoporosis. Objective: The objective of the study was to compare the effect of transitioning from oral bisphosphonates to denosumab or ZOL on bone mineral density (BMD) and bone turnover. Design and Setting: This was an international, multicenter, randomized, double-blind trial. Participants: A total of 643 postmenopausal women with osteoporosis previously treated with oral bisphosphonates participated in the study. Interventions: Subjects were randomized 1:1 to sc denosumab 60 mg every 6 months plus iv placebo once or ZOL 5 mg iv once plus sc placebo every 6 months for 12 months. Main Outcome Measures: Changes in BMD and bone turnover markers were measured. Results: BMD change from baseline at month 12 was significantly greater with denosumab compared with ZOL at the lumbar spine (primary end point; 3.2% vs 1.1%; P < .0001), total hip (1.9% vs 0.6%; P < .0001), femoral neck (1.2% vs −0.1%; P < .0001), and one-third radius (0.6% vs 0.0%; P < .05). The median decrease from baseline was greater with denosumab than ZOL for serum C-telopeptide of type 1 collagen at all time points after day 10 and for serum procollagen type 1 N-terminal propeptide at month 1 and at all time points after month 3 (all P < .05). Median percentage changes from baseline in serum intact PTH were significantly greater at months 3 and 9 with denosumab compared with ZOL (all P < .05). Adverse events were similar between groups. Three events consistent with the definition of atypical femoral fracture were observed (two denosumab and one ZOL). Conclusions: In postmenopausal women with osteoporosis previously treated with oral bisphosphonates, denosumab was associated with greater BMD increases at all measured skeletal sites and greater inhibition of bone remodeling compared with ZOL. PMID:27270237

  4. Chemical and isotopic compositions in acid residues from various meteorites

    NASA Technical Reports Server (NTRS)

    Kano, N.; Yamakoshi, K.; Matsuzaki, H.; Nogami, K.

    1993-01-01

    We are planning to carry out systematic isotopic investigations of Ru, Mg, etc., in primordial samples. The investigations will be pursued in the context of a study of the pre-history of the solar system. It is hoped that the study will yield direct evidence for processes of nucleosynthesis in the pre-solar stage and detection of extinct radioactive nuclides. In this paper, we present the results of chemical compositions of acid residues obtained from three types of meteorites: Canyon Diablo (IA), Allende (CV3), and Nuevo Mercuro (H5); and the preliminary results of Ru isotopic compositions.

  5. Oxolinic acid in the trout: bioavailability and tissue residues.

    PubMed

    Archimbault, P; Ambroggi, G; Nicolas, S

    1988-01-01

    A study was performed on the serum bioavailability and tissue elimination of oxolinic acid in the trout. The compound was added to the diet and administered at a dosage-level of 12 mg/kg/day for 7 consecutive days. The study utilized an analytical technique, high performance liquid chromatography, which has been described in detail here. The results obtained demonstrate that serum concentrations higher than the MIC for the control of the target pathogens (Aeromonas and Yersinia) can by sustained throughout the treatment period. The same positive results were observed in the tissues. Besides, on the base of a tolerance level of 0.05 ppm for the residue levels of oxolinic acid in the edible tissues (muscle mass and skin), a withdrawal time of six days after interruption of the prescribed treatment can be proposed. PMID:3400972

  6. A Soluble, Folded Protein without Charged Amino Acid Residues.

    PubMed

    Højgaard, Casper; Kofoed, Christian; Espersen, Roall; Johansson, Kristoffer Enøe; Villa, Mara; Willemoës, Martin; Lindorff-Larsen, Kresten; Teilum, Kaare; Winther, Jakob R

    2016-07-19

    Charges are considered an integral part of protein structure and function, enhancing solubility and providing specificity in molecular interactions. We wished to investigate whether charged amino acids are indeed required for protein biogenesis and whether a protein completely free of titratable side chains can maintain solubility, stability, and function. As a model, we used a cellulose-binding domain from Cellulomonas fimi, which, among proteins of more than 100 amino acids, presently is the least charged in the Protein Data Bank, with a total of only four titratable residues. We find that the protein shows a surprising resilience toward extremes of pH, demonstrating stability and function (cellulose binding) in the pH range from 2 to 11. To ask whether the four charged residues present were required for these properties of this protein, we altered them to nontitratable ones. Remarkably, this chargeless protein is produced reasonably well in Escherichia coli, retains its stable three-dimensional structure, and is still capable of strong cellulose binding. To further deprive this protein of charges, we removed the N-terminal charge by acetylation and studied the protein at pH 2, where the C-terminus is effectively protonated. Under these conditions, the protein retains its function and proved to be both soluble and have a reversible folding-unfolding transition. To the best of our knowledge, this is the first time a soluble, functional protein with no titratable side chains has been produced. PMID:27307139

  7. Removal of coagulant aluminum from water treatment residuals by acid.

    PubMed

    Okuda, Tetsuji; Nishijima, Wataru; Sugimoto, Mayo; Saka, Naoyuki; Nakai, Satoshi; Tanabe, Kazuyasu; Ito, Junki; Takenaka, Kenji; Okada, Mitsumasa

    2014-09-01

    Sediment sludge during coagulation and sedimentation in drinking water treatment is called "water treatment residuals (WTR)". Polyaluminum chloride (PAC) is mainly used as a coagulant in Japan. The recycling of WTR has been desired; one method for its reuse is as plowed soil. However, WTR reuse in this way is inhibited by the aluminum from the added PAC, because of its high adsorption capacity for phosphate and other fertilizer components. The removal of such aluminum from WTR would therefore be advantageous for its reuse as plowed soil; this research clarified the effect of acid washing on aluminum removal from WTR and on plant growth in the treated soil. The percentage of aluminum removal from raw WTR by sulphuric acid solution was around 90% at pH 3, the percentage decreasing to 40% in the case of a sun-dried sample. The maximum phosphate adsorption capacity was decreased and the available phosphorus was increased by acid washing, with 90% of aluminum removal. The enhancement of Japanese mustard spinach growth and the increased in plant uptake of phosphates following acid washing were observed. PMID:24835954

  8. Amino acid sequences around the cysteine residues of rabbit muscle triose phosphate isomerase

    PubMed Central

    Miller, Janet C.; Waley, S. G.

    1971-01-01

    1. The nature of the subunits in rabbit muscle triose phosphate isomerase has been investigated. 2. Amino acid analyses show that there are five cysteine residues and two methionine residues/subunit. 3. The amino acid sequences around the cysteine residues have been determined; these account for about 75 residues. 4. Cleavage at the methionine residues with cyanogen bromide gave three fragments. 5. These results show that the subunits correspond to polypeptide chains, containing about 230 amino acid residues. The chains in triose phosphate isomerase seem to be shorter than those of other glycolytic enzymes. PMID:5165707

  9. Amino acid residue doublet propensity in the protein-RNA interface and its application to RNA interface prediction.

    PubMed

    Kim, Oanh T P; Yura, Kei; Go, Nobuhiro

    2006-01-01

    Protein-RNA interactions play essential roles in a number of regulatory mechanisms for gene expression such as RNA splicing, transport, translation and post-transcriptional control. As the number of available protein-RNA complex 3D structures has increased, it is now possible to statistically examine protein-RNA interactions based on 3D structures. We performed computational analyses of 86 representative protein-RNA complexes retrieved from the Protein Data Bank. Interface residue propensity, a measure of the relative importance of different amino acid residues in the RNA interface, was calculated for each amino acid residue type (residue singlet interface propensity). In addition to the residue singlet propensity, we introduce a new residue-based propensity, which gives a measure of residue pairing preferences in the RNA interface of a protein (residue doublet interface propensity). The residue doublet interface propensity contains much more information than the sum of two singlet propensities alone. The prediction of the RNA interface using the two types of propensities plus a position-specific multiple sequence profile can achieve a specificity of about 80%. The prediction method was then applied to the 3D structure of two mRNA export factors, TAP (Mex67) and UAP56 (Sub2). The prediction enables us to point out candidate RNA interfaces, part of which are consistent with previous experimental studies and may contribute to elucidation of atomic mechanisms of mRNA export. PMID:17130160

  10. The Nature of Intermolecular Interactions Between Aromatic Amino Acid Residues

    SciTech Connect

    Gervasio, Francesco; Chelli, Riccardo; Procacci, Piero; Schettino, Vincenzo

    2002-05-01

    The nature of intermolecular interactions between aromatic amino acid residues has been investigated by a combination of molecular dynamics and ab initio methods. The potential energy surface of various interacting pairs, including tryptophan, phenilalanine, and tyrosine, was scanned for determining all the relevant local minima by a combined molecular dynamics and conjugate gradient methodology with the AMBER force field. For each of these minima, single-point correlated ab initio calculations of the binding energy were performed. The agreement between empirical force field and ab initio binding energies of the minimum energy structures is excellent. Aromatic-aromatic interactions can be rationalized on the basis of electrostatic and van der Waals interactions, whereas charge transfer or polarization phenomena are small for all intermolecular complexes and, particularly, for stacked structures.

  11. 40 CFR 180.325 - 2-(m-Chlorophenoxy) propionic acid; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false 2-(m-Chlorophenoxy) propionic acid... Tolerances § 180.325 2-(m-Chlorophenoxy) propionic acid; tolerances for residues. (a) General. A tolerance is established for negligible residues of the plant regulator 2-(m-chlorophenoxy) propionic acid from...

  12. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false p-Chlorophenoxyacetic acid; tolerances... Tolerances § 180.202 p-Chlorophenoxyacetic acid; tolerances for residues. (a) General. A tolerance is established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite...

  13. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false p-Chlorophenoxyacetic acid; tolerances... Tolerances § 180.202 p-Chlorophenoxyacetic acid; tolerances for residues. (a) General. A tolerance is established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite...

  14. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false p-Chlorophenoxyacetic acid; tolerances... Tolerances § 180.202 p-Chlorophenoxyacetic acid; tolerances for residues. (a) General. A tolerance is established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite...

  15. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false p-Chlorophenoxyacetic acid; tolerances... Tolerances § 180.202 p-Chlorophenoxyacetic acid; tolerances for residues. (a) General. A tolerance is established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite...

  16. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false p-Chlorophenoxyacetic acid; tolerances... Tolerances § 180.202 p-Chlorophenoxyacetic acid; tolerances for residues. (a) General. A tolerance is established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite...

  17. Vapor pressures of substituted polycarboxylic acids are much lower than previously reported

    NASA Astrophysics Data System (ADS)

    Huisman, A. J.; Krieger, U. K.; Zuend, A.; Marcolli, C.; Peter, T.

    2013-07-01

    The partitioning of compounds between the aerosol and gas phase is a primary focus in the study of the formation and fate of secondary organic aerosol. We present measurements of the vapor pressure of 2-methylmalonic (isosuccinic) acid, 2-hydroxymalonic (tartronic) acid, 2-methylglutaric acid, 3-hydroxy-3-carboxy-glutaric (citric) acid and DL-2,3-dihydroxysuccinic (DL-tartaric) acid, which were obtained from the evaporation rate of supersaturated liquid particles levitated in an electrodynamic balance. Our measurements indicate that the pure component liquid vapor pressures at 298.15 K for tartronic, citric and tartaric acids are much lower than the same quantity that was derived from solid state measurements in the only other room temperature measurement of these materials (made by Booth et al., 2010). This strongly suggests that empirical correction terms in a recent vapor pressure estimation model to account for the inexplicably high vapor pressures of these and similar compounds should be revisited, and that due caution should be used when the estimated vapor pressures of these and similar compounds are used as inputs for other studies.

  18. Vapor pressures of substituted polycarboxylic acids are much lower than previously reported

    NASA Astrophysics Data System (ADS)

    Huisman, A. J.; Krieger, U. K.; Zuend, A.; Marcolli, C.; Peter, T.

    2013-01-01

    The partitioning of compounds between the aerosol and gas phase is a primary focus in the study of the formation and fate of secondary organic aerosol. We present measurements of the vapor pressure of 2-Methylmalonic (isosuccinic) acid, 2-Hydroxymalonic (tartronic) acid, 2-Methylglutaric acid, 3-Hydroxy-3-carboxy-glutaric (citric) acid and 2,3-Dihydroxysuccinic (tartaric) acid which were obtained from the evaporation rate of supersaturated liquid particles levitated in an electrodynamic balance. Our measurements indicate that the pure component liquid vapor pressures at 298.15 K for tartronic, citric and tartaric acids are much lower than the same quantity which was derived from solid state measurements in the only other room temperature measurement of these materials (made by Booth et al., 2010). This strongly suggests that empirical correction terms in vapor pressure estimation models to account for the inexplicably high vapor pressures of these and similar compounds should be revisited, and that due caution should be used when the estimated vapor pressures of these and similar compounds are used as inputs for other studies.

  19. Degradation of articular cartilage keratan sulphates using hydrazinolysis and nitrous acid. Environment of fucose residues.

    PubMed Central

    Brown, G M; Huckerby, T N; Morris, H G; Nieduszynski, I A

    1992-01-01

    Alkaline borohydride-reduced keratan sulphate (KS) chains from bovine articular cartilage (6-8-year-old animals) were fragmented by an anhydrous hydrazine/nitrous acid procedure, previously used on KS by Hopwood & Elliott to isolate the major disaccharides from the poly-N-acetyl-lactosamine repeat sequence [Hopwood & Elliott (1983) Carbohydr. Res. 117, 263-274]. The resulting oligosaccharides were reduced with NaB3H4 or NaBH4 and subjected to ion-exchange chromatography on a Nucleosil 5SB column. In addition to the major disaccharides, two fucose-containing oligosaccharides were examined by high-field 1H n.m.r. spectroscopy, and shown to have the following structures (where AnManOH is 2,5-anhydro-D-mannitol): [formula: see text] It is evident that the presence of fucose protects the N-acetylglucosamine residue from de-N-acetylation, and therefore fragments are produced which preserve the immediate environment of the fucose residue. It may be of biosynthetic significance that these two oligosaccharides contain an unsulphated galactose on the non-reducing side of the fucose residue. The hydrazine/nitrous acid/NaB3H4 method followed by h.p.l.c. provides a sensitive fingerprinting technique for the assay of KS composition and sub-populations. PMID:1520275

  20. 40 CFR 180.318 - 4-(2-Methyl-4-chlorophenoxy) butyric acid; tolerance for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... established for the combined residues, free and conjugated, of the herbicide MCPB, 4-(4-chloro-2-methylphenoxy)butanoic acid, and its metabolite MCPA, (4-chloro-2-methylphenoxy)acetic acid, in or on the following...

  1. 40 CFR 180.155 - 1-Naphthaleneacetic acid; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... for residues. 180.155 Section 180.155 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Tolerances § 180.155 1-Naphthaleneacetic acid; tolerances for residues. (a) General. Tolerances are... Avocado 0.05 12/31/12 (c) Tolerances with regional registrations. (d) Indirect or inadvertent residues....

  2. Pyrolytic characteristics of biomass acid hydrolysis residue rich in lignin.

    PubMed

    Huang, Yanqin; Wei, Zhiguo; Yin, Xiuli; Wu, Chuangzhi

    2012-01-01

    Pyrolytic characteristics of acid hydrolysis residue (AHR) of corncob and pinewood (CAHR, WAHR) were investigated using a thermo-gravimetric analyzer (TGA) and a self-designed pyrolysis apparatus. Gasification reactivity of CAHR char was then examined using TGA and X-ray diffractometer. Result of TGA showed that thermal degradation curves of AHR descended smoothly along with temperature increasing from 150 °C to 850 °C, while a "sharp mass loss stage" for original biomass feedstock (OBF) was observed. Char yield from AHR (42.64-30.35 wt.%) was found to be much greater than that from OBF (26.4-19.15 wt.%). In addition, gasification reactivity of CAHR char was lower than that of corncob char, and there was big difference in micro-crystallite structure. It was also found that CAHR char reactivity decreased with pyrolysis temperature, but increased with pyrolysis heating rate and gasification temperature at 850-950 °C. Furthermore, CAHR char reactivity performed better under steam atmosphere than under CO2 atmosphere. PMID:22055106

  3. Oxidation in Acidic Medium of Lignins from Agricultural Residues

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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

  5. 40 CFR 180.318 - 4-(2-Methyl-4-chlorophenoxy) butyric acid; tolerance for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...)butanoic acid, and its metabolite MCPA, (4-chloro-2-methylphenoxy)acetic acid, in or on the following food... acid; tolerance for residues. 180.318 Section 180.318 Protection of Environment ENVIRONMENTAL... FOOD Specific Tolerances § 180.318 4-(2-Methyl-4-chlorophenoxy) butyric acid; tolerance for...

  6. Radiolytic Modification and Reactivity of Amino Acid Residues Serving as Structural Probes for Protein Footprinting

    SciTech Connect

    Xu,G.; Chance, M.

    2005-01-01

    Hydroxyl radical-mediated protein footprinting is a convenient and sensitive technique for mapping solvent-accessible surfaces of proteins and examining the structure and dynamics of biological assemblies. In this study, the reactivities and tendencies to form easily detectible products for all 20 (common) amino acid side chains along with cystine are directly compared using various standards. Although we have previously reported on the oxidation of many of these residues, this study includes a detailed examination of the less reactive residues and better defines their usefulness in hydroxyl radical-mediated footprinting experiments. All 20 amino amides along with cystine and a few tripeptides were irradiated by -rays, the products were analyzed by electrospray mass spectrometry, and rate constants of modification were measured. The reactivities of amino acid side chains were compared based on their loss of mass spectral signal normalized to the rate of loss for Phe or Pro that were radiolyzed simultaneously to serve as internal standards. In this way, accurate quantitation of relative rates could be assured. A reactivity order of amino acid side chains was obtained as Cys > Met > Trp > Tyr > Phe > cystine > His > Leu, Ile > Arg, Lys, Val > Ser, Thr, Pro > Gln, Glu > Asp, Asn > Ala > Gly. Ala and Gly are far too unreactive to be useful probes in typical experiments and Asp and Asn are unlikely to be useful as well. Although Ser and Thr are more reactive than Pro, which is known to be a useful probe, their oxidation products are not easily detectible. Thus, it appears that 14 of the 20 side chains (plus cystine) are most likely to be useful in typical experiments. Since these residues comprise 65% of the sequence of a typical protein, the footprinting approach provides excellent coverage of the side-chain reactivity for proteins.

  7. Chlorine residuals and haloacetic acid reduction in rapid sand filtration.

    PubMed

    Chuang, Yi-Hsueh; Wang, Gen-Shuch; Tung, Hsin-hsin

    2011-11-01

    It is quite rare to find biodegradation in rapid sand filtration for drinking water treatment. This might be due to frequent backwashes and low substrate levels. High chlorine concentrations may inhibit biofilm development, especially for plants with pre-chlorination. However, in tropical or subtropical regions, bioactivity on the sand surface may be quite significant due to high biofilm development--a result of year-round high temperature. The objective of this study is to explore the correlation between biodegradation and chlorine concentration in rapid sand filters, especially for the water treatment plants that practise pre-chlorination. In this study, haloacetic acid (HAA) biodegradation was found in conventional rapid sand filters practising pre-chlorination. Laboratory column studies and field investigations were conducted to explore the association between the biodegradation of HAAs and chlorine concentrations. The results showed that chlorine residual was an important factor that alters bioactivity development. A model based on filter influent and effluent chlorine was developed for determining threshold chlorine for biodegradation. From the model, a temperature independent chlorine concentration threshold (Cl(threshold)) for biodegradation was estimated at 0.46-0.5mgL(-1). The results imply that conventional filters with adequate control could be conducive to bioactivity, resulting in lower HAA concentrations. Optimizing biodegradable disinfection by-product removal in conventional rapid sand filter could be achieved with minor variation and a lower-than-Cl(threshold) influent chlorine concentration. Bacteria isolation was also carried out, successfully identifying several HAA degraders. These degraders are very commonly seen in drinking water systems and can be speculated as the main contributor of HAA loss. PMID:21974919

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

    PubMed

    Cook, Naomi L; Moeke, Cassidy H; Fantoni, Luca I; Pattison, David I; Davies, Michael J

    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 (SCN(-)) oxidation by H2O2 to form hypothiocyanous acid (HOSCN), an oxidant that targets Cys residues. Dysregulated phosphorylation and elevated MPO levels have been associated with chronic inflammatory diseases where HOSCN can be generated. Previous studies have shown that HOSCN inhibits isolated PTP1B and induces cellular dysfunction in cultured macrophage-like cells. The present study extends this previous work and shows that physiologically-relevant concentrations of HOSCN alter the activity and structure of other members of the wider PTP family (including leukocyte antigen-related PTP, PTP-LAR; T-cell PTP, TC-PTP; CD45 and Src homology phosphatase-1, Shp-1) by targeting Cys residues. Isolated PTP activity, and activity in lysates of human monocyte-derived macrophages (HMDM) was inhibited by 0-100 µM HOSCN with this being accompanied by reversible oxidation of Cys residues, formation of sulfenic acids or sulfenyl-thiocyanates (detected by Western blotting, and LC-MS as dimedone adducts), and structural changes. LC-MS/MS peptide mass-mapping has provided data on the modified Cys residues in PTP-LAR. This study indicates that inflammation-induced oxidants, and particularly myeloperoxidase-derived species, can modulate the activity of multiple members of the PTP superfamily via oxidation of Cys residues to sulfenic acids. This alteration of the balance of PTP/kinase activity may perturb protein phosphorylation and disrupt cell signaling with subsequent induction of apoptosis at sites of inflammation. PMID:26616646

  9. The acid lability of the glycosidic bonds of L-iduronic acid residues in glycosaminoglycans.

    PubMed Central

    Conrad, H E

    1980-01-01

    Heparan sulphate, heparin and dermatan sulphate were hydrolysed in 0.5M-H2SO4 at 100 degrees C. At intervals portions of the hydrolysate were removed and treated with HNO2 at pH 4.0 to cleave the glycosidic bonds of the N-unsubstituted hexosamine residues and to convert both free and combined hexosamines into anhydrohexoses. These hydrolysis/deamination mixtures were reduced with NaB3H4 and analysed by radiochromatography for alpha-L-iduronosylanhydrohexose, beta-D-glucuronosylanhydrohexose, and the free uronic acids and anhydrohexose. These data gave a kinetic profile of the cleavage of the alpha-L-iduronosyl and the beta-D-glucuronosyl bonds in these glycosaminoglycans. The beta-D-glucuronosyl bonds showed the expected resistance to acid hydrolysis, but the alpha-L-iduronosyl bonds were found to be as labile to acid as some neutral sugar glycosides. This unusual lability of alpha-D-iduronosyl-anhydromannitol and beta-D-glucuronosylanhydromannitol. The procedures used to follow the kinetics of glycosaminoglycan hydrolysis can also be sued to obtain quantitative analyses of L-iduronic acid, D-glucuronic acid and hexosamine in these polymers. PMID:6453583

  10. Degradation and enantiomeric fractionation of mecoprop in soil previously exposed to phenoxy acid herbicides - New insights for bioremediation.

    PubMed

    Frková, Zuzana; Johansen, Anders; de Jonge, Lis Wollesen; Olsen, Preben; Gosewinkel, Ulrich; Bester, Kai

    2016-11-01

    Phenoxy acid-contaminated subsoils are common as a result of irregular disposal of residues and production wastes in the past. For enhancing in situ biodegradation at reducing conditions, biostimulation may be an effective option. Some phenoxy acids were marketed in racemic mixtures, and biodegradation rates may differ between enantiomers. Therefore, enantio-preferred degradation of mecoprop (MCPP) in soil was measured to get in-depth information on whether amendment with glucose (BOD equivalents as substrate for microbial growth) and nitrate (redox equivalents for oxidation) can stimulate bioremediation. The degradation processes were studied in soil sampled at different depths (3, 4.5 and 6m) at a Danish urban site with a history of phenoxy acid contamination. We observed preferential degradation of the R-enantiomer only under aerobic conditions in the soil samples from 3- and 6-m depth at environmentally relevant (nM) MCPP concentrations: enantiomer fraction (EF)<0.5. On the other hand, we observed preferential degradation of the S-enantiomer in all samples and treatments at elevated (μM) MCPP concentrations: EF>0.5. Three different microbial communities were discriminated by enantioselective degradation of MCPP: 1) aerobic microorganisms with little enantioselectivity, 2) aerobic microorganisms with R-selectivity and 3) anaerobic denitrifying organisms with S-selectivity. Glucose-amendment did not enhance MCPP degradation, while nitrate amendment enhanced the degradation of high concentrations of the herbicide. PMID:27432728

  11. Role of surface lysine residues of adipocyte fatty acid-binding protein in fatty acid transfer to phospholipid vesicles.

    PubMed

    Liou, H L; Storch, J

    2001-05-29

    The tertiary structure of murine adipocyte fatty acid-binding protein (AFABP) is a flattened 10-stranded beta-barrel capped by a helix-turn-helix segment. This helical domain is hypothesized to behave as a "lid" or portal for ligand entry into and exit from the binding cavity. Previously, we demonstrated that anthroyloxy-labeled fatty acid (AOFA) transfer from AFABP to phospholipid membranes occurs by a collisional process, in which ionic interactions between positively charged lysine residues on the protein surface and negatively charged phospholipid headgroups are involved. In the present study, the role of specific lysine residues located in the portal and other regions of AFABP was directly examined using site-directed mutagenesis. The results showed that isoleucine replacement for lysine in the portal region, including the alphaI- and alphaII-helices and the beta C-D turn, resulted in much slower 2-(9-anthroyloxy)palmitate (2AP) transfer rates to acidic membranes than those of native AFABP. An additive effect was found for mutant K22,59I, displaying the slowest rates of FA transfer. Rates of 2AP transfer from "nonportal" mutants on the beta-G and I strands were affected only moderately; however, a lysine --> isoleucine mutation in the nonportal beta-A strand decreased the 2AP transfer rate. These studies suggest that lysines in the helical cap domain are important for governing ionic interactions between AFABP and membranes. Furthermore, it appears that more than one distinct region, including the alphaI-helix, alphaII-helix, beta C-D turn, and the beta-A strand, is involved in these charge-charge interactions. PMID:11371211

  12. The acid and enzymic hydrolysis of O-acetylated sialic acid residues from rabbit Tamm–Horsfall glycoprotein

    PubMed Central

    Neuberger, A.; Ratcliffe, Wendy A.

    1972-01-01

    Rabbit Tamm–Horsfall glycoprotein and bovine submaxillary glycoprotein were both found to contain sialic acid residues which are released at a slow rate by the standard conditions of acid hydrolysis. These residues are also resistant to neuraminidases from Vibrio cholerae and Clostridium perfringens. This behaviour was attributed to the presence of O-acetylated sialic acid, since the removal of O-acetyl groups by mild alkaline treatment normalized the subsequent release of sialic acid from rabbit Tamm–Horsfall glycoprotein by acid and by enzymic hydrolysis. Determination of the O-acetyl residues in rabbit Tamm–Horsfall glycoprotein indicated that on average two hydroxyl groups of sialic acid are O-acetylated, and these were located on the polyhydroxy side-chain of sialic acid or on C-4 and C-8. These findings confirm the assumption that certain O-acetylated forms of sialic acid are not substrates for bacterial neuraminidases. Several explanations have been suggested to explain the effect of O-acetylation of the side-chain on the rate of acidcatalysed hydrolysis of sialic acid residues. PMID:4349114

  13. Evaluation of the methanogenic step of a two-stage anaerobic digestion process of acidified olive mill solid residue from a previous hydrolytic-acidogenic step.

    PubMed

    Rincón, B; Borja, R; Martín, M A; Martín, A

    2009-09-01

    A study of the second step or methanogenic stage of a two-stage anaerobic digestion process treating two-phase olive oil mill solid residue (OMSR) was conducted at mesophilic temperature (35 degrees C). The substrate fed to the methanogenic step was the effluent from a hydrolytic-acidogenic reactor operating at an organic loading rate (OLR) of 12.9 g chemical oxygen demand (COD) L(-1) d(-1) and at a hydraulic retention time (HRT) of 12.4 days; these OLR and HRT were found to be the best values to achieve the maximum total volatile fatty acid concentration (14.5 g L(-1) expressed as acetic acid) with a high concentration in acetic acid (57.5% of the total concentration) as the principal precursor of methane. The methanogenic stage was carried out in an anaerobic stirred tank reactor containing saponite as support media for the immobilization of microorganisms. OLRs of between 0.8 and 22.0 g COD L(-1) d(-1) were studied. These OLRs corresponded to HRTs of between 142.9 and 4.6 days. The methanogenic reactor operated with high stability for OLRs lower than 20.0 g COD L(-1) d(-1). This behaviour was shown by the total volatile fatty acids/total alkalinity ratio, whose values were always kept 0.12 for HRTs>4.6 days. The total COD (T-COD) removed was in the range of 94.3-61.3% and the volatile solids (VS) removed between 92.8% and 56.1% for OLRs between 0.8 and 20.0 g COD L(-1) d(-1). In the same way, a reduction of 43.8% was achieved for phenolic content. The low concentration of total volatile fatty acids (TVFA) observed (below 1 g L(-1) expressed as CH(3)COOH) in the methanogenic reactor effluents showed the high percentage of consumption and conversion of these acids to methane. A methane yield of 0.268+/-0.003 L CH(4) at standard temperature and pressure conditions (STP) g(-1) COD eliminated was achieved. PMID:19450962

  14. 40 CFR 180.155 - 1-Naphthaleneacetic acid; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false 1-Naphthaleneacetic acid; tolerances for residues. 180.155 Section 180.155 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.155 1-Naphthaleneacetic...

  15. 40 CFR 180.155 - 1-Naphthaleneacetic acid; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false 1-Naphthaleneacetic acid; tolerances for residues. 180.155 Section 180.155 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.155 1-Naphthaleneacetic...

  16. 40 CFR 180.155 - 1-Naphthaleneacetic acid; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false 1-Naphthaleneacetic acid; tolerances for residues. 180.155 Section 180.155 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.155 1-Naphthaleneacetic...

  17. 40 CFR 180.155 - 1-Naphthaleneacetic acid; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false 1-Naphthaleneacetic acid; tolerances for residues. 180.155 Section 180.155 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.155 1-Naphthaleneacetic...

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

    PubMed

    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

  19. Structure of the genetic code suggested by the hydropathy correlation between anticodons and amino acid residues

    NASA Astrophysics Data System (ADS)

    Farias, Sávio Torres De; Moreira, Carlos Henrique Costa; Guimarães, Romeu Cardoso

    2007-02-01

    The correlation between hydropathies of anticodons and amino acids, detected by other authors utilizing scales of amino acid molecules in solution, was improved with the utilization of scales of amino acid residues in proteins. Three partitions were discerned in the correlation plot with the principal dinucleotides of anticodons (pDiN, excluding the wobble position). (a) The set of outliers of the correlation: Gly-CC, Pro-GG, Ser-GA and Ser-CU. The amino acids are consistently small, hydro-apathetic, stabilizers of protein N-ends, preferred in aperiodic protein conformations and belong to synthetases class II. The pDiN sequences are representative of the homogeneous sector (triplets N RR and N YY), distinguished from the mixed sector (triplets N RY and N YR), that depict a 70% correspondence to the synthetases class II and I, respectively. The triplet pairs proposed to be responsible for the coherence in the set of outliers are of the palindromic kind, where the lateral bases are the same, C CC: G GG and A GA: U CU. This suggests that U CU previously belonged to Ser, adding to other indications that the attribution of Arg to Y CU was due to an expansion of the Arg- tRNA synthetase specificity. The other attributions produced two correlation sets. (b) One corresponds to the remaining pDiN of the homogeneous sector, containing both synthetase classes; its regression line overlapped the one formed by the remaining attributions to class II. (c) The other contains the pDiN of the mixed sector and produced steeper slopes, especially with the class I attributions. It is suggested that the correlation was established when the amino acid composition of the protein synthetases became progressively enriched and that the set of outliers were the earliest to have been fixed.

  20. Intra-molecular cross-linking of acidic residues for protein structure studies.

    SciTech Connect

    Kruppa, Gary Hermann; Young, Malin M.; Novak, Petr; Schoeniger, Joseph S.

    2005-03-01

    Intra-molecular cross-linking has been suggested as a method of obtaining distance constraints that would be useful in developing structural models of proteins. Recent work published on intra-molecular cross-linking for protein structural studies has employed commercially available primary amine selective reagents that can cross-link lysine residues to other lysine residues or the amino terminus. Previous work using these cross-linkers has shown that for several proteins of known structure, the number of cross-links that can be obtained experimentally may be small compared to what would be expected from the known structure, due to the relative reactivity, distribution, and solvent accessibility of the lysines in the protein sequence. To overcome these limitations we have investigated the use of cross-linking reagents that can react with other reactive sidechains in proteins. We used 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to activate the carboxylic acid containing residues, aspartic acid (D), glutamic acid (E), and the carboxy terminus (O), for cross-linking reactions. Once activated, the DEO sidechains can react to form 'zero-length' cross-links with nearby primary amine containing resides, lysines (K) and the amino terminus (X), via the formation of a new amide bond. We also show that the EDC-activated DEO sidechains can be cross-linked to each other using dihydrazides, two hydrazide moieties connected by an alkyl cross-linker ann of variable length. Using these reagents, we have found three new 'zero-length' cross-links in ubiquitin consistent with its known structure (M1-E16, M1-E18, and K63-E64). Using the dihydrazide cross-linkers, we have identified 2 new cross-links (D21-D32 and E24-D32) unambiguously. Using a library of dihydrazide cross-linkers with varying arm length, we have shown that there is a minimum arm length required for the DEO-DEO cross-links of 5.8 angstroms. These results show that additional structural information

  1. Residual deformity in congenital radial club hands after previous centralisation of the wrist. Ulnar lengthening and correction by the Ilizarov method.

    PubMed

    Kawabata, H; Shibata, T; Masatomi, T; Yasui, N

    1998-09-01

    We used the Ilizarov method in seven patients with severe congenital radial club hands who had had previous wrist surgery, to correct residual shortening and bowing of the ulna together with recurrent wrist deformity. The mean age at operation was 6.5 years. The mean ulnar shortening was 5.3 cm and the mean angular deformity 42 degrees. The mean length gained was 51% of the original ulna. The mean healing index was 46.9 days (29.8 to 64.0). The ratio of the length of the lengthened ulna to the normal side improved on average from 64% to 95%. The angular deformity was initially completely corrected in six out of seven patients. The length ratio, however, decreased to 83% at the final follow-up. In four patients, the angular deformity partially recurred. We recommend correction of congenital radial club hand by staged procedures. The first is centralisation and stabilisation of the wrist and the second lengthening of the ulna and correction of the angular deformity using the Ilizarov method. PMID:9768881

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

    PubMed

    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

  3. Residual Activity of Thermally Denatured Transforming Deoxyribonucleic Acid from Haemophilus influenzae

    PubMed Central

    Barnhart, Benjamin J.

    1965-01-01

    Barnhart, Benjamin J. (Johns Hopkins University School of Hygiene and Public Health, Baltimore, Md.). Residual activity of thermally denatured transforming deoxyribonucleic acid from Haemophilus influenzae. J. Bacteriol. 89:1271–1279. 1965.—The level of residual transforming activity of heated deoxyribonucleic acid (DNA) (i.e., 1 to a few per cent of native DNA-transforming activity) was found to be independent of the heating and quenching temperatures and less susceptible than native or renatured DNA to heat inactivation upon prolonged heating above or below the critical melting temperature. Similar dose-response curves were obtained for inactivation by formamide of native and renatured DNA, but the residual-active material was much more resistant. Heating DNA above the Tm in the presence of 1% formaldehyde resulted in a level of residual activity 4 logs lower than that obtained without formaldehyde. Residual-active material was not inactivated by Escherichia coli phosphodiesterase, but it was susceptible to snake venom phosphodiesterase. A new genetic marker was induced in heated-quenched DNA but not in purified residual-active material following nitrous acid treatment. Residual activity was found to be less susceptible to ultraviolet inactivation and to band at a higher density region in CsCl than native DNA. In conclusion, it is suggested that the residual-active material is a structure formed by intrastrand hydrogen bonding of the separated units of heated-quenched DNA. Such a configuration would result in at least a partially double-stranded structure, which is probably the essential characteristic of the residual-active material endowing it with biological activity. PMID:14292997

  4. Active-site amino acid residues in γ-glutamyltransferase and the nature of the γ-glutamyl-enzyme bond

    PubMed Central

    Elce, John S.

    1980-01-01

    Active-site residues in rat kidney γ-glutamyltransferase (EC 2.3.2.2) were investigated by means of chemical modification. 1. In the presence of maleate, the activity was inhibited by phenylmethanesulphonyl fluoride, and the inhibition was not reversed by β-mercaptoethanol, suggesting that a serine residue is close to the active site, but is shielded except in the presence of maleate. 2. Treatment of the enzyme with N-acetylimidazole modified an amino group, exposed a previously inaccessible cysteine residue and inhibited hydrolysis of the γ-glutamyl-enzyme intermediate, but not its formation. 3. After reaction of the enzyme successively with N-acetylimidazole and with non-radioactive iodoacetamide/serine/borate, two active-site residues reacted with iodo[14C]acetamide. One of these possessed a carboxy group, which formed a [14C]glycollamide ester, and the other was cysteine, shown by isolation of S-[14C]carboxymethylcysteine after acid hydrolysis. When N-acetylimidazole treatment was omitted, only the carboxy group reacted with iodo[14C]acetamide. 4. Isolation of the γ-[14C]glutamyl-enzyme intermediate was made easier by prior treatment of the enzyme with N-acetylimidazole. The γ-glutamyl-enzyme bond was stable to performic acid, and to hydroxylamine/urea at pH10, but was hydrolysed slowly at pH12, indicating attachment of the γ-[14C]glutamyl group in amide linkage to an amino group on the enzyme. Proteolysis of the γ-[14C]glutamyl-enzyme after performic acid oxidation gave rise to a small acidic radioactive peptide that was resistant to further proteolysis and was not identical with γ-glutamyl-ε-lysine. 5. A scheme for the catalytic mechanism is proposed. PMID:6104953

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

    PubMed Central

    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

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

    PubMed

    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

  7. Intra-molecular cross-linking of acidic residues for protein structure studies.

    PubMed

    Novak, Petr; Kruppa, Gary H

    2008-01-01

    Intra-molecular cross-linking has been suggested as a method of obtaining distance constraints that would help to develop structural models of proteins. Recent work published on intra-molecular cross-linking for protein structural studies has employed commercially available primary amine (lysine, the amino terminus) selective reagents. Previous work using these cross-linkers has shown that for several proteins of known structure, the number of cross-links that can be obtained experimentally may be small compared to what would be expected from the known structure, due to the relative reactivity, distribution and solvent accessibility of the lysines in the protein sequence. To overcome these limitations, we have investigated the use of cross-linking reagents that can react with other reactive side chains in proteins. We used 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to activate the carboxylic acid containing residues, aspartic acid (D), glutamic acid (E) and the carboxy terminus (O), for cross-linking reactions. Once activated, the DEO side chains can react to form "zero-length" cross-links with nearby primary amine containing residues, lysines (K) and the amino terminus (X), via the formation of a new amide bond. We also show that the EDC-activated DEO side chains can be cross-linked to each other using dihydrazides, two hydrazide moieties connected by an alkyl cross-linker arm of variable length. Using these reagents, we have found three new "zero-length" cross-links in ubiquitin consistent with its known structure (M1-E16, M1-E18 and K63-E64). Using the dihydrazide cross-linkers, we have identified two new cross-links (D21-D32 and E24-D32) unambiguously. Using a library of dihydrazide cross-linkers with varying arm length, we have shown that there is a minimum arm length required for the DEO-DEO cross-links of 5.8 A. These results show that additional structural information can be obtained by exploiting new cross-linker chemistry

  8. New charge-bearing amino acid residues that promote β-sheet secondary structure.

    PubMed

    Maynard, Stacy J; Almeida, Aaron M; Yoshimi, Yasuharu; Gellman, Samuel H

    2014-11-26

    Proteinogenic amino acid residues that promote β-sheet secondary structure are hydrophobic (e.g., Ile or Val) or only moderately polar (e.g., Thr). The design of peptides intended to display β-sheet secondary structure in water typically requires one set of residues to ensure conformational stability and an orthogonal set, with charged side chains, to ensure aqueous solubility and discourage self-association. Here we describe new amino acids that manifest substantial β-sheet propensity, by virtue of β-branching, and also bear an ionizable group in the side chain. PMID:25393077

  9. Effect of co-evolving amino acid residues on topology of phylogenetic trees.

    PubMed

    Sherbakov, D Yu; Triboy, T I

    2007-12-01

    The presence in proteins of amino acid residues that change in concert during evolution is associated with keeping constant the protein spatial structure and functions. As in the case with morphological features, correlated substitutions may become the cause of homoplasies--the independent evolution of identical non-homological adaptations. Our data obtained on model phylogenetic trees and corresponding sets of sequences have shown that the presence of correlated substitutions distorts the results of phylogenetic reconstructions. A method for accounting for co-evolving amino acid residues in phylogenetic analysis is proposed. According to this method, only a single site from the group of correlated amino acid positions should remain, whereas other positions should not be used in further phylogenetic analysis. Simulations performed have shown that replacement on the average of 8% of variable positions in a pair of model sequences by coordinately evolving amino acid residues is able to change the tree topology. The removal of such amino acid residues from sequences before phylogenetic analysis restores the correct topology. PMID:18205620

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

    PubMed

    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

  11. Prediction of nucleic acid binding probability in proteins: a neighboring residue network based score.

    PubMed

    Miao, Zhichao; Westhof, Eric

    2015-06-23

    We describe a general binding score for predicting the nucleic acid binding probability in proteins. The score is directly derived from physicochemical and evolutionary features and integrates a residue neighboring network approach. Our process achieves stable and high accuracies on both DNA- and RNA-binding proteins and illustrates how the main driving forces for nucleic acid binding are common. Because of the effective integration of the synergetic effects of the network of neighboring residues and the fact that the prediction yields a hierarchical scoring on the protein surface, energy funnels for nucleic acid binding appear on protein surfaces, pointing to the dynamic process occurring in the binding of nucleic acids to proteins. PMID:25940624

  12. Prediction of nucleic acid binding probability in proteins: a neighboring residue network based score

    PubMed Central

    Miao, Zhichao; Westhof, Eric

    2015-01-01

    We describe a general binding score for predicting the nucleic acid binding probability in proteins. The score is directly derived from physicochemical and evolutionary features and integrates a residue neighboring network approach. Our process achieves stable and high accuracies on both DNA- and RNA-binding proteins and illustrates how the main driving forces for nucleic acid binding are common. Because of the effective integration of the synergetic effects of the network of neighboring residues and the fact that the prediction yields a hierarchical scoring on the protein surface, energy funnels for nucleic acid binding appear on protein surfaces, pointing to the dynamic process occurring in the binding of nucleic acids to proteins. PMID:25940624

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

    PubMed

    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

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

    PubMed

    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

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

    PubMed

    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

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

    ERIC Educational Resources Information Center

    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.)

  17. Transcription of the Streptococcus pyogenes hyaluronic acid capsule biosynthesis operon is regulated by previously unknown upstream elements.

    PubMed

    Falaleeva, Marina; Zurek, Oliwia W; Watkins, Robert L; Reed, Robert W; Ali, Hadeel; Sumby, Paul; Voyich, Jovanka M; Korotkova, Natalia

    2014-12-01

    The important human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]) produces a hyaluronic acid (HA) capsule that plays critical roles in immune evasion. Previous studies showed that the hasABC operon encoding the capsule biosynthesis enzymes is under the control of a single promoter, P1, which is negatively regulated by the two-component regulatory system CovR/S. In this work, we characterize the sequence upstream of P1 and identify a novel regulatory region controlling transcription of the capsule biosynthesis operon in the M1 serotype strain MGAS2221. This region consists of a promoter, P2, which initiates transcription of a novel small RNA, HasS, an intrinsic transcriptional terminator that inefficiently terminates HasS, permitting read-through transcription of hasABC, and a putative promoter which lies upstream of P2. Electrophoretic mobility shift assays, quantitative reverse transcription-PCR, and transcriptional reporter data identified CovR as a negative regulator of P2. We found that the P1 and P2 promoters are completely repressed by CovR, and capsule expression is regulated by the putative promoter upstream of P2. Deletion of hasS or of the terminator eliminates CovR-binding sequences, relieving repression and increasing read-through, hasA transcription, and capsule production. Sequence analysis of 44 GAS genomes revealed a high level of polymorphism in the HasS sequence region. Most of the HasS variations were located in the terminator sequences, suggesting that this region is under strong selective pressure. We discovered that the terminator deletion mutant is highly resistant to neutrophil-mediated killing and is significantly more virulent in a mouse model of GAS invasive disease than the wild-type strain. Together, these results are consistent with the naturally occurring mutations in this region modulating GAS virulence. PMID:25287924

  18. Transcription of the Streptococcus pyogenes Hyaluronic Acid Capsule Biosynthesis Operon Is Regulated by Previously Unknown Upstream Elements

    PubMed Central

    Falaleeva, Marina; Zurek, Oliwia W.; Watkins, Robert L.; Reed, Robert W.; Ali, Hadeel; Sumby, Paul; Voyich, Jovanka M.

    2014-01-01

    The important human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]) produces a hyaluronic acid (HA) capsule that plays critical roles in immune evasion. Previous studies showed that the hasABC operon encoding the capsule biosynthesis enzymes is under the control of a single promoter, P1, which is negatively regulated by the two-component regulatory system CovR/S. In this work, we characterize the sequence upstream of P1 and identify a novel regulatory region controlling transcription of the capsule biosynthesis operon in the M1 serotype strain MGAS2221. This region consists of a promoter, P2, which initiates transcription of a novel small RNA, HasS, an intrinsic transcriptional terminator that inefficiently terminates HasS, permitting read-through transcription of hasABC, and a putative promoter which lies upstream of P2. Electrophoretic mobility shift assays, quantitative reverse transcription-PCR, and transcriptional reporter data identified CovR as a negative regulator of P2. We found that the P1 and P2 promoters are completely repressed by CovR, and capsule expression is regulated by the putative promoter upstream of P2. Deletion of hasS or of the terminator eliminates CovR-binding sequences, relieving repression and increasing read-through, hasA transcription, and capsule production. Sequence analysis of 44 GAS genomes revealed a high level of polymorphism in the HasS sequence region. Most of the HasS variations were located in the terminator sequences, suggesting that this region is under strong selective pressure. We discovered that the terminator deletion mutant is highly resistant to neutrophil-mediated killing and is significantly more virulent in a mouse model of GAS invasive disease than the wild-type strain. Together, these results are consistent with the naturally occurring mutations in this region modulating GAS virulence. PMID:25287924

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

    PubMed Central

    Sawada, Kazutaka

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

    PubMed

    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

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

    PubMed

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

    NASA Astrophysics Data System (ADS)

    Takahashi, Ohgi; Kirikoshi, Ryota

    2014-01-01

    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.

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

    PubMed

    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

  4. The cyst wall of Colpoda steinii. A substance rich in glutamic acid residues

    PubMed Central

    Tibbs, J.

    1966-01-01

    1. The cyst wall of Colpoda steinii has been isolated and its chemical nature examined. It had a nitrogen content 13·9±0·2% (s.d.) and an ash 8·6±1·6% (s.d.). After lipid and hot-acid extraction there was a variable residual phosphorus of 0·19–0·64%. The protein nature, indicated by infrared and ultraviolet absorption, was confirmed when 100μg. of hydrolysed wall gave a ninhydrin colour equivalent to that given by 0·88–1·01μmoles of glycine. Hexosamine, hexose, pentose, lipid and dipicolinic acid were absent. 2. Paper chromatography of hydrolysates, besides showing the presence of the usual protein amino acids and three unidentified ninhydrin-reacting spots, indicated the presence of large amounts of glutamic acid. Estimated by chromatography, the amount present was 52·9±0·6 (s.d.) g./100g. of ash-free wall; manometric estimation of l-glutamic acid with l-glutamate 1-carboxy-lyase gave 46·5±0·9 (s.d.) g./100g. 3. Free carboxyl groups were estimated by titration as 0·159±0·011 (s.d.) mole/100g. and those present as amide as 0·154±0·004 (s.d.) mole/100g., and the total was compared with the dicarboxylic acid content 0·360±0·010 (s.d.) mole/100g. 4. After treatment with 98% formic acid 25–30% of the wall material could be extracted by 0·05m-sodium carbonate solution (extract 1); after treatment of the residue with performic acid a further 62–63% based on the original weight could be extracted by 0·05m-sodium carbonate (extract 2). 5. The average values found for the glutamic acid contents were 21·7g./100g. for extract 1 and 58·0g./100g. for extract 2. The cysteic acid content of whole oxidized wall was about 5·8g./100g. and of extract 2 also about 5·8g./100g. The glutamic acid and cysteic acid contents of the final residue were also investigated. 6. The significance of these extraction experiments in relation to the wall structure is discussed. ImagesPlate 1. PMID:4957913

  5. Identification of Catalytic Amino Acid Residues by Chemical Modification in Dextranase.

    PubMed

    Ko, Jin-A; Nam, Seung-Hee; Kim, Doman; Lee, Jun-Ho; Kim, Young-Min

    2016-05-28

    A novel endodextranase isolated from Paenibacillus sp. was found to produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides with a degree of polymerization of 7-14 from dextran. To determine the active site, the enzyme was modified with 1-ethyl-3-[3- (dimethylamino)-propyl]-carbodiimide (EDC) and α-epoxyalkyl α-glucosides (EAGs), an affinity labeling reagent. The inactivation followed pseudo first-order kinetics. Kinetic analysis and chemical modification using EDC and EAGs indicated that carboxyl groups are essential for the enzymatic activity. Three Asp and one Glu residues were identified as candidate catalytic amino acids, since these residues are completely conserved across the GH family of 66 enzymes. Replacement of Asp189, Asp340, or Glu412 completely abolished the enzyme activity, indicating that these residues are essential for catalytic activity. PMID:26907761

  6. Acetic Acid Can Catalyze Succinimide Formation from Aspartic Acid Residues by a Concerted Bond Reorganization Mechanism: A Computational Study

    PubMed Central

    Takahashi, Ohgi; Kirikoshi, Ryota; Manabe, Noriyoshi

    2015-01-01

    Succinimide formation from aspartic acid (Asp) residues is a concern in the formulation of protein drugs. Based on density functional theory calculations using Ace-Asp-Nme (Ace = acetyl, Nme = NHMe) as a model compound, we propose the possibility that acetic acid (AA), which is often used in protein drug formulation for mildly acidic buffer solutions, catalyzes the succinimide formation from Asp residues by acting as a proton-transfer mediator. The proposed mechanism comprises two steps: cyclization (intramolecular addition) to form a gem-diol tetrahedral intermediate and dehydration of the intermediate. Both steps are catalyzed by an AA molecule, and the first step was predicted to be rate-determining. The cyclization results from a bond formation between the amide nitrogen on the C-terminal side and the side-chain carboxyl carbon, which is part of an extensive bond reorganization (formation and breaking of single bonds and the interchange of single and double bonds) occurring concertedly in a cyclic structure formed by the amide NH bond, the AA molecule and the side-chain C=O group and involving a double proton transfer. The second step also involves an AA-mediated bond reorganization. Carboxylic acids other than AA are also expected to catalyze the succinimide formation by a similar mechanism. PMID:25588215

  7. Camphorquinone-10-sulfonic acid and derivatives: convenient reagents for reversible modification of arginine residues

    SciTech Connect

    Pande, C.S.; Pelzig, M.; Glass, J.D.

    1980-02-01

    Camphorquinone-10-sulfonic acid hydrate was prepared by the action of selenous acid on camphor-10-sulfonic acid. Camphorquinone-10-sulfonylnorleucine was prepared either from the sulfonic acid via the sulfonyl chloride or by selenous acid oxidation of camphor-10-sulfonylnorleucine. These reagents are useful for specific, reversible modification of the guanidino groups of arginine residues. Camphorquinonsulfonic acid is a crystalline water-soluble reagent that is especially suitable for use with small arginine-containing molecules, because the sulfonic acid group of the reagent is a convenient handle for analytical and preparative separation of products. Camphorquinonesulfonylnorleucine is more useful for work with large polypeptides and proteins, because hydrolysates of modified proteins may be analyzed for norleucine to determine the extent of arginine modification. The adducts of the camphorquinone derivatives with the guanidino group are stable to 0.5 M hydroxylamine solutions at pH 7, the recommended conditions for cleavage of the corresponding cyclohexanedione adducts. At pH 8-9 the adducts of the camphorquinone derivatives with the guanidino group are cleaved by o-phenylenediamine. The modification and regeneration of arginine, of the dipeptide arginylaspartic acid, of ribonuclease S-peptide, and of soybean trypsin inhibitor are presented as demonstrations of the use of the reagents.The use of camphorquinonesulfonyl chloride to prepare polymers containing arginine-specific ligands is discussed.

  8. Genetic analysis of L123 of the tRNA-mimicking eukaryote release factor eRF1, an amino acid residue critical for discrimination of stop codons

    PubMed Central

    Saito, Kazuki; Ito, Koichi

    2015-01-01

    In eukaryotes, the tRNA-mimicking polypeptide-chain release factor, eRF1, decodes stop codons on the ribosome in a complex with eRF3; this complex exhibits striking structural similarity to the tRNA–eEF1A–GTP complex. Although amino acid residues or motifs of eRF1 that are critical for stop codon discrimination have been identified, the details of the molecular mechanisms involved in the function of the ribosomal decoding site remain obscure. Here, we report analyses of the position-123 amino acid of eRF1 (L123 in Saccharomyces cerevisiae eRF1), a residue that is phylogenetically conserved among species with canonical and variant genetic codes. In vivo readthrough efficiency analysis and genetic growth complementation analysis of the residue-123 systematic mutants suggested that this amino acid functions in stop codon discrimination in a manner coupled with eRF3 binding, and distinctive from previously reported adjacent residues. Furthermore, aminoglycoside antibiotic sensitivity analysis and ribosomal docking modeling of eRF1 in a quasi-A/T state suggested a functional interaction between the side chain of L123 and ribosomal residues critical for codon recognition in the decoding site, as a molecular explanation for coupling with eRF3. Our results provide insights into the molecular mechanisms underlying stop codon discrimination by a tRNA-mimicking protein on the ribosome. PMID:25897120

  9. Identification by mutational analysis of amino acid residues essential in the chaperone function of calreticulin.

    PubMed

    Martin, Virginie; Groenendyk, Jody; Steiner, Simone S; Guo, Lei; Dabrowska, Monika; Parker, J M Robert; Müller-Esterl, Werner; Opas, Michal; Michalak, Marek

    2006-01-27

    Calreticulin is a Ca2+ -binding chaperone that resides in the lumen of the endoplasmic reticulum and is involved in the regulation of intracellular Ca2+ homeostasis and in the folding of newly synthesized glycoproteins. In this study, we have used site-specific mutagenesis to map amino acid residues that are critical in calreticulin function. We have focused on two cysteine residues (Cys(88) and Cys(120)), which form a disulfide bridge in the N-terminal domain of calreticulin, on a tryptophan residue located in the carbohydrate binding site (Trp(302)), and on certain residues located at the tip of the "hairpin-like" P-domain of the protein (Glu(238), Glu(239), Asp(241), Glu(243), and Trp(244)). Calreticulin mutants were expressed in crt(-/-) fibroblasts, and bradykinin-dependent Ca2+ release was measured as a marker of calreticulin function. Bradykinin-dependent Ca2+ release from the endoplasmic reticulum was rescued by wild-type calreticulin and by the Glu(238), Glu(239), Asp(241), and Glu(243) mutants. The Cys(88) and Cys(120) mutants rescued the calreticulin-deficient phenotype only partially ( approximately 40%), and the Trp(244) and Trp(302) mutants did not rescue it at all. We identified four amino acid residues (Glu(239), Asp(241), Glu(243), and Trp(244)) at the hairpin tip of the P-domain that are critical in the formation of a complex between ERp57 and calreticulin. Although the Glu(239), Asp(241), and Glu(243) mutants did not bind ERp57 efficiently, they fully restored bradykinin-dependent Ca2+ release in crt(-/-) cells. This indicates that binding of ERp57 to calreticulin may not be critical for the chaperone function of calreticulin with respect to the bradykinin receptor. PMID:16291754

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

    PubMed

    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

  11. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    PubMed Central

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea

    2015-01-01

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. This study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin. PMID:25724962

  12. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    SciTech Connect

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea; Wall, Judy D.

    2015-02-27

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. Ultimately, this study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.

  13. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    DOE PAGESBeta

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea; Wall, Judy D.

    2015-02-27

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential formore » mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. Ultimately, this study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.« less

  14. Surface lysine residues modulate the collisional transfer of fatty acid from adipocyte fatty acid binding protein to membranes.

    PubMed

    Herr, F M; Matarese, V; Bernlohr, D A; Storch, J

    1995-09-19

    The transfer of unesterified fatty acids (FA) from adipocyte fatty acid binding protein (A-FABP) to phospholipid membranes is proposed to occur via a collisional mechanism involving transient ionic and hydrophobic interactions [Wootan & Storch (1994) J. Biol. Chem. 269, 10517-10523]. In particular, it was suggested that membrane acidic phospholipids might specifically interact with basic residues on the surface of A-FABP. Here we addressed whether lysine residues on the surface of the protein are involved in this collisional transfer mechanism. Recombinant A-FABP was acetylated to neutralize all positively charged surface lysine residues. Protein fluorescence, CD spectra, and chemical denaturant data indicate that acetylation did not substantially alter the conformational integrity of the protein, and nearly identical affinities were obtained for binding of the fluorescently labeled FA [12-(9-anthroyloxy)oleate] to native and acetylated protein. Transfer of 2-(9-anthroyloxy)palmitate (2AP) from acetylated A-FABP to small unilamellar vesicles (SUV) was 35-fold slower than from native protein. In addition, whereas the 2AP transfer rate from native A-FABP was directly dependent on SUV concentration, 2AP transfer from acetylated protein was independent on the concentration of acceptor membranes. Factors which alter aqueous-phase solubility of FA, such as ionic strength and acyl chain length and saturation, affected the AOFA transfer rate from acetylated but not native A-FABP. Finally, an increase in the negative charge density of the acceptor SUV resulted in a marked increase in the rate of transfer from native A-FABP but did not increase the rate from acetylated A-FABP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7547918

  15. A Conserved Acidic Residue in Phenylalanine Hydroxylase Contributes to Cofactor Affinity and Catalysis

    PubMed Central

    2015-01-01

    The catalytic domains of aromatic amino acid hydroxylases (AAAHs) contain a non-heme iron coordinated to a 2-His-1-carboxylate facial triad and two water molecules. Asp139 from Chromobacterium violaceum PAH (cPAH) resides within the second coordination sphere and contributes key hydrogen bonds with three active site waters that mediate its interaction with an oxidized form of the cofactor, 7,8-dihydro-l-biopterin, in crystal structures. To determine the catalytic role of this residue, various point mutants were prepared and characterized. Our isothermal titration calorimetry (ITC) analysis of iron binding implies that polarity at position 139 is not the sole criterion for metal affinity, as binding studies with D139E suggest that the size of the amino acid side chain also appears to be important. High-resolution crystal structures of the mutants reveal that Asp139 may not be essential for holding the bridging water molecules together, because many of these waters are retained even in the Ala mutant. However, interactions via the bridging waters contribute to cofactor binding at the active site, interactions for which charge of the residue is important, as the D139N mutant shows a 5-fold decrease in its affinity for pterin as revealed by ITC (compared to a 16-fold loss of affinity in the case of the Ala mutant). The Asn and Ala mutants show a much more pronounced defect in their kcat values, with nearly 16- and 100-fold changes relative to that of the wild type, respectively, indicating a substantial role of this residue in stabilization of the transition state by aligning the cofactor in a productive orientation, most likely through direct binding with the cofactor, supported by data from molecular dynamics simulations of the complexes. Our results indicate that the intervening water structure between the cofactor and the acidic residue masks direct interaction between the two, possibly to prevent uncoupled hydroxylation of the cofactor before the arrival of

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

    PubMed

    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

  17. A Novel Treatment for Acid Mine Drainage Utilizing Reclaimed Limestone Residual

    SciTech Connect

    Horace K. Moo-Young; Charles E. Ochola

    2004-08-31

    The viability of utilizing Reclaimed Limestone Residual (RLR) to remediate Acid Mine Drainage (AMD) was investigated. Physical and chemical characterization of RLR showed that it is composed of various minerals that contain significant quantities of limestone or calcium bearing compounds that can be exploited for acid neutralization. Acid Neutralization Potential (ANP) test results showed that RLR has a neutralization potential of approximately 83% as calcium carbonate (CaCO{sub 3}). Neutralization tests with most of the heavy metals associated with AMD showed removal efficiencies of over 99%. An unexpected benefit of utilizing RLR was the removal of hexavalent chromium Cr (VI) from the aqueous phase. Due to an elevation in pH by RLR most AMD heavy metals are removed from solution by precipitation as their metal hydroxides. Cr (VI) however is not removed by pH elevation and therefore subsequent ongoing tests to elucidate the mechanism responsible for this reaction were conducted.

  18. A role for residue 151 of LamB in bacteriophage lambda adsorption: possible steric effect of amino acid substitutions.

    PubMed

    Charbit, A; Werts, C; Michel, V; Klebba, P E; Quillardet, P; Hofnung, M

    1994-06-01

    LamB is the cell surface receptor for bacteriophage lambda. LamB missense mutations yielding resistance to lambda have been previously grouped in two classes. Class I mutants block growth of lambda with wild-type host range (lambda h+) but support growth of one-step extended-host-range mutants (lambda h). Class II mutants block lambda h but support growth of two-step extended host range mutants (lambda hh*). While Class I mutations occur at 11 different amino acid sites, in five distinct portions of LamB, all the Class II mutations analyzed previously correspond to the same G-to-D change at amino acid 151. We generated by in vitro mutagenesis four different new substitutions at site 151 (to S, V, R, and C). Two of the mutants (G-151-->V [G151V] and G151R) were of Class II, while the two others (G151S and G151C) were of Class I, demonstrating that not only the site but also the nature of the substitutions at residue 151 was critical for the phage sensitivity phenotypes. The introduction of a negatively charged, a positively charged, or an aliphatic nonpolar residue at site 151 of LamB prevented both lambda h+ and lambda h adsorption, indicating that the block is not due to a charge effect. In contrast to G151D, which was sensitive to all the lambda hh* phages, G151V and G151R conferred sensitivity to only four of the five lambda hh* phages. Thus, G151V and G151R represent a new subclass of Class II LamB mutations that is more restrictive with respect to the growth of lambda hh*. Our results agree with the hypothesis that residue 151 belongs to an accessibility gate controlling the access to the phage tight-binding site and that substitutions at this residue affect the access of the phage to the binding site in relation to the size of the substitute side chain (surface area): the most restrictive changes are G151V and G151R, followed to a lesser extent by G151D and they by G151S and G151C. PMID:8195074

  19. Conformational characterization of the 1-aminocyclobutane-1-carboxylic acid residue in model peptides.

    PubMed

    Gatos, M; Formaggio, F; Crisma, M; Toniolo, C; Bonora, G M; Benedetti, Z; Di Blasio, B; Iacovino, R; Santini, A; Saviano, M; Kamphuis, J

    1997-01-01

    A series of N- and C-protected, monodispersed homo-oligopeptides (to the dodecamer level) from the small-ring alicyclic C alpha, alpha-dialkylated glycine 1-aminocyclobutane-1-carboxylic acid (Ac4c) and two Ala/Ac4c tripeptides were synthesized by solution methods and fully characterized. The conformational preferences of all the model peptides were determined in deuterochloroform solution by FT-IR absorption and 1H-NMR. The molecular structures of the amino acid derivatives Z-Ac4c-OH and Z2-Ac4c-OH, the tripeptides Z-(Ac4c)3-OtBu, Z-Ac4c-(L-Ala)2-OMe and Z-L-Ala-Ac4c-L-Ala-OMe, and the tetrapeptide Z-(Ac4c)4-OtBu were determined in the crystal state by X-ray diffraction. The average geometry of the cyclobutyl moiety of the Ac4c residue was assessed and the tau(N-C alpha-C') bond angle was found to be significantly expanded from the regular tetrahedral value. The conformational data are strongly in favour of the conclusion that the Ac4c residue is an effective beta-turn and helix former. A comparison with the structural propensities of alpha-aminoisobutyric acid, the prototype of C alpha, alpha-dialkylated glycines, and the other extensively investigated members of the family of 1-aminocycloalkane-1-carboxylic acids (Acnc, with n = 3, 5-8) is made and the implications for the use of the Ac4c residue in conformationally constrained peptide analogues are briefly examined. PMID:9230476

  20. Functional role of polar amino acid residues in Na+/H+ exchangers.

    PubMed Central

    Wiebe, C A; Dibattista, E R; Fliegel, L

    2001-01-01

    Na(+)/H(+) exchangers are a family of ubiquitous membrane proteins. In higher eukaryotes they regulate cytosolic pH by removing an intracellular H(+) in exchange for an extracellular Na(+). In yeast and Escherichia coli, Na(+)/H(+) exchangers function in the opposite direction to remove intracellular Na(+) in exchange for extracellular H(+). Na(+)/H(+) exchangers display an internal pH-sensitivity that varies with the different antiporter types. Only recently have investigations examined the amino acids involved in pH-sensitivity and in cation binding and transport. Histidine residues are good candidates for H(+)-sensing amino acids, since they can ionize within the physiological pH range. Histidine residues have been shown to be important in the function of the E. coli Na(+)/H(+) exchanger NhaA and in the yeast Na(+)/H(+) exchanger sod2. In E. coli, His(225) of NhaA may function to interact with, or regulate, the pH-sensory region of NhaA. In sod2, His(367) is also critical to transport and may be a functional analogue of His(225) of NhaA. Histidine residues are not critical for the function of the mammalian Na(+)/H(+) exchanger, although an unusual histidine-rich sequence of the C-terminal tail has some influence on activity. Other amino acids involved in cation binding and transport by Na(+)/H(+) exchangers are only beginning to be studied. Amino acids with polar side chains such as aspartate and glutamate have been implicated in transport activity of NhaA and sod2, but have not been studied in the mammalian Na(+)/H(+) exchanger. Further studies are needed to elucidate the mechanisms involved in pH-sensitivity and cation binding and transport by Na(+)/H(+) exchangers. PMID:11415429

  1. Particulates in hydrometallurgy: Part III. Dewatering behavior of flocculated laterite acid leach residues

    NASA Astrophysics Data System (ADS)

    Briceno, A.; Osseo-Asare, K.

    1995-02-01

    Three polyacrylamide-based polymers of different chemical properties (polymer A, 34 pct anionic, 11×106 mol wt; polymer B, 7 pct anionic, 7.5×106 mol wt; polymer C, nonionic, 13.5×106 mol wt) were used to evaluate the flocculation behavior of laterite acid leach residues. The solid-liquid separation characteristics of the leach residues were investigated with the aid of settling rate, supernatant turbidity, and slurry filtrability measurements. The polymeric flocculants were found to be effective in improving the dewatering properties of the acid leach residues. Polymer effectiveness increased with increasing polymer dosage for all the polymers, but an optimum polymer dose was only found for polymer A (34 pct anionic, 11×106 mol wt) in the studied range of polymer addition. Similarly, the dewatering behavior was improved at higher polymer molecular weight. In addition, it was found that the flocculation performance was adversely affected by an increase in the degree of polymer hydrolysis which, in turn, increases the ratio of carboxylic to amide functional groups in the polymer chain. Polymer C (nonionic ˜0 pct hydrolysis, 13.5×106 mol wt) was found to be the most efficient flocculant in terms of all the performance criteria investigated. The preceding results were rationalized in terms of bridging flocculation, the ionization and molecular configuration of the polymers, hydrogen bonding, and the solid/aqueous interfacial charge.

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

    SciTech Connect

    Delegard, C.H.; Rinehart, D.E.; Soderquist, C.Z.; Fadeff, S.K.

    1999-04-02

    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 (HNO{sub 3}). 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 {sup 239}Pu by a factor of 200, and for {sup 241}Am by a factor of 50. The solids also exceed the ERDF criterion for {sup 137}Cs by a factor of 2 and uranium by a factor of 5. Therefore, the radionuclides of greatest interest in this leaching study are first {sup 239}Pu and {sup 241}Am (both components of TRU) and then uranium and {sup 137}Cs.

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

    NASA Astrophysics Data System (ADS)

    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.

  4. Volatile fatty acids distribution during acidogenesis of algal residues with pH control.

    PubMed

    Li, Yan; Hua, Dongliang; Zhang, Jie; Zhao, Yuxiao; Xu, Haipeng; Liang, Xiaohui; Zhang, Xiaodong

    2013-06-01

    The anaerobic acidification of protein-rich algal residues with pH control (4, 6, 8, 10) was studied in batch reactors, which was operated at mesophilic(35 °C) condition. The distribution of major volatile fatty acids (VFAs) during acidogenesis was emphasized in this paper. The results showed that the acidification efficiency and VFAs distribution in the acid reactor strongly depended on the pH. The main product for all the runs involved acetic acid except that the proportion of butyric acid acidified at pH 6 was relatively higher. The other organic acids remained at lower levels. The VFAs yield reached the maximum value with about 0.6 g VFAs/g volatile solid (VS) added as pH was 8, and also the content of total ammonia nitrogen (TAN) reached the highest values of 9,629 mg/l. Low acidification degrees were obtained under the conditions at pH 4 and 10, which was not suitable for the metabolism of acidogens. Hydralic retention time (HRT) required for different conditions varied. As a consequence, it was indicated that pH was crucial to the acidification efficiency and products distribution. The investigation of acidogenesis process, which was producing the major substrates, short-chain fatty acids, would play the primary role in the efficient operation of methanogenesis. PMID:23381617

  5. 75 FR 1773 - Notice of Receipt of a Pesticide Petition Filed for Residues of Polymeric Polyhydroxy Acid in or...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-13

    ...This notice announces the Agency's receipt of an initial filing of a pesticide petition proposing the establishment of a regulation for residues of the plant growth regulator, polymeric polyhydroxy acid, in or on all food...

  6. A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases.

    PubMed

    Salmon, Melissa; Thimmappa, Ramesha B; Minto, Robert E; Melton, Rachel E; Hughes, Richard K; O'Maille, Paul E; Hemmings, Andrew M; Osbourn, Anne

    2016-07-26

    Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply. PMID:27412861

  7. Determination of thymine glycol residues in irradiated or oxidized DNA by formation of methylglyceric acid

    SciTech Connect

    Schellenberg, K.A.; Shaeffer, J.

    1986-05-01

    Treatment of DNA solutions with X-irradiation various oxidants including hydrogen peroxide plus ferrous ion, hydrogen peroxide plus copper ion and ascorbate, permanganate, or sonication in the presence of dissolved oxygen all produced varying amounts of thymine glycol residues. After denaturing the DNA with heat, the glycol residues were reduced and labeled at the 6 position with tritium- labeled sodium borohydride. Subsequent reaction with anhydrous methanolic HCl gave a quantitative yield of the methyl ester of methylglyceric acid, which was determined by thin layer chromatography. The method, developed using thymidine as a model, was used to ascertain the requirements for glycol formation in DNA. It was shown that hydroxyl radical generating systems, permanganate, X-irradiation, or sonication in presence of oxygen were required, but hydrogen peroxide in the absence of iron or copper and ascorbate was inactive. Application to determination of DNA damage in vivo is being explored.

  8. A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases

    PubMed Central

    Salmon, Melissa; Thimmappa, Ramesha B.; Minto, Robert E.; Melton, Rachel E.; O’Maille, Paul E.; Hemmings, Andrew M.; Osbourn, Anne

    2016-01-01

    Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply. PMID:27412861

  9. Toxicity of boric acid to Blattella germanica (Dictyoptera: Blattellidae) and analysis of residues in several organs.

    PubMed

    Habes, D; Kilani-Morakchi, S; Aribi, N; Farine, J P; Soltani, N

    2001-01-01

    Pestiferous cockroach species are associated closely with humans and are important from medical and public health points of view. Conventional insecticides have been used widely to control cockroaches which have developed resistance to these compounds. Thus, interest has again centered on lesser-used compounds such as boric acid. Boric acid has been used as an insecticide for many years, especially against cockroach. Its mode of action on insects has not been satisfactorily established. In Algeria, Blattella germanica (Dictyoptera: Blattellidae) is a serious pest in the urban environment and their infestation were controlled for many years by organophosphate, carbamate or pyrethroid insecticides. In order to obtain more information on the mode of action of boric acid, we first evaluated the oral toxicity of boric acid on B. germanica adults. Then, the compound was determined in several organs by an colorimetric method. This insecticide was incorporated into the diet and orally administered at different concentrations ranging from 1 to 40% (w/w) to newly emerged adults. Mortality was recorded at different times during treatment (24, 48, 72 and 144 h). Treatment resulted in a dose-dependent mortality since the LD50 (%) recorded are 85 at 24 h, 67 at 48 h, 39 at 72 h and 8 at 144 h, respectively. Then the quantity of boric acid accumulated in several organs (hemolymph, gut, ovaries, testicles and fat body) was determined as function the duration of treatment (1 to 5 days) for two doses (LD50 and LD90). Results revealed that bioaccumulation of residues in these organs increased as function the duration of treatment. In addition, relatively important amounts of residues, are detected in fat body. PMID:12425074

  10. Effect of lime on the availability of residual phosphorus and its extractability by dilute acid

    SciTech Connect

    Rhue, R.D.; Hensel, D.R.

    1983-01-01

    The objective of this study was to determine the long-term effects of liming an acid, P-deficient Placid sand (sandy, siliceous, hyperthermic Typic Humaquept) on the availability of residual fertilizer P to potatoes (Solanum tuberosum L.). Dolomitic limestone was applied in November 1977, at rates of 0, 2240, 4480, and 8960 kg/ha in a split-plot design with lime as main plots and P treatments as subplots. Phosphorus was applied at rates of 0, 56, 112, and 168 kg/ha in 1978. In 1979 and 1980, P plots were split with one-half fertilized with 56 kg P/ha and the other one-half not fertilized with P (residual). In 1978, maximum tuber yields and top dry weights occurred at the 2240 kg/ha lime rate which resulted in a soil pH of 5.8. Plant P concentrations were unaffected by lime at any sampling rate. In 1979, availability of residual soil P decreased with lime rates > 2240 kg/ha but not enough to significantly affect yields. However, in 1980, overliming injury was observed for tuber yields at the higher lime rates which was the result of P deficiency. Application of P at planting eliminated the overliming injury with maximum yields occurring in the pH range of 6.0 to 6.5. It appears that liming to pH 6.5 in this study resulted in fertilizer reaction products that were more soluble in dilute acid but less plant available than those formed under more acid conditions. However, the Mehlich I extractant appeared to be a suitable extractant for P on this soil if pH was taken into account when interpreting soil-test P. 23 references, 4 figures, 2 tables.

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

    PubMed

    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

  12. Identification of essential amino acid residues of an alpha-amylase inhibitor from Phaseolus vulgaris white kidney beans.

    PubMed

    Takahashi, T; Hiramoto, S; Wato, S; Nishimoto, T; Wada, Y; Nagai, K; Yamaguchi, H

    1999-11-01

    Kidney bean (Phaseolus vulgaris) alpha-amylase inhibitors, which are bivalent inhibitors with the subunit stoichiometry of (alphabeta)(2) complex, have been inferred to contain unique arginine, tryptophan, and tyrosine residues essential for the inhibitory activity. To test the validity of this inference, an attempt was made to identify the essential amino acid residues of a white kidney bean (P. vulgaris) alpha-amylase inhibitor (PHA-I) by using the chemical modification technique combined with amino acid sequencing and mass spectrometry. Exhaustive modification of the arginine residues by phenylglyoxal did not lead to a marked loss of activity, suggesting that no arginine residue is directly associated with the inhibitory activity. N-Bromosuccinimide treatment of PHA-I in the presence or absence of a substrate alpha-amylase revealed the involvement of two tryptophan residues in alpha-amylase inhibition, and they were identified as Trp188 of the beta-subunit by amino acid sequencing and mass spectrometry of lysylendopeptidase peptides. Further, two tyrosine residues were preferentially modified either by N-acetylimidazole or by tetranitromethane, resulting in a concomitant loss of most of the PHA-I activity. Amino acid sequencing of the lysylendopeptidase peptides from a tetranitromethane-modified PHA-I identified Tyr186 of the beta-subunit as an essential residue. PMID:10544275

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

    PubMed Central

    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

  14. Identification of Key Amino Acid Residues Modulating Intracellular and In vitro Microcin E492 Amyloid Formation

    PubMed Central

    Aguilera, Paulina; Marcoleta, Andrés; Lobos-Ruiz, Pablo; Arranz, Rocío; Valpuesta, José M.; Monasterio, Octavio; Lagos, Rosalba

    2016-01-01

    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 Escherichia coli cells, that were visualized as round-shaped inclusion bodies recognized by two amyloidophilic 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

  15. Identification of three critical acidic residues of poly(ADP-ribose) glycohydrolase involved in catalysis: determining the PARG catalytic domain

    PubMed Central

    Patel, Chandra N.; Koh, David W.; Jacobson, Myron K.; Oliveira, Marcos A.

    2005-01-01

    PARG [poly(ADP-ribose) glycohydrolase] catalyses the hydrolysis of α(1″→2′) or α(1‴→2″) O-glycosidic linkages of ADP-ribose polymers to produce free ADP-ribose. We investigated possible mechanistic similarities between PARG and glycosidases, which also cleave O-glycosidic linkages. Glycosidases typically utilize two acidic residues for catalysis, thus we targeted acidic residues within a conserved region of bovine PARG that has been shown to contain an inhibitor-binding site. The targeted glutamate and aspartate residues were changed to asparagine in order to minimize structural alterations. Mutants were purified and assayed for catalytic activity, as well as binding, to an immobilized PARG inhibitor to determine ability to recognize substrate. Our investigation revealed residues essential for PARG catalytic activity. Two adjacent glutamic acid residues are found in the conserved sequence Gln755-Glu-Glu757, and a third residue found in the conserved sequence Val737-Asp-Phe-Ala-Asn741. Our functional characterization of PARG residues, along with recent identification of an inhibitor-binding residue Tyr796 and a glycine-rich region Gly745-Gly-Gly747 important for PARG function, allowed us to define a PARG ‘signature sequence’ [vDFA-X3-GGg-X6–8-vQEEIRF-X3-PE-X14-E-X12-YTGYa], which we used to identify putative PARG sequences across a range of organisms. Sequence alignments, along with our mapping of PARG functional residues, suggest the presence of a conserved catalytic domain of approx. 185 residues which spans residues 610–795 in bovine PARG. PMID:15658938

  16. Improving the acidic stability of Staphylococcus aureus α-acetolactate decarboxylase in Bacillus subtilis by changing basic residues to acidic residues.

    PubMed

    Zhang, Xian; Rao, Zhiming; Li, Jingjing; Zhou, Junping; Yang, Taowei; Xu, Meijuan; Bao, Teng; Zhao, Xiaojing

    2015-04-01

    The α-acetolactate decarboxylase (ALDC) can reduce diacetyl fleetly to promote mature beer. A safe strain Bacillus subtilis WB600 for high-yield production of ALDC was constructed with the ALDC gene saald from Staphylococcus aureus L3-15. SDS-PAGE analysis revealed that S. aureus α-acetolactate decarboxylase (SaALDC) was successfully expressed in recombinant B. siutilis strain. The enzyme SaALDC was purified using Ni-affinity chromatography and showed a maximum activity at 45 °C and pH 6.0. The values of K m and V max were 17.7 μM and 2.06 mM min(-1), respectively. Due to the unstable property of SaALDC at low pH conditions that needed in brewing process, site-directed mutagenesis was proposed for improving the acidic stability of SaALDC. Homology comparative modeling analysis showed that the mutation (K52D) gave rise to the negative-electrostatic potential on the surface of protein while the numbers of hydrogen bonds between the mutation site (N43D) and the around residues increased. Taken together the effect of mutation N43D-K52D, recombinant SaALDCN43D-K52D showed dramatically improved acidic stability with prolonged half-life of 3.5 h (compared to the WT of 1.5 h) at pH 4.0. In a 5-L fermenter, the recombinant B. subtilis strain that could over-express SaALDCN43D-K52D exhibited a high yield of 135.8 U mL(-1) of SaALDC activity, about 320 times higher comparing to 0.42 U mL(-1) of S. aureus L3-15. This work proposed a  strategy for improving the acidic stability of SaALDC in the  B. subtilis host. PMID:25543264

  17. Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes.

    PubMed

    Leitner, Alexander; Joachimiak, Lukasz A; Unverdorben, Pia; Walzthoeni, Thomas; Frydman, Judith; Förster, Friedrich; Aebersold, Ruedi

    2014-07-01

    The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches. PMID:24938783

  18. Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes

    PubMed Central

    Leitner, Alexander; Joachimiak, Lukasz A.; Unverdorben, Pia; Walzthoeni, Thomas; Frydman, Judith; Förster, Friedrich; Aebersold, Ruedi

    2014-01-01

    The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches. PMID:24938783

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

    PubMed

    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

  20. Newly identified essential amino acid residues affecting ^8-sphingolipid desaturase activity revealed by site-directed mutagenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to identify amino acid residues crucial for the enzymatic activity of ^8-sphingolipid desaturases, a sequence comparison was performed among ^8-sphingolipid desaturases and ^6-fatty acid desaturase from various plants. In addition to the known conserved cytb5 (cytochrome b5) HPGG motif and...

  1. The Dual NOD1/NOD2 Agonism of Muropeptides Containing a Meso-Diaminopimelic Acid Residue

    PubMed Central

    Dagil, Yulia A.; Arbatsky, Nikolai P.; Alkhazova, Biana I.; L’vov, Vyacheslav L.; Mazurov, Dmitriy V.; Pashenkov, Mikhail V.

    2016-01-01

    Muropeptides are fragments of peptidoglycan that trigger innate immune responses by activating nucleotide-binding oligomerization domain (NOD) 1 and NOD2. Muropeptides from Gram-negative bacteria contain a meso-diaminopimelic acid (meso-DAP) residue in either a terminal or a non-terminal position. While the former ones are known to be recognized by NOD1, much less is known about recognition of muropeptides with non-terminal meso-DAP, which are most abundant moieties of Gram-negative peptidoglycans. Here, we developed a novel system to assess biological activity of muropeptides, based on CRISPR/Cas9-mediated knockout (KO) of NOD1 and NOD2 genes in modified HEK293T cells. Using NOD1/NOD2 knockout and overexpression systems, as well as human monocytes and macrophages, we refine the current view of muropeptide recognition. We show that NOD2 can recognize different natural muropeptides containing a meso-DAP residue (preferably in a non-terminal position), provided they are present at micromolar concentrations. NOD2 accepts muropeptides with long and branched peptide chains and requires an intact N-acetylmuramyl residue. Muropeptides with non-terminal meso-DAP can activate NOD1 as well, but, in this case, probably require peptidase pre-processing to expose the meso-DAP residue. Depending on NOD1/NOD2 ratio in specific cell types, meso-DAP-containing muropeptides can be recognized either primarily via NOD2 (in monocytes) or via NOD1 (in monocyte-derived macrophages and HEK293T-derived cells). The dual NOD1/NOD2 agonism of meso-DAP-containing muropeptides should be taken into account when assessing cellular responses to muropeptides and designing muropeptide immunostimulants and vaccine adjuvants. PMID:27513337

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

    PubMed

    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 tacid 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

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

    PubMed

    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

  4. Acidic Residues in the Hfq Chaperone Increase the Selectivity of sRNA Binding and Annealing.

    PubMed

    Panja, Subrata; Santiago-Frangos, Andrew; Schu, Daniel J; Gottesman, Susan; Woodson, Sarah A

    2015-11-01

    Hfq facilitates gene regulation by small non-coding RNAs (sRNAs), thereby affecting bacterial attributes such as biofilm formation and virulence. Escherichia coli Hfq recognizes specific U-rich and AAN motifs in sRNAs and target mRNAs, after which an arginine patch on the rim promotes base pairing between their complementary sequences. In the cell, Hfq must discriminate between many similar RNAs. Here, we report that acidic amino acids lining the sRNA binding channel between the inner pore and rim of the Hfq hexamer contribute to the selectivity of Hfq's chaperone activity. RNase footprinting, in vitro binding and stopped-flow fluorescence annealing assays showed that alanine substitution of D9, E18 or E37 strengthened RNA interactions with the rim of Hfq and increased annealing of non-specific or U-tailed RNA oligomers. Although the mutants were less able than wild-type Hfq to anneal sRNAs with wild-type rpoS mRNA, the D9A mutation bypassed recruitment of Hfq to an (AAN)4 motif in rpoS, both in vitro and in vivo. These results suggest that acidic residues normally modulate access of RNAs to the arginine patch. We propose that this selectivity limits indiscriminate target selection by E. coli Hfq and enforces binding modes that favor genuine sRNA and mRNA pairs. PMID:26196441

  5. Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1

    PubMed Central

    2009-01-01

    Background Antibacterial activity is a novel function of high-mobility group box 1 (HMGB1). However, the functional site for this new effect is presently unknown. Methods and Results In this study, recombinant human HMGB1 A box and B box (rHMGB1 A box, rHMGB1 B box), recombinant human HMGB1 (rHMGB1) and the truncated C-terminal acidic tail mutant (tHMGB1) were prepared by the prokaryotic expression system. The C-terminal acidic tail (C peptide) was synthesized, which was composed of 30 amino acid residues. Antibacterial assays showed that both the full length rHMGB1 and the synthetic C peptide alone could efficiently inhibit bacteria proliferation, but rHMGB1 A box and B box, and tHMGB1 lacking the C-terminal acidic tail had no antibacterial function. These results suggest that C-terminal acidic tail is the key region for the antibacterial activity of HMGB1. Furthermore, we prepared eleven different deleted mutants lacking several amino acid residues in C-terminal acidic tail of HMGB1. Antibacterial assays of these mutants demonstrate that the amino acid residues 201-205 in C-terminal acidic tail region is the core functional site for the antibacterial activity of the molecule. Conclusion In sum, these results define the key region and the crucial site in HMGB1 for its antibacterial function, which is helpful to illustrating the antibacterial mechanisms of HMGB1. PMID:19751520

  6. Determination of the amino acid residue involved in [3H]beta-funaltrexamine covalent binding in the cloned rat mu-opioid receptor.

    PubMed

    Chen, C; Yin, J; Riel, J K; DesJarlais, R L; Raveglia, L F; Zhu, J; Liu-Chen, L Y

    1996-08-30

    We previously demonstrated that [3H]beta-funaltrexamine ([3H]beta-FNA) labeled the rat mu opioid receptor expressed in Chinese hamster ovary cells with high specificity, and [3H]beta-FNA-labeled receptors migrated as one broad band with a mass of 80 kDa. In this study, we determined the region and then the amino acid residue of the mu receptor involved in the covalent binding of [3H]beta-FNA. [3H]beta-FNA-labeled receptors were solubilized and purified to approximately 10% purity by immunoaffinity chromatography with antibodies against a C-terminal domain peptide. The site of covalent bond formation was determined to be within Ala206-Met243 by CNBr cleavage of partially purified labeled mu receptors and determinations of sizes of labeled receptor fragments. The amino acid residue of beta-FNA covalent incorporation was then determined by site-directed mutagenesis studies within this region. Mutation of Lys233 to Ala, Arg, His, and Leu completely eliminated covalent binding of [3H]beta-FNA, although these mutants bound beta-FNA with high affinity. Mutations of other amino acid residues did not affect covalent binding of [3H]beta-FNA. These results indicate that [3H]beta-FNA binds covalently to Lys233. Since [3H]beta-FNA is a rigid molecule, the information will be very useful for molecular modeling of interaction between morphinans and the mu receptor. PMID:8702924

  7. Cloning and identification of amino acid residues of human phospholipase C delta 1 essential for catalysis.

    PubMed

    Cheng, H F; Jiang, M J; Chen, C L; Liu, S M; Wong, L P; Lomasney, J W; King, K

    1995-03-10

    In vitro single point mutagenesis, inositol phospholipid hydrolysis, and substrate protection experiments were used to identify catalytic residues of human phosphatidylinositide-specific phospholipase C delta 1 (PLC delta 1) isolated from a human aorta cDNA library. Invariant amino acid residues containing a functional side chain in the highly conserved X region were changed by in vitro mutagenesis. Most of the mutant enzymes were still able to hydrolyze inositol phospholipid with activity ranging from 10 to 100% of levels in the wild type enzyme. Exceptions were mutants with the conversion of Arg338 to Leu (R338L), Glu341 to Gly (E341G), or His356 to Leu (H356L), which made the enzyme severely defective in hydrolyzing inositol phospholipid. Phospholipid vesicle binding experiments showed that these three cleavage-defective mutant forms of PLC delta 1 could specifically bind to phosphatidylinositol 4,5-bisphosphate (PIP2) with an affinity similar to that of wild type enzyme. Western blotting analysis of trypsin-treated enzyme-PIP2 complexes revealed that a 67-kDa major protein fragment survived trypsin digestion if the wild type enzyme, E341G, or H356L mutant PLC delta 1 was preincubated with 7.5 microM PIP2, whereas if it was preincubated with 80 microM PIP2, the size of major protein surviving was comparable to that of intact enzyme. However, mutant enzyme R338L was not protected from trypsin degradation by PIP2 binding. These observations suggest that PLC delta 1 can recognize PIP2 through a high affinity and a low affinity binding site and that residues Glu341 and His356 are not involved in either high affinity or low affinity PIP2 binding but rather are essential for the Ca(2+)-dependent cleavage activity of PLC. PMID:7890667

  8. Characterisation of the products from pyrolysis of residues after acid hydrolysis of Miscanthus.

    PubMed

    Melligan, F; Dussan, K; Auccaise, R; Novotny, E H; Leahy, J J; Hayes, M H B; Kwapinski, W

    2012-03-01

    Platform chemicals such as furfural and hydroxymethylfurfural are major products formed during the acid hydrolysis of lignocellulosic biomass in second generation biorefining processes. Solid hydrolysis residues (HR) can amount to 50 wt.% of the starting biomass materials. Pyrolysis of the HRs gives rise to biochar, bio-liquids, and gases. Time and temperature were variables during the pyrolysis of HRs in a fixed bed tubular reactor, and both parameters have major influences on the amounts and properties of the products. Biochar, with potential for carbon sequestration and soil conditioning, composed about half of the HR pyrolysis product. The amounts (11-20 wt.%) and compositions (up to 77% of phenols in organic fraction) of the bio-liquids formed suggest that these have little value as fuels, but could be sources of phenols, and the gas can have application as a fuel. PMID:22281143

  9. A Mutational Analysis of Active Site Residues in trans-3-Chloroacrylic Acid Dehalogenase

    PubMed Central

    Poelarends, Gerrit J.; Serrano, Hector; Huddleston, Jamison P.; Johnson, William H.; Whitman, Christian P.

    2013-01-01

    trans -3-Chloroacrylic acid dehalogenase (CaaD) catalyzes the hydrolytic dehalogenation of trans-3-haloacrylates to yield malonate semialdehyde by a mechanism utilizing βPro-1, αArg-8, αArg-11, and αGlu-52. These residues are implicated in a promiscuous hydratase activity where 2-oxo-3-pentynoate is processed to acetopyruvate. The roles of three nearby residues (βAsn-39, αPhe-39, and αPhe-50) are unexplored. Mutants were constructed at these positions (βN39A, αF39A, αF39T, αF50A and αF50Y) and kinetic parameters determined along with those of the αR8K and αR11K mutants. Analysis indicates that αArg-8, αArg-11, and βAsn-39 are critical for dehalogenase activity whereas αArg-11 and αPhe-50 are critical for hydratase activity. Docking studies suggest structural bases for these observations. PMID:23851010

  10. Selective conversion of cellulose in corncob residue to levulinic acid in an aluminum trichloride-sodium chloride system.

    PubMed

    Li, Jianmei; Jiang, Zhicheng; Hu, Libin; Hu, Changwei

    2014-09-01

    Increased energy consumption and environmental concerns have driven efforts to produce chemicals from renewable biomass with high selectivity. Here, the selective conversion of cellulose in corncob residue, a process waste from the production of xylose, to levulinic acid was carried out using AlCl3 as catalyst and NaCl as promoter by a hydrothermal method at relatively low temperature. A levulinic acid yield of 46.8 mol% was obtained, and the total selectivity to levulinic acid with formic acid was beyond 90%. NaCl selectively promoted the dissolution of cellulose from corncob residue, and significantly improved the yield and selectivity to levulinic acid by inhibiting lactic acid formation in the subsequent dehydration process. Owing to the salt effect of NaCl, the obtained levulinic acid could be efficiently extracted to tetrahydrofuran from aqueous solution. The aqueous solution with AlCl3 and NaCl could be recycled 4 times. Because of the limited conversion of lignin, this process allows for the production of levulinic acid with high selectivity directly from corncob residue in a simple separation process. PMID:25045141

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

    PubMed

    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

  12. Differential contribution of basic residues to HIV-1 nucleocapsid protein’s nucleic acid chaperone function and retroviral replication

    PubMed Central

    Wu, Hao; Mitra, Mithun; Naufer, M. Nabuan; McCauley, Micah J.; Gorelick, Robert J.; Rouzina, Ioulia; Musier-Forsyth, Karin; Williams, Mark C.

    2014-01-01

    The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein contains 15 basic residues located throughout its 55-amino acid sequence, as well as one aromatic residue in each of its two CCHC-type zinc finger motifs. NC facilitates nucleic acid (NA) rearrangements via its chaperone activity, but the structural basis for this activity and its consequences in vivo are not completely understood. Here, we investigate the role played by basic residues in the N-terminal domain, the N-terminal zinc finger and the linker region between the two zinc fingers. We use in vitro ensemble and single-molecule DNA stretching experiments to measure the characteristics of wild-type and mutant HIV-1 NC proteins, and correlate these results with cell-based HIV-1 replication assays. All of the cationic residue mutations lead to NA interaction defects, as well as reduced HIV-1 infectivity, and these effects are most pronounced on neutralizing all five N-terminal cationic residues. HIV-1 infectivity in cells is correlated most strongly with NC’s NA annealing capabilities as well as its ability to intercalate the DNA duplex. Although NC’s aromatic residues participate directly in DNA intercalation, our findings suggest that specific basic residues enhance these interactions, resulting in optimal NA chaperone activity. PMID:24293648

  13. Radiogenic Ar retention in residual silica from acid-treated micas

    NASA Astrophysics Data System (ADS)

    Derkowski, Arkadiusz; Szczerba, Marek; Środoń, Jan; Banaś, Michał

    2014-03-01

    In sedimentary basins, immediate equilibration with surface and pore waters of Ar, released from K-bearing minerals during their diagenesis or weathering, has been a paradigm for geochemistry and geochronology. Consequently, K-Ar and Ar-Ar isotope geochronology techniques applied to sedimentary rocks are based on an assumption that no measurable external radiogenic 40Ar (“excess argon”) has been locked in the rock components during their formation and alteration. Our results indicate that the reaction of micaceous sedimentary and diagenetic clay minerals (illite, glauconite) with acid produces microporous silica that retains a great fraction of the initial argon, releasing potassium to the solution. In all tested cases the evolution of K-Ar isotope ages followed the very same pattern: the apparent K-Ar isotope age increased enormously after acid treatment and dropped significantly after silica removal (with hot Na2CO3), but never decreased lower than the initial K-Ar isotope age of the untreated sample. The amorphous silica content and the apparent K-Ar age increased with the acid reaction time. Using the molecular dynamics simulations, the clay-acid reaction by-product was shown to bend and wrap, producing three-dimensional, protonated and hydrated silica. As a consequence of dramatically different hydration energies of Ar and K, potassium is instantaneously released and hydrated outside the residual structure while Ar atoms remain inside the silica network, adsorbed on the surface. This is, to our knowledge, the first experimental evidence that the excess argon can be retained in solid mineral reaction products formed under pressure and temperature close to those of the Earth surface (1 atm, <80 °C).

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

    PubMed

    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

  15. Polyunsaturated fatty acids inhibit Kv1.4 by interacting with positively charged extracellular pore residues.

    PubMed

    Farag, N E; Jeong, D; Claydon, T; Warwicker, J; Boyett, M R

    2016-08-01

    Polyunsaturated fatty acids (PUFAs) modulate voltage-gated K(+) channel inactivation by an unknown site and mechanism. The effects of ω-6 and ω-3 PUFAs were investigated on the heterologously expressed Kv1.4 channel. PUFAs inhibited wild-type Kv1.4 during repetitive pulsing as a result of slowing of recovery from inactivation. In a mutant Kv1.4 channel lacking N-type inactivation, PUFAs reversibly enhanced C-type inactivation (Kd, 15-43 μM). C-type inactivation was affected by extracellular H(+) and K(+) as well as PUFAs and there was an interaction among the three: the effect of PUFAs was reversed during acidosis and abolished on raising K(+) Replacement of two positively charged residues in the extracellular pore (H508 and K532) abolished the effects of the PUFAs (and extracellular H(+) and K(+)) on C-type inactivation but had no effect on the lipoelectric modulation of voltage sensor activation, suggesting two separable interaction sites/mechanisms of action of PUFAs. Charge calculations suggest that the acidic head group of the PUFAs raises the pKa of H508 and this reduces the K(+) occupancy of the selectivity filter, stabilizing the C-type inactivated state. PMID:27281482

  16. Degradation of carbohydrates during dilute sulfuric acid pretreatment can interfere with lignin measurements in solid residues.

    PubMed

    Katahira, Rui; Sluiter, Justin B; Schell, Daniel J; Davis, Mark F

    2013-04-01

    The lignin content measured after dilute sulfuric acid pretreatment of corn stover indicates more lignin than could be accounted for on the basis of the untreated corn stover lignin content. This phenomenon was investigated using a combination of (13)C cross-polarization/magic-angle spinning (CP/MAS) solid-state nuclear magnetic resonance (NMR) spectroscopy and lignin removal using acid chlorite bleaching. Only minimal contamination with carbohydrates and proteins was observed in the pretreated corn stover. Incorporating degradation products from sugars was also investigated using (13)C-labeled sugars. The results indicate that sugar degradation products are present in the pretreatment residue and may be intimately associated with the lignin. Studies comparing whole corn stover (CS) to extractives-free corn stover [CS(Ext)] clearly demonstrated that extractives are a key contributor to the high-lignin mass balance closure (MBC). Sugars and other low molecular weight compounds present in plant extractives polymerize and form solids during pretreatment, resulting in apparent Klason lignin measurements that are biased high. PMID:23428141

  17. Tri-peptide reference structures for the calculation of relative solvent accessible surface area in protein amino acid residues.

    PubMed

    Topham, Christopher M; Smith, Jeremy C

    2015-02-01

    Relative amino acid residue solvent accessibility values allow the quantitative comparison of atomic solvent-accessible surface areas in different residue types and physical environments in proteins and in protein structural alignments. Geometry-optimised tri-peptide structures in extended solvent-exposed reference conformations have been obtained for 43 amino acid residue types at a high level of quantum chemical theory. Significant increases in side-chain solvent accessibility, offset by reductions in main-chain atom solvent exposure, were observed for standard residue types in partially geometry-optimised structures when compared to non-minimised models built from identical sets of proper dihedral angles abstracted from the literature. Optimisation of proper dihedral angles led most notably to marked increases of up to 54% in proline main-chain atom solvent accessibility compared to literature values. Similar effects were observed for fully-optimised tri-peptides in implicit solvent. The relief of internal strain energy was associated with systematic variation in N, C(α) and C(β) atom solvent accessibility across all standard residue types. The results underline the importance of optimisation of 'hard' degrees of freedom (bond lengths and valence bond angles) and improper dihedral angle values from force field or other context-independent reference values, and impact on the use of standardised fixed internal co-ordinate geometry in sampling approaches to the determination of absolute values of protein amino acid residue solvent accessibility. Quantum chemical methods provide a useful and accurate alternative to molecular mechanics methods to perform energy minimisation of peptides containing non-standard (chemically modified) amino acid residues frequently present in experimental protein structure data sets, for which force field parameters may not be available. Reference tri-peptide atomic co-ordinate sets including hydrogen atoms are made freely available

  18. SeqX: a tool to detect, analyze and visualize residue co-locations in protein and nucleic acid structures

    PubMed Central

    Biro, Jan C; Fördös, Gergely

    2005-01-01

    Background The interacting residues of protein and nucleic acid sequences are close to each other – they are co-located. Structure databases (like Protein Data Bank, PDB and Nucleic Acid Data Bank, NDB) contain all information about these co-locations; however it is not an easy task to penetrate this complex information. We developed a JAVA tool, called SeqX for this purpose. Results SeqX tool is useful to detect, analyze and visualize residue co-locations in protein and nucleic acid structures. The user a. selects a structure from PDB; b. chooses an atom that is commonly present in every residues of the nucleic acid and/or protein structure(s) c. defines a distance from these atoms (3–15 Å). The SeqX tool detects every residue that is located within the defined distances from the defined "backbone" atom(s); provides a DotPlot-like visualization (Residues Contact Map), and calculates the frequency of every possible residue pairs (Residue Contact Table) in the observed structure. It is possible to exclude +/- 1 to 10 neighbor residues in the same polymeric chain from detection, which greatly improves the specificity of detections (up to 60% when tested on dsDNA). Results obtained on protein structures showed highly significant correlations with results obtained from literature (p < 0.0001, n = 210, four different subsets). The co-location frequency of physico-chemically compatible amino acids is significantly higher than is calculated and expected in random protein sequences (p < 0.0001, n = 80). Conclusion The tool is simple and easy to use and provides a quick and reliable visualization and analyses of residue co-locations in protein and nucleic acid structures. Availability and requirements SeqX, Java J2SE Runtime Environment 5.0 (available from [see Additional file 1] ) and at least a 1 GHz processor and with a minimum 256 Mb RAM. Source codes are available from the authors. PMID:16011796

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

    PubMed

    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

  20. National Diet and Nutrition Survey: fat and fatty acid intake from the first year of the rolling programme and comparison with previous surveys.

    PubMed

    Pot, Gerda K; Prynne, Celia J; Roberts, Caireen; Olson, Ashley; Nicholson, Sonja K; Whitton, Clare; Teucher, Birgit; Bates, Beverley; Henderson, Helen; Pigott, Sarah; Swan, Gillian; Stephen, Alison M

    2012-02-01

    High saturated fat intake is an established risk factor for several chronic diseases. The objective of the present study is to report dietary intakes and main food sources of fat and fatty acids (FA) from the first year of the National Diet and Nutrition Survey (NDNS) rolling programme in the UK. Dietary data were collected using 4 d estimated food diaries (n 896) and compared with dietary reference values (DRV) and previous NDNS results. Total fat provided 34-36 % food energy (FE) across all age groups, which was similar to previous surveys for adults. Men (19-64 years) and older girls (11-18 years) had mean intakes just above the DRV, while all other groups had mean total fat intakes of < 35 % FE. SFA intakes were lower compared with previous surveys, ranging from 13 to 15 % FE, but still above the DRV. Mean MUFA intakes were 12·5 % FE for adults and children aged 4-18 years and all were below the DRV. Mean n-3 PUFA intake represented 0·7-1·1 % FE. Compared with previous survey data, the direction of change for n-3 PUFA was upwards for all age groups, although the differences in absolute terms were very small. Trans-FA intakes were lower than in previous NDNS and were less than 2 g/d for all age groups, representing 0·8 % FE and lower than the DRV in all age groups. In conclusion, dietary intake of fat and FA is moving towards recommended levels for the UK population. However, there remains room for considerable further improvement. PMID:21767448

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

    PubMed

    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

  2. Mutational analysis of nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of critical amino acid residues involved in exopolysaccharide alginate synthesis.

    PubMed Central

    Sundin, G W; Shankar, S; Chakrabarty, A M

    1996-01-01

    We report the utilization of site-directed and random mutagenesis procedures in the gene encoding nucleoside diphosphate kinase (ndk) from Pseudomonas aeruginosa in order to examine the role of Ndk in the production of alginate by this organism. Cellular levels of the 16-kDa form of the Ndk enzyme are greatly reduced in P. aeruginosa 8830 with a knockout mutation in the algR2 gene (8830R2::Cm); this strain is also defective in the production of the exopolysaccharide alginate. In this study, we isolated four mutations in ndk (Ala-14-->Pro [Ala14Pro], Gly21Val, His117Gln, and Ala125Arg) which resulted in the loss of Ndk biochemical activity; hyperexpression of any of these four mutant genes did not restore alginate production to 8830R2::Cm. We identified six additional amino acid residues (Ser-43, Ala-56, Ser-69, Glu-80, Gly-91, and Asp-135) whose alteration resulted in the inability of Ndk to complement alginate production. After hyperproduction in 8830R2::Cm, it was determined that each of these six mutant Ndks was biochemically active. However, in four cases, the in vivo levels of Ndk were reduced, which consequently affected the growth of 8830R2::Cm in the presence of Tween 20. Two mutant Ndk proteins which could not complement the alginate synthesis defect in 8830R2::Cm were not affected in any characteristic examined in the present study. All of the mutant Ndks characterized which were still biochemically active formed membrane complexes with Pk, resulting in GTP synthesis. Two of the four Ndk activity mutants (His117Gln and Ala125Arg) identified were capable of being truncated to 12 kDa and formed a membrane complex with Pk; however, the complexes formed were inactive for GTP synthesis. The other two Ndk activity mutants could be truncated to 12 kDa but were not detected in membrane fractions. These results further our understanding of the role of Ndk in alginate synthesis and identify amino acid residues in Ndk which have not previously been studied as

  3. Use of methanol for the efficient extraction and analysis of melamine and cyanuric acid residues in dairy products and pet foods.

    PubMed

    Tran, Buu N; Okoniewski, Richard; Storm, Robin; Jansing, Robert; Aldous, Kenneth M

    2010-01-13

    The recent worldwide shortage of acetonitrile has prompted the development of a new method using methanol as an alternative organic solvent in the extraction and liquid chromatographic analysis of melamine and cyanuric acid that may be present as contaminants in dairy products and pet foods. A simple extraction of melamine and cyanuric acid residues in fortified samples was successfully achieved, using a methanol-water mixture and analysis by isotopic dilution high-performance liquid chromatography-triple-quadrupole mass spectrometry (HPLC-MS/MS). A two-step centrifugation procedure was employed to remove matrix components from extracts. The separation of melamine and cyanuric acid was carried out on a Dionex Acclaim Trinity P1 column, with a methanol and ammonium acetate buffer used as the mobile phase. Excellent linearity was achieved for both the melamine and cyanuric acid calibrations. A variety of dairy products and pet foods were fortified with melamine and cyanuric acid at three levels, 1, 2.5, and 10 microg/g, producing recovery yields of 101-119% for melamine and 84-123% for cyanuric acid. The lower limit of quantification (LLOQ) of melamine was 0.03 microg/g for liquid milk and 0.05 microg/g for dry infant milk formula. The quantitative results were comparable with those derived from previous methods that have been proposed by the U.S. Food and Drug Administration for the screening of melamine and its analogues in foods. PMID:19904985

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

    PubMed

    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

  5. Preparation of a novel carbon-based solid acid from cassava stillage residue and its use for the esterification of free fatty acids in waste cooking oil.

    PubMed

    Wang, Lingtao; Dong, Xiuqin; Jiang, Haoxi; Li, Guiming; Zhang, Minhua

    2014-04-01

    A novel carbon-based solid acid catalyst was prepared by the sulfonation of incompletely carbonized cassava stillage residue (CSR) with concentrated sulfuric acid, and employed to catalyze the esterification of methanol and free fatty acids (FFAs) in waste cooking oil (WCO). The effects of the carbonization and the sulfonation temperatures on the pore structure, acid density and catalytic activity of the CSR-derived catalysts were systematically investigated. Low temperature carbonization and high temperature sulfonation can cause the collapse of the carbon framework, while high temperature carbonization is not conducive to the attachment of SO3H groups on the surface. The catalyst showed high catalytic activity for esterification, and the acid value for WCO is reduced to below 2mg KOH/g after reaction. The activity of catalyst can be well maintained after five cycles. CSR can be considered a promising raw material for the production of a new eco-friendly solid acid catalyst. PMID:24661813

  6. Solid acid catalysts pretreatment and enzymatic hydrolysis of macroalgae cellulosic residue for the production of bioethanol.

    PubMed

    Tan, Inn Shi; Lee, Keat Teong

    2015-06-25

    The aim of this study is to investigate the technical feasibility of converting macroalgae cellulosic residue (MCR) into bioethanol. An attempt was made to present a novel, environmental friendly and economical pretreatment process that enhances enzymatic conversion of MCR to sugars using Dowex (TM) Dr-G8 as catalyst. The optimum yield of glucose reached 99.8% under the optimal condition for solid acid pretreatment (10%, w/v biomass loading, 4%, w/v catalyst loading, 30min, 120°C) followed by enzymatic hydrolysis (45FPU/g of cellulase, 52CBU/g of β-glucosidase, 50°C, pH 4.8, 30h). The yield of sugar obtained was found more superior than conventional pretreatment process using H2SO4 and NaOH. Biomass loading for the subsequent simultaneous saccharification and fermentation (SSF) of the pretreated MCR was then optimized, giving an optimum bioethanol yield of 81.5%. The catalyst was separated and reused for six times, with only a slight drop in glucose yield. PMID:25839825

  7. Mutagenesis identifies the critical amino acid residues of human endonuclease G involved in catalysis, magnesium coordination, and substrate specificity

    PubMed Central

    Wu, Shih-Lu; Li, Chia-Cheng; Chen, Jaw-Chyun; Chen, Yi-Jin; Lin, Ching-Ting; Ho, Tin-Yun; Hsiang, Chien-Yun

    2009-01-01

    Background Endonuclease G (EndoG), a member of DNA/RNA nonspecific ββα-Me-finger nucleases, is involved in apoptosis and normal cellular proliferation. In this study, we analyzed the critical amino acid residues of EndoG and proposed the catalytic mechanism of EndoG. Methods To identify the critical amino acid residues of human EndoG, we replaced the conserved histidine, asparagine, and arginine residues with alanine. The catalytic efficacies of Escherichia coli-expressed EndoG variants were further analyzed by kinetic studies. Results Diethyl pyrocarbonate modification assay revealed that histidine residues were involved in EndoG activity. His-141, Asn-163, and Asn-172 in the H-N-H motif of EndoG were critical for catalysis and substrate specificity. H141A mutant required a higher magnesium concentration to achieve its activity, suggesting the unique role of His-141 in both catalysis and magnesium coordination. Furthermore, an additional catalytic residue (Asn-251) and an additional metal ion binding site (Glu-271) of human EndoG were identified. Conclusion Based on the mutational analysis and homology modeling, we proposed that human EndoG shared a similar catalytic mechanism with nuclease A from Anabaena. PMID:19272175

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

    PubMed Central

    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 strains screened for amylolytic activity and the ability to form lactic acid as the major product of the anaerobic metabolism. In total more than 30 batches at 3–l scale were run at 60 °C under non-sterile conditions. The process developed produced 37 g L−1 optically pure (98%) L-lactic acid in 20 h from 50 g L−1 raw potato starch. As co-metabolites smaller amounts (<7% w/v) of acetate, formate and ethanol were formed. Yields of lactic acid increased from 66% to 81% when potato residues from food processing were used as a starchy substrate in place of raw potato starch. CONCLUSIONS Potato starch and residues were successfully converted to lactic acid by G. stearothermophilus. The process described in this study provides major benefits in industrial applications and for the valorization of starch-rich waste streams. © 2015 The Authors.Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. PMID:25937690

  9. Trace metal occurrences in acid-insoluble residues of the Ordovician Galena Group, southeastern Minnesota

    SciTech Connect

    Lively, R.S.; Mossler, J.H.; Morey, G.B. ); Hauck, S.A. . Natural Resources Research Inst.)

    1993-03-01

    Regional geochemical, studies on insoluble residues from Paleozoic carbonate rocks have become an integral part of the search for new Upper Mississippi Valley-type mineral deposits in the northern Midcontinent. The authors have extended these studies to southeastern Minnesota, an area well to the north of known lead-zinc deposits of commercial size and grade. In this region, a thin sequence of Upper Cambrian to Middle Ordovician strata unconformably overlies a complex Precambrian basement. More than 500 samples of limestone and dolomite from 40 drill holes and outcrops were analyzed for 29 related trace elements. Preliminary interpretations are based on the analysis of 380 samples of the Ordovician Galena Group from 37 localities. Results indicate that anomalous concentrations of Pb, Cu, zn, As, Cd, and Ni are confined to the southern half of the Galena subgroup area and extend less than 30 miles north of the Iowa border. The anomalous areas, as well as saddles between the, have a distinct northwest trend, coincident with structural features previously recognized in the Precambrian basement. The spatial relationships of the anomalies and the lack of direct correlation imply deposition from fluids moving north out of the main lead-zinc district along structural pathways. The lack of significant anomalies in the northern part of the subcrop area implies northwest weakening of the forces driving the metal-bearing fluids, as well as a decrease over distance in the absolute metal content of the migrating fluids.

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

    PubMed

    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

  11. Thermostability Improvement of a Streptomyces Xylanase by Introducing Proline and Glutamic Acid Residues

    PubMed Central

    Wang, Kun; Tian, Jian; Turunen, Ossi; Huang, Huoqing; Shi, Pengjun; Hua, Huifang; Wang, Caihong; Wang, Shuanghe

    2014-01-01

    Protein engineering is commonly used to improve the robustness of enzymes for activity and stability at high temperatures. In this study, we identified four residues expected to affect the thermostability of Streptomyces sp. strain S9 xylanase XynAS9 through multiple-sequence analysis (MSA) and molecular dynamic simulations (MDS). Site-directed mutagenesis was employed to construct five mutants by replacing these residues with proline or glutamic acid (V81P, G82E, V81P/G82E, D185P/S186E, and V81P/G82E/D185P/S186E), and the mutant and wild-type enzymes were expressed in Pichia pastoris. Compared to the wild-type XynAS9, all five mutant enzymes showed improved thermal properties. The activity and stability assays, including circular dichroism and differential scanning calorimetry, showed that the mutations at positions 81 and 82 increased the thermal performance more than the mutations at positions 185 and 186. The mutants with combined substitutions (V81P/G82E and V81P/G82E/D185P/S186E) showed the most pronounced shifts in temperature optima, about 17°C upward, and their half-lives for thermal inactivation at 70°C and melting temperatures were increased by >9 times and approximately 7.0°C, respectively. The mutation combination of V81P and G82E in adjacent positions more than doubled the effect of single mutations. Both mutation regions were at the end of long secondary-structure elements and probably rigidified the local structure. MDS indicated that a long loop region after positions 81 and 82 located at the end of the inner β-barrel was prone to unfold. The rigidified main chain and filling of a groove by the mutations on the bottom of the active site canyon may stabilize the mutants and thus improve their thermostability. PMID:24463976

  12. A 13C{31P} REDOR NMR Investigation of the Role of Glutamic Acid Residues in Statherin-Hydroxyapatite Recognition

    PubMed Central

    Ndao, Moise; Ash, Jason T.; Breen, Nicholas F.; Goobes, Gil; Stayton, Patrick S.; Drobny, Gary P.

    2011-01-01

    The side chain carboxyl groups of acidic proteins found in the extra-cellular matrix (ECM) of mineralized tissues play a key role in promoting or inhibiting the growth of minerals such as hydroxyapatite (HAP), the principal mineral component of bone and teeth. Among the acidic proteins found in the saliva is statherin, a 43-residue tyrosine-rich peptide that is a potent lubricant in the salivary pellicle and an inhibitor of both HAP crystal nucleation and growth. Three acidic amino acids – D1, E4, and E5 – are located in the N-terminal 15 amino acid segment, with a fourth amino acid, E26, located outside the N-terminus. We have utilized 13C{31P} REDOR NMR to analyze the role played by acidic amino acids in the binding mechanism of statherin to the HAP surface by measuring the distance between the δ-carboxyl 13C spins of the three glutamic acid side chains of statherin (residues E4, E5, E26) and 31P spins of the phosphate groups at the HAP surface. 13C{31P} REDOR studies of glutamic-5-13C acid incorporated at positions E4 and E26 indicate a 13C–31P distance of more than 6.5 Å between the side chain carboxyl 13C spin of E4 and the closest 31P in the HAP surface. In contrast, the carboxyl 13C spin at E5 has a much shorter 13C–31P internuclear distance of 4.25±0.09 Å, indicating that the carboxyl group of this side chain interacts directly with the surface. 13C T1ρ and slow-spinning MAS studies indicate that the motions of the side chains of E4 and E5 are more restricted than that of E26. Together, these results provide further insight into the molecular interactions of statherin with HAP surfaces. PMID:19678690

  13. T Cell Determinants Incorporating [beta]-Amino Acid Residues Are Protease Resistant and Remain Immunogenic In Vivo

    SciTech Connect

    Webb, Andrew I.; Dunstone, Michelle A.; Williamson, Nicholas A.; Price, Jason D.; Kauwe, Andreade; Chen, Weisan; Oakley, Aaron; Perlmutter, Patrick; McCluskey, James; Aguilar, Marie-Isabel; Rossjohn, Jamie; Purcell, Anthony W.

    2010-07-20

    A major hurdle in designing successful epitope-based vaccines resides in the delivery, stability, and immunogenicity of the peptide immunogen. The short-lived nature of unmodified peptide-based vaccines in vivo limits their therapeutic application in the immunotherapy of cancers and chronic viral infections as well as their use in generating prophylactic immunity. The incorporation of {beta}-amino acids into peptides decreases proteolysis, yet its potential application in the rational design of T cell mimotopes is poorly understood. To address this, we have replaced each residue of the SIINFEKL epitope individually with the corresponding {beta}-amino acid and examined the resultant efficacy of these mimotopes. Some analogs displayed similar MHC binding and superior protease stability compared with the native epitope. Importantly, these analogs were able to generate cross-reactive CTLs in vivo that were capable of lysing tumor cells that expressed the unmodified epitope as a surrogate tumor Ag. Structural analysis of peptides in which anchor residues were substituted with {beta}-amino acids revealed the basis for enhanced MHC binding and retention of immunogenicity observed for these analogs and paves the way for future vaccine design using {beta}-amino acids. We conclude that the rational incorporation of {beta}-amino acids into T cell determinants is a powerful alternative to the traditional homologous substitution of randomly chosen naturally occurring {alpha}-amino acids, and these mimotopes may prove particularly useful for inclusion in epitope-based vaccines.

  14. Is Uric Acid a Missing Link between Previous Gestational Diabetes Mellitus and the Development of Type 2 Diabetes at a Later Time of Life?

    PubMed Central

    Fronczyk, Aneta; Safranow, Krzysztof; Majkowska, Lilianna

    2016-01-01

    Introduction A high level of uric acid (UA) is a strong, independent risk factor for type 2 diabetes mellitus. The relationship between UA levels and the development of type 2 diabetes in women with previous gestational diabetes mellitus (pGDM) remains unclear. The aim of study was to evaluate the UA levels in pGDM women in relation to their current nutritional status and carbohydrate metabolism. Material and Methods 199 women with pGDM diagnoses based on oral glucose tolerance tests (OGTTs) 5–12 years previously and a control group of 50 women without pGDM. The assessment included anthropometric parameters, body composition (Tanita SC-330S), current OGTT, insulin resistance index (HOMA-IR), β-cell function (HOMA-%B), HbA1c, lipids, and uric acid. Results No differences between groups were found in terms of age, time from the index pregnancy, anthropometric parameters, lipids or creatinine levels. The incidences of overweight and obesity were similar. Carbohydrate abnormalities were more frequent in the pGDM group than the control group (43.2% vs 12.0% p<0.001). The women with pGDM had significantly higher fasting glucose, HbA1c, glucose and insulin levels in the OGTTs, but similar HOMA-IR values. Their UA levels were significantly higher (258±58 vs 230±50 μmol/L, p<0.005) and correlated with BMI and the severity of carbohydrate disorders. The normal weight and normoglycemic pGDM women also demonstrated higher UA levels than a similar control subgroup (232±48 vs 208±48 μmol/L, p<0.05). Multivariate analysis revealed significant correlations of UA level with BMI (β = 0.38, 95% CI 0.25–0.51, p<0.0001), creatinine level (β = 0.23, 95% CI 0.11–0.35, p<0.0005), triglycerides (β = 0.20, 95% CI 0.07–0.33, p<0.005) and family history of diabetes (β = 0.13, 95% CI 0.01–0.25, p<0.05). In logistic regression analysis, the association between higher UA level (defined as value ≥297 μmol/L) and presence of any carbohydrate metabolism disorder (IFG, IGT or

  15. Residual cadmium forms in acid-extracted anaerobically digested sewage sludge

    SciTech Connect

    Feltz, R.E.; Logan, T.J.

    1985-01-01

    The effects of phosphorus and lime additions after acid extraction on residual Cd solubility and chemical forms in an anaerobically digested sewage sludge were investigated. High Cd content anaerobically digested sewage sludge was aerated and then acidified to pH 2 to solubilize Cd. After 18 h of acidification, the sludge was dewatered and the supernatant and solids separated. Seventy or more percent of the Cd was removed from the solids. Similar amounts of Ni, Mn and Zn were also removed, but Cu removal was only 26% and that of Pb was < 5%. Before liming the sludge was amended with rock phosphate (RP) or monocalcium phosphate (MCP). The RP was insoluble while MCP dissolved, providing a high level of phosphate ligand for Cd precipitation or coprecipitation. Estimated sludge solution solubility products for major Fe, Al and Ca phosphates showed that several of these minerals could have precipitated with P addition, especially with MCP, and Cd may have coprecipitated with these solid phases. Cadmium phosphate may also have been formed in the MCP sludge. Chemical fractionation indicated that 50% of the Cd in the aerated unextracted sludge existed as inorganic precipitates with another 40% Na/sub 4/P/sub 2/O/sub 7/ extractable. Acidification solubilized 98% of the inorganic Cd and 86% of the organically bound Cd. Seventy-nine percent of the Cd remaining in the dewatered acidified sludge was in the KNO/sub 3/ extractable (exchangeable) fraction. Liming redistributed the Cd with 13 to 19% as inorganic precipitates, 70 to 85% organically bound and < 3% in the exchangeable fraction. Phosphate addition had no significant effect on Cd fractionation.

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

    NASA Astrophysics Data System (ADS)

    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.

  17. Computational study on the roles of amino acid residues in the active site formation mechanism of blue-light photoreceptors

    NASA Astrophysics Data System (ADS)

    Sato, Ryuma; Kitoh-Nishioka, Hirotaka; Ando, Koji; Yamato, Takahisa

    2015-07-01

    To examine the functional roles of the active site methionine (M-site) and glutamic acid (E-site) residues of blue-light photoreceptors, we performed in silico mutation at the M-site in a systematic manner and focused on the hydrogen bonding between the E-site and the substrate: the cyclobutane-pyrimidine dimer (CPD). Fragment molecular orbital calculations with electron correlations demonstrated that substitution of the M-site methionine with either alanine or glutamine always destabilizes the interaction energy between the E-site and the CPD by more than 12.0 kcal/mol, indicating that the methionine and glutamic acid residues cooperatively facilitate the enzymatic reaction in the active site.

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

    PubMed

    Shiheido, Hirokazu; 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 (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. PMID:25600804

  19. Identification of highly conserved residues involved in inhibition of HIV-1 RNase H function by Diketo acid derivatives.

    PubMed

    Corona, Angela; Di Leva, Francesco Saverio; Thierry, Sylvain; Pescatori, Luca; Cuzzucoli Crucitti, Giuliana; Subra, Frederic; Delelis, Olivier; Esposito, Francesca; Rigogliuso, Giuseppe; Costi, Roberta; Cosconati, Sandro; Novellino, Ettore; Di Santo, Roberto; Tramontano, Enzo

    2014-10-01

    HIV-1 reverse transcriptase (RT)-associated RNase H activity is an essential function in viral genome retrotranscription. RNase H is a promising drug target for which no inhibitor is available for therapy. Diketo acid (DKA) derivatives are active site Mg(2+)-binding inhibitors of both HIV-1 RNase H and integrase (IN) activities. To investigate the DKA binding site of RNase H and the mechanism of action, six couples of ester and acid DKAs, derived from 6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester (RDS1643), were synthesized and tested on both RNase H and IN functions. Most of the ester derivatives showed selectivity for HIV-1 RNase H versus IN, while acids inhibited both functions. Molecular modeling and site-directed mutagenesis studies on the RNase H domain demonstrated different binding poses for ester and acid DKAs and proved that DKAs interact with residues (R448, N474, Q475, Y501, and R557) involved not in the catalytic motif but in highly conserved portions of the RNase H primer grip motif. The ester derivative RDS1759 selectively inhibited RNase H activity and viral replication in the low micromolar range, making contacts with residues Q475, N474, and Y501. Quantitative PCR studies and fluorescence-activated cell sorting (FACS) analyses showed that RDS1759 selectively inhibited reverse transcription in cell-based assays. Overall, we provide the first demonstration that RNase H inhibition by DKAs is due not only to their chelating properties but also to specific interactions with highly conserved amino acid residues in the RNase H domain, leading to effective targeting of HIV retrotranscription in cells and hence offering important insights for the rational design of RNase H inhibitors. PMID:25092689

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

    PubMed

    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 pH<10, possibly due to the dissolution of ettringite (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

  1. Impaired Acid Catalysis by Mutation of a Protein Loop Hinge Residue in a YopH Mutant Revealed by Crystal Structures

    SciTech Connect

    Brandao, T.; Robinson, H; Johnson, S; Hengge, A

    2009-01-01

    Catalysis by the Yersinia protein-tyrosine phosphatase YopH is significantly impaired by the mutation of the conserved Trp354 residue to Phe. Though not a catalytic residue, this Trp is a hinge residue in a conserved flexible loop (the WPD-loop) that must close during catalysis. To learn why this seemingly conservative mutation reduces catalysis by 2 orders of magnitude, we have solved high-resolution crystal structures for the W354F YopH in the absence and in the presence of tungstate and vanadate. Oxyanion binding to the P-loop in W354F is analogous to that observed in the native enzyme. However, the WPD-loop in the presence of oxyanions assumes a half-closed conformation, in contrast to the fully closed state observed in structures of the native enzyme. This observation provides an explanation for the impaired general acid catalysis observed in kinetic experiments with Trp mutants. A 1.4 Angstroms structure of the W354F mutant obtained in the presence of vanadate reveals an unusual divanadate species with a cyclic [VO]2 core, which has precedent in small molecules but has not been previously reported in a protein crystal structure.

  2. Effect of 3' terminal adenylic acid residue on the uridylation of human small RNAs in vitro and in frog oocytes.

    PubMed Central

    Chen, Y; Sinha, K; Perumal, K; Reddy, R

    2000-01-01

    It is known that several small RNAs including human and Xenopus signal recognition particle (SRP) RNA, U2 small nuclear RNA (snRNA) and 7SK RNAs are posttranscriptionally adenylated, whereas U6 snRNA and ribosomal 5S RNA are posttranscriptionally uridylated on their 3' ends. In this study, we provide evidence that a small fraction of U6 snRNA and 5S ribosomal RNA molecules from human as well as Xenopus oocytes contain a single posttranscriptionally added adenylic acid residue on their 3' ends. These data show that U6 snRNA and 5S rRNAs are posttranscriptionally modified on their 3' ends by both uridylation and adenylation. Although the SRP RNA, 7SK RNA, 5S RNA, and U6 snRNA with the uridylic acid residue on their 3' ends were readily uridylated, all these RNAs with posttranscriptionally added adenylic acid residue on their 3' ends were not uridylated in vitro, or when U6 snRNA with 3' A(OH) was injected into Xenopus oocytes. These results show that the presence of a single posttranscriptionally added adenylic acid residue on the 3' end of SRP RNA, U6 snRNA, 5S rRNA, or 7SK RNA prevents 3' uridylation. These data also show that adenylation and uridylation are two competing processes that add nucleotides on the 3' end of some small RNAs and suggest that one of the functions of the 3' adenylation may be to negatively affect the 3' uridylation of small RNAs. PMID:10999605

  3. Biosynthesis of D-alanyl-lipoteichoic acid by Lactobacillus casei: interchain transacylation of D-alanyl ester residues

    SciTech Connect

    Childs, W.C. 3d.; Taron, D.J.; Neuhaus, F.C.

    1985-06-01

    Lipoteichoic acid (LTA) from Lactobacillus casei contains poly(glycerophosphate) substituted with D-alanyl ester residues. The distribution of these residues in the in vitro-synthesized polymer is uniform. Esterification of LTA with D-alanine may occur in one of two modes: (i) addition at random or (ii) addition at a defined locus in the poly(glycerophosphate) chain followed by redistribution of the ester residues. A time-dependent transacylation of these residues from D-(/sup 14/C)alanyl-lipophilic LTA to hydrophilic acceptor was observed. The hydrophilic acceptor was characterized as D-alanyl-hydrophilic LTA. This transacylation requires neither ATP nor the D-alanine incorporation system, i.e., the D-alanine activating enzyme and D-alanine:membrane acceptor ligase. No evidence for an enzyme-catalyzed transacylation reaction was observed. The authors propose that this process of transacylation may be responsible for the redistribution of D-alanyl residues after esterification to the poly(glycerophosphate). As a result, it is difficult to distinguish between these proposed modes of addition.

  4. Amino acid residues required for fast Na(+)-channel inactivation: charge neutralizations and deletions in the III-IV linker.

    PubMed Central

    Patton, D E; West, J W; Catterall, W A; Goldin, A L

    1992-01-01

    The cytoplasmic linker connecting domains III and IV of the voltage-gated Na+ channel is thought to be involved in fast inactivation. This linker is highly conserved among the various Na+ channels that have been cloned. In the rat brain IIA Na+ channel, it consists of 53 amino acids of which 15 are charged. To investigate the role of this linker in inactivation, we mutated all 15 of the charged residues in various combinations. All but one of these mutants expressed functional channels, and all of these inactivated with kinetics similar to the wild-type channel. We then constructed a series of deletion mutations that span the III-IV linker to determine if any region of the linker is essential for fast inactivation. Deletion of the first 10 amino acids completely eliminated fast inactivation in the channel, whereas deletion of the last 10 amino acids had no substantial effect on inactivation. These results demonstrate that some residues in the amino end of the III-IV linker are critical for fast Na(+)-channel inactivation, but that the highly conserved positively charged and paired negatively charged residues are not essential. PMID:1332059

  5. Role of acidic residues in helices TH8-TH9 in membrane interactions of the diphtheria toxin T domain.

    PubMed

    Ghatak, Chiranjib; Rodnin, Mykola V; Vargas-Uribe, Mauricio; McCluskey, Andrew J; Flores-Canales, Jose C; Kurnikova, Maria; Ladokhin, Alexey S

    2015-04-01

    The pH-triggered membrane insertion of the diphtheria toxin translocation domain (T domain) results in transferring the catalytic domain into the cytosol, which is relevant to potential biomedical applications as a cargo-delivery system. Protonation of residues is suggested to play a key role in the process, and residues E349, D352 and E362 are of particular interest because of their location within the membrane insertion unit TH8-TH9. We have used various spectroscopic, computational and functional assays to characterize the properties of the T domain carrying the double mutation E349Q/D352N or the single mutation E362Q. Vesicle leakage measurements indicate that both mutants interact with the membrane under less acidic conditions than the wild-type. Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts. Fluorescence experiments show that removal of the charge in E362Q, and not in E349Q/D352N, is important for insertion of TH8-TH9. Both mutants adopt a final functional state upon further acidification. We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions. PMID:25875295

  6. Efficient hydrolysis of corncob residue through cellulolytic enzymes from Trichoderma strain G26 and L-lactic acid preparation with the hydrolysate.

    PubMed

    Xie, Lulu; Zhao, Jin; Wu, Jian; Gao, Mingfu; Zhao, Zhewei; Lei, Xiangyun; Zhao, Yi; Yang, Wei; Gao, Xiaoxue; Ma, Cuiyun; Liu, Huanfei; Wu, Fengjuan; Wang, Xingxing; Zhang, Fengwei; Guo, Pengyuan; Dai, Guifu

    2015-10-01

    To prepare fermentable hydrolysate from corncob residue (CCR), Trichoderma strain G26 was cultured on medium containing CCR for production of cellulolytic enzymes through solid-state fermentation (SSF), resulting in 71.3 IU/g (FPA), 136.2 IU/g (CMCase), 85.1 IU/g (β-glucosidase) and 11,344 IU/g (xylanase), respectively. Through a three-stage saccharification strategy, CCR was hydrolyzed by the enzymatic solution (6.5 FPU/ml) into fermentable hydrolysate containing 60.1g/l glucose (81.2% cellulose was converted at solid loading of 12.5%), 21.4% higher than that by the one-stage method. And then the hydrolysate was used to produce L-lactic acid by a previous screened strain Bacillus coagulans ZX25 in the submerged fermentation. 52.0 g/l L-lactic acid was obtained after fermentation for 44 h, with 86.5% glucose being converted to L-lactic acid. The results indicate that the strains and the hydrolysis strategy are promising for commercial production of L-lactic acid from CCR and other biomass. PMID:26143000

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

    SciTech Connect

    Shiheido, Hirokazu 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.

  8. Urea, glycolic acid, and glycerol in an organic residue produced by ultraviolet irradiation of interstellar/pre-cometary ice analogs.

    PubMed

    Nuevo, Michel; Bredehöft, Jan Hendrik; Meierhenrich, Uwe J; d'Hendecourt, Louis; Thiemann, Wolfram H-P

    2010-03-01

    More than 50 stable organic molecules have been detected in the interstellar medium (ISM), from ground-based and onboard-satellite astronomical observations, in the gas and solid phases. Some of these organics may be prebiotic compounds that were delivered to early Earth by comets and meteorites and may have triggered the first chemical reactions involved in the origin of life. Ultraviolet irradiation of ices simulating photoprocesses of cold solid matter in astrophysical environments have shown that photochemistry can lead to the formation of amino acids and related compounds. In this work, we experimentally searched for other organic molecules of prebiotic interest, namely, oxidized acid labile compounds. In a setup that simulates conditions relevant to the ISM and Solar System icy bodies such as comets, a condensed CH(3)OH:NH(3) = 1:1 ice mixture was UV irradiated at approximately 80 K. The molecular constituents of the nonvolatile organic residue that remained at room temperature were separated by capillary gas chromatography and identified by mass spectrometry. Urea, glycolic acid, and glycerol were detected in this residue, as well as hydroxyacetamide, glycerolic acid, and glycerol amide. These organics are interesting target molecules to be searched for in space. Finally, tentative mechanisms of formation for these compounds under interstellar/pre-cometary conditions are proposed. PMID:20402585

  9. Multiple Roles of the Extracellular Vestibule Amino Acid Residues in the Function of the Rat P2X4 Receptor

    PubMed Central

    Rokic, Milos B.; Stojilkovic, Stanko S.; Vavra, Vojtech; Kuzyk, Pavlo; Tvrdonova, Vendula; Zemkova, Hana

    2013-01-01

    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. PMID:23555667

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

    PubMed

    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

  11. Identification of carboxylic acid residues in glucoamylase G2 from Aspergillus niger that participate in catalysis and substrate binding.

    PubMed

    Svensson, B; Clarke, A J; Svendsen, I; Møller, H

    1990-02-22

    Functionally important carboxyl groups in glucoamylase G2 from Aspergillus niger were identified using a differential labelling approach which involved modification of the acarbose-inhibited enzyme with 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) and inactivation by [3H]EAC following removal of acarbose. Subsequent sequence localization of the substituted acidic residues was facilitated by specific phenylthiohydantoins. The acid cluster Asp176, Glu179 and Glu180 reacted exclusively with [3H]EAC, while Asp112, Asp153, Glu259 and Glu389 had incorporated both [3H]EAC and EAC. It is conceivable that one or two of the [3H]EAC-labelled side chains act in catalysis while the other fully protected residue(s) participates in substrate binding probably together with the partially protected ones. Twelve carboxyl groups that reacted with EAC in the enzyme-acarbose complex were also identified. Asp176, Glu179 and Glu180 are all invariant in fungal glucoamylases. Glu180 was tentatively identified as a catalytic group on the basis of sequence alignments to catalytic regions in isomaltase and alpha-amylase. The partially radiolabelled Asp112 corresponds in Taka-amylase A to Tyr75 situated in a substrate binding loop at a distance from the site of cleavage. A possible correlation between carbodiimide modification of an essential carboxyl group and its role in the glucoamylase catalysis is discussed. PMID:2108020

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

    PubMed

    Xu, Weiying; Gao, Baoyu; Du, Bin; Xu, Zhenghe; Zhang, Yongfang; Wei, Dong

    2014-04-30

    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=40.7-178.3s(-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 under the same shear condition. PMID:24583809

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

    PubMed Central

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

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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

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

    SciTech Connect

    Li, Sai Kam; Ho, Sai Fan; Tsui, Kwok Wing; Fung, Kwok Pui; Waye, M.Y. Mary

    2008-11-10

    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.

  18. Influence of diet or intrarectal bile acid injections on colon epithelial cell proliferation in rats previously injected with 1,2-dimethylhydrazine

    SciTech Connect

    Glauert, H.P.; Bennink, M.R.

    1983-03-01

    The effects of varying colon bile acid concentrations on rat colon epithelial cell proliferation were studied. Bile acid concentrations were altered by intrarectally injecting either deoxycholic or lithocholic acid for 4 weeks or by increasing the dietary fat or fiber (wheat bran, agar, or carrageenan) intake for 4 weeks. 1,2-Dimethylhydrazine (DMH) was s.c. injected into half of the rats 1 week before treatments began. Colon epithelial cell proliferation was measured by (/sup 3/H)thymidine autoradiography of colon crypts. Rats injected with DMH had more DNA-synthesizing cells per crypt. Neither bile acid injection nor any of the diets altered the number of DNA-synthesizing cells per crypt. DMH injections, deoxycholic and lithocholic acid intrarectal injections, and dietary agar and wheat bran all increased the total number of cells per crypt. High fat diets and dietary carrageenan did not affect cell number. All diets containing fiber lowered total fecal bile acid concentrations, but increasing the fat content of the diet did not affect them. These results indicate that the bile acid injections and dietary agar and wheat bran induce a slight hyperplasia in the colon.

  19. Identification of important amino acid residues that modulate binding of Escherichia coli GroEL to its various cochaperones.

    PubMed Central

    Klein, G; Georgopoulos, C

    2001-01-01

    Genetic experiments have shown that the GroEL/GroES chaperone machine of Escherichia coli is absolutely essential, not only for bacterial growth but also for the propagation of many bacteriophages including lambda. The virulent bacteriophages T4 and RB49 are independent of the host GroES function, because they encode their own cochaperone proteins, Gp31 and CocO, respectively. E. coli groEL44 mutant bacteria do not form colonies above 42 degrees nor do they propagate bacteriophages lambda, T4, or RB49. We found that the vast majority (40/46) of spontaneous groEL44 temperature-resistant colonies at 43 degrees were due to the presence of an intragenic suppressor mutation. These suppressors define 21 different amino acid substitutions in GroEL, each affecting one of 13 different amino acid residues. All of these amino acid residues are located at or near the hinge, which regulates the large en bloc movements of the GroEL apical domain. All of these intragenic suppressors support bacteriophages lambda, T4, and RB49 growth to various extents in the presence of the groEL44 allele. Since it is known that the GroEL44 mutant protein does not interact effectively with Gp31, the suppressor mutations should enhance cochaperone binding. Analogous intragenic suppressor studies were conducted with the groEL673 temperature-sensitive allele. PMID:11404317

  20. Toxin acidic residue evolutionary function-guided design of de novo peptide drugs for the immunotherapeutic target, the Kv1.3 channel

    PubMed Central

    Chen, Zongyun; Hu, Youtian; Hong, Jing; Hu, Jun; Yang, Weishan; Xiang, Fang; Yang, Fan; Xie, Zili; Cao, Zhijian; Li, Wenxin; Lin, Donghai; Wu, Yingliang

    2015-01-01

    During the long-term evolution of animal toxins acting on potassium channels, the acidic residues can orientate the toxin binding interfaces by adjusting the molecular polarity. Based on the evolutionary function of toxin acidic residues, de novo peptide drugs with distinct binding interfaces were designed for the immunotherapeutic target, the Kv1.3 channel. Using a natural basic toxin, BmKTX, as a template, which contains 2 acidic residues (Asp19 and Asp33), we engineered two new peptides BmKTX-19 with 1 acidic residue (Asp33), and BmKTX-196 with 2 acidic residues (Asp6 and Asp33) through only adjusting acidic residue distribution for reorientation of BmKTX binding interface. Pharmacological experiments indicated that BmKTX-19 and BmKTX-196 peptides were specific inhibitors of the Kv1.3 channel and effectively suppressed cytokine secretion. In addition to the structural similarity between the designed and native peptides, both experimental alanine-scanning mutagenesis and computational simulation further indicated that the binding interface of wild-type BmKTX was successfully reoriented in BmKTX-19 and BmKTX-196, which adopted distinct toxin surfaces as binding interfaces. Together, these findings indicate not only the promising prospect of BmKTX-19 and BmKTX-196 as drug candidates but also the desirable feasibility of the evolution-guided peptide drug design for discovering numerous peptide drugs for the Kv1.3 channel. PMID:25955787

  1. Preparation of a modified flue gas desulphurization residue and its effect on pot sorghum growth and acidic soil amelioration.

    PubMed

    Shi, Lin; Xu, Peizhi; Xie, Kaizhi; Tang, Shuanhu; Li, Yongli

    2011-09-15

    A modified flue gas desulphurization residue (MFGDR) was prepared and its effects on sorghum growth and acidic soil amelioration were evaluated in this paper. The MFGDR was prepared by calcining a mixture of dry/semi-dry flue gas desulphurization (FGD) residue from a coal-fired power plant, sorted potash feldspar and/or limestone powder. The available nutrients from the MFGDR were determined with 4.91 wt% K(+), 1.15 wt% Mg(2+), 22.4 wt% Ca(2+), 7.01 wt% Si(4+) and 2.07 wt% SO(4)(2-)-S in 0.1 mol L(-1) citric acid solution. Its pH value was held at 9.60 displaying slightly alkaline. The results of sorghum pot growth in both red and crimson acidic soil for 30 days indicated that adding the MFGDR at a dosage of 2 g kg(-1) in total soil weight would increase the growth rate of biomass by 24.3-149% (wet weight basis) and 47.3-157% (dry weight), the stem length and thickness increase by 5.75-22.1% and 4.76-30.9% in contrast with CK treatment for two test cuttings, respectively. The effect on sorghum growth was attributed to the increase of available nutrients, the enhancement of soil pH value and the reduction of aluminum toxicity in acidic soil due to the addition of the MFGDR. The experimental results also suggested that the MFGDR could be effectively used to ameliorate the acidic soil which is widely distributed throughout the southern China. PMID:21763070

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

    NASA Astrophysics Data System (ADS)

    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. Amino acid residues in the Ler protein critical for derepression of the LEE5 promoter in enteropathogenic E. coli.

    PubMed

    Choi, Su-Mi; Jeong, Jae-Ho; Choy, Hyon E; Shin, Minsang

    2016-08-01

    Enteropathogenic E. coli causes attaching and effacing (A/E) intestinal lesions. The genes involved in the formation of A/E lesions are encoded within a chromosomal island comprising of five major operons, LEE1-5. The global regulator H-NS represses the expression of these operons. Ler, a H-NS homologue, counteracts the H-NS-mediated repression. Using a novel genetic approach, we identified the amino acid residues in Ler that are involved in the interaction with H-NS: I20 and L23 in the C-terminal portion of α-helix 3, and I42 in the following unstructured linker region. PMID:27480636

  4. Highly Amino Acid Selective Hydrolysis of Myoglobin at Aspartate Residues as Promoted by Zirconium(IV)-Substituted Polyoxometalates.

    PubMed

    Ly, Hong Giang T; Absillis, Gregory; Janssens, Rik; Proost, Paul; Parac-Vogt, Tatjana N

    2015-06-15

    SDS-PAGE/Edman degradation and HPLC MS/MS showed that zirconium(IV)-substituted Lindqvist-, Keggin-, and Wells-Dawson-type polyoxometalates (POMs) selectively hydrolyze the protein myoglobin at Asp-X peptide bonds under mildly acidic and neutral conditions. This transformation is the first example of highly sequence selective protein hydrolysis by POMs, a novel class of protein-hydrolyzing agents. The selectivity is directed by Asp residues located on the surface of the protein and is further assisted by electrostatic interactions between the negatively charged POMs and positively charged surface patches in the vicinity of the cleavage site. PMID:25950869

  5. Influence of bleaching on flavor of 34% whey protein concentrate and residual benzoic acid concentration in dried whey products

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous studies have shown that bleaching negatively affects the flavor of 70% whey protein concentrate (WPC70), but bleaching effects on lower-protein products have not been established. Benzoyl peroxide (BP), a whey bleaching agent, degrades to benzoic acid (BA) and may elevate BA concentrations...

  6. Influence of Bleaching on Flavor of 34% Whey Protein Concentrate and Residual Benzoic Acid Concentration in Dried Whey Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous studies have shown that bleaching negatively affects the flavor of 70% whey protein concentrate (WPC70), but bleaching effects on lower-protein products have not been established. Benzoyl peroxide (BP), a whey bleaching agent, degrades to benzoic acid (BA) and may elevate BA concentrations...

  7. Sequence Polymorphism, Segmental Recombination and Toggling Amino Acid Residues within the DBL3X Domain of the VAR2CSA Placental Malaria Antigen

    PubMed Central

    Talundzic, Eldin; Shah, Sheel; Fawole, Ope; Owino, Simon; Moore, Julie M.; Peterson, David S.

    2012-01-01

    Plasmodium falciparum malaria remains one of the world's foremost health problems, primarily in highly endemic regions such as Sub-Saharan Africa, where it is responsible for substantial morbidity, mortality and economic losses. Malaria is a significant cause of severe disease and death in pregnant women and newborns, with pathogenesis being associated with expression of a unique variant of the multidomain Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) called VAR2CSA. Here, we characterize the polymorphism of the DBL3X domain of VAR2CSA and identify regions under selective pressure among placental parasites from women living in endemic western Kenya. In addition to significant levels of polymorphism, our analysis reveals evidence for diversification through intra-segmental recombination and novel mutations that likely contributed to the high number of unique VAR2CSA sequence types identified in this study. Interestingly, we also identified a number of critical residues that may be implicated in immune evasion through switching (or toggling) to alternative amino acids, including an arginine residue within the predicted binding pocket in subdomain III, which was previously implicated in binding to placental CSA. Overall, these findings are important for understanding parasite diversity in pregnant women and will be useful for identifying epitopes and variants of DBL3X to be included in a vaccine against placental malaria. PMID:22347496

  8. Identity of the amino acid residues involved in C3bi binding to the I-domain supports a mosaic model to explain the broad ligand repertoire of integrin alpha M beta 2.

    PubMed

    Ustinov, Valentin A; Plow, Edward F

    2005-03-22

    Interactions between the complement degradation product C3bi and leukocyte integrin alpha(M)beta(2) are critical for host defense against foreign pathogens and in tumor cell surveillance. To gain insight into the mechanism by which the alpha(M)I-domain of the integrin interacts with C3bi, detailed mapping of the C3bi binding site was undertaken. Previous mutagenesis studies had implicated five small structural segments within the alpha(M)I-domain in recognition of this ligand. Sets of three amino acids within the five implicated segments were mutated to the corresponding alpha(L)I-domain residues. Then, within the affected mutants, single point mutations were introduced to precisely define the requisite residues. Ultimately, H148, F150, Q204, L205, R208, T211, T213, I256, P257 were identified as being critical for C3bi binding. A synthetic peptide approach confirmed the involvement of the specified residues with the complex midsegment, Q204-I215, in C3bi recognition. Furthermore, the alpha(D)I-domain, which has a low intrinsic affinity for C3bi, acquired high affinity for the ligand when the implicated residues were inserted. The residues necessary to engage C3bi reside on or adjacent to the cation binding MIDAS site of the alpha(M)I-domain. The amino acids involved in C3bi binding are distinct from those involved in interaction of previously mapped ligands with the alpha(M)I-domain. This divergence supports a mosaic model, in which different ligands engage different amino acids to bind to alpha(M)I-domain, accounting for the broad recognition capacity of integrin alpha(M)beta(2). PMID:15766265

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

    NASA Astrophysics Data System (ADS)

    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.

  10. Tsetse Salivary Gland Proteins 1 and 2 Are High Affinity Nucleic Acid Binding Proteins with Residual Nuclease Activity

    PubMed Central

    Caljon, Guy; Ridder, Karin De; Stijlemans, Benoît; Coosemans, Marc; Magez, Stefan; De Baetselier, Patrick; Van Den Abbeele, Jan

    2012-01-01

    Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans) saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2) display DNA/RNA non-specific, high affinity nucleic acid binding with KD values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents. PMID:23110062

  11. Controlling fine particulate and acid mist emissions from a residual oil fired utility boiler with an EDV{trademark} system

    SciTech Connect

    Olen, K.R.; Vincent, H.B.; Jones, G.

    1995-06-01

    Florida Power & Light Company (FPL), in cooperation with the Electric Power Research Institute (EPRI) and Belco Technologies Corporation, evaluated the performance of an EDV system to remove fine particulate and acid mist from untreated flue gas from a residual oil-fired utility boiler. The cosponsored project was carried out using a full-scale EDV module in a slip stream from one of the 400 MW wall-fired boilers at FPL`s Sanford Plant. Particulate, acid gas and chemical analytical data are presented, and used to illustrate the effects of operating variables on EDV performance. EDV system efficiencies of 90% were achieved, which resulted in controlled particulate and SO{sub 3} emissions of less than 10 mg/Nm{sup 3} (0.0065 lbs/10{sup 6}Btu) and 1 ppmv, respectively.

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

    SciTech Connect

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

    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.

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

    NASA Technical Reports Server (NTRS)

    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.

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

    SciTech Connect

    Delegard, C.H.; Rinehart, D.E.; Carlson, C.D.; Soderquist, C.Z.; Fadeff, S.K.

    1999-04-02

    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 HNO{sub 3}. 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 {sup 239}Pu by a factor of 10, and for {sup 241}Am by a factor of 1.6. It meets the ERDF criterion for {sup 137}Cs 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 {sup 239}Pu, and then {sup 241}Am, {sup 137}Cs, and uranium.

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

    PubMed Central

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

    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 detected at 50°C/pH 4. rEG A retained 100% of activity when incubated at 45 and 55°C for 72 h. Purified rEG A kinetic parameters towards CMC were determined as Km = 27.5 ± 4.33 mg/mL, Vmax = 1.185 ± 0.11 mmol/min, and 55.8 IU (international units)/mg specific activity. Recombinant P. pastoris supernatant presented hydrolytic activity towards lignocellulosic residues such as banana stalk, sugarcane bagasse, soybean residues, and corn straw. These data indicate that rEG A is suitable for plant biomass conversion into products of commercial importance, such as second-generation fuel ethanol. PMID:23936633

  16. Synthesis and application of boronic acid-functionalized magnetic adsorbent for sensitive analysis of salbutamol residues in pig tissues.

    PubMed

    Fan, Hua; Wang, Chaozhan; Wei, Yinmao

    2015-12-01

    Salbutamol (SAL) is the most widely used β2 -agonist drug for asthma and chronic obstructive pulmonary patients, but it is also often abused as feed additive. In recent years, the abuse of SAL has led to a large number of food safety incidents. Therefore, the monitoring of SAL residues in animal products is very important. A highly selective boronate affinity magnetic adsorbent was synthesized and developed for detection of trace levels of SAL residues in pig tissue samples. The obtained Fe3O4@SiO2@FPBA(4-formylphenylboronic acid) magnetic adsorbent showed good adsorption ability to catechol and SAL, and then it was successfully applied as special magnetic solid-phase phase extraction adsorbent coupled with high-performance liquid chromatography (HPLC) for simultaneous isolation and determination of cis-diol compounds. The binding capacity of catechol and SAL reached 96 and 50 µmol/g, respectively. The method was successfully established for the detection of trace levels of SAL in pig tissue samples. The linear range extended from 0.32 to 800 µg/kg (R(2) = 0.9994). The limit of detection of SAL was 0.19 µg/kg. The recoveries were satisfactory (89.5-108.0%) at three spiked levels with RSD between 2.1 and 11.3%. These results indicated that the method has potential for enrichment and detection of trace levels of SAL residual in animal food products. PMID:26061980

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

    PubMed

    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

  18. Developing an Acidic Residue Reactive and Sulfoxide-Containing MS-Cleavable Homobifunctional Cross-Linker for Probing Protein-Protein Interactions.

    PubMed

    Gutierrez, Craig B; Yu, Clinton; Novitsky, Eric J; Huszagh, Alexander S; Rychnovsky, Scott D; Huang, Lan

    2016-08-16

    Cross-linking mass spectrometry (XL-MS) has become a powerful strategy for defining protein-protein interactions and elucidating architectures of large protein complexes. However, one of the inherent challenges in MS analysis of cross-linked peptides is their unambiguous identification. To facilitate this process, we have previously developed a series of amine-reactive sulfoxide-containing MS-cleavable cross-linkers. These MS-cleavable reagents have allowed us to establish a common robust XL-MS workflow that enables fast and accurate identification of cross-linked peptides using multistage tandem mass spectrometry (MS(n)). Although amine-reactive reagents targeting lysine residues have been successful, it remains difficult to characterize protein interaction interfaces with little or no lysine residues. To expand the coverage of protein interaction regions, we present here the development of a new acidic residue-targeting sulfoxide-containing MS-cleavable homobifunctional cross-linker, dihydrazide sulfoxide (DHSO). We demonstrate that DHSO cross-linked peptides display the same predictable and characteristic fragmentation pattern during collision induced dissociation as amine-reactive sulfoxide-containing MS-cleavable cross-linked peptides, thus permitting their simplified analysis and unambiguous identification by MS(n). Additionally, we show that DHSO can provide complementary data to amine-reactive reagents. Collectively, this work not only enlarges the range of the application of XL-MS approaches but also further demonstrates the robustness and applicability of sulfoxide-based MS-cleavability in conjunction with various cross-linking chemistries. PMID:27417384

  19. Developing an Acidic Residue Reactive and Sulfoxide-Containing MS-Cleavable Homobifunctional Cross-Linker for Probing Protein–Protein Interactions

    PubMed Central

    2016-01-01

    Cross-linking mass spectrometry (XL-MS) has become a powerful strategy for defining protein–protein interactions and elucidating architectures of large protein complexes. However, one of the inherent challenges in MS analysis of cross-linked peptides is their unambiguous identification. To facilitate this process, we have previously developed a series of amine-reactive sulfoxide-containing MS-cleavable cross-linkers. These MS-cleavable reagents have allowed us to establish a common robust XL-MS workflow that enables fast and accurate identification of cross-linked peptides using multistage tandem mass spectrometry (MSn). Although amine-reactive reagents targeting lysine residues have been successful, it remains difficult to characterize protein interaction interfaces with little or no lysine residues. To expand the coverage of protein interaction regions, we present here the development of a new acidic residue-targeting sulfoxide-containing MS-cleavable homobifunctional cross-linker, dihydrazide sulfoxide (DHSO). We demonstrate that DHSO cross-linked peptides display the same predictable and characteristic fragmentation pattern during collision induced dissociation as amine-reactive sulfoxide-containing MS-cleavable cross-linked peptides, thus permitting their simplified analysis and unambiguous identification by MSn. Additionally, we show that DHSO can provide complementary data to amine-reactive reagents. Collectively, this work not only enlarges the range of the application of XL-MS approaches but also further demonstrates the robustness and applicability of sulfoxide-based MS-cleavability in conjunction with various cross-linking chemistries. PMID:27417384

  20. Proteomic Investigation of Protein Profile Changes and Amino Acid Residue Level Modification in Cooked Lamb Meat: The Effect of Boiling.

    PubMed

    Yu, Tzer-Yang; Morton, James D; Clerens, Stefan; Dyer, Jolon M

    2015-10-21

    Hydrothermal treatment (heating in water) is a common method of general food processing and preparation. For red-meat-based foods, boiling is common; however, how the molecular level effects of this treatment correlate to the overall food properties is not yet well-understood. The effects of differing boiling times on lamb meat and the resultant cooking water were here examined through proteomic evaluation. The longer boiling time was found to result in increased protein aggregation involving particularly proteins such as glyceraldehyde-3-phosphate dehydrogenase, as well as truncation in proteins such as in α-actinin-2. Heat-induced protein backbone cleavage was observed adjacent to aspartic acid and asparagine residues. Side-chain modifications of amino acid residues resulting from the heating, including oxidation of phenylalanine and formation of carboxyethyllysine, were characterized in the cooked samples. Actin and myoglobin bands from the cooked meat per se remained visible on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, even after significant cooking time. These proteins were also found to be the major source of observed heat-induced modifications. This study provides new insights into molecular-level modifications occurring in lamb meat proteins during boiling and a protein chemistry basis for better understanding the effect of this common treatment on the nutritional and functional properties of red-meat-based foods. PMID:26381020

  1. Solution structure of gamma-bungarotoxin: the functional significance of amino acid residues flanking the RGD motif in integrin binding.

    PubMed

    Shiu, Jia-Hau; Chen, Chiu-Yueh; Chang, Long-Sen; Chen, Yi-Chun; Chen, Yen-Chin; Lo, Yu-Hui; Liu, Yu-Chen; Chuang, Woei-Jer

    2004-12-01

    Gamma-bungarotoxin, a snake venom protein isolated from Bungarus multicinctus, contains 68 amino acids, including 10 cysteine residues and a TAVRGDGP sequence at positions 30-37. The solution structure of gamma-bungarotoxin has been determined by nuclear magnetic resonance (NMR) spectroscopy. The structure is similar to that of the short-chain neurotoxins that contain three loops extending from a disulfide-bridged core. The tripeptide Arg-Gly-Asp (RGD) sequence is located at the apex of the flexible loop and is similar to that of other RGD-containing proteins. However, gamma-bungarotoxin only inhibits platelet aggregations with an IC50 of 34 microM. To understand its weak activity in inhibiting platelet aggregation, we mutated the RGD loop sequences of rhodostomin, a potent platelet aggregation inhibitor, from RIPRGDMP to TAVRGDGP, resulting in a 196-fold decrease in activity. In addition, the average Calpha-to-Calpha distance between R33 and G36 of gamma-bungarotoxin is 6.02 A, i.e., shorter than that of other RGD-containing proteins that range from 6.55 to 7.46 A. These results suggested that the amino acid residues flanking the RGD motif might control the width of the RGD loop. This structural difference may be responsible for its decrease in platelet aggregation inhibition compared with other RGD-containing proteins. PMID:15390258

  2. Nuclear localization of the Hermes transposase depends on basic amino acid residues at the N-terminus of the protein.

    PubMed

    Michel, K; Atkinson, P W

    2003-07-01

    For the Hermes transposable element to be mobilized in its eukaryotic host, the transposase, encoded by the element, must make contact with its DNA. After synthesis in the cytoplasm, the transposase has to be actively imported into the nucleus because its size of 70.1 kDa prevents passive diffusion through the nuclear pore. Studies in vitro using transient expression of a Hermes-EGFP fusion protein in Drosophila melanogaster Schneider 2 cells showed the transposase was located predominantly in the nucleus. In silico sequence analysis, however, did not reveal any nuclear localization signal (NLS). To identify the sequence(s) responsible for localization of Hermes transposase in the nucleus, truncated or mutated forms of the transposase were examined for their influence on sub-cellular localization of marker proteins fused to the transposase. Using the same expression system and a GFP-GUS fusion double marker, residues 1-110 were recognized as sufficient, and residues 1-32 as necessary, for nuclear localization. Amino acid K25 greatly facilitated nuclear localization, indicating that at least this basic amino acid plays a significant role in this process. This sequence overlaps the proposed DNA binding region of the Hermes transposase and is not necessarily conserved in all members of the hAT transposable element family. PMID:12858343

  3. Enrichment of anhydrous milk fat in polyunsaturated fatty acid residues from linseed and rapeseed oils through enzymatic interesterification.

    PubMed

    Aguedo, Mario; Hanon, Emilien; Danthine, Sabine; Paquot, Michel; Lognay, Georges; Thomas, Annick; Vandenbol, Micheline; Thonart, Philippe; Wathelet, Jean-Paul; Blecker, Christophe

    2008-03-12

    Lipozyme TL IM was used in a solvent-free batch and microaqueous system for enzymatic interesterification of anhydrous milkfat (AMF) with linseed oil (LO) in binary blends and with rapeseed oil (RO) in one ternary blend. The aim was to obtain and characterize physicochemically fats enriched with unsaturated C 18 fatty acids (oleic, linoleic, and, especially, linolenic acids) from natural vegetable oils. Binary blends of AMF/LO 100/0, 90/10, 80/20, 70/30, and 60/40 (w/w) were interesterified. The change in triacylglycerol (TAG) profiles showed that quasi-equilibrium conditions were reached after 4-6 h of reaction. Free fatty acid contents <1%. The decrease in solid fat content and in dropping point temperature obtained with increasing content of LO and interesterification resulted in good plastic properties for the products originating from the blends 70/30 and 60/40. This was confirmed by textural measurements. Melting profiles determined by differential scanning calorimetry showed complete disappearance of low-melting TAGs from LO and the formation of intermediary species with a lower melting temperature. Oxidative stability of the interesterified products was diminished with increasing LO content, resulting in low oxidation induction times. A ternary blend composed of AMF/RO/LO 70/20/10 gave satisfactory rheological and oxidative properties, fulfilling the requirements for a marketable spread and, moreover, offering increased potential health benefits due to the enriched content in polyunsaturated fatty acid residues. PMID:18271538

  4. Radiolytic Modification of Sulfur Containing Acidic Amino Residues in Model Peptides: Fundamental Studies for Protein Footprinting

    SciTech Connect

    Xu,G.; Chance, M.

    2005-01-01

    Protein footprinting based on hydroxyl radical-mediated modification and quantitative mass spectroscopic analysis is a proven technique for examining protein structure, protein-ligand interactions, and structural allostery upon protein complex formation. The reactive and solvent-accessible amino acid side chains function as structural probes; however, correct structural analysis depends on the identification and quantification of all the relevant oxidative modifications within the protein sequence. Sulfur-containing amino acids are oxidized readily and the mechanisms of oxidation are particularly complex, although they have been extensively investigated by EPR and other spectroscopic methods. Here we have undertaken a detailed mass spectrometry study (using electrospray ionization mass spectrometry and tandem mass spectrometry) of model peptides containing cysteine (Cys-SH), cystine (disulfide bonded Cys), and methionine after oxidation using {gamma}-rays or synchrotron X-rays and have compared these results to those expected from oxidation mechanisms proposed in the literature. Radiolysis of cysteine leads to cysteine sulfonic acid (+48 Da mass shift) and cystine as the major products; other minor products including cysteine sulfinic acid (+32 Da mass shift) and serine (-16 Da mass shift) are observed. Radiolysis of cystine results in the oxidative opening of the disulfide bond and generation of cysteine sulfonic acid and sulfinic acid; however, the rate of oxidation is significantly less than that for cysteine. Radiolysis of methionine gives rise primarily to methionine sulfoxide (+16 Da mass shift); this can be further oxidized to methionine sulfone (+32 Da mass shift) or another product with a -32 Da mass shift likely due to aldehyde formation at the {gamma}-carbon. Due to the high reactivity of sulfur-containing amino acids, the extent of oxidation is easily influenced by secondary oxidation events or the presence of redox reagents used in standard proteolytic

  5. Characterization of amino acid residues within the N-terminal region of Ubc9 that play a role in Ubc9 nuclear localization

    SciTech Connect

    Sekhri, Palak; Tao, Tao; Kaplan, Feige; Zhang, Xiang-Dong

    2015-02-27

    As the sole E2 enzyme for SUMOylation, Ubc9 is predominantly nuclear. However, the underlying mechanisms of Ubc9 nuclear localization are still not well understood. Here we show that RNAi-depletion of Imp13, an importin known to mediate Ubc9 nuclear import, reduces both Ubc9 nuclear accumulation and global SUMOylation. Furthermore, Ubc9-R13A or Ubc9-H20D mutation previously shown to interrupt the interaction of Ubc9 with nucleus-enriched SUMOs reduces the nuclear enrichment of Ubc9, suggesting that the interaction of Ubc9 with the nuclear SUMOs may enhance Ubc9 nuclear retention. Moreover, Ubc9-R17E mutation, which is known to disrupt the interaction of Ubc9 with both SUMOs and Imp13, causes a greater decrease in Ubc9 nuclear accumulation than Ubc9-R13A or Ubc9-H20D mutation. Lastly, Ubc9-K74A/S89D mutations that perturb the interaction of Ubc9 with nucleus-enriched SUMOylation-consensus motifs has no effect on Ubc9 nuclear localization. Altogether, our results have elucidated that the amino acid residues within the N-terminal region of Ubc9 play a pivotal role in regulation of Ubc9 nuclear localization. - Highlights: • Imp13-mediated nuclear import of Ubc9 is critical for global SUMOylation. • Ubc9 mutations disrupting Ubc9-SUMO interaction decrease Ubc9 nuclear accumulation. • N-terminal amino acid residues of Ubc9 are critical for Ubc9 nuclear enrichment.

  6. Nonenzymatic oligomerization reactions on templates containing inosinic acid or diaminopurine nucleotide residues

    NASA Technical Reports Server (NTRS)

    Kozlov, I. A.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1999-01-01

    The template-directed oligomerization of nucleoside-5'-phosphoro-2-methyl imidazolides on standard oligonucleotide templates has been studied extensively. Here, we describe experiments with templates in which inosinic acid (I) is substituted for guanylic acid, or 2,6-diaminopurine nucleotide (D) for adenylic acid. We find that the substitution of I for G in a template is strongly inhibitory and prevents any incorporation of C into internal positions in the oligomeric products of the reaction. The substitution of D for A, on the contrary, leads to increased incorporation of U into the products. We found no evidence for the template-directed facilitation of oligomerization of A or I through A-I base pairing. The significance of these results for prebiotic chemistry is discussed.

  7. Characterization of Protective Epitopes in a Highly Conserved Plasmodium falciparum Antigenic Protein Containing Repeats of Acidic and Basic Residues

    PubMed Central

    Sharma, Pawan; Kumar, Anil; Singh, Balwan; Bharadwaj, Ashima; Sailaja, V. Naga; Adak, T.; Kushwaha, Ashima; Malhotra, Pawan; Chauhan, V. S.

    1998-01-01

    The delineation of putatively protective and immunogenic epitopes in vaccine candidate proteins constitutes a major research effort towards the development of an effective malaria vaccine. By virtue of its role in the formation of the immune clusters of merozoites, its location on the surface of merozoites, and its highly conserved nature both at the nucleotide sequence level and the amino acid sequence level, the antigen which contains repeats of acidic and basic residues (ABRA) of the human malaria parasite Plasmodium falciparum represents such an antigen. Based upon the predicted amino acid sequence of ABRA, we synthesized eight peptides, with six of these (AB-1 to AB-6) ranging from 12 to 18 residues covering the most hydrophilic regions of the protein, and two more peptides (AB-7 and AB-8) representing its repetitive sequences. We found that all eight constructs bound an appreciable amount of antibody in sera from a large proportion of P. falciparum malaria patients; two of these peptides (AB-1 and AB-3) also elicited a strong proliferation response in peripheral blood mononuclear cells from all 11 human subjects recovering from malaria. When used as carrier-free immunogens, six peptides induced a strong, boostable, immunoglobulin G-type antibody response in rabbits, indicating the presence of both B-cell determinants and T-helper-cell epitopes in these six constructs. These antibodies specifically cross-reacted with the parasite protein(s) in an immunoblot and in an immunofluorescence assay. In another immunoblot, rabbit antipeptide sera also recognized recombinant fragments of ABRA expressed in bacteria. More significantly, rabbit antibodies against two constructs (AB-1 and AB-5) inhibited the merozoite reinvasion of human erythrocytes in vitro up to ∼90%. These results favor further studies so as to determine possible inclusion of these two constructs in a multicomponent subunit vaccine against asexual blood stages of P. falciparum. PMID:9596765

  8. Early region 1B of adenovirus 2 encodes two coterminal proteins of 495 and 155 amino acid residues.

    PubMed Central

    Anderson, C W; Schmitt, R C; Smart, J E; Lewis, J B

    1984-01-01

    Partial sequence analysis of tryptic peptides has identified the E1B-495R (E1b-57K) (early transcription region 1B of 495 amino acid residues, with an approximate molecular weight of 57,000) protein of adenovirus 2 as encoded by the 495 amino acid open reading frame located in the adenovirus 2 DNA sequence between nucleotides 2016 and 3500. Additional proteins of 16,000 Mr and 18,000 Mr that are related to the E1B-495R protein were identified by cell-free translation of hybridization-selected mRNA. Analysis of [35S]methionine-containing amino terminal tryptic peptides by thin-layer chromatography showed that the E1B-495R, E1B-18K, and E1B-16K proteins all begin at the same initiation codon. The E1B-495R protein from 293 cells also has the same initial tryptic peptide, acetyl-methionyl-glutamyl-arginine. Sequence analysis of E1B-18K tryptic peptides indicated that this protein also has the same carboxy terminus as the E1B-495R protein and that it is derived from an mRNA that is spliced to remove sequences between nucleotides 2250 and 3269, resulting in a protein product of 155 amino acid residues. Analysis of E1B-16K tryptic peptides has not yet revealed the carboxy terminal structure of this protein. Both the E1B-495R and the E1B-155R (E1B-18K) proteins, as well as the E1B-16K protein, were precipitated from cell-free translations and from extracts of infected cells by antiserum against an amino terminal nonapeptide common to these proteins. Images PMID:6323739

  9. Residual dipolar couplings measured in unfolded proteins are sensitive to amino-acid-specific geometries as well as local conformational sampling.

    PubMed

    Huang, Jie-rong; Gentner, Martin; Vajpai, Navratna; Grzesiek, Stephan; Blackledge, Martin

    2012-10-01

    Many functional proteins do not have well defined folded structures. In recent years, both experimental and computational approaches have been developed to study the conformational behaviour of this type of protein. It has been shown previously that experimental RDCs (residual dipolar couplings) can be used to study the backbone sampling of disordered proteins in some detail. In these studies, the backbone structure was modelled using a common geometry for all amino acids. In the present paper, we demonstrate that experimental RDCs are also sensitive to the specific geometry of each amino acid as defined by energy-minimized internal co-ordinates. We have modified the FM (flexible-Meccano) algorithm that constructs conformational ensembles on the basis of a statistical coil model, to account for these differences. The modified algorithm inherits the advantages of the FM algorithm to efficiently sample the potential energy landscape for coil conformations. The specific geometries incorporated in the new algorithm result in a better reproduction of experimental RDCs and are generally applicable for further studies to characterize the conformational properties of intrinsically disordered proteins. In addition, the internal-co-ordinate-based algorithm is an order of magnitude more efficient, and facilitates side-chain construction, surface osmolyte simulation, spin-label distribution sampling and proline cis/trans isomer simulation. PMID:22988852

  10. Chemical fragmentation by o-iodosobenzoic acid of. cap alpha. -chain of histidine decarboxylase from Micrococcus sp. n. at tryptophan residues

    SciTech Connect

    Alekseeva, E.A.; Grebenshchikova, O.G.; Prozorovskii, V.N.

    1987-02-10

    The carboxymethylated ..cap alpha..-chain of histidine decarboxylase from Micrococcus sp. n., which contains four tryptophan residues, was cleaved by o-iodosobenzoic acid. Five fragments were isolated in homogeneous form by means of gel filtration on Sephadex, rechromatography, and high-voltage paper electrophoresis. The molecular weight, amino acid composition, and N-terminal amino acid sequence were determined for all the peptides isolated.

  11. Glutamic Acid Residues in HIV-1 p6 Regulate Virus Budding and Membrane Association of Gag

    PubMed Central

    Friedrich, Melanie; Setz, Christian; Hahn, Friedrich; Matthaei, Alina; Fraedrich, Kirsten; Rauch, Pia; Henklein, Petra; Traxdorf, Maximilian; Fossen, Torgils; Schubert, Ulrich

    2016-01-01

    The HIV-1 Gag p6 protein regulates the final abscission step of nascent virions from the cell membrane by the action of its two late (l-) domains, which recruit Tsg101 and ALIX, components of the ESCRT system. Even though p6 consists of only 52 amino acids, it is encoded by one of the most polymorphic regions of the HIV-1 gag gene and undergoes various posttranslational modifications including sumoylation, ubiquitination, and phosphorylation. In addition, it mediates the incorporation of the HIV-1 accessory protein Vpr into budding virions. Despite its small size, p6 exhibits an unusually high charge density. In this study, we show that mutation of the conserved glutamic acids within p6 increases the membrane association of Pr55 Gag followed by enhanced polyubiquitination and MHC-I antigen presentation of Gag-derived epitopes, possibly due to prolonged exposure to membrane bound E3 ligases. The replication capacity of the total glutamic acid mutant E0A was almost completely impaired, which was accompanied by defective virus release that could not be rescued by ALIX overexpression. Altogether, our data indicate that the glutamic acids within p6 contribute to the late steps of viral replication and may contribute to the interaction of Gag with the plasma membrane. PMID:27120610

  12. Influence of washing time on residual contamination of carcasses sprayed with lauric acid-potassium hydroxide

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A series of experiments were conducted to examine reductions in bacterial contamination of broiler carcasses washed for various times in a spray cabinet with a 2% lauric acid (LA)-1% potassium hydroxide (KOH) solution (w/v). Forty eviscerated carcasses and 5 ceca were obtained from the processing li...

  13. Influence of washing time on residual contamination of carcasses sprayed with lauric acid-potassium hydroxide.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A series of experiments were conducted to examine reductions in bacterial contamination of broiler carcasses washed for various times in a spray cabinet with a 2% lauric acid (LA)-1% potassium hydroxide (KOH) (w/v) solution. Forty eviscerated carcasses and 5 ceca were obtained from the processing l...

  14. Factors contributing to decreased protein stability when aspartic acid residues are in {beta}-sheet regions.

    SciTech Connect

    Pokkuluri, P. R.; Cai, X.; Raffen, R.; Gu, M.; Stevens, F. J.; Schiffer, M.

    2002-07-01

    Asp residues are significantly under represented in {beta}-sheet regions of proteins, especially in the middle of {beta}-strands, as found by a number of studies using statistical, modeling, or experimental methods. To further understand the reasons for this under representation of Asp, we prepared and analyzed mutants of a {beta}-domain. Two Gln residues of the immunoglobulin light-chain variable domain (V{sub L}) of protein Len were replaced with Asp, and then the effects of these changes on protein stability and protein structure were studied. The replacement of Q38D, located at the end of a {beta}-strand, and that of Q89D, located in the middle of a {beta}-strand, reduced the stability of the parent immunoglobulin VL domain by 2.0 kcal/mol and 5.3 kcal/mol, respectively. Because the Q89D mutant of the wild-type V{sub L}-Len domain was too unstable to be expressed as a soluble protein, we prepared the Q89D mutant in a triple mutant background, V{sub L}-Len M4L/Y27dD/T94H, which was 4.2 kcal/mol more stable than the wild-type V{sub L}-Len domain. The structures of mutants V{sub L}-Len Q38D and V{sub L}-Len Q89D/M4L/Y27dD/T94H were determined by X-ray diffraction at 1.6 A resolution. We found no major perturbances in the structures of these QD mutant proteins relative to structures of the parent proteins. The observed stability changes have to be accounted for by cumulative effects of the following several factors: (1) by changes in main-chain dihedral angles and in side-chain rotomers, (2) by close contacts between some atoms, and, most significantly, (3) by the unfavorable electrostatic interactions between the Asp side chain and the carbonyls of the main chain. We show that the Asn side chain, which is of similar size but neutral, is less destabilizing. The detrimental effect of Asp within a {beta}-sheet of an immunoglobulin-type domain can have very serious consequences. A somatic mutation of a {beta}-strand residue to Asp could prevent the expression of the

  15. Effect of the replacement of aspartic acid/glutamic acid residues with asparagine/glutamine residues in RNase He1 from Hericium erinaceus on inhibition of human leukemia cell line proliferation.

    PubMed

    Kobayashi, Hiroko; Motoyoshi, Naomi; Itagaki, Tadashi; Suzuki, Mamoru; Inokuchi, Norio

    2015-01-01

    RNase He1 from Hericium erinaceus, a member of the RNase T1 family, has high identity with RNase Po1 from Pleurotus ostreatus with complete conservation of the catalytic sequence. However, the optimal pH for RNase He1 activity is lower than that of RNase Po1, and the enzyme shows little inhibition of human tumor cell proliferation. Hence, to investigate the potential antitumor activity of recombinant RNase He1 and to possibly enhance its optimum pH, we generated RNase He1 mutants by replacing 12 Asn/Gln residues with Asp/Glu residues; the amino acid sequence of RNase Po1 was taken as reference. These mutants were then expressed in Escherichia coli. Using site-directed mutagenesis, we successfully modified the optimal pH for enzyme activity and generated a recombinant RNase He1 that inhibited the proliferation of cells in the human leukemia cell line. These properties are extremely important in the production of anticancer biologics that are based on RNase activity. PMID:25338779

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

    PubMed

    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

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

    PubMed

    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

  18. Macromitophagy, neutral lipids synthesis, and peroxisomal fatty acid oxidation protect yeast from "liponecrosis", a previously unknown form of programmed cell death.

    PubMed

    Sheibani, Sara; Richard, Vincent R; Beach, Adam; Leonov, Anna; Feldman, Rachel; Mattie, Sevan; Khelghatybana, Leila; Piano, Amanda; Greenwood, Michael; Vali, Hojatollah; Titorenko, Vladimir I

    2014-01-01

    We identified a form of cell death called "liponecrosis." It can be elicited by an exposure of the yeast Saccharomyces cerevisiae to exogenous palmitoleic acid (POA). Our data imply that liponecrosis is: (1) a programmed, regulated form of cell death rather than an accidental, unregulated cellular process and (2) an age-related form of cell death. Cells committed to liponecrotic death: (1) do not exhibit features characteristic of apoptotic cell death; (2) do not display plasma membrane rupture, a hallmark of programmed necrotic cell death; (3) akin to cells committed to necrotic cell death, exhibit an increased permeability of the plasma membrane for propidium iodide; (4) do not display excessive cytoplasmic vacuolization, a hallmark of autophagic cell death; (5) akin to cells committed to autophagic death, exhibit a non-selective en masse degradation of cellular organelles and require the cytosolic serine/threonine protein kinase Atg1p for executing the death program; and (6) display a hallmark feature that has not been reported for any of the currently known cell death modalities-namely, an excessive accumulation of lipid droplets where non-esterified fatty acids (including POA) are deposited in the form of neutral lipids. We therefore concluded that liponecrotic cell death subroutine differs from the currently known subroutines of programmed cell death. Our data suggest a hypothesis that liponecrosis is a cell death module dynamically integrated into a so-called programmed cell death network, which also includes the apoptotic, necrotic, and autophagic modules of programmed cell death. Based on our findings, we propose a mechanism underlying liponecrosis. PMID:24196447

  19. The Last C-Terminal Residue of VP3, Glutamic Acid 257, Controls Capsid Assembly of Infectious Bursal Disease Virus

    PubMed Central

    Chevalier, Christophe; Lepault, Jean; Da Costa, Bruno; Delmas, Bernard

    2004-01-01

    Infectious bursal disease virus (IBDV) is a nonenveloped virus with an icosahedral capsid composed of two proteins, VP2 and VP3, that derive from the processing of the polyprotein NH2-pVP2-VP4-VP3-COOH. The virion contains VP1, the viral polymerase, which is both free and covalently linked to the two double-stranded RNA (dsRNA) genomic segments. In this study, the virus assembly process was studied further with the baculovirus expression system. While expression of the wild-type polyprotein was not found to be self-sufficient to give rise to virus-like particles (VLPs), deletion or replacement of the five C-terminal residues of VP3 was observed to promote capsid assembly. Indeed, the single deletion of the C-terminal glutamic acid was sufficient to induce VLP formation. Moreover, fusion of various peptides or small proteins (a green fluorescent protein or a truncated form of ovalbumin) at the C terminus of VP3 also promoted capsid assembly, suggesting that assembly required screening of the negative charges at the C terminus of VP3. The fused polypeptides mimicked the effect of VP1, which interacts with VP3 to promote VLP assembly. The C-terminal segment of VP3 was found to contain two functional domains. While the very last five residues of VP3 mainly controlled both assembly and capsid architecture, the five preceding residues constituted the VP1 (and possibly the pVP2/VP2) binding domain. Finally, we showed that capsid formation is associated with VP2 maturation, demonstrating that the protease VP4 is involved in the virus assembly process. PMID:15016850

  20. Purification, Cloning, Characterization and Essential Amino Acid Residues Analysis of a New ι-Carrageenase from Cellulophaga sp. QY3

    PubMed Central

    Ma, Su; Duan, Gaofei; Chai, Wengang; Geng, Cunliang; Tan, Yulong; Wang, Lushan; Le Sourd, Frédéric; Michel, Gurvan; Yu, Wengong; Han, Feng

    2013-01-01

    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. PMID:23741363

  1. Prediction of Residue Status to Be Protected or Not Protected From Hy-drogen Exchange Using Amino Acid Sequence Only.

    PubMed

    Nikita V, Dovidchenko; Oxana V, Galzitskaya

    2008-01-01

    We have outlined here some structural aspects of local flexibility. Important functional properties are related to flexible segments. We try to predict regions that have been shown to exhibit the highest probability of being folded in the equilibrium intermediate or native state and will be protected from hydrogen exchange using amino acid sequence only. Our approach FoldUnfold for the prediction of unstructured regions has been applied to seven different proteins. For 80% of the residues considered in this paper we can predict correctly their status: will they be protected or not from hydrogen exchange. An additional goal of our study is to assess whether properties inferred using the bioinformatics approach are easily applicable to predict behavior of proteins in solution. PMID:18949078

  2. Prediction of Residue Status to Be Protected or Not Protected From Hy-drogen Exchange Using Amino Acid Sequence Only

    PubMed Central

    Dovidchenko, Nikita V; Galzitskaya, Oxana V

    2008-01-01

    We have outlined here some structural aspects of local flexibility. Important functional properties are related to flexible segments. We try to predict regions that have been shown to exhibit the highest probability of being folded in the equilibrium intermediate or native state and will be protected from hydrogen exchange using amino acid sequence only. Our approach FoldUnfold for the prediction of unstructured regions has been applied to seven different proteins. For 80% of the residues considered in this paper we can predict correctly their status: will they be protected or not from hydrogen exchange. An additional goal of our study is to assess whether properties inferred using the bioinformatics approach are easily applicable to predict behavior of proteins in solution. PMID:18949078

  3. [Determination of glyphosate and aminomethylphosphonic acid residues in foods using high performance liquid chromatography-mass spectrometry/mass spectrometry].

    PubMed

    Li, Bo; Deng, Xiaojun; Guo, Dehua; Jin, Shuping

    2007-07-01

    A method for the determination of glyphosate (PMG) and aminomethylphosphonic acid (AMPA) residues in plant products, such as rice, wheat, vegetables, fruits and tea, pig and chicken muscles, aquatic products, chestnut, honey, etc., was developed using high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). In this method, PMG and AMPA were extracted with water from samples, defatted using an extraction step with dichloromethane, and purified using a cation-exchange (CAX) solid phase extraction cartridge. Then, these were derived using fluorenylmethylchloroformate (FMOC-Cl) in borate buffer for subsequent HPLC-MS/MS analysis. Isotope-labeled PMG 1, 2(13)- C(15) N was used as the internal standard for the quantitative analysis of two residues. For all samples, the recoveries ranged from 80.0% to 104% and the relative standard deviations (RSDs) ranged from 6.7% to 18.2%. The limit of quantification (LOQ) was determined to be 0.05 mg/kg with a linear range of 0.20-10 microg/L. It is demonstrated that this method is reliable and sensitive for the analysis of PMG and APMA with low concentrations in foods. PMID:17970103

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

    NASA Technical Reports Server (NTRS)

    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.

  5. Molecular mechanism of long-range synergetic color tuning between multiple amino acid residues in conger rhodopsin

    PubMed Central

    Watanabe, Hiroshi C.; Mori, Yoshiharu; Tada, Takashi; Yokoyama, Shozo; Yamato, Takahisa

    2011-01-01

    The synergetic effects of multiple rhodopsin mutations on color tuning need to be completely elucidated. Systematic genetic studies and spectroscopy have demonstrated an interesting example of synergetic color tuning between two amino acid residues in conger rhodopsin's ancestral pigment (p501): —a double mutation at one nearby and one distant residue led to a significant λmax blue shift of 13 nm, whereas neither of the single mutations at these two sites led to meaningful shifts. To analyze the molecular mechanisms of this synergetic color tuning, we performed homology modeling, molecular simulations, and electronic state calculations. For the double mutant, N195A/A292S, in silico mutation analysis demonstrated conspicuous structural changes in the retinal chromophore, whereas that of the single mutant, A292S, was almost unchanged. Using statistical ensembles of QM/MM optimized structures, the excitation energy of retinal chromophore was evaluated for the three visual pigments. As a result, the λmax shift of double mutant (DM) from p501 was –8 nm, while that of single mutant (SM) from p501 was +1 nm. Molecular dynamics simulation for DM demonstrated frequent isomerization between 6-s-cis and 6-s-trans conformers. Unexpectedly, however, the two conformers exhibited almost identical excitation energy, whereas principal component analysis (PCA) identified the retinal-counterion cooperative change of BLA (bond length alternation) and retinal-counterion interaction lead to the shift. PMID:21297892

  6. Keys to Lipid Selection in Fatty Acid Amide Hydrolase Catalysis: Structural Flexibility, Gating Residues and Multiple Binding Pockets

    PubMed Central

    Palermo, Giulia; Bauer, Inga; Campomanes, Pablo; Cavalli, Andrea; Armirotti, Andrea; Girotto, Stefania; Rothlisberger, Ursula; De Vivo, Marco

    2015-01-01

    The fatty acid amide hydrolase (FAAH) regulates the endocannabinoid system cleaving primarily the lipid messenger anandamide. FAAH has been well characterized over the years and, importantly, it represents a promising drug target to treat several diseases, including inflammatory-related diseases and cancer. But its enzymatic mechanism for lipid selection to specifically hydrolyze anandamide, rather than similar bioactive lipids, remains elusive. Here, we clarify this mechanism in FAAH, examining the role of the dynamic paddle, which is formed by the gating residues Phe432 and Trp531 at the boundary between two cavities that form the FAAH catalytic site (the “membrane-access” and the “acyl chain-binding” pockets). We integrate microsecond-long MD simulations of wild type and double mutant model systems (Phe432Ala and Trp531Ala) of FAAH, embedded in a realistic membrane/water environment, with mutagenesis and kinetic experiments. We comparatively analyze three fatty acid substrates with different hydrolysis rates (anandamide > oleamide > palmitoylethanolamide). Our findings identify FAAH’s mechanism to selectively accommodate anandamide into a multi-pocket binding site, and to properly orient the substrate in pre-reactive conformations for efficient hydrolysis that is interceded by the dynamic paddle. Our findings therefore endorse a structural framework for a lipid selection mechanism mediated by structural flexibility and gating residues between multiple binding cavities, as found in FAAH. Based on the available structural data, this exquisite catalytic strategy for substrate specificity seems to be shared by other lipid-degrading enzymes with similar enzymatic architecture. The mechanistic insights for lipid selection might assist de-novo enzyme design or drug discovery efforts. PMID:26111155

  7. The Loss and Gain of Functional Amino Acid Residues Is a Common Mechanism Causing Human Inherited Disease

    PubMed Central

    Lugo-Martinez, Jose; Pejaver, Vikas; Pagel, Kymberleigh A.; Mort, Matthew; Cooper, David N.; Mooney, Sean D.; Radivojac, Predrag

    2016-01-01

    Elucidating the precise molecular events altered by disease-causing genetic variants represents a major challenge in translational bioinformatics. To this end, many studies have investigated the structural and functional impact of amino acid substitutions. Most of these studies were however limited in scope to either individual molecular functions or were concerned with functional effects (e.g. deleterious vs. neutral) without specifically considering possible molecular alterations. The recent growth of structural, molecular and genetic data presents an opportunity for more comprehensive studies to consider the structural environment of a residue of interest, to hypothesize specific molecular effects of sequence variants and to statistically associate these effects with genetic disease. In this study, we analyzed data sets of disease-causing and putatively neutral human variants mapped to protein 3D structures as part of a systematic study of the loss and gain of various types of functional attribute potentially underlying pathogenic molecular alterations. We first propose a formal model to assess probabilistically function-impacting variants. We then develop an array of structure-based functional residue predictors, evaluate their performance, and use them to quantify the impact of disease-causing amino acid substitutions on catalytic activity, metal binding, macromolecular binding, ligand binding, allosteric regulation and post-translational modifications. We show that our methodology generates actionable biological hypotheses for up to 41% of disease-causing genetic variants mapped to protein structures suggesting that it can be reliably used to guide experimental validation. Our results suggest that a significant fraction of disease-causing human variants mapping to protein structures are function-altering both in the presence and absence of stability disruption. PMID:27564311

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

    NASA Astrophysics Data System (ADS)

    Takahashi, O.

    2014-03-01

    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.

  9. Distributions of amino acids suggest that certain residue types more effectively determine protein secondary structure.

    PubMed

    Saraswathi, S; Fernández-Martínez, J L; Koliński, A; Jernigan, R L; Kloczkowski, A

    2013-10-01

    Exponential growth in the number of available protein sequences is unmatched by the slower growth in the number of structures. As a result, the development of efficient and fast protein secondary structure prediction methods is essential for the broad comprehension of protein structures. Computational methods that can efficiently determine secondary structure can in turn facilitate protein tertiary structure prediction, since most methods rely initially on secondary structure predictions. Recently, we have developed a fast learning optimized prediction methodology (FLOPRED) for predicting protein secondary structure (Saraswathi et al. in JMM 18:4275, 2012). Data are generated by using knowledge-based potentials combined with structure information from the CATH database. A neural network-based extreme learning machine (ELM) and advanced particle swarm optimization (PSO) are used with this data to obtain better and faster convergence to more accurate secondary structure predicted results. A five-fold cross-validated testing accuracy of 83.8 % and a segment overlap (SOV) score of 78.3 % are obtained in this study. Secondary structure predictions and their accuracy are usually presented for three secondary structure elements: α-helix, β-strand and coil but rarely have the results been analyzed with respect to their constituent amino acids. In this paper, we use the results obtained with FLOPRED to provide detailed behaviors for different amino acid types in the secondary structure prediction. We investigate the influence of the composition, physico-chemical properties and position specific occurrence preferences of amino acids within secondary structure elements. In addition, we identify the correlation between these properties and prediction accuracy. The present detailed results suggest several important ways that secondary structure predictions can be improved in the future that might lead to improved protein design and engineering. PMID:23907551

  10. His-311 and Arg-559 are key residues involved in fatty acid oxygenation in pathogen-inducible oxygenase.

    PubMed

    Koszelak-Rosenblum, Mary; Krol, Adam C; Simmons, Danielle M; Goulah, Christopher C; Wroblewski, Liliana; Malkowski, Michael G

    2008-09-01

    Pathogen-inducible oxygenase (PIOX) oxygenates fatty acids into 2R-hydroperoxides. PIOX belongs to the fatty acid alpha-dioxygenase family, which exhibits homology to cyclooxygenase enzymes (COX-1 and COX-2). Although these enzymes share common catalytic features, including the use of a tyrosine radical during catalysis, little is known about other residues involved in the dioxygenase reaction of PIOX. We generated a model of linoleic acid (LA) bound to PIOX based on computational sequence alignment and secondary structure predictions with COX-1 and experimental observations that governed the placement of carbon-2 of LA below the catalytic Tyr-379. Examination of the model identified His-311, Arg-558, and Arg-559 as potential molecular determinants of the dioxygenase reaction. Substitutions at His-311 and Arg-559 resulted in mutant constructs that retained virtually no oxygenase activity, whereas substitutions of Arg-558 caused only moderate decreases in activity. Arg-559 mutant constructs exhibited increases of greater than 140-fold in Km, whereas no substantial change in Km was observed for His-311 or Arg-558 mutant constructs. Thermal shift assays used to measure ligand binding affinity show that the binding of LA is significantly reduced in a Y379F/R559A mutant construct compared with that observed for Y379F/R558A construct. Although Oryza sativa PIOX exhibited oxygenase activity against a variety of 14-20-carbon fatty acids, the enzyme did not oxygenate substrates containing modifications at the carboxylate, carbon-1, or carbon-2. Taken together, these data suggest that Arg-559 is required for high affinity binding of substrates to PIOX, whereas His-311 is involved in optimally aligning carbon-2 below Tyr-379 for catalysis. PMID:18596034

  11. G glycoprotein amino acid residues required for human monoclonal antibody RAB1 neutralization are conserved in rabies virus street isolates.

    PubMed

    Wang, Yang; Rowley, Kirk J; Booth, Brian J; Sloan, Susan E; Ambrosino, Donna M; Babcock, Gregory J

    2011-08-01

    Replacement of polyclonal anti-rabies immunoglobulin (RIG) used in rabies post-exposure prophylaxis (PEP) with a monoclonal antibody will eliminate cost and availability constraints that currently exist using RIG in the developing world. The human monoclonal antibody RAB1 has been shown to neutralize all rabies street isolates tested; however for the laboratory-adapted fixed strain, CVS-11, mutation in the G glycoprotein of amino acid 336 from asparagine (N) to aspartic acid (D) resulted in resistance to neutralization. Interestingly, this same mutation in the G glycoprotein of a second laboratory-adapted fixed strain (ERA) did not confer resistance to RAB1 neutralization. Using cell surface staining and lentivirus pseudotyped with rabies virus G glycoprotein (RABVpp), we identified an amino acid alteration in CVS-11 (K346), not present in ERA (R346), which was required in combination with D336 to confer resistance to RAB1. A complete analysis of G glycoprotein sequences from GenBank demonstrated that no identified rabies isolates contain the necessary combination of G glycoprotein mutations for resistance to RAB1 neutralization, consistent with the broad neutralization of RAB1 observed in direct viral neutralization experiments with street isolates. All combinations of amino acids 336 and 346 reported in the sequence database were engineered into the ERA G glycoprotein and RAB1 was able to neutralize RABVpp bearing ERA G glycoprotein containing all known combinations at these critical residues. These data demonstrate that RAB1 has the capacity to neutralize all identified rabies isolates and a minimum of two distinct mutations in the G glycoprotein are required for abrogation of RAB1 neutralization. PMID:21693135

  12. Dendronylation: Residue-specific chemoselective attachment of oligoglycerol dendrimers on proteins with noncanonical amino acids.

    PubMed

    Ma, Ying; Thota, Bala N S; Haag, Rainer; Budisa, Nediljko

    2015-11-15

    Polyglycerol dendrimers as an important class of polymeric materials especially attractive for covalent attachment to therapeutic proteins as a useful alternative to traditional PEGylation procedures. Herein, we combine in vivo noncanonical amino acid (ncAA) incorporation and chemoselective conjugation in vitro to produce novel hybrid protein-dendrimer conjugates with the defined architectures. We incorporated Azidohomoalanine (Aha) as methionine substitute in vivo into various protein scaffolds to allow non-invasive dendrimer conjugations (dendronylation). Our approach makes recombinant proteins accessible for the design of multivalent dendrimer conjugates since it enables the preparation of many sequences with various positions for regioselective dendronylation. PMID:26483199

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

    SciTech Connect

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    SciTech Connect

    South, T.L.; Kim, B.; Summers, M.F.

    1989-01-04

    The Zn/sup 2+/ and Cd/sup 2+/ 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. /sup 1/H NMR data indicate that the metal adducts are 1:1 compounds that are stable in aqueous solutions for at least a month. The /sup 113/Cd NMR spectral results for the adduct are presented and analyzed. 26 references, 3 figures.

  16. Role of portal region lysine residues in electrostatic interactions between heart fatty acid binding protein and phospholipid membranes.

    PubMed

    Herr, F M; Aronson, J; Storch, J

    1996-01-30

    The structure of heart fatty acid binding protein (HFABP) is a flattened beta-barrel comprising 10 antiparallel beta-sheets capped by two alpha-helical segments. The helical cap region is hypothesized to behave as a portal "lid" for the entry and release of ligand from the binding pocket. The transfer of fatty acid from HFABP is thought to occur via effective collisional interactions with membranes, and these interactions are enhanced when transfer is to membranes of net negative charge, thus implying that specific basic residues on the surface of HFABP may govern the transfer process [Wootan, M. G., & Storch, J. (1994) J. Biol. Chem. 269, 10517-10523]. To directly examine the role of charged lysine residues on the HFABP surface in specific interactions with membranes, chemical modification and selective mutagenesis of HFABP were used. All surface lysine residues were neutralized by acetylation of recombinant HFABP with acetic anhydride. In addition, seven mutant HFABPs were generated that resulted in charge alterations in five distinct sites of HFABP. Modification of the protein did not significantly alter the structural or ligand binding properties of HFABP, as assessed by circular dichroism, fluorescence quantum yield, and ligand binding analyses. By using a resonance energy transfer assay, transfer of 2-(9-anthroyloxy)palmitate (2AP) from acetylated HFABP to membranes was significantly slower than transfer from native HFABP. In addition, in distinct contrast to transfer from native protein, the 2AP transfer rate from acetylated HFABP was not increased to acceptor membranes of increased negative charge. Transfer of 2AP from HFABP mutants involving K22, located on alpha-helix I (alpha-I) of the helical cap region, was 3-fold slower than transfer from wild-type protein, whereas rates from a mutant involving the K59 residue, located on the beta 2-turn of the barrel near the helical cap, were 2-fold faster than those of wild type. A double mutant involving K22 and K

  17. Role of the glutamic acid 54 residue in transthyretin stability and thyroxine binding.

    PubMed

    Miyata, Masanori; Sato, Takashi; Mizuguchi, Mineyuki; Nakamura, Teruya; Ikemizu, Shinji; Nabeshima, Yuko; Susuki, Seiko; Suwa, Yoshiaki; Morioka, Hiroshi; Ando, Yukio; Suico, Mary Ann; Shuto, Tsuyoshi; Koga, Tomoaki; Yamagata, Yuriko; Kai, Hirofumi

    2010-01-12

    Transthyretin (TTR) is a tetrameric protein associated with amyloidosis caused by tetramer dissociation and monomer misfolding. The structure of two TTR variants (E54G and E54K) with Glu54 point mutation that cause clinically aggressive amyloidosis remains unclear, although amyloidogenicity of artificial triple mutations (residues 53-55) in beta-strand D had been investigated. Here we first analyzed the crystal structures and biochemical and biophysical properties of E54G and E54K TTRs. The direction of the Lys15 side chain in E54K TTR and the surface electrostatic potential in the edge region in both variants were different from those of wild-type TTR. The presence of Lys54 leads to destabilization of tetramer structure due to enhanced electrostatic repulsion between Lys15 of two monomers. Consistent with structural data, the biochemical analyses demonstrated that E54G and E54K TTRs were more unstable than wild-type TTR. Furthermore, the entrance of the thyroxine (T(4)) binding pocket in TTR was markedly narrower in E54K TTR and wider in E54G TTR compared with wild-type TTR. The tetramer stabilization and amyloid fibril formation assays in the presence of T(4) showed lower tetramer stability and more fibril formation in E54K and E54G TTRs than in wild-type TTR, suggesting decreased T(4) binding to the TTR variants. These findings indicate that structural modification by Glu54 point mutation may sufficiently alter tetramer stability and T(4) binding. PMID:19950966

  18. Predicting HLA Class I Non-Permissive Amino Acid Residues Substitutions

    PubMed Central

    Binkowski, T. Andrew; Marino, Susana R.; Joachimiak, Andrzej

    2012-01-01

    Prediction of peptide binding to human leukocyte antigen (HLA) molecules is essential to a wide range of clinical entities from vaccine design to stem cell transplant compatibility. Here we present a new structure-based methodology that applies robust computational tools to model peptide-HLA (p-HLA) binding interactions. The method leverages the structural conservation observed in p-HLA complexes to significantly reduce the search space and calculate the system’s binding free energy. This approach is benchmarked against existing p-HLA complexes and the prediction performance is measured against a library of experimentally validated peptides. The effect on binding activity across a large set of high-affinity peptides is used to investigate amino acid mismatches reported as high-risk factors in hematopoietic stem cell transplantation. PMID:22905104

  19. N-terminal basic amino acid residues of Beet black scorch virus capsid protein play a critical role in virion assembly and systemic movement

    PubMed Central

    2013-01-01

    Background Beet black scorch virus (BBSV) is a small single-stranded, positive-sense RNA plant virus belonging to the genus Necrovirus, family Tombusviridae. Its capsid protein (CP) contains a 13 amino acid long basic region at the N-terminus, rich in arginine and lysine residues, which is thought to interact with viral RNA to initiate virion assembly. Results In the current study, a series of BBSV mutants containing amino acid substitutions as well as deletions within the N-terminal region were generated and examined for their effects on viral RNA replication, virion assembly, and long distance spread in protoplasts and whole host plants of BBSV. The RNA-binding activities of the mutated CPs were also evaluated in vitro. These experiments allowed us to identify two key basic amino acid residues in this region that are responsible for initiating virus assembly through RNA-binding. Proper assembly of BBSV particles is in turn needed for efficient viral systemic movement. Conclusions We have identified two basic amino acid residues near the N-terminus of the BBSV CP that bind viral RNA with high affinity to initiate virion assembly. We further provide evidence showing that systemic spread of BBSV in infected plants requires intact virions. This study represents the first in-depth investigation of the role of basic amino acid residues within the N-terminus of a necroviral CP. PMID:23786675

  20. Variable clinical manifestations of a glycine to glutamic acid substitution of the COL3A1 gene at residue 736

    SciTech Connect

    Pope, F.M.; Narcisi, P.; Richards, A.J.

    1994-09-01

    Glycine substitutions at the 3{prime} end of the COL3A1 gene generally produce a characteristic clinical phenotype including acrogeria and severe vascular fragility. Here we report a three generation British family in which the propositus presented with aneurysms of the groins. He, his mother, sister and elder daughter all had the external clinical phenotype of vascular EDS IV whilst another daughter and nephew were clinically normal. Cultured skin fibroblasts from the propositus and his clinically affected relatives poorly secreted normal and overmodified collagen III species. Normal components of secreted proteins predominated whilst overmodified molecules were prominent in intracellular material. Surprisingly the normal children also secreted less collagen type III than expected (though more than their clinically abnormal relatives). cDNA from bases 2671 to 3714 were amplified as four overlapping PCR fragments and analysed by DGGE. The region between 2671 and 3015 was heterozygous. Sequencing showed a mutation of glycine to glutamic acid at residue 736. This mutation created an extra Apa 1 restriction site which was suitable for family studies. These showed inheritance of the mutant gene by both vascular and non-vascular clinical phenotypes. This family therefore illustrates that replacement of glycine to glutamic acid at position 736 produces variable clinical and biochemical phenotypes ranging from easily recognizable vascular EDS IV with very poor collagen secretion to an EDS III-like picture and with less severe protein disturbance. The reasons for these differences are at present unexplained.

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

    PubMed

    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

  2. The adsorption of chromium (VI) from industrial wastewater by acid and base-activated lignocellulosic residues.

    PubMed

    Alvarez, Patricia; Blanco, Clara; Granda, Marcos

    2007-06-01

    This study deals with the adsorption of Cr(VI) from synthetic and industrial wastewater, produced by a sewage plant. The activated carbons were prepared from a lignocellulosic raw material by thermal treatment at 450 and 650 degrees C in the presence of acid (AlCl(3), HCl, H(3)PO(4) and H(2)SO(4)) and base (NaOH) agents. To optimize the adsorption of Cr(VI), the chemical modifications caused by each activating agent (related to the capability of Cr(VI) removal), and the optimal experimental conditions of the pH, Cr(VI) concentration, adsorbent dose and residence time, were studied. Thus, treatment with H(3)PO(4) gives rise to carbons with a high surface area and high efficiency for Cr(VI) removal at short equilibrium times. In contrast, the generation of active surface sites by means of NaOH requires longer equilibrium times, the adsorption being less effective than in the former case. The adsorption isotherms obey the Langmuir equation only in the first stages of the reaction but fit the Freundlich equations over the whole range studied, so the heat of adsorption can be easily calculated. The results also show that the activated carbons obtained can be recovered by filtration with an efficiency of 30% in the third cycle. PMID:17126488

  3. Influence of calcium and phosphorus, lactose, and salt-to-moisture ratio on Cheddar cheese quality: changes in residual sugars and water-soluble organic acids during ripening.

    PubMed

    Upreti, P; McKay, L L; Metzger, L E

    2006-02-01

    Cheddar cheese ripening involves the conversion of lactose to glucose and galactose or galactose-6-phosphate by starter and nonstarter lactic acid bacteria. Under ideal conditions (i.e., where bacteria grow under no stress of pH, water activity, and salt), these sugars are mainly converted to lactic acid. However, during ripening of cheese, survival and growth of bacteria occurs under the stressed condition of low pH, low water activity, and high salt content. This forces bacteria to use alternate biochemical pathways resulting in production of other organic acids. The objective of this study was to determine if the level and type of organic acids produced during ripening was influenced by calcium (Ca) and phosphorus (P), residual lactose, and salt-to-moisture ratio (S/M) of cheese. Eight cheeses with 2 levels of Ca and P (0.67 and 0.47% vs. 0.53 and 0.39%, respectively), lactose at pressing (2.4 vs. 0.78%), and S/M (6.4 vs. 4.8%) were manufactured. The cheeses were analyzed for organic acids (citric, orotic, pyruvic, lactic, formic, uric, acetic, propanoic, and butyric acids) and residual sugars (lactose, galactose) during 48 wk of ripening using an HPLC-based method. Different factors influenced changes in concentration of residual sugars and organic acids during ripening and are discussed in detail. Our results indicated that the largest decrease in lactose and the largest increase in lactic acid occurred between salting and d 1 of ripening. It was interesting to observe that although the lactose content in cheese was influenced by several factors (Ca and P, residual lactose, and S/M), the concentration of lactic acid was influenced only by S/M. More lactic acid was produced in low S/M treatments compared with high S/M treatments. Although surprising for Cheddar cheese, a substantial amount (0.2 to 0.4%) of galactose was observed throughout ripening in all treatments. Minor changes in the levels of citric, uric, butyric, and propanoic acids were observed during

  4. Hepatotoxicity of Pentavalent Antimonial Drug: Possible Role of Residual Sb(III) and Protective Effect of Ascorbic Acid

    PubMed Central

    Kato, Kelly C.; Morais-Teixeira, Eliane; Reis, Priscila G.; Silva-Barcellos, Neila M.; Salaün, Pascal; Campos, Paula P.; Dias Corrêa-Junior, José; Rabello, Ana; Demicheli, Cynthia

    2014-01-01

    Pentavalent antimonial drugs such as meglumine antimoniate (Glucantime [Glu; Sanofi-Aventis, São Paulo, Brazil]) produce severe side effects, including cardiotoxicity and hepatotoxicity, during the treatment of leishmaniasis. We evaluated the role of residual Sb(III) in the hepatotoxicity of meglumine antimoniate, as well as the protective effect of the antioxidant ascorbic acid (AA) during antimonial chemotherapy in a murine model of visceral leishmaniasis. BALB/c mice infected with Leishmania infantum were treated intraperitoneally at 80 mg of Sb/kg/day with commercial meglumine antimoniate (Glu) or a synthetic meglumine antimoniate with lower Sb(III) level (MA), in association or not with AA (15 mg/kg/day), for a 20-day period. Control groups received saline or saline plus AA. Livers were evaluated for hepatocytes histological alterations, peroxidase activity, and apoptosis. Increased proportions of swollen and apoptotic hepatocytes were observed in animals treated with Glu compared to animals treated with saline or MA. The peroxidase activity was also enhanced in the liver of animals that received Glu. Cotreatment with AA reduced the extent of histological changes, the apoptotic index, and the peroxidase activity to levels corresponding to the control group. Moreover, the association with AA did not affect the hepatic uptake of Sb and the ability of Glu to reduce the liver and spleen parasite loads in infected mice. In conclusion, our data supports the use of pentavalent antimonials with low residue of Sb(III) and the association of pentavalent antimonials with AA, as effective strategies to reduce side effects in antimonial therapy. PMID:24189251

  5. ASCORBIC ACID TREATMENT TO REDUCE RESIDUAL HALOGEN-BASED OXIDANTS PRIOR TO THE DETERMINATION OF HALOGENATED DISINFECTION BYPRODUCTS IN POTABLE WATER

    EPA Science Inventory

    Treatment of potable water samples with ascorbic acid has been investigated as a means for reducing residual halogen-based oxidants (disinfectants)i.e., HOCl, Cl2, Brw and BrCl, prior to determination of EPA Method 551.1A and 551.1B analytes. These disinfection byproducts include...

  6. Chemical structures of corn stover and its residue after dilute acid prehydrolysis and enzymatic hydrolysis: Insight into factors limiting enzymatic hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Advanced solid-state NMR techniques and wet chemical analyses were applied to investigate untreated corn stover (UCS) and its residues after dilute acid prehydrolysis (DAP) and enzymatic hydrolysis (RES) to provide evidence for the limitations to the effectiveness of enzyme hydrolysis. Advanced soli...

  7. Identification of amino acid residues of AcMNPV P143 protein involved in rRNA degradation and restricted viral replication in BM-N cells from the silkworm Bombyx mori.

    PubMed

    Hamajima, Rina; Kobayashi, Michihiro; Ikeda, Motoko

    2015-11-01

    We previously demonstrated that rRNA undergoes rapid and extensive degradation in Bombyx mori BM-N cells upon infection with AcMNPV, which is triggered by AcMNPV P143 (Ac-P143) protein. Here, we showed that six amino acid residues of Ac-P143 protein, distributing between positions 514 and 599, are involved in rRNA degradation in BM-N cells. The six residues are highly conserved among P143 proteins from AcMNPV, HycuMNPV, SeMNPV and SpltMNPV, which trigger rRNA degradation in BM-N cells upon infection, but are only partially conserved in Bm-P143 protein, which does not induce rRNA degradation in BM-N cells. We also demonstrated that substitution of only two selected residues (N565S/L578F) of Bm-P143 protein with the corresponding Ac-P143 protein residues generates a mutant Bm-P143 protein that is capable of triggering rRNA degradation in BM-N cells. These results indicate that BmNPV evolved a unique P143 protein to evade the antiviral response and allow replication in B. mori cells. PMID:26313611

  8. Identification and Functions of Amino Acid Residues in PotB and PotC Involved in Spermidine Uptake Activity*

    PubMed Central

    Higashi, Kyohei; Sakamaki, Yoshiharu; Herai, Emiko; Demizu, Risa; Uemura, Takeshi; Saroj, Sunil D.; Zenda, Risa; Terui, Yusuke; Nishimura, Kazuhiro; Toida, Toshihiko; Kashiwagi, Keiko; Igarashi, Kazuei

    2010-01-01

    Amino acid residues on PotB and PotC involved in spermidine uptake were identified by random and site-directed mutagenesis. It was found that Trp8, Tyr43, Trp100, Leu110, and Tyr261 in PotB and Trp46, Asp108, Glu169, Ser196, Asp198, and Asp199 in PotC were strongly involved in spermidine uptake and that Tyr160, Glu172, and Leu274 in PotB and Tyr19, Tyr88, Tyr148, Glu160, Leu195, and Tyr211 in PotC were moderately involved in spermidine uptake. Among 11 amino acid residues that were strongly involved in spermidine uptake, Trp8 in PotB was important for insertion of PotB and PotC into membranes. Tyr43, Trp100, and Leu110 in PotB and Trp46, Asp108, Ser196, and Asp198 in PotC were found to be involved in the interaction with PotD. Leu110 and Tyr261 in PotB and Asp108, Asp198, and Asp199 in PotC were involved in the recognition of spermidine, and Trp100 and Tyr261 in PotB and Asp108, Glu169, and Asp198 in PotC were involved in ATPase activity of PotA. Accordingly, Trp100 in PotB was involved in both PotD recognition and ATPase activity, Leu110 in PotB was involved in both PotD and spermidine recognition, and Tyr261 in PotB was involved in both spermidine recognition and ATPase activity. Asp108 and Asp198 in PotC were involved in PotD and spermidine recognition as well as ATPase activity. These results suggest that spermidine passage from PotD to the cytoplasm is coupled to the ATPase activity of PotA through a structural change of PotA by its ATPase activity. PMID:20937813

  9. Biochemical characterization of the water-soluble squalene synthase from Methylococcus capsulatus and the functional analyses of its two DXXD(E)D motifs and the highly conserved aromatic amino acid residues.

    PubMed

    Ohtake, Kana; Saito, Naoki; Shibuya, Satoshi; Kobayashi, Wakako; Amano, Ryosuke; Hirai, Takumi; Sasaki, Shinji; Nakano, Chiaki; Hoshino, Tsutomu

    2014-12-01

    Information regarding squalene synthases (SQSs) from prokaryotes is scarce. We aimed to characterize the SQS from Methylococcus capsulatus. We studied its reaction mechanism by kinetic analysis and evaluated the structure of the substrate/inhibitor-binding sites via homology modeling. The cloned M. capsulatus SQS was expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid column chromatography. Interestingly, M. capsulatus SQS was water-soluble and did not require any detergent for its higher activity, unlike other SQSs studied previously; supplementation of any type of detergent inhibited enzyme activity. The specific activity and the kinetic values (Km and kcat ) for the substrate farnesyl diphosphate and NADPH are reported. The substrate analog farnesyl methylenediphosphonate showed potent inhibition toward the enzyme. We prepared the site-specific mutants directed at potential active-site residues (58) DXX(61) E(62) D (S1 site) and (213) DXX(216) D(217) D (S2 site), which were assumed to be involved in the binding of the substrate farnesyl diphosphate through the Mg(2+) ion. We first demonstrated that the S1 site and the two basic residues (R55 and K212) were responsible for the binding of farnesyl diphosphate. Furthermore, we examined the catalytic roles of the highly conserved aromatic residues and demonstrated that the Y164 residue abstracts the proton of cation 5, which is produced during the first half-reaction (Scheme 1), to afford presqualene diphosphate, and that the W224 residue stabilizes the intermediary cation 5 via the cation-π interaction. Furthermore, we confirm for the first time that the F32 and the Y51 residues also stabilize the carbocation intermediate(s) generated during the second half-reaction. PMID:25283713

  10. Interaction of acid mine drainage with Ordinary Portland Cement blended solid residues generated from active treatment of acid mine drainage with coal fly ash.

    PubMed

    Gitari, Wilson M; Petrik, Leslie F; Key, David L; Okujeni, Charles

    2011-01-01

    Fly ash (FA) has been investigated as a possible treatment agent for Acid mine drainage (AMD) and established to be an alternative, cheap and economically viable agent compared to the conventional alkaline agents. However, this treatment option also leads to generation of solid residues (SR) that require disposal and one of the proposed disposal method is a backfill in coal mine voids. In this study, the interaction of the SR with AMD that is likely to be present in such backfill scenario was simulated by draining columns packed with SR and SR + 6% Ordinary Portland Cement (OPC) unsaturated with simulated AMD over a 6 month period. The evolving geochemistry of the liquid/solid (L/S) system was evaluated in-terms of the mineral phases likely or controlling contaminants attenuation at the different pH regimes generated. Stepwise acidification of the percolates was observed as the drainage progressed. Two pH buffer zones were observed (7.5-9 and 3-4) for SR and (11.2-11.3 and 3.5-4) for SR + 6% OPC. The solid residue cores (SR) appeared to have a significant buffering capacity, maintaining a neutral to slightly alkaline pH in the leachates for an extended period of time (97 days: L/S 4.3) while SR + 6% OPC reduced this neutralization capacity to 22 days (L/S 1.9). Interaction of AMD with SR or SR + 6% OPC generated alkaline conditions that favored precipitation of Fe, Al, Mn-(oxy) hydroxides, Fe and Ca-Al hydroxysulphates that greatly contributed to the contaminants removal. However, precipitation of these phases was restricted to the pH of the leachates remaining at neutral to circum-neutral levels. Backfill of mine voids with SR promises to be a feasible technology for the disposal of the SR but its success will greatly depend on the disposal scenario, AMD generated and the alkalinity generating potential of the SR. A disadvantage would be the possible re-dissolution of the precipitated phases at pH < 4 that would release the contaminants back to the water column

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

    PubMed

    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

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

    PubMed

    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

  13. Identifying the Types of Ion Channel-Targeted Conotoxins by Incorporating New Properties of Residues into Pseudo Amino Acid Composition

    PubMed Central

    Wu, Yun

    2016-01-01

    Conotoxins are a kind of neurotoxin which can specifically interact with potassium, sodium type, and calcium channels. They have become potential drug candidates to treat diseases such as chronic pain, epilepsy, and cardiovascular diseases. Thus, correctly identifying the types of ion channel-targeted conotoxins will provide important clue to understand their function and find potential drugs. Based on this consideration, we developed a new computational method to rapidly and accurately predict the types of ion-targeted conotoxins. Three kinds of new properties of residues were proposed to use in pseudo amino acid composition to formulate conotoxins samples. The support vector machine was utilized as classifier. A feature selection technique based on F-score was used to optimize features. Jackknife cross-validated results showed that the overall accuracy of 94.6% was achieved, which is higher than other published results, demonstrating that the proposed method is superior to published methods. Hence the current method may play a complementary role to other existing methods for recognizing the types of ion-target conotoxins.

  14. The apparent absence of involvement of biotin in the vitamin K-dependent carboxylation of glutamic acid residues of proteins.

    PubMed Central

    Friedman, P A; Shia, M A

    1977-01-01

    The mechanism of the vitamin K-dependent post-translational carboxylation of the gamma-carbon atom of glutamic acid residues in proteins remains obscure. Experiments were performed in vivo and in vitro in an attempt to establish a role for biotin in the transfer of the carboxyl group. Weanling male rats were fed on a biotin-deficient diet until severe biotin deficiency was induced. Their degree of biotin deficiency was documented by assaying for liver acetyl-CoA carboxylase activity, which was about 15% of normal. However, one-stage and two-stage prothrombin times measured on the plasmas were normal. In addition, the liver microsomal fraction did not contain any more prothrombin precursor than did that of normal rat liver. Experiments were done in vitro in which vitamin K-dependent fixing of 14CO2 was measured in the liver microsomal fraction from vitamin K-deficient male rats in the presence or absence of avidin. No evidence for an avidin-sensitive critical biotin-containing site was obtained. Thus neither series of experiments suggests a role for biotin; the data are compatible with carboxyl transfer occurring either through a carboxylated vitamin K intermediate; or via a yet to be identified intermediate, or perhaps via CO2 itself. PMID:17395

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

    PubMed

    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

  16. Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules

    SciTech Connect

    Su, Xiaoyun; Agarwal, Vinayak; Dodd, Dylan; Bae, Brian; Mackie, Roderick I.; Nair, Satish K.; Cann, Isaac K.O.

    2010-11-22

    Carbohydrate binding modules (CBMs) are specialized proteins that bind to polysaccharides and oligosaccharides. Caldanaerobius polysaccharolyticus Man5ACBM16-1/CBM16-2 bind to glucose-, mannose-, and glucose/mannose-configured substrates. The crystal structures of the two proteins represent the only examples in CBM family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking. In this study, we probed the roles of amino acids (selected based on CBM16-1/ligand co-crystal structures) on substrate binding. Two tryptophan (Trp-20 and Trp-125) and two glutamine (Gln-81 and Gln-93) residues are shown to be critical in ligand binding. Additionally, several polar residues that flank the critical residues also contribute to ligand binding. The CBM16-1 Q121E mutation increased affinity for all substrates tested, whereas the Q21G and N97R mutants exhibited decreased substrate affinity. We solved CBM/substrate co-crystal structures to elucidate the molecular basis of the increased substrate binding by CBM16-1 Q121E. The Gln-121, Gln-21, and Asn-97 residues can be manipulated to fine-tune ligand binding by the Man5A CBMs. Surprisingly, none of the eight residues investigated was absolutely conserved in CBM family 16. Thus, the critical residues in the Man5A CBMs are either not essential for substrate binding in the other members of this family or the two CBMs are evolutionarily distinct from the members available in the current protein database. Man5A is dependent on its CBMs for robust activity, and insights from this study should serve to enhance our understanding of the interdependence of its catalytic and substrate binding modules.

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

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

    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

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

    PubMed

    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

  20. Ligand binding site of tear lipocalin: contribution of a trigonal cluster of charged residues probed by 8-anilino-1-naphthalenesulfonic acid.

    PubMed

    Gasymov, Oktay K; Abduragimov, Adil R; Glasgow, Ben J

    2008-02-01

    Human tear lipocalin (TL) exhibits diverse functions, most of which are linked to ligand binding. To map the binding site of TL for some amphiphilic ligands, we capitalized on the hydrophobic and hydrophilic properties of 8-anilino-1-naphthalenesulfonic acid (ANS). In single Trp mutants, resonance energy transfer from Trp to ANS indicates that the naphthalene group of ANS is proximate to Leu105 in the cavity. Binding energies of TL to ANS and its analogues reveal contributions from electrostatic interactions. The sulfonate group of ANS interacts strongly with the nonconserved intracavitary residue Lys114 and less with neighboring residues His84 and Glu34. This trigonal cluster of residues may play a role in the ligand recognition site for some negatively charged ligands. Because many drugs possess sulfonate groups, the trigonal cluster-sulfonate interaction can also be exploited as a lipocalin-based drug delivery mechanism. The binding of lauric acid and its analogues shows that fatty acids assume heterogeneous orientations in the cavity of TL. Predominantly, the hydrocarbon tail is buried in the cavity of TL and the carboxyl group is oriented toward the mouth. However, TL can also interact, albeit relatively weakly, with fatty acids oriented in the opposite direction. As the major lipid binding protein of tears, the ability to accommodate fatty acids in two opposing orientations may have functional implications for TL. At the aqueous-lipid interface, fatty acids whose carboxyl groups are positioned toward the aqueous phase are available for interaction with TL that could augment stability of the tear film. PMID:18179255

  1. Deletion of the last five C-terminal amino acid residues of connexin43 leads to lethal ventricular arrhythmias in mice without affecting coupling via gap junction channels.

    PubMed

    Lübkemeier, Indra; Requardt, Robert Pascal; Lin, Xianming; Sasse, Philipp; Andrié, René; Schrickel, Jan Wilko; Chkourko, Halina; Bukauskas, Feliksas F; Kim, Jung-Sun; Frank, Marina; Malan, Daniela; Zhang, Jiong; Wirth, Angela; Dobrowolski, Radoslaw; Mohler, Peter J; Offermanns, Stefan; Fleischmann, Bernd K; Delmar, Mario; Willecke, Klaus

    2013-05-01

    The cardiac intercalated disc harbors mechanical and electrical junctions as well as ion channel complexes mediating propagation of electrical impulses. Cardiac connexin43 (Cx43) co-localizes and interacts with several of the proteins located at intercalated discs in the ventricular myocardium. We have generated conditional Cx43D378stop mice lacking the last five C-terminal amino acid residues, representing a binding motif for zonula occludens protein-1 (ZO-1), and investigated the functional consequences of this mutation on cardiac physiology and morphology. Newborn and adult homozygous Cx43D378stop mice displayed markedly impaired and heterogeneous cardiac electrical activation properties and died from severe ventricular arrhythmias. Cx43 and ZO-1 were co-localized at intercalated discs in Cx43D378stop hearts, and the Cx43D378stop gap junction channels showed normal coupling properties. Patch clamp analyses of isolated adult Cx43D378stop cardiomyocytes revealed a significant decrease in sodium and potassium current densities. Furthermore, we also observed a significant loss of Nav1.5 protein from intercalated discs in Cx43D378stop hearts. The phenotypic lethality of the Cx43D378stop mutation was very similar to the one previously reported for adult Cx43 deficient (Cx43KO) mice. Yet, in contrast to Cx43KO mice, the Cx43 gap junction channel was still functional in the Cx43D378stop mutant. We conclude that the lethality of Cx43D378stop mice is independent of the loss of gap junctional intercellular communication, but most likely results from impaired cardiac sodium and potassium currents. The Cx43D378stop mice reveal for the first time that Cx43 dependent arrhythmias can develop by mechanisms other than impairment of gap junction channel function. PMID:23558439

  2. Deletion of the last five C-terminal amino acid residues of connexin43 leads to lethal ventricular arrhythmias in mice without affecting coupling via gap junction channels

    PubMed Central

    Lübkemeier, Indra; Requardt, Robert Pascal; Lin, Xianming; Sasse, Philipp; Andrié, René; Schrickel, Jan Wilko; Chkourko, Halina; Bukauskas, Feliksas F.; Kim, Jung-Sun; Frank, Marina; Malan, Daniela; Zhang, Jiong; Wirth, Angela; Dobrowolski, Radoslaw; Mohler, Peter J.; Offermanns, Stefan; Fleischmann, Bernd K.; Delmar, Mario

    2013-01-01

    The cardiac intercalated disc harbors mechanical and electrical junctions as well as ion channel complexes mediating propagation of electrical impulses. Cardiac connexin43 (Cx43) co-localizes and interacts with several of the proteins located at intercalated discs in the ventricular myocardium. We have generated conditional Cx43D378stop mice lacking the last five C-terminal amino acid residues, representing a binding motif for zonula occludens protein-1 (ZO-1), and investigated the functional consequences of this mutation on cardiac physiology and morphology. Newborn and adult homozygous Cx43D378stop mice displayed markedly impaired and heterogeneous cardiac electrical activation properties and died from severe ventricular arrhythmias. Cx43 and ZO-1 were co-localized at intercalated discs in Cx43D378stop hearts, and the Cx43D378stop gap junction channels showed normal coupling properties. Patch clamp analyses of isolated adult Cx43D378stop cardiomyocytes revealed a significant decrease in sodium and potassium current densities. Furthermore, we also observed a significant loss of Nav1.5 protein from intercalated discs in Cx43D378stop hearts. The phenotypic lethality of the Cx43D378stop mutation was very similar to the one previously reported for adult Cx43 deficient (Cx43KO) mice. Yet, in contrast to Cx43KO mice, the Cx43 gap junction channel was still functional in the Cx43D378stop mutant. We conclude that the lethality of Cx43D378stop mice is independent of the loss of gap junctional intercellular communication, but most likely results from impaired cardiac sodium and potassium currents. The Cx43D378stop mice reveal for the first time that Cx43 dependent arrhythmias can develop by mechanisms other than impairment of gap junction channel function. PMID:23558439

  3. 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.

    PubMed

    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

  4. Examination of acylated 4-aminopiperidine-4-carboxylic acid residues in the phosphotyrosyl+1 position of Grb2 SH2 domain-binding tripeptides.

    PubMed

    Kang, Sang-Uk; Choi, Won Jun; Oishi, Shinya; Lee, Kyeong; Karki, Rajeshri G; Worthy, Karen M; Bindu, Lakshman K; Nicklaus, Marc C; Fisher, Robert J; Burke, Terrence R

    2007-04-19

    A 4-aminopiperidine-4-carboxylic acid residue was placed in the pTyr+1 position of a Grb2 SH2 domain-binding peptide to form a general platform, which was then acylated with a variety of groups to yield a library of compounds designed to explore potential binding interactions, with protein features lying below the betaD strand. The highest affinities were obtained using phenylethyl carbamate and phenylbutyrylamide functionalities. PMID:17371004

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

    PubMed

    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

  6. Simultaneous screening analysis of 3-methyl-quinoxaline-2-carboxylic acid and quinoxaline-2-carboxylic acid residues in edible animal tissues by a competitive indirect immunoassay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Immunoassays contribute greatly to veterinary drug residue analysis and food safety, but there are no reported immunoassays on simultaneously detecting MQCA and QCA, the marker residues for carbadox and olaquindox. It is extremely difficult to produce broad-specificity antibodies that bind both res...

  7. Efficacy of citric acid denture cleanser on the Candida albicans biofilm formed on poly(methyl methacrylate): effects on residual biofilm and recolonization process

    PubMed Central

    2014-01-01

    Background It is well known that the use of denture cleansers can reduce Candida albicans biofilm accumulation; however, the efficacy of citric acid denture cleansers is uncertain. In addition, the long-term efficacy of this denture cleanser is not well established, and their effect on residual biofilms is unknown. This in vitro study evaluated the efficacy of citric acid denture cleanser treatment on C. albicans biofilm recolonization on poly(methyl methacrylate) (PMMA) surface. Methods C. albicans biofilms were developed for 72 h on PMMA resin specimens (n = 168), which were randomly assigned to 1 of 3 cleansing treatments (CTs) overnight (8 h). CTs included purified water as a control (CTC) and two experimental groups that used either a 1:5 dilution of citric acid denture cleanser (CT5) or a 1:8 dilution of citric acid denture cleanser (CT8). Residual biofilms adhering to the specimens were collected and quantified at two time points: immediately after CTs (ICT) and after cleaning and residual biofilm recolonization (RT). Residual biofilms were analyzed by quantifying the viable cells (CFU/mL), and biofilm architecture was evaluated by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Denture cleanser treatments and evaluation periods were considered study factors. Data were analyzed using two-way ANOVA and Tukey’s Honestly Significant Difference (HSD) test (α = 0.05). Results Immediately after treatments, citric acid denture cleansing solutions (CT5 and CT8) reduced the number of viable cells as compared with the control (p < 0.01). However, after 48 h, both CT groups (CT5 and CT8) showed biofilm recolonization (p < 0.01). Residual biofilm recolonization was also detected by CLSM and SEM analysis, which revealed a higher biomass and average biofilm thickness for the CT8 group (p < 0.01). Conclusion Citric acid denture cleansers can reduce C. albicans biofilm accumulation and cell viability. However, this

  8. Role of Acidic Residues in Helices TH8–TH9 in Membrane Interactions of the Diphtheria Toxin T Domain

    PubMed Central

    Ghatak, Chiranjib; Rodnin, Mykola V.; Vargas-Uribe, Mauricio; McCluskey, Andrew J.; Flores-Canales, Jose C.; Kurnikova, Maria; Ladokhin, Alexey S.

    2015-01-01

    The pH-triggered membrane insertion of the diphtheria toxin translocation domain (T domain) results in transferring the catalytic domain into the cytosol, which is relevant to potential biomedical applications as a cargo-delivery system. Protonation of residues is suggested to play a key role in the process, and residues E349, D352 and E362 are of particular interest because of their location within the membrane insertion unit TH8–TH9. We have used various spectroscopic, computational and functional assays to characterize the properties of the T domain carrying the double mutation E349Q/D352N or the single mutation E362Q. Vesicle leakage measurements indicate that both mutants interact with the membrane under less acidic conditions than the wild-type. Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts. Fluorescence experiments show that removal of the charge in E362Q, and not in E349Q/D352N, is important for insertion of TH8–TH9. Both mutants adopt a final functional state upon further acidification. We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions. PMID:25875295

  9. D-Lactic acid production by Sporolactobacillus inulinus YBS1-5 with simultaneous utilization of cottonseed meal and corncob residue.

    PubMed

    Bai, Zhongzhong; Gao, Zhen; Sun, Junfei; Wu, Bin; He, Bingfang

    2016-05-01

    d-Lactic acid, is an important organic acid produced from agro-industrial wastes by Sporolactobacillus inulinus YBS1-5 was investigated to reduce the raw material cost of fermentation. The YBS1-5 strain could produce d-lactic acid by using cottonseed meal as the sole nitrogen source. For efficient utilization, the cottonseed meal was enzymatically hydrolyzed and simultaneously utilized during d-lactic acid fermentation. Corncob residues are rich in cellulose and can be enzymatically hydrolyzed without pretreatment. The hydrolysate of this lignocellulosic waste could be utilized by strain YBS1-5 as a carbon source for d-lactic acid production. Under optimal conditions, a high d-lactic acid concentration (107.2g/L) was obtained in 7-L fed-batch fermenter, with an average productivity of 1.19g/L/h and a yield of 0.85g/g glucose. The optical purity of d-lactic acid in the broth was 99.2%. This study presented a new approach for low-cost production of d-lactic acid for an industrial application. PMID:26897413

  10. Significance of amino acid variation at human immunodeficiency virus type 1 reverse transcriptase residue 210 for zidovudine susceptibility.

    PubMed Central

    Harrigan, P R; Kinghorn, I; Bloor, S; Kemp, S D; Nájera, I; Kohli, A; Larder, B A

    1996-01-01

    Amino acid variation at reverse transcriptase (RT) codon 210 (generally Leu-210 to Trp [L210W], TTG-->TGG) is occasionally detected after the initiation of azidothymidine (AZT) therapy. The impact of this variation on AZT resistance and viral replication was addressed by four different approaches. The frequency and genetic background of the L210W mutation in vivo were assessed by analyzing sera of AZT-naive and AZT-experienced patients by RT-PCR and DNA sequencing. The degree of AZT resistance (50% infective concentration [IC50]) of recombinant viruses constructed by using the RT of 21 clinical isolates was stratified by the presence or absence of the 210 mutation. The AZT IC50S of a panel of mutant viruses (with or without W-210) constructed by site-directed mutagenesis in an HXB2 background were assayed by using a HeLa CD4 plaque reduction assay. Finally, the effect of the 210 mutation on viral replication was assessed by replication competition of an AZT-resistant virus, RTMN (L-41/Y-215), and RTMN with the W-210 mutation in the presence and in the absence of AZT. In AZT-naive patients, tryptophan at RT residue 210 was rare. After AZT exposure, W-210 appeared in a minority of those patients, most commonly in association with L-41 and Y-215. The presence of W-210 increased the AZTIC50 by two- to fourfold, as determined by both the recombinant virus assay and site-directed mutagenesis. A significant replication advantage in favor of the wild-type L-210 over W-210 was observed, although the selection against the 210 mutant was two- to threefold lower when the viruses were grown in the presence of 5 microM AZT. In summary, the L210W mutation appears to be of marginal significance, conferring approximately two- to fourfold-reduced sensitivity to AZT compared with similar AZT-resistant genomes with L-210. The selection pressure against W-210 may account for the modest proportion of patients in which W-210 appears in vivo. PMID:8709214

  11. Conserved Amino Acid Residues of the NuoD Segment Important for Structure and Function of Escherichia coli NDH-1 (Complex I)

    PubMed Central

    2015-01-01

    The NuoD segment (homologue of mitochondrial 49 kDa subunit) of the proton-translocating NADH:quinone oxidoreductase (complex I/NDH-1) from Escherichia coli is in the hydrophilic domain and bears many highly conserved amino acid residues. The three-dimensional structural model of NDH-1 suggests that the NuoD segment, together with the neighboring subunits, constitutes a putative quinone binding cavity. We used the homologous DNA recombination technique to clarify the role of selected key amino acid residues of the NuoD segment. Among them, residues Tyr273 and His224 were considered candidates for having important interactions with the quinone headgroup. Mutant Y273F retained partial activity but lost sensitivity to capsaicin-40. Mutant H224R scarcely affected the activity, suggesting that this residue may not be essential. His224 is located in a loop near the N-terminus of the NuoD segment (Gly217–Phe227) which is considered to form part of the quinone binding cavity. In contrast to the His224 mutation, mutants G217V, P218A, and G225V almost completely lost the activity. One region of this loop is positioned close to a cytosolic loop of the NuoA subunit in the membrane domain, and together they seem to be important in keeping the quinone binding cavity intact. The structural role of the longest helix in the NuoD segment located behind the quinone binding cavity was also investigated. Possible roles of other highly conserved residues of the NuoD segment are discussed. PMID:25545070

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

    PubMed

    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

  13. Predicting the mobility of Zn, Fe, Cu, Pb, Cd from roasted sulfide (pyrite) residues -- A case study of wastes from the sulfuric acid industry in Sweden

    SciTech Connect

    Lin, Z.; Qvarfort, U.

    1996-12-31

    Leachates from roasted sulfide residues, which are the wastes from the production of sulfuric acid at Falun, Sweden, have low pH and high concentrations of Zn, Fe, and SO{sub 4}. The minerals are mainly hematite and maghemite and, because the various sulfides in the feed behave differently during the roasting process, the residual sulfides minerals are pyrrhotite and sphalerite. Oxidation of the residual sulfides contributes acidity, Zn, Fe, Cu, Cd, and sulfate to the effluents from the waste deposits. The dissolution of sphalerite is most likely accelerated in acid solution rich in Fe(III). The formation of Pb-sulfate coatings on galena may provide an armoring effect which slows the oxidation of the galena. Residual sulfides are source phases controlling long-term contaminant release. Other source minerals for Zn, Fe, Pb, Cu, Cd and SO{sub 4} in the effluents are iron oxides which retained percentage quantities of SO{sub 4}, roast-derived alteration rims of Zn oxides on sphalerite, altered silicates formed during the roasting process, and secondary minerals (e.g., Zn, Fe, Cu sulfates, iron hydroxides) that were precipitated by in-site oxidation in the waste dumps. The Zn, Fe, and Cu sulfates most likely control short-term changes in the chemistry of the leachate, while Pb concentration in the leachates may be controlled predominantly by Pb-release from the altered silicates. The mineralogical and geochemical data provide fundamental information essential for the remedial management of this type of industrial waste.

  14. Lysine residues direct the chlorination of tyrosines in YXXK motifs of apolipoprotein A-I when hypochlorous acid oxidizes high density lipoprotein.

    PubMed

    Bergt, Constanze; Fu, Xiaoyun; Huq, Nabiha P; Kao, Jeff; Heinecke, Jay W

    2004-02-27

    Oxidized lipoproteins may play an important role in the pathogenesis of atherosclerosis. Elevated levels of 3-chlorotyrosine, a specific end product of the reaction between hypochlorous acid (HOCl) and tyrosine residues of proteins, have been detected in atherosclerotic tissue. Thus, HOCl generated by the phagocyte enzyme myeloperoxidase represents one pathway for protein oxidation in humans. One important target of the myeloperoxidase pathway may be high density lipoprotein (HDL), which mobilizes cholesterol from artery wall cells. To determine whether activated phagocytes preferentially chlorinate specific sites in HDL, we used tandem mass spectrometry (MS/MS) to analyze apolipoprotein A-I that had been oxidized by HOCl. The major site of chlorination was a single tyrosine residue located in one of the protein's YXXK motifs (where X represents a nonreactive amino acid). To investigate the mechanism of chlorination, we exposed synthetic peptides to HOCl. The peptides encompassed the amino acid sequences YKXXY, YXXKY, or YXXXY. MS/MS analysis demonstrated that chlorination of tyrosine in the peptides that contained lysine was regioselective and occurred in high yield if the substrate was KXXY or YXXK. NMR and MS analyses revealed that the N(epsilon) amino group of lysine was initially chlorinated, which suggests that chloramine formation is the first step in tyrosine chlorination. Molecular modeling of the YXXK motif in apolipoprotein A-I demonstrated that these tyrosine and lysine residues are adjacent on the same face of an amphipathic alpha-helix. Our observations suggest that HOCl selectively targets tyrosine residues that are suitably juxtaposed to primary amino groups in proteins. This mechanism might enable phagocytes to efficiently damage proteins when they destroy microbial proteins during infection or damage host tissue during inflammation. PMID:14660678

  15. Effect of pH on the coagulation performance of Al-based coagulants and residual aluminum speciation during the treatment of humic acid-kaolin synthetic water.

    PubMed

    Yang, Zhong Lian; Gao, Bao Yu; Yue, Qin Yan; Wang, Yan

    2010-06-15

    The fractionation and measurement of residual aluminum was conducted during the treatment of humic (HA)-kaolin synthetic water with Al(2)(SO(4))(3), AlCl(3) and polyaluminum chloride (PAC) in order to investigate the effect of pH on the coagulation performance as well as residual aluminum speciation. Experimental results suggested that turbidity removal performance varied according to the following order: AlCl(3)>PAC>Al(2)(SO(4))(3). HA removal performance of PAC was better than that of AlCl(3) under acidic condition. The optimum pH range for AlCl(3) and Al(2)(SO(4))(3) was between 6.0 and 7.0 while PAC showed stable HA and UV(254) removal capacity with broader pH variation (5.0-8.0). For the three coagulants, majority of residual aluminum existed in the form of total dissolved Al (60-80%), which existed mostly in oligomers or complexes formed between Al and natural organic matter or polymeric colloidal materials. PAC exhibited the least concentration for each kind of residual aluminum species as well as their percentage in total residual aluminum, followed by AlCl(3) and Al(2)(SO(4))(3) (in increasing order). Moreover, PAC could effectively reduce the concentration of dissolved monomeric Al and its residual aluminum ratio was the least among the three coagulants and varied little at an initial pH between 7.0 and 9.0. PMID:20188465

  16. Non-equivalence of Key Positively Charged Residues of the Free Fatty Acid 2 Receptor in the Recognition and Function of Agonist Versus Antagonist Ligands*

    PubMed Central

    Sergeev, Eugenia; Hansen, Anders Højgaard; Pandey, Sunil K.; MacKenzie, Amanda E.; Hudson, Brian D.; Ulven, Trond; Milligan, Graeme

    2016-01-01

    Short chain fatty acids (SCFAs) are produced in the gut by bacterial fermentation of poorly digested carbohydrates. A key mediator of their actions is the G protein-coupled free fatty acid 2 (FFA2) receptor, and this has been suggested as a therapeutic target for the treatment of both metabolic and inflammatory diseases. However, a lack of understanding of the molecular determinants dictating how ligands bind to this receptor has hindered development. We have developed a novel radiolabeled FFA2 antagonist to probe ligand binding to FFA2, and in combination with mutagenesis and molecular modeling studies, we define how agonist and antagonist ligands interact with the receptor. Although both agonist and antagonist ligands contain negatively charged carboxylates that interact with two key positively charged arginine residues in transmembrane domains V and VII of FFA2, there are clear differences in how these interactions occur. Specifically, although agonists require interaction with both arginine residues to bind the receptor, antagonists require an interaction with only one of the two. Moreover, different chemical series of antagonist interact preferentially with different arginine residues. A homology model capable of rationalizing these observations was developed and provides a tool that will be invaluable for identifying improved FFA2 agonists and antagonists to further define function and therapeutic opportunities of this receptor. PMID:26518871

  17. Non-equivalence of Key Positively Charged Residues of the Free Fatty Acid 2 Receptor in the Recognition and Function of Agonist Versus Antagonist Ligands.

    PubMed

    Sergeev, Eugenia; Hansen, Anders Højgaard; Pandey, Sunil K; MacKenzie, Amanda E; Hudson, Brian D; Ulven, Trond; Milligan, Graeme

    2016-01-01

    Short chain fatty acids (SCFAs) are produced in the gut by bacterial fermentation of poorly digested carbohydrates. A key mediator of their actions is the G protein-coupled free fatty acid 2 (FFA2) receptor, and this has been suggested as a therapeutic target for the treatment of both metabolic and inflammatory diseases. However, a lack of understanding of the molecular determinants dictating how ligands bind to this receptor has hindered development. We have developed a novel radiolabeled FFA2 antagonist to probe ligand binding to FFA2, and in combination with mutagenesis and molecular modeling studies, we define how agonist and antagonist ligands interact with the receptor. Although both agonist and antagonist ligands contain negatively charged carboxylates that interact with two key positively charged arginine residues in transmembrane domains V and VII of FFA2, there are clear differences in how these interactions occur. Specifically, although agonists require interaction with both arginine residues to bind the receptor, antagonists require an interaction with only one of the two. Moreover, different chemical series of antagonist interact preferentially with different arginine residues. A homology model capable of rationalizing these observations was developed and provides a tool that will be invaluable for identifying improved FFA2 agonists and antagonists to further define function and therapeutic opportunities of this receptor. PMID:26518871

  18. Application of Ganghwa Mugwort in Combination with Ascorbic Acid for the Reduction of Residual Nitrite in Pork Sausage during Refrigerated Storage

    PubMed Central

    Hwang, Ko-Eun; Kim, Hyun-Wook; Song, Dong-Heon; Kim, Yong-Jae; Ham, Youn-Kyung; Lee, Choong-Hee; Choi, Yun-Sang; Kim, Cheon-Jei

    2014-01-01

    The application of ganghwa mugwort (GM), ascorbic acid (AC), and their combinations for reduction of residual nitrite contents was analyzed in pork sausages during storage of 28 d. Six treatments of pork sausages contained the following: Control (no antioxidant added), AC (0.05% AC), GM 0.1 (0.1% GM), GM 0.2 (0.2% GM), AC+GM 0.1 (0.05% AC + 0.1% GM) and AC+GM 0.2 (0.05% AC + 0.2% GM). Results showed that the mixture of 0.05% AC and 0.2% GM was most effective for reducing thiobarbituric acid reactive substances (TBARS) and residual nitrite contents than the control and GM added sausages alone (p<0.05). The color values of all treatments were significantly affected by adding GM (either alone or with AC). Additionally, the total color difference (ΔE) and hue angle (H°) values of treatments added with GM were higher than those of the control as the amount of GM increased (p<0.05). However, there were no significant differences in the pH values between the control and all treatments during the storage period (p>0.05). Our results showed possible applications of antioxidant combination, for preventing the lipid oxidation and decreasing the residual nitrite levels of meat products. PMID:26760936

  19. Exploring the structure of the 100 amino-acid residue long N-terminus of the plant antenna protein CP29.

    PubMed

    Shabestari, Maryam Hashemi; Wolfs, Cor J A M; Spruijt, Ruud B; van Amerongen, Herbert; Huber, Martina

    2014-03-18

    The structure of the unusually long (∼100 amino-acid residues) N-terminal domain of the light-harvesting protein CP29 of plants is not defined in the crystal structure of this membrane protein. We studied the N-terminus using two electron paramagnetic resonance (EPR) approaches: the rotational diffusion of spin labels at 55 residues with continuous-wave EPR, and three sets of distances with a pulsed EPR method. The N-terminus is relatively structured. Five regions that differ considerably in their dynamics are identified. Two regions have low rotational diffusion, one of which shows α-helical character suggesting contact with the protein surface. This immobile part is flanked by two highly dynamic, unstructured regions (loops) that cover residues 10-22 and 82-91. These loops may be important for the interaction with other light-harvesting proteins. The region around residue 4 also has low rotational diffusion, presumably because it attaches noncovalently to the protein. This section is close to a phosphorylation site (Thr-6) in related proteins, such as those encoded by the Lhcb4.2 gene. Phosphorylation might influence the interaction with other antenna complexes, thereby regulating the supramolecular organization in the thylakoid membrane. PMID:24655510

  20. Exploring the Structure of the 100 Amino-Acid Residue Long N-Terminus of the Plant Antenna Protein CP29

    PubMed Central

    Shabestari, Maryam Hashemi; Wolfs, Cor J.A.M.; Spruijt, Ruud B.; van Amerongen, Herbert; Huber, Martina

    2014-01-01

    The structure of the unusually long (∼100 amino-acid residues) N-terminal domain of the light-harvesting protein CP29 of plants is not defined in the crystal structure of this membrane protein. We studied the N-terminus using two electron paramagnetic resonance (EPR) approaches: the rotational diffusion of spin labels at 55 residues with continuous-wave EPR, and three sets of distances with a pulsed EPR method. The N-terminus is relatively structured. Five regions that differ considerably in their dynamics are identified. Two regions have low rotational diffusion, one of which shows α-helical character suggesting contact with the protein surface. This immobile part is flanked by two highly dynamic, unstructured regions (loops) that cover residues 10–22 and 82–91. These loops may be important for the interaction with other light-harvesting proteins. The region around residue 4 also has low rotational diffusion, presumably because it attaches noncovalently to the protein. This section is close to a phosphorylation site (Thr-6) in related proteins, such as those encoded by the Lhcb4.2 gene. Phosphorylation might influence the interaction with other antenna complexes, thereby regulating the supramolecular organization in the thylakoid membrane. PMID:24655510

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

    PubMed

    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

  2. Conversion of agroindustrial residues for high poly(γ-glutamic acid) production by Bacillus subtilis NX-2 via solid-state fermentation.

    PubMed

    Tang, Bao; Xu, Hong; Xu, Zongqi; Xu, Cen; Xu, Zheng; Lei, Peng; Qiu, Yibin; Liang, Jinfeng; Feng, Xiaohai

    2015-04-01

    Poly(γ-glutamic acid) (γ-PGA) production by Bacillus subtilis NX-2 was carried out through solid-state fermentation with dry mushroom residues (DMR) and monosodium glutamate production residues (MGPR; a substitute of glutamate) for the first time. Dry shiitake mushroom residue (DSMR) was found to be the most suitable solid substrate among these DMRs; the optimal DSMR-to-MGPR ratio was optimized as 12:8. To increase γ-PGA production, industrial waste glycerol was added as a carbon source supplement to the solid-state medium. As a result, γ-PGA production increased by 34.8%. The batch fermentation obtained an outcome of 115.6 g kg(-1) γ-PGA and 39.5×10(8) colony forming units g(-1) cells. Furthermore, a satisfactory yield of 107.7 g kg(-1) γ-PGA was achieved by compost experiment on a scale of 50 kg in open air, indicating that economically large-scale γ-PGA production was feasible. Therefore, this study provided a novel method to produce γ-PGA from abundant and low-cost agroindustrial residues. PMID:25670398

  3. Full-Quantum chemical calculation of the absorption maximum of bacteriorhodopsin: a comprehensive analysis of the amino acid residues contributing to the opsin shift

    PubMed Central

    Hayashi, Tomohiko; Matsuura, Azuma; Sato, Hiroyuki; Sakurai, Minoru

    2012-01-01

    Herein, the absorption maximum of bacteriorhodopsin (bR) is calculated using our recently developed method in which the whole protein can be treated quantum mechanically at the level of INDO/S-CIS//ONIOM (B3LYP/6-31G(d,p): AMBER). The full quantum mechanical calculation is shown to reproduce the so-called opsin shift of bR with an error of less than 0.04 eV. We also apply the same calculation for 226 different bR mutants, each of which was constructed by replacing any one of the amino acid residues of the wild-type bR with Gly. This substitution makes it possible to elucidate the extent to which each amino acid contributes to the opsin shift and to estimate the inter-residue synergistic effect. It was found that one of the most important contributions to the opsin shift is the electron transfer from Tyr185 to the chromophore upon excitation. We also indicate that some aromatic (Trp86, Trp182) and polar (Ser141, Thr142) residues, located in the vicinity of the retinal polyene chain and the β-ionone ring, respectively, play an important role in compensating for the large blue-shift induced by both the counterion residues (Asp85, Asp212) and an internal water molecule (W402) located near the Schiff base linkage. In particular, the effect of Trp86 is comparable to that of Tyr185. In addition, Ser141 and Thr142 were found to contribute to an increase in the dipole moment of bR in the excited state. Finally, we provide a complete energy diagram for the opsin shift together with the contribution of the chromophore-protein steric interaction. PMID:27493528

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

    PubMed

    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

  5. Amino acid residues that control pH modulation of transport-associated current in mammalian serotonin transporters.

    PubMed

    Cao, Y; Li, M; Mager, S; Lester, H A

    1998-10-01

    The rat and human serotonin transporters (rSERT and hSERT, respectively) were expressed in Xenopus oocytes and studied using site-directed mutagenesis, electrophysiological recordings, and [3H]5-HT uptake measurements. rSERT, but not hSERT, displayed increased transport-associated current at low pH. Chimeras and point mutations showed that, of the 52 nonidentical residues, a single residue at position 490 (threonine in rSERT and lysine in hSERT) governs this difference. Furthermore, potentiation required the glutamate residue at position 493. Cysteine substitution and alkylation experiments showed that residue 493 is extracellular. Cysteine at 493 increased, whereas aspartate decreased, the net charge movement per transported 5-HT molecule. The mutations at this region did not significantly affect other aspects of SERT function, including agonist-independent leakage current, voltage-dependent transient current, and H+ current. This region may therefore be part of an external gate required for rSERT function. The data and analyses show that, in the absence of detailed structural information, a gate-lumen-gate scheme is useful for interpreting results from mutations that alter functional properties of neurotransmitter transporters. PMID:9742144

  6. Mutational analysis defines the roles of conserved amino acid residues in the predicted catalytic pocket of the rRNA:m6A methyltransferase ErmC'.

    PubMed

    Maravić, Gordana; Feder, Marcin; Pongor, Sándor; Flögel, Mirna; Bujnicki, Janusz M

    2003-09-01

    Methyltransferases (MTases) from the Erm family catalyze S-adenosyl-L-methionine-dependent modification of a specific adenine residue in bacterial 23S rRNA, thereby conferring resistance to clinically important macrolide, lincosamide and streptogramin B antibiotics. Despite the available structural data and functional analyses on the level of the RNA substrate, still very little is known about the mechanism of rRNA:adenine-N(6) methylation. Only predictions regarding various aspects of this reaction have been made based on the analysis of the crystal structures of methyltransferase ErmC' (without the RNA) and their comparison with the crystallographic and biochemical data for better studied DNA:m(6)A MTases. To validate the structure-based predictions of presumably essential residues in the catalytic pocket of ErmC', we carried out the site-directed mutagenesis and studied the function of the mutants in vitro and in vivo. Our results indicate that the active site of rRNA:m(6)A MTases is much more tolerant to amino acid substitutions than the active site of DNA:m(6)A MTases. Only the Y104 residue implicated in stabilization of the target base was found to be indispensable. Remarkably, the N101 residue from the "catalytic" motif IV and two conserved residues that form the floor (F163) and one of the walls (N11) of the base-binding site are not essential for catalysis in ErmC'. This somewhat surprising result is discussed in the light of the available structural data and in the phylogenetic context of the Erm family. PMID:12946350

  7. Effect of charged amino acid side chain length on lateral cross-strand interactions between carboxylate- and guanidinium-containing residues in a β-hairpin.

    PubMed

    Kuo, Hsiou-Ting; Liu, Shing-Lung; Chiu, Wen-Chieh; Fang, Chun-Jen; Chang, Hsien-Chen; Wang, Wei-Ren; Yang, Po-An; Li, Jhe-Hao; Huang, Shing-Jong; Huang, Shou-Ling; Cheng, Richard P

    2015-05-01

    β-Sheet is one of the major protein secondary structures. Oppositely charged residues are frequently observed across neighboring strands in antiparallel sheets, suggesting the importance of cross-strand ion pairing interactions. The charged amino acids Asp, Glu, Arg, and Lys have different numbers of hydrophobic methylenes linking the charged functionality to the backbone. To investigate the effect of side chain length of guanidinium- and carboxylate-containing residues on lateral cross-strand ion pairing interactions at non-hydrogen-bonded positions, β-hairpin peptides containing Zbb-Agx (Zbb = Asp, Glu, Aad in increasing length; Agx = Agh, Arg, Agb, Agp in decreasing length) sequence patterns were studied by NMR methods. The fraction folded population and folding energy were derived from the chemical shift deviation data. Peptides with high fraction folded populations involved charged residue side chain lengths that supported high strand propensity. Double mutant cycle analysis was used to determine the interaction energy for the potential lateral ion pairs. Minimal interaction was observed between residues with short side chains, most likely due to the diffused positive charge on the guanidinium group, which weakened cross-strand electrostatic interactions with the carboxylate side chain. Only the Aad-Arg/Agh interactions with long side chains clearly exhibited stabilizing energetics, possibly relying on hydrophobics. A survey of a non-redundant protein structure database revealed that the statistical sheet pair propensity followed the trend Asp-Arg < Glu-Arg, implying the need for matching long side chains. This suggested the need for long side chains on both guanidinium-bearing and carboxylate-bearing residues to stabilize the β-hairpin motif. PMID:25646959

  8. Identification and Modulation of the Key Amino Acid Residue Responsible for the pH Sensitivity of Neoculin, a Taste-Modifying Protein

    PubMed Central

    Nakajima, Ken-ichiro; Yokoyama, Kanako; Koizumi, Taichi; Koizumi, Ayako; Asakura, Tomiko; Terada, Tohru; Masuda, Katsuyoshi; Ito, Keisuke; Shimizu-Ibuka, Akiko; Misaka, Takumi; Abe, Keiko

    2011-01-01

    Neoculin occurring in the tropical fruit of Curculigo latifolia is currently the only protein that possesses both a sweet taste and a taste-modifying activity of converting sourness into sweetness. Structurally, this protein is a heterodimer consisting of a neoculin acidic subunit (NAS) and a neoculin basic subunit (NBS). Recently, we found that a neoculin variant in which all five histidine residues are replaced with alanine elicits intense sweetness at both neutral and acidic pH but has no taste-modifying activity. To identify the critical histidine residue(s) responsible for this activity, we produced a series of His-to-Ala neoculin variants and evaluated their sweetness levels using cell-based calcium imaging and a human sensory test. Our results suggest that NBS His11 functions as a primary pH sensor for neoculin to elicit taste modification. Neoculin variants with substitutions other than His-to-Ala were further analyzed to clarify the role of the NBS position 11 in the taste-modifying activity. We found that the aromatic character of the amino acid side chain is necessary to elicit the pH-dependent sweetness. Interestingly, since the His-to-Tyr variant is a novel taste-modifying protein with alternative pH sensitivity, the position 11 in NBS can be critical to modulate the pH-dependent activity of neoculin. These findings are important for understanding the pH-sensitive functional changes in proteinaceous ligands in general and the interaction of taste receptor–taste substance in particular. PMID:21559382

  9. Active-Site Residues of Escherichia coli DNA Gyrase Required in Coupling ATP Hydrolysis to DNA Supercoiling and Amino Acid Substitutions Leading to Novobiocin Resistance

    PubMed Central

    Gross, Christian H.; Parsons, Jonathan D.; Grossman, Trudy H.; Charifson, Paul S.; Bellon, Steven; Jernee, James; Dwyer, Maureen; Chambers, Stephen P.; Markland, William; Botfield, Martyn; Raybuck, Scott A.

    2003-01-01

    DNA gyrase is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP · PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis. Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A2B2 gyrase enzyme. Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis. Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities. All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected. Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E. coli gyrB mutant, and the activities correlated well with the in vitro activities. We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay. Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E. coli. PMID:12604539

  10. 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.

    PubMed

    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

  11. A haemagglutinin in the tissue fluid of the Pacific oyster, Crassostrea gigas, with specificity for sialic acid residues in glycoproteins.

    PubMed

    Hardy, S W; Grant, P T; Fletcher, T C

    1977-06-15

    An agglutinin for human red cells has a specificity for sialic acid and a high affinity for bovine salivary glycoprotein. Digestion of the glycoprotein with Pronase or neuraminidas indicated that binding of sialic acid to receptors in the agglutinin is the first step in the mechanism of formation of a stable complex between ligand and receptor. PMID:891745

  12. RBscore&NBench: a high-level web server for nucleic acid binding residues prediction with a large-scale benchmarking database.

    PubMed

    Miao, Zhichao; Westhof, Eric

    2016-07-01

    RBscore&NBench combines a web server, RBscore and a database, NBench. RBscore predicts RNA-/DNA-binding residues in proteins and visualizes the prediction scores and features on protein structures. The scoring scheme of RBscore directly links feature values to nucleic acid binding probabilities and illustrates the nucleic acid binding energy funnel on the protein surface. To avoid dataset, binding site definition and assessment metric biases, we compared RBscore with 18 web servers and 3 stand-alone programs on 41 datasets, which demonstrated the high and stable accuracy of RBscore. A comprehensive comparison led us to develop a benchmark database named NBench. The web server is available on: http://ahsoka.u-strasbg.fr/rbscorenbench/. PMID:27084939

  13. Extraction and separation of nickel(II) using bis(2,4,4-trimethylpentyl) dithiophosphinic acid (Cyanex 301) and its recovery from spent catalyst and electroplating bath residue

    SciTech Connect

    Singh, R.; Khwaja, A.R.; Gupta, B.; Tandon, S.N.

    1999-03-01

    The paper embodies the details on the extraction behavior of Ni(II) along with Cr(III), Fe(III), Mn(II), Co(II), Cu(II) and Zn(II) from sulfuric acid media employing Cyanex 301-toluene system. The effect of various parameters like concentration of acid, metal ion and extractant and nature of diluent on the extraction of Ni(II) has been studied. On the basis of the distribution data the extracting species has been proposed. The recycling capacity of the extractant has been assessed. Some binary and ternary separations have also been achieved. The practical utility of the extractant has been demonstrated by recovering Ni(II) from spent catalyst and electroplating bath residue.

  14. Forensic applications of sodium rhodizonate and hydrochloric acid: a new histological technique for detection of gunshot residues.

    PubMed

    Andreola, Salvatore; Gentile, Guendalina; Battistini, Alessio; Cattaneo, Cristina; Zoja, Riccardo

    2011-05-01

    Demonstration of the presence of lead residues deriving from gunshot in skin and underlying tissues is essential for the correct forensic analysis of numerous legal cases. Optical microscopy remains the fastest, cheapest diagnostic technique, even though its sensitivity and specificity are poor because of the scarce quantity of histological tissue that can be examined and possible environmental lead pollution. To confirm the presence of lead from gunshot residues, we applied to histological sections of human skin a technique proposed by Owens and George in 1991 for macroscopic detection of lead on the clothing of shooting victims, involving a reaction with sodium rhodizonate and subsequent confirmation by color change on application of HCl. Our results demonstrate the technical possibility of using this macroscopic technique even on histological samples and support the need for further studies on a larger series of cases correlated with the type of ammunition and firing distance. PMID:21521219

  15. Comparative analysis of core amino acid residues of H-2D(b)-restricted cytotoxic T-lymphocyte recognition epitopes in simian virus 40 T antigen.

    PubMed Central

    Deckhut, A M; Lippolis, J D; Tevethia, S S

    1992-01-01

    Simian virus 40 (SV40) tumor (T) antigen expressed in H-2b SV40-transformed cells induces the generation of Lyt-2+ (CD8+) cytotoxic T lymphocytes (CTL), which are involved in tumor rejection, in syngeneic mice. Five CTL recognition sites on T antigen have been described by using mutant T antigens. Four of the sites (I, II, III, and V) are H-2Db restricted and have been broadly mapped with synthetic peptides of 15 amino acids in length overlapping by 5 residues at the amino and carboxy termini. The goal of this study was to define the minimal and optimal amino acid sequences of T antigen which would serve as recognition elements for the H-2Db-restricted CTL clones Y-1, Y-2, Y-3, and Y-5, which recognizes sites I, II, III, and V, respectively. The minimal and optimal residues of T antigen recognized by the four CTL clones were determined by using synthetic peptides truncated at the amino or carboxy terminus and an H-2Db peptide-binding motif. The minimal site recognized by CTL clone Y-1 was defined as amino acids 207 to 215 of SV40 T antigen. However, the optimal sequence recognized by CTL clone Y-1 spanned T-antigen amino acids 205 to 215. The T-antigen peptide sequence LT223-231 was the optimal and minimal sequence recognized by both CTL clones Y-2 and Y-3. Site V was determined to be contained within amino acids 489 to 497 of T antigen. The lytic activities of CTL clones Y-2 and Y-3, which recognize a single nonamer peptide, LT223-231, were affected differently by anti-Lyt-2 antibody, suggesting that the T-cell receptors of these two CTL clones differ in their avidities. As the minimal and optimal H-2Db-restricted CTL recognition sites have been defined by nonamer synthetic peptides, it is now possible to search for naturally processed H-2Db-restricted epitopes of T antigen and identify critical residues involved in processing, presentation, and recognition by SV40-specific CTL. PMID:1370091

  16. Conformational study reveals amino acid residues essential for hemagglutinating and anti-proliferative activities of Clematis montana lectin.

    PubMed

    Lu, Bangmin; Zhang, Bin; Qi, Wei; Zhu, Yanan; Zhao, Yan; Zhou, Nan; Sun, Rong; Bao, Jinku; Wu, Chuanfang

    2014-11-01

    Clematis montana lectin (CML), a novel mannose-binding lectin purified from C. montana Buch.-Ham stem (Ranunculaceae), has been proved to have hemagglutinating activity in rabbit erythrocytes and apoptosis-inducing activity in tumor cells. However, the biochemical properties of CML have not revealed and its structural information still needs to be elucidated. In this study, it was found that CML possessed quite good thermostability and alkaline resistance, and its hemagglutinating activity was bivalent metal cation dependent. In addition, hemagglutination test and fluorescence spectroscopy proved that GuHCl, urea, and sodium dodecyl sulfate could change the conformation of CML and further caused the loss of hemagglutination activity. Moreover, the changes of fluorescence spectrum indicated that the tryptophan (Trp) microenvironment conversion might be related to the conformation and bioactivities of CML. In addition, it was also found that Trp residues, arginine (Arg) residues, and sulfhydryl were important for the hemagglutinating activity of CML, but only Trp was proved to be crucial for the CML conformation. Furthermore, the Trp, Arg, and sulfhydryl-modified CML exhibited 97.17%, 76.99%, and 49.64% loss of its anti-proliferative activity, respectively, which was consistent with the alterations of its hemagglutinating activity. Given these findings, Trp residues on the surface of CML are essential for the active center to form substrate-accessible conformation and suitable environment for carbohydrate binding. PMID:25239139

  17. Amino acid residues in the GerAB protein important in the function and assembly of the alanine spore germination receptor of Bacillus subtilis 168.

    PubMed

    Cooper, Gareth R; Moir, Anne

    2011-05-01

    The paradigm gerA operon is required for endospore germination in response to c-alanine as the sole germinant, and the three protein products, GerAA, GerAB, and GerAC are predicted to form a receptor complex in the spore inner membrane. GerAB shows homology to the amino acid-polyamine-organocation (APC) family of single-component transporters and is predicted to be an integral membrane protein with 10 membrane-spanning helices. Site-directed mutations were introduced into the gerAB gene at its natural location on the chromosome. Alterations to some charged or potential helix-breaking residues within membrane spans affected receptor function dramatically. In some cases, this is likely to reflect the complete loss of the GerA receptor complex, as judged by the absence of the germinant receptor protein GerAC, which suggests that the altered GerAB protein itself may be unstable or that the altered structure destabilizes the complex. Mutants that have a null phenotype for Instituto de Biotecnología de León, INBIOTEC, Parque Científico de León, Av. Real, 1, 24006 León, Spain-alanine germination but retain GerAC protein at near-normal levels are more likely to define amino acid residues of functional, rather than structural, importance. Single-amino-acid substitutions in each of the GerAB and GerAA proteins can prevent incorporation of GerAC protein into the spore; this provides strong evidence that the proteins within a specific receptor interact and that these interactions are required for receptor assembly. The lipoprotein nature of the GerAC receptor subunit is also important; an amino acid change in the prelipoprotein signal sequence in the gerAC1 mutant results in the absence of GerAC protein from the spore. PMID:21378181

  18. Effect of second coagulant addition on coagulation efficiency, floc properties and residual Al for humic acid treatment by Al13 polymer and polyaluminum chloride (PACl).

    PubMed

    Xu, Weiying; Gao, Baoyu; Wang, Yan; Yue, Qinyan; Ren, Haijing

    2012-05-15

    Influence of second dose on coagulation efficiency, floc re-growth, fractal structure and residual Al of the effluent in humic acid (HA) coagulation with Al(13) polymer ([Al(13)O(4)(OH)(24)(H(2)O)(12)](7+)) and PACl were comparatively investigated in this study. Effects of breakage shear on the floc properties generated in the coagulation with and without additional dose were also investigated. The results indicated that additional dose during breakage could essentially improve the HA removal efficiency and floc re-growth in both Al(13) and PACl coagulations. Second doses of Al(13) at 0.5 and 1.0mg/L resulted in better turbidity and UV(254) removal as well as floc re-growth rather than higher additional dose of 1.5 and 2.0mg/L; while in PACl coagulation, more efficient HA removal and better floc re-growth were obtained at higher additional doses (1.0, 1.5 and 2.0mg/L). Small additional Al(13) could apparently increase the D(f) of re-formed flocs while the additional PACl displayed inconspicuous effect on floc D(f). The additional coagulant dose could alleviate the further decrease of re-grown floc size with increased breakage shear for both coagulants. The residual Al analysis implied that two-stage addition contributed to lower residual Al in effluent than one-time addition mode with the same total coagulant concentration. PMID:22410719

  19. Hydrogen peroxide and hypochlorous acid influx through the major S. Typhimurium porin OmpD is affected by substitution of key residues of the channel.

    PubMed

    Aguayo, Daniel; Pacheco, Nicolás; Morales, Eduardo H; Collao, Bernardo; Luraschi, Roberto; Cabezas, Carolina; Calderón, Paulina; González-Nilo, Fernando; Gil, Fernando; Calderón, Iván L; Saavedra, Claudia P

    2015-02-15

    OmpD is the major Salmonella enterica serovar Typhimurium (S. Typhimurium) porin and mediates hydrogen peroxide (H2O2) influx. The results described herein extend this finding to hypochlorous acid (HOCl), another reactive oxygen species that is also part of the oxidative burst generated by the phagosome. S. Typhimurium cells lacking OmpD show decreased HOCl influx, and OmpD-reconstituted proteoliposomes show an increase in the uptake of the toxic compound. To understand this physiologically relevant process, we investigated the role of key OmpD residues in H2O2 and NaOCl transport. Using a theoretical approach, residue K16 was defined as a major contributor to the channel electrostatic properties, and E111 was shown to directly participate in the size-exclusion limit of the channel. Together, we provide theoretical, genetic, and biochemical evidence that OmpD mediates H2O2 and NaOCl uptake, and that key residues of the channel are implicated in this process. PMID:25600570

  20. NPPD: A Protein-Protein Docking Scoring Function Based on Dyadic Differences in Networks of Hydrophobic and Hydrophilic Amino Acid Residues

    PubMed Central

    Shih, Edward S. C.; Hwang, Ming-Jing

    2015-01-01

    Protein-protein docking (PPD) predictions usually rely on the use of a scoring function to rank docking models generated by exhaustive sampling. To rank good models higher than bad ones, a large number of scoring functions have been developed and evaluated, but the methods used for the computation of PPD predictions remain largely unsatisfactory. Here, we report a network-based PPD scoring function, the NPPD, in which the network consists of two types of network nodes, one for hydrophobic and the other for hydrophilic amino acid residues, and the nodes are connected when the residues they represent are within a certain contact distance. We showed that network parameters that compute dyadic interactions and those that compute heterophilic interactions of the amino acid networks thus constructed allowed NPPD to perform well in a benchmark evaluation of 115 PPD scoring functions, most of which, unlike NPPD, are based on some sort of protein-protein interaction energy. We also showed that NPPD was highly complementary to these energy-based scoring functions, suggesting that the combined use of conventional scoring functions and NPPD might significantly improve the accuracy of current PPD predictions. PMID:25811640

  1. Acid decomposition and thiourea leaching of silver from hazardous jarosite residues: Effect of some cations on the stability of the thiourea system.

    PubMed

    Calla-Choque, D; Nava-Alonso, F; Fuentes-Aceituno, J C

    2016-11-01

    The recovery of silver from hazardous jarosite residues was studied employing thiourea as leaching agent at acid pH and 90°C. The stability of the thiourea in synthetic solutions was evaluated in the presence of some cations that can be present in this leaching system: cupric and ferric ions as oxidant species, and zinc, lead and iron as divalent ions. Two silver leaching methods were studied: the simultaneous jarosite decomposition-silver leaching, and the jarosite decomposition followed by the silver leaching. The study with synthetic solutions demonstrated that cupric and ferric ions have a negative effect on thiourea stability due to their oxidant properties. The effect of cupric ions is more significant than the effect of ferric ions; other studied cations (Fe(2+), Zn(2+), Pb(2+)) had no effect on the stability of thiourea. When the decomposition of jarosite and the silver leaching are carried out simultaneously, 70% of the silver can be recovered. When the acid decomposition was performed at pH 0.5 followed by the leaching step at pH 1, total silver recovery increased up to 90%. The zinc is completely dissolved with any of these processes while the lead is practically insoluble with these systems producing a lead-rich residue. PMID:27322901

  2. Multi-residue analysis of 26 organochlorine pesticides in Alpinia oxyphylla by GC-ECD after solid phase extraction and acid cleanup.

    PubMed

    Zhao, Xiangsheng; Zhou, Yakui; Kong, Weijun; Gong, Bao; Chen, Deli; Wei, Jianhe; Yang, Meihua

    2016-04-01

    A simple and effective multi-residue method was developed and validated for the analysis of 26 organochlorine pesticide residues in Alpinia oxyphylla by a gas chromatography with an electron capture detector (GC-ECD). The target pesticides were extracted by sonication and cleaned up with florisil solid phase extraction and sulphuric acid. Some crucial parameters, including extraction solvent and time, sorbent type, elute solvent and concentration of sulphuric acid were optimized to improve the performance of sample preparation procedure. The optimized method gave high sensitivity with detection limit ranging from 0.1 to 2.0μg/kg. Matrix-matched calibration was employed for the quantification, and a wide linear range (from 1.0 to 1000μg/kg) with r(2) values ranging from 0.9971 to 0.9998 was obtained. For the majority of the tested pesticides, the average recoveries were in acceptable range (between 70% and 110%) with relative standard deviation values below 15.0%. Matrix effect was evaluated for target compounds through the study of ratio of peak area obtained in the solvent and blank matrix. The proposed method was applied to simultaneously analyze 26 pesticides in 55 batches of Alpinia oxyphylla samples. 3 samples were found to be positive with four pesticides (α-BHC, quintozene, trans-chlordane and op'-DDD), which were confirmed by gas chromatography-mass spectrometry (GC-MS) in selective ion monitoring (SIM) mode. PMID:26990736

  3. A replacement of the active-site aspartic acid residue 293 in mouse cathepsin D affects its intracellular stability, processing and transport in HEK-293 cells.

    PubMed Central

    Partanen, Sanna; Storch, Stephan; Löffler, Hans-Gerhard; Hasilik, Andrej; Tyynelä, Jaana; Braulke, Thomas

    2003-01-01

    The substitution of an active-site aspartic acid residue by asparagine in the lysosomal protease cathepsin D (CTSD) results in a loss of enzyme activity and severe cerebrocortical atrophy in a novel form of neuronal ceroid lipofuscinosis in sheep [Tyynelä, Sohar, Sleat, Gin, Donnelly, Baumann, Haltia and Lobel (2000) EMBO J. 19, 2786-2792]. In the present study we have introduced the corresponding mutation by replacing aspartic acid residue 293 with asparagine (D293N) into the mouse CTSD cDNA to analyse its effect on synthesis, transport and stability in transfected HEK-293 cells. The complete inactivation of mutant D293N mouse CTSD was confirmed by a newly developed fluorimetric quantification system. Moreover, in the heterologous overexpression systems used, mutant D293N mouse CTSD was apparently unstable and proteolytically modified during early steps of the secretory pathway, resulting in a loss of mass by about 1 kDa. In the affected sheep, the endogenous mutant enzyme was stable but also showed the shift in its molecular mass. In HEK-293 cells, the transport of the mutant D293N mouse CTSD to the lysosome was delayed and associated with a low secretion rate compared with wild-type CTSD. These data suggest that the mutation may result in a conformational change which affects stability, processing and transport of the enzyme. PMID:12350228

  4. Enhanced stability of Cu(2+)-ATCUN complexes under physiologically relevant conditions by insertion of structurally bulky and hydrophobic amino acid residues into the ATCUN motif.

    PubMed

    Miyamoto, Takaaki; Fukino, Yuta; Kamino, Shinichiro; Ueda, Masashi; Enomoto, Shuichi

    2016-06-21

    Copper complexes formed by an amino terminal Cu(2+)- and Ni(2+)-binding (ATCUN) motif have attracted attention as metallodrug candidates that cleave DNA or RNA and inactivate enzymes. Although the stability of the Cu(2+)-ATCUN complex under physiologically relevant conditions is a key factor for medical applications, it has remained unclear. Here we prepared a series of ATCUN peptides by inserting various amino acid residues into positions 1 and 2, and investigated the stability of the Cu(2+)-ATCUN complexes in aqueous solution, blood plasma, and living animals. Systematic pH titration showed that the low basicity of the N-terminal amine of the peptide stabilized the Cu(2+)-ATCUN complex in aqueous solution. Interestingly, the stability of (64)Cu-labeled ATCUN complexes in blood plasma was significantly enhanced by the structural bulkiness and hydrophobicity of the amino acid residues at positions 1 and 2. To validate the in vivo stability, six ATCUN motifs (YYH, VVH, NNH, TTH, GGH, and DDH) were conjugated to a tumor-targeting peptide, octreotide (Oct). The stability of the (64)Cu-ATCUN-Oct complexes in blood plasma showed a similar trend to that of the (64)Cu-ATCUN complexes. The (64)Cu-YYH-Oct complex exhibited the highest stability in blood plasma. According to the positron emission tomography and competitive blocking studies of a tumor-bearing mouse model, (64)Cu-YYH-Oct specifically accumulated in tumors, suggesting that the complex was sufficiently stable to reach its target in vivo. The results show that the structural bulkiness and hydrophobicity of the residues at positions 1 and 2 are key parameters for designing metallodrugs on the basis of the Cu(2+)-ATCUN complex. PMID:27184978

  5. Usefulness of 18F-Fluorodeoxyglucose Positron Emission Tomography for Follow-Up of 13-cis-Retinoic Acid Treatment for Residual Neuroblastoma After Myeloablative Chemotherapy

    PubMed Central

    Sato, Yuya; Kurosawa, Hidemitsu; Sakamoto, Setsu; Kuwashima, Shigeko; Hashimoto, Teisuke; Okamoto, Kentaro; Tsuchioka, Takashi; Fukushima, Keitaro; Arisaka, Osamu

    2015-01-01

    Abstract 13-cis-retinoic acid (13-cis-RA) treatment is used as a second-line treatment for residual or recurrent neuroblastoma. However, determining the duration of 13-cis-RA treatment for residual and recurrent neuroblastoma can be a problem because it is difficult to evaluate the effectiveness of the treatment. We performed 13-cis-RA treatment to remove residual active neuroblastoma cells in an 8-year-old boy with stage 4 neuroblastoma that developed from a left sympathetic ganglion and had been treated with chemotherapy, surgery, autologous peripheral blood stem-cell transplantation, and radiotherapy. 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET) and iodine-123 metaiodobenzylguanidine (123I-MIBG) scintigraphy obtained immediately before 13-cis-RA treatment both showed positive findings in the area of the primary lesion. At 18 months after 13-cis-RA treatment, there was accumulation on 123I-MIBG scintigraphy but no uptake on 18F-FDG-PET, and 13-cis-RA treatment was suspended. The patient has been in complete remission for 3 years. In comparing the effectiveness of the 2 imaging modalities for monitoring the response to 13-cis-RA treatment, we considered that 18F-FDG-PET was superior to 123I-MIBG scintigraphy because 18F-FDG-PET images were not affected by the cell differentiation induced by 13-cis-RA treatment in our case. Thus, 18F-FDG-PET was useful for determining the treatment response and outcomes. We have reported a case of residual neuroblastoma treated with differentiation-inducing 13-cis-RA therapy. Different results were produced with 18F-FDG-PET and 123I-MIBG scintigraphy. The cessation of 13-cis-RA treatment was based on 18F-FDG-PET findings and there has been no relapse for 3 years. PMID:26252303

  6. Usefulness of 18F-Fluorodeoxyglucose Positron Emission Tomography for Follow-Up of 13-cis-Retinoic Acid Treatment for Residual Neuroblastoma After Myeloablative Chemotherapy.

    PubMed

    Sato, Yuya; Kurosawa, Hidemitsu; Sakamoto, Setsu; Kuwashima, Shigeko; Hashimoto, Teisuke; Okamoto, Kentaro; Tsuchioka, Takashi; Fukushima, Keitaro; Arisaka, Osamu

    2015-08-01

    13-cis-retinoic acid (13-cis-RA) treatment is used as a second-line treatment for residual or recurrent neuroblastoma. However, determining the duration of 13-cis-RA treatment for residual and recurrent neuroblastoma can be a problem because it is difficult to evaluate the effectiveness of the treatment.We performed 13-cis-RA treatment to remove residual active neuroblastoma cells in an 8-year-old boy with stage 4 neuroblastoma that developed from a left sympathetic ganglion and had been treated with chemotherapy, surgery, autologous peripheral blood stem-cell transplantation, and radiotherapy. F-fluorodeoxyglucose positron emission tomography (F-FDG-PET) and iodine-123 metaiodobenzylguanidine (I-MIBG) scintigraphy obtained immediately before 13-cis-RA treatment both showed positive findings in the area of the primary lesion. At 18 months after 13-cis-RA treatment, there was accumulation on I-MIBG scintigraphy but no uptake on F-FDG-PET, and 13-cis-RA treatment was suspended. The patient has been in complete remission for 3 years. In comparing the effectiveness of the 2 imaging modalities for monitoring the response to 13-cis-RA treatment, we considered that F-FDG-PET was superior to I-MIBG scintigraphy because F-FDG-PET images were not affected by the cell differentiation induced by 13-cis-RA treatment in our case. Thus, F-FDG-PET was useful for determining the treatment response and outcomes.We have reported a case of residual neuroblastoma treated with differentiation-inducing 13-cis-RA therapy. Different results were produced with F-FDG-PET and I-MIBG scintigraphy. The cessation of 13-cis-RA treatment was based on F-FDG-PET findings and there has been no relapse for 3 years. PMID:26252303

  7. Production of bio-oil rich in acetic acid and phenol from fast pyrolysis of palm residues using a fluidized bed reactor: Influence of activated carbons.

    PubMed

    Jeong, Jae-Yong; Lee, Uen-Do; Chang, Won-Seok; Jeong, Soo-Hwa

    2016-11-01

    In this study, palm residues were pyrolyzed in a bench-scale (3kg/h) fast pyrolysis plant equipped with a fluidized bed reactor and bio-oil separation system for the production of bio-oil rich in acetic acid and phenol. Pyrolysis experiments were performed to investigate the effects of reaction temperature and the types and amounts of activated carbon on the bio-oil composition. The maximum bio-oil yield obtained was approximately 47wt% at a reaction temperature of 515°C. The main compounds produced from the bio-oils were acetic acid, hydroxyacetone, phenol, and phenolic compounds such as cresol, xylenol, and pyrocatechol. When coal-derived activated carbon was applied, the acetic acid and phenol yields in the bio-oils reached 21 and 19wt%, respectively. Finally, bio-oils rich in acetic acid and phenol could be produced separately by using an in situ bio-oil separation system and activated carbon as an additive. PMID:27501032

  8. Gas chromatographic mass spectrometric detection of dihydroxy fatty acids preserved in the 'bound' phase of organic residues of archaeological pottery vessels.

    PubMed

    Hansel, Fabricio A; Bull, Ian D; Evershed, Richard P

    2011-07-15

    A methodology is presented for the determination of dihydroxy fatty acids preserved in the 'bound' phase of organic residues preserved in archaeological potsherds. The method comprises saponification, esterification, silica gel column chromatographic fractionation, and analysis by gas chromatography/mass spectrometry. The electron ionisation mass spectra of the trimethylsilyl ether methyl ester derivatives are characterised by fragment ions arising from cleavage of the bond between the two vicinal trimethylsiloxy groups. Other significant fragment ions are [M-15](+.), [M-31](+.), m/z 147 and ions characteristic of vicinal disubstituted (trimethylsiloxy) TMSO- groups (Δ(7,8), Δ(9,10), Δ(11,12) and Δ(13,14): m/z 304, 332, 360 and 388, respectively). The dihydroxy fatty acids identified in archaeological extracts exhibited carbon numbers ranging from C(16) to C(22) and concentrations varying from 0.05 to 14.05 µg g(-1) . The wide range of dihydroxy fatty acids observed indicates that this approach may be applied confidently in screening archaeological potsherds for the degradation products of monounsaturated fatty acids derived from commodities processed in archaeological pottery vessels. PMID:21638365

  9. The crystal structure of a xyloglucan-specific endo-beta-1,4-glucanase from Geotrichum sp. M128 xyloglucanase reveals a key amino acid residue for substrate specificity.

    PubMed

    Yaoi, Katsuro; Kondo, Hidemasa; Hiyoshi, Ayako; Noro, Natsuko; Sugimoto, Hiroshi; Tsuda, Sakae; Miyazaki, Kentaro

    2009-09-01

    Geotrichum sp. M128 possesses two xyloglucan-specific glycoside hydrolases belonging to family 74, xyloglucan-specific endo-beta-1,4-glucanase (XEG) and oligoxyloglucan reducing-end-specific cellobiohydrolase (OXG-RCBH). Despite their similar amino acid sequences (48% identity), their modes of action and substrate specificities are distinct. XEG catalyzes the hydrolysis of xyloglucan polysaccharides in endo mode, while OXG-RCBH acts on xyloglucan oligosaccharides at the reducing end in exo mode. Here, we determined the crystal structure of XEG at 2.5 A resolution, and compared it to a previously determined structure of OXG-RCBH. For the most part, the amino acid residues that interact with substrate are conserved between the two enzymes. However, there are notable differences at subsite positions -1 and +2. OXG-RCBH has a loop around the +2 site that blocks one end of the active site cleft, which accounts for its exo mode of action. In contrast, XEG lacks a corresponding loop at this site, thereby allowing binding to the middle of the main chain of the substrate. At the -1 site in OXG-RCBH, Asn488 interacts with the xylose side chain of the substrate, whereas the -1 site is occupied by Tyr457 in XEG. To confirm the contribution of this residue to substrate specificity, Tyr457 was substituted by Gly in XEG. The wild-type XEG cleaved the oligoxyloglucan at a specific site; the Y457G variant cleaved the same substrate, but at various sites. Together, the absence of a loop in the cleft and the presence of bulky Tyr457 determine the substrate specificity of XEG. PMID:19682300

  10. Site-directed mutagenesis of amino acid residues of D1 protein interacting with phosphatidylglycerol affects the function of plastoquinone QB in photosystem II.

    PubMed

    Endo, Kaichiro; Mizusawa, Naoki; Shen, Jian-Ren; Yamada, Masato; Tomo, Tatsuya; Komatsu, Hirohisa; Kobayashi, Masami; Kobayashi, Koichi; Wada, Hajime

    2015-12-01

    Recent X-ray crystallographic analysis of photosystem (PS) II at 1.9-Å resolution identified 20 lipid molecules in the complex, five of which are phosphatidylglycerol (PG). In this study, we mutagenized amino acid residues S232 and N234 of D1, which interact with two of the PG molecules (PG664 and PG694), by site-directed mutagenesis in Synechocystis sp. PCC 6803 to investigate the role of the interaction in PSII. The serine and asparagine residues at positions 232 and 234 from the N-terminus were mutagenized to alanine and aspartic acid, respectively, and a mutant carrying both amino acid substitutions was also produced. Although the obtained mutants, S232A, N234D, and S232AN234D, exhibited normal growth, they showed decreased photosynthetic activities and slower electron transport from QA to QB than the control strain. Thermoluminescence analysis suggested that this slower electron transfer in the mutants was caused by more negative redox potential of QB, but not in those of QA and S2. In addition, the levels of extrinsic proteins, PsbV and PsbU, were decreased in PSII monomer purified from the S232AN234D mutant, while that of Psb28 was increased. In the S232AN234D mutant, the content of PG in PSII was slightly decreased, whereas that of monogalactosyldiacylglycerol was increased compared with the control strain. These results suggest that the interactions of S232 and N234 with PG664 and PG694 are important to maintain the function of QB and to stabilize the binding of extrinsic proteins to PSII. PMID:25921208

  11. Effect of adding amino acids residues in N- and C-terminus of Vip3Aa16 (L121I) toxin.

    PubMed

    Sellami, Sameh; Cherif, Marwa; Jamoussi, Kaïs

    2016-06-01

    To study the importance of N- and C-terminus of Bacillus thuringiensis Vip3Aa16 (L121I) toxin (88 kDa), a number of mutants were generated. The addition of two (2R: RS) or eleven (11R: RSRPGHHHHHH) amino acid residues at the Vip3Aa16 (L121I) C-terminus allowed to an unappropriated folding illustrated by the abundant presence of the 62 kDa proteolytic form. The produced Vip3Aa16 (L121I) full length form was less detected when increasing the number of amino acids residues in the C-terminus. Bioassays demonstrated that the growth of the lepidopteran Ephestia kuehniella was slightly affected by Vip3Aa16 (L121I)-2R and not affected by Vip3Aa16 (L121I)-11R. Additionally, the fusion at the Vip3Aa16 (L121I) N-terminus of 39 amino acids harboring the E. coli OmpA leader peptide and the His-tag sequence allowed to the increase of protease sensitivity of Vip3Aa16 (L121I) full length form, as only the 62 kDa proteolysis form was detected. Remarkably, this fused protein produced in Escherichia coli (E. coli) was biologically inactive toward Ephestia kuehniella larvae. Thus, the N-terminus of the protein is required to the accomplishment of the insecticidal activity of Vip3 proteins. This report serves as guideline for the study of Vip3Aa16 (L121I) protein stability and activity. PMID:26876111

  12. AtNHX5 and AtNHX6 Control Cellular K+ and pH Homeostasis in Arabidopsis: Three Conserved Acidic Residues Are Essential for K+ Transport

    PubMed Central

    Wang, Liguang; Wu, Xuexia; Liu, Yafen; Qiu, Quan-Sheng

    2015-01-01

    AtNHX5 and AtNHX6, the endosomal Na+,K+/H+ antiporters in Arabidopsis, play an important role in plant growth and development. However, their function in K+ and pH homeostasis remains unclear. In this report, we characterized the function of AtNHX5 and AtNHX6 in K+ and H+ homeostasis in Arabidopsis. Using a yeast expression system, we found that AtNHX5 and AtNHX6 recovered tolerance to high K+ or salt. We further found that AtNHX5 and AtNHX6 functioned at high K+ at acidic pH while AtCHXs at low K+ under alkaline conditions. In addition, we showed that the nhx5 nhx6 double mutant contained less K+ and was sensitive to low K+ treatment. Overexpression of AtNHX5 or AtNHX6 gene in nhx5 nhx6 recovered root growth to the wild-type level. Three conserved acidic residues, D164, E188, and D193 in AtNHX5 and D165, E189, and D194 in AtNHX6, were essential for K+ homeostasis and plant growth. nhx5 nhx6 had a reduced vacuolar and cellular pH as measured with the fluorescent pH indicator BCECF or semimicroelectrode. We further show that AtNHX5 and AtNHX6 are localized to Golgi and TGN. Taken together, AtNHX5 and AtNHX6 play an important role in K+ and pH homeostasis in Arabidopsis. Three conserved acidic residues are essential for K+ transport. PMID:26650539

  13. Human recombinant endopeptidase PHEX has a strict S1' specificity for acidic residues and cleaves peptides derived from fibroblast growth factor-23 and matrix extracellular phosphoglycoprotein.

    PubMed Central

    Campos, Marcelo; Couture, Constance; Hirata, Izaura Y; Juliano, Maria A; Loisel, Thomas P; Crine, Philippe; Juliano, Luiz; Boileau, Guy; Carmona, Adriana K

    2003-01-01

    The PHEX gene (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) encodes a protein (PHEX) with structural homologies to members of the M13 family of zinc metallo-endopeptidases. Mutations in the PHEX gene are responsible for X-linked hypophosphataemia in humans. However, the mechanism by which loss of PHEX function results in the disease phenotype, and the endogenous PHEX substrate(s) remain unknown. In order to study PHEX substrate specificity, combinatorial fluorescent-quenched peptide libraries containing o -aminobenzoic acid (Abz) and 2,4-dinitrophenyl (Dnp) as the donor-acceptor pair were synthesized and tested as PHEX substrates. PHEX showed a strict requirement for acidic amino acid residues (aspartate or glutamate) in S(1)' subsite, with a strong preference for aspartate. Subsites S(2)', S(1) and S(2) exhibited less defined specificity requirements, but the presence of leucine, proline or glycine in P(2)', or valine, isoleucine or histidine in P(1) precluded hydrolysis of the substrate by the enzyme. The peptide Abz-GFSDYK(Dnp)-OH, which contains the most favourable residues in the P(2) to P(2)' positions, was hydrolysed by PHEX at the N-terminus of aspartate with a k(cat)/ K(m) of 167 mM(-1) x s(-1). In addition, using quenched fluorescence peptides derived from fibroblast growth factor-23 and matrix extracellular phosphoglycoprotein sequences flanked by Abz and N -(2,4-dinitrophenyl)ethylenediamine, we showed that these physiologically relevant proteins are potential PHEX substrates. Finally, our results clearly indicate that PHEX does not have neprilysin-like substrate specificity. PMID:12678920

  14. Filtrates & Residues. Acid Pickling with Amines: An Experiment in Applied Chemistry for High School or Freshman Chemistry.

    ERIC Educational Resources Information Center

    Spears, Steven G.; And Others

    1988-01-01

    This article gives a brief description of the process of the removal of corrosion and millscale from the surfaces of ferrous metals by acid pickling. It suggests an experiment to illustrate this process including the procedure and a discussion of the results. (CW)

  15. Contribution of post-harvest agricultural paddy residue fires in the N.W. Indo-Gangetic Plain to ambient carcinogenic benzenoids, toxic isocyanic acid and carbon monoxide

    NASA Astrophysics Data System (ADS)

    Praphulla Chandra, Boggarapu; Sinha, Vinayak

    2016-04-01

    In the North West Indo-Gangetic Plain (N.W.IGP), large scale post-harvest paddy residue fires occur every year during the months of October-November. This anthropogenic perturbation causes contamination of the atmospheric environment with adverse impacts on regional air quality posing health risks for the population exposed to high concentrations of carcinogens such as benzene and toxic VOCs such as isocyanic acid. These gases and carbon monoxide are known to be emitted from biomass fires along with acetonitrile. Yet no long-term in-situ measurements quantifying the impact of this activity have been carried out in the N.W. IGP. Using high quality continuous online in-situ measurements of these gases at a strategic downwind site over a three year period from 2012 to 2014, we demonstrate the strong impact of this anthropogenic emission activity on ambient concentrations of these gases. In contrast to the pre-paddy harvest period, excellent correlation of benzenoids, isocyanic acid and CO with acetonitrile (a biomass burning chemical tracer); (r ≥ 0.82) and distinct VOC/acetonitrile emission ratios were observed for the post-paddy harvest period which was also characterized by high ambient concentrations of these species. The average concentrations of acetonitrile (1.62 ± 0.18 ppb), benzene (2.51 ± 0.28 ppb), toluene (3.72 ± 0.41 ppb), C8-aromatics (2.88 ± 0.30 ppb), C9-aromatics (1.55 ± 0.19 ppb) and CO (552 ± 113 ppb) in the post-paddy harvest periods were about 1.5 times higher than the annual average concentrations. For isocyanic acid, a compound with both primary and secondary sources, the concentration in the post-paddy harvest period was 0.97 ± 0.17 ppb. The annual average concentrations of benzene, a class A carcinogen, exceeded the annual exposure limit of 1.6 ppb at NTP mandated by the National Ambient Air Quality Standard of India (NAAQS). We show that mitigating the post-harvest paddy residue fires can lower the annual average concentration of

  16. Both foliar and residual applications of herbicides that inhibit amino acid biosynthesis induce alternative respiration and aerobic fermentation in pea roots.

    PubMed

    Armendáriz, O; Gil-Monreal, M; Zulet, A; Zabalza, A; Royuela, M

    2016-05-01

    The objective of this work was to ascertain whether there is a general pattern of carbon allocation and utilisation in plants following herbicide supply, independent of the site of application: sprayed on leaves or supplied to nutrient solution. The herbicides studied were the amino acid biosynthesis-inhibiting herbicides (ABIH): glyphosate, an inhibitor of aromatic amino acid biosynthesis, and imazamox, an inhibitor of branched-chain amino acid biosynthesis. All treated plants showed impaired carbon metabolism; carbohydrate accumulation was detected in both leaves and roots of the treated plants. The accumulation in roots was due to lack of use of available sugars as growth was arrested, which elicited soluble carbohydrate accumulation in the leaves due to a decrease in sink strength. Under aerobic conditions, ethanol fermentative metabolism was enhanced in roots of the treated plants. This fermentative response was not related to a change in total respiration rates or cytochrome respiratory capacity, but an increase in alternative oxidase capacity was detected. Pyruvate accumulation was detected after most of the herbicide treatments. These results demonstrate that both ABIH induce the less-efficient, ATP-producing pathways, namely fermentation and alternative respiration, by increasing the key metabolite, pyruvate. The plant response was similar not only for the two ABIH but also after foliar or residual application. PMID:26560850

  17. Role of certain amino acid residues of the coelenterazine-binding cavity in bioluminescence of light-sensitive Ca(2+)-regulated photoprotein berovin.

    PubMed

    Burakova, Ludmila P; Stepanyuk, Galina A; Eremeeva, Elena V; Vysotski, Eugene S

    2016-05-11

    Bright bioluminescence of ctenophores is caused by Ca(2+)-regulated photoproteins. Although these photoproteins are functionally identical to and share many properties of cnidarian photoproteins, like aequorin and obelin, and retain the same spatial architecture, they are extremely sensitive to light, i.e. lose the ability to bioluminesce on exposure to light over the entire absorption spectrum. In addition, the degree of identity of their amino acid sequences with those of cnidarian photoproteins is only 29.4%. This suggests that the residues involved in bioluminescence of ctenophore and cnidarian photoproteins significantly differ. Here we describe the bioluminescent properties of berovin mutants with substitution of the residues located in the photoprotein internal cavity. Since the spatial structure of berovin bound with a substrate is not determined yet, to identify these residues we have modeled it with an accommodated substrate using the structures of some cnidarian Ca(2+)-regulated photoproteins with bound coelenterazine or coelenteramide as templates in order to obtain an adequate sampling and to take into account all possible conformers and variants for ligand-protein docking. Based on the impact of substitutions on the bioluminescent properties and model structures we speculate that within the internal cavity of ctenophore photoproteins, coelenterazine is bound as a 2-peroxy anion adduct which is stabilized owing to Coulomb interaction with a positively charged guanidinium group of Arg41 paired with Tyr204. In this case, the bioluminescence reaction is triggered by only calcium-induced conformational changes leading to the disturbance of charge-charge interaction. PMID:27117544

  18. Evidence for the vitamin K-dependent gamma-carboxylation of the first glutamic acid residue in peptide substrates containing a diglutamyl sequence.

    PubMed Central

    Burgess, A I; Esnouf, M P; Rose, K; Offord, R E

    1983-01-01

    The peptide substrate commonly used in vitamin K-dependent carboxylation, Phe-Leu-Glu-Glu-Val, has been shown, by the use of high-voltage paper electrophoresis, to be degraded from the N-terminus by a microsomal leucine amino-peptidase. The replacement of phenylalanine with a N-t-butoxycarbonyl group resulted in a tetrapeptide substrate with a blocked N-terminus resistant to enzymic degradation. Vitamin K-dependent carboxylation of this non-degradable substrate gave a unique carboxylated product, which was separated from microsomal protein and unchanged substrate by using DEAE-Sephadex A25 as a final step. The carboxylated product was subsequently decarboxylated in 2HCl and analysed by using g.l.c. coupled to a mass spectrometer. This showed that only the first glutamic acid residue in the peptide substrate was carboxylated. PMID:6138032

  19. Evaluation of soluble fraction and enzymatic residual fraction of dilute dry acid, ethylenediamine, and steam explosion pretreated corn stover on the enzymatic hydrolysis of cellulose.

    PubMed

    Qin, Lei; Liu, Li; Li, Wen-Chao; Zhu, Jia-Qing; Li, Bing-Zhi; Yuan, Ying-Jin

    2016-06-01

    This study is aimed to examine the inhibition of soluble fraction (SF) and enzymatic residual fraction (ERF) in dry dilute acid (DDA), ethylenediamine (EDA) and steam explosion (SE) pretreated corn stover (CS) on the enzymatic digestibility of cellulose. SF of DDA, EDA and SE pretreated CS has high xylose, soluble lignin and xylo-oligomer content, respectively. SF of EDA pretreated CS leads to the highest inhibition, followed by SE and DDA pretreated CS. Inhibition of ERF of DDA and SE pretreated CS is higher than that of EDA pretreated CS. The inhibition degree (A0/A) of SF is 1.76 and 1.21 times to that of ERF for EDA and SE pretreated CS, respectively. The inhibition degree of ERF is 1.05 times to that of SF in DDA pretreated CS. The quantitative analysis shows that SF of EDA pretreated CS, SF and ERF of SE pretreated CS cause significant inhibition during enzymatic hydrolysis. PMID:26970919

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

    PubMed

    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

  1. Enhanced photocatalytic activity of Cl-residual rutile TiO2 nanorods after targeted co-modification with phosphoric and boric acids.

    PubMed

    Wu, Jing; Cui, Haiqin; Zhang, Xuliang; Luan, Yunbo; Jing, Liqiang

    2015-06-28

    The promotion of O2 adsorption on semiconductor surfaces for effectively capturing photogenerated electrons in the photocatalytic degradation of pollutants is highly desired. In this study, the targeted co-modification of residual chlorine rutile TiO2 nanorods with phosphoric and boric acids has been accomplished for the first time by simple wet chemical processes. The key to targeted co-modification is to connect -P-OH and -B-OH to the Cl-residual TiO2 surfaces by -Ti-OH and -Ti-Cl, respectively, consequently forming -Ti-O-P-OH and -Ti-Cl:B-OH ends. By means of the atmosphere-controlled surface photovoltage spectroscopy, the degrees for capturing photogenerated electrons by the adsorbed O2 as receptors on the resulting TiO2 nanorods are quantitatively analyzed. It is confirmed that the targeted co-modification could greatly promote the capture of the photogenerated electrons compared to the phosphate and borate modification alone. This is attributed to increased amounts of adsorbed O2 based on electrochemical O2 reduction and O2 temperature-programmed desorption measurements, further leading to the enhanced separation of photogenerated charges, characterized by an increase in the amount of produced hydroxyl radicals. This is responsible for the obviously enhanced photocatalytic activity of TiO2 nanorods towards the degradation of colorless gas-phase acetaldehyde and liquid-phase phenol. This work would provide us a feasible route for the co-modification with inorganic acids to synthesize efficient nanosized TiO2-based photocatalysts. PMID:26017969

  2. Differential Recognition of Influenza A Viruses by M158–66 Epitope-Specific CD8+ T Cells Is Determined by Extraepitopic Amino Acid Residues

    PubMed Central

    van de Sandt, Carolien E.; Kreijtz, Joost H. C. M.; Geelhoed-Mieras, Martina M.; Nieuwkoop, Nella J.; Spronken, Monique I.; van de Vijver, David A. M. C.; Fouchier, Ron A. M.; Osterhaus, Albert D. M. E.

    2015-01-01

    ABSTRACT Natural influenza A virus infections elicit both virus-specific antibody and CD4+ and CD8+ T cell responses. Influenza A virus-specific CD8+ cytotoxic T lymphocytes (CTLs) contribute to clearance of influenza virus infections. Viral CTL epitopes can display variation, allowing influenza A viruses to evade recognition by epitope-specific CTLs. Due to functional constraints, some epitopes, like the immunodominant HLA-A*0201-restricted matrix protein 1 (M158–66) epitope, are highly conserved between influenza A viruses regardless of their subtype or host species of origin. We hypothesized that human influenza A viruses evade recognition of this epitope by impairing antigen processing and presentation by extraepitopic amino acid substitutions. Activation of specific T cells was used as an indication of antigen presentation. Here, we show that the M158–66 epitope in the M1 protein derived from human influenza A virus was poorly recognized compared to the M1 protein derived from avian influenza A virus. Furthermore, we demonstrate that naturally occurring variations at extraepitopic amino acid residues affect CD8+ T cell recognition of the M158–66 epitope. These data indicate that human influenza A viruses can impair recognition by M158–66-specific CTLs while retaining the conserved amino acid sequence of the epitope, which may represent a yet-unknown immune evasion strategy for influenza A viruses. This difference in recognition may have implications for the viral replication kinetics in HLA-A*0201 individuals and spread of influenza A viruses in the human population. The findings may aid the rational design of universal influenza vaccines that aim at the induction of cross-reactive virus-specific CTL responses. IMPORTANCE Influenza viruses are an important cause of acute respiratory tract infections. Natural influenza A virus infections elicit both humoral and cellular immunity. CD8+ cytotoxic T lymphocytes (CTLs) are directed predominantly against

  3. Key role of cysteine residues and sulfenic acids in thermal- and H2O2-mediated modification of β-lactoglobulin.

    PubMed

    Krämer, Anna C; Thulstrup, Peter W; Lund, Marianne N; Davies, Michael J

    2016-08-01

    Oxidation results in protein deterioration in mammals, plants, foodstuffs and pharmaceuticals, via changes in amino acid composition, fragmentation, aggregation, solubility, hydrophobicity, conformation, function and susceptibility to digestion. This study investigated whether and how individual or combined treatment with heat, a commonly encountered factor in industrial processing, and H2O2 alters the structure and composition of the major whey protein β-lactoglobulin. Thermal treatment induced reducible cross-links, with this being enhanced by low H2O2 concentrations, but decreased by high concentrations, where fragmentation was detected. Cross-linking was prevented when the single free Cys121 residue was blocked with iodoacetamide. Low concentrations of H2O2 added before heating depleted thiols, with H2O2 alone, or H2O2 added after heating, having lesser effects. A similar pattern was detected for methionine loss and methionine sulfoxide formation. Tryptophan loss was only detected with high levels of H2O2, and no other amino acid was affected, indicating that sulfur-centered amino acids are critical targets. No protection against aggregation was provided by high concentrations of the radical scavenger 5, 5-dimethyl-1-pyrroline N-oxide (DMPO), consistent with molecular oxidation, rather than radical reactions, being the major process. Sulfenic acid formation was detected by Western blotting and LC-MS/MS peptide mass-mapping of dimedone-treated protein, consistent with these species being significant intermediates in heat-induced cross-linking, especially in the presence of H2O2. Studies using circular dichroism and intrinsic fluorescence indicate that H2O2 increases unfolding during heating. These mechanistic insights provide potential strategies for modulating the extent of modification of proteins exposed to thermal and oxidant treatment. PMID:27430598

  4. Prediction of DNA-binding residues in proteins from amino acid sequences using a random forest model with a hybrid feature

    PubMed Central

    Wu, Jiansheng; Liu, Hongde; Duan, Xueye; Ding, Yan; Wu, Hongtao; Bai, Yunfei; Sun, Xiao

    2009-01-01

    Motivation: In this work, we aim to develop a computational approach for predicting DNA-binding sites in proteins from amino acid sequences. To avoid overfitting with this method, all available DNA-binding proteins from the Protein Data Bank (PDB) are used to construct the models. The random forest (RF) algorithm is used because it is fast and has robust performance for different parameter values. A novel hybrid feature is presented which incorporates evolutionary information of the amino acid sequence, secondary structure (SS) information and orthogonal binary vector (OBV) information which reflects the characteristics of 20 kinds of amino acids for two physical–chemical properties (dipoles and volumes of the side chains). The numbers of binding and non-binding residues in proteins are highly unbalanced, so a novel scheme is proposed to deal with the problem of imbalanced datasets by downsizing the majority class. Results: The results show that the RF model achieves 91.41% overall accuracy with Matthew's correlation coefficient of 0.70 and an area under the receiver operating characteristic curve (AUC) of 0.913. To our knowledge, the RF method using the hybrid feature is currently the computationally optimal approach for predicting DNA-binding sites in proteins from amino acid sequences without using three-dimensional (3D) structural information. We have demonstrated that the prediction results are useful for understanding protein–DNA interactions. Availability: DBindR web server implementation is freely available at http://www.cbi.seu.edu.cn/DBindR/DBindR.htm. Contact: xsun@seu.edu.cn Supplementary information: Supplementary data are available at Bioinformatics online. PMID:19008251

  5. An integrated process for the production of platform chemicals and diesel miscible fuels by acid-catalyzed hydrolysis and downstream upgrading of the acid hydrolysis residues with thermal and catalytic pyrolysis.

    PubMed

    Girisuta, Buana; Kalogiannis, Konstantinos G; Dussan, Karla; Leahy, James J; Hayes, Michael H B; Stefanidis, Stylianos D; Michailof, Chrysa M; Lappas, Angelos A

    2012-12-01

    This study evaluates an integrated process for the production of platform chemicals and diesel miscible biofuels. An energy crop (Miscanthus) was treated hydrothermally to produce levulinic acid (LA). Temperatures ranging between 150 and 200 °C, sulfuric acid concentrations 1-5 wt.% and treatment times 1-12 h were applied to give different combined severity factors. Temperatures of 175 and 200 °C and acid concentration of 5 wt.% were found to be necessary to achieve good yield (17 wt.%) and selectivities of LA while treatment time did not have an effect. The acid hydrolysis residues were characterized for their elemental, cellulose, hemicellulose and lignin contents, and then tested in a small-scale pyrolyzer using silica sand and a commercial ZSM-5 catalyst. Milder pretreatment yielded more oil (43 wt.%) and oil O(2) (37%) while harsher pretreatment and catalysis led to more coke production (up to 58 wt.%), less oil (12 wt.%) and less oil O(2) (18 wt.%). PMID:23073094

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

    NASA Astrophysics Data System (ADS)

    Hu, Xuebing; Yu, Yun; Wang, Yongqing; Zhou, Jianer; Song, Lixin

    2015-02-01

    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.

  7. Mutations affecting two adjacent amino acid residues in the alpha subunit of RNA polymerase block transcriptional activation by the bacteriophage P2 Ogr protein.

    PubMed Central

    Ayers, D J; Sunshine, M G; Six, E W; Christie, G E

    1994-01-01

    The bacteriophage P2 ogr gene product is a positive regulator of transcription from P2 late promoters. The ogr gene was originally defined by compensatory mutations that overcame the block to P2 growth imposed by a host mutation, rpoA109, in the gene encoding the alpha subunit of RNA polymerase. DNA sequence analysis has confirmed that this mutation affects the C-terminal region of the alpha subunit, changing a leucine residue at position 290 to a histidine (rpoAL290H). We have employed a reporter plasmid system to screen other, previously described, rpoA mutants for effects on activation of a P2 late promoter and have identified a second allele, rpoA155, that blocks P2 late transcription. This mutation lies just upstream of rpoAL290H, changing the leucine residue at position 289 to a phenylalanine (rpoAL289F). The effect of the rpoAL289F mutation is not suppressed by the rpoAL290H-compensatory P2 ogr mutation. P2 ogr mutants that overcome the block imposed by rpoAL289F were isolated and characterized. Our results are consistent with a direct interaction between Ogr and the alpha subunit of RNA polymerase and support a model in which transcription factor contact sites within the C terminus of alpha are discrete and tightly clustered. PMID:8002564

  8. Specially-Made Lipid-Based Assemblies for Improving Transmembrane Gene Delivery: Comparison of Basic Amino Acid Residue Rich Periphery.

    PubMed

    Jiang, Qian; Yue, Dong; Nie, Yu; Xu, Xianghui; He, Yiyan; Zhang, Shiyong; Wagner, Ernst; Gu, Zhongwei

    2016-06-01

    Cationic lipid based assemblies provide a promising platform for effective gene condensation into nanosized particles, and the peripheral properties of the assemblies are vital for complexation and interaction with physical barriers. Here, we report three cationic twin head lipids, and each of them contains a dioleoyl-glutamate hydrophobic tail and a twin polar head of lysine, arginine, or histidine. Such lipids were proven to self-assemble in aqueous solution with well-defined nanostructures and residual amino-, guanidine-, or imidazole-rich periphery, showing strong buffering capacity and good liquidity. The assemblies with arginine (RL) or lysine (KL) periphery exhibited positive charges (∼+35 mV) and complete condensation of pDNA into nanosized complexes (∼120 nm). In contrast, assemblies composed of histidine-rich lipids (HL) showed relatively low cationic electric potential (∼+10 mV) and poor DNA binding ability. As expected, the designed RL assemblies with guanidine-rich periphery enhanced the in vitro gene transfection up to 190-fold as compared with the golden standard PEI25k and Lipofectamine 2000, especially in the presence of serum. Meanwhile, interaction with cell and endo/lysosome membrane also revealed the superiority of RL complexes, that the guanidine-rich surface efficiently promoted transmembrane process in cellular internalization and endosomal disruption. More importantly, RL complexes also succeeded beyond others in vivo with significantly (∼7-fold) enhanced expression in HepG2 tumor xenografts in mice, as well as stronger green fluorescence protein imaging in isolated tumors and tumor frozen sections. PMID:27097286

  9. Amino Acid Residues of RegA Important for Interactions with the CbbR-DNA Complex of Rhodobacter sphaeroides

    PubMed Central

    Dangel, Andrew W.; Luther, Amanda

    2014-01-01

    CbbR and RegA (PrrA) are transcriptional regulators of the Calvin-Benson-Bassham (CBB) CO2 fixation pathway (cbbI and cbbII) operons of Rhodobacter sphaeroides. The CbbR and RegA proteins interact, but CbbR must be bound to the promoter DNA in order for RegA-CbbR protein-protein interactions to occur. RegA greatly enhances the ability of CbbR to bind the cbbI promoter or greatly enhances the stability of the CbbR/promoter complex. The N-terminal receiver domain and the DNA binding domain of RegA were shown to interact with CbbR. Residues in α-helix 7 and α-helix 8 of the DNA binding domain (helix-turn-helix) of RegA directly interacted with CbbR, with α-helix 7 positioned immediately above the DNA and α-helix 8 located in the major groove of the DNA. A CbbR protein containing only the DNA binding motif and the linker helix was capable of binding to RegA. In contrast, a truncated CbbR containing only the linker helix and recognition domains I and II (required for effector binding) was not able to interact with RegA. The accumulated results strongly suggest that the DNA binding domains of both proteins interact to facilitate optimal transcriptional control over the cbb operons. In vivo analysis, using constitutively active mutant CbbR proteins, further indicated that CbbR must interact with phosphorylated RegA in order to accomplish transcriptional activation. PMID:24957624

  10. Identification of amino acid residues involved in 4-chloroindole 3-hydroxylation by cytochrome P450 2A6 using screening of random libraries

    PubMed Central

    Zhang, Zhi-Gang; Liu, Yan; Guengerich, F. Peter; Matse, Johannes H.; Chen, Jun; Wu, Zhong-Liu

    2016-01-01

    Cytochrome P450 (P450) 2A6 is able to catalyze indole hydroxylation to form the blue dye indigo. The wild type P450 2A6 enzyme was randomly mutated throughout the whole open reading frame and screened using 4-chloroindole hydroxylation, a substituted indole selected from 30 indole compounds for enhanced color development. Mutants with up to 5-fold increases of catalytic efficiency (kcat/Km) and 2-fold increases in kcat were selected after two rounds of screening. Important residues located both in (e.g., Thr305) and outside the active site (e.g., Ser224) were identified. The study utilized a better substrate for "indigo assay" to obtain new information on the structure-functional relationship of P450 2A6 that was not revealed by previous mutagenesis studies with this enzyme. PMID:18984015

  11. Isolation of key amino acid residues at the N-terminal end of the core region Streptococcus downei glucansucrase, GTF-I.

    PubMed

    Monchois, V; Vignon, M; Russell, R R

    1999-11-01

    Related streptococcal and Leuconostoc mesenteroides glucansucrases are enzymes of medical and biotechnological interest. Molecular modelling has suggested that the catalytic domain contains a circularly permuted version of the (beta/alpha)8 barrel structure found in the amylase superfamily, and site-directed mutagenesis has identified critical amino acids in this region. In this study, sequential N-terminal truncations of Streptococcus downei GTF-I showed that key amino acids are also present in the first one-third of the core domain. Mutations were introduced at Trp-344, Glu-349 and His-355, residues that are conserved in all glucansucrases and lie within a region which is a target for inhibitory antibodies. W344L, E349L and H355V substitutions were assayed for their effect on mutan synthesis and also on oligosaccharide synthesis with various acceptors. It appeared that Trp-344 and His-355 are involved in the action mechanism of GTF-I; His-355 may also play a role in a binding subsite necessary for oligosaccharide and glucan elongation. PMID:10570812

  12. Lactic Acid Bacteria in Total Mixed Ration Silage Containing Soybean Curd Residue: Their Isolation, Identification and Ability to Inhibit Aerobic Deterioration

    PubMed Central

    Li, Y.; Wang, F.; Nishino, N.

    2016-01-01

    We investigated the effects of the predominant lactic acid bacteria (LAB) on the fermentation characteristics and aerobic stability of total mixed ration (TMR) silage containing soybean curd residue (SC-TMR silage). The SC-TMR materials were ensiled in laboratory silos for 14 or 56 days. LAB predominant in SC-TMR silage were identified (Exp. 1). Lactobacillus fermentum (L. fermentum) and Streptococcus bovis (S. bovis) were found in the untreated materials, Leuconostoc pseudomesenteroides (L. pseudomesenteroides) in 14-day silage and Lactobacillus plantarum (L. plantarum) in all silages. Pediococcus acidilactici (P. acidilactici), Lactobacillus paracasei (L. paracasei), and Lactobacillus brevis (L. brevis) formed more than 90% of the isolates in 56-day silage. Italian ryegrass and whole crop maize were inoculated with P. acidilactici and L. brevis isolates and the fermentation and aerobic stability determined (Exp. 2). Inoculation with P. acidilactici and L. brevis alone or combined improved the fermentation products in ryegrass silage and markedly enhanced its aerobic stability. In maize silage, P. acidilactici and L. brevis inoculation caused no changes and suppressed deterioration when combined with increases in acetic acid content. The results indicate that P. acidilactici and L. brevis may produce a synergistic effect to inhibit SC-TMR silage deterioration. Further studies are needed to identify the inhibitory substances, which may be useful for developing potential antifungal agents. PMID:26949952

  13. Functionally important amino acid residues in the transient receptor potential vanilloid 1 (TRPV1) ion channel – an overview of the current mutational data

    PubMed Central

    2013-01-01

    This review aims to create an overview of the currently available results of site-directed mutagenesis studies on transient receptor potential vanilloid type 1 (TRPV1) receptor. Systematization of the vast number of data on the functionally important amino acid mutations of TRPV1 may provide a clearer picture of this field, and may promote a better understanding of the relationship between the structure and function of TRPV1. The review summarizes information on 112 unique mutated sites along the TRPV1, exchanged to multiple different residues in many cases. These mutations influence the effect or binding of different agonists, antagonists, and channel blockers, alter the responsiveness to heat, acid, and voltage dependence, affect the channel pore characteristics, and influence the regulation of the receptor function by phosphorylation, glycosylation, calmodulin, PIP2, ATP, and lipid binding. The main goal of this paper is to publish the above mentioned data in a form that facilitates in silico molecular modelling of the receptor by promoting easier establishment of boundary conditions. The better understanding of the structure-function relationship of TRPV1 may promote discovery of new, promising, more effective and safe drugs for treatment of neurogenic inflammation and pain-related diseases and may offer new opportunities for therapeutic interventions. PMID:23800232

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

    PubMed

    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

  15. Lactic Acid Bacteria in Total Mixed Ration Silage Containing Soybean Curd Residue: Their Isolation, Identification and Ability to Inhibit Aerobic Deterioration.

    PubMed

    Li, Y; Wang, F; Nishino, N

    2016-04-01

    We investigated the effects of the predominant lactic acid bacteria (LAB) on the fermentation characteristics and aerobic stability of total mixed ration (TMR) silage containing soybean curd residue (SC-TMR silage). The SC-TMR materials were ensiled in laboratory silos for 14 or 56 days. LAB predominant in SC-TMR silage were identified (Exp. 1). Lactobacillus fermentum (L. fermentum) and Streptococcus bovis (S. bovis) were found in the untreated materials, Leuconostoc pseudomesenteroides (L. pseudomesenteroides) in 14-day silage and Lactobacillus plantarum (L. plantarum) in all silages. Pediococcus acidilactici (P. acidilactici), Lactobacillus paracasei (L. paracasei), and Lactobacillus brevis (L. brevis) formed more than 90% of the isolates in 56-day silage. Italian ryegrass and whole crop maize were inoculated with P. acidilactici and L. brevis isolates and the fermentation and aerobic stability determined (Exp. 2). Inoculation with P. acidilactici and L. brevis alone or combined improved the fermentation products in ryegrass silage and markedly enhanced its aerobic stability. In maize silage, P. acidilactici and L. brevis inoculation caused no changes and suppressed deterioration when combined with increases in acetic acid content. The results indicate that P. acidilactici and L. brevis may produce a synergistic effect to inhibit SC-TMR silage deterioration. Further studies are needed to identify the inhibitory substances, which may be useful for developing potential antifungal agents. PMID:26949952

  16. Supercritical Extraction from Vinification Residues: Fatty Acids, α-Tocopherol, and Phenolic Compounds in the Oil Seeds from Different Varieties of Grape

    PubMed Central

    Agostini, F.; Bertussi, R. A.; Agostini, G.; Atti dos Santos, A. C.; Rossato, M.; Vanderlinde, R.

    2012-01-01

    Supercritical fluid extraction has been widely employed in the extraction of high purity substances. In this study, we used the technology to obtain oil from seeds from a variety of grapes, from vinification residues generated in the Southern region of the state of Rio Grande do Sul, Brazil. This work encompasses three varieties of Vitis vinifera (Moscato Giallo, Merlot, and Cabernet Sauvignon) and two of Vitis labrusca (Bordô e Isabel), harvested in 2005 and 2006. We obtained the highest oil content from Bordô (15.40%) in 2005 and from Merlot (14.66%), 2006. The biggest concentration of palmitic, stearic, and linoleic acids was observed in Bordô, 2005, and in Bordô, Merlot, and Moscato Giallo, 2006. Bordô showed the highest concentration of oleic acid and α-tocopherol in both seasons too. For the equivalent of procyanidins, we did not notice significant difference among the varieties from the 2005 harvest. In 2006, both varieties Isabel and Cabernet Sauvignon showed a value slightly lower than the other varieties. The concentration of total phenolics was higher in Bordô and Cabernet Sauvignon. The presence of these substances is related to several important pharmacological properties and might be an alternative to conventional processes to obtain these bioactives. PMID:22593706

  17. Contribution of post-harvest agricultural paddy residue fires in the N.W. Indo-Gangetic Plain to ambient carcinogenic benzenoids, toxic isocyanic acid and carbon monoxide.

    PubMed

    Chandra, B P; Sinha, Vinayak

    2016-03-01

    In the north west Indo-Gangetic Plain (N.W.IGP), large scale post-harvest paddy residue fires occur every year during the months of October-November. This anthropogenic perturbation causes contamination of the atmospheric environment with adverse impacts on regional air quality posing health risks for the population exposed to high concentrations of carcinogens such as benzene and toxic VOCs such as isocyanic acid. These gases and carbon monoxide are known to be emitted from biomass fires along with acetonitrile. Yet no long-term in-situ measurements quantifying the impact of this activity have been carried out in the N.W. IGP. Using high quality continuous online in-situ measurements of these gases at a strategic downwind site over a three year period from 2012 to 2014, we demonstrate the strong impact of this anthropogenic emission activity on ambient concentrations of these gases. In contrast to the pre-paddy harvest period, excellent correlation of benzenoids, isocyanic acid and CO with acetonitrile (a biomass burning chemical tracer); (r≥0.82) and distinct VOC/acetonitrile emission ratios were observed for the post-paddy harvest period which was also characterized by high ambient concentrations of these species. The average concentrations of acetonitrile (1.62±0.18ppb), benzene (2.51±0.28ppb), toluene (3.72±0.41ppb), C8-aromatics (2.88±0.30ppb), C9-aromatics (1.55±0.19ppb) and CO (552±113ppb) in the post-paddy harvest periods were about 1.5 times higher than the annual average concentrations. For isocyanic acid, a compound with both primary and secondary sources, the concentration in the post-paddy harvest period was 0.97±0.17ppb. The annual average concentrations of benzene, a class A carcinogen, exceeded the annual exposure limit of 1.6ppb at NTP mandated by the National Ambient Air Quality Standard of India (NAAQS). We show that mitigating the post-harvest paddy residue fires can lower the annual average concentration of benzene and ensure

  18. Stabilization of the previously unknown tautomer HP(OH)2 of hypophosphorous acid as ligand; preparation of [W3(Ni(HP(OH)2))Q4(H2O)9]4+ (Q = S, Se) complexes.

    PubMed

    Sokolov, Maxim N; Hernández-Molina, Rita; Clegg, William; Fedin, Vladimir P; Mederos, Alfredo

    2003-01-01

    Bis(hydroxy)phosphine, the isomer of hypophosphorous acid which remained elusive for a long time, coordinates to the Ni site of heterometallic clusters with a W3NiQ4 core (Q = S, Se) to give [W3(Ni(HP(OH)2))Q4(H2O)9]4+ (Q = S, Se). PMID:12611005

  19. Unexpected functional diversity in the fatty acid desaturases of the flour beetle Tribolium castaneum and identification of key residues determining activity.

    PubMed

    Haritos, Victoria S; Horne, Irene; Damcevski, Katherine; Glover, Karen; Gibb, Nerida

    2014-08-01

    Desaturases catalyse modifications to fatty acids which are essential to homeostasis and for pheromone and defensive chemical production. All desaturases of the flour beetle Tribolium castaneum were investigated via query of the sequenced genome which yielded 15 putative acyl-Coenzyme A genes. Eleven desaturase mRNA were obtained in full length and functionally expressed in yeast. Phylogenetic analysis separated the desaturases into 4 distinct clades; one clade contained conserved beetle Δ9 desaturases, second clade was Tribolium-specific having diverse activities including Δ5, Δ9 and Δ12 desaturation and the other 2 clades had mixed insect representatives. Three members of this clade contained unusual inserted sequences of ∼20 residues in the C-terminal region and were related to desaturases that all contained similar inserts. Deletion of the entirety of the insert in the flour beetle Δ12 desaturase abolished its activity but this was partially restored by the reintroduction of two histidine residues, suggesting the histidine(s) are required for activity but the full length insert is not. Five new desaturase activities were discovered: Δ9 desaturation of C12:0-C16:0 substrates; two unprecedented Δ5 enzymes acting on C18:0 and C16:0; Δ9 activity exclusively on C16:0 and a further stearate Δ9 desaturase. qPCR analysis ruled out a role in sex pheromone synthesis for the Δ5 and Δ9/C16:0 desaturases. The flour beetle genome has underpinned an examination of all transcribed desaturases in the organism and revealed a diversity of novel and unusual activities, an improved understanding of the evolutionary relationships among insect desaturases and sequence determinants of activity. PMID:24880119

  20. Inhibition of amyloid fibril formation of human amylin by N-alkylated amino acid and alpha-hydroxy acid residue containing peptides.

    PubMed

    Rijkers, Dirk T S; Höppener, Jo W M; Posthuma, George; Lips, Cornelis J M; Liskamp, Rob M J

    2002-09-16

    Amyloid deposits are formed as a result of uncontrolled aggregation of (poly)peptides or proteins. Today several diseases are known, for example Alzheimer's disease, Creutzfeldt-Jakob disease, mad cow disease, in which amyloid formation is involved. Amyloid fibrils are large aggregates of beta-pleated sheets and here a general method is described to introduce molecular mutations in order to achieve disruption of beta-sheet formation. Eight backbone-modified amylin derivatives, an amyloidogenic peptide involved in maturity onset diabetes, were synthesized. Their beta-sheet forming properties were studied by IR spectroscopy and electron microscopy. Modification of a crucial amide NH by an alkyl chain led to a complete loss of the beta-sheet forming capacity of amylin. The resulting molecular mutated amylin derivative could be used to break the beta-sheet thus retarding beta-sheet formation of unmodified amylin. Moreover, it was found that the replacement of this amide bond by an ester moiety suppressed fibrillogenesis significantly. Introduction of N-alkylated amino acids and/or ester functionalities-leading to depsipeptides-into amyloidogenic peptides opens new avenues towards novel peptidic beta-sheet breakers for inhibition of beta-amyloid aggregation. PMID:12298020

  1. The Role of VP1 Amino Acid Residue 145 of Enterovirus 71 in Viral Fitness and Pathogenesis in a Cynomolgus Monkey Model

    PubMed Central

    Kataoka, Chikako; Suzuki, Tadaki; Kotani, Osamu; Iwata-Yoshikawa, Naoko; Nagata, Noriyo; Ami, Yasushi; Wakita, Takaji; Nishimura, Yorihiro; Shimizu, Hiroyuki

    2015-01-01

    Enterovirus 71 (EV71), a major causative agent of hand, foot, and mouth disease, occasionally causes severe neurological symptoms. We identified P-selectin glycoprotein ligand-1 (PSGL-1) as an EV71 receptor and found that an amino acid residue 145 in the capsid protein VP1 (VP1-145) defined PSGL-1-binding (PB) and PSGL-1-nonbinding (non-PB) phenotypes of EV71. However, the role of PSGL-1-dependent EV71 replication in neuropathogenesis remains poorly understood. In this study, we investigated viral replication, genetic stability, and the pathogenicity of PB and non-PB strains of EV71 in a cynomolgus monkey model. Monkeys were intravenously inoculated with cDNA-derived PB and non-PB strains of EV71, EV71-02363-EG and EV71-02363-KE strains, respectively, with two amino acid differences at VP1-98 and VP1-145. Mild neurological symptoms, transient lymphocytopenia, and inflammatory cytokine responses, were found predominantly in the 02363-KE-inoculated monkeys. During the early stage of infection, viruses were frequently detected in clinical samples from 02363-KE-inoculated monkeys but rarely in samples from 02363-EG-inoculated monkeys. Histopathological analysis of central nervous system (CNS) tissues at 10 days postinfection revealed that 02363-KE induced neuropathogenesis more efficiently than that induced by 02363-EG. After inoculation with 02363-EG, almost all EV71 variants detected in clinical samples, CNS, and non-CNS tissues, possessed a G to E amino acid substitution at VP1-145, suggesting a strong in vivo selection of VP1-145E variants and CNS spread presumably in a PSGL-1-independent manner. EV71 variants with VP1-145G were identified only in peripheral blood mononuclear cells in two out of four 02363-EG-inoculated monkeys. Thus, VP1-145E variants are mainly responsible for the development of viremia and neuropathogenesis in a non-human primate model, further suggesting the in vivo involvement of amino acid polymorphism at VP1-145 in cell-specific viral

  2. Iterative Saturation Mutagenesis of −6 Subsite Residues in Cyclodextrin Glycosyltransferase from Paenibacillus macerans To Improve Maltodextrin Specificity for 2-O-d-Glucopyranosyl-l-Ascorbic Acid Synthesis

    PubMed Central

    Han, Ruizhi; Shin, Hyun-dong; Chen, Rachel R.; Li, Jianghua; Chen, Jian

    2013-01-01

    2-O-d-Glucopyranosyl-l-ascorbic acid (AA-2G), a stable l-ascorbic acid derivative, is usually synthesized by cyclodextrin glycosyltransferase (CGTase), which contains nine substrate-binding subsites (from +2 to −7). In this study, iterative saturation mutagenesis (ISM) was performed on the −6 subsite residues (Y167, G179, G180, and N193) in the CGTase from Paenibacillus macerans to improve its specificity for maltodextrin, which is a cheap and easily soluble glycosyl donor for AA-2G synthesis. Site saturation mutagenesis of four sites—Y167, G179, G180, and N193—was first performed and revealed that four mutants—Y167S, G179R, N193R, and G180R—produced AA-2G yields higher than those of other mutant and wild-type CGTases. ISM was then conducted with the best positive mutant as a template. Under optimal conditions, mutant Y167S/G179K/N193R/G180R produced the highest AA-2G titer of 2.12 g/liter, which was 84% higher than that (1.15 g/liter) produced by the wild-type CGTase. Kinetics analysis of AA-2G synthesis using mutant CGTases confirmed the enhanced maltodextrin specificity and showed that compared to the wild-type CGTase, the mutants had no cyclization activity but high hydrolysis and disproportionation activities. A possible mechanism for the enhanced substrate specificity was also analyzed through structure modeling of the mutant and wild-type CGTases. These results indicated that the −6 subsite played crucial roles in the substrate binding and catalytic reactions of CGTase and that the obtained CGTase mutants, especially Y167S/G179K/N193R/G180R, are promising starting points for further development through protein engineering. PMID:24077706

  3. 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).

    PubMed

    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

  4. Proteomic investigation of protein profile changes and amino acid residue-level modification in cooked lamb longissimus thoracis et lumborum: The effect of roasting.

    PubMed

    Yu, Tzer-Yang; Morton, James D; Clerens, Stefan; Dyer, Jolon M

    2016-09-01

    Protein modifications of meat cooked by typical dry-heat methods (e.g., roasting) are currently not well understood. The present study utilised a shotgun proteomic approach to examine the molecular-level effect of roasting on thin lamb longissimus thoracis et lumborum patties, in terms of changes to both the protein profile and amino acid residue side-chain modifications. Cooking caused aggregation of actin, myosin heavy chains and sarcoplasmic proteins. Longer roasting time resulted in significantly reduced protein extractability as well as protein truncation involving particularly a number of myofibrillar and sarcoplasmic proteins, e.g., 6-phosphofructokinase, beta-enolase, l-lactate dehydrogenase A chain, alpha-actinin-3, actin and possibly myosin heavy chains. Modifications that have potential influence on nutritional properties, including carboxyethyllysine and a potentially glucose-derived N-terminal Amadori compound, were observed in actin and myoglobin after roasting. This study provided new insights into molecular changes resulting from the dry-heat treatment of meat, such as commonly used in food preparation. PMID:27150797

  5. Identification of amino acid residues of a designed ankyrin repeat protein potentially involved in intermolecular interactions with CD4: analysis by molecular dynamics simulations.

    PubMed

    Nimmanpipug, Piyarat; Khampa, Chalermpon; Lee, Vannajan Sanghiran; Nangola, Sawitree; Tayapiwatana, Chatchai

    2011-11-01

    We applied molecular dynamics simulations to investigate the binding properties of a designed ankyrin repeat protein, the DARPin-CD4 complex. DARPin 23.2 has been reported to disturb the human immunodeficiency virus (HIV) viral entry process by Schweizer et al. The protein docking simulation was analysed by comparing the specific ankyrin binder (DARPin 23.2) to an irrelevant control (2JAB) in forming a composite with CD4. To determine the binding free energy of both ankyrins, the MM/PBSA and MM/GBSA protocols were used. The free energy decomposition of both complexes were analysed to explore the role of certain amino acid residues in complex configuration. Interestingly, the molecular docking analysis of DARPin 23.2 revealed a similar CD4 interaction regarding the gp120 theoretical anchoring motif. In contrast, the binding of control ankyrin to CD4 occurred at a different location. This observation suggests that there is an advantage to the molecular modification of DARPin 23.2, an enhanced affinity for CD4. PMID:21962990

  6. Growth performance, carcass traits, physiochemical characteristics and intramuscular fatty acid composition of finishing Japanese black steers fed soybean curd residue and soy sauce cake.

    PubMed

    Yasuda, Kaori; Kitagawa, Masayuki; Oishi, Kazato; Hirooka, Hiroyuki; Tamura, Takemi; Kumagai, Hajime

    2016-07-01

    This study was conducted to determine the effects of dietary soybean curd residue (SCR) and soy sauce cake (SSC) on the growth performance, carcass traits and physiochemical and intramuscular fatty acid (FA) characteristics in Japanese Black steers. Ten steers (29.7 ± 0.3 months old, 856.6 ± 24.4 kg body weight) were assigned to either treatment C, fed a conventional concentrate or T, fed the test diet including dried SCR and SSC for 3 months. In growth performance, dry matter (DM) intake and average daily gain, and carcass traits did not differ significantly between the treatments. Color of beef was affected by the dietary treatments and meat samples from T showed higher a(*) value and chroma than those in C. On FA composition, there was no significant difference between the treatments in neutral lipids, whereas in polar lipids, meat samples from T had higher C16:1 (P < 0.05) and tended to have higher C16:0 (P = 0.05) and C18:1 (P = 0.08), but lower C17:0 (P = 0.098), C18:2 (P = 0.06) and C20:4 (P = 0.07) than those from C. The study suggested that SCR and SSC could be used as a substitute for conventional concentrate and would influence meat color and intramuscular FA composition of polar lipids. PMID:26663641

  7. Analysis of SAT Type Foot-And-Mouth Disease Virus Capsid Proteins and the Identification of Putative Amino Acid Residues Affecting Virus Stability

    PubMed Central

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

    2013-01-01

    Foot-and-mouth disease virus (FMDV) initiates infection by adhering to integrin receptors on target cells, followed by cell entry and disassembly of the virion through acidification within endosomes. Mild heating of the virions also leads to irreversible dissociation into pentamers, a characteristic linked to reduced vaccine efficacy. In this study, the structural stability of intra- and inter-serotype chimeric SAT2 and SAT3 virus particles to various conditions including low pH, mild temperatures or high ionic strength, was compared. Our results demonstrated that while both the SAT2 and SAT3 infectious capsids displayed different sensitivities in a series of low pH buffers, their stability profiles were comparable at high temperatures or high ionic strength conditions. Recombinant vSAT2 and intra-serotype chimeric viruses were used to map the amino acid differences in the capsid proteins of viruses with disparate low pH stabilities. Four His residues at the inter-pentamer interface were identified that change protonation states at pH 6.0. Of these, the H145 of VP3 appears to be involved in interactions with A141 in VP3 and K63 in VP2, and may be involved in orientating H142 of VP3 for interaction at the inter-pentamer interfaces. PMID:23717387

  8. Analysis of SAT type foot-and-mouth disease virus capsid proteins and the identification of putative amino acid residues affecting virus stability.

    PubMed

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

    2013-01-01

    Foot-and-mouth disease virus (FMDV) initiates infection by adhering to integrin receptors on target cells, followed by cell entry and disassembly of the virion through acidification within endosomes. Mild heating of the virions also leads to irreversible dissociation into pentamers, a characteristic linked to reduced vaccine efficacy. In this study, the structural stability of intra- and inter-serotype chimeric SAT2 and SAT3 virus particles to various conditions including low pH, mild temperatures or high ionic strength, was compared. Our results demonstrated that while both the SAT2 and SAT3 infectious capsids displayed different sensitivities in a series of low pH buffers, their stability profiles were comparable at high temperatures or high ionic strength conditions. Recombinant vSAT2 and intra-serotype chimeric viruses were used to map the amino acid differences in the capsid proteins of viruses with disparate low pH stabilities. Four His residues at the inter-pentamer interface were identified that change protonation states at pH 6.0. Of these, the H145 of VP3 appears to be involved in interactions with A141 in VP3 and K63 in VP2, and may be involved in orientating H142 of VP3 for interaction at the inter-pentamer interfaces. PMID:23717387

  9. Histidine residues in the Na+-coupled ascorbic acid transporter-2 (SVCT2) are central regulators of SVCT2 function, modulating pH sensitivity, transporter kinetics, Na+ cooperativity, conformational stability, and subcellular localization.

    PubMed

    Ormazabal, Valeska; Zuñiga, Felipe A; Escobar, Elizabeth; Aylwin, Carlos; Salas-Burgos, Alexis; Godoy, Alejandro; Reyes, Alejandro M; Vera, Juan Carlos; Rivas, Coralia I

    2010-11-19

    Na(+)-coupled ascorbic acid transporter-2 (SVCT2) activity is impaired at acid pH, but little is known about the molecular determinants that define the transporter pH sensitivity. SVCT2 contains six histidine residues in its primary sequence, three of which are exofacial in the transporter secondary structure model. We used site-directed mutagenesis and treatment with diethylpyrocarbonate to identify histidine residues responsible for SVCT2 pH sensitivity. We conclude that five histidine residues, His(109), His(203), His(206), His(269), and His(413), are central regulators of SVCT2 function, participating to different degrees in modulating pH sensitivity, transporter kinetics, Na(+) cooperativity, conformational stability, and subcellular localization. Our results are compatible with a model in which (i) a single exofacial histidine residue, His(413), localized in the exofacial loop IV that connects transmembrane helices VII-VIII defines the pH sensitivity of SVCT2 through a mechanism involving a marked attenuation of the activation by Na(+) and loss of Na(+) cooperativity, which leads to a decreased V(max) without altering the transport K(m); (ii) exofacial histidine residues His(203), His(206), and His(413) may be involved in maintaining a functional interaction between exofacial loops II and IV and influence the general folding of the transporter; (iii) histidines 203, 206, 269, and 413 affect the transporter kinetics by modulating the apparent transport K(m); and (iv) histidine 109, localized at the center of transmembrane helix I, might be fundamental for the interaction of SVCT2 with the transported substrate ascorbic acid. Thus, histidine residues are central regulators of SVCT2 function. PMID:20843809

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

    PubMed

    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

  11. Functional role of residues corresponding to helical domain II (amino acids 35 to 46) of human immunodeficiency virus type 1 Vpr.

    PubMed

    Singh, S P; Tomkowicz, B; Lai, D; Cartas, M; Mahalingam, S; Kalyanaraman, V S; Murali, R; Srinivasan, A

    2000-11-01

    Vpr, encoded by the human immunodeficiency virus type 1 genome, contains 96 amino acids and is a multifunctional protein with features which include cell cycle arrest at G(2), nuclear localization, participation in transport of the preintegration complex, cation channel activity, oligomerization, and interaction with cellular proteins, in addition to its incorporation into the virus particles. Recently, structural studies based on nuclear magnetic resonance and circular dichroism spectroscopy showed that Vpr contains a helix (HI)-turn-helix (HII) core at the amino terminus and an amphipathic helix (HIII) in the middle region. Though the importance of helical domains HI and HIII has been defined with respect to Vpr functions, the role of helical domain HII is not known. To address this issue, we constructed a series of mutants in which the HII domain was altered by deletion, insertion, and/or substitution mutagenesis. To enable the detection of Vpr, the sequence corresponding to the Flag epitope (DYKDDDDK) was added, in frame, to the Vpr coding sequences. Mutants, expressed through the in vitro transcription/translation system and in cells, showed an altered migration corresponding to deletions in Vpr. Substitution mutational analysis of residues in HII showed reduced stability for VprW38S-FL, VprL42G-FL, and VprH45W-FL. An assay involving cotransfection of NLDeltaVpr proviral DNA and a Vpr expression plasmid was employed to analyze the virion incorporation property of Vpr. Mutant Vpr containing deletions and specific substitutions (VprW38S-FL, VprL39G-FL, VprL42G-FL, VprG43P-FL, and VprI46G-FL) exhibited a negative virion incorporation phenotype. Further, mutant Vpr-FL containing deletions also failed to associate with wild-type Vpr, indicating a possible defect in the oligomerization feature of Vpr. Subcellular localization studies indicated that mutants VprDelta35-50-H-FL, VprR36W-FL, VprL39G-FL, and VprI46G-FL exhibited both cytoplasmic and nuclear localization

  12. Functional Role of Residues Corresponding to Helical Domain II (Amino Acids 35 to 46) of Human Immunodeficiency Virus Type 1 Vpr

    PubMed Central

    Singh, Satya P.; Tomkowicz, Brian; Lai, Derhsing; Cartas, Maria; Mahalingam, Sundarasamy; Kalyanaraman, Vaniambadi S.; Murali, Ramachandran; Srinivasan, Alagarsamy

    2000-01-01

    Vpr, encoded by the human immunodeficiency virus type 1 genome, contains 96 amino acids and is a multifunctional protein with features which include cell cycle arrest at G2, nuclear localization, participation in transport of the preintegration complex, cation channel activity, oligomerization, and interaction with cellular proteins, in addition to its incorporation into the virus particles. Recently, structural studies based on nuclear magnetic resonance and circular dichroism spectroscopy showed that Vpr contains a helix (HI)-turn-helix (HII) core at the amino terminus and an amphipathic helix (HIII) in the middle region. Though the importance of helical domains HI and HIII has been defined with respect to Vpr functions, the role of helical domain HII is not known. To address this issue, we constructed a series of mutants in which the HII domain was altered by deletion, insertion, and/or substitution mutagenesis. To enable the detection of Vpr, the sequence corresponding to the Flag epitope (DYKDDDDK) was added, in frame, to the Vpr coding sequences. Mutants, expressed through the in vitro transcription/translation system and in cells, showed an altered migration corresponding to deletions in Vpr. Substitution mutational analysis of residues in HII showed reduced stability for VprW38S-FL, VprL42G-FL, and VprH45W-FL. An assay involving cotransfection of NLΔVpr proviral DNA and a Vpr expression plasmid was employed to analyze the virion incorporation property of Vpr. Mutant Vpr containing deletions and specific substitutions (VprW38S-FL, VprL39G-FL, VprL42G-FL, VprG43P-FL, and VprI46G-FL) exhibited a negative virion incorporation phenotype. Further, mutant Vpr-FL containing deletions also failed to associate with wild-type Vpr, indicating a possible defect in the oligomerization feature of Vpr. Subcellular localization studies indicated that mutants VprΔ35-50-H-FL, VprR36W-FL, VprL39G-FL, and VprI46G-FL exhibited both cytoplasmic and nuclear localization, unlike

  13. Evaluation of local structure alphabets based on residue burial.

    PubMed

    Karchin, Rachel; Cline, Melissa; Karplus, Kevin

    2004-05-15

    Residue burial, which describes a protein residue's exposure to solvent and neighboring atoms, is key to protein structure prediction, modeling, and analysis. We assessed 21 alphabets representing residue burial, according to their predictability from amino acid sequence, conservation in structural alignments, and utility in one fold-recognition scenario. This follows upon our previous work in assessing nine representations of backbone geometry.1 The alphabet found to be most effective overall has seven states and is based on a count of C(beta) atoms within a 14 A-radius sphere centered at the C(beta) of a residue of interest. When incorporated into a hidden Markov model (HMM), this alphabet gave us a 38% performance boost in fold recognition and 23% in alignment quality. PMID:15103615

  14. Evolutionary Divergence of Plant Borate Exporters and Critical Amino Acid Residues for the Polar Localization and Boron-Dependent Vacuolar Sorting of AtBOR1.

    PubMed

    Wakuta, Shinji; Mineta, Katsuhiko; Amano, Taro; Toyoda, Atsushi; Fujiwara, Toru; Naito, Satoshi; Takano, Junpei

    2015-05-01

    Boron (B) is an essential micronutrient for plants but is toxic when accumulated in excess. The plant BOR family encodes plasma membrane-localized borate exporters (BORs) that control translocation and homeostasis of B under a wide range of conditions. In this study, we examined the evolutionary divergence of BORs among terrestrial plants and showed that the lycophyte Selaginella moellendorffii and angiosperms have evolved two types of BOR (clades I and II). Clade I includes AtBOR1 and homologs previously shown to be involved in efficient transport of B under conditions of limited B availability. AtBOR1 shows polar localization in the plasma membrane and high-B-induced vacuolar sorting, important features for efficient B transport under low-B conditions, and rapid down-regulation to avoid B toxicity. Clade II includes AtBOR4 and barley Bot1 involved in B exclusion for high-B tolerance. We showed, using yeast complementation and B transport assays, that three genes in S. moellendorffii, SmBOR1 in clade I and SmBOR3 and SmBOR4 in clade II, encode functional BORs. Furthermore, amino acid sequence alignments identified an acidic di-leucine motif unique in clade I BORs. Mutational analysis of AtBOR1 revealed that the acidic di-leucine motif is required for the polarity and high-B-induced vacuolar sorting of AtBOR1. Our data clearly indicated that the common ancestor of vascular plants had already acquired two types of BOR for low- and high-B tolerance, and that the BOR family evolved to establish B tolerance in each lineage by adapting to their environments. PMID:25619824

  15. Identification of essential residues for the catalytic function of 85-kDa cytosolic phospholipase A2. Probing the role of histidine, aspartic acid, cysteine, and arginine.

    PubMed

    Pickard, R T; Chiou, X G; Strifler, B A; DeFelippis, M R; Hyslop, P A; Tebbe, A L; Yee, Y K; Reynolds, L J; Dennis, E A; Kramer, R M; Sharp, J D

    1996-08-01

    Cytosolic phospholipase A2 (cPLA2) hydrolyzes the sn-2-acyl ester bond of phospholipids and shows a preference for arachidonic acid-containing substrates. We found previously that Ser-228 is essential for enzyme activity and is likely to function as a nucleophile in the catalytic center of the enzyme (Sharp, J. D., White, D. L., Chiou, X. G., Goodson, T., Gamboa, G. C., McClure, D., Burgett, S., Hoskins, J., Skatrud, P. L., Sportsman, J. R., Becker, G. W., Kang, L. H., Roberts, E. F., and Kramer, R. M.(1991) J. Biol. Chem. 266, 14850-14853). cPLA2 contains a catalytic aspartic acid motif common to the subtilisin family of serine proteases. Substitution within this motif of Ala for Asp-549 completely inactivated the enzyme, and substitutions with either glutamic acid or asparagine reduced activity 2000- and 300-fold, respectively. Additionally, using mutants with cysteine replaced by alanine, we found that Cys-331 is responsible for the enzyme's sensitivity to N-ethylmaleimide. Surprisingly, substituting alanine for any of the 19 histidines did not produce inactive enzyme, demonstrating that a classical serine-histidine-aspartate mechanism does not operate in this hydrolase. We found that substituting alanine or histidine for Arg-200 did produce inactive enzyme, while substituting lysine reduced activity 200-fold. Results obtained with the lysine mutant (R200K) and a coumarin ester substrate suggest no specific interaction between Arg-200 and the phosphoryl group of the phospholipid substrate. Arg-200, Ser-228, and Asp-549 are conserved in cPLA2 from six species and also in four nonmammalian phospholipase B enzymes. Our results, supported by circular dichroism, provide evidence that Asp-549 and Arg-200 are critical to the enzyme's function and suggest that the cPLA2 catalytic center is novel. PMID:8702602

  16. Previously unknown species of Aspergillus.

    PubMed

    Gautier, M; Normand, A-C; Ranque, S

    2016-08-01

    The use of multi-locus DNA sequence analysis has led to the description of previously unknown 'cryptic' Aspergillus species, whereas classical morphology-based identification of Aspergillus remains limited to the section or species-complex level. The current literature highlights two main features concerning these 'cryptic' Aspergillus species. First, the prevalence of such species in clinical samples is relatively high compared with emergent filamentous fungal taxa such as Mucorales, Scedosporium or Fusarium. Second, it is clearly important to identify these species in the clinical laboratory because of the high frequency of antifungal drug-resistant isolates of such Aspergillus species. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been shown to enable the identification of filamentous fungi with an accuracy similar to that of DNA sequence-based methods. As MALDI-TOF MS is well suited to the routine clinical laboratory workflow, it facilitates the identification of these 'cryptic' Aspergillus species at the routine mycology bench. The rapid establishment of enhanced filamentous fungi identification facilities will lead to a better understanding of the epidemiology and clinical importance of these emerging Aspergillus species. Based on routine MALDI-TOF MS-based identification results, we provide original insights into the key interpretation issues of a positive Aspergillus culture from a clinical sample. Which ubiquitous species that are frequently isolated from air samples are rarely involved in human invasive disease? Can both the species and the type of biological sample indicate Aspergillus carriage, colonization or infection in a patient? Highly accurate routine filamentous fungi identification is central to enhance the understanding of these previously unknown Aspergillus species, with a vital impact on further improved patient care. PMID:27263029

  17. Americium recovery from reduction residues

    DOEpatents

    Conner, W.V.; Proctor, S.G.

    1973-12-25

    A process for separation and recovery of americium values from container or bomb'' reduction residues comprising dissolving the residues in a suitable acid, adjusting the hydrogen ion concentration to a desired level by adding a base, precipitating the americium as americium oxalate by adding oxalic acid, digesting the solution, separating the precipitate, and thereafter calcining the americium oxalate precipitate to form americium oxide. (Official Gazette)

  18. Protein Thermostability Is Owing to Their Preferences to Non-Polar Smaller Volume Amino Acids, Variations in Residual Physico-Chemical Properties and More Salt-Bridges

    PubMed Central

    Panja, Anindya Sundar; Bandopadhyay, Bidyut; Maiti, Smarajit

    2015-01-01

    Introduction Protein thermostability is an important field for its evolutionary perspective of mesophilic versus thermophilic relationship and for its industrial/ therapeutic applications. Methods Presently, a total 400 (200 thermophilic and 200 mesophilic homologue) proteins were studied utilizing several software/databases to evaluate their amino acid preferences. Randomly selected 50 homologous proteins with available PDB-structure of each group were explored for the understanding of the protein charges, isoelectric-points, hydrophilicity, hydrophobicity, tyrosine phosphorylation and salt-bridge occurrences. These 100 proteins were further probed to generate Ramachandran plot/data for the gross secondary structure prediction in and comparison between the thermophilic and mesophilic proteins. Results Present results strongly suggest that nonpolar smaller volume amino acids Ala (χ2 = 238.54, p<0.001) and Gly (χ2 = 73.35, p<0.001) are highly and Val moderately (χ2 = 144.43, p<0.001) occurring in the 85% of thermophilic proteins. Phospho-regulated Tyr and redox-sensitive Cys are also moderately distributed (χ2~20.0, p<0.01) in a larger number of thermophilic proteins. A consistent lower distribution of thermophilicity and discretely higher distribution of hydrophobicity is noticed in a large number of thermophilic versus their mesophilic protein homolog. The mean differences of isoelectric points and charges are found to be significantly less (7.11 vs. 6.39, p<0.05 and 1 vs. -0.6, p<0.01, respectively) in thermophilic proteins compared to their mesophilic counterpart. The possible sites for Tyr phosphorylation are noticed to be 25% higher (p<0.05) in thermophilic proteins. The 60% thermophiles are found with higher number of salt bridges in this study. The average percentage of salt-bridge of thermophiles is found to be higher by 20% than their mesophilic homologue. The GLU-HIS and GLU-LYS salt-bridge dyads are calculated to be significantly higher (p<0.05 and p

  19. 40 CFR 180.339 - MCPA; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...; tolerances for residues. (a) General. (1) Tolerances are established for residues of the herbicide MCPA ((4... for residues of the herbicide MCPA ((4-chloro-2-methylphenoxy)acetic acid) resulting from the...

  20. 40 CFR 180.339 - MCPA; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...; tolerances for residues. (a) General. (1) Tolerances are established for residues of the herbicide MCPA ((4... for residues of the herbicide MCPA ((4-chloro-2-methylphenoxy)acetic acid) resulting from the...

  1. 40 CFR 180.339 - MCPA; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...; tolerances for residues. (a) General. (1) Tolerances are established for residues of the herbicide MCPA ((4... for residues of the herbicide MCPA ((4-chloro-2-methylphenoxy)acetic acid) resulting from the...

  2. 40 CFR 180.339 - MCPA; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...; tolerances for residues. (a) General. (1) Tolerances are established for residues of the herbicide MCPA ((4... for residues of the herbicide MCPA ((4-chloro-2-methylphenoxy)acetic acid) resulting from the...

  3. 40 CFR 180.339 - MCPA; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...; tolerances for residues. (a) General. (1) Tolerances are established for residues of the herbicide MCPA ((4... for residues of the herbicide MCPA ((4-chloro-2-methylphenoxy)acetic acid) resulting from the...

  4. Differential responses of needle and branch order-based root decay to nitrogen addition: dominant effects of acid-unhydrolyzable residue and microbial enzymes

    NASA Astrophysics Data System (ADS)

    Kou, Liang; Chen, Weiwei; Zhang, Xinyu; Gao, Wenlong; Yang, Hao; Li, Dandan; Li, Shenggong

    2016-04-01

    Both chemical differences between foliage and different orders of fine roots and their contrasting decomposing microenvironments may affect their decomposition. However, little is known about how foliage and branch order-based root decomposition responds to increased N availability and the response mechanisms behind. The effects of different doses of N addition on the decomposition of needles and order-based roots of Pinus elliottii (slash pine) were monitored using the litterbag method for 524 days in a subtropical slash pine plantation in south China. The acid-unhydrolyzable residue (AUR) concentration and microbial extracellular enzymatic activities (EEA) in decomposing needles and roots were also determined. Our results indicate that the responses of needle and order-based root decomposition were N-dose-specific. The decomposition of both needles and lower-order roots was inhibited under the high N dose rate. The retarded decomposition of lower-order roots could be explained more by the increased binding of AUR to inorganic N ions, while the retarded decomposition of needles could be explained more by the reduced microbial EEA. Further, in contrast to lower-order roots, N addition had no effect on the decomposition of higher-order roots. We conclude that the decomposition of foliage and fine roots may fail to mirror each other at ambient conditions or in response to N deposition due to their contrasting decomposition microenvironments and tissue chemistry. Given the differential effects of N addition on order-based roots, our findings highlight the need to consider the tissue chemistry heterogeneity within branching fine root systems when predicting the responses of root decomposition to N loading.

  5. The dapE-encoded N-succinyl-L,L-Diaminopimelic Acid Desuccinylase from Haemophilus influenzae Contains two Active Site Histidine Residues

    PubMed Central

    Gillner, Danuta M.; Bienvenue, David L.; Nocek, Boguslaw P.; Joachimiak, Andrzej; Zachary, Vincentos; Bennett, Brian; Holz, Richard C.

    2009-01-01

    The catalytic and structural properties of the H67A and H349A altered dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from H. influenzae were investigated. Based on sequence alignment with CPG2 both H67 and H349 were predicted to be Zn(II) ligands. Catalytic activity was observed for the H67A altered DapE enzyme which exhibited kcat = 1.5 ± 0.5 sec−1 and Km = 1.4 ± 0.3 mM. No catalytic activity was observed for H349A under the experimental conditions used. The EPR and electronic absorption data indicate that the Co(II) ion bound to H349A-DapE is analogous to WT DapE after the addition of a single Co(II) ion. The addition of one equivalent of Co(II) to H67A altered DapE provides spectra that are very different from the first Co(II) binding site of the WT enzyme, but similar to the second binding site. The EPR and electronic absorption data, in conjunction with the kinetic data, are consistent with the assignment of H67 and H349 as active site metal ligands for the DapE from H. influenzae. Furthermore, the data suggest that H67 is a ligand in the first metal binding site while H349 resides in the second metal binding site. A three-dimensional homology structure of the DapE from H. influenzae was generated using the X-ray crystal structure of the DapE from N. meningitidis as a template and superimposed on the structure of AAP. This homology structure confirms the assignment of H67 and H349 as active site ligands. The superimposition of the homology model of DapE with the dizinc(II) structure of AAP indicates that within 4.0 Å of the Zn(II) binding sites of AAP, all of the amino acid residues of DapE are nearly identical. PMID:18712420

  6. Mapping of the active site of Escherichia coli methionyl-tRNA synthetase: Identification of amino acid residues labeled by periodate-oxidized tRNA sup fMet molecules having modified lengths at the 3 prime -acceptor end

    SciTech Connect

    Hountondji, C.; Schmitter, J.M.; Beauvallet, C.; Blanquet, S. )

    1990-09-04

    Initiator tRNA molecules modified at the 3{prime}-end and lacking either A{sub 76} (tRNA-C{sub 75}), the C{sub 75}-A{sub 76} (tRNA-C{sub 74}), the C{sub 74}-C{sub 75}-A{sub 76} (tRNA-A{sub 73}), or the A{sub 73}-C{sub 74}-C{sub 75}-A{sub 76} (tRNA-A{sub 72}) nucleotides were prepared stepwise by repeated periodate, lysine, and alkaline phosphatase treatments. When incubated with trypsin-modified methionyl-tRNA synthetase (MTS{sub T}), excess amounts of the dialdehyde derivative of each of these shortened tRNAs (tRNA-C{sub 75}ox, tRNA-A{sub 73}ox, and tRNA-A{sub 72}ox) abolished both the isotopic ({sup 32}P)PP{sub i}ATP exchange and the tRNA aminoacylation activities of the enzyme. In the presence of limiting concentrations of the various tRNAox species, the relative extents of inactivation of the enzyme were consistent with the formation of 1:1 complexes of the reacting tRNAs with the monomeric modified synthetase. Specificity of the labeling was further established by demonstrating that tRNA-C{sub 75}ox binds the enzyme with an equilibrium constant and stoichiometry values in good agreement with those for the binding of nonoxidized tRNA-C{sub 75}. The peptides of MTS{sub T} labeled with either tRNA-C{sub 75}ox or tRNA-C{sub 74}ox were identified. In a previous work all these peptides but one (peptide D) had been already found labeled upon MTS{sub T} incubation with ({sup 14}C)tRNA-A{sub 76}ox. According to the crystallographic structure of MTS{sub T}, the labeled residues K335, K61, K142, K147, and K149 are within a sphere of about 5.5-{angstrom} radius. The present results therefore argue for a marked flexibility of the 3{prime}-end of the enzyme-bound tRNA, enabling it to contact any of the identified reacting residues. Such a cluster of basic amino acids may reflect ionic requirements in the guiding of the negatively charged CCA arm of tRNA toward enzyme-bound methionyl-adenylate.

  7. Effect of fly ash, organic wastes and chemical fertilizers on yield, nutrient uptake, heavy metal content and residual fertility in a rice-mustard cropping sequence under acid lateritic soils.

    PubMed

    Rautaray, S K; Ghosh, B C; Mittra, B N

    2003-12-01

    A field experiment was conducted for two years in sandy loam acid lateritic soil to study the direct effect of fly ash, organic wastes and chemical fertilizers on rice (Oryza sativa) and their residual effect on mustard (Brassica napus var glauca) grown in sequence. Rice yields were higher when fly ash, organic wastes and chemical fertilizers were used in an integrated manner as compared to sole application of chemical fertilizers. Yields of mustard were also higher under the residual effect of the former rather than the latter. However, this beneficial residual effect under integrated nutrient sources was inadequate for the mustard crop in the low fertility test soil. Hence, direct application of fertilizers was needed, in addition to residual fertility. The effect of fly ash on mean rice equivalent yield of the rice-mustard cropping sequence was highest (up to 14%) when it was used in combination with organic wastes and chemical fertilizers. While the yield increase was 10% when it was used in combination with only chemical fertilizers. The minimum yield advantage, 3%, occurred when fly ash was applied alone. The equivalent yield of the rice-mustard cropping sequence was equally influenced by either of the organic wastes. Cadmium and Ni content in rice grain and straw were less under the direct effect of fly ash. The residual effect on mustard was similar for Ni content in seed and stover; however, Cd content was increased. Beneficial residual soil chemical properties in terms of pH, organic carbon and available N, P and K were noted for integrated nutrient treatments involved fly ash, organic wastes and chemical fertilizers as compared to continuous use of only chemical fertilizers. Application of fly ash alone was effective in raising soil available P. Thus, integrated use of fly ash, organic wastes and chemical fertilizers was beneficial in improving crop yield, soil pH, organic carbon and available N, P and K in sandy loam acid lateritic soil. PMID:14575950

  8. Conformation-Specific IR and UV Spectroscopy of the Amino Acid Glutamine: Amide-Stacking and Hydrogen Bonding in AN Important Residue in Neurodegenerative Diseases

    NASA Astrophysics Data System (ADS)

    Walsh, Patrick S.; Dean, Jacob C.; Zwier, Timothy S.

    2014-06-01

    Glutamine plays an important role in several neurodegenerative diseases including Huntington's disease (HD) and Alzheimer's disease (AD). An intriguing aspect of the structure of glutamine is its incorporation of an amide group in its side chain, thereby opening up the possibility of forming amide-amide H-bonds between the peptide backbone and side chain. In this study the conformational preferences of two capped gluatamines Z(carboxybenzyl)-Glutamine-X (X=OH, NHMe) are studied under jet-cooled conditions in the gas phase in order to unlock the intrinsic structural motifs that are favored by this flexible sidechain. Conformational assignments are made by comparing the hydride stretch ( 3100-3700 cm-1) and amide I and II ( 1400-1800 cm-1) resonant ion-dip infrared spectra with predictions from harmonic frequency calculations. Assigned structures will be compared to previously published results on both natural and unnatural residues. Particular emphasis will be placed on the comparison between glutamine and unconstrained γ-peptides due to the similar three-carbon spacing between backbone and side chain in glutamine to the backbone spacing in γ-peptides. The ability of the glutamine side-chain to form amide stacked conformations will be a main focus, along with the prevalence of extended backbone type structures. W. H. James, III, C W. Müller, E. G. Buchanan, M. G. D. Nix, L. Guo, L. Roskop, M. S. Gordon, L. V. Slipchenko, S. H. Gellman, and T. S. Zwier, J. Am. Chem. Soc., 2009, 131(40), 14243-14245.

  9. D-amino acid peptide residualizing agents bearing N-hydroxysuccinimido-and maleimido- functional groups and their application for trastuzumab radioiodination

    PubMed Central

    Pruszynski, Marek; Koumarianou, Eftychia; Vaidyanathan, Ganesan; Chitneni, Satish; Zalutsky, Michael R.

    2014-01-01

    Introduction Proteins that undergo receptor-mediated endocytosis are subject to lysosomal degradation, requiring radioiodination methods that minimize loss of radioactivity from tumor cells after this process occurs. To accomplish this, we developed the residualizing radioiodination agent N∊-(3-[*I]iodobenzoyl)-Lys5-Nα-maleimido-Gly1-d-GEEEK (Mal-d-GEEEK-[*I]IB), which enhanced tumor uptake but also increased kidney activity and necessitates generation of sulfhydryl moieties on the protein. The purpose of the current study was to synthesize and evaluate a new d-amino acid based agent that might avoid these potential problems. Methods Nα-(3-iodobenzoyl)-(5-succinimidyloxycarbonyl)-d-EEEG (NHS-IB-d-EEEG), which contains 3 d-glutamates to provide negative charge and a N-hydroxysuccinimide function to permit conjugation to unmodified proteins, and the corresponding tin precursor were produced by solid phase peptide synthesis and subsequent conjugation with appropriate reagents. Radioiodination of the anti-HER2 antibody trastuzumab using NHS-IB-d-EEEG and Mal-d-GEEEK-IB were compared. Paired-label internalization assays on BT474 breast carcinoma cells and biodistribution studies in athymic mice bearing BT474M1 xenografts were performed to evaluate the two radioiodinated d-peptide trastuzumab conjugates. Results NHS-[131I]IB-d-EEEG was produced in 53.8 ± 13.4 % and conjugated to trastuzumab in 39.5 ± 7.6 % yield. Paired-label internalization assays with trastuzumab-NHS-[131I]IB-d-EEEG and trastuzumab-Mal-d-GEEEK-[125I]IB demonstrated similar intracellular trapping for both conjugates at 1 h (131I, 84.4 ± 6.1%; 125I, 88.6 ± 5.2%) through 24 h (131I, 60.7 ± 6.8%; 125I, 64.9 ± 6.9 %). In the biodistribution experiment, tumor uptake peaked at 48 h (trastuzumab-NHS-[131I]IB-d-EEEG, 29.8 ± 3.6 %ID/g; trastuzumab-Mal-d-GEEEK-[125I]IB, 45.3 ± 5.3 %ID/g) and was significantly higher for 125I at all time points. In general, normal tissue levels were lower for

  10. Chemical modifications of therapeutic proteins induced by residual ethylene oxide.

    PubMed

    Chen, Louise; Sloey, Christopher; Zhang, Zhongqi; Bondarenko, Pavel V; Kim, Hyojin; Ren, Da; Kanapuram, Sekhar

    2015-02-01

    Ethylene oxide (EtO) is widely used in sterilization of drug product primary containers and medical devices. The impact of residual EtO on protein therapeutics is of significant interest in the biopharmaceutical industry. The potential for EtO to modify individual amino acids in proteins has been previously reported. However, specific identification of EtO adducts in proteins and the effect of residual EtO on the stability of therapeutic proteins has not been reported to date. This paper describes studies of residual EtO with two therapeutic proteins, a PEGylated form of the recombinant human granulocyte colony-stimulating factor (Peg-GCSF) and recombinant human erythropoietin (EPO) formulated with human serum albumin (HSA). Peg-GCSF was filled in an EtO sterilized delivery device and incubated at accelerated stress conditions. Glu-C peptide mapping and LC-MS analyses revealed residual EtO reacted with Peg-GCSF and resulted in EtO modifications at two methionine residues (Met-127 and Met-138). In addition, tryptic peptide mapping and LC-MS analyses revealed residual EtO in plastic vials reacted with HSA in EPO formulation at Met-328 and Cys-34. This paper details the work conducted to understand the effects of residual EtO on the chemical stability of protein therapeutics. PMID:25407640

  11. Residue determination of glyphosate, glufosinate and aminomethylphosphonic acid in water and soil samples by liquid chromatography coupled to electrospray tandem mass spectrometry.

    PubMed

    Ibáñez, María; Pozo, Oscar J; Sancho, Juan V; López, Francisco J; Hernández, Félix

    2005-07-22

    This paper describes a method for the sensitive and selective determination of glyphosate, glufosinate and aminomethylphosphonic acid (AMPA) residues in water and soil samples. The method involves a derivatization step with 9-fluorenylmethylchloroformate (FMOC) in borate buffer and detection based on liquid chromatography coupled to electrospray tandem mass spectrometry (LC-ESI-MS/MS). In the case of water samples a volume of 10 mL was derivatized and then 4.3 mL of the derivatized mixture was directly injected in an on-line solid phase extraction (SPE)-LC-MS/MS system using an OASIS HLB cartridge column and a Discovery chromatographic column. Soil samples were firstly extracted with potassium hydroxide. After that, the aqueous extract was 10-fold diluted with water and 2 mL were derivatized. Then, 50 microL of the derivatized 10-fold diluted extract were injected into the LC-MS/MS system without pre-concentration into the SPE cartridge. The method has been validated in both ground and surface water by recovery studies with samples spiked at 50 and 500 ng/L, and also in soil samples, spiked at 0.05 and 0.5 mg/kg. In water samples, the mean recovery values ranged from 89 to 106% for glyphosate (RSD <9%), from 97 to 116% for AMPA (RSD < 10%), and from 72 to 88% in the case of glufosinate (RSD < 12%). Regarding soil samples, the mean recovery values ranged from 90 to 92% for glyphosate (RSD <7%), from 88 to 89% for AMPA (RSD <5%) and from 83 to 86% for glufosinate (RSD <6%). Limits of quantification for all the three compounds were 50 ng/L and 0.05 mg/kg in water and soil, respectively, with limits of detection as low as 5 ng/L, in water, and 5 microg/kg, in soil. The use of labelled glyphosate as internal standard allowed improving the recovery and precision for glyphosate and AMPA, while it was not efficient for glufosinate, that was quantified by external standards calibration. The method developed has been applied to the determination of these compounds in real

  12. Cloning of the non-neuronal intermediate filament protein of the gastropod Aplysia californica; identification of an amino acid residue essential for the IFA epitope.

    PubMed

    Riemer, D; Dodemont, H; Weber, K

    1991-12-01

    We describe the isolation and characterization of a full-length cDNA corresponding to the larger non-neuronal (nn) intermediate filament (IF) protein of the gastropod Aplysia californica. Comparison of the sequences of the nn-IF proteins from Aplysia californica and Helix aspersa shows a strong evolutionary drift. At a 72% sequence identity level, the IF proteins of Opisthobranchia and Pulmonata show a larger distance than vimentins from Xenopus and mammals. The sequence comparison of the two snail proteins provides an important step in understanding the epitope of the monoclonal antibody IFA mapped by previous studies to the consensus sequence at the carboxy-terminal end of the rod domain of IF proteins. We identify for the first time in a naturally occurring IF protein a single amino acid exchange which leads to the loss of the epitope. The consensus sequence YRKLLEGEE present in IFA-positive proteins such as the Helix IF protein is changed in the IFA-negative Aplysia protein only by the conservative substitution of the arginine (R) by a lysine (K). Thus, the IFA epitope is not a necessity of IF structure, and its presence or absence on different IF proteins reflects only small changes in an otherwise conserved consensus sequence. Consequently, lack of IFA reactivity does not exclude the presence of IF. This result predicts that IF are much more universally expressed in lower eukaryotes than currently expected from immunological results with the monoclonal antibody IFA. PMID:1724961

  13. Mutation of G234 amino acid residue in candida albicans drug-resistance-related protein Rta2p is associated with fluconazole resistance and dihydrosphingosine transport.

    PubMed

    Zhang, Shi-Qun; Miao, Qi; Li, Li-Ping; Zhang, Lu-Lu; Yan, Lan; Jia, Yu; Cao, Yong-Bing; Jiang, Yuan-Ying

    2015-01-01

    Widespread and repeated use of azoles has led to the rapid development of drug resistance in Candida albicans. Our previous study found Rta2p, a membrane protein with 7 transmembrane domains, was involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans. Conserved amino acids in the transmembrane domain of Rta2p were subjected to site-directed mutagenesis. The sensitivity of C. albicans to fluconazole in vitro was examined by minimum inhibitory concentration and killing assay, and the therapeutic efficacy of fluconazole in vivo was performed by systemic mice candidiasis model. Furthermore, dihydrosphingosine transport activity was detected by NBD labeled D-erythro-dihydrosphingosine uptake and release assay, and the sensitivity to sphingolipid biosynthesis inhibitors. We successfully constructed 14 mutant strains of Rta2p, screened them by minimum inhibitory concentration and found Ca(2+) did not completely induce fluconazole resistance with G158E and G234S mutations. Furthermore, we confirmed that G234S mutant enhanced the therapeutic efficacy of fluconazole against systemic candidiasis and significantly increased the accumulation of dihydrosphingosine by decreasing its release. However, G158E mutant didn't affect drug therapeutic efficacy in vivo and dihydrosphingosine transport in C. albicans. G234 of Rta2p in C. albicans is crucial in calcineurin-mediated fluconazole resistance and dihydrosphingosine transport. PMID:26220356

  14. Mutation of G234 amino acid residue in Candida albicans drug-resistance-related protein Rta2p is associated with fluconazole resistance and dihydrosphingosine transport

    PubMed Central

    Zhang, Shi-Qun; Miao, Qi; Li, Li-Ping; Zhang, Lu-lu; Yan, Lan; Jia, Yu; Cao, Yong-Bing; Jiang, Yuan-Ying

    2015-01-01

    Widespread and repeated use of azoles has led to the rapid development of drug resistance in Candida albicans. Our previous study found Rta2p, a membrane protein with 7 transmembrane domains, was involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans. Conserved amino acids in the transmembrane domain of Rta2p were subjected to site-directed mutagenesis. The sensitivity of C. albicans to fluconazole in vitro was examined by minimum inhibitory concentration and killing assay, and the therapeutic efficacy of fluconazole in vivo was performed by systemic mice candidiasis model. Furthermore, dihydrosphingosine transport activity was detected by NBD labeled D-erythro-dihydrosphingosine uptake and release assay, and the sensitivity to sphingolipid biosynthesis inhibitors. We successfully constructed 14 mutant strains of Rta2p, screened them by minimum inhibitory concentration and found Ca2+ did not completely induce fluconazole resistance with G158E and G234S mutations. Furthermore, we confirmed that G234S mutant enhanced the therapeutic efficacy of fluconazole against systemic candidiasis and significantly increased the accumulation of dihydrosphingosine by decreasing its release. However, G158E mutant didn't affect drug therapeutic efficacy in vivo and dihydrosphingosine transport in C. albicans. G234 of Rta2p in C. albicans is crucial in calcineurin-mediated fluconazole resistance and dihydrosphingosine transport. PMID:26220356

  15. Crop residues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Crop residues [e.g., corn (Zea mays) stover and small grain straw] are sometimes excluded when discussing cellulosic energy crops per se, but because of the vast area upon which they are grown and their current role in the development of cellulosic energy systems. This chapter focuses on current cor...

  16. Amino acid substitutions of conserved residues in the carboxyl-terminal domain of the [alpha]I(X) chain of type X collagen occur in two unrelated families with metaphyseal chondrodysplasia type Schmid

    SciTech Connect

    Wallis, G.A.; Rash, B.; Sweetman, W.A.; Thomas, J.T.; Grant, M.E.; Boot-Handford, R.P. ); Super, M. ); Evans, G. )

    1994-02-01

    Type X collagen is a homotrimeric, short-chain, nonfibrillar extracellular-matrix component that is specifically and transiently synthesized by hypertrophic chondrocytes at the site of endochondral ossification. The precise function of type X collagen is not known, but its specific pattern of expression suggests that mutations within the encoding gene (COL10A1) that alter the structure or synthesis of the protein may cause heritable forms of chondrodysplasia. The authors used the PCR and the SSCP techniques to analyze the coding and upstream promoter regions of the COL10A1 gene in a number of individuals with forms of chondrodysplasia. Using this approach, they identified two individuals with metaphyseal chondrodysplasia type Schmid (MCDS) with SSCP changes in the region of the gene encoding the carboxyl-terminal domain. Sequence analysis demonstrated that the individuals were heterozygous for two unique single-base-pair transitions that led to the substitution of the highly conserved amino acid residue tyrosine at position 598 by aspartic acid in one person and of leucine at position 614 by proline in the other. The substitution at residue 598 segregated with the phenotype in a family of eight (five affected and three unaffected) related persons. The substitutions at residue 614 occurred in a sporadically affected individual but not in her unaffected mother and brother. Additional members of this family were not available for further study. These results suggest that certain amino acid substitutions within the carboxyl-terminal domain of the chains of the type X collagen molecule cause MCDS. These amino acid substitutions are likely to alter either chain recognition or assembly of the type X collagen molecule, thereby depleting the amount of normal type X collagen deposited in the extracellular matrix, with consequent aberrations in bone growth and development. 36 refs., 5 figs.

  17. Key Amino Acid Residues within the Third Membrane Domains of NR1 and NR2 Subunits Contribute to the Regulation of the Surface Delivery of N-methyl-d-aspartate Receptors*

    PubMed Central

    Kaniakova, Martina; Krausova, Barbora; Vyklicky, Vojtech; Korinek, Miloslav; Lichnerova, Katarina; Vyklicky, Ladislav; Horak, Martin

    2012-01-01

    N-methyl-d-aspartate (NMDA) receptors are glutamate ionotropic receptors that play critical roles in synaptic transmission, plasticity, and excitotoxicity. The functional NMDA receptors, heterotetramers composed mainly of two NR1 and two NR2 subunits, likely pass endoplasmic reticulum quality control before they are released from the endoplasmic reticulum and trafficked to the cell surface. However, the mechanism underlying this process is not clear. Using truncated and mutated NMDA receptor subunits expressed in heterologous cells, we found that the M3 domains of both NR1 and NR2 subunits contain key amino acid residues that contribute to the regulation of the number of surface functional NMDA receptors. These key residues are critical neither for the interaction between the NR1 and NR2 subunits nor for the formation of the functional receptors, but rather they regulate the early trafficking of the receptors. We also found that the identified key amino acid residues within both NR1 and NR2 M3 domains contribute to the regulation of the surface expression of unassembled NR1 and NR2 subunits. Thus, our data identify the unique role of the membrane domains in the regulation of the number of surface NMDA receptors. PMID:22711533

  18. RECOVERY OF URANIUM VALUES FROM RESIDUES

    DOEpatents

    Schaap, W.B.

    1959-08-18

    A process is described for the recovery of uranium from insoluble oxide residues resistant to repeated leaching with mineral acids. The residue is treated with gaseous hydrogen fluoride, then with hydrogen and again with hydrogen fluoride, preferably at 500 to 700 deg C, prior to the mineral acid leaching.

  19. A 24-residue presequence localizes human factor B to mitochondria.

    PubMed

    Belogrudov, Grigory I

    2007-05-01

    We reported previously that the human factor B precursor is a 215-amino acid polypeptide, the first 40 amino acid residues of which function as a mitochondrial targeting presequence [G.I. Belogrudov, Y. Hatefi, J. Biol. Chem. 277 (2002) 6097-6103]. Confocal microscopy of live HEK293 cells, transiently transfected with factor B constructs tagged at the C-terminus with green fluorescent protein (GFP) revealed that either a 40- or 25-residue presequence localized factor B to mitochondria. Indirect immunofluorescent labeling of fixed, permeabilized HEK293 cells that were transiently transfected with a construct lacking a presequence, showed diffuse, intracellular staining that was consistent with targeting of ectopically expressed factor B to cellular compartments distinct from the mitochondria. Mutants in which either Met(-25) or both Met(-25)/Met(-24) residues of the presequence were deleted exhibited decreased or undetectable levels, respectively, of the GFP-tagged factor B. The factor B presequence alone was shown to target a reporter polypeptide GFP to mitochondria. Our studies, therefore, demonstrate that a 24-residue presequence is sufficient to localize factor B to mitochondria, and suggest that the human factor B precursor is a 199-amino acid polypeptide. PMID:17359931

  20. Key amino acid residues involved in multi-point binding interactions between brazzein, a sweet protein, and the T1R2-T1R3 human sweet receptor

    PubMed Central

    Assadi-Porter, Fariba M.; Maillet, Emeline L.; Radek, James T.; Quijada, Jeniffer; Markley, John L.; Max, Marianna

    2010-01-01

    The sweet protein brazzein activates the human sweet receptor, a heterodimeric G-protein coupled receptor (GPCR) composed of subunits T1R2 and T1R3. In order to elucidate the key amino acid(s) responsible for this interaction, we mutated residues in brazzein and each of the two subunits of the receptor. The effects of brazzein mutations were assayed by a human taste panel and by an in vitro assay involving receptor subunits expressed recombinantly in human embryonic kidney cells; the effects of the receptor mutations were assayed by the in vitro assay. We mutated surface residues of brazzein at three putative interaction sites: Site 1 (Loop43), Site 2 (N- and C-terminus and adjacent Glu36, Loop33), and Site 3 (Loop9–19). Basic residues in Site 1 and acidic residues in Site 2 were essential for positive responses from each assay. Mutation of Y39A (Site 1) greatly reduced positive responses. A bulky side chain at position 54 (Site 2), rather than a side chain with hydrogen bonding potential, was required for positive responses as was the presence of the native disulfide bond in Loop 9–19 (Site 3). Results from mutagenesis and chimeras of the receptor indicated that brazzein interacts with both T1R2 and T1R3 and that the Venus fly trap module of T1R2 is important for brazzein agonism. With one exception, all mutations of receptor residues at putative interaction sites predicted by wedge models failed to yield the expected decrease in the brazzein response. The exception, hT1R2:R217A-hT1R3, which contained a substitution in lobe 2 at the interface between the two subunits, exhibited a small selective decrease in brazzein activity. However, because the mutation was found to increase the positive cooperativity of binding by multiple ligands proposed to bind both T1R subunits (brazzein, monellin, and sucralose) but not those that bind to a single subunit (neotame and cyclamate), we suggest that this site in involved in subunit-subunit interaction rather than direct

  1. In-frame amber stop codon replacement mutagenesis for the directed evolution of proteins containing non-canonical amino acids: identification of residues open to bio-orthogonal modification.

    PubMed

    Arpino, James A J; Baldwin, Amy J; McGarrity, Adam R; Tippmann, Eric M; Jones, D Dafydd

    2015-01-01

    Expanded genetic code approaches are a powerful means to add new and useful chemistry to proteins at defined residues positions. One such use is the introduction of non-biological reactive chemical handles for site-specific biocompatible orthogonal conjugation of proteins. Due to our currently limited information on the impact of non-canonical amino acids (nAAs) on the protein structure-function relationship, rational protein engineering is a "hit and miss" approach to selecting suitable sites. Furthermore, dogma suggests surface exposed native residues should be the primary focus for introducing new conjugation chemistry. Here we describe a directed evolution approach to introduce and select for in-frame codon replacement to facilitate engineering proteins with nAAs. To demonstrate the approach, the commonly reprogrammed amber stop codon (TAG) was randomly introduced in-frame in two different proteins: the bionanotechnologically important cyt b(562) and therapeutic protein KGF. The target protein is linked at the gene level to sfGFP via a TEV protease site. In absence of a nAA, an in-frame TAG will terminate translation resulting in a non-fluorescent cell phenotype. In the presence of a nAA, TAG will encode for nAA incorporation so instilling a green fluorescence phenotype on E. coli. The presence of endogenously expressed TEV proteases separates in vivo target protein from its fusion to sfGFP if expressed as a soluble fusion product. Using this approach, we incorporated an azide reactive handle and identified residue positions amenable to conjugation with a fluorescence dye via strain-promoted azide-alkyne cycloaddition (SPAAC). Interestingly, best positions for efficient conjugation via SPAAC were residues whose native side chain were buried through analysis of their determined 3D structures and thus may not have been chosen through rational protein engineering. Molecular modeling suggests these buried native residues could become partially exposed on

  2. Second Order Rate Constants of Donor-Strand Exchange Reveal Individual Amino Acid Residues Important in Determining the Subunit Specificity of Pilus Biogenesis

    NASA Astrophysics Data System (ADS)

    Leney, Aneika C.; Phan, Gilles; Allen, William; Verger, Denis; Waksman, Gabriel; Radford, Sheena E.; Ashcroft, Alison E.

    2011-07-01

    P pili are hair-like adhesive structures that are assembled on the outer membrane (OM) of uropathogenic Escherichia coli by the chaperone-usher pathway. In this pathway, chaperone-subunit complexes are formed in the periplasm and targeted to an OM assembly platform, the usher. Pilus subunits display a large groove caused by a missing β-strand which, in the chaperone-subunit complex, is provided by the chaperone. At the usher, pilus subunits are assembled in a mechanism termed "donor-strand exchange (DSE)" whereby the β-strand provided by the chaperone is exchanged by the incoming subunit's N-terminal extension (Nte). This occurs in a zip-in-zip-out fashion, starting with a defined residue, P5, in the Nte inserting into a defined site in the groove, the P5 pocket. Here, electrospray ionization-mass spectrometry (ESI-MS) has been used to measure DSE rates in vitro. Second order rate constants between the chaperone-subunit complex and a range of Nte peptides substituted at different residues confirmed the importance of the P5 residue of the Nte in determining the rate of DSE. In addition, residues either side of the P5 residue (P5 + 1 and P5 - 1), the side-chains of which are directed away from the subunit groove, also modulate the rates of DSE, most likely by aiding the docking of the Nte into the P5 pocket on the accepting subunit prior to DSE. The ESI-MS approach developed is applicable to the measurement of rates of DSE in pilus biogenesis in general and demonstrates the scope of ESI-MS in determining biomolecular processes in molecular detail.

  3. A previously undescribed pathway for pyrimidine catabolism

    PubMed Central

    Loh, Kevin D.; Gyaneshwar, Prasad; Markenscoff Papadimitriou, Eirene; Fong, Rebecca; Kim, Kwang-Seo; Parales, Rebecca; Zhou, Zhongrui; Inwood, William; Kustu, Sydney

    2006-01-01

    The b1012 operon of Escherichia coli K-12, which is composed of seven unidentified ORFs, is one of the most highly expressed operons under control of nitrogen regulatory protein C. Examination of strains with lesions in this operon on Biolog Phenotype MicroArray (PM3) plates and subsequent growth tests indicated that they failed to use uridine or uracil as the sole nitrogen source and that the parental strain could use them at room temperature but not at 37°C. A strain carrying an ntrB(Con) mutation, which elevates transcription of genes under nitrogen regulatory protein C control, could also grow on thymidine as the sole nitrogen source, whereas strains with lesions in the b1012 operon could not. Growth-yield experiments indicated that both nitrogens of uridine and thymidine were available. Studies with [14C]uridine indicated that a three-carbon waste product from the pyrimidine ring was excreted. After trimethylsilylation and gas chromatography, the waste product was identified by mass spectrometry as 3-hydroxypropionic acid. In agreement with this finding, 2-methyl-3-hydroxypropionic acid was released from thymidine. Both the number of available nitrogens and the waste products distinguished the pathway encoded by the b1012 operon from pyrimidine catabolic pathways described previously. We propose that the genes of this operon be named rutA–G for pyrimidine utilization. The product of the divergently transcribed gene, b1013, is a tetracycline repressor family regulator that controls transcription of the b1012 operon negatively. PMID:16540542

  4. Maximization of organic acids production by Aspergillus niger in a bubble column bioreactor for V and Ni recovery enhancement from power plant residual ash in spent-medium bioleaching experiments.

    PubMed

    Rasoulnia, P; Mousavi, S M

    2016-09-01

    Spent-medium bioleaching of V and Ni from a power plant residual ash (PPR ash) was conducted using organic acids produced by Aspergillus niger. The production of organic acids in a bubble column bioreactor was optimized through selecting three most influencing factors. Under optimum condition of aeration rate of 762.5(ml/min), sucrose concentration of 101.9(g/l) and inoculum size of 40(ml/l), respectively 17,185, 4539, 1042 and 502(ppm) of oxalic, gluconic, citric and malic acids were produced. Leaching experiments were carried out using biogenic produced organic acids under leaching environment temperature of 60°C and rotary shaking speed of 135rpm, with various pulp densities of 1, 2, 3, 5, 7 and 9(%w/v). The results showed that biogenic produced organic acids leached V much more efficiently than Ni so that even at high pulp density of 9(%w/v), 83% of V was recovered while Ni recovery yield was 30%. PMID:27295250

  5. Identification of hormone-interacting amino acid residues within the steroid-binding domain of the glucocorticoid receptor in relation to other steroid hormone receptors

    SciTech Connect

    Carlstedt-Duke, J.; Stroemstedt, P.E.; Persson, B.; Cederlund, E.; Gustafsson, J.A.; Joernvall, H.

    1988-05-15

    Purified rat liver glucocorticoid receptor was covalently charged with (/sup 3/H)glucocorticoid by photoaffinity labeling (UV irradiation of (/sup 3/H)triamcinolone acetonide-glucocorticoid receptor) or affinity labeling (incubation with (/sup 3/H)dexamethasone mesylate). After labeling, separate samples of the denatured receptor were cleaved with trypsin (directly or after prior succinylation), chymotrypsin, and cyanogen bromide. Labeled residues in the peptides obtained were identified by radiosequence analysis. The peaks of radioactivity corresponded to Met-622 and Cys-754 after photoaffinity labeling with (/sup 3/H)triamcinolone acetonide and Cys-656 after affinity labeling with (/sup 3/H)dexamethasone mesylate. The labeled residues are all positioned within hydrophobic segments of the steroid-binding domain. The patterns of hydropathy and secondary structure for the glucocorticoid receptor are highly similar to those for the progestin receptor and similar but less so to those for the estrogen receptor and to those for c-erb A.

  6. Characterization of the Functional Roles of Amino Acid Residues in Acceptor-binding Subsite +1 in the Active Site of the Glucansucrase GTF180 from Lactobacillus reuteri 180.

    PubMed

    Meng, Xiangfeng; Pijning, Tjaard; Dobruchowska, Justyna M; Gerwig, Gerrit J; Dijkhuizen, Lubbert

    2015-12-11

    α-Glucans produced by glucansucrase enzymes hold strong potential for industrial applications. The exact determinants of the linkage specificity of glucansucrase enzymes have remained largely unknown, even with the recent elucidation of glucansucrase crystal structures. Guided by the crystal structure of glucansucrase GTF180-ΔN from Lactobacillus reuteri 180 in complex with the acceptor substrate maltose, we identified several residues (Asp-1028 and Asn-1029 from domain A, as well as Leu-938, Ala-978, and Leu-981 from domain B) near subsite +1 that may be critical for linkage specificity determination, and we investigated these by random site-directed mutagenesis. First, mutants of Ala-978 (to Leu, Pro, Phe, or Tyr) and Asp-1028 (to Tyr or Trp) with larger side chains showed reduced degrees of branching, likely due to the steric hindrance by these bulky residues. Second, Leu-938 mutants (except L938F) and Asp-1028 mutants showed altered linkage specificity, mostly with increased (α1 → 6) linkage synthesis. Third, mutation of Leu-981 and Asn-1029 significantly affected the transglycosylation reaction, indicating their essential roles in acceptor substrate binding. In conclusion, glucansucrase product specificity is determined by an interplay of domain A and B residues surrounding the acceptor substrate binding groove. Residues surrounding the +1 subsite thus are critical for activity and specificity of the GTF180 enzyme and play different roles in the enzyme functions. This study provides novel insights into the structure-function relationships of glucansucrase enzymes and clearly shows the potential of enzyme engineering to produce tailor-made α-glucans. PMID:26507662

  7. CO2CRC's Otway Residual Saturation and Dissolution Test: Using Reactive Ester Tracers to Determine Residual CO2 Saturation

    NASA Astrophysics Data System (ADS)

    Myers, M.; Stalker, L.; LaForce, T.; Pejcic, B.; Dyt, C.; Ho, K.; Ennis-King, J.

    2013-12-01

    Residual trapping, that is CO2 held in the rock pore space due to capillarity, is an important storage mechanism in geo-sequestration of over the short to medium term (up to 1000 years). As such residual CO2 saturation is a critical reservoir parameter for assessing the storage capacity and security of carbon capture and storage (CCS). As a component of the CO2CRC's Residual Gas Saturation and Dissolution Test at the CO2CRC Otway Project site in Victoria (Australia), we have recently tested a suite of reactive esters (triacetin, tripropionin and propylene glycol diacetate) in a single well chemical tracer test to determine residual CO2 saturation. The goal of this project was to assess and validate a suite of possible tests that could be implemented to determine residual CO2 saturation. For this test, the chemical tracers were injected with a saturated CO2/water mixture into the formation (that is already at residual CO2 saturation) where they were allowed to 'soak' for approximately 10 days allowing for the partial hydrolysis of the esters to their corresponding carboxylic acids and alcohols. Water containing the tracers was then produced from the well resulting in over 600 tracer samples over a period of 12 hours. A selection of these samples were analysed for tracer content and to establish tracer breakthrough curves. To understand the behaviour of these chemical tracers in the downhole environment containing residually trapped supercritical CO2 and formation water, it is necessary to determine the supercritical CO2/water partition coefficients. We have previously determined these in the laboratory (Myers et al., 2012) and they are used here to model the tracer behaviour and provide an estimate of the residual CO2 saturation. Two different computational simulators were used to analyse the tracer breakthrough profiles. The first is based on simple chromatographic retardation and has been used extensively in single well chemical tracer tests to determine residual

  8. On tide-induced lagrangian residual current and residual transport: 1. Lagrangian residual current

    USGS Publications Warehouse

    Feng, Shizuo; Cheng, Ralph T.; Pangen, Xi

    1986-01-01

    Residual currents in tidal estuaries and coastal embayments have been recognized as fundamental factors which affect the long-term transport processes. It has been pointed out by previous studies that it is more relevant to use a Lagrangian mean velocity than an Eulerian mean velocity to determine the movements of water masses. Under weakly nonlinear approximation, the parameter k, which is the ratio of the net displacement of a labeled water mass in one tidal cycle to the tidal excursion, is assumed to be small. Solutions for tides, tidal current, and residual current have been considered for two-dimensional, barotropic estuaries and coastal seas. Particular attention has been paid to the distinction between the Lagrangian and Eulerian residual currents. When k is small, the first-order Lagrangian residual is shown to be the sum of the Eulerian residual current and the Stokes drift. The Lagrangian residual drift velocity or the second-order Lagrangian residual current has been shown to be dependent on the phase of tidal current. The Lagrangian drift velocity is induced by nonlinear interactions between tides, tidal currents, and the first-order residual currents, and it takes the form of an ellipse on a hodograph plane. Several examples are given to further demonstrate the unique properties of the Lagrangian residual current.

  9. Independent of Their Localization in Protein the Hydrophobic Amino Acid Residues Have No Effect on the Molten Globule State of Apomyoglobin and the Disulfide Bond on the Surface of Apomyoglobin Stabilizes This Intermediate State

    PubMed Central

    Melnik, Tatiana N.; Majorina, Maria A.; Larina, Daria S.; Kashparov, Ivan A.; Samatova, Ekaterina N.; Glukhov, Anatoly S.; Melnik, Bogdan S.

    2014-01-01

    At present it is unclear which interactions in proteins reveal the presence of intermediate states, their stability and formation rate. In this study, we have investigated the effect of substitutions of hydrophobic amino acid residues in the hydrophobic core of protein and on its surface on a molten globule type intermediate state of apomyoglobin. It has been found that independent of their localization in protein, substitutions of hydrophobic amino acid residues do not affect the stability of the molten globule state of apomyoglobin. It has been shown also that introduction of a disulfide bond on the protein surface can stabilize the molten globule state. However in the case of apomyoglobin, stabilization of the intermediate state leads to relative destabilization of the native state of apomyoglobin. The result obtained allows us not only to conclude which mutations can have an effect on the intermediate state of the molten globule type, but also explains why the introduction of a disulfide bond (which seems to “strengthen” the protein) can result in destabilization of the protein native state of apomyoglobin. PMID:24892675

  10. Low resolution X-ray structure of γ-glutamyltranspeptidase from Bacillus licheniformis: opened active site cleft and a cluster of acid residues potentially involved in the recognition of a metal ion.

    PubMed

    Lin, Long-Liu; Chen, Yi-Yu; Chi, Meng-Chun; Merlino, Antonello

    2014-09-01

    γ-Glutamyltranspeptidases (γ-GTs) cleave the γ-glutamyl amide bond of glutathione and transfer the released γ-glutamyl group to water (hydrolysis) or acceptor amino acids (transpeptidation). These ubiquitous enzymes play a key role in the biosynthesis and degradation of glutathione, and in xenobiotic detoxification. Here we report the 3Å resolution crystal structure of Bacillus licheniformis γ-GT (BlGT) and that of its complex with l-Glu. X-ray structures confirm that BlGT belongs to the N-terminal nucleophilic hydrolase superfamily and reveal that the protein possesses an opened active site cleft similar to that reported for the homologous enzyme from Bacillus subtilis, but different from those observed for human γ-GT and for γ-GTs from other microorganisms. Data suggest that the binding of l-Glu induces a reordering of the C-terminal tail of BlGT large subunit and allow the identification of a cluster of acid residues that are potentially involved in the recognition of a metal ion. The role of these residues on the conformational stability of BlGT has been studied by characterizing the autoprocessing, enzymatic activity, chemical and thermal denaturation of four new Ala single mutants. The results show that replacement of Asp568 with an Ala affects both the autoprocessing and structural stability of the protein. PMID:24780583

  11. Two Cytoplasmic Acylation Sites and an Adjacent Hydrophobic Residue, but No Other Conserved Amino Acids in the Cytoplasmic Tail of HA from Influenza A Virus Are Crucial for Virus Replication.

    PubMed

    Siche, Stefanie; Brett, Katharina; Möller, Lars; Kordyukova, Larisa V; Mintaev, Ramil R; Alexeevski, Andrei V; Veit, Michael

    2015-12-01

    Recruitment of the matrix protein M1 to the assembly site of the influenza virus is thought to be mediated by interactions with the cytoplasmic tail of hemagglutinin (HA). Based on a comprehensive sequence comparison of all sequences present in the database, we analyzed the effect of mutating conserved residues in the cytosol-facing part of the transmembrane region and cytoplasmic tail of HA (A/WSN/33 (H1N1) strain) on virus replication and morphology of virions. Removal of the two cytoplasmic acylation sites and substitution of a neighboring isoleucine by glutamine prevented rescue of infectious virions. In contrast, a conservative exchange of the same isoleucine, non-conservative exchanges of glycine and glutamine, deletion of the acylation site at the end of the transmembrane region and shifting it into the tail did not affect virus morphology and had only subtle effects on virus growth and on the incorporation of M1 and Ribo-Nucleoprotein Particles (RNPs). Thus, assuming that essential amino acids are conserved between HA subtypes we suggest that, besides the two cytoplasmic acylation sites (including adjacent hydrophobic residues), no other amino acids in the cytoplasmic tail of HA are indispensable for virus assembly and budding. PMID:26670246

  12. Two Cytoplasmic Acylation Sites and an Adjacent Hydrophobic Residue, but No Other Conserved Amino Acids in the Cytoplasmic Tail of HA from Influenza A Virus Are Crucial for Virus Replication

    PubMed Central

    Siche, Stefanie; Brett, Katharina; Möller, Lars; Kordyukova, Larisa V.; Mintaev, Ramil R.; Alexeevski, Andrei V.; Veit, Michael

    2015-01-01

    Recruitment of the matrix protein M1 to the assembly site of the influenza virus is thought to be mediated by interactions with the cytoplasmic tail of hemagglutinin (HA). Based on a comprehensive sequence comparison of all sequences present in the database, we analyzed the effect of mutating conserved residues in the cytosol-facing part of the transmembrane region and cytoplasmic tail of HA (A/WSN/33 (H1N1) strain) on virus replication and morphology of virions. Removal of the two cytoplasmic acylation sites and substitution of a neighboring isoleucine by glutamine prevented rescue of infectious virions. In contrast, a conservative exchange of the same isoleucine, non-conservative exchanges of glycine and glutamine, deletion of the acylation site at the end of the transmembrane region and shifting it into the tail did not affect virus morphology and had only subtle effects on virus growth and on the incorporation of M1 and Ribo-Nucleoprotein Particles (RNPs). Thus, assuming that essential amino acids are conserved between HA subtypes we suggest that, besides the two cytoplasmic acylation sites (including adjacent hydrophobic residues), no other amino acids in the cytoplasmic tail of HA are indispensable for virus assembly and budding. PMID:26670246

  13. Independent of their localization in protein the hydrophobic amino acid residues have no effect on the molten globule state of apomyoglobin and the disulfide bond on the surface of apomyoglobin stabilizes this intermediate state.

    PubMed

    Melnik, Tatiana N; Majorina, Maria A; Larina, Daria S; Kashparov, Ivan A; Samatova, Ekaterina N; Glukhov, Anatoly S; Melnik, Bogdan S

    2014-01-01

    At present it is unclear which interactions in proteins reveal the presence of intermediate states, their stability and formation rate. In this study, we have investigated the effect of substitutions of hydrophobic amino acid residues in the hydrophobic core of protein and on its surface on a molten globule type intermediate state of apomyoglobin. It has been found that independent of their localization in protein, substitutions of hydrophobic amino acid residues do not affect the stability of the molten globule state of apomyoglobin. It has been shown also that introduction of a disulfide bond on the protein surface can stabilize the molten globule state. However in the case of apomyoglobin, stabilization of the intermediate state leads to relative destabilization of the native state of apomyoglobin. The result obtained allows us not only to conclude which mutations can have an effect on the intermediate state of the molten globule type, but also explains why the introduction of a disulfide bond (which seems to "strengthen" the protein) can result in destabilization of the protein native state of apomyoglobin. PMID:24892675

  14. Residual Cap

    NASA Technical Reports Server (NTRS)

    2006-01-01

    10 May 2006 This Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) image shows a summertime view of the south polar residual cap of Mars. In this image, mesas composed largely of solid carbon dioxide are separated from one another by irregularly-shaped depressions. The variation in brightness across this scene is a function of several factors including, but not limited to, varying proportions of dust and solid carbon dioxide, undulating topography, and differences in the roughness of the slopes versus the flat surfaces.

    Location near: 86.7oS, 343.3oW Image width: 3 km (1.9 mi) Illumination from: upper left Season: Southern Summer

  15. Identification of Conserved Amino Acid Residues of the Salmonella σS Chaperone Crl Involved in Crl-σS Interactions ▿

    PubMed Central

    Monteil, Véronique; Kolb, Annie; D'Alayer, Jacques; Beguin, Pierre; Norel, Françoise

    2010-01-01

    Proteins that bind σ factors typically attenuate the function of the σ factor by restricting its access to the RNA polymerase (RNAP) core enzyme. An exception to this general rule is the Crl protein that binds the stationary-phase sigma factor σS (RpoS) and enhances its affinity for the RNAP core enzyme, thereby increasing expression of σS-dependent genes. Analyses of sequenced bacterial genomes revealed that crl is less widespread and less conserved at the sequence level than rpoS. Seventeen residues are conserved in all members of the Crl family. Site-directed mutagenesis of the crl gene from Salmonella enterica serovar Typhimurium and complementation of a Δcrl mutant of Salmonella indicated that substitution of the conserved residues Y22, F53, W56, and W82 decreased Crl activity. This conclusion was further confirmed by promoter binding and abortive transcription assays. We also used a bacterial two-hybrid system (BACTH) to show that the four substitutions in Crl abolish Crl-σS interaction and that residues 1 to 71 in σS are dispensable for Crl binding. In Escherichia coli, it has been reported that Crl also interacts with the ferric uptake regulator Fur and that Fur represses crl transcription. However, the Salmonella Crl and Fur proteins did not interact in the BACTH system. In addition, a fur mutation did not have any significant effect on the expression level of Crl in Salmonella. These results suggest that the relationship between Crl and Fur is different in Salmonella and E. coli. PMID:20008066

  16. Interplay among side chain sequence, backbone composition, and residue rigidification in polypeptide folding and assembly

    PubMed Central

    Horne, W. Seth; Price, Joshua L.; Gellman, Samuel H.

    2008-01-01

    The extent to which polypeptide conformation depends on side-chain composition and sequence has been widely studied, but less is known about the importance of maintaining an α-amino acid backbone. Here, we examine a series of peptides with backbones that feature different repeating patterns of α- and β-amino acid residues but an invariant side-chain sequence. In the pure α-backbone, this sequence corresponds to the previously studied peptide GCN4-pLI, which forms a very stable four-helix bundle quaternary structure. Physical characterization in solution and crystallographic structure determination show that a variety of α/β-peptide backbones can adopt sequence-encoded quaternary structures similar to that of the α prototype. There is a loss in helix bundle stability upon β-residue incorporation; however, stability of the quaternary structure is not a simple function of β-residue content. We find that cyclically constrained β-amino acid residues can stabilize the folds of α/β-peptide GCN4-pLI analogues and restore quaternary structure formation to backbones that are predominantly unfolded in the absence of cyclic residues. Our results show a surprising degree of plasticity in terms of the backbone compositions that can manifest the structural information encoded in a sequence of amino acid side chains. These findings offer a framework for the design of nonnatural oligomers that mimic the structural and functional properties of proteins. PMID:18587049

  17. Identification of an Amino Acid Domain Encoded by the Capsid Protein Gene of Porcine Circovirus Type 2 that Modulates Viral Protein Distribution During Replication

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous work showed that distinct amino acid motifs are encoded by the Rep, Cap and ORF3 genes of two subgroups of porcine circoviruses (PCV), PCV2a and PCV2b. At a specific location of the gene, a certain amino acid residue or sequence is preferred. Specifically, two amino acid domains located in ...

  18. Tissue deposition and residue depletion in rainbow trout following continuous voluntary feeding with various levels of melamine or a blend of melamine and cyanuric acid.

    PubMed

    Liu, Haiyan; Xue, Min; Wang, Jia; Qiu, Jing; Wu, Xiufeng; Zheng, Yinhua; Li, Junguo; Qin, Yuchang

    2014-11-01

    This study determined the deposition and depletion in rainbow trout after continuous administration of melamine (MEL) alone or a blend of MEL and cyanuric acid (CYA). The plasma, muscles, kidneys, liver and gills were sampled at 0, 3, 7, 13, 21, 28 and 42d. After the final sampling at 42d, fish from the MEL0.05, MEL20 and MCA groups were fed the control diet (MEL0) for the depletion test. Co-administration with cyanuric acid accelerated the deposition time to the Css for melamine; during the withdrawal phrase, the melamine and CYA concentrations in the tissues decreased exponentially. Compared to the t(½) for single oral administration, the t(½) for melamine and cyanuric acid after 42d continuous feeding was prolonged. The presence of trace CYA in the plasma and kidneys of trout was detected in the MEL20 group, indicating that MEL can convert into CYA in rainbow trout. PMID:25038476

  19. Systematic Analysis of the Amino Acid Residues of Human Papillomavirus Type 16 E7 Conserved Region 3 Involved in Dimerization and Transformation ▿

    PubMed Central

    Todorovic, Biljana; Massimi, Paola; Hung, Katherine; Shaw, Gary S.; Banks, Lawrence; Mymryk, Joe S.

    2011-01-01

    The human papillomavirus (HPV) E7 oncoprotein exists as a dimer and acts by binding to many cellular factors, preventing or retargeting their function and thereby making the infected cell conducive for viral replication. Dimerization of E7 is attributed primarily to the C-terminal domain, referred to as conserved region 3 (CR3). CR3 is highly structured and is necessary for E7's transformation ability. It is also required for binding of numerous E7 cellular targets. To systematically analyze the molecular mechanisms by which HPV16 E7 CR3 contributes to carcinogenesis, we created a comprehensive panel of mutations in residues predicted to be exposed on the surface of CR3. We analyzed our novel collection of mutants, as well as mutants targeting predicted hydrophobic core residues of the dimer, for the ability to dimerize. The same set of mutants was also assessed functionally for transformation capability in a baby rat kidney cell assay in conjugation with activated ras. We show that some mutants of HPV16 E7 CR3 failed to dimerize yet were still able to transform baby rat kidney cells. Our results identify several novel E7 mutants that abrogate transformation and also indicate that E7 does not need to exist as a stable dimer in order to transform cells. PMID:21775462

  20. Efficient identification of photolabelled amino acid residues by combining immunoaffinity purification with MS: revealing the semotiadil-binding site and its relevance to binding sites for myristates in domain III of human serum albumin.

    PubMed Central

    Kawahara, Kohichi; Kuniyasu, Akihiko; Masuda, Katsuyoshi; Ishiguro, Masaji; Nakayama, Hitoshi

    2002-01-01

    To identify photoaffinity-labelled amino acid residue(s), we devised an effective method utilizing immunoaffinity purification of photolabelled fragments, followed by matrix-assisted laser-desorption ionization-time of flight (MALDI-TOF) MS and nanoelectrospray ionization tandem MS (nano-ESI-MS/MS) analysis. Human serum albumin (HSA) was photolabelled with an azidophenyl derivative of semotiadil, FNAK [(+)-(R)-3,4-dihydro-2-[5-methoxy-2-[3-[N-methyl-N-[2-(3-azidophenoxy)-ethyl]amino]propoxyl]phenyl]-4-methyl-2H-1,4-benzothiazin-3-(4H)-one], since HSA is a major binding protein for semotiadil in serum. After lysyl endopeptidase digestion, photolabelled HSA fragments were adsorbed selectively on to Sepharose beads on which an anti-semotiadil antibody was immobilized, and fractions were eluted quantitatively by 50% acetonitrile/10 mM HCl. MALDI-TOF MS analysis of the eluted fraction showed that it contained two photolabelled fragments of m/z 2557.54 (major) and 1322.44 (minor), corresponding to Lys-414-Lys-432 and Ala-539-Lys-545, respectively. Further nano-ESI-MS/MS analysis revealed that Lys-414 was the photolabelled amino acid residue in fragment 414-432 and Lys-541 was a likely candidate in fragment 539-545. Based on the photolabelling results, we constructed a three-dimensional model of the FNAK-HSA complex, revealing that FNAK resides in a pocket that overlaps considerably with myristate (Myr)-binding sites, Myr-3 and -4, by comparison with crystallographic data of HSA-Myr complexes described in Curry, Mandelkow, Brick and Franks (1998) Nat. Struct. Biol. 5, 827-835. Moreover, addition of Myr increased photo-incorporation into Lys-414, whereas incorporation into Lys-541 decreased under conditions of [Myr]/[HSA]<1. Further addition of Myr, however, uniformly decreased photo-incorporation into both Lys residues. These results indicate that FNAK labelling can also be used to monitor Myr binding in domain III. An interpretation for the concomitant local

  1. Separation and quantitation of three acidic herbicide residues in tobacco and soil by dispersive solid-phase extraction and UPLC-MS/MS.

    PubMed

    Xiong, Wei; Tao, Xiaoqiu; Pang, Su; Yang, Xue; Tang, GangLing; Bian, Zhaoyang

    2014-01-01

    A method for the determination of three acidic herbicides, dicamba, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) in tobacco and soil has been developed based on the use of liquid-liquid extraction and dispersive solid-phase extraction (dispersive-SPE) followed by UPLC-MS/MS. Two percentage of (v/v) formic acid in acetonitrile as the extraction helped partitioning of analytes into the acetonitrile phase. The extract was then cleaned up by dispersive-SPE using primary secondary amine as selective sorbents. Quantitative analysis was done in the multiple-reaction monitoring mode using stable isotope-labeled internal standards for each compound. A separate internal standard for each analyte is required to minimize sample matrix effects on each analyte, which can lead to poor analyte recoveries and decreases in method accuracy and precision. The total analysis time was <4 min. The linear range of the method was from 1 to 100 ng mL(-1) with a limit of detection of each herbicide varied from 0.012 to 0.126 ng g(-1). The proposed method is faster, more sensitive and selective than the traditional methods and more accurate and robust than the published LC-MS/MS methods. PMID:24366907

  2. Amino acid substitutions of cysteine residues near the amino terminus of Wheat streak mosaic virus HC-Pro abolishes virus transmission by the wheat curl mite

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The amino-terminal half of HC-Pro of Wheat streak mosaic virus (WSMV) is required for semi-persistent transmission by the wheat curl mite (Aceria tosichella Keifer). The amino-proximal region of WSMV HC-Pro is cysteine-rich with a zinc finger-like motif. Amino acid substitutions were made in this re...

  3. The C-terminal 18 Amino Acid Region of Dengue Virus NS5 Regulates its Subcellular Localization and Contains a Conserved Arginine Residue Essential for Infectious Virus Production.

    PubMed

    Tay, Moon Y F; Smith, Kate; Ng, Ivan H W; Chan, Kitti W K; Zhao, Yongqian; Ooi, Eng Eong; Lescar, Julien; Luo, Dahai; Jans, David A; Forwood, Jade K; Vasudevan, Subhash G

    2016-09-01

    Dengue virus NS5 is the most highly conserved amongst the viral non-structural proteins and is responsible for capping, methylation and replication of the flavivirus RNA genome. Interactions of NS5 with host proteins also modulate host immune responses. Although replication occurs in the cytoplasm, an unusual characteristic of DENV2 NS5 is that it localizes to the nucleus during infection with no clear role in replication or pathogenesis. We examined NS5 of DENV1 and 2, which exhibit the most prominent difference in nuclear localization, employing a combination of functional and structural analyses. Extensive gene swapping between DENV1 and 2 NS5 identified that the C-terminal 18 residues (Cter18) alone was sufficient to direct the protein to the cytoplasm or nucleus, respectively. The low micromolar binding affinity between NS5 Cter18 and the nuclear import receptor importin-alpha (Impα), allowed their molecular complex to be purified, crystallised and visualized at 2.2 Å resolution using x-ray crystallography. Structure-guided mutational analysis of this region in GFP-NS5 clones of DENV1 or 2 and in a DENV2 infectious clone reveal residues important for NS5 subcellular localization. Notably, the trans conformation adopted by Pro-884 allows proper presentation for binding Impα and mutating this proline to Thr, as present in DENV1 NS5, results in mislocalizaion of NS5 to the cytoplasm without compromising virus fitness. In contrast, a single mutation to alanine at NS5 position R888, a residue conserved in all flaviviruses, resulted in a completely non-viable virus, and the R888K mutation led to a severely attenuated phentoype, even though NS5 was located in the nucleus. R888 forms a hydrogen bond with Y838 that is also conserved in all flaviviruses. Our data suggests an evolutionarily conserved function for NS5 Cter18, possibly in RNA interactions that are critical for replication, that is independent of its role in subcellular localization. PMID:27622521

  4. 40 CFR 180.447 - Imazethapyr; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... of the herbicide imazethapyr, 2- -5-ethyl-3-pyridine carboxylic acid, applied as its acid or ammonium... 21, 2003. (2) Tolerances are established for the sum of the residues of the herbicide imazethapyr, 2... sum of residues of the herbicide imazethapyr, 2- -5-ethyl-3-pyridine carboxylic acid, and...

  5. 40 CFR 180.447 - Imazethapyr; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... of the herbicide imazethapyr, 2- -5-ethyl-3-pyridine carboxylic acid, applied as its acid or ammonium... 21, 2003. (2) Tolerances are established for the sum of the residues of the herbicide imazethapyr, 2... sum of residues of the herbicide imazethapyr, 2- -5-ethyl-3-pyridine carboxylic acid, and...

  6. 40 CFR 180.441 - Quizalofop ethyl; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... combined residues of the herbicide quizalofop (2- propanoic acid) and quizalofop ethyl (ethyl-2- propanoate...) Tolerances are established for the combined residues of the herbicide quizalofop (2- propanoic acid... herbicide quizalofop-p ethyl ester , and its acid metabolite quizalofop-p , and the S enantiomers of...

  7. 40 CFR 180.447 - Imazethapyr; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... of the herbicide imazethapyr, 2- -5-ethyl-3-pyridine carboxylic acid, applied as its acid or ammonium... 21, 2003. (2) Tolerances are established for the sum of the residues of the herbicide imazethapyr, 2... sum of residues of the herbicide imazethapyr, 2- -5-ethyl-3-pyridine carboxylic acid, and...

  8. 40 CFR 180.447 - Imazethapyr; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... of the herbicide imazethapyr, 2- -5-ethyl-3-pyridine carboxylic acid, applied as its acid or ammonium... 21, 2003. (2) Tolerances are established for the sum of the residues of the herbicide imazethapyr, 2... sum of residues of the herbicide imazethapyr, 2- -5-ethyl-3-pyridine carboxylic acid, and...

  9. 40 CFR 180.447 - Imazethapyr; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... of the herbicide imazethapyr, 2- -5-ethyl-3-pyridine carboxylic acid, applied as its acid or ammonium... 21, 2003. (2) Tolerances are established for the sum of the residues of the herbicide imazethapyr, 2... sum of residues of the herbicide imazethapyr, 2- -5-ethyl-3-pyridine carboxylic acid, and...

  10. Accessibility and mobility of lysine residues in. beta. -lactoglobulin

    SciTech Connect

    Brown, E.M.; Pfeffer, P.E.; Kumosinski, T.F.; Greenberg, R.

    1988-07-26

    N/sup epsilon/-(/sup 2/H/sub 6/)Isopropyllysyl-..beta..-lactoglobulin was prepared by reductive alkylation of ..beta..-lactoglobulin with (/sup 2/H/sub 6/)acetone and NaBH/sub 4/ to provide a /sup 2/H (NMR) probe for the study of lysine involvement in lipid-protein interactions. Amino acid analysis showed 80% of the protein's 15 lysine residues to be labeled. Unmodified lysine residues were located through peptide maps produced from CNBr, tryptic, and chymotryptic digests of the labeled protein. Average correlation times calculated from /sup 2/H NMR spectra were 20 and 320 ps for 8.7 and 3.3 residues, respectively, in 6 M guanidine hydrochloride; in nondenaturing solution, values of 70 and 320 ps were obtained for 6.5 and 3.2 residues, respectively, with the remaining 2.3 modified residues not observed, suggesting that side chains of lysine residues in unordered or flexible regions were more mobile than those in stable periodic structures. /sup 2/H NMR spectra of the protein complexed with dipalmitoylphosphatidylcholine confirmed the extrinsic membrane protein type behavior of ..beta..-lactoglobulin previously reported from /sup 31/P NMR studies of the phospholipids complexed with ..beta..-lactoglobulin. Although no physiological function has yet been identified, comparison of these results with the X-ray structure supports the hypothesis that residues not accessible for modification may help to stabilize the cone-shaped ..beta..-barrel thought to contain binding sites for small lipid-soluble molecules.

  11. Amino-Acid Sequence of Porcine Pepsin

    PubMed Central

    Tang, J.; Sepulveda, P.; Marciniszyn, J.; Chen, K. C. S.; Huang, W-Y.; Tao, N.; Liu, D.; Lanier, J. P.

    1973-01-01

    As the culmination of several years of experiments, we propose a complete amino-acid sequence for porcine pepsin, an enzyme containing 327 amino-acid residues in a single polypeptide chain. In the sequence determination, the enzyme was treated with cyanogen bromide. Five resulting fragments were purified. The amino-acid sequence of four of the fragments accounted for 290 residues. Because the structure of a 37-residue carboxyl-terminal fragment was already known, it was not studied. The alignment of these fragments was determined from the sequence of methionyl-peptides we had previously reported. We also discovered the locations of activesite aspartyl residues, as well as the pairing of the three disulfide bridges. A minor component of commercial crystalline pepsin was found to contain two extra amino-acid residues, Ala-Leu-, at the amino-terminus of the molecule. This minor component was apparently derived from a different site of cleavage during the activation of porcine pepsinogen. PMID:4587252

  12. Effect of Suberoylanilide Hydroxamic Acid (SAHA) Administration on the Residual Virus Pool in a Model of Combination Antiretroviral Therapy-Mediated Suppression in SIVmac239-Infected Indian Rhesus Macaques

    PubMed Central

    Del Prete, Gregory Q.; Shoemaker, Rebecca; Oswald, Kelli; Lara, Abigail; Trubey, Charles M.; Fast, Randy; Schneider, Douglas K.; Kiser, Rebecca; Coalter, Vicky; Wiles, Adam; Wiles, Rodney; Freemire, Brandi; Keele, Brandon F.; Estes, Jacob D.; Quiñones, Octavio A.; Smedley, Jeremy; Macallister, Rhonda; Sanchez, Rosa I.; Wai, John S.; Tan, Christopher M.; Alvord, W. Gregory; Hazuda, Daria J.; Piatak, Michael

    2014-01-01

    Nonhuman primate models are needed for evaluations of proposed strategies targeting residual virus that persists in HIV-1-infected individuals receiving suppressive combination antiretroviral therapy (cART). However, relevant nonhuman primate (NHP) models of cART-mediated suppression have proven challenging to develop. We used a novel three-class, six-drug cART regimen to achieve durable 4.0- to 5.5-log reductions in plasma viremia levels and declines in cell-associated viral RNA and DNA in blood and tissues of simian immunodeficiency virus SIVmac239-infected Indian-origin rhesus macaques, then evaluated the impact of treatment with the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA; Vorinostat) on the residual virus pool. Ex vivo SAHA treatment of CD4+ T cells obtained from cART-suppressed animals increased histone acetylation and viral RNA levels in culture supernatants. cART-suppressed animals each received 84 total doses of oral SAHA. We observed SAHA dose-dependent increases in acetylated histones with evidence for sustained modulation as well as refractoriness following prolonged administration. In vivo virologic activity was demonstrated based on the ratio of viral RNA to viral DNA in peripheral blood mononuclear cells, a presumptive measure of viral transcription, which significantly increased in SAHA-treated animals. However, residual virus was readily detected at the end of treatment, suggesting that SAHA alone may be insufficient for viral eradication in the setting of suppressive cART. The effects observed were similar to emerging data for repeat-dose SAHA treatment of HIV-infected individuals on cART, demonstrating the feasibility, utility, and relevance of NHP models of cART-mediated suppression for in vivo assessments of AIDS virus functional cure/eradication approaches. PMID:25182644

  13. Profiling and characterisation by liquid chromatography/multi-stage mass spectrometry of the chlorogenic acids in Gardeniae Fructus.

    PubMed

    Clifford, Michael N; Wu, Weiguo; Kirkpatrick, Jo; Jaiswal, Rakesh; Kuhnert, Nikolai

    2010-11-15

    The chlorogenic acids of Gardeniae Fructus used traditionally as a Chinese herbal medicine (zhizi) have been investigated qualitatively by liquid chromatography/multi-stage mass spectrometry (LC/MS(4)). Twenty-nine chlorogenic acids were detected and twenty-five characterised to regioisomer level on the basis of their fragmentation, twenty-four for the first time from this source. Assignment to the level of individual regioisomers was possible for three caffeoylquinic acids, three dicaffeoylquinic acids, three sinapoylquinic acids, four caffeoyl-sinapoylquinic acids, two feruloyl-sinapoylquinic acids, one p-coumaroyl-sinapoylquinic acid, three (3-hydroxy, 3-methyl)glutaroylquinic acids, two (3-hydroxy, 3-methyl)glutaroyl-feruloylquinic acids, one (3-hydroxy, 3-methyl)glutaroyl-dicaffeoylquinic acid, and one (3-hydroxy, 3-methyl)glutaroyl-caffeoyl-feruloylquinic acid. Six (3-hydroxy, 3-methyl)glutaroyl-caffeoylquinic acids were detected and two were tentatively assigned as 3-caffeoyl-4-(3-hydroxy, 3-methyl)glutaroylquinic acid and 3-caffeoyl-5-(3-hydroxy, 3-methyl)glutaroylquinic acid. The (3-hydroxy, 3-methyl)glutaroyl residue modifies the mass spectral fragmentation behavior and elution sequence compared with the chlorogenic acids that contain only a cinnamic acid residue(s). Fourteen of these twenty-nine chlorogenic acids have not previously been reported from any source. PMID:20941757

  14. Amino Acid Residues 489–503 of Dihydropyridine Receptor (DHPR) β1a Subunit Are Critical for Structural Communication between the Skeletal Muscle DHPR Complex and Type 1 Ryanodine Receptor*

    PubMed Central

    Eltit, Jose M.; Franzini-Armstrong, Clara; Perez, Claudio F.

    2014-01-01

    The β1a subunit is a cytoplasmic component of the dihydropyridine receptor (DHPR) complex that plays an essential role in skeletal muscle excitation-contraction (EC) coupling. Here we investigate the role of the C-terminal end of this auxiliary subunit in the functional and structural communication between the DHPR and the Ca2+ release channel (RyR1). Progressive truncation of the β1a C terminus showed that deletion of amino acid residues Gln489 to Trp503 resulted in a loss of depolarization-induced Ca2+ release, a severe reduction of L-type Ca2+ currents, and a lack of tetrad formation as evaluated by freeze-fracture analysis. However, deletion of this domain did not affect expression/targeting or density (Qmax) of the DHPR-α1S subunit to the plasma membrane. Within this motif, triple alanine substitution of residues Leu496, Leu500, and Trp503, which are thought to mediate direct β1a-RyR1 interactions, weakened EC coupling but did not replicate the truncated phenotype. Therefore, these data demonstrate that an amino acid segment encompassing sequence 489QVQVLTSLRRNLSFW503 of β1a contains critical determinant(s) for the physical link of DHPR and RyR1, further confirming a direct correspondence between DHPR positioning and DHPR/RyR functional interactions. In addition, our data strongly suggest that the motif Leu496-Leu500-Trp503 within the β1a C-terminal tail plays a nonessential role in the bidirectional DHPR/RyR1 signaling that supports skeletal-type EC coupling. PMID:25384984

  15. Amino acid residues that flank core peptide epitopes and the extracellular domains of CD4 modulate differential signaling through the T cell receptor

    PubMed Central

    1994-01-01

    Hen egg lysozyme 52-61-specific CD4+ T cells responded by interleukin 2 (IL-2) secretion to any peptide containing this epitope regardless of length of NH2- and COOH-terminal composition. However, CD4- variants could only respond to peptides containing the two COOH-terminal tryptophans at positions 62 and 63. Substitutions at these positions defined patterns of reactivity that were specific for individual T cells inferring a T cell receptor (TCR)-based phenomenon. Thus, the fine specificity of major histocompatibility complex (MHC)-peptide recognition by the TCR was dramatically affected by CD4 and the COOH- terminal peptide composition. Peptides that failed to induce IL-2 secretion in the CD4- variants nevertheless induced strong tyrosine phosphorylation of CD3 zeta. Thus, whereas the TCR still recognized and bound to the MHC class II-peptide complex resulting in protein phosphorylation, this interaction failed to induce effective signal transduction manifested by IL-2 secretion. This provides a clear example of differential signaling mediated by peptides known to be naturally processed. In addition, the external domains of CD4, rather than its cytoplasmic tail, were critical in aiding TCR recognition of all peptides derived from a single epitope. These data suggest that the nested flanking residues, which are present on MHC class II but not class I bound peptides, are functionally relevant. PMID:7515103

  16. Introduction of {alpha}-hydroxymethyamino acid residues in substrate specificity P1 position of trypsin inhibitor SFTI-1 from sunflower seeds retains its activity

    SciTech Connect

    Zablotna, Ewa; Kret, Agnieszka; Jaskiewicz, Anna; Olma, Aleksandra; Leplawy, Miroslaw T.; Rolka, Krzysztof . E-mail: krzys@chem.univ.gda.pl

    2006-02-17

    In many complexes formed by serine proteinases and their inhibitors, the hydroxyl group provided by water molecule or by the inhibitor Ser residue is located close to the inhibitor P{sub 1}-P{sub 1}{sup '} reactive site. In order to investigate the role of this group, we synthesized analogues of trypsin inhibitor SFTI-1 isolated from the seeds of sunflower modified in P{sub 1} by {alpha}-hydroxymethylserine (HmSer) and both enantiomers of {alpha}-hydroxymethylvaline (HmVal). All the synthesized analogues inhibited bovine {beta}-trypsin and human leukocyte elastase. SFTI-1 analogues with HmVal and HmSer appear to be potent inhibitors of bovine {beta}-trypsin, whereas [Val{sup 5}]SFTI-1 is practically inactive. Also trypsin inhibitory activity of [Ser{sup 5}]SFTI-1 is significantly lower. Since the electrostatic interaction between protonated {epsilon}-NH{sub 2} group of the inhibitor P{sub 1} position and {beta}-carboxylate of trypsin Asp{sup 189} is the main driving force for interaction of both molecules, the results obtained are very interesting. We believe that these SFTI-1 analogues belong to a novel class of serine proteinase inhibitors.

  17. Identification of key neoculin residues responsible for the binding and activation of the sweet taste receptor

    PubMed Central

    Koizumi, Taichi; Terada, Tohru; Nakajima, Ken-ichiro; Kojima, Masaki; Koshiba, Seizo; Matsumura, Yoshitaka; Kaneda, Kohei; Asakura, Tomiko; Shimizu-Ibuka, Akiko; Abe, Keiko; Misaka, Takumi

    2015-01-01

    Neoculin (NCL) is a heterodimeric protein isolated from the edible fruit of Curculigo latifolia. It exerts a taste-modifying activity by converting sourness to sweetness. We previously demonstrated that NCL changes its action on the human sweet receptor hT1R2-hT1R3 from antagonism to agonism as the pH changes from neutral to acidic values, and that the histidine residues of NCL molecule play critical roles in this pH-dependent functional change. Here, we comprehensively screened key amino acid residues of NCL using nuclear magnetic resonance (NMR) spectroscopy and alanine scanning mutagenesis. We found that the mutations of Arg48, Tyr65, Val72 and Phe94 of NCL basic subunit increased or decreased both the antagonist and agonist activities. The mutations had only a slight effect on the pH-dependent functional change. These residues should determine the affinity of NCL for the receptor regardless of pH. Their locations were separated from the histidine residues responsible for the pH-dependent functional change in the tertiary structure. From these results, we concluded that NCL interacts with hT1R2-hT1R3 through a pH-independent affinity interface including the four residues and a pH-dependent activation interface including the histidine residues. Thus, the receptor activation is induced by local structural changes in the pH-dependent interface. PMID:26263392

  18. Gene cloning of cold-adapted isocitrate lyase from a psychrophilic bacterium, Colwellia psychrerythraea, and analysis of amino acid residues involved in cold adaptation of this enzyme.

    PubMed

    Sato, Yuhya; Watanabe, Seiya; Yamaoka, Naoto; Takada, Yasuhiro

    2008-01-01

    The gene (icl) encoding cold-adapted isocitrate lyase (ICL) of a psychrophilic bacterium, Colwellia psychrerythraea, was cloned and sequenced. Open reading frame of the gene was 1,587 bp in length and corresponded to a polypeptide composed of 528 amino acids. The deduced amino acid sequence showed high homology with that of cold-adapted ICL from other psychrophilic bacterium, C. maris (88% identity), but the sequential homology with that of the Escherichia coli ICL was low (28% identity). Primer extension analysis revealed that transcriptional start site for the C. psychrerythraea icl gene was guanine, located at 87 bases upstream of translational initiation codon. The expression of this gene in the cells of an E. coli mutant defective in ICL was induced by not only low temperature but also acetate. However, cis-acting elements for cold-inducible expression known in the several other bacterial genes were absent in the promoter region of the C. psychrerythraea icl gene. The substitution of Ala214 for Ser in the C. psychrerythraea ICL introduced by point mutation resulted in the increased thermostability and lowering of the specific activity at low temperature, indicating that Ala214 is important for psychrophilic properties of this enzyme. PMID:17965824

  19. Thermal and thermo-chemical pre-treatment of four waste residues and the effect on acetic acid production and methane synthesis.

    PubMed

    Strong, P J; Gapes, D J

    2012-09-01

    In this study four diverse solid waste substrates (coal, Kraft pulp solids, chicken feathers and chicken processing waste) were thermally pre-treated (70, 140 and 200 °C), under an inert (nitrogen) or oxidative (oxygen) atmosphere, and then anaerobically digested. Membrane inlet mass spectrometry during the thermal and thermo-chemical reactions was successfully used to establish oxygen and carbon dioxide gas fluxes and product formation (acetic acid). There was significant solids hydrolysis pre-treatment at 200 °C under an oxidative atmosphere, as indicated by a decrease in the volatile suspended solids and an increase in dissolved organic carbon. Greater concentrations of volatile fatty acids were produced under oxidative conditions at higher temperatures. The methane yield more than tripled for feathers after pre-treatment at 140 °C (under both atmospheres), but decreased after oxidative pre-treatment at 200 °C, due to the destruction of available carbon by the thermo-chemical reaction. Methane yield more than doubled for the Kraft pulp solids with the 200 °C pre-treatment under oxidative conditions. This study illustrated the power of wet oxidation for solids destruction and its potential to improve methane yields generated during anaerobic digestion. PMID:22609530

  20. Solder Flux Residues and Humidity-Related Failures in Electronics: Relative Effects of Weak Organic Acids Used in No-Clean Flux Systems

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

    This paper presents the results of humidity testing of weak organic acids (WOAs), namely adipic, succinic, glutaric, dl-malic, and palmitic acids, which are commonly used as activators in no-clean solder fluxes. The study was performed under humidity conditions varying from 60% relative humidity (RH) to ˜99%RH at 25°C. The following parameters were used for characterization of WOAs: mass gain due to water adsorption and deliquescence of the WOA (by quartz crystal microbalance), resistivity of the water layer formed on the printed circuit board (by impedance spectroscopy), and leakage current measured using the surface insulation resistance pattern in the potential range from 0 V to 10 V. The combined results indicate the importance of the WOA chemical structure for the water adsorption and therefore conductive water layer formation on the printed circuit board assembly (PCBA). A substantial increase of leakage currents and probability of electrochemical migration was observed at humidity levels above the RH corresponding to the deliquescence point of WOAs present as contaminants on the printed circuit boards. The results suggest that use of solder fluxes with WOAs having higher deliquescence point could improve the reliability of electronics operating under circumstances in which exposure to high humidity is likely to occur.

  1. Identification of amino acid residues of the coat protein of Sri Lankan cassava mosaic virus affecting symptom production and viral titer in Nicotiana benthamiana.

    PubMed

    Kelkar, Vaishali; Kushawaha, Akhilesh Kumar; Dasgupta, Indranil

    2016-06-01

    Sri Lankan cassava mosaic virus (SLCMV) is bipartite begomovirus infecting cassava in India and Sri Lanka. Interestingly, the DNA-A component of the SLCMV alone is able to infect Nicotiana benthamiana causing symptoms of upward leaf rolling and stunting. One of the differences between monopartite and bipartite begomoviruses is the requirement of Coat Protein (CP) for infectivity; CP being essential for the former, but dispensable in the latter. This investigation was aimed to determine the importance of CP in the infectivity of the bipartite SLCMV, behaving as a monopartite virus in N. benthamiana. We tested CP-null mutants, single amino acid replacement mutants and double, triple and quadruple combinations of the above in SLCMV DNA-A, for infectivity, symptom development and viral DNA accumulation in N. benthamiana. While CP-null mutants were non-infectious, a majority of the single amino acid replacement mutants and their combinations retained infectivity, some with attenuated symptoms and reduced viral titers. Some of the combined mutations restored the attenuated symptoms to wild type levels. Some of the mutations were predicted to cause changes in the secondary structure of the CP, which roughly correlated with the attenuation of symptoms and the reduction in viral titers. PMID:26948262

  2. 77 FR 70176 - Previous Participation Certification

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-23

    ... URBAN DEVELOPMENT Previous Participation Certification AGENCY: Office of the Chief Information Officer... submitted to the Office of Management and Budget (OMB) for review, as required by the Paperwork Reduction... programs. The information will be used to evaluate participants' previous participation in...

  3. Influence of steam explosion pretreatment on the anaerobic digestion of vinegar residue.

    PubMed

    Feng, Jiayu; Zhang, Jiyu; Zhang, Jiafu; He, Yanfeng; Zhang, Ruihong; Liu, Guangqing; Chen, Chang

    2016-07-01

    Vinegar residue is the by-product in the vinegar production process. The large amount of vinegar residue has caused a serious environmental problem owing to its acidity and corrosiveness. Anaerobic digestion is an effective way to convert agricultural waste into bioenergy, and a previous study showed that vinegar residue could be treated by anaerobic digestion but still had room to improve digestion efficiency. In this study, steam explosion at pressure of 0.8, 1.2, and 1.5 MPa and residence time of 5, 10, 15, and 20 min were used to pretreat vinegar residue to improve methane production, respectively. Scanning electron microscopy and X-ray diffraction analyses were applied to validate structural changes of vinegar residue after steam explosion. Results showed that steam explosion pretreatment could destroy the structure of lignocellulose by removing the hemicellulose and lignin, and improve the methane yield effectively. Steam explosion-treated vinegar residue at 0.8 MPa for 5 min produced the highest methane yield of 153.58 mL gVS (-1), which was 27.65% (significant, α < 0.05) more than untreated vinegar residue (120.31 mL gVS (-1)). The analyses of pH, total ammonia-nitrogen, total alkalinity, and volatile fatty acids showed that steam explosion did not influence the stability of anaerobic digestion. This study suggested that steam explosion pretreatment on vinegar residue might be a promising approach and it is worth further study to improve the efficiency of vinegar residue waste utilisation. PMID:27154975

  4. Influence of the size and protonation state of acidic residue 85 on the absorption spectrum and photoreaction of the bacteriorhodopsin chromophore

    NASA Technical Reports Server (NTRS)

    Lanyi, J. K.; Tittor, J.; Varo, G.; Krippahl, G.; Oesterhelt, D.

    1992-01-01

    The consequences of replacing Asp-85 with glutamate in bacteriorhodopsin, as expressed in Halobacterium sp. GRB, were investigated. Similarly to the in vitro mutated and in Escherichia coli expressed protein, the chromophore was found to exist as a mixture of blue (absorption maximum 615 nm) and red (532 nm) forms, depending on the pH. However, we found two widely separated pKa values (about 5.4 and 10.4 without added salt), arguing for two blue and two red forms in separate equilibria. Both blue and red forms of the protein are in the two-dimensional crystalline state. A single pKa, such as in the E. coli expressed protein, was observed only after solubilization with detergent. The photocycle of the blue forms was determined at pH 4.0 with 610 nm photoexcitation, and that of the red forms at pH 10.5 and with 520 nm photoexcitation, in the time-range of 100 ns to 1 s. The blue forms produced no M, but a K- and an L-like intermediate, whose spectra and kinetics resembled those of blue wild-type bacteriorhodopsin below pH 3. The red forms produced a K-like intermediate, as well as M and N. Only the red forms transported protons. Specific perturbation of the neighborhood of the Schiff base by the replacement of Asp-85 with glutamate was suggested by (1) the shift and splitting of the pKa for what is presumably the protonation of residue 85, (2) a 36 nm blue-shift in the absorption of the all-trans red chromophore and a 25 nm red-shift of the 13-cis N chromophore, as compared to wild-type bacteriorhodopsin and its N intermediate, and (3) significant acceleration of the deprotonation of the Schiff base at pH 7, but not of its reprotonation and the following steps in the photocycle.

  5. Identification of the basic amino acid residues on the PsbP protein involved in the electrostatic interaction with photosystem II.

    PubMed

    Nishimura, Taishi; Uno, Chihiro; Ido, Kunio; Nagao, Ryo; Noguchi, Takumi; Sato, Fumihiko; Ifuku, Kentaro

    2014-09-01

    The PsbP protein is an extrinsic subunit of photosystem II (PSII) that is essential for photoautotrophic growth in higher plants. Several crystal structures of PsbP have been reported, but the binding topology of PsbP in PSII has not yet been clarified. In this study, we report that the basic pocket of PsbP, which consists of conserved Arg48, Lys143, and Lys160, is important for the electrostatic interaction with the PSII complex. Our release-reconstitution experiment showed that the binding affinities of PsbP-R48A, -K143A, and -K160A mutated proteins to PSII were lower than that of PsbP-WT, and triple mutations of these residues greatly diminished the binding affinity to PSII. Even when maximum possible binding had occurred, the R48A, K143A, and K160A proteins showed a reduced ability to restore the rate of oxygen evolution at low chloride concentrations. Fourier transform infrared resonance (FTIR) difference spectroscopy results were consistent with the above finding, and suggested that these mutated proteins were not able to induce the normal conformational change around the Mn cluster during S1 to S2 transition. Finally, chemical cross-linking experiments suggested that the interaction between the N-terminus of PsbP with PsbE was inhibited by these mutations. These data suggest that the basic pocket of PsbP is important for proper association and interaction with PSII. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy. PMID:24388917

  6. 40 CFR 180.595 - Flufenpyr-ethyl; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... residues of the herbicide, flufenpyr-ethyl; acetic acid, -phenoxy]-ethyl ester], in or on the following...) Tolerances are established for residues of the herbicide flufenpyr-ethyl; acetic acid, -phenoxy]-ethyl...

  7. 40 CFR 180.595 - Flufenpyr-ethyl; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... residues of the herbicide, flufenpyr-ethyl; acetic acid, -phenoxy]-ethyl ester], in or on the following...) Tolerances are established for residues of the herbicide flufenpyr-ethyl; acetic acid, -phenoxy]-ethyl...

  8. 40 CFR 180.595 - Flufenpyr-ethyl; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... residues of the herbicide, flufenpyr-ethyl; acetic acid, -phenoxy]-ethyl ester], in or on the following...) Tolerances are established for residues of the herbicide flufenpyr-ethyl; acetic acid, -phenoxy]-ethyl...

  9. 40 CFR 180.595 - Flufenpyr-ethyl; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... residues of the herbicide, flufenpyr-ethyl; acetic acid, -phenoxy]-ethyl ester], in or on the following...) Tolerances are established for residues of the herbicide flufenpyr-ethyl; acetic acid, -phenoxy]-ethyl...

  10. 40 CFR 180.595 - Flufenpyr-ethyl; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... residues of the herbicide, flufenpyr-ethyl; acetic acid, -phenoxy]-ethyl ester], in or on the following...) Tolerances are established for residues of the herbicide flufenpyr-ethyl; acetic acid, -phenoxy]-ethyl...

  11. Synthesis of poly(alkenoic acid) with L-leucine residue and methacrylate photopolymerizable groups useful in formulating dental restorative materials.

    PubMed

    Buruiana, Tinca; Nechifor, Marioara; Melinte, Violeta; Podasca, Viorica; Buruiana, Emil C

    2014-01-01

    To develop resin-modified glass ionomer materials, we synthesized methacrylate-functionalized acrylic copolymer (PAlk-LeuM) derived from acrylic acid, itaconic acid and N-acryloyl-L-leucine using (N-methacryloyloxyethylcarbamoyl-N'-4-hydroxybutyl) urea as the modifying agent. The spectroscopic (proton/carbon nuclear magnetic resonance, Fourier transform infrared spectroscopy) characteristics, and the gel permeation chromatography/Brookfield viscosity measurements were analysed and compared with those of the non-modified copolymer (PAlk-Leu). The photocurable copolymer (PAlk-LeuM, ~14 mol% methacrylate groups) and its precursor (PAlk-Leu) were incorporated in dental ionomer compositions besides diglycidyl methacrylate of bisphenol A (Bis-GMA) or an analogue of Bis-GMA (Bis-GMA-1), triethylene glycol dimethacrylate and 2-hydroxyethyl methacrylate. The kinetic data obtained by photo-differential scanning calorimetry showed that both the degree of conversion (60.50-75.62%) and the polymerization rate (0.07-0.14 s(-1)) depend mainly on the amount of copolymer (40-50 wt.%), and conversions over 70% were attained in the formulations with 40 wt.% PAlk-LeuM. To formulate light-curable cements, each organic composition was mixed with filler (90 wt.% fluoroaluminosilicate/10 wt.% hydroxyapatite) into a 2.7:1 ratio (powder/liquid ratio). The light-cured specimens exhibited flexural strength (FS), compressive strength (CS) and diametral tensile strength (DTS) varying between 28.08 and 64.79 MPa (FS), 103.68-147.13 MPa (CS) and 16.89-31.87 MPa (DTS). The best values for FS, CS and DTS were found for the materials with the lowest amount of PAlk-LeuM. Other properties such as the surface hardness, water sorption/water solubility, surface morphology and fluorescence caused by adding the fluorescein monomer were also evaluated. PMID:24701975

  12. Vegetable fiber fermentation by human fecal bacteria: cell wall polysaccharide disappearance and short-chain fatty acid production during in vitro fermentation and water-holding capacity of unfermented residues.

    PubMed

    Bourquin, L D; Titgemeyer, E C; Fahey, G C

    1993-05-01

    Dietary fiber from eight vegetables (broccoli, carrot, cauliflower, celery, cucumber, lettuce, onion and radish) was analyzed for chemical composition and potential in vitro fermentation by human fecal bacteria. Total dietary fiber concentration of substrates ranged from 34.9 (broccoli) to 5.8 (cucumber) g/kg edible matter. Substrate fiber fractions were composed primarily of pectic substances and cellulose with smaller concentrations of hemicelluloses and lignin. Total dietary fiber residues isolated from substrates were fermented in vitro for 24 h with fecal bacteria obtained from each of three human volunteers. Substrate dry matter disappearance during fermentation was highest for carrot (63.7%) and lowest for cucumber (49.4%). Averaged across all substrates, disappearances of arabinose, galactose, glucose, mannose, xylose and uronic acids during fermentation were 96, 90, 54, 68, 51 and 97%, respectively. Short-chain fatty acid (SCFA) production during substrate fermentation averaged 10.5 mmol SCFA/g dry matter fermented. Averaged across all substrates, production of the major SCFA, acetate, propionate and butyrate, occurred in the molar ratio 76:14:10. Potential water-holding capacity of substrates was not influenced by fiber source and averaged 2.04 g H2O/g original substrate dry matter. Extent of substrate fermentation, SCFA production and substrate potential water-holding capacity were significantly different among inoculum donors, indicating that considerable inter-individual variation exists in the potential in vivo fermentation of vegetable fiber. PMID:8387579

  13. Residues contributing to the Na(+)-binding pocket of the SLC24 Na(+)/Ca(2+)-K(+) Exchanger NCKX2.

    PubMed

    Altimimi, Haider F; Fung, Eric H; Winkfein, Robert J; Schnetkamp, Paul P M

    2010-05-14

    Na(+)/Ca(2+)-K(+) exchangers (NCKX; gene family SLC24) are plasma membrane Ca(2+) transporters that mediate the extrusion of one Ca(2+) ion and one K(+) ion in exchange for four Na(+) ions. NCKX is modeled to have two sets of five transmembrane segments separated by a large cytosolic loop; within each set of transmembrane segments are regions of internal symmetry termed alpha(1) and alpha(2) repeats. The central residues that are important for Ca(2+) and K(+) liganding and transport have been identified in NCKX2, and they comprise three central acidic residues, Glu(188) in alpha(1) and Asp(548) and Asp(575) in alpha(2), as well as Ser/Thr residues one-helical turn away from these residues. In this study, we have scanned through more than 100 single-residue substitutions of NCKX2 for shifts in Na(+) affinity using a fluorescence assay to monitor changes in free Ca(2+) in HEK293 cells treated with gramicidin to control intracellular Na(+). We have identified 31 residues that, when substituted, result in shifts in Na(+) affinity, either toward higher or lower K(m) values when compared with wild type NCKX2 (K(m) for Na(+) 58 mm). These residues include the central acidic residues Glu(188), Asp(548), and Asp(575), and their neighboring residues in alpha(1) and alpha(2), in addition to a number of newly investigated residues in transmembrane segment 3. Our results relate the identification of residues important for Na(+) transport in this study to those previously identified as important in the counter-transport of Ca(2+) and K(+), lending support to the alternating access model of transmembrane transport. PMID:20231282

  14. Composition of the amino acid and amino sugar for molecular weight fractions of hot-water extractable soil organic matters from soils with plant residue compost or mineral fertilization

    NASA Astrophysics Data System (ADS)

    Moriizumi, M.; Matsunaga, T.; Uezono, I.; Kato, N.

    2009-04-01

    The hot-water extractable organic nitrogen is well known as a laboratory index of mineralizable nitrogen. This available nitrogen is indispensable for growth of plants because of being absorbed in crops. We measured the composition of the amino acid and amino sugar for molecular weight fractions in hot-water extractable organic matters to understand the source of the available nitrogen in soils inserted a compost. Two soil samples were collected from fields (Soil Type; Andosol) in National Agricultural Research Center in Tsukuba, Japan. A plant residue compost of 2 kga-1y-1 during 25 year has been applied to a soil and another soil was under the mineral fertilization. Organic matters were extracted from the soils of 3 g in the water of 50 ml at 80 degree centigrade for16 hours. The molecular size distribution of the hot-water extractable organic matters was analyzed by HPSCE (column YMC Diol-120, elution; 50mM phosphate buffer under pH=7.0, flow rate 1 mlmin-1), and 20 fractions were collected at regular intervals in the retention time. The chromatograms were monitored under the absorbance at 280 nm and fluorescence intensity at Ex.280 nm: Em.330nm. The concentrations of the 15 amino acids and three amino sugars (muramic acid, glucosamine, and galactosamine) for the molecule weight fractions were measured by HPLC as o-phthaldialdehyde (OPA) derivatives after the vapor HCl hydrolysis. Organic nitrogen concentrations of the hot-water extractable organic matters in the soil inserted the compost (C-soil) and the mineral fertilization soil (M-soil) were 133 and 35 mgkg-1, respectively. The extracted organic matters had the variable molecule weight (103- 104 Da). The concentrations of the amino acid and amino sugar of organic nitrogen in the C-soil were higher than those in the M-soil in all fractions. The fractions were classified into 3 groups (LW, MW, and SW) based on the molecule weight and spectroscopic characteristics. Each group had unique composition of the amino

  15. 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.

    PubMed

    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

  16. Effect of D-amino acids at Asp{sup 23} and Ser{sup 26} residues on the conformational preference of A{beta}{sub 20-29} peptides

    SciTech Connect

    Shanmugam, Ganesh; Polavarapu, Prasad L. . E-mail: Prasad.L.Polavarapu@Vanderbilt.edu; Hallgas, Balazs; Majer, Zsuzsa

    2005-09-30

    The effects of D-amino acids at Asp{sup 23} and Ser{sup 26} residues on the conformational preference of {beta}-amyloid (A{beta}) peptide fragment (A{beta}{sub 20-29}) have been studied using different spectroscopic techniques, namely vibrational circular dichroism (VCD), vibrational absorption, and electronic circular dichroism. To study the structure of the A{beta}{sub 20-29}, [D-Asp{sup 23}]A{beta}{sub 20-29}, and [D-Ser{sup 26}]A{beta}{sub 20-29} peptides under different conditions, the spectra were measured in 10 mM acetate buffer (pH 3) and in 2,2,2-trifluoroethanol (TFE). The spectroscopic results indicated that at pH 3, A{beta}{sub 20-29} peptide takes random coil with {beta}-turn structure, while [D-Ser{sup 26}]A{beta}{sub 20-29} peptide adopts significant amount of polyproline II (PPII) type structure along with {beta}-turn contribution and D-Asp-substituted peptide ([D-Asp{sup 23}]A{beta}{sub 20-29}) adopts predominantly PPII type structure. The increased propensity for PPII conformation upon D-amino acid substitution, in acidic medium, has important biological implications. In TFE, A{beta}{sub 20-29}, [D-Asp{sup 23}]A{beta}{sub 20-29}, and [D-Ser{sup 26}]A{beta}{sub 20-29} peptides adopt 3{sub 10}-helix, {alpha}-helix, and random coil with some {beta}-turn structures, respectively. The VCD data obtained for the A{beta} peptide films suggested that the secondary structures for the peptide films are not the same as those for corresponding solution and are also different among the A{beta} peptides studied here. This observation suggests that dehydration can have a significant influence on the structural preferences of these peptides.

  17. Emergent Biomarkers of Residual Cardiovascular Risk in Patients with Low HDL-c and/or High Triglycerides and Average LDL-c Concentrations: Focus on HDL Subpopulations, Oxidized LDL, Adiponectin, and Uric Acid

    PubMed Central

    Mascarenhas-Melo, Filipa; Sereno, José; Freitas, Isabel; Isabel-Mendonça, Maria; Pinto, Rui; Teixeira, Frederico

    2013-01-01

    This study intended to determine the impact of HDL-c and/or TGs levels on patients with average LDL-c concentration, focusing on lipidic, oxidative, inflammatory, and angiogenic profiles. Patients with cardiovascular risk factors (n = 169) were divided into 4 subgroups, combining normal and low HDL-c with normal and high TGs patients. The following data was analyzed: BP, BMI, waist circumference and serum glucose, Total-c, TGs, LDL-c, oxidized-LDL, total HDL-c and HDL subpopulations, paraoxonase-1 (PON1) activity, hsCRP, uric acid, TNF-α, adiponectin, VEGF, and iCAM1. The two populations with increased TGs levels, regardless of the normal or low HDL-c, presented obesity and higher waist circumference, Total-c, LDL-c, Ox-LDL, and uric acid. Adiponectin concentration was significantly lower and VEGF was higher in the population with cumulative low values of HDL-c and high values of TGs, while HDL quality was reduced in the populations with impaired values of HDL-c and/or TGs, viewed by reduced large and increased small HDL subfractions. In conclusion, in a population with cardiovascular risk factors, low HDL-c and/or high TGs concentrations seem to be associated with a poor cardiometabolic profile, despite average LDL-c levels. This condition, often called residual risk, is better evidenced by using both traditional and nontraditional CV biomarkers, including large and small HDL subfractions, Ox-LDL, adiponectin, VEGF, and uric acid. PMID:24319364

  18. Human antibody response to fragments A and B of diphtheria toxin and a synthetic peptide of amino acid residues 141-157 of fragment A.

    PubMed Central

    Perera, V. Y.; Corbel, M. J.

    1990-01-01

    Examination of a selection of serum samples from adults from two regions of England showed that 50% of men in the 16-24 years and over 55 years age groups had high titres of antibody to diphtheria toxin (DT). In contrast, only 11% of women aged 16 to over 55 years had high titres of antibody to DT. All human antisera with high anti-DT titres reacted with a synthetic peptide (SP) corresponding to the amino acids 141-157 of DT fragment A, with sera from men aged 35 to over 55 years showing the highest titres. High antibody titres to fragment A paralleled those to SP in both sexes. Titres of antibody to DT fragment B were highest in individuals with high titres to DT. In sera from both sexes immunoglobulin G1 was the predominant subclass reactive with all three antigens. However, both IgG1 and IgG4 and to a lesser extent IgG2 and IgG3 were present in immunoglobulin concentrates. Images Fig. 1 Fig. 2 PMID:2249709

  19. Competing addition and hydrolysis of the cytidylylcytidylyladenosine terminal residues of transfer ribonucleic acid isolated from the non-lactating bovine mammary gland

    PubMed Central

    Herrington, M. D.; Hawtrey, A. O.

    1970-01-01

    1. The enzyme fraction obtained from the pH5 enzyme of non-lactating bovine mammary gland between 40 and 100% ammonium sulphate saturation markedly inhibited the AMP-incorporating activity of rat liver nucleotide-incorporating enzyme. This inhibitory effect has been attributed to high nuclease activity which can be partially removed by adsorption of the enzyme fraction on to calcium phosphate gel. 2. The degradation action of the calcium phosphate-purified enzyme is confined mainly to the terminal trinucleotide sequence -pCpCpA of tRNA, its effect being analogous to that of venom phosphodiesterase. This enzyme is heat labile and very readily loses its degradative activity. 3. Treatment of the enzyme fraction with Macaloid results in complete removal of the phosphodiesterase, leaving an enzyme capable of incorporating AMP into tRNA. 4. Transfer RNA extracted from non-lactating bovine mammary gland in the presence of polyvinyl sulphate and Macaloid is able to accept amino acids with an efficiency 30% of that shown by lactating bovine mammary-gland tRNA isolated under identical conditions. PMID:4321270

  20. Phosphorylation of serine residue modulates cotton Di19-1 and Di19-2 activities for responding to high salinity stress and abscisic acid signaling

    PubMed Central

    Qin, Li-Xia; Nie, Xiao-Ying; Hu, Rong; Li, Gang; Xu, Wen-Liang; Li, Xue-Bao

    2016-01-01

    Di19 (drought-induced protein 19) family is a novel type of Cys2/His2 zinc-finger proteins. In this study, we demonstrated that cotton Di19-1 and Di19-2 (GhDi19-1/-2) proteins could be phosphorylated in vitro by the calcium-dependent protein kinase (CDPK). Mutation of Ser to Ala in N-terminus of GhDi19-1/-2 led to the altered subcellular localization of the two proteins, but the constitutively activated form (Ser was mutated to Asp) of GhDi19-1/-2 still showed the nuclear localization. GhDi19-1/-2 overexpression transgenic Arabidopsis seedlings displayed the hypersensitivity to high salinity and abscisic acid (ABA). However, Ser site-mutated GhDi19-1(S116A) and GhDi19-2(S114A), and Ser and Thr double sites-mutated GhDi19-1(S/T-A/A) and GhDi19-2(S/T-A/A) transgenic Arabidopsis did not show the salt- and ABA-hypersensitive phenotypes. In contrast, overexpression of Thr site-mutated GhDi19-1(T114A) and GhDi19-2(T112A) in Arabidopsis still resulted in salt- and ABA-hypersensitivity phenotypes, like GhDi19-1/-2 transgenic lines. Overexpression of GhDi19-1/-2 and their constitutively activated forms in Atcpk11 background could recover the salt- and ABA-insensitive phenotype of the mutant. Thus, our results demonstrated that Ser phosphorylation (not Thr phosphorylation) is crucial for functionally activating GhDi19-1/-2 in response to salt stress and ABA signaling during early plant development, and GhDi19-1/-2 proteins may be downstream targets of CDPKs in ABA signal pathway. PMID:26829353

  1. Site specific oxidation of amino acid residues in rat lens γ-crystallin induced by low-dose γ-irradiation.

    PubMed

    Kim, Ingu; Saito, Takeshi; Fujii, Norihiko; Kanamoto, Takashi; Chatake, Toshiyuki; Fujii, Noriko

    2015-10-30

    Although cataracts are a well-known age-related disease, the mechanism of their formation is not well understood. It is currently thought that eye lens proteins become abnormally aggregated, initially causing clumping that scatters the light and interferes with focusing on the retina, and ultimately resulting in a cataract. The abnormal aggregation of lens proteins is considered to be triggered by various post-translational modifications, such as oxidation, deamidation, truncation and isomerization, that occur during the aging process. Such modifications, which are also generated by free radical and reactive oxygen species derived from γ-irradiation, decrease crystallin solubility and lens transparency, and ultimately lead to the development of a cataract. In this study, we irradiated young rat lenses with low-dose γ-rays and extracted the water-soluble and insoluble protein fractions. The water-soluble and water-insoluble lens proteins were digested with trypsin, and the resulting peptides were analyzed by LC-MS. Specific oxidation sites of methionine, cysteine and tryptophan in rat water-soluble and -insoluble γE and γF-crystallin were determined by one-shot analysis. The oxidation sites in rat γE and γF-crystallin resemble those previously identified in γC and γD-crystallin from human age-related cataracts. Our study on modifications of crystallins induced by ionizing irradiation may provide useful information relevant to human senile cataract formation. PMID:26385181

  2. Identification of functionally important negatively charged residues in the carboxy end of mouse hepatitis coronavirus A59 nucleocapsid protein.

    PubMed

    Verma, Sandhya; Bednar, Valerie; Blount, Andrew; Hogue, Brenda G

    2006-05-01

    The coronavirus nucleocapsid (N) protein is a multifunctional viral gene product that encapsidates the RNA genome and also plays some as yet not fully defined role in viral RNA replication and/or transcription. A number of conserved negatively charged amino acids are located within domain III in the carboxy end of all coronavirus N proteins. Previous studies suggested that the negatively charged residues are involved in virus assembly by mediating interaction between the membrane (M) protein carboxy tail and nucleocapsids. To determine the importance of these negatively charged residues, a series of alanine and other charged-residue substitutions were introduced in place of those in the N gene within a mouse hepatitis coronavirus A59 infectious clone. Aspartic acid residues 440 and 441 were identified as functionally important. Viruses could not be isolated when both residues were replaced by positively charged amino acids. When either amino acid was replaced by a positively charged residue or both were changed to alanine, viruses were recovered that contained second-site changes within N, but not in the M or envelope protein. The compensatory role of the new changes was confirmed by the construction of new viruses. A few viruses were recovered that retained the D441-to-arginine change and no compensatory changes. These viruses exhibited a small-plaque phenotype and produced significantly less virus. Overall, results from our analysis of a large panel of plaque-purified recovered viruses indicate that the negatively charged residues at positions 440 and 441 are key residues that appear to be involved in virus assembly. PMID:16611893

  3. The pKa values of acidic and basic residues buried at the same internal location in a protein are governed by different factors

    PubMed Central

    Harms, Michael J.; Castañeda, Carlos A.; Schlessman, Jamie L.; Sue, Gloria R.; Bertrand García-Moreno, E

    2009-01-01

    Summary The pKa values of internal ionizable groups are usually very different than the normal pKa values of ionizable groups in water. To examine the molecular determinants of pKa values of internal groups, we compared the properties of Lys, Asp and Glu at internal position 38 in staphylococcal nuclease. Lys-38 titrates with a normal or elevated pKa whereas Asp-38 and Glu-38 titrate with elevated pKa values of 7.0 and 7.2, respectively. In the structure of the L38K variant, the buried amino group of the Lys-38 side chain makes an ion pair with Glu-122; whereas, in the structure of the L38E variant, the buried carboxyl group of Glu-38 interacts with two backbone amides and has several nearby carboxyl oxygen atoms. Previously we showed that the pKa of Lys-38 is normal owing to structural reorganization and water penetration concomitant with ionization of the Lys side chain. In contrast, the pKa of Asp-38 and Glu-38 are perturbed significantly owing to an imbalance between favorable polar interactions and unfavorable contributions from dehydration and from Coulomb interactions with surface carboxylic groups. Their ionization is also coupled to subtle structural reorganization. These results illustrate the complex interplay between local polarity, Coulomb interactions and structural reorganization as determinants of pKa values of internal groups in proteins. This study suggests that improvements to computational methods for pKa calculations will require explicit treatment of the conformational reorganization that can occur when internal groups ionize. PMID:19324049

  4. DISSOLUTION OF NEPTUNIUM OXIDE RESIDUES

    SciTech Connect

    Kyser, E

    2009-01-12

    This report describes the development of a dissolution flowsheet for neptunium (Np) oxide (NpO{sub 2}) residues (i.e., various NpO{sub 2} sources, HB-Line glovebox sweepings, and Savannah River National Laboratory (SRNL) thermogravimetric analysis samples). Samples of each type of materials proposed for processing were dissolved in a closed laboratory apparatus and the rate and total quantity of off-gas were measured. Samples of the off-gas were also analyzed. The quantity and type of solids remaining (when visible) were determined after post-dissolution filtration of the solution. Recommended conditions for dissolution of the NpO{sub 2} residues are: Solution Matrix and Loading: {approx}50 g Np/L (750 g Np in 15 L of dissolver solution), using 8 M nitric acid (HNO{sub 3}), 0.025 M potassium fluoride (KF) at greater than 100 C for at least 3 hours. Off-gas: Analysis of the off-gas indicated nitric oxide (NO), nitrogen dioxide (NO{sub 2}) and nitrous oxide (N{sub 2}O) as the only identified components. No hydrogen (H{sub 2}) was detected. The molar ratio of off-gas produced per mole of Np dissolved ranged from 0.25 to 0.4 moles of gas per mole of Np dissolved. A peak off-gas rate of {approx}0.1 scfm/kg bulk oxide was observed. Residual Solids: Pure NpO{sub 2} dissolved with little or no residue with the proposed flowsheet but the NpCo and both sweepings samples left visible solid residue after dissolution. For the NpCo and Part II Sweepings samples the residue amounted to {approx}1% of the initial material, but for the Part I Sweepings sample, the residue amounted to {approx}8 % of the initial material. These residues contained primarily aluminum (Al) and silicon (Si) compounds that did not completely dissolve under the flowsheet conditions. The residues from both sweepings samples contained minor amounts of plutonium (Pu) particles. Overall, the undissolved Np and Pu particles in the residues were a very small fraction of the total solids.

  5. The Pseudomonas aeruginosa antimetabolite L -2-amino-4-methoxy-trans-3-butenoic acid (AMB) is made from glutamate and two alanine residues via a thiotemplate-linked tripeptide precursor

    PubMed Central

    Rojas Murcia, Nelson; Lee, Xiaoyun; Waridel, Patrice; Maspoli, Alessandro; Imker, Heidi J.; Chai, Tiancong; Walsh, Christopher T.; Reimmann, Cornelia

    2015-01-01

    The Pseudomonas aeruginosa toxin L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) is a non-proteinogenic amino acid which is toxic for prokaryotes and eukaryotes. Production of AMB requires a five-gene cluster encoding a putative LysE-type transporter (AmbA), two non-ribosomal peptide synthetases (AmbB and AmbE), and two iron(II)/α-ketoglutarate-dependent oxygenases (AmbC and AmbD). Bioinformatics analysis predicts one thiolation (T) domain for AmbB and two T domains (T1 and T2) for AmbE, suggesting that AMB is generated by a processing step from a precursor tripeptide assembled on a thiotemplate. Using a combination of ATP-PPi exchange assays, aminoacylation assays, and mass spectrometry-based analysis of enzyme-bound substrates and pathway intermediates, the AmbB substrate was identified to be L-alanine (L-Ala), while the T1 and T2 domains of AmbE were loaded with L-glutamate (L-Glu) and L-Ala, respectively. Loading of L-Ala at T2 of AmbE occurred only in the presence of AmbB, indicative of a trans loading mechanism. In vitro assays performed with AmbB and AmbE revealed the dipeptide L-Glu-L-Ala at T1 and the tripeptide L-Ala-L-Glu-L-Ala attached at T2. When AmbC and AmbD were included in the assay, these peptides were no longer detected. Instead, an L-Ala-AMB-L-Ala tripeptide was found at T2. These data are in agreement with a biosynthetic model in which L-Glu is converted into AMB by the action of AmbC, AmbD, and tailoring domains of AmbE. The importance of the flanking L-Ala residues in the precursor tripeptide is discussed. PMID:25814981

  6. Back pain in a previously healthy teenager.

    PubMed

    Fitzgerald, Felicity; Howard, Julia; Bailey, Fiona; Soleimanian, Sorrush

    2013-01-01

    We present the case of a 14-year-old previously healthy boy who presented to his general practitioner with back pain and fever after rugby training. He was initially treated for suspected discitis but during the course of his admission he rapidly deteriorated and developed severe necrotising pneumonia. He was intubated, ventilated and transferred to a paediatric intensive care unit. Panton-Valentine leukocidin Staphylococcus aureus was suspected and subsequently identified in blood cultures. PMID:23867883

  7. Reutilization of previously hybridized slides for fluorescence in situ hybridization

    SciTech Connect

    Epstein, L.; DeVries, S.; Waldman, F.M.

    1995-12-01

    Application of fluorescence in situ hybridization (FISH) to clinical material is sometimes limited by sample size. In addition, heterogeneity among slides prepared from a single sample may lead to variation in FISH analyses. Reutilization of material for repeated FISH analyses would help to alleviate these problems. We have developed a simple procedure for repeated FISH analyses with directly conjugated probes. Previously hybridized probes are removed by incubation in denaturing solution, and slides can then be rehybridized without residual signals remaining. Several cycles of this procedure allow a full complement of chromosomal loci to be analyzed on the same population of cells. Advantages of this protocol include gaining more cytogenetic information from small samples and eliminating the problem of intratumorvariability. 5 refs., 4 figs.

  8. Synthesis of a novel zwitterionic biodegradable poly (α,β-L-aspartic acid) derivative with some L-histidine side-residues and its resistance to non-specific protein adsorption.

    PubMed

    Wang, Xiaojuan; Wu, Guolin; Lu, Caicai; Wang, Yinong; Fan, Yunge; Gao, Hui; Ma, Jianbiao

    2011-08-01

    A novel zwitterionic polypeptide derivative, denoted as His-PAsp/PAsp, was successfully synthesized by amidation of Poly (α,β-L-aspartic acid) with L-histidine methyl ester. Turbidity, zeta potential and ¹H NMR measurements were used to study the aggregation behaviors of His-PAsp/PAsp under different pH values. The modified polypeptide derivative composed of electro-negatively carboxylic and electro-positively imidazole residues randomly so as to bear opposite charges at pH values above or below the isoelectric point. When the zwitterionic polypeptide was coated on silicon wafer as a model substrate material, the absorption resistance of fibrinogen, a blood protein resulting in the blood coagulation cascade, on the coated surface was measured. It was found that the adsorption amount of fibrinogen on the polypeptide-coated surface depended on the dose of the polypeptide on silicon wafer. Obvious resistance of the fibrinogen adsorption on the polypeptide-coated surface was observed. Since its good biodegradability and superior anti-protein-fouling property, this pH-responsive zwitterionic polypeptide is a promising candidate for surface modification in many biomedical applications, including medical implants, drug delivery carriers, and biosensors. PMID:21536417

  9. Selective adsorption and recovery of Au(III) from three kinds of acidic systems by persimmon residual based bio-sorbent: a method for gold recycling from e-wastes.

    PubMed

    Fan, Ruiyi; Xie, Feng; Guan, Xueliang; Zhang, Qinglin; Luo, Zhengrong

    2014-07-01

    A low cost bio-sorbent, named "PPF resin", was prepared by crosslinking the persimmon residual with formaldehyde. The adsorption behavior of PPF resin towards Au(III) from varied HCl and HNO3 concentration solutions was studied. PPF resin could adsorb almost complete Au(III) from high acidic systems. The influence of dilution ratio, solid-liquid ratio and time towards Au(III) from aqua regia leached PCBs liquor was censored in detail by batch and continuous adsorption methods. The PPF resin before and after adsorption was characterized by FT-IR, XRD and XPS spectra which provided evidences for the reduction of Au(III) to Au(0) with a proposed mechanism of Au(III) adsorption-reduction process. After saturated column adsorption of 0.1g PPF resin, 0.0506 g gold (purity: 99.9%) was obtained by the method of incineration. The present results provide a new approach for gold recovery from the secondary resources. PMID:24811444

  10. Involvement of hydrophobic amino acid residues in C7-C8 loop of Aspergillus oryzae hydrophobin RolA in hydrophobic interaction between RolA and a polyester.

    PubMed

    Tanaka, Takumi; Tanabe, Hiroki; Uehara, Kenji; Takahashi, Toru; Abe, Keietsu

    2014-01-01

    Hydrophobins are amphipathic secretory proteins with eight conserved cysteine residues and are ubiquitous among filamentous fungi. The Cys3-Cys4 and Cys7-Cys8 loops of hydrophobins are thought to form hydrophobic segments involved in adsorption of hydrophobins on hydrophobic surfaces. When the fungus Aspergillus oryzae is grown in a liquid medium containing the polyester polybutylene succinate-co-adipate (PBSA), A. oryzae produces hydrophobin RolA, which attaches to PBSA. Here, we analyzed the kinetics of RolA adsorption on PBSA by using a PBSA pull-down assay and a quartz crystal microbalance (QCM) with PBSA-coated electrodes. We constructed RolA mutants in which hydrophobic amino acids in the two loops were replaced with serine, and we examined the kinetics of mutant adsorption on PBSA. QCM analysis revealed that mutants with replacements in the Cys7-Cys8 loop had lower affinity than wild-type RolA for PBSA, suggesting that this loop is involved in RolA adsorption on PBSA. PMID:25273133

  11. Previous Open Rotor Research in the US

    NASA Technical Reports Server (NTRS)

    VanZante, Dale

    2011-01-01

    Previous Open Rotor noise experience in the United States, current Open Rotor noise research in the United States and current NASA prediction methods activities were presented at a European Union (EU) X-Noise seminar. The invited attendees from EU industries, research establishments and universities discussed prospects for reducing Open Rotor noise and reviewed all technology programs, past and present, dedicated to Open Rotor engine concepts. This workshop was particularly timely because the Committee on Aviation Environmental Protection (CAEP) plans to involve Independent Experts in late 2011 in assessing the noise of future low-carbon technologies including the open rotor.

  12. The tify family previously known as ZIM.

    PubMed

    Vanholme, Bartel; Grunewald, Wim; Bateman, Alex; Kohchi, Takayuki; Gheysen, Godelieve

    2007-06-01

    The ZIM domain was originally identified in the ZIM protein (BAA97679; Zinc-finger protein expressed in Inflorescence Meristem). Since then it has been found in other proteins and the corresponding genes have been grouped into a plant-specific family. However, the family lacks consistency in its classification among different databases. Here, we try to clarify this incongruity by presenting an overview of the Arabidopsis proteins having this domain. The presented genome-wide survey can be seen as a start point to reveal the unknown function of these proteins. Furthermore, because of the confusing ZIM nomenclature being used at present, we propose to rename the domain and family as tify, after the most conserved amino acid motif characterizing the members of this family. PMID:17499004

  13. Sequence-Specific Solvent Accessibilities of Protein Residues in Unfolded Protein Ensembles

    PubMed Central

    Bernadó, Pau; Blackledge, Martin; Sancho, Javier

    2006-01-01

    Protein stability cannot be understood without the correct description of the unfolded state. We present here an efficient method for accurate calculation of atomic solvent exposures for denatured protein ensembles. The method used to generate the ensembles has been shown to reproduce diverse biophysical experimental data corresponding to natively and chemically unfolded proteins. Using a data set of 19 nonhomologous proteins containing from 98 to 579 residues, we report average accessibilities for all residue types. These averaged accessibilities are considerably lower than those previously reported for tripeptides and close to the lower limit reported by Creamer and co-workers. Of importance, we observe remarkable sequence dependence for the exposure to solvent of all residue types, which indicates that average residue solvent exposures can be inappropriate to interpret mutational studies. In addition, we observe smaller influences of both protein size and protein amino acid composition in the averaged residue solvent exposures for individual proteins. Calculating residue-specific solvent accessibilities within the context of real sequences is thus necessary and feasible. The approach presented here may allow a more precise parameterization of protein energetics as a function of polar- and apolar-area burial and opens new ways to investigate the energetics of the unfolded state of proteins. PMID:17012314

  14. Rheumatic pains of previously undiagnosed diabetic subjects.

    PubMed

    Qiao, Q; Keinänen-Kiukaanniemi, S; Rajala, U; Uusimäki, A; Kivelä, S L

    1995-01-01

    To identify the early diabetic musculoskeletal symptoms of previously undiagnosed diabetic subjects, a case-control study was carried out. The cases and controls were recruited from a population aged 55 years. Questions concerning the symptoms were asked before the 2-h oral glucose tolerance tests (OGTT). The results show that pain in the right hand was the most prominent symptom among the diabetic women. Pains in the left hand and the shoulders in the diabetic women and pains in the right knee and the right hip joint in the diabetic men tended to be more prevalent than the corresponding symptoms in the controls. The highest prevalence of most musculoskeletal pains occurred in the highest tertile of 2-h OGTT values among women. The conclusion is that the hand pain is closely associated with the development of diabetes and may give clues to an early diagnosis of diabetes in a middle-aged population. PMID:7481588

  15. Books average previous decade of economic misery.

    PubMed

    Bentley, R Alexander; Acerbi, Alberto; Ormerod, Paul; Lampos, Vasileios

    2014-01-01

    For the 20(th) century since the Depression, we find a strong correlation between a 'literary misery index' derived from English language books and a moving average of the previous decade of the annual U.S. economic misery index, which is the sum of inflation and unemployment rates. We find a peak in the goodness of fit at 11 years for the moving average. The fit between the two misery indices holds when using different techniques to measure the literary misery index, and this fit is significantly better than other possible correlations with different emotion indices. To check the robustness of the results, we also analysed books written in German language and obtained very similar correlations with the German economic misery index. The results suggest that millions of books published every year average the authors' shared economic experiences over the past decade. PMID:24416159

  16. Books Average Previous Decade of Economic Misery

    PubMed Central

    Bentley, R. Alexander; Acerbi, Alberto; Ormerod, Paul; Lampos, Vasileios

    2014-01-01

    For the 20th century since the Depression, we find a strong correlation between a ‘literary misery index’ derived from English language books and a moving average of the previous decade of the annual U.S. economic misery index, which is the sum of inflation and unemployment rates. We find a peak in the goodness of fit at 11 years for the moving average. The fit between the two misery indices holds when using different techniques to measure the literary misery index, and this fit is significantly better than other possible correlations with different emotion indices. To check the robustness of the results, we also analysed books written in German language and obtained very similar correlations with the German economic misery index. The results suggest that millions of books published every year average the authors' shared economic experiences over the past decade. PMID:24416159

  17. Congruency sequence effects are driven by previous-trial congruency, not previous-trial response conflict

    PubMed Central

    Weissman, Daniel H.; Carp, Joshua

    2013-01-01

    Congruency effects in distracter interference tasks are often smaller after incongruent trials than after congruent trials. However, the sources of such congruency sequence effects (CSEs) are controversial. The conflict monitoring model of cognitive control links CSEs to the detection and resolution of response conflict. In contrast, competing theories attribute CSEs to attentional or affective processes that vary with previous-trial congruency (incongruent vs. congruent). The present study sought to distinguish between conflict monitoring and congruency-based accounts of CSEs. To this end, we determined whether CSEs are driven by previous-trial reaction time (RT)—a putative measure of response conflict—or by previous-trial congruency. In two experiments using a face-word Stroop task (n = 49), we found that current-trial congruency effects did not vary with previous-trial RT independent of previous-trial congruency. In contrast, current-trial congruency effects were influenced by previous-trial congruency independent of previous-trial RT. These findings appear more consistent with theories that attribute CSEs to non-conflict processes whose recruitment varies with previous-trial congruency than with theories that link CSEs to previous-trial response conflict. PMID:24027550

  18. Identification of kinetically hot residues in proteins.

    PubMed Central

    Demirel, M. C.; Atilgan, A. R.; Jernigan, R. L.; Erman, B.; Bahar, I.

    1998-01-01

    A number of recent studies called attention to the presence of kinetically important residues underlying the formation and stabilization of folding nuclei in proteins, and to the possible existence of a correlation between conserved residues and those participating in the folding nuclei. Here, we use the Gaussian network model (GNM), which recently proved useful in describing the dynamic characteristics of proteins for identifying the kinetically hot residues in folded structures. These are the residues involved in the highest frequency fluctuations near the native state coordinates. Their high frequency is a manifestation of the steepness of the energy landscape near their native state positions. The theory is applied to a series of proteins whose kinetically important residues have been extensively explored: chymotrypsin inhibitor 2, cytochrome c, and related C2 proteins. Most of the residues previously pointed out to underlie the folding process of these proteins, and to be critically important for the stabilization of the tertiary fold, are correctly identified, indicating a correlation between the kinetic hot spots and the early forming structural elements in proteins. Additionally, a strong correlation between kinetically hot residues and loci of conserved residues is observed. Finally, residues that may be important for the stability of the tertiary structure of CheY are proposed. PMID:9865946

  19. A sub-population of keratan sulphates derived from bovine articular cartilage is capped with alpha(2-6)-linked N-acetylneuraminic acid residues. Affinity chromatography using immobilized Sambucus nigra lectin and characterization using 1H n.m.r. spectroscopy.

    PubMed Central

    Tai, G H; Morris, H G; Brown, G M; Huckerby, T N; Nieduszynski, I A

    1992-01-01

    Alkaline borohydride-reduced keratan sulphate (KS) chains derived from bovine femoral head cartilage were fractionated by lectin affinity chromatography with Sambucus nigra agglutinin (SNA) into binding and non-binding populations. Analysis of the SNA-binding and non-binding KS chains using 600 MHz 1H n.m.r. spectroscopy showed that the former population contained alpha(2-6)-N-acetylneuraminic acid residues and the latter contained primarily alpha(2-3)-N-acetylneuraminic acid residues as chain terminators. Both populations contained a similar proportion of alpha(2-3)-N-acetylneuraminic acid residues within their protein-linkage regions, and similar sulphation and fucosylation levels. Analysis of these two fractions by gel-permeation chromatography (g.p.c.) on a TSK-30 XL column showed them to have the same size distributions. It was concluded from the n.m.r. spectra and g.p.c. data that the populations differed primarily in the mode of linkage of the chain-terminating sialic acids. PMID:1520274

  20. Saturation Mutagenesis of TsrA Ala4 Unveils a Highly Mutable Residue of Thiostrepton A

    PubMed Central

    Zhang, Feifei; Kelly, Wendy L.

    2016-01-01

    Thiopeptides are posttranslationally processed macrocyclic peptide metabolites, characterized by extensive backbone and side chain modifications that include a six-membered nitrogeneous ring, thiazol(in)e/oxazol(in)e rings, and dehydrated amino acid residues. Thiostrepton A, one of the more structurally complex and well-studied thiopeptides, contains a second macrocycle bearing a quinaldic acid moiety. Antibacterial, antimalarial, and anticancer properties have been described for thiostrepton A and other thiopeptides, although the molecular details for binding the cellular target in each case are not fully elaborated. We previously demonstrated that a mutation of the TsrA core peptide, Ala4Gly, supported the successful production of the corresponding thiostrepton variant. To more thoroughly probe the thiostrepton biosynthetic machinery’s tolerance toward structural variation at the fourth position of the TsrA core peptide, we report here the saturation mutagenesis of this residue using a fosmid-dependent biosynthetic engineering method and the isolation of 16 thiostrepton analogs. Several types of side chain substitutions at the fourth position of TsrA, including those that introduce polar or branched, hydrophobic residues, are accepted, albeit with varied preferences. In contrast, proline and amino acid residues inherently charged at physiological pH are not well-tolerated at the queried site by the thiostrepton biosynthetic system. These newly generated thiostrepton analogs were assessed for their antibacterial activities and abilities to inhibit the proteolytic functions of the eukaryotic 20S proteasome. We demonstrate that the identity of the fourth amino acid residue in the thiostrepton scaffold is not critical for either ribosome or proteasome inhibition. PMID:25572285

  1. [Electronic cigarettes - effects on health. Previous reports].

    PubMed

    Napierała, Marta; Kulza, Maksymilian; Wachowiak, Anna; Jabłecka, Katarzyna; Florek, Ewa

    2014-01-01

    Currently very popular in the market of tobacco products have gained electronic cigarettes (ang. E-cigarettes). These products are considered to be potentially less harmful in compared to traditional tobacco products. However, current reports indicate that the statements of the producers regarding to the composition of the e- liquids not always are sufficient, and consumers often do not have reliable information on the quality of the product used by them. This paper contain a review of previous reports on the composition of e-cigarettes and their impact on health. Most of the observed health effects was related to symptoms of the respiratory tract, mouth, throat, neurological complications and sensory organs. Particularly hazardous effects of the e-cigarettes were: pneumonia, congestive heart failure, confusion, convulsions, hypotension, aspiration pneumonia, face second-degree burns, blindness, chest pain and rapid heartbeat. In the literature there is no information relating to passive exposure by the aerosols released during e-cigarette smoking. Furthermore, the information regarding to the use of these products in the long term are not also available. PMID:25799862

  2. Previous gastric bypass surgery complicating total thyroidectomy.

    PubMed

    Alfonso, Bianca; Jacobson, Adam S; Alon, Eran E; Via, Michael A

    2015-03-01

    Hypocalcemia is a well-known complication of total thyroidectomy. Patients who have previously undergone gastric bypass surgery may be at increased risk of hypocalcemia due to gastrointestinal malabsorption, secondary hyperparathyroidism, and an underlying vitamin D deficiency. We present the case of a 58-year-old woman who underwent a total thyroidectomy for the follicular variant of papillary thyroid carcinoma. Her history included Roux-en-Y gastric bypass surgery. Following the thyroid surgery, she developed postoperative hypocalcemia that required large doses of oral calcium carbonate (7.5 g/day), oral calcitriol (up to 4 μg/day), intravenous calcium gluconate (2.0 g/day), calcium citrate (2.0 g/day), and ergocalciferol (50,000 IU/day). Her serum calcium levels remained normal on this regimen after hospital discharge despite persistent hypoparathyroidism. Bariatric surgery patients who undergo thyroid surgery require aggressive supplementation to maintain normal serum calcium levels. Preoperative supplementation with calcium and vitamin D is strongly recommended. PMID:25738720

  3. Sebacinales everywhere: previously overlooked ubiquitous fungal endophytes.

    PubMed

    Weiss, Michael; Sýkorová, Zuzana; Garnica, Sigisfredo; Riess, Kai; Martos, Florent; Krause, Cornelia; Oberwinkler, Franz; Bauer, Robert; Redecker, Dirk

    2011-01-01

    Inconspicuous basidiomycetes from the order Sebacinales are known to be involved in a puzzling variety of mutualistic plant-fungal symbioses (mycorrhizae), which presumably involve transport of mineral nutrients. Recently a few members of this fungal order not fitting this definition and commonly referred to as 'endophytes' have raised considerable interest by their ability to enhance plant growth and to increase resistance of their host plants against abiotic stress factors and fungal pathogens. Using DNA-based detection and electron microscopy, we show that Sebacinales are not only extremely versatile in their mycorrhizal associations, but are also almost universally present as symptomless endophytes. They occurred in field specimens of bryophytes, pteridophytes and all families of herbaceous angiosperms we investigated, including liverworts, wheat, maize, and the non-mycorrhizal model plant Arabidopsis thaliana. They were present in all habitats we studied on four continents. We even detected these fungi in herbarium specimens originating from pioneering field trips to North Africa in the 1830s/40s. No geographical or host patterns were detected. Our data suggest that the multitude of mycorrhizal interactions in Sebacinales may have arisen from an ancestral endophytic habit by specialization. Considering their proven beneficial influence on plant growth and their ubiquity, endophytic Sebacinales may be a previously unrecognized universal hidden force in plant ecosystems. PMID:21347229

  4. CAMS confirmation of previously reported meteor showers

    NASA Astrophysics Data System (ADS)

    Jenniskens, P.; Nénon, Q.; Gural, P. S.; Albers, J.; Haberman, B.; Johnson, B.; Holman, D.; Morales, R.; Grigsby, B. J.; Samuels, D.; Johannink, C.

    2016-03-01

    Leading up to the 2015 IAU General Assembly, the International Astronomical Union's Working List of Meteor Showers included 486 unconfirmed showers, showers that are not certain to exist. If confirmed, each shower would provide a record of past comet or asteroid activity. Now, we report that 41 of these are detected in the Cameras for Allsky Meteor Surveillance (CAMS) video-based meteor shower survey. They manifest as meteoroids arriving at Earth from a similar direction and orbit, after removing the daily radiant drift due to Earth's motion around the Sun. These showers do exist and, therefore, can be moved to the IAU List of Established Meteor Showers. This adds to 31 previously confirmed showers from CAMS data. For each shower, finding charts are presented based on 230,000 meteors observed up to March of 2015, calculated by re-projecting the drift-corrected Sun-centered ecliptic coordinates into more familiar equatorial coordinates. Showers that are not detected, but should have, and duplicate showers that project to the same Sun-centered ecliptic coordinates, are recommended for removal from the Working List.

  5. Pyrotechnic reaction residue particle analysis.

    PubMed

    Kosanke, Kenneth L; Dujay, Richard C; Kosanke, Bonnie J

    2006-03-01

    Pyrotechnic reaction residue particle (PRRP) production, sampling and analysis are all very similar to that for primer gunshot residue. In both cases, the preferred method of analysis uses scanning electron microscopy to locate suspect particles and then uses energy dispersive x-ray spectroscopy to characterize the particle's constituent chemical elements. There are relatively few times when standard micro-analytical chemistry performed on pyrotechnic residues may not provide sufficient information for forensic investigators. However, on those occasions, PRRP analysis provides a greatly improved ability to discriminate between materials of pyrotechnic origin and other unrelated substances also present. The greater specificity of PRRP analysis is the result of its analyzing a large number of individual micron-sized particles, rather than producing only a single integrated result such as produced using standard micro-analytical chemistry. For example, PRRP analyses are used to demonstrate its ability to successfully (1) discriminate between pyrotechnic residues and unrelated background contamination, (2) identify that two different pyrotechnic compositions had previously been exploded within the same device, and (3) establish the chronology of an incident involving two separate and closely occurring explosions. PMID:16566762

  6. The selectivity of conantokin-G for ion channel inhibition of NR2B subunit-containing NMDA receptors is regulated by amino acid residues in the S2 region of NR2B.

    PubMed

    Sheng, Zhenyu; Liang, Zhong; Geiger, James H; Prorok, Mary; Castellino, Francis J

    2009-08-01

    The conantokins are short, naturally occurring peptides that inhibit ion flow through N-methyl-d-aspartate receptor (NMDAR) channels. One member of this peptide family, conantokin-G (con-G), shows high selectivity for antagonism of NR2B-containing NMDAR channels, whereas other known conantokins are less selective inhibitors with regard to the nature of the NR2 subunit of the NMDAR complex. In order to define the molecular determinants of NR2B that govern con-G selectivity, we evaluated the ability of con-G to inhibit NMDAR ion channels expressed in human embryonic kidney (HEK)293 cells transfected with NR1, in combination with various NR2A/2B chimeras and point mutants, by electrophysiology using cells voltage-clamped in the whole-cell configuration. We found that a variant of the con-G-insensitive subunit, NR2A, in which the 158 residues comprising the S2 peptide segment (E(657)-I(814)) were replaced by the corresponding S2 region of NR2B (E(658)-I(815)), results in receptors that are highly sensitive to inhibition by con-G. Of the 22 amino acids that are different between the NR2A-S2 and the NR2B-S2 regions, exchange of one of these, M(739) of NR2B for the equivalent K(738) of NR2A, was sufficient to completely import the inhibitory activity of con-G into NR1b/NR2A-containing NMDARs. Some reinforcement of this effect was found by substitution of a second amino acid, K(755) of NR2B for Y(754) of NR2A. The discovery of the molecular determinants of NR2B selectivity with con-G has implications for the design of subunit-selective neurobiological probes and drug therapies, in addition to advancing our understanding of NR2B- versus NR2A-mediated neurological processes. PMID:19427876

  7. Characterization by LC-MS(n) of four new classes of chlorogenic acids in green coffee beans: dimethoxycinnamoylquinic acids, diferuloylquinic acids, caffeoyl-dimethoxycinnamoylquinic acids, and feruloyl-dimethoxycinnamoylquinic acids.

    PubMed

    Clifford, Michael N; Knight, Susan; Surucu, Birgul; Kuhnert, Nikolai

    2006-03-22

    LC-MS4 has been used to detect and characterize in green coffee beans 12 chlorogenic acids not previously reported in nature. These comprise three isomeric dimethoxycinnamoylquinic acids (7-9) (Mr 382), three caffeoyl-dimethoxycinnamoylquinic acids (22, 24, and 26) (Mr 544), three diferuloylquinic acids (13-15) (Mr 544), and three feruloyl-dimethoxycinnamoylquinic acids (28, 30, and 32) (Mr 558). Structures have been assigned on the basis of LC-MS4 patterns of fragmentation and relative hydrophobicity and, in the case of the dimethoxycinnamoylquinic acids, by comparison with authentic standards. Several new structure-diagnostic fragmentations have been identified for use with diacyl-chlorogenic acids, for example, m/z 299 and 255 for C4 caffeoyl, m/z 313 and 269 for C4 feruloyl, nearly equal elimination of both cinnamoyl residues for vic-3,4-diacyl, and an increasing ratio of "dehydrated" ions to "non-dehydrated" ions at MS2 with increasing methylation of those cinnamoyl residues. Possible mechanisms have been proposed to account for the fragmentations observed. The mass spectrometric resolution of six isomeric chlorogenic acids (Mr 544) in a crude plant extract by fragment-targeted LC-MS2 and LC-MS3 experiments illustrates the analytical power and advantage of ion trap mass spectroscopy. PMID:16536562

  8. Mycobacterium microti: More diverse than previously thought.

    PubMed

    Smith, N H; Crawshaw, T; Parry, J; Birtles, R J

    2009-08-01

    Mycobacterium microti is a member of the Mycobacterium tuberculosis complex of bacteria. This species was originally identified as a pathogen of small rodents and shrews and was associated with limited diversity and a much reduced spoligotype pattern. More recently, specific deletions of chromosomal DNA have been shown to define this group of organisms, which can be identified by the absence of chromosomal region RD1(mic). We describe here the molecular characteristics of 141 strains of the Mycobacterium tuberculosis complex isolated in Great Britain over a 14-year period. All strains have characteristic loss of some spoligotype spacers and characteristic alleles at the ETR-E and ETR-F variable-number tandem-repeat (VNTR) loci, and a sample of these strains was deleted for regions RD7, RD9, and RD1(mic) but intact for regions RD4 and RD12. We therefore identified these strains as M. microti and show that they have much more diverse spoligotype patterns and VNTR types than previously thought. The most common source of these strains was domestic cats, and we show that the molecular types of M. microti are geographically localized in the same way that molecular types of Mycobacterium bovis are geographically localized in cattle in the United Kingdom. We describe the pathology of M. microti infection in cats and suggest that the feline disease is a spillover from a disease maintained in an unknown wild mammal, probably field voles. The location of the cats with M. microti infection suggests that they do not overlap geographically with the strains of Mycobacterium bovis in Great Britain. PMID:19535520

  9. Mycobacterium microti: More Diverse than Previously Thought▿

    PubMed Central

    Smith, N. H.; Crawshaw, T.; Parry, J.; Birtles, R. J.

    2009-01-01

    Mycobacterium microti is a member of the Mycobacterium tuberculosis complex of bacteria. This species was originally identified as a pathogen of small rodents and shrews and was associated with limited diversity and a much reduced spoligotype pattern. More recently, specific deletions of chromosomal DNA have been shown to define this group of organisms, which can be identified by the absence of chromosomal region RD1mic. We describe here the molecular characteristics of 141 strains of the Mycobacterium tuberculosis complex isolated in Great Britain over a 14-year period. All strains have characteristic loss of some spoligotype spacers and characteristic alleles at the ETR-E and ETR-F variable-number tandem-repeat (VNTR) loci, and a sample of these strains was deleted for regions RD7, RD9, and RD1mic but intact for regions RD4 and RD12. We therefore identified these strains as M. microti and show that they have much more diverse spoligotype patterns and VNTR types than previously thought. The most common source of these strains was domestic cats, and we show that the molecular types of M. microti are geographically localized in the same way that molecular types of Mycobacterium bovis are geographically localized in cattle in the United Kingdom. We describe the pathology of M. microti infection in cats and suggest that the feline disease is a spillover from a disease maintained in an unknown wild mammal, probably field voles. The location of the cats with M. microti infection suggests that they do not overlap geographically with the strains of Mycobacterium bovis in Great Britain. PMID:19535520

  10. A highly conserved arginine residue of the chitosanase from Streptomyces sp. N174 is involved both in catalysis and substrate binding

    PubMed Central

    2013-01-01

    Background Streptomyces sp. N174 chitosanase (CsnN174), a member of glycoside hydrolases family 46, is one of the most extensively studied chitosanases. Previous studies allowed identifying several key residues of this inverting enzyme, such as the two catalytic carboxylic amino acids as well as residues that are involved in substrate binding. In spite of the progress in understanding the catalytic mechanism of this chitosanase, the function of some residues highly conserved throughout GH46 family has not been fully elucidated. This study focuses on one of such residues, the arginine 42. Results Mutation of Arg42 into any other amino acid resulted in a drastic loss of enzyme activity. Detailed investigations of R42E and R42K chitosanases revealed that the mutant enzymes are not only impaired in their catalytic activity but also in their mode of interaction with the substrate. Mutated enzymes were more sensitive to substrate inhibition and were altered in their pattern of activity against chitosans of various degrees of deacetylation. Our data show that Arg42 plays a dual role in CsnN174 activity. Conclusions Arginine 42 is essential to maintain the enzymatic function of chitosanase CsnN174. We suggest that this arginine is influencing the catalytic nucleophile residue and also the substrate binding mode of the enzyme by optimizing the electrostatic interaction between the negatively charged carboxylic residues of the substrate binding cleft and the amino groups of GlcN residues in chitosan. PMID:24041306

  11. EVALUATING THE USE OF SNOW-COVERED RANGES TO ESTIMATE THE EXPLOSIVES RESIDUES THAT RESULT FROM DETONATION OF ARMY MUNITIONS

    EPA Science Inventory

    Estimating the amounts of residues remaining after munitions detonate is complicated by the presence of residues from previous detonations and the difficulty in easily obtaining adequately sized samples to overcome spatial heterogeneity in residue deposition. This study was cond...

  12. [Migrants from disposable gloves and residual acrylonitrile].

    PubMed

    Wakui, C; Kawamura, Y; Maitani, T

    2001-10-01

    Disposable gloves made from polyvinyl chloride with and without di(2-ethylhexyl) phthalate (PVC-DEHP, PVC-NP), polyethylene (PE), natural rubber (NR) and nitrile-butadiene rubber (NBR) were investigated with respect to evaporation residue, migrated metals, migrants and residual acrylonitrile. The evaporation residue found in n-heptane was 870-1,300 ppm from PVC-DEHP and PVC-NP, which was due to the plasticizers. Most of the PE gloves had low evaporation residue levels and migrants, except for the glove designated as antibacterial, which released copper and zinc into 4% acetic acid. For the NR and NBR gloves, the evaporation residue found in 4% acetic acid was 29-180 ppm. They also released over 10 ppm of calcium and 6 ppm of zinc into 4% acetic acid, and 1.68-8.37 ppm of zinc di-ethyldithiocarbamate and zinc di-n-butyldithiocarbamate used as vulcanization accelerators into n-heptane. The acrylonitrile content was 0.40-0.94 ppm in NBR gloves. PMID:11775358

  13. 40 CFR 180.473 - Glufosinate ammonium; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ..., pulp 0.20 Beet, sugar, molasses 5.0 Beet, sugar, roots 0.9 Beet, sugar, tops (leaves) 1.5 Bushberry... residues of the herbicide glufosinate-ammonium (butanoic acid, 2-amino-4-(hydroxymethylphosphinyl...-propionic acid, expressed as 2-amino-4-(hydroxymethylphosphinyl)butanoic acid equivalents, in or on...

  14. Ionization characteristics and chemical influences of aspartic acid residue 158 of papain and caricain determined by structure-related kinetic and computational techniques: multiple electrostatic modulators of active-centre chemistry.

    PubMed

    Noble, M A; Gul, S; Verma, C S; Brocklehurst, K

    2000-11-01

    The pK(a) of (Asp(158))-CO(2)H of papain (EC 3.4.22.2) was determined as 2.8 by using 4-chloro-7-nitrobenzofurazan (Nbf-Cl) as a reactivity probe targeted on the thiolate anion component of the Cys(25)/His(159) nucleophilic-acid/base motif of the catalytic site. The possibility of using Nbf-Cl for this purpose was established by modelling the papain-Nbf-Cl Meisenheimer intermediate by using QUANTA/CHARMM and performing molecular orbital calculations with MOPAC interfaced with Cerius 2. A pH-dependent stopped-flow kinetic study of the reaction of papain with Nbf-Cl established that the striking rate maximum at pH 3 results from reaction in a minor ionization state comprising (Cys(25))-S(-)/(His(159))-Im(+)H (in which Im represents imidazole) produced by protonic dissociation of (Cys(25))-SH/(His(159))-Im(+)H with pK(a) 3.3 and (Asp(158))-CO(2)H. Although the analogous intermediate in the reaction of caricain (EC 3.4.22.30) with Nbf-Cl has similar geometry, the pH-k profile (k being the second-order rate constant) lacks a rate maximum under acidic conditions. This precludes the experimental determination of the pK(a) value of (Asp(158))-CO(2)H of caricain, which was calculated to be 2.0 by solving the linearized Poisson-Boltzmann equation with the program UHBD ('University of Houston Brownian dynamics'). A value lower than 2.8 had been predicted by consideration of the hydrogen-bonded networks involving Asp(158) and its microenvironments in both enzymes. The difference between these pK(a) values (values not previously detected in reactions of either enzyme) accounts for the lack of the rate maximum in the caricain reaction and for the differences in the electronic absorption spectra of the two S-Nbf-enzymes under acidic conditions. The concept of control of cysteine proteinase activity by multiple electrostatic modulators, including (Asp(158))-CO(2)(-), which modifies traditional mechanistic views, is discussed. PMID:11042128

  15. Ionization characteristics and chemical influences of aspartic acid residue 158 of papain and caricain determined by structure-related kinetic and computational techniques: multiple electrostatic modulators of active-centre chemistry.

    PubMed Central

    Noble, M A; Gul, S; Verma, C S; Brocklehurst, K

    2000-01-01

    The pK(a) of (Asp(158))-CO(2)H of papain (EC 3.4.22.2) was determined as 2.8 by using 4-chloro-7-nitrobenzofurazan (Nbf-Cl) as a reactivity probe targeted on the thiolate anion component of the Cys(25)/His(159) nucleophilic-acid/base motif of the catalytic site. The possibility of using Nbf-Cl for this purpose was established by modelling the papain-Nbf-Cl Meisenheimer intermediate by using QUANTA/CHARMM and performing molecular orbital calculations with MOPAC interfaced with Cerius 2. A pH-dependent stopped-flow kinetic study of the reaction of papain with Nbf-Cl established that the striking rate maximum at pH 3 results from reaction in a minor ionization state comprising (Cys(25))-S(-)/(His(159))-Im(+)H (in which Im represents imidazole) produced by protonic dissociation of (Cys(25))-SH/(His(159))-Im(+)H with pK(a) 3.3 and (Asp(158))-CO(2)H. Although the analogous intermediate in the reaction of caricain (EC 3.4.22.30) with Nbf-Cl has similar geometry, the pH-k profile (k being the second-order rate constant) lacks a rate maximum under acidic conditions. This precludes the experimental determination of the pK(a) value of (Asp(158))-CO(2)H of caricain, which was calculated to be 2.0 by solving the linearized Poisson-Boltzmann equation with the program UHBD ('University of Houston Brownian dynamics'). A value lower than 2.8 had been predicted by consideration of the hydrogen-bonded networks involving Asp(158) and its microenvironments in both enzymes. The difference between these pK(a) values (values not previously detected in reactions of either enzyme) accounts for the lack of the rate maximum in the caricain reaction and for the differences in the electronic absorption spectra of the two S-Nbf-enzymes under acidic conditions. The concept of control of cysteine proteinase activity by multiple electrostatic modulators, including (Asp(158))-CO(2)(-), which modifies traditional mechanistic views, is discussed. PMID:11042128

  16. Permethylation Linkage Analysis Techniques for Residual Carbohydrates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Permethylation analysis is the classic approach to establishing the position of glycosidic linkages between sugar residues. Typically, the carbohydrate is derivatized to form acid-stable methyl ethers, hydrolyzed, peracetylated, and analyzed by gas chromatography-mass spectrometry (GC-MS). The pos...

  17. Residual macrovascular risk in 2013: what have we learned?

    PubMed Central

    2014-01-01

    Cardiovascular disease poses a major challenge for the 21st century, exacerbated by the pandemics of obesity, metabolic syndrome and type 2 diabetes. While best standards of care, including high-dose statins, can ameliorate the risk of vascular complications, patients remain at high risk of cardiovascular events. The Residual Risk Reduction Initiative (R3i) has previously highlighted atherogenic dyslipidaemia, defined as the imbalance between proatherogenic triglyceride-rich apolipoprotein B-containing-lipoproteins and antiatherogenic apolipoprotein A-I-lipoproteins (as in high-density lipoprotein, HDL), as an important modifiable contributor to lipid-related residual cardiovascular risk, especially in insulin-resistant conditions. As part of its mission to improve awareness and clinical management of atherogenic dyslipidaemia, the R3i has identified three key priorities for action: i) to improve recognition of atherogenic dyslipidaemia in patients at high cardiometabolic risk with or without diabetes; ii) to improve implementation and adherence to guideline-based therapies; and iii) to improve therapeutic strategies for managing atherogenic dyslipidaemia. The R3i believes that monitoring of non-HDL cholesterol provides a simple, practical tool for treatment decisions regarding the management of lipid-related residual cardiovascular risk. Addition of a fibrate, niacin (North and South America), omega-3 fatty acids or ezetimibe are all options for combination with a statin to further reduce non-HDL cholesterol, although lacking in hard evidence for cardiovascular outcome benefits. Several emerging treatments may offer promise. These include the next generation peroxisome proliferator-activated receptorα agonists, cholesteryl ester transfer protein inhibitors and monoclonal antibody therapy targeting proprotein convertase subtilisin/kexin type 9. However, long-term outcomes and safety data are clearly needed. In conclusion, the R3i believes that ongoing trials with

  18. Acid fog

    SciTech Connect

    Hileman, B.

    1983-03-01

    Fog in areas of southern California previously thought to be pollution-free has been shown to have a pH as low as 1.69. It has been found to be most acidic after smoggy days, suggesting that it forms on the aerosol associated with the previously exiting smog. Studies on Whiteface Mountain in the Adirondacks show that fog water is often 10 times as acidic as rainwater. As a result of their studies, California plans to spend $4 million on acid deposition research in the coming year. (JMT)

  19. A determinative and confirmatory method for residues of the metabolites of carbadox and olaquindox in porcine tissues.

    PubMed

    Boison, Joe O; Lee, Stephen C; Gedir, Ron G

    2009-04-01

    Carbadox (CBX) and olaquindox (OLQ) are used in swine feed for growth promotion, to improve feed efficiency, increase the rate of weight gain, control swine dysentery and bacterial enteritis in young swine. In 1991, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) recommended maximum residue limits (MRLs) of 30 and 5mugkg(-1) in liver and muscle tissues of pigs, respectively, based on the concentration of, and expressed as, quinoxaline-2-carboxylic acid (QCA) as marker residue. In 1998, the European Commission (EC) banned the use of CBX and OLQ in food animal production together with four other feed additives, following reports that CBX and desoxycarbadox (DCBX) are suspect carcinogens and mutagens. In 2001, the sale of CBX was halted in Canada. In 2003, JECFA recommended the withdrawal of the previously recommended acceptable daily intake (ADI) and MRLs and concluded that QCA was not a suitable marker residue for CBX, based on new sponsor studies reporting that DCBX, the suspect carcinogen, persisted in animal tissues much longer than had previously been thought. This paper presents a very sensitive LC-MS/MS method that was developed by CFIA scientists for the simultaneous determination and confirmation of DCBX residues at concentrations >/=0.050 ngkg(-1) and QCA and mQCA residues at concentrations >/=0.50 ngkg(-1)in bovine muscle, pork liver and muscle tissues. PMID:19286021

  20. Conserved residues Asp16 and Pro24 of TnaC-tRNAPro participate in tryptophan induction of Tna operon expression.

    PubMed

    Cruz-Vera, Luis R; Yanofsky, Charles

    2008-07-01

    In Escherichia coli, interactions between the nascent TnaC-tRNA(Pro) peptidyl-tRNA and the translating ribosome create a tryptophan binding site in the ribosome where bound tryptophan inhibits TnaC-tRNA(Pro) cleavage. This inhibition delays ribosome release, thereby inhibiting Rho factor binding and action, resulting in increased tna operon transcription. Replacing Trp12 of TnaC with any other amino acid residue was previously shown to prevent tryptophan binding and induction of tna operon expression. Genome-wide comparisons of TnaC amino acid sequences identify Asp16 and Pro24, as well as Trp12, as highly conserved TnaC residues. Replacing these residues with other residues was previously shown to influence tryptophan induction of tna operon expression. In this study, in vitro analyses were performed to examine the potential roles of Asp16 and Pro24 in tna operon induction. Replacing Asp16 or Pro24 of TnaC of E. coli with other amino acids established that these residues are essential for free tryptophan binding and inhibition of TnaC-tRNA(Pro) cleavage at the peptidyl transferase center. Asp16 and Pro24 are in fact located in spatial positions corresponding to critical residues of AAP, another ribosome regulatory peptide. Sparsomycin-methylation protection studies further suggested that segments of 23S RNA were arranged differently in ribosomes bearing TnaCs with either the Asp16Ala or the Pro24Ala change. Thus, features of the amino acid sequence of TnaC of the nascent TnaC-tRNA(Pro) peptidyl-tRNA, in addition to the presence of Trp12, are necessary for the nascent peptide to create a tryptophan binding/inhibition site in the translating ribosome. PMID:18424524

  1. Numerical simulation of the shot peening process under previous loading conditions

    NASA Astrophysics Data System (ADS)

    Romero-Ángeles, B.; Urriolagoitia-Sosa, G.; Torres-San Miguel, C. R.; Molina-Ballinas, A.; Benítez-García, H. A.; Vargas-Bustos, J. A.; Urriolagoitia-Calderón, G.

    2015-01-01

    This research presents a numerical simulation of the shot peening process and determines the residual stress field induced into a component with a previous loading history. The importance of this analysis is based on the fact that mechanical elements under shot peening are also subjected to manufacturing processes, which convert raw material into finished product. However, material is not provided in a virgin state, it has a previous loading history caused by the manner it is fabricated. This condition could alter some beneficial aspects of the residual stress induced by shot peening and could accelerate the crack nucleation and propagation progression. Studies were performed in beams subjected to strain hardening in tension (5epsilony) before shot peening was applied. Latter results were then compared in a numerical assessment of an induced residual stress field by shot peening carried out in a component (beam) without any previous loading history. In this paper, it is clearly shown the detrimental or beneficial effect that previous loading history can bring to the mechanical component and how it can be controlled to improve the mechanical behavior of the material.

  2. Effects of Single α-to-β Residue Replacements on Structure and Stability in a Small Protein: Insights from Quasiracemic Crystallization.

    PubMed

    Kreitler, Dale F; Mortenson, David E; Forest, Katrina T; Gellman, Samuel H

    2016-05-25

    Synthetic peptides that contain backbone modifications but nevertheless adopt folded structures similar to those of natural polypeptides are of fundamental interest and may provide a basis for biomedical applications. Such molecules can, for example, mimic the ability of natural prototypes to bind to specific target macromolecules but resist degradation by proteases. We have previously shown that oligomers containing mixtures of α- and β-amino acid residues ("α/β-peptides") can mimic the α-helix secondary structure, and that properly designed α/β-peptides can bind to proteins that evolved to bind to α-helical partners. Here we report fundamental studies that support the long-range goal of extending the α/β approach to tertiary structures. We have evaluated the impact of single α → β modifications on the structure and stability of the small and well-studied villin headpiece subdomain (VHP). The native state of this 35-residue polypeptide contains several α-helical segments packed around a small hydrophobic core. We examined α → β substitution at four solvent-exposed positions, Asn19, Trp23, Gln26 and Lys30. In each case, both the β(3) homologue of the natural α residue and a cyclic β residue were evaluated. All α → β(3) substitutions caused significant destabilization of the tertiary structure as measured by variable-temperature circular dichroism, although at some of these positions, replacing the β(3) residue with a cyclic β residue led to improved stability. Atomic-resolution structures of four VHP analogues were obtained via quasiracemic crystallization. These findings contribute to a fundamental