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Sample records for acid residues previously

  1. Critical aspartic acid residues in pseudouridine synthases.

    PubMed

    Ramamurthy, V; Swann, S L; Paulson, J L; Spedaliere, C J; Mueller, E G

    1999-08-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411-2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998) Biochemistry 37, 344-351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function.

  2. 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.

  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. Amino acid residues modulating the activities of staphylococcal glutamyl endopeptidases.

    PubMed

    Ono, Toshio; Ohara-Nemoto, Yuko; Shimoyama, Yu; Okawara, Hisami; Kobayakawa, Takeshi; Baba, Tomomi T; Kimura, Shigenobu; Nemoto, Takayuki K

    2010-10-01

    The glutamyl endopeptidase family of enzymes from staphylococci has been shown to be important virulence determinants of pathogenic family members, such as Staphylococcus aureus. Previous studies have identified the N-terminus and residues from positions 185-195 as potentially important regions that determine the activity of three members of the family. Cloning and sequencing of the new family members from Staphylococcus caprae (GluScpr) and Staphylococcus cohnii (GluScoh) revealed that the N-terminal Val residue is maintained in all family members. Mutants of the GluV8 enzyme from S. aureus with altered N-terminal residues, including amino acids with similar properties, were inactive, indicating that the Val residue is specifically required at the N-terminus of this enzyme family in order for them to function correctly. Recombinant GluScpr was found to have peptidase activity intermediate between GluV8 and GluSE from Staphylococcus epidermis and to be somewhat less specific in its substrate requirements than other family members. The 185-195 region was found to contribute to the activity of GluScpr, although other regions of the enzyme must also play a role in defining the activity. Our results strongly indicate the importance of the N-terminal and the 185-195 region in the activity of the glutamyl endopeptidases of staphylococci. PMID:20707600

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. Amino acid network for prediction of catalytic residues in enzymes: a comparison survey.

    PubMed

    Zhou, Jianhong; Yan, Wenying; Hu, Guang; Shen, Bairong

    2016-01-01

    Catalytic residues play a significant role in enzyme functions. With the recent accumulation of experimentally determined enzyme 3D structures and network theory on protein structures, the prediction of catalytic residues by amino acid network (AAN, where nodes are residues and links are residue interactions) has gained much interest. Computational methods of identifying catalytic residues are traditionally divided into two groups: sequence-based and structure-based methods. Two new structure- based methods are proposed in current advances: AAN and Elastic Network Model (ENM) of enzyme structures. By concentrating on AAN-based approach, we herein summarized network properties for predictions of catalytic residues. AAN attributes were showed responsible for performance improvement, and therefore the combination of AAN with previous sequence and structural information will be a promising direction for further improvement. Advantages and limitations of AAN-based methods, future perspectives on the application of AAN to the study of protein structure-function relationships are discussed.

  13. 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.

  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. Core Amino Acid Residues in the Morphology-Regulating Protein, Mms6, for Intracellular Magnetite Biomineralization

    PubMed Central

    Yamagishi, Ayana; Narumiya, Kaori; Tanaka, Masayoshi; Matsunaga, Tadashi; Arakaki, Atsushi

    2016-01-01

    Living organisms produce finely tuned biomineral architectures with the aid of biomineral-associated proteins. The functional amino acid residues in these proteins have been previously identified using in vitro and in silico experimentation in different biomineralization systems. However, the investigation in living organisms is limited owing to the difficulty in establishing appropriate genetic techniques. Mms6 protein, isolated from the surface of magnetite crystals synthesized in magnetotactic bacteria, was shown to play a key role in the regulation of crystal morphology. In this study, we have demonstrated a defect in the specific region or substituted acidic amino acid residues in the Mms6 protein for observing their effect on magnetite biomineralization in vivo. Analysis of the gene deletion mutants and transformants of Magnetospirillum magneticum AMB-1 expressing partially truncated Mms6 protein revealed that deletions in the N-terminal or C-terminal regions disrupted proper protein localization to the magnetite surface, resulting in a change in the crystal morphology. Moreover, single amino acid substitutions at Asp123, Glu124, or Glu125 in the C-terminal region of Mms6 clearly indicated that these amino acid residues had a direct impact on magnetite crystal morphology. Thus, these consecutive acidic amino acid residues were found to be core residues regulating magnetite crystal morphology. PMID:27759096

  16. Ligatin binds phosphohexose residues on acidic hydrolases.

    PubMed

    Jakoi, E R; Kempe, K; Gaston, S M

    1981-01-01

    Ligatin, a receptor that recognizes phosphorylated sugars, was isolated from plasma membranes of mouse macrophages, rat ileum, and rat brain. Several acidic hydrolases including N-acetyl beta-D-glucosaminidase (beta-NAG) were solubilized with this receptor. The solubilized beta-NAG bound to ligatin in vitro as demonstrated by affinity chromatography using the immobilized receptor. beta-N-Acetyl D-glucosaminidase-ligatin complexes were dissociated by low concentrations of mannose 6-phosphate (Man6P) and/or glucose 1-phosphate (Glc 1P). The effectiveness of these two phosphomonosaccharides varied depending on the source of the enzyme: ileal beta-NAG-ligatin complexes showed a four-fold preferential dissociation with Man6P; macrophage complexes showed a 160-fold preferential dissociation with Glc 1P. Brain complexes dissociated with nearly equal preference for Man6P and Glc 1P. Heterologous complexes displayed the specificity characteristic of the source of the enzyme regardless of the source of the ligatin. Treatment of the solubilized hydrolases with endoglucosaminidase H released phosphorous-32 label from these enzymes and prevented binding of beta-NAG to ligatin. However, treatment of the solubilized hydrolases with alkaline phosphatase reduced the binding of beta-NAG to ligatin by no more than 30%. This apparent resistance of beta-NAG to dephosphorylation was consistent with the chromatographic behavior of QAE of 3H-labeled acidic oligosaccharides isolated from the solubilized hydrolases. The oligosaccharides that contain phosphorylated hexose were less acidic than phosphomonoesters and were insensitive to alkaline phosphatase until subjected to acid hydrolysis. These results suggested the presence of a phosphodiester on beta-NAG analogous to the NAC glucosamine 1 P6 mannose present on beta-glucuronidase isolated from mouse lymphoma cells (Tabas I, Kornfield, S: J Biol Chem 255: 6633, 1980). PMID:7299841

  17. 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.

  18. Electron microscopy of carbonaceous matter in Allende acid residues

    NASA Astrophysics Data System (ADS)

    Lumpkin, G. R.

    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.

  19. 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.

  20. 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

  1. A color-determining amino acid residue of proteorhodopsin.

    PubMed

    Ozaki, Yuya; Kawashima, Takayoshi; Abe-Yoshizumi, Rei; Kandori, Hideki

    2014-09-30

    Proteorhodopsin (PR) is a light-driven proton pump found in marine bacteria. More than 1000 PRs are classified as blue-absorbing (λmax ∼ 490 nm) and green-absorbing (λmax ∼ 525 nm) PRs. The color determinant is known to be at position 105, where blue-absorbing and green-absorbing PRs possess Gln and Leu, respectively. This suggests hydrophobicity at position 105 plays a key role in color tuning. Here we successfully introduced 19 amino acid residues into position 105 of green-absorbing PR in the membrane environment and investigated the absorption properties. High-performance liquid chromatography analysis shows that the isomeric composition of the all-trans form is >70% for all mutants, indicating little influence of different isomers on color tuning. Absorption spectra of the wild-type and 19 mutant proteins were well-characterized by the pH-dependent equilibria of the protonated and deprotonated counterion (Asp97) of the Schiff base, whereas the λmax values of these two states and the pKa value differed significantly among mutants. Although Gln and Leu are hydrophilic and hydrophobic residues, respectively, the λmax values of the two states and the pKa value did not correlate with the hydropathy index of residues. In contrast, the λmax and pKa were correlated with the volume of residues, though Gln and Leu possess similar volumes. This observation concludes that the λmax and pKa of Asp97 are determined by local and specific interactions in the Schiff base moiety, in which the volume of the residue at position 105 is more influential than its hydrophobicity. We suggest that the hydrogen-bonding network in the Schiff base moiety plays a key role in the λmax and pKa of Asp97, and the hydrogen-bonding network is significantly perturbed by large amino acid residues but may be preserved by additional water molecule(s) for small amino acid residues at position 105. PMID:25180875

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. Involvement of Acidic Amino Acid Residues in Zn(2+) Binding to Respiratory Complex I.

    PubMed

    Kriegel, Sébastien; Srour, Batoul; Steimle, Stefan; Friedrich, Thorsten; Hellwig, Petra

    2015-09-21

    Proton transfer across membranes and membrane proteins is a central process in biological systems. Zn(2+) ions are capable of binding to acidic residues, often found within such specific pathways, thereby leading to a blockage. Here we probed Zn(2+) inhibition of the proton-pumping NADH:ubiquinone oxidoreductase from Escherichia coli by means of electrochemically induced FTIR difference spectroscopy. Numerous conformational changes were identified including those that arise from the reorganization of the membrane arm upon electron transfer in the peripheral arm of the protein. Signals at very high wavenumbers (1781 and 1756 cm(-1)) point to the perturbation of acidic residues in a highly hydrophobic environment upon Zn(2+) binding. In variant D563N(L), which lacks part of the proton pumping activity (residue located on the horizontal amphipathic helix), the spectral signature of Zn(2+) binding is changed. Our data support a role for this residue in proton translocation.

  7. 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.

  8. 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

  9. Identification of the amino acid residues rendering TI-VAMP insensitive toward botulinum neurotoxin B.

    PubMed

    Sikorra, Stefan; Henke, Tina; Swaminathan, Subramanyam; Galli, Thierry; Binz, Thomas

    2006-03-24

    Botulinum neurotoxins types B, D, F, and G, and tetanus neurotoxin inhibit vesicular fusion via proteolytic cleavage of VAMP/Synaptobrevin, a core component of the membrane fusion machinery. Thus, these neurotoxins became widely used tools for investigating vesicular trafficking routes. Except for VAMP-1, VAMP-2, and Cellubrevin, no other member of the VAMP family represents a substrate for these neurotoxins. The molecular basis for this discrepancy is not known. A 34 amino acid residue segment of VAMP-2 was previously suggested to mediate the interaction with botulinum neurotoxin B, but the validity of the data was later questioned. To check whether this segment alone controls the susceptibility toward botulinum neurotoxin B, it was used to replace the corresponding segment in TI-VAMP. The resulting VAMP hybrid and VAMP-2 were hydrolysed at virtually identical rates. Resetting the VAMP-2 portion in the hybrid from either end to TI-VAMP residues gradually reduced the cleavability. A hybrid encompassing merely the VAMP-2 segment 71-80 around the Gln76/Phe77 scissile bond was still hydrolysed, albeit at a approximately tenfold lower cleavage rate. The contribution of each non-conserved amino acid of the whole 34-mer segment to the interaction was investigated employing VAMP-2. We find that the eight non-conserved residues of the 71-80 segment are all necessary for efficient cleavage. Mutation of an additional six residues located upstream and downstream of this segment affects substrate hydrolysis as well. Vice versa, a readily cleavable TI-VAMP molecule requires at the least the replacement of Ile158, Thr161, and the section 165-174 by Asp64, Ala67, and the 71-80 segment of VAMP-2, respectively. However, the insensitivity of TI-VAMP to botulinum neurotoxin B relies on at least 12 amino acid changes versus VAMP-2. These are scattered along an interface of 22 amino acid residues in length.

  10. 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.

  11. Chondroitin 4-sulphotransferase-1 and chondroitin 6-sulphotransferase-1 are affected differently by uronic acid residues neighbouring the acceptor GalNAc residues

    PubMed Central

    2004-01-01

    C4ST-1 (chondroitin 4-sulphotransferase-1) and C6ST-1 (chondroitin 6-sulphotransferase-1) transfer sulphate from PAPS (adenosine 3′-phosphate 5′-phosphosulphate) to positions 4 and 6 respectively of the GalNAc residues of chondroitin. We showed previously that C4ST-1 purified from rat chondrosarcoma and recombinant C4ST-1 both transfer sulphate efficiently to position 4 of the GalNAc residues of DSDS (desulphated dermatan sulphate). We report here the specificity of C4ST-1 and C6ST-1 in terms of uronic acid residue recognition around the GalNAc residue to which sulphate is transferred. When [35S]glycosaminoglycans formed from DSDS after incubation with [35S]PAPS and C4ST-1 were digested with chondroitinase ACII, a major part of the radioactivity was recovered in disaccharide fractions and the remainder distributed to tetrasaccharides and larger fractions, indicating that C4ST-1 mainly transferred sulphate to position 4 of the GalNAc residue located at the GlcA-GalNAc-GlcA sequence. Structural analysis of tetrasaccharide and larger oligosaccharide fractions indicated that C4ST-1 mainly transferred sulphate to the GalNAc residue adjacent to the reducing side of the GlcA residue. On the other hand, when [35S]glycosaminoglycans formed from DSDS after incubation with [35S]PAPS and C6ST-1 were digested with chondroitinase ACII, a major part of the radioactivity was recovered in fractions larger than hexasaccharides, indicating that C6ST-1 transferred sulphate to the GalNAc residues located in the L-iduronic acid-rich region. Structural analysis of the tetrasaccharide and larger oligosaccharide fractions indicated that C6ST-1 showed very little preference for the GalNAc residue neighbouring the GlcA residue. These results indicate that C4ST-1 and C6ST-1 differ from each other in the recognition of uronic acid residues adjacent to the targeted GalNAc residue. PMID:15324304

  12. 40 CFR 180.155 - 1-Naphthaleneacetic acid; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Tolerances § 180.155 1-Naphthaleneacetic acid; tolerances for residues. (a) General. Tolerances are established for the combined residues of the plant growth regulator 1-naphthaleneacetic acid and its... 40 Protection of Environment 23 2010-07-01 2010-07-01 false 1-Naphthaleneacetic acid;...

  13. 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.

  14. 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.

  15. 40 CFR 180.318 - 4-(2-Methyl-4-chlorophenoxy) butyric acid; tolerance for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...)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...

  16. 40 CFR 180.318 - 4-(2-Methyl-4-chlorophenoxy) butyric acid; tolerance for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...)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...

  17. 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...

  18. 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

  19. Amino-acid residues involved in the expression of the activity of Escherichia coli TolC.

    PubMed

    Yamanaka, Hiroyasu; Morisada, Naoyuki; Miyano, Masaya; Tsuge, Hideaki; Shinoda, Sumio; Takahashi, Eizo; Okamoto, Keinosuke

    2004-01-01

    The Escherichia coli TolC, composed of 471 amino-acid residues, functions as a channel tunnel in the transport of various molecules across the outer membrane. We found previously that Leu-412, the 60th amino-acid residue from the carboxy terminal end, was crucial to the transport activity of TolC. Leu-412 is located in a domain which protrudes from the main body of TolC into the periplasm. Subsequent study indicated that the hydrophobicity generated by Leu-412 played an important role in the activity of TolC (H. Yamanaka, T. Nomura, N. Morisada, S. Shinoda, and K. Okamoto, Microb. Pathog. 33: 81-89, 2002). We predicted that other hydrophobic amino-acid residues around Leu-412 were also involved in the expression of the activity of TolC. To test this possibility, we substituted several hydrophobic residues around Leu-412, (Leu-3, Val-6, Leu-212, Leu-213, Leu-223, and Leu-224), with serine and examined the activity of these mutant TolCs. The result showed that Leu-3 is involved in the activity of TolC, but the other residues are not. The involvement of Leu-3 was confirmed by the residue deletion experiment. A subsequent point-mutational analysis of the residue showed that a hydrophobic side chain is required at position 3 for TolC to express its activity. As the distance between the alpha-carbons of Leu-3 and Leu-412 is just 7.45 angstroms, hydrophobic interaction between the two leucine residues might be involved in the activity of TolC. PMID:15502403

  20. Newly identified essential amino acid residues affecting Δ8-sphingolipid desaturase activity revealed by site-directed mutagenesis.

    PubMed

    Li, Shu-Fen; Song, Li-Ying; Zhang, Guo-Jun; Yin, Wei-Bo; Chen, Yu-Hong; Wang, Richard R-C; Hu, Zan-Min

    2011-12-01

    In order to identify amino acid residues crucial for the enzymatic activity of Δ(8)-sphingolipid desaturases, a sequence comparison was performed among Δ(8)-sphingolipid desaturases and Δ(6)-fatty acid desaturases from various plants. In addition to the known conserved cytb(5) (cytochrome b(5)) HPGG motif and three conserved histidine boxes, they share additional 15 completely conserved residues. A series of site-directed mutants were generated using our previously isolated Δ(8)-sphingolipid desaturase gene from Brassica rapa to evaluate the importance of these residues to the enzyme function. The mutants were functionally characterized by heterologous expression in yeast, allowing the identification of the products of the enzymes. The results revealed that residues H63, N203, D208, D210, and G368 were obligatorily required for the enzymatic activity, and substitution of the residues F59, W190, W345, L369 and Q372 markedly decreased the enzyme activity. Among them, replacement of the residues W190, L369 and Q372 also has significant influence on the ratio of the two enzyme products. Information obtained in this work provides the molecular basis for the Δ(8)-sphingolipid desaturase activity and aids in our understanding of the structure-function relationships of the membrane-bound desaturases.

  1. 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

  2. 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.

  3. 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...

  4. 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...

  5. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite p... million Bean, mung, sprouts 0.2 (b) Section 18 emergency exemptions. (c) Tolerances with...

  6. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite p... million Bean, mung, sprouts 0.2 (b) Section 18 emergency exemptions. (c) Tolerances with...

  7. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite p... million Bean, mung, sprouts 0.2 (b) Section 18 emergency exemptions. (c) Tolerances with...

  8. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite p... million Bean, mung, sprouts 0.2 (b) Section 18 emergency exemptions. (c) Tolerances with...

  9. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite p... million Bean, mung, sprouts 0.2 (b) Section 18 emergency exemptions. (c) Tolerances with...

  10. The isotopic composition of zinc, palladium, silver, cadmium, tin, and tellurium in acid-etched residues of the Allende meteorite

    SciTech Connect

    Loss, R.D.; Rosman, K.J.R.; De Laeter, J.R. )

    1990-12-01

    The isotopic and elemental abundances of Zn, Pd, Ag, Cd, Sn, and Te have been measured in three acid-resistant residues extracted from the Allende meteorite. High-efficiency, low-contamination ion-exchange procedures were developed to separate and purify the nanogram amounts of these elements present. Elemental-abundance determinations performed by Mass Spectrometric Isotope Dilution agree with previously published work for similarly derived residues. No isotope anomalies similar to those found for Xe (Xe-HL) in these samples were detected for any of these elements, which is consistent with the residues not being derived directly from the Xe-HL carriers. The lack of major Te-isotope anomalies does not support earlier reports of {sup 126}Te and {sup 130}Te excesses which were measured by neutron activation in similar samples. Small excesses were detected in the minor isotopes of Sn and Te, but these may be due to measurement problems associated with the small ion currents obtained for these samples. Two of the residue solutions contain Cd with up to several percent excesses for {sup 106}Cd and {sup 108}Cd. Interpretations of these results are limited by the unknown nature of the carrier minerals in the residues but may indicate the presence of a p-process component in Allende residues.

  11. Entropy reduction in unfolded peptides (and proteins) due to conformational preferences of amino acid residues.

    PubMed

    Schweitzer-Stenner, Reinhard; Toal, Siobhan E

    2014-11-01

    As established by several groups over the last 20 years, amino acid residues in unfolded peptides and proteins do not exhibit the unspecific random distribution as assumed by the classical random coil model. Individual amino acid residues in small peptides were found to exhibit different conformational preferences. Here, we utilize recently obtained conformational distributions of guest amino acid residues in GxG peptides to estimate their conformational entropy, which we find to be significantly lower than the entropy of an assumed random coil like distribution. Only at high temperature do backbone entropies approach random coil like values. We utilized the obtained backbone entropies of the investigated amino acid residues to estimate the loss of conformational entropy caused by a coil → helix transition and identified two subsets of amino acid residues for which the thus calculated entropy losses correlate well with the respective Gibbs energy of helix formation obtained for alanine based host-guest systems. Calculated and experimentally derived entropic losses were found to be in good agreement. For most of the amino acid residues investigated entropic losses derived from our GxG distributions correlate very well with corresponding values recently obtained from MD simulations biased by conformational propensities derived from truncated coil libraries. Both, conformational entropy and the entropy of solvation exhibit a strong, residue specific temperature dependence, which can be expected to substantially affect the stability of unfolded states. Altogether, our results provide strong evidence for the notion that conformational preferences of amino acid residues matter with regard to the thermodynamics of peptide and protein folding.

  12. 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

  13. 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

  14. [Oxytetracycline and oxolinic acid residues in kuruma prawn (Penaeus japonicus) and the effect of cooking procedures on the residues].

    PubMed

    Uno, Kazuaki

    2002-04-01

    Tissue distribution and residue depletion of oxytetracycline (OTC) and oxolinic acid (OA) were studied in the kuruma prawn (Penaeus japonicus). The prawn were kept in tanks with recirculated artificial seawater at a salinity of 22-23@1000. The water temperature was maintained at 25 degrees C. The average body weight was 22.9 +/- 4.9 g for OTC and 22.5 +/- 3.6 g for OA. The drug was mixed with the diet and orally administered through a catheter to the prawn. The doses of OTC and OA, respectively, were 50 mg/kg body weight. At each sample time, four prawns were sacrificed and tissues were sampled. OTC and OA levels were determined by high-performance liquid chromatography. At the highest levels, the concentrations of OTC were in the other: shell (13.57 micrograms/g) > hemolymph (12.20 micrograms/mL) > muscle (8.30 micrograms/g). For OA, the order was: shell (20.74 micrograms/g) > hemolymph (7.06 micrograms/mL) > muscle (2.05 micrograms/g). The elimination half-lives of hemolymph and muscle were 44.7 and 46.8 hours for OTC and 55.0 and 107.9 hours for OA, respectively. Residual OTC could not be detected in hemolymph and muscle at 20 days after dosing. Residual OA disappeared from hemolymph and muscle at 25 days after dosing. A 25-day period for OTC and 30-day period for OA could be regarded as the proper withdrawal time established for kuruma prawn by the Pharmaceutical Law in Japan. However, the elimination half-lives of shell for OTC and OA could not be calculated because both drug residues persisted in shell tissues, and the elimination phase was not completed during the experimental period. Residual OTC (14.10 +/- 2.26 micrograms/g, n = 6) and OA (0.32 +/- 0.06 microgram/g, n = 7) were detected in exuviae at 3 days and 4 days after dosing, respectively. Residual OTC was reduced to 50-70% in muscle by the usual methods of cooking (boiling, baking at 200 degrees C and frying at 180 degrees C), whereas reduction levels in shell were only 20-30%. Residual OA was

  15. A complete enzymatic recovery of ferulic acid from corn residues with extracellular enzymes from Neosartorya spinosa NRRL185.

    PubMed

    Shin, Hyun-Dong; McClendon, Shara; Le, Tien; Taylor, Frank; Chen, Rachel Ruizhen

    2006-12-20

    An economic ferulic acid recovery from biomass via biological methods is of interest for a number of reasons. Ferulic acid is a precursor to vanillin synthesis. It is also a known antioxidant with potential food and medical applications. Despite its universal presence in all plant cell wall material, the complex structure of the plant cell wall makes ferulic acid recovery from biomass a challenging bioprocess. Previously, without pretreatment, very low (3-13%) recovery of ferulic acid from corn residues was achieved. We report here the discovery of a filamentous fungus Neosartorya spinosa NRRL185 capable of producing a full complement of enzymes to release ferulic acid and the development of an enzymatic process for a complete recovery of ferulic acid from corn bran and corn fibers. A partial characterization of the extracellular proteome of the microbe revealed the presence of at least seven cellulases and hemicellulases activities, including multiple iso-forms of xylanase and ferulic acid esterase. The recovered ferulic acid was bio-converted to vanillin, demonstrating its potential application in natural vanillin synthesis. The enzymatic ferulic acid recovery accompanied a significant release of reducing sugars (76-100%), suggesting much broader applications of the enzymes and enzyme mixtures from this organism.

  16. Dynamics of linker residues modulate the nucleic acid binding properties of the HIV-1 nucleocapsid protein zinc fingers.

    PubMed

    Zargarian, Loussiné; Tisné, Carine; Barraud, Pierre; Xu, Xiaoqian; Morellet, Nelly; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

    2014-01-01

    The HIV-1 nucleocapsid protein (NC) is a small basic protein containing two zinc fingers (ZF) separated by a short linker. It is involved in several steps of the replication cycle and acts as a nucleic acid chaperone protein in facilitating nucleic acid strand transfers occurring during reverse transcription. Recent analysis of three-dimensional structures of NC-nucleic acids complexes established a new property: the unpaired guanines targeted by NC are more often inserted in the C-terminal zinc finger (ZF2) than in the N-terminal zinc finger (ZF1). Although previous NMR dynamic studies were performed with NC, the dynamic behavior of the linker residues connecting the two ZF domains remains unclear. This prompted us to investigate the dynamic behavior of the linker residues. Here, we collected 15N NMR relaxation data and used for the first time data at several fields to probe the protein dynamics. The analysis at two fields allows us to detect a slow motion occurring between the two domains around a hinge located in the linker at the G35 position. However, the amplitude of motion appears limited in our conditions. In addition, we showed that the neighboring linker residues R29, A30, P31, R32, K33 displayed restricted motion and numerous contacts with residues of ZF1. Our results are fully consistent with a model in which the ZF1-linker contacts prevent the ZF1 domain to interact with unpaired guanines, whereas the ZF2 domain is more accessible and competent to interact with unpaired guanines. In contrast, ZF1 with its large hydrophobic plateau is able to destabilize the double-stranded regions adjacent to the guanines bound by ZF2. The linker residues and the internal dynamics of NC regulate therefore the different functions of the two zinc fingers that are required for an optimal chaperone activity.

  17. 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.

  18. 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

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

    PubMed

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

    1994-08-01

    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.

  20. A microalgae residue based carbon solid acid catalyst for biodiesel production.

    PubMed

    Fu, Xiaobo; Li, Dianhong; Chen, Jie; Zhang, Yuanming; Huang, Weiya; Zhu, Yi; Yang, Jun; Zhang, Chengwu

    2013-10-01

    Biodiesel production from microalgae is recognized as one of the best solutions to deal with the energy crisis issues. However, after the oil extraction from the microalgae, the microalgae residue was generally discarded or burned. Here a novel carbon-based solid acid catalyst derived from microalgae residue by in situ hydrothermal partially carbonization were synthesized. The obtained catalyst was characterized and subjected to both the esterification of oleic acid and transesterification of triglyceride to produce biodiesel. The catalyst showed high catalytic activity and can be regenerated while its activity can be well maintained after five cycles.

  1. 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

  2. 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

  3. Evolutionary diversification of aminopeptidase N in Lepidoptera by conserved clade-specific amino acid residues.

    PubMed

    Hughes, Austin L

    2014-07-01

    Members of the aminopepidase N (APN) gene family of the insect order Lepidoptera (moths and butterflies) bind the naturally insecticidal Cry toxins produced by the bacterium Bacillus thuringiensis. Phylogenetic analysis of amino acid sequences of seven lepidopteran APN classes provided strong support for the hypothesis that lepidopteran APN2 class arose by gene duplication prior to the most recent common ancestor of Lepidoptera and Diptera. The Cry toxin-binding region (BR) of lepidopteran and dipteran APNs was subject to stronger purifying selection within APN classes than was the remainder of the molecule, reflecting conservation of catalytic site and adjoining residues within the BR. Of lepidopteran APN classes, APN2, APN6, and APN8 showed the strongest evidence of functional specialization, both in expression patterns and in the occurrence of conserved derived amino acid residues. The latter three APN classes also shared a convergently evolved conserved residue close to the catalytic site. APN8 showed a particularly strong tendency towards class-specific conserved residues, including one of the catalytic site residues in the BR and ten others in close vicinity to the catalytic site residues. The occurrence of class-specific sequences along with the conservation of enzymatic function is consistent with the hypothesis that the presence of Cry toxins in the environment has been a factor shaping the evolution of this multi-gene family.

  4. Differentiating N-terminal aspartic and isoaspartic acid residues in peptides.

    PubMed

    Sargaeva, Nadezda P; Lin, Cheng; O'Connor, Peter B

    2011-09-01

    Formation of isoaspartic acid (isoAsp) is a common modification of aspartic acid (Asp) or asparagine (Asn) residue in proteins. Differentiation of isoAsp and Asp residues is a challenging task owing to their similar properties and identical molecular mass. It was recently shown that they can be differentiated using ion-electron or ion-ion interaction fragmentation methods (ExD) because these methods provide diagnostic fragments c + 57 and z(•) - 57 specific to the isoAsp residue. To date, however, the presence of such fragments has not been explored on peptides with an N-terminal isoAsp residue. To address this question, several N-terminal isoAsp-containing peptides were analyzed using ExD methods alone or combined with chromatography. A diagnostic fragment [M + 2H - 74](+•) was observed for the doubly charged precursor ions with N-terminal isoAsp residues. For some peptides, identification of the N-terminal isoAsp residue was challenging because of the low diagnostic ion peak intensity and the presence of interfering peaks. Supplemental activation was used to improve diagnostic ion detection. Further, N-terminal acetylation was offered as a means to overcome the interference problem by shifting the diagnostic fragment peak to [M + 2H - 116](+•).

  5. Evolutionary Diversifaction of Aminopeptidase N in Lepidoptera by Conserved Clade-specific Amino Acid Residues

    PubMed Central

    Hughes, Austin L.

    2015-01-01

    Members of the aminopepidase N (APN) gene family of the insect order Lepidoptera (moths and butterflies) bind the naturally insecticidal Cry toxins produced by the bacterium Bacillus thuringiensis. Phylogenetic analysis of amino acid sequences of seven lepidopteran APN classes provided strong support for the hypothesis that lepidopteran APN2 class arose by gene duplication prior to the most recent common ancestor of Lepidoptera and Diptera. The Cry toxin-binding region (BR) of lepidopteran and dipteran APNs was subject to stronger purifying selection within APN classes than was the remainder of the molecule, reflecting conservation of catalytic site and adjoining residues within the BR. Of lepidopteran APN classes, APN2, APN6, and APN8 showed the strongest evidence of functional specialization, both in expression patterns and in the occurrence of conserved derived amino acid residues. The latter three APN classes also shared a convergently evolved conserved residue close to the catalytic site. APN8 showed a particularly strong tendency towards class-specific conserved residues, including one of the catalytic site residues in the BR and ten others in close vicinity to the catalytic site residues. The occurrence of class-specific sequences along with the conservation of enzymatic function is consistent with the hypothesis that the presence of Cry toxins in the environment has been a factor shaping the evolution of this multi-gene family. PMID:24675701

  6. Revised Backbone-Virtual-Bond-Angle Potentials to Treat the l- and d-Amino Acid Residues in the Coarse-Grained United Residue (UNRES) Force Field

    PubMed Central

    2015-01-01

    Continuing our effort to introduce d-amino-acid residues in the united residue (UNRES) force field developed in our laboratory, in this work the Cα ··· Cα ··· Cα backbone-virtual-bond-valence-angle (θ) potentials for systems containing d-amino-acid residues have been developed. The potentials were determined by integrating the combined energy surfaces of all possible triplets of terminally blocked glycine, alanine, and proline obtained with ab initio molecular quantum mechanics at the MP2/6-31G(d,p) level to calculate the corresponding potentials of mean force (PMFs). Subsequently, analytical expressions were fitted to the PMFs to give the virtual-bond-valence potentials to be used in UNRES. Alanine represented all types of amino-acid residues except glycine and proline. The blocking groups were either the N-acetyl and N′,N′-dimethyl or N-acetyl and pyrrolidyl group, depending on whether the residue next in sequence was an alanine-type or a proline residue. A total of 126 potentials (63 symmetry-unrelated potentials for each set of terminally blocking groups) were determined. Together with the torsional, double-torsional, and side-chain-rotamer potentials for polypeptide chains containing d-amino-acid residues determined in our earlier work (Sieradzan et al. J. Chem. Theory Comput., 2012, 8, 4746), the new virtual-bond-angle (θ) potentials now constitute the complete set of physics-based potentials with which to run coarse-grained simulations of systems containing d-amino-acid residues. The ability of the extended UNRES force field to reproduce thermodynamics of polypeptide systems with d-amino-acid residues was tested by comparing the experimentally measured and the calculated free energies of helix formation of model KLALKLALxxLKLALKLA peptides, where x denotes any d- or l- amino-acid residue. The obtained results demonstrate that the UNRES force field with the new potentials reproduce the changes of free energies of helix formation upon d

  7. 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.)

  8. 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.

  9. 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

  10. 40 CFR 180.550 - Arsanilic acid [(4-aminophenyl) arsonic acid]; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances... connection with the use of the pesticide under section 5 experimental use permit. The tolerance will...

  11. 40 CFR 180.550 - Arsanilic acid [(4-aminophenyl) arsonic acid]; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances... connection with the use of the pesticide under section 5 experimental use permit. The tolerance will...

  12. 40 CFR 180.550 - Arsanilic acid [(4-aminophenyl) arsonic acid]; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances... connection with the use of the pesticide under section 5 experimental use permit. The tolerance will...

  13. 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

  14. 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

  15. 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

  16. 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.

  17. Catalytic residues are shared between two pseudosubunits of the dehydratase domain of the animal fatty acid synthase.

    PubMed

    Pasta, Saloni; Witkowski, Andrzej; Joshi, Anil K; Smith, Stuart

    2007-12-01

    Expression, characterization, and mutagenesis of a series of N-terminal fragments of an animal fatty acid synthase, containing the beta-ketoacyl synthase, acyl transferase, and dehydratase domains, demonstrate that the dehydratase domain consists of two pseudosubunits, derived from contiguous regions of the same polypeptide, in which a single active site is formed by the cooperation of the catalytic histidine 878 residue of the first pseudosubunit with aspartate 1032 of the second pseudosubunit. Mutagenesis and modeling studies revealed an essential role for glutamine 1036 in anchoring the position of the catalytic aspartate. These findings establish that sequence elements previously assigned to a central structural core region of the type I fatty acid synthases and some modular polyketide synthase counterparts play an essential catalytic role as part of the dehydratase domain.

  18. Physicochemical pretreatments and hydrolysis of furfural residues via carbon-based sulfonated solid acid.

    PubMed

    Ma, Bao Jun; Sun, Yuan; Lin, Ke Ying; Li, Bing; Liu, Wan Yi

    2014-03-01

    Potential commercial physicochemical pretreatment methods, NaOH/microwave and NaOH/ultrasound were developed, and the carbon-based sulfonated solid acid catalysts were prepared for furfural residues conversion into reducing sugars. After the two optimum pretreatments, both the content of cellulose increased (74.03%, 72.28%, respectively) and the content of hemicellulose (94.11%, 94.17% of removal rate, respectively) and lignin (91.75%, 92.09% of removal rate, respectively) decreased in furfural residues. The reducing sugar yields of furfural residues with the two physicochemical pretreatments on coal tar-based solid acid reached 33.94% and 33.13%, respectively, higher than that pretreated via NaOH alone (27%) and comparable to that pretreated via NaOH/H2O2 (35.67%). The XRD patterns, IR spectra and SEM images show microwave and ultrasound improve the pretreatment effect. The results demonstrate the carbon-based sulfonated solid acids and the physicochemical pretreatments are green, effective, low-cost for furfural residues conversion.

  19. 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

  20. Colanic Acid Intermediates Prevent De Novo Shape Recovery of Escherichia coli Spheroplasts, Calling into Question Biological Roles Previously Attributed to Colanic Acid

    PubMed Central

    Ranjit, Dev K.

    2016-01-01

    ABSTRACT After losing their protective peptidoglycan, bacterial spheroplasts can resynthesize a cell wall to recreate their normal shape. In Escherichia coli, this process requires the Rcs response. In its absence, spheroplasts do not revert to rod shapes but instead form enlarged spheroids and lyse. Here, we investigated the reason for this Rcs requirement. Rcs-deficient spheroids exhibited breaks and bulges in their periplasmic spaces and failed to synthesize a complete peptidoglycan cell wall, indicating that the bacterial envelope was defective. To determine the Rcs-dependent gene(s) required for shape recovery, we tested spheroplasts lacking selected RcsB-regulated genes and found that colanic acid (CA) biosynthesis appeared to be involved. Surprisingly, though, extracellular CA was not required for recovery. Instead, lysis was caused by mutations that interrupted CA biosynthesis downstream of the initial glycosyl transferase, WcaJ. Deleting wcaJ prevented lysis of spheroplasts lacking ensuing steps in the pathway, and providing WcaJ in trans to a mutant lacking the entire CA operon triggered spheroplast enlargement and lysis. Thus, CA is not required for spheroplast recovery. Instead, CA intermediates accumulate as dead-end products which inhibit recovery of wall-less cells. The results strongly imply that CA may not be required for the survival E. coli L-forms. More broadly, these findings mandate that previous conclusions about the role of colanic acid in biofilm formation or virulence must be reevaluated. IMPORTANCE Wall-less bacteria can resynthesize their walls and recreate a normal shape, which in Escherichia coli requires the Rcs response. While attempting to identify the Rcs-dependent gene required for shape recovery, we found that colanic acid (CA) biosynthesis appeared to be involved. Surprisingly, though, cell death was caused by mutations that interrupted CA biosynthesis downstream of the initial step in the pathway, creating dead-end compounds

  1. 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

  2. Determination of free acidic and alkaline residues of protein via moving reaction boundary titration in microdevice electrophoresis.

    PubMed

    Wang, Hou-yu; Li, Si; Tang, Yun-yun; Dong, Jing-yu; Fan, Liu-yin; Cao, Cheng-xi

    2013-06-21

    As two important physico-chemical parameters, the acidic and alkaline residues of protein are of evident significance for the evaluation of protein properties and the design of relevant separation and analysis. However, there is still no electrophoretic method used for the direct detection of free acidic and alkaline residues of protein. Herein, we developed the concepts of moving reaction boundary (MRB) and MRB titration, relevant MRB titration theory, and the method of microdevice electrophoresis for the determination of free acidic and alkaline residues of protein. In the MRB titration, the boundary was created with acid or alkali and target protein immobilized via highly cross-linked polyacrylamide gel (PAG). It was theoretically revealed that the number of free acidic or alkaline residues of protein was as a function of MRB displacement in the electrophoretic titration system. As a proof of concept, seven model proteins were chosen for the determination of acidic or alkaline residues of protein via MRB titration. The results showed that the numbers of free acidic and alkaline residues of proteins detected were in good agreement with those obtained from the relevant amino sequences in the NCBI database, demonstrating the feasibility of the developed concept, theory and technique. The general methodology of MRB titration has potential application for inexpensive, facilitative and informative protein structure analysis of free acidic or alkaline residues of protein.

  3. Comparison between liquid and solid acids catalysts on reducing sugars conversion from furfural residues via pretreatments.

    PubMed

    Lin, Keying; Ma, Baojun; Sun, Yuan; Liu, Wanyi

    2014-09-01

    Liquid sulphuric acid is adopted and compared with carbon-based sulfonated solid acids (coal tar-based and active carbon-based) for furfural residues conversion into reducing sugars. The optimum hydrolysis conditions of liquid acid are at 4% of sulphuric acid, 25:1 of liquid and solid ratio, 175°C of reaction temperature and 120 min of reaction time. The reducing sugar yields are reached over 60% on liquid acid via NaOH/H2O2, NaOH/microwave and NaOH/ultrasonic pretreatments, whereas only over 30% on solid acids. The TOFs (turnover number frequency) via NaOH/H2O2 pretreatments are 0.093, 0.020 and 0.023 h(-1) for liquid sulphuric acid, coal tar-based and active carbon-based solid acids catalysts, respectively. Considering the efficiency, cost and environment factors, the liquid and solid acids have their own advantages of potential commercial application values.

  4. Identification of amino acid residues important for the function of Agrobacterium tumefaciens Irr protein.

    PubMed

    Bhubhanil, Sakkarin; Ruangkiattikul, Nantaporn; Niamyim, Phettree; Chamsing, Jareeya; Ngok-Ngam, Patchara; Sukchawalit, Rojana; Mongkolsuk, Skorn

    2012-10-01

    The key amino acid residues that influence the function of the Agrobacterium tumefaciens iron response regulator protein (Irr(At) ) were investigated. Several Irr(At) mutant proteins containing substitutions in amino acids corresponding to candidate metal- and haem-binding sites were constructed. The ability of the mutant proteins to repress the promoter of the membrane bound ferritin (mbfA) gene was investigated using a promoter-lacZ fusion assay. A single mutation at residue H94 significantly decreased the repressive activity of Irr(At) . Multiple mutation analysis revealed the importance of H45, H65, the HHH motif (H92, H93 and H94) and H127 for the repressor function of Irr(At) . H94 is essential for the iron responsiveness of Irr(At) . Furthermore, the Irr(At) mutant proteins showed differential abilities to complement the H(2) O(2) -hyper-resistant phenotype of an irr mutant. PMID:22817265

  5. Key Amino Acid Residues of Ankyrin-Sensitive Phosphatidylethanolamine/Phosphatidylcholine-Lipid Binding Site of βI-Spectrin

    PubMed Central

    Wolny, Marcin; Grzybek, Michał; Bok, Ewa; Chorzalska, Anna; Lenoir, Marc; Czogalla, Aleksander; Adamczyk, Klaudia; Kolondra, Adam; Diakowski, Witold; Overduin, Michael; Sikorski, Aleksander F.

    2011-01-01

    It was shown previously that an ankyrin-sensitive, phosphatidylethanolamine/phosphatidylcholine (PE/PC) binding site maps to the N-terminal part of the ankyrin-binding domain of β-spectrin (ankBDn). Here we have identified the amino acid residues within this domain which are responsible for recognizing monolayers and bilayers composed of PE/PC mixtures. In vitro binding studies revealed that a quadruple mutant with substituted hydrophobic residues W1771, L1775, M1778 and W1779 not only failed to effectively bind PE/PC, but its residual PE/PC-binding activity was insensitive to inhibition with ankyrin. Structure prediction and analysis, supported by in vitro experiments, suggests that “opening” of the coiled-coil structure underlies the mechanism of this interaction. Experiments on red blood cells and HeLa cells supported the conclusions derived from the model and in vitro lipid-protein interaction results, and showed the potential physiological role of this binding. We postulate that direct interactions between spectrin ankBDn and PE-rich domains play an important role in stabilizing the structure of the spectrin-based membrane skeleton. PMID:21738695

  6. The role of amino acid residues in the active site of L-methionine γ-lyase from Pseudomonas putida.

    PubMed

    Fukumoto, Mitsuki; Kudou, Daizou; Murano, Shouko; Shiba, Tomoo; Sato, Dan; Tamura, Takashi; Harada, Shigeharu; Inagaki, Kenji

    2012-01-01

    Cys116, Lys240*, and Asp241* (asterisks indicate residues from the second subunit of the active dimer) at the active site of L-methionine γ-lyase of Pseudomonas putida (MGL_Pp) are highly conserved among heterologous MGLs. In a previous study, we found that substitution of Cys116 for His led to a drastic increase in activity toward L-cysteine and a decrease in that toward L-methionine. In this study, we examined some properties of the C116H mutant by kinetic analysis and 3D structural analysis. We assumed that substitution of Cys116 for His broke the original hydrogen-bond network and that this induced a significant effect of Tyr114 as a general acid catalyst, possibly due to the narrow space in the active site. The C116H mutant acquired a novel β-elimination activity and lead a drastic conformation change in the histidine residue at position 116 by binding the substrate, suggesting that this His residue affects the reaction specificity of C116H. Furthermore, we suggest that Lys240* is important for substrate recognition and structural stability and that Asp241* is also involved in substrate specificity in the elimination reaction. Based on this, we suggest that the hydrogen-bond network among Cys116, Lys240*, and Asp241* contributes to substrate specificity that is, to L-methionine recognition at the active site in MGL_Pp.

  7. Role of interfacial amino acid residues in assembly, stability, and conformation of a spherical virus capsid

    PubMed Central

    Reguera, Juan; Carreira, Aura; Riolobos, Laura; Almendral, José María; Mateu, Mauricio G.

    2004-01-01

    Twenty-eight amino acid residues involved in most noncovalent interactions between trimeric protein subunits in the capsid of the parvovirus minute virus of mice were truncated individually to alanine, and the effects on capsid assembly, thermostability, and conformation were analyzed. Only seven side chains were essential for protein subunit recognition. These side chains virtually corresponded with those that either buried a large hydrophobic surface on trimer association or formed buried intertrimer hydrogen bonds or salt bridges. The seven residues are evolutionarily conserved, and they define regularly spaced spots on a thin equatorial belt surrounding each trimer. Truncation of the many side chains that were dispensable for assembly, including those participating in solvent-accessible polar interactions, did not substantially affect capsid thermostability either. However, the interfacial residues located at the base of the pores delineating the capsid five-fold axes participated in a heat-induced conformational rearrangement associated with externalization of the capsid protein N terminus, and they were needed for infectivity. Thus, at the subunit interfaces of this model virus capsid, only key residues involved in the strongest interactions are critical for assembly and stability, but additional residues fulfill other important biological roles. PMID:14981262

  8. 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

  9. Removal of copper from acid wastewater of bioleaching by adsorption onto ramie residue and uptake by Trichoderma viride.

    PubMed

    Wang, Buyun; Wang, Kai

    2013-05-01

    A continuous batch bioleaching was built to realize the bioleaching of sewage sludge in large scale. In the treatment, heavy metal in acid wastewater of bioleaching was removed by adsorption onto ramie residue. Then, acid wastewater was reused in next bioleaching batch. In this way, most time and water of bioleaching was saved and leaching efficiency of copper, lead and chromium kept at a high level in continuous batch bioleaching. It was found that residual heavy metal in sewage sludge is highly related to that in acid wastewater after bioleaching. To get a high leaching efficiency, concentration of heavy metal in acid wastewater should be low. Adsorption of copper from acid wastewater onto ramie residue can be described by pseudo first-order kinetics equation and Freundlich isotherm model. Trichoderma viride has the potential to be used for the concentration and recovery of heavy metal adsorbed onto ramie residue. PMID:23567687

  10. Extension of UNRES force field to treat polypeptide chains with D-amino-acid residues

    PubMed Central

    Sieradzan, Adam K.; Hansmann, Ulrich H.E.; Scheraga, Harold A.; Liwo, Adam

    2013-01-01

    Coarse-grained force fields for protein simulations are usually designed and parameterized to treat proteins composed of natural L-amino-acid residues. However, D-amino-acid residues occur in bacterial, fungal (e.g., gramicidins), as well as human-designed proteins. For this reason, we have extended the UNRES coarse-grained force field developed in our laboratory to treat systems with D-amino-acid residues. We developed the respective virtual-bond-torsional and double-torsional potentials for rotation about the Cα · · · Cα virtual-bond axis and two consecutive Cα · · · Cα virtual-bond axes, respectively, as functions of virtual-bond-dihedral angles γ. In turn, these were calculated as potentials of mean force (PMFs) from the diabatic energy surfaces of terminally-blocked model compounds for glycine, alanine, and proline. The potential-energy surfaces were calculated by using the ab initio method of molecular quantum mechanics at the Møller-Plesset (MP2) level of theory and the 6-31G(d,p) basis set, with the rotation angles of the peptide groups about Ci-1α⋯Ciα(λ(1)) and Ciα⋯Ci+1α(λ(2)) used as variables, and the energy was minimized with respect to the remaining degrees of freedom. The PMFs were calculated by numerical integration for all pairs and triplets with all possible combinations of types (glycine, alanine, and proline) and chirality (D or L); however, symmetry relations reduce the number of non-equivalent torsional potentials to 13 and the number of double-torsional potentials to 63 for a given C-terminal blocking group. Subsequently, one- (for torsional) and two-dimensional (for double-torsional potentials) Fourier series were fitted to the PMFs to obtain analytical expressions. It was found that the torsional potentials of the x-Y and X-y types, where X and Y are Ala or Pro, respectively, and a lowercase letter denotes D-chirality, have global minima for small absolute values of γ, accounting for the double-helical structure of

  11. 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

  12. 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.

  13. Study of TATP: method for determination of residual acids in TATP.

    PubMed

    Matyáš, Robert; Chýlková, Jaromíra

    2013-05-10

    Triacetone triperoxide (3,3,6,6,9,9-hexamethyl-1,2,4,5,7,8-hexoxonane, TATP) is nowadays one of the most commonly used improvised explosives. It is prepared by the action of hydrogen peroxide on acetone in an acidic environment. Easily available mineral acids - hydrochloric, sulfuric, nitric and perchloric - are the most often recommended on the extremist web pages dealing with improvised production of explosives. The various TATP producers' choice of acid mainly depends on the author's experiences and the local availability of the acid. A knowledge of the kind of acid used for TATP production can help in detecting the person who has made the TATP, or who has committed a criminal act using TATP. Therefore, a capillary isotachophoretic method was developed for determination of residual anions (originating from the acid used during TATP synthesis) in the resulting TATP crystals. This analytical method has proved to be reliable; the acid used for TATP synthesis was correctly identified in all samples analyzed. PMID:23542054

  14. Amino Acid Residues in the ω-Minus Region Participate in Cellular Localization of Yeast Glycosylphosphatidylinositol-Attached Proteins

    PubMed Central

    Hamada, Kenji; Terashima, Hiromichi; Arisawa, Mikio; Yabuki, Nami; Kitada, Kunio

    1999-01-01

    The final destination of glycosylphosphatidylinositol (GPI)-attached proteins in Saccharomyces cerevisiae is the plasma membrane or the cell wall. Two kinds of signals have been proposed for their cellular localization: (i) the specific amino acid residues V, I, or L at the site 4 or 5 amino acids upstream of the GPI attachment site (the ω site) and Y or N at the site 2 amino acids upstream of the ω site for cell wall localization and (ii) dibasic residues in the region upstream of the ω site (the ω-minus region) for plasma membrane localization. The relationships between these amino acid residues and efficiencies of cell wall incorporation were examined by constructing fusion reporter proteins from open reading frames encoding putative GPI-attached proteins. The levels of incorporation were high in the constructs containing the specific amino acid residues and quite low in those containing two basic amino acid residues in the ω-minus region. With constructs that contained neither specific residues nor two basic residues, levels of incorporation were moderate. These correlations clearly suggest that GPI-attached proteins have two different signals which act positively or negatively in cell wall incorporation for their cellular localization. PMID:10383953

  15. Site-directed mutagenesis of HgcA and HgcB reveals amino acid residues important for mercury methylation.

    PubMed

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

    2015-05-01

    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.

  16. 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

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

    DOE PAGES

    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

  18. 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.

  19. A conserved acidic residue in phenylalanine hydroxylase contributes to cofactor affinity and catalysis.

    PubMed

    Ronau, Judith A; Paul, Lake N; Fuchs, Julian E; Liedl, Klaus R; Abu-Omar, Mahdi M; Das, Chittaranjan

    2014-11-01

    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

  20. Acid hydrolysis of Curcuma longa residue for ethanol and lactic acid fermentation.

    PubMed

    Nguyen, Cuong Mai; Nguyen, Thanh Ngoc; Choi, Gyung Ja; Choi, Yong Ho; Jang, Kyoung Soo; Park, Youn-Je; Kim, Jin-Cheol

    2014-01-01

    This research examines the acid hydrolysis of Curcuma longa waste, to obtain the hydrolysate containing lactic acid and ethanol fermentative sugars. A central composite design for describing regression equations of variables was used. The selected optimum condition was 4.91% sulphuric acid, 122.68°C and 50 min using the desirability function under the following conditions: the maximum reducing sugar (RS) yield is within the limited range of the 5-hydroxymethylfurfural (HMF) and furfural concentrations. Under the condition, the obtained solution contained 144 g RS/L, 0.79 g furfural/L and 2.59 g HMF/L and was directly fermented without a detoxification step. The maximum product concentration, average productivity, RS conversion and product yield were 115.36 g/L, 2.88 g/L/h, 89.43% and 64% for L-lactic acid; 113.92 g/L, 2.59 g/L/h, 88.31% and 63.29% for D-lactic acid; and 55.03 g/L, 1.38 g/L/h, 42.66 and 30.57%, respectively, for ethanol using a 7-L jar fermenter.

  1. Acid hydrolysis of Curcuma longa residue for ethanol and lactic acid fermentation.

    PubMed

    Nguyen, Cuong Mai; Nguyen, Thanh Ngoc; Choi, Gyung Ja; Choi, Yong Ho; Jang, Kyoung Soo; Park, Youn-Je; Kim, Jin-Cheol

    2014-01-01

    This research examines the acid hydrolysis of Curcuma longa waste, to obtain the hydrolysate containing lactic acid and ethanol fermentative sugars. A central composite design for describing regression equations of variables was used. The selected optimum condition was 4.91% sulphuric acid, 122.68°C and 50 min using the desirability function under the following conditions: the maximum reducing sugar (RS) yield is within the limited range of the 5-hydroxymethylfurfural (HMF) and furfural concentrations. Under the condition, the obtained solution contained 144 g RS/L, 0.79 g furfural/L and 2.59 g HMF/L and was directly fermented without a detoxification step. The maximum product concentration, average productivity, RS conversion and product yield were 115.36 g/L, 2.88 g/L/h, 89.43% and 64% for L-lactic acid; 113.92 g/L, 2.59 g/L/h, 88.31% and 63.29% for D-lactic acid; and 55.03 g/L, 1.38 g/L/h, 42.66 and 30.57%, respectively, for ethanol using a 7-L jar fermenter. PMID:24240182

  2. Measuring Residual Dipolar Couplings in Excited Conformational States of Nucleic Acids by CEST NMR Spectroscopy.

    PubMed

    Zhao, Bo; Zhang, Qi

    2015-10-28

    Nucleic acids undergo structural transitions to access sparsely populated and transiently lived conformational states--or excited conformational states--that play important roles in diverse biological processes. Despite ever-increasing detection of these functionally essential states, 3D structure determination of excited states (ESs) of RNA remains elusive. This is largely due to challenges in obtaining high-resolution structural constraints in these ESs by conventional structural biology approaches. Here, we present nucleic-acid-optimized chemical exchange saturation transfer (CEST) NMR spectroscopy for measuring residual dipolar couplings (RDCs), which provide unique long-range angular constraints in ESs of nucleic acids. We demonstrate these approaches on a fluoride riboswitch, where one-bond (13)C-(1)H RDCs from both base and sugar moieties provide direct structural probes into an ES of the ligand-free riboswitch.

  3. 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.

  4. 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.

  5. [Determination of clavulanic acid residue in milk by high performance liquid chromatography-tandem mass spectrometry].

    PubMed

    Yang, Gang; Huang, Xianhui; Guo, Chunna; Fang, Qiuhua; He, Limin

    2012-06-01

    An analytical method was developed for the determination of clavulanic acid (CLAV) in milk by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). A 2 g milk sample was deproteinized by ethanol. The supernatant was transferred into a pear-shaped bottle to be evaporated to about 0.5 mL, and the residue was dissolved with ammonium acetate solution. The sample was determined by HPLC-MS/MS after the purification. The chromatographic separation was achieved on a Luna 5u C8 column using 0.1% formic acid in water and acetonitrile as mobile phases with gradient elution. The identification of CLAV was carried out by MS/MS equipped with electrospray ionization in negative scanning and multiple reaction monitoring (MRM) modes. Matrix-matched calibration standard was used for the quantification. The calibration curve showed perfect linear in the range of 10 - 400 microg/kg with the correlation coefficient of 0.999. The limit of detection (LOD, S/N > or = 3) was 10 microg/kg in milk, and the limit of quantification (LOQ, S/N > or = 10) was 20 microg/kg. The mean recoveries varied from 80.00% to 91.25% at the four spiked levels of LOQ, 1/2MRL (the maximum residue limit), MRL, and 2MRL with the relative standard deviations of 5.60% -8.77%. In conclusion, the established method can be applied for the determination of CLAV residues in milk.

  6. 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.

  7. 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.

  8. Cysteine residues in the zinc finger and amino acids adjacent to the finger are necessary for DNA binding by the LAC9 regulatory protein of Kluyveromyces lactis.

    PubMed Central

    Witte, M M; Dickson, R C

    1988-01-01

    LAC9 is a positive regulatory protein that controls transcription of the lactose-galactose regulon in Kluyveromyces lactis. LAC9 is homologous to the GAL4 protein of Saccharomyces cerevisiae. Both proteins have a single "zinc finger" which plays a role in DNA binding. We previously hypothesized (L. V. Wray, M. M. Witte, R. C. Dickson, and M. I. Riley, Mol. Cell. Biol. 7:1111-1121, 1987) that the DNA-binding domain of the LAC9 protein consisted of the zinc finger as well as a region of amino acids on the carboxyl-terminal side of the zinc finger. In this study we used oligonucleotide-directed mutagenesis to introduce 13 single-amino-acid changes into the proposed DNA-binding domain of the LAC9 protein. Variant LAC9 proteins carrying an amino acid substitution in any one of the four highly conserved Cys residues of the zinc finger had reduced DNA-binding activity, suggesting that each Cys is necessary for DNA binding. Three of four variant LAC9 proteins with amino acid substitutions located on the carboxyl-terminal side of the zinc finger had reduced DNA-binding activity. These results support our hypothesis that the DNA-binding domain of the LAC9 protein is composed of the zinc finger and the adjacent region on the carboxyl side of the zinc finger, a region that has the potential to form an alpha-helix. Finally, LAC9 proteins containing His residues substituted for the conserved Cys residues also had reduced DNA-binding activity, indicating that His residues are not equivalent to Cys residues, as had been previously thought. Images PMID:3146691

  9. [Nitrate nitrogen leaching and residue of humic acid fertilizer in field soil].

    PubMed

    Liu, Fang-chun; Xing, Shang-jun; Duan, Chun-hua; Du, Zhen-yu; Ma, Hai-lin; Ma, Bing-yao

    2010-07-01

    To elucidate the potential influence of humic acidfertilizer on groundwater and soil quality in clay soil (CS) and sandy soil (SS), nitrate nitrogen leaching and residue of different fertilizers in field soil were studied using a self-made leaching field device. Nitrate nitrogen concentration in leaching water of fertilizer treatments was 28.1%-222.2% higher than that of non-nitrogen treatment in different times, but humic acid fertilizer could prevent nitrate nitrogen leaching both in CS and SS, especially in CS. Nitrate nitrogen concentration of leaching water in CS was 41.2%-59.1% less than that in SS and the inhibiting effect in CS was greater than that in SS. Nitrate nitrogen could be accumulated in soil profile by fertilizer application. The residue of nitrate nitrogen retained in 0-40 cm soil layer of humic acid fertilizer treatment was 59.8% and 54.4% respectively, higher than that of urea and compound fertilizer treatments. Nitrate nitrogen amount of humic acid, urea and compound fertilizer treatments in SS was significantly less than that in CS, being 81.7%, 81.1% and 47.6% respectively. Compared with the conventional fertilizer, humic acid fertilizer treatment improved the contents of organic matter, available nitrogen, phosphorus, and potassium of upper layer soil as well as cation exchange capacity. Besides, total amount of water-soluble salts in humic acid fertilizer treatment was decreased by 24.8% and 22.5% in comparison to urea and compound fertilizer treatments in CS, respectively. In summary, the application of humic acid fertilizer could improve physical and chemical properties of upper layer soil and reduce the risk of potential pollution to groundwater.

  10. 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

  11. 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

  12. Peptide hairpins with strand segments containing alpha- and beta-amino acid residues: cross-strand aromatic interactions of facing Phe residues.

    PubMed

    Roy, Rituparna S; Gopi, Hosahudya N; Raghothama, S; Gilardi, Richard D; Karle, Isabella L; Balaram, Padmanabhan

    2005-01-01

    The incporation of beta-amino acid residues into the strand segments of designed beta-hairpin leads to the formation of polar sheets, since in the case of beta-peptide strands, all adjacent carbonyl groups point in one direction and the amide groups orient in the opposite direction. The conformational analysis of two designed peptide hairpins composed of alpha/beta-hybrid segments are described: Boc-Leu-betaPhe-Val-(D)-Pro-Gly-Leu-betaPhe-Val-OMe (1) and Boc-betaLeu-Phe-betaVal-D-Pro-Gly-betaLeu-Phe-betaVal-OMe (2). A 500-MHz 1H-NMR (nuclear magnetic resonance) analysis in methanol supports a significant population of hairpin conformations in both peptides. Diagnostic nuclear Overhauser effects (NOEs) are observed in both cases. X-ray diffraction studies on single crystals of peptide 1 reveal a beta-hairpin conformation in both the molecules, which constitute the crystallographic asymmetric unit. Three cross-strand hydrogen bonds and a nucleating type II' beta-turn at the D-Pro-Gly segment are observed in the two independent molecules. In peptide 1, the betaPhe residues at positions 2 and 7 occur at the nonhydrogen-bonding position, with the benzyl side chains pointing on opposite faces of the beta-sheet. The observed aromatic centroid-to-centroid distances are 8.92 A (molecule A) and 8.94 A (molecule B). In peptide 2, the aromatic rings must occupy facing positions in antiparallel strands, in the NMR-derived structure. Peptide 1 yields a normal "hairpin-like" CD spectrum in methanol with a minimum at 224 nm. The CD spectrum of peptide 2 reveals a negative band at 234 nm and a positive band at 221 nm, suggestive of an exciton split doublet. Modeling of the facing Phe side chains at the hydrogen-bonding position of a canonical beta-hairpin suggests that interring separation is approximately 4.78 A for the gauche+ gauche- (g+ g-) rotamer. A previously reported peptide beta-hairpin composed of only alpha-amino acids, Boc-Leu-Phe-Val-D-Pro-Gly-Leu-Phe-Val-OMe also

  13. Zinc-Mediated Binding of Nucleic Acids to Amyloid-β Aggregates: Role of Histidine Residues.

    PubMed

    Khmeleva, Svetlana A; Radko, Sergey P; Kozin, Sergey A; Kiseleva, Yana Y; Mezentsev, Yuri V; Mitkevich, Vladimir A; Kurbatov, Leonid K; Ivanov, Alexis S; Makarov, Alexander A

    2016-09-01

    Amyloid-β peptide (Aβ) plays a central role in Alzheimer's disease (AD) pathogenesis. Besides extracellular Aβ, intraneuronal Aβ (iAβ) has been suggested to contribute to AD onset and development. Based on reported in vitro Aβ-DNA interactions and nuclear localization of iAβ, the interference of iAβ with the normal DNA expression has recently been proposed as a plausible pathway by which Aβ can exert neurotoxicity. Employing the sedimentation assay, thioflavin T fluorescence, and dynamic light scattering we have studied effects of zinc ions on binding of RNA and single- and double-stranded DNA molecules to Aβ42 aggregates. It has been found that zinc ions significantly enhance the binding of RNA and DNA molecules to pre-formed β-sheet rich Aβ42 aggregates. Another type of Aβ42 aggregates, the zinc-induced amorphous aggregates, was demonstrated to also bind all types of nucleic acids tested. To evaluate the role of the Aβ metal-binding domain's histidine residues in Aβ-nucleic acid interactions mediated by zinc, Aβ16 mutants with substitutions H6R and H6A-H13A and rat Aβ16 lacking histidine residue 13 were used. The zinc-induced interaction of Aβ16 with DNA was shown to critically depend on histidine residues 6 and 13. However, the inclusion of H6R mutation in Aβ42 peptide did not affect DNA binding to Aβ42 aggregates. Since oxidative and/or nitrosative stresses implicated in AD pathogenesis are known to release zinc ions from metallothioneins in cytoplasm and cell nuclei, our findings suggest that intracellular zinc can be an important player in iAβ-nucleic acid interactions. PMID:27567853

  14. 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.

  15. Amino acid residues 56 to 69 of HLA-A2 specify an antigenic determinant shared by HLA-A2 and HLA-B17.

    PubMed

    Ways, J P; Rothbard, J B; Parham, P

    1986-07-01

    The mouse monoclonal antibody MA2.1 was previously used to define an epitope shared by native HLA-A2 and HLA-B17 molecules and amino acid sequence comparison of nine HLA-A,B,C molecules identified residues 62 to 65 as the region most likely to form this epitope. An unabsorbed rabbit antiserum raised against a peptide corresponding to residues 56 to 69 of HLA-A2 gives highly specific reactions with HLA-A2 and HLA-B17 heavy chains in Western blots. No interactions with native HLA-A2 and B17 molecules were detected in a variety of assays. Although the topographic relationship between the epitopes recognized by the rabbit antiserum and the monoclonal antibody could not be determined, the results show that residues 56 to 69 of HLA-A2 can form epitopes with specificity for HLA-A2 and HLA-B17.

  16. 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

  17. Technique development for characterization of metalloorganics in acid-base-neutral fractions of heavy petroleum residues: Topical report

    SciTech Connect

    Pearson, C.D.; Green, J.B.

    1988-01-01

    A novel approach for the characterization of metallorganic compounds in heavy petroleum residues has been developed. Wilmington 1000/sup 0/ F+ and Mayan 925/sup 0/ F+ residues and hydrotreated products were separated into acid-base-neutral (ABN) fractions by a unique nonaqueous ion-exchange technique developed at NIPER. The metal complexes in the feeds, hydrotreated products and ABN fractions were then characterized by determining the total vanadium and nickel and by measuring the vanadium and nickel porphyrin content of each fraction. Molecular weight distribution profiles of the vanadium and nickel compounds in the feed, 400/sup 0/C hydrotreated product and corresponding ABN fractions were obtained by size exclusion chromatography/inductively coupled plasma. The majority of the metal appeared to be in non-porphyrinic form. The vanadium and nickel complexes were distributed into all of the ABN fractions. In the feed and the whole hydrotreated products the porphyrin levels decreased as hydrotreating temperatures increased. In contrast to previously reported work, porphyrins do not always decrease when hydrotreated. The amount of porphyrins in certain ABN fractions increased after hydrotreating at moderate temperatures. The Mayan V and Ni complexes were more resistant to hydrotreating than the Wilmington metal complexes; in particular, the high molecular weight Mayan metal complexes were more resistant to hydrotreating than the high molecular weight Wilmington metal complexes. 15 refs., 11 figs., 10 tabs.

  18. 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.

  19. 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

  20. A Nitrogen-concentrated Phase in IA Iron Meteorite Acid Residue

    NASA Astrophysics Data System (ADS)

    Hashizume, K.; Sugiura, N.

    1993-07-01

    Introduction: Iron meteorites are considered to have experienced a complex history, which is indicated by the variations in trace element chemistry (e.g., [1]). Among iron meteorite groups, the so called nonmagmatic groups, such as IAB, IIE, and IIICD, may have passed through different formation paths compared to others. Nitrogen isotopes can be a useful tool to understand the origin and formation processes of iron meteorites. Nikogen isotopes in a number of iron meteorites are measured [2,3], although trapping sites of nitrogen in iron meteorites are not yet clear. This is an important issue because nitrogen, a typical mobile element, may well reflect thermal history of their parent bodies (c.f., [4]). Generally, a major portion of nitrogen in iron meteorites is expected to be in a solid solution in Fe-Ni, especially in f.c.c. Fe-Ni (taenite). Franchi et al. [3] report that at least 25 to 35% of nitrogen in magmatic iron meteorites is in acid insoluble phases, however, not in those of non-magmatic meteorites. This result contradicts with the result [5] who report that a significant portion of nitrogen seems to be trapped in acid residues not only of magmatic meteorites but also of non- magmatic meteorites. To resolve the contradiction described above, and to identify the trapping site, we started measuring nitrogen isotopes in acid residues of iron metcorites. We report here preliminary results on acid residues of Canyon Diablo (IA). Procedures: Acid residues were prepared by Dr. J.-I. Matsuda and his colleagues. Different blocks of Canyon Diablo, "Can-1" and "Can-2" were treated by 14M HCl, 10M-HF + 1M-HCl, 1M-HCl, and by aqua regia, which destroyed Fe-Ni, sulfides, silicates, and shreibersite. Acid residues of these two blocks, "Can-1bn" and "Can-2b," yielded 0.102 wt% and 0.299 wt% of their original masses, respectively These residues seem to consist mostly of graphite No diamond was detected by powder X-ray analysis [6]. Preliminary Results: A predominant

  1. Differentiating amino acid residues and side chain orientations in peptides using scanning tunneling microscopy.

    PubMed

    Claridge, Shelley A; Thomas, John C; Silverman, Miles A; Schwartz, Jeffrey J; Yang, Yanlian; Wang, Chen; Weiss, Paul S

    2013-12-11

    Single-molecule measurements of complex biological structures such as proteins are an attractive route for determining structures of the large number of important biomolecules that have proved refractory to analysis through standard techniques such as X-ray crystallography and nuclear magnetic resonance. We use a custom-built low-current scanning tunneling microscope to image peptide structures at the single-molecule scale in a model peptide that forms β sheets, a structural motif common in protein misfolding diseases. We successfully differentiate between histidine and alanine amino acid residues, and further differentiate side chain orientations in individual histidine residues, by correlating features in scanning tunneling microscope images with those in energy-optimized models. Beta sheets containing histidine residues are used as a model system due to the role histidine plays in transition metal binding associated with amyloid oligomerization in Alzheimer's and other diseases. Such measurements are a first step toward analyzing peptide and protein structures at the single-molecule level.

  2. 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

  3. Optimization of thermal-dilute sulfuric acid pretreatment for enhancement of methane production from cassava residues.

    PubMed

    Zhang, Qinghua; Tang, Lei; Zhang, Jianhua; Mao, Zhonggui; Jiang, Li

    2011-02-01

    In this study, the pretreatment of cassava residues by thermal-dilute sulfuric acid (TDSA) hydrolysis was investigated by means of a statistically designed set of experiments. A three-factor central composite design (CCD) was employed to identify the optimum pretreatment condition of cassava residues for methane production. The individual and interactive effects of temperature, H(2)SO(4) concentration and reaction time on increase of methane yield (IMY) were evaluated by applying response surface methodology (RSM). After optimization, the resulting optimum pretreatment condition was 157.84°C, utilizing 2.99% (w/w TS) H(2)SO(4) for 20.15 min, where the maximum methane yield (248 mL/g VS) was 56.96% higher than the control (158 mL/g VS), which was very close to the predict value 56.53%. These results indicate the model obtained through RSM analysis is suit to predict the optimum pretreatment condition and there is great potential of using TDSA pretreatment of cassava residues to enhance methane yield.

  4. Amino-terminal basic residues of Src mediate membrane binding through electrostatic interaction with acidic phospholipids.

    PubMed Central

    Sigal, C T; Zhou, W; Buser, C A; McLaughlin, S; Resh, M D

    1994-01-01

    Membrane targeting of pp60src (Src) is mediated by its myristoylated amino terminus. We demonstrate that, in addition to myristate, six basic residues in the amino terminus are essential for high-affinity binding to the lipid bilayer via electrostatic interaction with acidic phospholipids. Specifically, c-Src was shown to bind 2500-fold more strongly to vesicles composed of the physiological ratio of 2:1 phosphatidylcholine (PC)/phosphatidylserine (PS) than to neutral PC bilayer vesicles. The apparent Kd for binding of c-Src to the PC/PS bilayer was 6 x 10(-7) M. This interaction is sufficiently strong to account for c-Src membrane targeting. Mutants of c-Src in which the amino-terminal basic residues were replaced by neutral asparagine residues exhibited binding isotherms approaching that of wild-type binding to neutral bilayers (apparent Kd of 2 x 10(-3) M). The transforming v-Src and activated c-Src (Y527F) proteins also bound more strongly to PC/PS bilayers (apparent Kd of approximately 1 x 10(-5) M) than to neutral PC bilayers. In vivo experiments with Src mutants confirmed the role of positive charge in mediating membrane binding and cellular transformation. Images PMID:7527558

  5. Effects of the number of fatty acid residues on the phase behaviors of decaglycerol fatty acid esters.

    PubMed

    Ai, Sakiko; Ishitobi, Masahiko

    2006-04-15

    The effects of the number of fatty acid residues (n) in decaglycerol fatty acid esters, i.e., decaglycerol laurates (abbreviated to (C11)nG10), on the phase behaviors of three laurate esters, (C11)1.9G10, (C11)2.7G10, and (C11)3.4G10, were investigated. The unreacted decaglycerol remaining in each ester was removed by liquid extraction before use. (C11)1.9G10 formed hexagonal liquid crystals in aqueous solutions, while (C11)2.7G10 and (C11)3.4G10, which are more hydrophobic than (C11)1.9G10, formed lamellar liquid crystals. The cloud point in aqueous solution was measured for mixtures of these three esters. The cloud phenomenon was observed when the weight ratio of hydrophilic groups to the total surfactant (WH/WS) was around 0.6. The cloud point shifted to a markedly higher temperature, even with a slight increase in the WH/WS ratio. The solubilization abilities of (C11)nG10 for the oils m-xylene and (R)-(+)-limonene were also examined. When the WH/WS ratio was between 0.60 and 0.64, (C11)nG10 formed microemulsions and lyotropic liquid crystals in the presence of water and the oils. These self-organized structures were stable, even above 90 degrees C. It is concluded that the phase behavior of (C11)nG10 are insensitive to temperature, but strongly dependent on both the WH/WS ratio and the number of fatty acid residues (n).

  6. Solid substrate fermentation of cassava fibrous residue for production of alpha-amylase, lactic acid and ethanol.

    PubMed

    Ray, Ramesh C; Mohapatra, Sabita; Panda, Shrutirupa; Kar, Shaktimay

    2008-01-01

    There is serious concern about the disposal of solid residues left after large scale extraction of starch from cassava. Owing to the high starch content (55-65% on dry weight basis) and organic matter of these wastes, an attempt has been made to utilize it for the production of three bioproducts, i.e. alpha-amylase, lactic acid and ethanol in solid substrate fermentation by incubating the solid residue at different moisture holding capacity (40-80%) and incubation period (12- 60 hr for alpha-amylase, 24-144 hr for ethanol and 2-10 days for lactic acid). The highest product yield was obtained at 60% moisture holding capacity of the residue and period of incubation varied from 36 hr (alpha-amylase), 120 hr (ethanol) to 6 days (lactic acid). This study showed that the solid residues from cassava starch factories could serve as a low-cost substrate for bioproducts production.

  7. Role of enthalpy-entropy compensation interactions in determining the conformational propensities of amino acid residues in unfolded peptides.

    PubMed

    Toal, Siobhan E; Verbaro, Daniel J; Schweitzer-Stenner, Reinhard

    2014-02-01

    The driving forces governing the unique and restricted conformational preferences of amino acid residues in the unfolded state are still not well understood. In this study, we experimentally determine the individual thermodynamic components underlying intrinsic conformational propensities of these residues. Thermodynamic analysis of ultraviolet-circular dichroism (UV-CD) and (1)H NMR data for a series of glycine capped amino acid residues (i.e., G-x-G peptides) reveals the existence of a nearly exact enthalpy-entropy compensation for the polyproline II-β strand equilibrium for all investigated residues. The respective ΔHβ, ΔSβ values exhibit a nearly perfect linear relationship with an apparent compensation temperature of 295 ± 2 K. Moreover, we identified iso-equilibrium points for two subsets of residues at 297 and 305 K. Thus, our data suggest that within this temperature regime, which is only slightly below physiological temperatures, the conformational ensembles of amino acid residues in the unfolded state differ solely with respect to their capability to adopt turn-like conformations. Such iso-equilibria are rarely observed, and their existence herein indicates a common physical origin behind conformational preferences, which we are able to assign to side-chain dependent backbone solvation. Conformational effects such as differences between the number of sterically allowed side chain rotamers can contribute to enthalpy and entropy but not to the Gibbs energy associated with conformational preferences. Interestingly, we found that alanine, aspartic acid, and threonine are the only residues which do not share these iso-equilbiria. The enthalpy-entropy compensation discovered as well as the iso-equilbrium and thermodynamics obtained for each amino acid residue provide a new and informative way of identifying the determinants of amino acid propensities in unfolded and disordered states.

  8. 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

  9. 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

  10. Glycolic acid-catalyzed deamidation of asparagine residues in degrading PLGA matrices: a computational study.

    PubMed

    Manabe, Noriyoshi; Kirikoshi, Ryota; Takahashi, Ohgi

    2015-03-31

    Poly(lactic-co-glycolic acid) (PLGA) is a strong candidate for being a drug carrier in drug delivery systems because of its biocompatibility and biodegradability. However, in degrading PLGA matrices, the encapsulated peptide and protein drugs can undergo various degradation reactions, including deamidation at asparagine (Asn) residues to give a succinimide species, which may affect their potency and/or safety. Here, we show computationally that glycolic acid (GA) in its undissociated form, which can exist in high concentration in degrading PLGA matrices, can catalyze the succinimide formation from Asn residues by acting as a proton-transfer mediator. A two-step mechanism was studied by quantum-chemical calculations using Ace-Asn-Nme (Ace = acetyl, Nme = NHCH3) as a model compound. The first step is cyclization (intramolecular addition) to form a tetrahedral intermediate, and the second step is elimination of ammonia from the intermediate. Both steps involve an extensive bond reorganization mediated by a GA molecule, and the first step was predicted to be rate-determining. The present findings are expected to be useful in the design of more effective and safe PLGA devices.

  11. Analysis of amino acids in latent fingerprint residue by capillary electrophoresis-mass spectrometry.

    PubMed

    Atherton, Tom; Croxton, Ruth; Baron, Mark; Gonzalez-Rodriguez, Jose; Gámiz-Gracia, Laura; García-Campaña, Ana M

    2012-11-01

    The analysis of the chemical composition of fingerprints is important for the development and improvement of existing fingerprint enhancement techniques. This study demonstrates the first analysis of a latent fingerprint sample, using an optimized CE-MS method. In total 12 amino acids were detected in the fingerprint sample. MS/MS fragmentation was used to provide additional identity confirmation, for which eight of the twelve detected amino acids generated confirmatory product ions. Nine amino acids were quantified and their relative abundances were consistent with previous studies with serine and glycine being the most abundant. The successful detection of amino acids from latent fingerprints demonstrates that CE-MS is a potential future technique for further study of such compounds in fingerprint samples.

  12. 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

  13. Acid-catalyzed hydrothermal severity on the fractionation of agricultural residues for xylose-rich hydrolyzates.

    PubMed

    Lee, Ji Ye; Ryu, Hyun Jin; Oh, Kyeong Keun

    2013-03-01

    The objective of this work was to investigate the feasibility of acid-catalyzed hydrothermal fractionation for maximum solubilization of the hemicellulosic portion of three agricultural residues. The fractionation conditions converted into combined severity factor (CS) in the range of 1.2-2.9. The highest hemicellulose yield of 87.88% was achieved when barley straw was fractionated at a CS of 2.19. However, the maximum glucose release of 15.29% was achieved for the case of rice straw. The maximum productions of various by-products were observed with the fractionation of rape straw: 0.88 g/L of 5-hydroxymethylfurfural (5-HMF), 2.16 g/L of furfural, 0.44 g/L of levulinic acid, 1.59 g/L of formic acid, and 3.06 g/L of acetic acid. The highest selectivities, a criterion for evaluating the fractionation of 21.55 for fractionated solid and 7.48 for liquid hydrolyzate were obtained from barley straw.

  14. Identification of acid-base catalytic residues of high-Mr thioredoxin reductase from Plasmodium falciparum.

    PubMed

    McMillan, Paul J; Arscott, L David; Ballou, David P; Becker, Katja; Williams, Charles H; Müller, Sylke

    2006-11-01

    High-M(r) thioredoxin reductase from the malaria parasite Plasmodium falciparum (PfTrxR) contains three redox active centers (FAD, Cys-88/Cys-93, and Cys-535/Cys-540) that are in redox communication. The catalytic mechanism of PfTrxR, which involves dithiol-disulfide interchanges requiring acid-base catalysis, was studied by steady-state kinetics, spectral analyses of anaerobic static titrations, and rapid kinetics analysis of wild-type enzyme and variants involving the His-509-Glu-514 dyad as the presumed acid-base catalyst. The dyad is conserved in all members of the enzyme family. Substitution of His-509 with glutamine and Glu-514 with alanine led to TrxR with only 0.5 and 7% of wild type activity, respectively, thus demonstrating the crucial roles of these residues for enzymatic activity. The H509Q variant had rate constants in both the reductive and oxidative half-reactions that were dramatically less than those of wild-type enzyme, and no thiolateflavin charge-transfer complex was observed. Glu-514 was shown to be involved in dithiol-disulfide interchange between the Cys-88/Cys-93 and Cys-535/Cys-540 pairs. In addition, Glu-514 appears to greatly enhance the role of His-509 in acid-base catalysis. It can be concluded that the His-509-Glu-514 dyad, in analogy to those in related oxidoreductases, acts as the acid-base catalyst in PfTrxR.

  15. A critical amino acid residue, asp446, in UDP-glucuronosyltransferase.

    PubMed Central

    Iwano, H; Yokota, H; Ohgiya, S; Yotumoto, N; Yuasa, A

    1997-01-01

    An amino acid residue, Asp446, was found to be essential for the enzymic activity of UDP-glucuronosyltransferase (UGT). We obtained a rat phenol UGT (UGT1*06) cDNA (named Ysh) from male rat liver by reverse-transcription (RT)-PCR using pfu polymerase. A mutant Ysh having two different bases, A1337G and G1384A (named Ysh A1337GC1384A), that result in two amino acid substitutions, D446G and V462M, was obtained by RT-PCR using Taq polymerase. Ysh was expressed functionally in microsomes of Saccharomyces cerevisiae strain AH22. However, the expressed protein from YshA1337GG1384A had no transferase activity. Two other mutant cDNAs with YshA1337G having one changed base, A1337G, resulting in one amino acid substitution, D446G, and YshG1384A having a changed base, G1384A, resulting in an amino acid substitution, V462M, were constructed and expressed in the yeast. The expressed protein from YshG1384A (named YshV462M) exhibited enzymic activity, but the one from YshA1337G (named YshD446G) did not show any activity at all. Asp446 was conserved in all UGTs and UDP-galactose:ceramide galactosyltransferases reported, suggesting that Asp446 plays a critical role in each enzyme. PMID:9271076

  16. Determination of residue-specific acid dissociation constants for peptides by band-selective homonuclear-decoupled (1)H NMR.

    PubMed

    Wang, Jing; Rabenstein, Dallas L

    2007-09-01

    Acid dissociation constants of side-chain acidic groups of amino acid residues in peptides can be determined by 1H NMR, provided resonances can be resolved for carbon-bonded reporter protons located near the acidic group. We report here that the increased resolution of the band-selective homonuclear-decoupled (BASHD) TOCSY experiment greatly extends the range of application of the NMR method for determination of residue-specific, side-chain acid dissociation constants of peptides that contain multiple residues of the same amino acid. Chemical shift-pH titration curves are obtained from cross-peaks for reporter protons in BASHD-TOCSY spectra measured as a function of pH. The method is based on using sequence-dependent differences in the chemical shifts of resonances for the backbone CalphaH protons and the increased resolution in BASHD-TOCSY spectra from collapse of CalphaH multiplets to singlets in the F1 dimension to resolve resonances for the side-chain reporter protons. Application of the method is demonstrated by determination of residue-specific pKA values for each of the side-chain ammonium groups of the six lysine residues in the hexadecapeptide Ac-SRGKAKVKAKVKDQTK-NH2. Chemical shift-pH titration curves were obtained for the lysine side-chain CepsilonH2 reporter protons from their resolved CalphaH-CepsilonH2 TOCSY cross-peaks in BASHD-TOCSY spectra. Relative acidities of the six ammonium groups were also determined from the residue specific chemical shift-pH titration data by a pH-independent method, and calculation of fractional concentrations of protonation microspecies using the residue-specific pKAs is also described.

  17. Identification of acidic and aromatic residues in the Zta activation domain essential for Epstein-Barr virus reactivation.

    PubMed

    Deng, Z; Chen, C J; Zerby, D; Delecluse, H J; Lieberman, P M

    2001-11-01

    Epstein-Barr virus (EBV) lytic cycle transcription and DNA replication require the transcriptional activation function of the viral immediate-early protein Zta. We describe a series of alanine substitution mutations in the Zta activation domain that reveal two functional motifs based on amino acid composition. Alanine substitution of single or paired hydrophobic aromatic amino acid residues resulted in modest transcription activation defects, while combining four substitutions of aromatic residues (F22/F26/W74/F75) led to more severe transcription defects. Substitution of acidic amino acid residue E27, D35, or E54 caused severe transcription defects on most viral promoters. Promoter- and cell-specific defects were observed for some substitution mutants. Aromatic residues were required for Zta interaction with TFIIA-TFIID and the CREB-binding protein (CBP) and for stimulation of CBP histone acetyltransferase activity in vitro. In contrast, acidic amino acid substitution mutants interacted with TFIIA-TFIID and CBP indistinguishably from the wild type. The nuclear domain 10 (ND10) protein SP100 was dispersed by most Zta mutants, but acidic residue mutations led to reduced, while aromatic substitution mutants led to increased SP100 nuclear staining. Acidic residue substitution mutants had more pronounced defects in transcription activation of endogenous viral genes in latently infected cells and for viral replication, as measured by the production of infectious virus. One mutant, K12/F13, was incapable of stimulating EBV lytic replication but had only modest transcription defects. These results indicate that Zta stimulates viral reactivation through two nonredundant structural motifs, one of which interacts with general transcription factors and coactivators, and the other has an essential but as yet not understood function in lytic transcription.

  18. New force field parameters for metalloproteins I: Divalent copper ion centers including three histidine residues and an oxygen-ligated amino acid residue.

    PubMed

    Wise, Olivia; Coskuner, Orkid

    2014-06-30

    Transition metal ion complexation with proteins is ubiquitous across such diverse fields as neurodegenerative and cardiovascular diseases and cancer. In this study, the structures of divalent copper ion centers including three histidine and one oxygen-ligated amino acid residues and the relative binding affinities of the oxygen-ligated amino acid residues with these metal ion centers, which are debated in the literature, are presented. Furthermore, new force field parameters, which are currently lacking for the full-length metal-ligand moieties, are developed for metalloproteins that have these centers. These new force field parameters enable investigations of metalloproteins possessing these binding sites using molecular simulations. In addition, the impact of using the atom equivalence and inequivalence atomic partial charge calculation procedures on the simulated structures of these metallopeptides, including hydration properties, is described.

  19. 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.

  20. 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

  1. Chemical modification of an alpha 3-fucosyltransferase; definition of amino acid residues essential for enzyme activity.

    PubMed

    Britten, C J; Bird, M I

    1997-02-11

    The biosynthesis of the carbohydrate antigen sialyl Lewis X (sLe(x)) is dependent on the activity of an alpha 3-fucosyltransferase (EC 2.4.1.152, GDP-fucose:Gal beta (1-4)GlcNAc-R alpha (1-3)fucosyltransferase). This enzyme catalyses the transfer of fucose from GDP-beta-fucose to the 3-OH of N-acetylglucosamine present in lactosamine acceptors. In this report, we have investigated the amino acids essential for the activity of a recombinant alpha 3-fucosyltransferase (FucT-VI) through chemical modification of the enzyme with group-selective reagents. FucT-VI activity was found to be particularly sensitive to the histidine-selective reagent diethylpyrocarbonate and the cysteine reagent N-ethylmaleimide, with IC50 values of less than 200 microM. Reagents selective for arginine and lysine had no effect on enzyme activity. The inclusion of GDP-beta-fucose during preincubation with NEM reduces the rate of inactivation whereas inclusion of an acceptor saccharide for the enzyme, Gal beta (1-4)GlcNAc, had no effect. No protective effect with either GDP-beta-fucose or Gal beta (1-4)GlcNAc was observed on treatment of the enzyme with diethylpyrocarbonate. These data suggest that in addition to an NEM-reactive cysteine in, or adjacent to, the substrate-binding site of the enzyme, FucT-VI possesses histidine residue(s) that are essential for enzyme activity.

  2. 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

  3. 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

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

    PubMed Central

    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

  5. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

    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.

  7. 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

  8. 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.

  9. 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

  10. 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).

  11. Amino acid residue 247 in canine sulphotransferase SULT1D1: a new determinant of substrate selectivity.

    PubMed Central

    Tsoi, Carrie; Widersten, Mikael; Morgenstern, Ralf; Swedmark, Stellan

    2004-01-01

    The SULT (sulphotransferase) family plays a critical role in the detoxification and activation of endogenous and exogenous compounds as well as in the regulation of steroid hormone actions and neurotransmitter functions. The structure-activity relationships of the human SULTs have been investigated with focus on the amino acid 146 in hSULT1A3 and its impact on dopamine/PNP (p-nitrophenol) specificity. In the present study, we have generated canine SULT1D1 (cSULT1D1) variants with mutations at amino acid residues in the substrate-binding pocket [A146E (Ala-146-->Glu), A146D, A146Q, I86D or D247L]. These mutation sites were chosen with regard to their possible contribution to the marked dopamine/PNP preference of cSULT1D1. After characterization, we found that the overall sulphation efficiencies for the cSULT1D1 A146 and the I86 mutants were strongly decreased for both substrates compared with wild-type cSULT1D1 but the substrate preference was unchanged. In contrast, the D247L mutant was found to be more than 21-fold better at sulphating PNP (120-fold decrease in K(m) value) but 54-fold less efficient in sulphating dopamine (8-fold increase in K(m) value) and the preference was switched from dopamine to PNP, indicating the importance of this amino acid in the dopamine/PNP preference in cSULT1D1. Our results show that Asp-247 has a pronounced effect on the substrate specificity of cSULT1D1 and thus we have identified a previously unrecognized contributor to active-site selectivity. PMID:14614767

  12. 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.

  13. Trace analysis of acidic pharmaceutical residues in waters with isotope dilution gas chromatography-mass spectrometry via methylation derivatization.

    PubMed

    Hu, Ruikang; Yang, Zhaoguang; Zhang, Lifeng

    2011-09-30

    Acidic pharmaceutical residues are pollutants of emerging concern and are generally monitored by HPLC-MS/MS. However, due to the limited separation efficiency of HPLC column and lack of suitable mass transition for confirmation analysis, some interference may not be separated completely and differentiated from ibuprofen, which may cause the results with interference, especially in sample with complex matrix. The objective of this study is to develop a sensitive and reliable method for the determination of acidic pharmaceutical residues in water samples by GC-MS with better resolution by using methylation derivatization and isotope dilution techniques. TMSDM, a mild reagent, was used as the derivatization reagent coupling with the isotope dilution technique, for the first time, to improve the precision and accuracy of the analytical method to determine the pharmaceutical residues in water. The MDLs for the five acidic organic compounds: ibuprofen, gemfibrozil, naproxen, ketoprofen and diclofenac were from 0.7 to 1.1 ng/L, with recoveries ranging from 93 to 110%. Alternative to the HPLC-MS/MS method, the developed GC-MS protocols provides an additional option for the analysis of acidic pharmaceutical residues in water, with better separation efficiency in reducing interferences from complicated sample matrix, for determination of ibuprofen residues.

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

    PubMed

    Listiyani, M A D; Campbell, R E; Miracle, R E; Dean, L O; Drake, M A

    2011-09-01

    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 in dried whey products. No legal limit exists in the United States for BP use in whey, but international concerns exist. The objectives of this study were to determine the effect of hydrogen peroxide (HP) or BP bleaching on the flavor of 34% WPC (WPC34) and to evaluate residual BA in commercial and experimental WPC bleached with and without BP. Cheddar whey was manufactured in duplicate. Pasteurized fat-separated whey was subjected to hot bleaching with either HP at 500 mg/kg, BP at 50 or 100 mg/kg, or no bleach. Whey was ultrafiltered and spray dried into WPC34. Color [L*(lightness), a* (red-green), and b* (yellow-blue)] measurements and norbixin extractions were conducted to compare bleaching efficacy. Descriptive sensory and instrumental volatile analyses were used to evaluate bleaching effects on flavor. Benzoic acid was extracted from experimental and commercial WPC34 and 80% WPC (WPC80) and quantified by HPLC. The b* value and norbixin concentration of BP-bleached WPC34 were lower than HP-bleached and control WPC34. Hydrogen peroxide-bleached WPC34 displayed higher cardboard flavor and had higher volatile lipid oxidation products than BP-bleached or control WPC34. Benzoyl peroxide-bleached WPC34 had higher BA concentrations than unbleached and HP-bleached WPC34 and BA concentrations were also higher in BP-bleached WPC80 compared with unbleached and HP-bleached WPC80, with smaller differences than those observed in WPC34. Benzoic acid extraction from permeate showed that WPC80 permeate contained more BA than did WPC34 permeate. Benzoyl peroxide is more effective in color removal of whey and results in fewer flavor side effects compared with HP and residual BA is

  15. 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

  16. Experimental test of the superheavy fission hypothesis in acid residues from the allende meteorite

    SciTech Connect

    Flynn, G.J.

    1982-01-01

    A description of a series of experiments to find evidence to confirm or contradict the hypothesis that isotopically anomalous Xe (called CCF-Xe) in carbonaceous chondrite meteorites results from the fission decay of a superheavy element is given. The first two experiments were searches for fossil evidence - fission tracks and isotopic anomalies - of superheavy fission decay in the Allende carbonaceous chondrite. It was demonstrated that chromite, a mineral rich in CCF-Xe, records fission tracks, and a search for such tracks in Allende chromite was performed with negative results. It was also demonstrated in certain CCF-XE rich phases of Allende, isotopic anomalies like those seen in Xe should be detectable in Ba and the light rare earths. Preliminary results from a collaborative measurement (with the University of Paris) show the Ba isotopic ratios to be normal in a CCF-Xe rich Allende sample. The third experiment was motivated by reports of the detection of a live, fissioning superheavy element, using a neutron counting technique, in bulk Allende (Flerov, 1978). Since almost all of the allegedly fissiogenic Xe in Allende is concentrated in certain acid insoluble phases, we developed a technique to detect low-level fission activity in these phases. Allende acid insoluble residue (provided by the University of Chicago) was dispersed in a 1 mg/cm/sup 2/ layer, between two track recording detectors. An automatic track locating system was developed to allow large detector areas to be scanned for rate fission events.

  17. 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

  18. Lysine Residues Are Not Required for Proteasome-Mediated Proteolysis of the Auxin/Indole Acidic Acid Protein IAA1.

    PubMed

    Gilkerson, Jonathan; Kelley, Dior R; Tam, Raymond; Estelle, Mark; Callis, Judy

    2015-06-01

    Although many ubiquitin-proteasome substrates have been characterized in plants, very little is known about the corresponding ubiquitin attachment(s) underlying regulated proteolysis. Current dogma asserts that ubiquitin is typically covalently attached to a substrate through an isopeptide bond between the ubiquitin carboxy terminus and a substrate lysyl amino group. However, nonlysine (non-Lys) ubiquitin attachment has been observed in other eukaryotes, including the N terminus, cysteine, and serine/threonine modification. Here, we investigate site(s) of ubiquitin attachment on indole-3-acetic acid1 (IAA1), a short-lived Arabidopsis (Arabidopsis thaliana) Auxin/indole-3-acetic acid (Aux/IAA) family member. Most Aux/IAA proteins function as negative regulators of auxin responses and are targeted for degradation after ubiquitination by the ubiquitin ligase SCF(TIR1/AFB) (for S-Phase Kinase-Associated Protein1, Cullin, F-box [SCF] with Transport Inhibitor Response1 [TIR1]/Auxin Signaling F-box [AFB]) by an interaction directly facilitated by auxin. Surprisingly, using a Histidine-Hemaglutinin (HIS(6x)-HA(3x)) epitope-tagged version expressed in vivo, Lys-less IAA1 was ubiquitinated and rapidly degraded in vivo. Lys-substituted versions of IAA1 localized to the nucleus as Yellow Fluorescent Protein fusions and interacted with both TIR1 and IAA7 in yeast (Saccharomyces cerevisiae) two-hybrid experiments, indicating that these proteins were functional. Ubiquitination on both HIS(6x)-HA(3x)-IAA1 and Lys-less HIS(6x)-HA(3x)-IAA1 proteins was sensitive to sodium hydroxide treatment, indicative of ubiquitin oxyester formation on serine or threonine residues. Additionally, base-resistant forms of ubiquitinated IAA1 were observed for HIS(6x)-HA(3x)-IAA1, suggesting additional lysyl-linked ubiquitin on this protein. Characterization of other Aux/IAA proteins showed that they have diverse degradation rates, adding additional complexity to auxin signaling. Altogether, these data

  19. Lysine Residues Are Not Required for Proteasome-Mediated Proteolysis of the Auxin/Indole Acidic Acid Protein IAA1.

    PubMed

    Gilkerson, Jonathan; Kelley, Dior R; Tam, Raymond; Estelle, Mark; Callis, Judy

    2015-06-01

    Although many ubiquitin-proteasome substrates have been characterized in plants, very little is known about the corresponding ubiquitin attachment(s) underlying regulated proteolysis. Current dogma asserts that ubiquitin is typically covalently attached to a substrate through an isopeptide bond between the ubiquitin carboxy terminus and a substrate lysyl amino group. However, nonlysine (non-Lys) ubiquitin attachment has been observed in other eukaryotes, including the N terminus, cysteine, and serine/threonine modification. Here, we investigate site(s) of ubiquitin attachment on indole-3-acetic acid1 (IAA1), a short-lived Arabidopsis (Arabidopsis thaliana) Auxin/indole-3-acetic acid (Aux/IAA) family member. Most Aux/IAA proteins function as negative regulators of auxin responses and are targeted for degradation after ubiquitination by the ubiquitin ligase SCF(TIR1/AFB) (for S-Phase Kinase-Associated Protein1, Cullin, F-box [SCF] with Transport Inhibitor Response1 [TIR1]/Auxin Signaling F-box [AFB]) by an interaction directly facilitated by auxin. Surprisingly, using a Histidine-Hemaglutinin (HIS(6x)-HA(3x)) epitope-tagged version expressed in vivo, Lys-less IAA1 was ubiquitinated and rapidly degraded in vivo. Lys-substituted versions of IAA1 localized to the nucleus as Yellow Fluorescent Protein fusions and interacted with both TIR1 and IAA7 in yeast (Saccharomyces cerevisiae) two-hybrid experiments, indicating that these proteins were functional. Ubiquitination on both HIS(6x)-HA(3x)-IAA1 and Lys-less HIS(6x)-HA(3x)-IAA1 proteins was sensitive to sodium hydroxide treatment, indicative of ubiquitin oxyester formation on serine or threonine residues. Additionally, base-resistant forms of ubiquitinated IAA1 were observed for HIS(6x)-HA(3x)-IAA1, suggesting additional lysyl-linked ubiquitin on this protein. Characterization of other Aux/IAA proteins showed that they have diverse degradation rates, adding additional complexity to auxin signaling. Altogether, these data

  20. Significance of the C-terminal amino acid residue in mengovirus RNA-dependent RNA polymerase.

    PubMed

    Dmitrieva, Tatiana M; Alexeevski, Andrei V; Shatskaya, Galina S; Tolskaya, Elena A; Gmyl, Anatoly P; Khitrina, Elena V; Agol, Vadim I

    2007-08-15

    Replication of picornavirus genomes is accomplished by the virally encoded RNA-dependent RNA polymerase (RdRP). Although the primary structure of this enzyme exhibits a high level of conservation, there are several significant differences among different picornavirus genera. In particular, a comparative alignment indicates that the C-terminal sequences of cardiovirus RdRP (known also as 3D(pol)), are 1-amino-acid residue (arginine or tryptophan) longer than that of the enterovirus or rhinovirus enzymes. Here, it is shown that alterations of the last codon of the RdRP-encoding sequence of mengovirus RNA leading to deletion of the C-terminal Trp460 or its replacement by Ala or Phe dramatically impaired viral RNA replication and, in the former case, resulted in a quasi-infectious phenotype (i.e., the mutant RNA might generate a low yield of pseudorevertants acquiring a Tyr residue in place of the deleted Trp460). The replacement of Trp460 by His or Tyr did not appreciably alter the viral growth potential. Homology modeling of three-dimensional structure of mengovirus RdRP suggested that Trp460 may be involved in interaction between the thumb and palm domains of the enzyme. Specifically, Trp460 of the thumb may form a hydrogen bond with Thr219 and hydrophobically interact with Val216 of the palm. The proposed interactions were consistent with the results of in vivo SELEX experiment, which demonstrated that infectious virus could contain Ser or Thr at position 219 and hydrophobic Val, Leu, Ile, as well as Arg (whose side chain has a nonpolar part) at position 216. A similar thumb-palm domain interaction may be a general feature of several RdRPs and its possible functional significance is discussed. PMID:17467026

  1. Identification of glutamic acid 646 as a zinc-coordinating residue in endopeptidase-24.11.

    PubMed

    Le Moual, H; Devault, A; Roques, B P; Crine, P; Boileau, G

    1991-08-25

    Neutral endopeptidase (EC 3.424.11, NEP) is a membrane-bound zinc-metallopeptidase. The substrate specificity and catalytic activity of NEP resemble those of thermolysin, a bacterial zinc-metalloprotease. Comparison of the primary structure of both enzymes suggests that several amino acids present in the active site of thermolysin are also found in NEP. Using site-directed mutagenesis of the cDNA encoding the NEP sequence, we have already shown that His residues 583 and 587 are two of the three zinc ligands. In order to identify the third zinc ligand, we have substituted Val or Asp for Glu616 or Glu646. Val616 NEP showed the same kinetic parameters as the non-mutated NEP. In contrast, the mutant Val646 NEP was almost completely devoid of catalytic activity and unable to bind the tritiated inhibitor [3H]N-[2(R,S)-3-hydroxyaminocarbonyl-2-benzyl-1-oxypropyl]gl ycine, the binding of which is dependent on the presence of the zinc ion. Replacing Glu for Asp at position 646 conserved the negative charge, and the mutant enzyme exhibited the same Km value as the non-mutated enzyme, but kCat was decreased to less than 3% of the value of the non-mutated enzyme. When compared to the non-mutated enzyme Asp646 NEP showed a higher susceptibility to chelating agents, but bound the tritiated inhibitor with the same affinity. Taken together, these observations strongly suggest that Glu646 of NEP is the third zinc-coordinating residue and is equivalent to Glu166 in thermolysin.

  2. Effect of low molecular weight organic acids on phosphorus adsorption by ferric-alum water treatment residuals.

    PubMed

    Wang, Changhui; Wang, Ziyuan; Lin, Lu; Tian, Binghui; Pei, Yuansheng

    2012-02-15

    Effects of low molecular weight organic acids (LMWOAs; citric acid, oxalic acid and tartaric acid) on phosphorus (P) adsorption by ferric-alum water treatment residuals (FARs) were studied. Both batch and column experiments indicated that the effects of LMWOAs on P adsorption were closely related to adsorption time. Initially, all acids presented inhibitory function on P adsorption. The inhibition became weaker with time, eventually promoting P adsorption for citric acid and tartaric acid. In the column experiment with a 61-day duration, high P adsorption rates (>55%) were observed for the test groups containing citric acid and tartaric acid. Interestingly, higher pH likely enhanced P adsorption with the effects of LMWOAs and a distinct relationship between LMWOAs' effects on P adsorption and their concentrations was not observed. Moreover, fractionation of the adsorbed P from the FARs demonstrated that oxalic acid reduced P adsorption capacity, while citric acid and tartaric acid increased. Based on the forms of Fe and Al existing in the FARs and Fourier transform infrared spectroscopy analyses, LMWOAs can promote P adsorption through activating crystalline Fe/Al and preventing crystallization of amorphous Fe/Al to increase P adsorption sites, and can also inhibit P adsorption by competition with adsorption sites.

  3. 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.

  4. 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

  5. A single amino acid residue controls ROS production in the respiratory Complex I from Escherichia coli.

    PubMed

    Knuuti, Juho; Belevich, Galina; Sharma, Vivek; Bloch, Dmitry A; Verkhovskaya, Marina

    2013-12-01

    Reactive oxygen species (ROS) production by respiratory Complex I from Escherichia coli was studied in bacterial membrane fragments and in the isolated and purified enzyme, either solubilized or incorporated in proteoliposomes. We found that the replacement of a single amino acid residue in close proximity to the nicotinamide adenine dinucleotide (NADH)-binding catalytic site (E95 in the NuoF subunit) dramatically increases the reactivity of Complex I towards dioxygen (O2 ). In the E95Q variant short-chain ubiquinones exhibit strong artificial one-electron reduction at the catalytic site, also leading to a stronger increase in ROS production. Two mechanisms can contribute to the observed kinetic effects: (a) a change in the reactivity of flavin mononucleotide (FMN) towards dioxygen at the catalytic site, and (b) a change in the population of the ROS-generating state. We propose the existence of two (closed and open) states of the NAD(+) -bound enzyme as one feature of the substrate-binding site of Complex I. The analysis of the kinetic model of ROS production allowed us to propose that the population of Complex I with reduced FMN is always low in the wild-type enzyme even at low ambient redox potentials, minimizing the rate of reaction with O2 in contrast to E95Q variant.

  6. 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

  7. 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.

  8. Electrostatic effects of surface acidic amino acid residues on the oxidation-reduction potentials of the flavodoxin from Desulfovibrio vulgaris (Hildenborough).

    PubMed

    Zhou, Z; Swenson, R P

    1995-03-14

    The flavodoxin from Desulfovibrio vulgaris (Hildenborough) is a member of a family of small, acidic proteins that contain a single noncovalently bound flavin mononucleotide (FMN) cofactor. These proteins function as low-potential one-electron transferases in bacteria. A distinguishing feature of these flavoproteins is the dramatic decrease in the midpoint potential of the semiquinone/hydroquinone couple of the FMN upon binding to the apoprotein (-172 mV for FMN free in solution versus -443 mV when bound), a perturbation thought to be essential for physiological function. The structural basis of this phenomenon is not yet thoroughly understood. In this study, the contribution of six acidic residues (Asp62, Asp63, Glu66, Asp95, Glu99, and Asp106) to the perturbation of the redox properties of the cofactor has been investigated. These residues are clustered about the FMN binding site within 13 A of the N(1) atom of the cofactor. Using oligonucleotide-directed mutagenesis, these residues were neutralized in various combinations through the substitution of asparagine for aspartate and glutamine for glutamate. Seventeen mutant flavodoxins were generated in which one to all six acidic residues were systematically neutralized, often in various spatial configurations. There was no obvious correlation between the midpoint potentials for the oxidized/semiquinone couple and general electrostatic environment, although some differences were noted. However, the midpoint potential for the semiquinone/hydroquinone couple for each of the mutants was less negative than that of the wild type. These increases are strongly correlated with the number of acid to amide substitutions, with an average contribution of about 15 mV per substitution. Collectively, the unfavorable electrostatic environment provided by these acidic residues accounts for approximately one-third of the large midpoint potential shift for the semiquinone/hydroquinone couple that typifies the flavodoxin family

  9. 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

  10. Acidic Residues Control the Dimerization of the N-terminal Domain of Black Widow Spiders’ Major Ampullate Spidroin 1

    PubMed Central

    Bauer, Joschka; Schaal, Daniel; Eisoldt, Lukas; Schweimer, Kristian; Schwarzinger, Stephan; Scheibel, Thomas

    2016-01-01

    Dragline silk is the most prominent amongst spider silks and comprises two types of major ampullate spidroins (MaSp) differing in their proline content. In the natural spinning process, the conversion of soluble MaSp into a tough fiber is, amongst other factors, triggered by dimerization and conformational switching of their helical amino-terminal domains (NRN). Both processes are induced by protonation of acidic residues upon acidification along the spinning duct. Here, the structure and monomer-dimer-equilibrium of the domain NRN1 of Latrodectus hesperus MaSp1 and variants thereof have been investigated, and the key residues for both could be identified. Changes in ionic composition and strength within the spinning duct enable electrostatic interactions between the acidic and basic pole of two monomers which prearrange into an antiparallel dimer. Upon naturally occurring acidification this dimer is stabilized by protonation of residue E114. A conformational change is independently triggered by protonation of clustered acidic residues (D39, E76, E81). Such step-by-step mechanism allows a controlled spidroin assembly in a pH- and salt sensitive manner, preventing premature aggregation of spider silk proteins in the gland and at the same time ensuring fast and efficient dimer formation and stabilization on demand in the spinning duct. PMID:27681031

  11. Acidic Residues Control the Dimerization of the N-terminal Domain of Black Widow Spiders’ Major Ampullate Spidroin 1

    NASA Astrophysics Data System (ADS)

    Bauer, Joschka; Schaal, Daniel; Eisoldt, Lukas; Schweimer, Kristian; Schwarzinger, Stephan; Scheibel, Thomas

    2016-09-01

    Dragline silk is the most prominent amongst spider silks and comprises two types of major ampullate spidroins (MaSp) differing in their proline content. In the natural spinning process, the conversion of soluble MaSp into a tough fiber is, amongst other factors, triggered by dimerization and conformational switching of their helical amino-terminal domains (NRN). Both processes are induced by protonation of acidic residues upon acidification along the spinning duct. Here, the structure and monomer-dimer-equilibrium of the domain NRN1 of Latrodectus hesperus MaSp1 and variants thereof have been investigated, and the key residues for both could be identified. Changes in ionic composition and strength within the spinning duct enable electrostatic interactions between the acidic and basic pole of two monomers which prearrange into an antiparallel dimer. Upon naturally occurring acidification this dimer is stabilized by protonation of residue E114. A conformational change is independently triggered by protonation of clustered acidic residues (D39, E76, E81). Such step-by-step mechanism allows a controlled spidroin assembly in a pH- and salt sensitive manner, preventing premature aggregation of spider silk proteins in the gland and at the same time ensuring fast and efficient dimer formation and stabilization on demand in the spinning duct.

  12. A single gamma-carboxyglutamic acid residue in a novel cysteine-rich secretory protein without propeptide.

    PubMed

    Hansson, Karin; Thämlitz, Ann-Marie; Furie, Bruce; Furie, Barbara C; Stenflo, Johan

    2006-10-24

    Gamma-glutamyl carboxylase catalyzes the modification of specific glutamyl residues to gamma-carboxyglutamyl (Gla) residues in precursor proteins that possess the appropriate gamma-carboxylation recognition signal within the propeptide region. We describe the immunopurification and first biochemical characterization of an invertebrate high molecular weight Gla-containing protein with homologues in mammals. The protein, named GlaCrisp, was isolated from the venom of the marine cone snail Conus marmoreus. GlaCrisp gave intense signals in Western blot experiments employing the Gla-specific antibody M3B, and the presence of Gla was chemically confirmed by amino acid analysis after alkaline hydrolysis. Characterization of a full-length cDNA clone encoding GlaCrisp deduced a precursor containing an N-terminal signal peptide but, unlike other Gla-containing proteins, no apparent propeptide. The predicted mature protein of 265 amino acid residues showed considerable sequence similarity to the widely distributed cysteine-rich secretory protein family and closest similarity (65% identity) to the recently described substrate-specific protease Tex31. In addition, two cDNA clones encoding the precursors of two isoforms of GlaCrisp were identified. The predicted precursor isoforms differed at three amino acid positions (-6, 9, and 25). Analysis by Edman degradation and nanoelectrospray ionization mass spectrometry, before and after methyl esterfication, identified a Gla residue at amino acid position 9 in GlaCrisp. This is the first example of a Gla-containing protein without an obvious gamma-carboxylation recognition site. The results define a new class of Gla proteins and support the notion that gamma-carboxylation of glutamyl residues is phylogenetically older than blood coagulation and the vertebrate lineage.

  13. Dimerization of a PACAP peptide analogue in DMSO via asparagine and aspartic acid residues.

    PubMed

    Severs, Joanne C; Froland, Wayne A

    2008-03-01

    To optimize the stability of a peptide development candidate for the treatment of type II diabetes, formulation studies were initiated in organic solvents and compared to results obtained in aqueous solutions. Stability was assessed by reversed phase liquid chromatography (RPLC) and electrospray ionization mass spectrometry (ESI-MS). Previous studies had shown deamidation and hydrolysis to be the primary mechanisms of degradation in aqueous formulations. Surprisingly, the use of an organic solvent did not decrease the rate of degradation and, as presented here, produced degradation products including dimers. We propose here that deamidation can readily occur in polar anhydrous organic solvents such as DMSO and that the dimer forms through intermolecular nucleophilic attack of an amino acid side chain on a stabilized cyclic imide intermediate.

  14. A Mutational Analysis of the Active Site Loop Residues in cis-3-Chloroacrylic Acid Dehalogenase

    PubMed Central

    Schroeder, Gottfried K.; Huddleston, Jamison P.; Johnson, William H.; Whitman, Christian P.

    2013-01-01

    cis -3-Chloroacrylic acid dehalogenase (cis-CaaD) from Pseudomonas pavonaceae 170 and a homologue from Corynebacterium glutamicum designated Cg10062 share 34% sequence identity (54% similarity). The former catalyzes a key step in a bacterial catabolic pathway for the nematocide 1,3-dichloropropene, whereas the latter has no known biological activity. Although Cg10062 has the six active site residues (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, Glu-114) that are critical for cis-CaaD activity, it shows only a low level cis-CaaD activity and lacks the specificity of cis-CaaD: Cg10062 processes both isomers of 3-chloroacrylate with a preference for the cis-isomer. Although the basis for these differences is unknown, a comparison of the crystal structures of the enzymes covalently modified by an adduct resulting from their incubation with the same inhibitor offers a possible explanation. A 6-residue active site loop in cis-CaaD shows a strikingly different conformation from that observed in Cg10062: the loop closes down on the active site of cis-CaaD, but not on that of Cg10062. In order to examine what this loop might contribute to cis-CaaD catalysis and specificity, the residues were changed individually to those found in Cg10062. Subsequent kinetic and mechanistic analysis suggests that the T34A mutant of cis-CaaD is more Cg10062-like. The mutant enzyme shows a 4-fold increase in Km (using cis-3-bromoacrylate), but not to the degree observed for Cg10062 (687-fold). The mutation also causes a 4-fold decrease in the burst rate (compared to the wild type cis-CaaD), whereas Cg10062 shows no burst rate. More telling is the reaction of the T34A mutant of cis-CaaD with the alternate substrate, 2,3-butadienoate. In the presence of NaBH4 and the allene, cis-CaaD is completely inactivated after one turnover due to the covalent modification of Pro-1. The same experiment with Cg10062 does not result in the covalent modification of Pro-1. The different outcomes are attributed to

  15. 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.

  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. Loss of Autonoetic Awareness of Recent Autobiographical Episodes and Accelerated Long-Term Forgetting in a Patient with Previously Unrecognized Glutamic Acid Decarboxylase Antibody Related Limbic Encephalitis

    PubMed Central

    Witt, Juri-Alexander; Vogt, Viola Lara; Widman, Guido; Langen, Karl-Josef; Elger, Christian Erich; Helmstaedter, Christoph

    2015-01-01

    We describe a 35-year-old male patient presenting with depressed mood and emotional instability, who complained about severe anterograde and retrograde memory deficits characterized by accelerated long-term forgetting and loss of autonoetic awareness regarding autobiographical memories of the last 3 years. Months before he had experienced two breakdowns of unknown etiology giving rise to the differential diagnosis of epileptic seizures after various practitioners and clinics had suggested different etiologies such as a psychosomatic condition, burnout, depression, or dissociative amnesia. Neuropsychological assessment indicated selectively impaired figural memory performance. Extended diagnostics confirmed accelerated forgetting of previously learned and retrievable verbal material. Structural imaging showed bilateral swelling and signal alterations of temporomesial structures (left >right). Video-EEG monitoring revealed a left temporal epileptic focus and subclincal seizure, but no overt seizures. Antibody tests in serum and liquor were positive for glutamic acid decarboxylase antibodies. These findings led to the diagnosis of glutamic acid decarboxylase antibody related limbic encephalitis. Monthly steroid pulses over 6 months led to recovery of subjective memory and to intermediate improvement but subsequent worsening of objective memory performance. During the course of treatment, the patient reported de novo paroxysmal non-responsive states. Thus, antiepileptic treatment was started and the patient finally became seizure free. At the last visit, vocational reintegration was successfully in progress. In conclusion, amygdala swelling, retrograde biographic memory impairment, accelerated long-term forgetting, and emotional instability may serve as indicators of limbic encephalitis, even in the absence of overt epileptic seizures. The monitoring of such patients calls for a standardized and concerted multilevel diagnostic approach with repeated assessments

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. Study of effect of substitution of the penultimate amino acid residue on expression, structure, and functional properties of Yersinia pseudotuberculosis OmpY porin.

    PubMed

    Solov'eva, T F; Tischenko, N M; Khomenko, V A; Portnyagina, O Y; Kim, N Y; Likhatskaya, G N; Novikova, O D; Isaeva, M P

    2014-07-01

    The purpose of the study was to compare the expression of two Yersinia pseudotuberculosis proteins, wild-type porin OmpY and the mutant porin OmpY designated as OmpY-Q having the uncharged amino acid residue Gln instead of positively charged Arg at the penultimate position in the same heterologous host. According to the literature, a similar substitution (Lys to Gln) of the penultimate amino acid residue in Neisseria meningitidis porin PorA drastically improved the assembly of the protein in the E. coli outer membrane in vivo. Site-directed mutagenesis was used to replace Arg by Gln (R338Q) in OmpY, and the conditions for optimal expression and maturation of OmpY-Q were selected. It was found that the growth rates of E. coli strains producing OmpY and OmpY-Q and the expression levels of the porins were approximately equal. Comparative analysis of recombinant OmpY and OmpY-Q did not show significant differences in structure, antigenic, and functional properties of the porins, or any noticeable effect of the R338Q substitution in OmpY on its assembly in the E. coli outer membrane in vivo. The probable causes of discrepancies between our results and the previous data on porin PorA are discussed considering the known mechanisms of biogenesis of porins at the periplasmic stage.

  8. 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

  9. Urea, Glycolic Acid, and Glycerol in an Organic Residue Produced by Ultraviolet Irradiation of Interstellar/Pre-Cometary Ice Analogs

    NASA Astrophysics Data System (ADS)

    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 CH3OH:NH3â = 1:1 ice mixture was UV irradiated at ˜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.

  10. 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.

  11. 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.

  12. 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

  13. Complete replacement of basic amino acid residues with cysteines in Rickettsia prowazekii ATP/ADP translocase.

    PubMed

    Alexeyev, Mikhail F; Winkler, Herbert H

    2002-09-20

    The ATP/ADP translocase (Tlc) of Rickettsia prowazekii is a basic protein with isoelectric point (pI)=9.84. It is conceivable, therefore, that basic residues in this protein are involved in electrostatic interactions with negatively charged substrates. We tested this hypothesis by individually mutating all basic residues in Tlc to Cys. Unexpectedly, mutations of only 20 out of 51 basic residues resulted in greater than 80% inhibition of transport activity. Moreover, 12 of 51Cys-substitution mutants exhibited higher than wild-type (WT) activity. At least in one case this up-effect was additive and the double mutant Lys422Cys Lys427Cys transported ATP five-fold better than WT protein. Since in these two single mutants and in the corresponding double mutant K(m)'s were similar to that of WT protein, we conclude that Tlc may have evolved a mechanism that limits the transporter's exchange rate and that at least these two basic residues play a key role in that mechanism. Based on the alignment of 16 Tlc homologs, the loss of activity in the mutants poorly correlates with charge conservation within the Tlc family. Also, despite the presence of three positively charged and one negatively charged intramembrane residues, we have failed to identify potential charge pairs (salt bridges) by either charge reversal or charge neutralization approaches. PMID:12225862

  14. 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.

  15. 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

  16. 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

  17. 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

  18. A facile route to preparation of high purity nanoporous silica from acid-leached residue of serpentine.

    PubMed

    Bai, Penn; Sharratt, Paul; Yeo, Tze Yuen; Bu, Jie

    2014-09-01

    As the current cost of mineral carbonation is too high for an economically viable industrial process, it is desirable to produce value-added products from CO2 mineralization process. In this work, a facile and cost-effective process was developed for the production of high purity SiO2 from acid-leached serpentine residue. The Si extraction rate is fast even under ambient conditions due to the highly defective structure of the residue. The reaction kinetics were studied and it was found that the Si extraction rate was under a combination of chemical reaction control and film diffusion control. The SiO2 sample prepared has high purity with a nanoporous structure, which renders it a potential candidate for applications such as an adsorbent and a catalyst support.

  19. 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.

  20. Activated protein C cofactor function of protein S: a novel role for a γ-carboxyglutamic acid residue.

    PubMed

    Ahnström, Josefin; Andersson, Helena M; Canis, Kevin; Norstrøm, Eva; Yu, Yao; Dahlbäck, Björn; Panico, Maria; Morris, Howard R; Crawley, James T B; Lane, David A

    2011-06-16

    Protein S has an important anticoagulant function by acting as a cofactor for activated protein C (APC). We recently reported that the EGF1 domain residue Asp95 is critical for APC cofactor function. In the present study, we examined whether additional interaction sites within the Gla domain of protein S might contribute to its APC cofactor function. We examined 4 residues, composing the previously reported "Face1" (N33S/P35T/E36A/Y39V) variant, as single point substitutions. Of these protein S variants, protein S E36A was found to be almost completely inactive using calibrated automated thrombography. In factor Va inactivation assays, protein S E36A had 89% reduced cofactor activity compared with wild-type protein S and was almost completely inactive in factor VIIIa inactivation; phospholipid binding was, however, normal. Glu36 lies outside the ω-loop that mediates Ca(2+)-dependent phospholipid binding. Using mass spectrometry, it was nevertheless confirmed that Glu36 is γ-carboxylated. Our finding that Gla36 is important for APC cofactor function, but not for phospholipid binding, defines a novel function (other than Ca(2+) coordination/phospholipid binding) for a Gla residue in vitamin K-dependent proteins. It also suggests that residues within the Gla and EGF1 domains of protein S act cooperatively for its APC cofactor function.

  1. Amino acid sequence of the 203-residue fragment of the heavy chain of chicken gizzard myosin containing the SH1-type cysteine residue.

    PubMed

    Onishi, H; Maita, T; Miyanishi, T; Watanabe, S; Matsuda, G

    1986-12-01

    A fluorescent fragment of Mr = 23,800 was obtained by the papain digestion of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylene diamine (abbreviated as IAEDANS)-modified chicken gizzard myosin. The fragment was isolated by gel filtration on a Sephadex G-100 column in the presence of 5 M guanidine-HCl followed by anion exchange chromatography on a QAE Sephadex A-50 column. This fragment contained 203 amino acid residues which could be assigned as a COOH-terminal part of the S-1 heavy chain based on the homology with the known sequence of rabbit skeletal myosin fragment. The amino acid sequence was K-G-M-F-R-T-V- G-Q-L-Y-K-E-Q-L-T-K-L-M-T-T-L-R-N-T-N-P-N-F-V-R-C-I-I-P-N-H-E-K-R-A- G-K-L-D-A-H-L-V-L-E-Q-L-R-C-N-G-V-L-E-G-I-R-I-C-R-Q-G-F-P-N-R-I-V-F-Q- E-F-R-Q-R-Y-E-I-L-A-A-N-A-I-P-K-G-F-M-D-G-K-Q-A-C-I-L-M -I-K-A-L-E-L- D-P-N-L-Y-R-I-G-Q-S-K-I-F-F-R-T-G-V-L-A-H-L-E-E-E-R-D-L-K- I-T-D-V-I-I-A- F-Q-A-Q-C-R-G-Y-L-A-R-K-A-F-A-K-R-Q-Q-Q-L-T-A-M-K-V-I-Q-R-N-C-A -A-Y-L-K-L-R-N-W-Q-W-W-R-L-F-T-K-V-K-P-L-L-Q-V-T-R. The cysteine residue which was modified with IAEDANS was of the SH1 type (Cys-65). Pro-197 was suggested to be the NH2-terminal boundary of the alpha-helical coiled-coil rod sequence of gizzard myosin, based on the homology with the nematode sequence reported by MacLachlan and Karn (Proc. Natl. Acad. Sci. U.S. 80, 4253-4257 (1983)). Three different COOH-terminal peptides (Val-Lys-Pro-Leu-Leu-Gln-Val-Thr-Arg, Val-Lys-Pro-Leu-Leu-Gln, and Val-Lys-Pro-Leu-Leu) were isolated from the tryptic digest of this fragment.(ABSTRACT TRUNCATED AT 400 WORDS)

  2. 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

  3. ASCORBIC ACID REDUCTION ON RESIDUAL ACTIVE CHLORINE IN POTABLE WATER PRIOR TO HALOCARBOXYLATE DETERMINATION

    EPA Science Inventory

    In studies on the formation of disinfection byproducts (DBPs), it is necessary to scavenge residual active (odxidizing) chlorine in order to fix the chlorination byproducts (such as haloethanoates) at a point in time . Such research projects often have distinct needs from requi...

  4. ASCORBIC ACID REDUCTION OF RESIDUAL ACTIVE CHLORINE IN POTABLE WATER PRIOR TO HALOCARBOXYLATE DETERMINATION

    EPA Science Inventory

    In studies on the formation of disinfection byproducts (DBPs), it is necessary to scavenge residual active (oxidizing) chlorine in order to fix the chlorination byproducts (such as haloethanoates) at a point in time. Thus, methods designed for compliance monitoring are not alway...

  5. 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.

  6. Function of aspartic acid residues in optimum pH control of L-arabinose isomerase from Lactobacillus fermentum.

    PubMed

    Xu, Zheng; Li, Sha; Feng, Xiaohai; Zhan, Yijing; Xu, Hong

    2014-05-01

    L-Arabinose isomerase (L-AI) catalyzes the isomerization of L-arabinose to L-ribulose and D-galactose to D-tagatose. Most reported L-AIs exhibit neutral or alkaline optimum pH, which is less beneficial than acidophilic ones in industrial D-tagatose production. Lactobacillus fermentum L-AI (LFAI) is a thermostable enzyme that can achieve a high conversion rate for D-galactose isomerization. However, its biocatalytic activity at acidic conditions can still be further improved. In this study, we report the single- and multiple-site mutagenesis on LFAI targeting three aspartic acid residues (D268, D269, and D299). Some of the lysine mutants, especially D268K/D269K/D299K, exhibited significant optimum pH shifts (from 6.5 to 5.0) and enhancement of pH stability (half-life time increased from 30 to 62 h at pH 6.0), which are more favorable for industrial applications. With the addition of borate, D-galactose was isomerized into D-tagatose by D268K/D269K/D299K at pH 5.0, resulting in a high conversion rate of 62 %. Based on the obtained 3.2-Å crystal structure of LFAI, the three aspartic acid residues were found to be distant from the active site and possibly did not participate in substrate catalysis. However, they were proven to possess similar optimum pH control ability in other L-AI, such as that derived from Escherichia coli. This study sheds light on the essential residues of L-AIs that can be modified for desired optimum pH and better pH stability, which are useful in D-tagatose bioproduction.

  7. KV4.3 expression and gating: S2 and S3 acidic and S4 innermost basic residues.

    PubMed

    Skerritt, Matthew R; Campbell, Donald L

    2009-11-01

    Effects of neutralizing individual negatively charged (acidic [E,D]) and innermost positively charged (basic [K,R]) residues in transmembrane segments S2 (D230Q, E240Q), S3 (D263Q) and S4 (K299A/Q, R302A/Q) of the K(V)4.3 putative voltage sensing domain (VSD) were determined. S2 D230Q generated large macroscopic currents, depolarized steady-state activation ("a(4)") and isochronal (1 sec) inactivation ("i") relationships, and significantly accelerated kinetics of deactivation and recovery (from both macroscopic and closed state inactivation [CSI]). D230Q thus stabilized non-inactivated closed states. These effects were attributable to structural perturbations, and indicated D230 is not primarily involved in voltage sensing. Both S2 E240Q and S3 D263Q failed to generate measurable ionic currents, suggesting deletion of negative charges at these putatively more intracellular acidic positions were functionally "lethal" to macroscopic K(V)4.3 function. S4 innermost positive charge deletion mutants K299A/Q and R032A/Q generated functional currents with reduced peak amplitudes. While reduced K299A/Q and R302A/Q currents prevented accurate determination of "a(4)" and estimates of potential electrostatic perturbations, both sets of mutants: (i) depolarized potentials at which currents could be macroscopically detected, consistent with stabilization of closed states (structural perturbations); and (ii) accelerated macroscopic recovery. These results provide further evidence that: (i) basic residues in S4 are involved not only in regulating K(V)4.3 activation and deactivation, but also CSI and recovery; and (ii) suggest putative electrostatic interactions between acidic S2/S3 and basic S4 residues may be different in K(V)4.3 from those proposed to exist in Shaker. Functional implications are discussed.

  8. Determination of organic acids in urine by solid-phase microextraction and gas chromatography-ion trap tandem mass spectrometry previous 'in sample' derivatization with trimethyloxonium tetrafluoroborate.

    PubMed

    Pacenti, Marco; Dugheri, Stefano; Villanelli, Fabio; Bartolucci, Gianluca; Calamai, Luca; Boccalon, Pierpaolo; Arcangeli, Giulio; Vecchione, Febe; Alessi, Paolo; Kikic, Ireneo; Cupelli, Vincenzo

    2008-10-01

    A method for the determination of the organic acids directly in the urine employing derivatization with trimethyloxonium tetrafluoroborate as a methylating agent and sequential extraction by head space and direct immersion/solid phase microextraction is reported. Furoic acid, hippuric acid, methylhippuric acid, mandelic acid, phenylglyoxylic acid and trans, trans muconic acid contained in urine and proposed by the American Conference of Governmental Industrial Hygienists as biological exposure indices were determined after a fast and economically convenient preparation step and sensitive gas chromatography-ion trap-mass spectrometry/tandem mass spectrometry analysis. Urine is rather a complex sample and hence the acquisition method required specific GC-MS instrumentation capable of supporting the changeover, fully automated during a single chromatographic separation, from mass to tandem mass spectrometry and both chemical and electron ionization modes. The automation of the analytical method provides a number of advantages, including reduced analysis time for both routine analysis and method development, and greater reproducibility. The equilibrium and kinetics of this substances vs head space/direct immersion-solid phase microextraction were investigated and evaluated theoretically.

  9. 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.

  10. Membrane topology and essential amino acid residues of Phs1, a 3-hydroxyacyl-CoA dehydratase involved in very long-chain fatty acid elongation.

    PubMed

    Kihara, Akio; Sakuraba, Hiroko; Ikeda, Mika; Denpoh, Aki; Igarashi, Yasuyuki

    2008-04-25

    Yeast Phs1 is the 3-hydroxyacyl-CoA dehydratase that catalyzes the third reaction of the four-step cycle in the elongation of very long-chain fatty acids (VLCFAs). In yeast, the hydrophobic backbone of sphingolipids, ceramide, consists of a long-chain base and an amide-linked C26 VLCFA. Therefore, defects in VLCFA synthesis would be expected to greatly affect sphingolipid synthesis. In fact, in this study we found that reduced Phs1 levels result in significant impairment of the conversion of ceramide to inositol phosphorylceramide. Phs1 proteins are conserved among eukaryotes, constituting a novel protein family. Phs1 family members exhibit no sequence similarity to other dehydratase families, so their active site sequence and catalytic mechanism have been completely unknown. Here, by mutating 22 residues conserved among Phs1 family members, we identified six amino acid residues important in Phs1 function, two of which (Tyr-149 and Glu-156) are indispensable. We also examined the membrane topology of Phs1 using an N-glycosylation reporter assay. Our results suggest that Phs1 is a membrane-spanning protein that traverses the membrane six times and has an N terminus and C terminus facing the cytosol. The important amino acids are concentrated in or near two of the six proposed transmembrane regions. Thus, we also propose a catalytic mechanism for Phs1 that is not unlike mechanisms used by other hydratases active in lipid synthesis.

  11. 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...

  12. 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...

  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. Delivery of a foreign epitope by sharing amino acid residues with the carrier matrix.

    PubMed

    Cheong, Wan-Shoo; Drummer, Heidi Edelgard; Netter, Hans-Jürgen

    2009-06-01

    A broad range of structural viral proteins has the ability to assemble into virus-like particles (VLPs). Under the condition that modified subunits are still competent to assemble into VLPs, they are epitope delivery platforms suitable for vaccination purposes. The insertion of foreign sequences can be detrimental for the formation of chimeric VLPs as a result of misfolded subunit proteins. Hence, a strategy was adopted to screen for locations allowing the use of shared residues between the wildtype subunit sequence and the foreign insert. The insertion of a cysteine-containing sequence of hepatitis C virus (HCV) envelope protein 2 (E2) without adding an additional cysteine residue retained the ability of recombinant small hepatitis B surface antigen (HBsAg-S) to form secretion competent VLPs. A cysteine residue shared by the insert and the template protein avoided the formation of non-native disulfide bonds, and allowed the formation of VLPs. The chimeric HBsAg-S VLPs were similar to wildtype VLPs in density exposing the inserted foreign epitope and being immunogenic. Overall, the use of shared sequences between the insert and the subunit will facilitate the design of chimeric VLPs carrying multiple epitopes.

  17. 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.

  18. 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.

  19. 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.

  20. Mutational analysis of amino acid residues involved in catalytic activity of a family 18 chitinase from tulip bulbs.

    PubMed

    Suzukawa, Keisuke; Yamagami, Takeshi; Ohnuma, Takayuki; Hirakawa, Hideki; Kuhara, Satoru; Aso, Yoichi; Ishiguro, Masatsune

    2003-02-01

    We expressed chitinase-1 (TBC-1) from tulip bulbs (Tulipa bakeri) in E. coli cells and used site-directed mutagenesis to identify amino acid residues essential for catalytic activity. Mutations at Glu-125 and Trp-251 completely abolished enzyme activity, and activity decreased with mutations at Asp-123 and Trp-172 when glycolchitin was the substrate. Activity changed with the mutations of Trp-251 to one of several amino acids with side-chains of little hydrophobicity, suggesting that hydrophobic interaction of Trp-251 is important for the activity. Molecular dynamics (MD) simulation analysis with hevamine as the model compound showed that the distance between Asp-123 and Glu-125 was extended by mutation of Trp-251. Kinetic studies of Trp-251-mutated chitinases confirmed these various phenomena. The results suggested that Glu-125 and Trp-251 are essential for enzyme activity and that Trp-251 had a direct role in ligand binding.

  1. 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

  2. Effects of amino acid sequence, buffers, and ionic strength on the rate and mechanism of deamidation of asparagine residues in small peptides.

    PubMed

    Tyler-Cross, R; Schirch, V

    1991-11-25

    The nonenzymatic rates of deamidation of Asn residues in a series of pentapeptides with the sequences VSNXV and VXNSV, where X is one of 10 different amino acids, were determined at neutral, alkaline, and acid pH values. The results demonstrate that in neutral and alkaline solutions the amino acid residue on the amino side of the Asn had little or no effect on the rate of deamidation regardless of its charge or size. The group on the carboxyl side of Asn affected the rate of deamidation significantly. Increasing size and branching in the side chain of this residue decreased the rate of deamidation by as much as 70-fold compared to glycine in the N-G sequence, which had the greatest rate of deamidation. In acidic solution, the rate of deamidation of the Asn residue was not affected by the amino acid sequence of the peptide. The products for each deamidation reaction were tested for the formation of isoAsp residues. In neutral and alkaline solutions, all products showed that the isoAsp:Asp peptide products were formed in about a 3:1 ratio. In acidic solution, the Asp peptide was the only deamidation product formed. All peptides in which a Ser residue follows the Asn residue were found to undergo a peptide cleavage reaction in neutral and alkaline solutions, yielding a tripeptide and a dipeptide. The rate of the cleavage reaction was about 10% of the rate of the deamidation pathway at neutral and alkaline pH values. The rates of deamidation of Asn residues in the peptides studied were not affected by ionic strength, and were not specific base catalyzed. General base catalysis was observed for small bases like ammonia. A model for the deamidation reaction is proposed to account for the observed effects. PMID:1939272

  3. Identification of functional amino acid residues involved in polyamine and agmatine transport by human organic cation transporter 2.

    PubMed

    Higashi, Kyohei; Imamura, Masataka; Fudo, Satoshi; Uemura, Takeshi; Saiki, Ryotaro; Hoshino, Tyuji; Toida, Toshihiko; Kashiwagi, Keiko; Igarashi, Kazuei

    2014-01-01

    Polyamine (putrescine, spermidine and spermine) and agmatine uptake by the human organic cation transporter 2 (hOCT2) was studied using HEK293 cells transfected with pCMV6-XL4/hOCT2. The Km values for putrescine and spermidine were 7.50 and 6.76 mM, and the Vmax values were 4.71 and 2.34 nmol/min/mg protein, respectively. Spermine uptake by hOCT2 was not observed at pH 7.4, although it inhibited both putrescine and spermidine uptake. Agmatine was also taken up by hOCT2, with Km value: 3.27 mM and a Vmax value of 3.14 nmol/min/mg protein. Amino acid residues involved in putrescine, agmatine and spermidine uptake by hOCT2 were Asp427, Glu448, Glu456, Asp475, and Glu516. In addition, Glu524 and Glu530 were involved in putrescine and spermidine uptake activity, and Glu528 and Glu540 were weakly involved in putrescine uptake activity. Furthermore, Asp551 was also involved in the recognition of spermidine. These results indicate that the recognition sites for putrescine, agmatine and spermidine on hOCT2 strongly overlap, consistent with the observation that the three amines are transported with similar affinity and velocity. A model of spermidine binding to hOCT2 was constructed based on the functional amino acid residues.

  4. Residual Host Cell Protein Promotes Polysorbate 20 Degradation in a Sulfatase Drug Product Leading to Free Fatty Acid Particles.

    PubMed

    Dixit, Nitin; Salamat-Miller, Nazila; Salinas, Paul A; Taylor, Katherine D; Basu, Sujit K

    2016-05-01

    This study investigated the root cause behind an observed free fatty acid particle formation and resulting Polysorbate 20 (PS20) loss for a sulfatase drug product upon long-term storage at 5 ± 3°C. Reversed- phase chromatography with mass spectrometric analysis as well as charged aerosol detection was used to characterize the peaks associated with the intact and degraded PS20. Additionally, a proteomics study was undertaken to identify the residual host cell proteins in the sulfatase drug substance. PS20 stability studies were conducted in the presence of sulfatase, a sulfatase inhibitor, putative phospholipase B-like 2, and mock drug substance produced using a null cell line vector under experimental conditions optimized for PS20 degradation. This study provides the first published evidence where the residual host cell protein present in the drug substance was identified and experimentally shown to catalyze the breakdown of PS20 in a protein formulation over time, resulting in free fatty acid particles and PS20 loss. This study demonstrates the importance of early detection of potential impurities in the protein drug substance that may contribute to polysorbate degradation to make a judicious selection of the surfactant and its optimized concentration for the final drug product. PMID:27032893

  5. 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.

  6. Energy compensation mechanism for charge-separated protonation states in aspartate-histidine amino acid residue pairs.

    PubMed

    Kamiya, Katsumasa; Boero, Mauro; Shiraishi, Kenji; Oshiyama, Atsushi; Shigeta, Yasuteru

    2010-05-20

    The initial stage of proton propagation in the D-path channel of bovine cytochrome c oxidase, consisting of the approach of an H(+) to the entrance of this specific pathway, is inspected via first-principles calculations. Our model, extracted from the X-ray crystallographic structure, includes the amino acid residue pair aspartate (Asp91) and histidine (His503) as protonatable sites. Our calculations show that an additional proton, corresponding to the H(+) uptake by the enzyme from the inner bulk water, is transferred to either Asp91 or His503, leading to the formation of a neutral or a charge-separated protonation state. The relative stability between the two states amounts to a total energy difference of about 5 kcal/mol; this indicates that both Asp91 and His503 are involved in the proton supply to the D-path, playing the role of proton acceptors. The hydrogen-bond environment around Asp91 and His503 has an important influence on both the energetics and the electronic structure of the system; for instance, it compensates the Coulomb-energy cost in the charge-separated protonation state. An energy partitioning analysis shows that the compensatory effect is mainly due to local electrostatic interactions among the charged Asp91 and His503 side chains and the surrounding polar residues. The energy compensation mechanism we found in this work balances the energetics of Asp-His pairs, hence permitting an efficient and selective regulation of the protonatable amino acid residues, where several protonation states are accessible within energy differences of the order of a few H-bonds. PMID:20411975

  7. Amino acid residues in the laminin G domains of protein S involved in tissue factor pathway inhibitor interaction.

    PubMed

    Somajo, Sofia; Ahnström, Josefin; Fernandez-Recio, Juan; Gierula, Magdalena; Villoutreix, Bruno O; Dahlbäck, Björn

    2015-05-01

    Protein S functions as a cofactor for tissue factor pathway inhibitor (TFPI) and activated protein C (APC). The sex hormone binding globulin (SHBG)-like region of protein S, consisting of two laminin G-like domains (LG1 and LG2), contains the binding site for C4b-binding protein (C4BP) and TFPI. Furthermore, the LG-domains are essential for the TFPI-cofactor function and for expression of full APC-cofactor function. The aim of the current study was to localise functionally important interaction sites in the protein S LG-domains using amino acid substitutions. Four protein S variants were created in which clusters of surface-exposed amino acid residues within the LG-domains were substituted. All variants bound normally to C4BP and were fully functional as cofactors for APC in plasma and in pure component assays. Two variants, SHBG2 (E612A, I614A, F265A, V393A, H453A), involving residues from both LG-domains, and SHBG3 (K317A, I330A, V336A, D365A) where residues in LG1 were substituted, showed 50-60 % reduction in enhancement of TFPI in FXa inhibition assays. For SHBG3 the decreased TFPI cofactor function was confirmed in plasma based thrombin generation assays. Both SHBG variants bound to TFPI with decreased affinity in surface plasmon resonance experiments. The TFPI Kunitz 3 domain is known to contain the interaction site for protein S. Using in silico analysis and protein docking exercises, preliminary models of the protein S SHBG/TFPI Kunitz domain 3 complex were created. Based on a combination of experimental and in silico data we propose a binding site for TFPI on protein S, involving both LG-domains.

  8. Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in vitro.

    PubMed Central

    Engelman, A; Craigie, R

    1992-01-01

    We have probed the structural organization of the human immunodeficiency virus type 1 integrase protein by limited proteolysis and the functional organization by site-directed mutagenesis of selected amino acid residues. A central region of the protein was relatively resistant to proteolysis. Proteins with altered amino acids in this region, or in the N-terminal part of the protein that includes a putative zinc-binding motif, were purified and assayed for 3' processing, DNA strand transfer, and disintegration activities in vitro. In general, these mutations had parallel effects on 3' processing and DNA strand transfer, suggesting that integrase may utilize a single active site for both reactions. The only proteins that were completely inactive in all three assays contained mutations at conserved amino acids in the central region, suggesting that this part of the protein may be involved in catalysis. In contrast, none of the mutations in the N-terminal region resulted in a protein that was inactive in all three assays, suggesting that this part of integrase may not be essential for catalysis. The disintegration reaction was particularly insensitive to these amino acid substitutions, indicating that some function that is important for 3' processing and DNA strand transfer may be dispensable for disintegration. Images PMID:1404595

  9. Residues in the extracellular loop 4 are critical for maintaining the conformational equilibrium of the gamma-aminobutyric acid transporter-1.

    PubMed

    MacAulay, Nanna; Meinild, Anne-Kristine; Zeuthen, Thomas; Gether, Ulrik

    2003-08-01

    We mutated residues Met345 and Thr349 in the rat gamma-aminobutyric acid transporter-1 (GAT-1) to histidines (M345H and T349H). These two residues are located four amino acids apart at the extracellular end of transmembrane segment 7 in a region of GAT-1 that we have previously suggested undergoes conformational changes critical for the transport process. The two single mutants and the double mutant (M345H/T349H) were expressed in Xenopus laevis oocytes, and their steady-state and presteady-state kinetics were examined and compared with wild type GAT-1 by using the two-electrode voltage clamp method. Oocytes expressing M345H showed a decrease in apparent GABA affinity, an increase in apparent affinity for Na+, a shift in the charge/voltage (Q/Vm) relationship to more positive membrane potentials, and an increased Li+-induced leak current. Oocytes expressing T349H showed an increase in apparent GABA affinity, a decrease in apparent Na+ affinity, a profound shift in the Q/Vm relationship to more negative potentials, and a decreased Li+-induced leak current. The data are consistent with a shift in the conformational equilibrium of the mutant transporters, with M345H stabilized in an outward-facing conformation and T349H in an inward-facing conformation. These data suggest that the extracellular end of transmembrane domain 7 not only undergoes conformational changes critical for the translocation process but also plays a role in regulating the conformational equilibrium between inward- and outward-facing conformations.

  10. 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.

  11. 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

  12. Effects of previous diet and duration of soybean oil supplementation on light lambs carcass composition, meat quality and fatty acid composition.

    PubMed

    Bessa, R J B; Lourenço, M; Portugal, P V; Santos-Silva, J

    2008-12-01

    Forty Merino Branco ram lambs were used to study the effects of initial diet and duration of supplementation with a conjugated linoleic acid (CLA) promoting diet, on carcass composition, meat quality and fatty acid composition of intramuscular fat. The experimental period was 6 weeks. The experimental design involved 2 initial diets (commercial concentrate (C); dehydrated lucerne (L)), and 2 finishing periods (2 and 4 weeks) on dehydrated lucerne plus 10% soybean oil (O). Data were analysed as a 2×2 factorial arrangement with initial diet and time on finishing (CLA promoting) diet as the main factors. The lambs were randomly assigned to four groups: CCO; COO; LLO; LOO according to the lamb's diet fed in each period. Lambs initially fed with concentrate showed higher hot carcass weights (11.2 vs 9.6kg) than lambs fed initially with lucerne. The increase of the duration of finishing period reduced the carcass muscle percentage (57.4% vs 55.5%) and increased the subcutaneous fat percentage (5.67% vs 7.03%). Meat colour was affected by initial diet. Lambs initially fed with concentrate showed a lower proportion of CLA (18:2cis-9, trans-11 isomer) (0.98% vs 1.38% of total fatty acids) and most of n-3 polyunsaturated fatty acids than lambs initially fed with lucerne. Initial diet did not compromise the response to the CLA-promoting diet and the proportion of 18:2cis-9, trans-11 in intramuscular fat increased with the duration of time on the CLA-promoting diet (1.02% vs 1.34% of total fatty acids).

  13. 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.

  14. 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

  15. 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

  16. 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

  17. Self-assembling properties of all γ-cyclic peptides containing sugar amino acid residues.

    PubMed

    Guerra, Arcadio; Brea, Roberto J; Amorín, Manuel; Castedo, Luis; Granja, Juan R

    2012-11-28

    In this study, a novel dimer-forming cyclic peptide composed exclusively by cyclic γ-amino acids with a saccharide-like outer surface is described. The antiparallel β-sheet type hydrogen bonding interactions responsible for the large association constant in non-polar solvents constitute a suitable model for a novel class of self-assembling peptide nanotubes.

  18. 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

  19. Self-assembling properties of all γ-cyclic peptides containing sugar amino acid residues.

    PubMed

    Guerra, Arcadio; Brea, Roberto J; Amorín, Manuel; Castedo, Luis; Granja, Juan R

    2012-11-28

    In this study, a novel dimer-forming cyclic peptide composed exclusively by cyclic γ-amino acids with a saccharide-like outer surface is described. The antiparallel β-sheet type hydrogen bonding interactions responsible for the large association constant in non-polar solvents constitute a suitable model for a novel class of self-assembling peptide nanotubes. PMID:23060041

  20. 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...

  1. 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...

  2. Identification of glutamic acid 204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity.

    PubMed

    Watanabe, T; Kobori, K; Miyashita, K; Fujii, T; Sakai, H; Uchida, M; Tanaka, H

    1993-09-01

    Prokaryotic chitinases, class III plant chitinases, yeast chitinases, and endo-beta-N-acetylglucosaminidases share weak amino acid sequence similarities at the certain region of each enzyme. These regions have been assumed to be important for catalytic activities of the enzymes. To verify this assumption, three amino acid residues (Ser-160, Asp-200, Glu-204) in chitinase A1 of Bacillus circulans WL-12 were chosen, based on the amino acid sequence alignment of the regions sharing sequence similarity, and were replaced by site-directed mutagenesis. Kinetic parameters for 4-methylumbelliferyl-N,N',N"-triacetylchitotriose hydrolysis were determined with wild-type and seven mutant chitinases. Chitinases with Glu-204-->Gln mutation and Glu-204-->Asp mutation were essentially inactive and kcat values of these chitinases were approximately 1/5,000 and 1/17,000 of that of wild-type chitinase, respectively. Asp-200-->Asn mutation decreased the kcat value to approximately 1/350 of that of the wild-type enzyme, while the Km value decreased only slightly. On the other hand, neither the kcat value nor the Km value was affected by Asp-200-->Glu mutation. Thus, it appeared that Glu-204 and Asp-200 are directly involved in the catalytic events of chitinase A1. The role of the carboxyl group of Asp-200 can be fully substituted by that of Glu residue. The Ser-160-->Ala mutant retained 10% activity of the wild-type chitinase indicating that the hydroxyl group of Ser-160 is not absolutely required for the catalytic activity. These results indicate a lysozyme-type catalytic mechanism of the chitinase.

  3. 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.

  4. Leaf Abscission Induced by Ethylene in Water-Stressed Intact Seedlings of Cleopatra Mandarin Requires Previous Abscisic Acid Accumulation in Roots.

    PubMed

    Gomez-Cadenas, A.; Tadeo, F. R.; Talon, M.; Primo-Millo, E.

    1996-09-01

    The involvement of abscisic acid (ABA) in the process of leaf abscission induced by 1-aminocyclopropane-1-carboxylic acid (ACC) transported from roots to shoots in Cleopatra mandarin (Citrus reshni Hort. ex Tan.) seedlings grown under water stress was studied using norflurazon (NF). Water stress induced both ABA (24-fold) and ACC (16-fold) accumulation in roots and arrested xylem flow. Leaf bulk ABA also increased (8-fold), although leaf abscission did not occur. Shortly after rehydration, root ABA and ACC returned to their prestress levels, whereas sharp and transitory increases of ACC (17-fold) and ethylene (10-fold) in leaves and high percentages of abscission (up to 47%) were observed. NF suppressed the ABA and ACC accumulation induced by water stress in roots and the sharp increases of ACC and ethylene observed after rewatering in leaves. NF also reduced leaf abscission (7-10%). These results indicate that water stress induces root ABA accumulation and that this is required for the process of leaf abscission to occur. It was also shown that exogenous ABA increases ACC levels in roots but not in leaves. Collectively, the data suggest that ABA, the primary sensitive signal to water stress, modulates the levels of ethylene, which is the hormonal activator of leaf abscission. This assumption implies that root ACC levels are correlated with root ABA amounts in a dependent way, which eventually links water status to an adequate, protective response such as leaf abscission.

  5. Reaction of unsaturated uronic acid residues with mercuric salts. Cleavage of the hyaluronic acid disaccharide 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose.

    PubMed Central

    Ludwigs, U; Elgavish, A; Esko, J D; Meezan, E; Rodén, L

    1987-01-01

    Degradation of connective-tissue polysaccharides with bacterial or fungal eliminases and subsequent characterization of the reaction products are now part of standard methodology for the analysis of these compounds. However, the scope of preparative and analytical work based on the use of eliminases has been limited by the lack of procedures for specific removal of the unsaturated uronic acid residues generated in the eliminase reactions. In the present investigation, we have shown that these residues are cleaved by mercuric salts under mild conditions that are not likely to affect other structures in an oligo- or poly-saccharide molecule. Thus the disaccharide generated from hyaluronic acid by digestion with chondroitinase AC or ABC was cleaved into a keto acid and free N-acetylglucosamine within 10 min at room temperature upon exposure to 14 mM-mercuric acetate at pH 5. The reaction of the disaccharide with mercuric salts was used for ready determination of the distribution of radioactivity between the glucuronic acid and N-acetylglucosamine moieties in radioactive hyaluronic acid that had been synthesized by IMR-90 fibroblasts from 3H-labelled monosaccharides. When the precursor was [3H]galactose, over 95% of the incorporated radioactivity was found in the glucuronic acid moiety. In contrast, cells grown in the presence of [3H]glucosamine synthesized a polysaccharide in which almost all of the label was located in the N-acetylglucosamine units. It is apparent from these experiments that the reaction of unsaturated uronic acid residues with mercuric salts provides a new tool with potential for many applications in the study of the structure and metabolism of connective-tissue polysaccharides. PMID:3663191

  6. 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

  7. [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.

  8. Amino acid residues 4425-4621 localized on the three-dimensional structure of the skeletal muscle ryanodine receptor.

    PubMed

    Benacquista, B L; Sharma, M R; Samsó, M; Zorzato, F; Treves, S; Wagenknecht, T

    2000-03-01

    We have localized a region contained within the sequence of amino acid residues 4425-4621 on the three-dimensional structure of the skeletal muscle ryanodine receptor (RyR). Mouse monoclonal antibodies raised against a peptide comprising these residues have been complexed with ryanodine receptors and imaged in the frozen-hydrated state by cryoelectron microscopy. These images, along with images of antibody-free ryanodine receptor, were used to compute two-dimensional averaged images and three-dimensional reconstructions. Two-dimensional averages of immunocomplexes in which the ryanodine receptor was in the fourfold symmetrical orientation disclosed four symmetrical regions of density located on the edges of the receptor's cytoplasmic assembly that were absent from control averages of receptor without added antibody. Three-dimensional reconstructions revealed the antibody-binding sites to be on the so-called handle domains of the ryanodine receptor's cytoplasmic assembly, near their junction with the transmembrane assembly. This study is the first to demonstrate epitope mapping on the three-dimensional structure of the ryanodine receptor.

  9. 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.

  10. Confronting the residual cardiovascular risk beyond statins: the role of fibrates, omega-3 fatty acids, or niacin, in diabetic patients.

    PubMed

    Christou, Georgios A; Rizos, Evangelos C; Mpechlioulis, Aris; Penzo, Carlo; Pacchioni, Andrea; Nikas, Dimitrios N

    2014-01-01

    Diabetics are regarded a special category of patients known to experience higher rates of cardiovascular complications as compared to the non-diabetic ones. Despite substantial efforts to minimize these risks, with aggressive antiplatelet and lipid lowering therapy, some of the diabetic patients still have a considerable residual risk for cardiovascular adverse events. Important preclinical data with potent lipid-lowering agents, like fibrates, omega-3-fatty acids, and niacin, have shown that they can provide sufficient help in reducing rates of cardiovascular events. In the present review, we are aim to explain their basic mechanisms of action, to present all the available clinical data regarding the efficacy of those agents, and to identify specific diabetic patients' subsets, in whom supplementary therapy with those agents could provide substantial benefit in terms of clinical outcome and not only lipid profile improvement.

  11. 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

  12. Aldehyde dehydrogenase enzyme ALDH3H1 from Arabidopsis thaliana: Identification of amino acid residues critical for cofactor specificity.

    PubMed

    Stiti, Naim; Podgórska, Karolina; Bartels, Dorothea

    2014-03-01

    The cofactor-binding site of the NAD(+)-dependent Arabidopsis thaliana aldehyde dehydrogenase ALDH3H1 was analyzed to understand structural features determining cofactor-specificity. Homology modeling and mutant analysis elucidated important amino acid residues. Glu149 occupies a central position in the cofactor-binding cleft, and its carboxylate group coordinates the 2'- and 3'-hydroxyl groups of the adenosyl ribose ring of NAD(+) and repels the 2'-phosphate moiety of NADP(+). If Glu149 is mutated to Gln, Asp, Asn or Thr the binding of NAD(+) is altered and rendered the enzyme capable of using NADP(+). This change is attributed to a weaker steric hindrance and elimination of the electrostatic repulsion force of the 2'-phosphate of NADP(+). Simultaneous mutations of Glu149 and Ile200, which is situated opposite of the cofactor binding cleft, improved the enzyme efficiency with NADP(+). The double mutant ALDH3H1Glu149Thr/Ile200Val showed a good catalysis with NADP(+). Subsequently a triple mutation was generated by replacing Val178 by Arg in order to create a "closed" cofactor binding site. The cofactor specificity was shifted even further in favor of NADP(+), as the mutant ALDH3H1E149T/V178R/I200V uses NADP(+) with almost 7-fold higher catalytic efficiency compared to NAD(+). Our experiments suggest that residues occupying positions equivalent to 149, 178 and 200 constitute a group of amino acids in the ALDH3H1 protein determining cofactor affinity.

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

    PubMed

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

    2016-08-01

    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

  14. 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

  15. Roles of basic amino acid residues in the activity of μ-conotoxin GIIIA and GIIIB, peptide blockers of muscle sodium channels.

    PubMed

    Sato, Kazuki; Yamaguchi, Yoko; Ishida, Yukisato; Ohizumi, Yasushi

    2015-04-01

    To study in detail the roles of basic amino acid residues in the activity of μ-conotoxin GIIIA (μ-GIIIA) and GIIIB (μ-GIIIB), specific blockers of muscle sodium channels, seven analogs of μ-GIIIA, and two analogs of μ-GIIIB were synthesized. μ-GIIIA analogs were synthesized by replacing systematically the three Arg residues (Arg1, Arg13, and Arg19) with one, two, and three Lys residues. μ-GIIIB analogs were synthesized by replacing simultaneously all four Lys residues (Lys9, Lys11, Lys16, and Lys19) with Arg residues and further replacement of acidic Asp residues with neutral Ala residues. Circular dichroism spectra of the synthesized analogs suggested that the replacement did not affect the three dimensional structure. The inhibitory effects on the twitch contractions of the rat diaphragm showed that the side chain guanidino group of Arg13 of μ-GIIIA was important for the activity, whereas that of Arg19 had little role for biological activity. Although [Arg9,11,16,19]μ-GIIIB showed higher activity than native μ-GIIIB, highly basic [Ala2,12, Arg9,11,16,19]μ-GIIIB showed lower activity, suggesting that there was an appropriate molecular basicity for the maximum activity.

  16. 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.

  17. Ruminal degradation, amino acid composition, and intestinal digestibility of the residual components of five protein supplements.

    PubMed

    Maiga, H A; Schingoethe, D J; Henson, J E

    1996-09-01

    Two ruminally cannulated Holstein cows (approximately 202 DIM) were used to determine the in situ degradability of five protein supplements: blood meal, meat and bone meal, corn gluten meal, expeller soybean meal, and solvent extracted soybean meal. Dacron bags containing 4 g of each supplement in duplicate were soaked in water and then incubated in the rumen for 0, 3, 6, 12, 18, and 24 h for 3 d. Four extra sample bags of each supplement were incubated in the rumen for 12 h to determine the in vitro intestinal digestibility and AA analysis of the residues. Protein supplements were also analyzed for their AA content. Ruminal degradability of individual supplements varied. Solvent soybean meal was the most degradable, and blood meal was the least degradable. Specific first-limiting essential AA were isoleucine for blood meal and meat and bone meal, lysine for corn gluten meal, and methionine for the soybean meals. The RUP fraction in solvent-extracted and expeller soybean meals tended to be more intestinally digestible than did the protein in blood meal and meat and bone meal. In general, all protein supplements, except solvent-extracted soybean meal, were high in RUP and had the potential to provide good quality AA to complement microbial AA for production.

  18. Peptide nucleic acids tagged with four lysine residues for amperometric genosensors

    PubMed Central

    Zanardi, Chiara; Terzi, Fabio; Seeber, Renato; Baldoli, Clara; Licandro, Emanuela; Maiorana, Stefano

    2012-01-01

    A homothymine PNA decamer bearing four lysine residues has been synthesized as a probe for the development of amperometric sensors. On one hand, the four amino groups introduced make this derivative nine times more soluble than the corresponding homothymine PNA decamer and, on the other hand, allow the stable anchoring of this molecule on Au nanostructured surface through the terminal -NH2 moieties. In particular, XPS and electrochemical investigations performed with hexylamine, as a model molecule, indicate that the stable deposition of primary amine derivatives on such a nanostructured surface is possible and involves the free electron doublet on the nitrogen atom. This finding indicates that this PNA derivative is suitable to act as the probe molecule for the development of amperometric sensors.   Thanks to the molecular probe chosen and to the use of a nanostructured surface as the substrate for the sensor assembly, the device proposed makes possible the selective recognition of the target oligonucleotide sequence with very high sensitivity. PMID:22772036

  19. 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

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

    PubMed Central

    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

  1. 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.

  2. 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.

  3. 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.

  4. /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.

  5. Toxicity of melamine and cyanuric acid in broilers and residues in tissues.

    PubMed

    Ding, X-M; Zhang, Ke-Ying; Wang, L; Bai, S-P

    2012-02-01

    The purpose of this study was to characterize the toxicity potential of melamine (MEL), cyanuric acid (CYA), and a combination of MEL and CYA in broilers. A total of 720 commercial 1-day-old COBB 500 male broilers were randomly allotted into 6 groups with 6 replicates each and 20 broilers in each replicate. The dietary treatments were as follows: group I was the control group, group II included 10 mg/kg MEL and 3.3 mg/kg CYA, group III included 30 mg/kg MEL and 10 mg/kg CYA, group IV included 100 mg/kg MEL and 33.3 mg/kg CYA, group V included 100 mg/kg MEL, and group VI included 33.3 mg/kg CYA. The trial lasted for 42 days. CYA alone and the combination of MEL and CYA had adverse effects on the performance, but MEL alone had no effects on the performance. On day 21, the uric acid (UA) content of group IV was increased in serum (p < 0.05); on day 42, the serum aspartate aminotransferase (AST) activity and the level of tumor necrosis factor (TNF)-α and interleukin (IL)-8 increased in group IV (p < 0.05); 100 mg/kg MEL alone increased the level of TNF-α and the rate of renal apoptosis (p < 0.05); and 33.3 mg/kg CYA alone increased the level of IL-8 and the rate of renal apoptosis (p < 0.05). The livers contained MEL concentrations of 17-125 μg/kg wet weight and CYA concentrations of 28-73 μg/kg, and the muscle contained MEL concentrations of 14-105 μg/kg wet weight. It was indicated that MEL alone, CYA alone, and a combination of MEL and CYA inhibit the growth and damage the kidney and liver.

  6. The C-terminal 50 amino acid residues of dengue NS3 protein are important for NS3-NS5 interaction and viral replication.

    PubMed

    Tay, Moon Y F; Saw, Wuan Geok; Zhao, Yongqian; Chan, Kitti W K; Singh, Daljit; Chong, Yuwen; Forwood, Jade K; Ooi, Eng Eong; Grüber, Gerhard; Lescar, Julien; Luo, Dahai; Vasudevan, Subhash G

    2015-01-23

    Dengue virus multifunctional proteins NS3 protease/helicase and NS5 methyltransferase/RNA-dependent RNA polymerase form part of the viral replication complex and are involved in viral RNA genome synthesis, methylation of the 5'-cap of viral genome, and polyprotein processing among other activities. Previous studies have shown that NS5 residue Lys-330 is required for interaction between NS3 and NS5. Here, we show by competitive NS3-NS5 interaction ELISA that the NS3 peptide spanning residues 566-585 disrupts NS3-NS5 interaction but not the null-peptide bearing the N570A mutation. Small angle x-ray scattering study on NS3(172-618) helicase and covalently linked NS3(172-618)-NS5(320-341) reveals a rigid and compact formation of the latter, indicating that peptide NS5(320-341) engages in specific and discrete interaction with NS3. Significantly, NS3:Asn-570 to alanine mutation introduced into an infectious DENV2 cDNA clone did not yield detectable virus by plaque assay even though intracellular double-stranded RNA was detected by immunofluorescence. Detection of increased negative-strand RNA synthesis by real time RT-PCR for the NS3:N570A mutant suggests that NS3-NS5 interaction plays an important role in the balanced synthesis of positive- and negative-strand RNA for robust viral replication. Dengue virus infection has become a global concern, and the lack of safe vaccines or antiviral treatments urgently needs to be addressed. NS3 and NS5 are highly conserved among the four serotypes, and the protein sequence around the pinpointed amino acids from the NS3 and NS5 regions are also conserved. The identification of the functionally essential interaction between the two proteins by biochemical and reverse genetics methods paves the way for rational drug design efforts to inhibit viral RNA synthesis.

  7. 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.

  8. 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

  9. Critical amino acid residues for the specific binding of the Ti-recognizing recombinant ferritin with oxide surfaces of titanium and silicon.

    PubMed

    Hayashi, Tomohiro; Sano, Ken-Ichi; Shiba, Kiyotaka; Iwahori, Kenji; Yamashita, Ichiro; Hara, Masahiko

    2009-09-15

    The interactions of ferritins fused with a Ti-recognizing peptide (RKLPDA) and their mutants with titanium oxide substrates were explored with an atomic force microscope (AFM). The amino acid sequence of the peptide was systematically modified to elucidate the role of each amino acid residue in the specific interaction. Force measurements revealed a clear correlation among the sequences in the N-terminal domain of ferritin, surface potentials, and long-range electrostatic interactions. Measurements of adhesion forces clearly revealed that hydrogen bonds take part in the specific binding as well as the electrostatic interaction between charged residues and surface charges of Ti oxides. Moreover, our results indicated that not only the charged and polar residues but also a neutral residue (proline) govern the strength of the specific binding, with the order of the residues also being significant. These results demonstrate that the local structure of the peptide governs the special arrangement of charged residues and strongly affects the strength of the bindings.

  10. 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

  11. 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.

  12. 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...

  13. Identification of amino acid residues essential to the activity of lyase CpcT1 from Nostoc sp. PCC7120.

    PubMed

    Zhang, Juan; Sun, Ya Fang; Zhao, Kai Hong; Zhou, Ming

    2012-12-10

    The phycocyanin lyase CpcT1 (encoded by gene all5339) and lyase CpcS1 (encoded by gene alr0617) are capable of catalyzing the phycocyanobilin (PCB) covalently bound to the different sites of phycocyanin's and phycoerythrocyanin's β subunits, respectively. Lyase CpcS1, whose catalytic mechanism had been researched clearly, participates in the covalent coupling of phycobilin and apoprotein in the form of chaperone, and its important amino acids have been confirmed. In order to identify the functional amino acid residues of CpcT1, chemical modification was conducted to arginine, histidine, tryptophan, lysine and amino acid carboxyl of CpcT1. The results indicated that the catalytic activity of the CpcT1 was changed. After the modification of arginine, tryptophan and histidine, site-directed mutations were performed to those highly conserved amino acids which were selected by means of homologous comparison. The mutated lyase, apoprotein and the enzymes that synthesize the phycobilins were recombined in Escherichia coli (E. coli) and in vitro, yielding chromoproteins, which were detected by fluorescence and UV absorption spectrometry. The spectra were compared with that of the chromoprotein catalyzed by wild type lyase CpcT1, achieving relative specific activities of the various mutants. Meanwhile, the mutants were expressed in E. coli, and then circular dichroism structure of near-UV region was determined. The results demonstrated that H33F, W175S, R97A, C137S and C116S influence the catalytic activity of CpcT1. Being different from wild CpcT1, a great deal of α helix was involved in the structure of circular dichroism of R97A and W13S. CpcT1 or its mutants and the enzymes that synthesize the phycobilins, were reconstituted in E. coli and detected by spectra to check the bounding of lyases and PCB. The results of spectra and SDS-PAGE confirm that CpcT1 and its mutants cannot bind phycobilin, differing from the catalytic mechanism of CpcS1. PMID:22982227

  14. 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

  15. Intrinsic propensities of amino acid residues in GxG peptides inferred from amide I' band profiles and NMR scalar coupling constants.

    PubMed

    Hagarman, Andrew; Measey, Thomas J; Mathieu, Daniel; Schwalbe, Harald; Schweitzer-Stenner, Reinhard

    2010-01-20

    A reliable intrinsic propensity scale of amino acid residues is indispensable for an assessment of how local conformational distributions in the unfolded state can affect the folding of peptides and proteins. Short host-guest peptides, such as GxG tripeptides, are suitable tools for probing such propensities. To explore the conformational distributions sampled by the central amino acid residue in these motifs, we combined vibrational (IR, Raman, and VCD) with NMR spectroscopy. The data were analyzed in terms of a superposition of two-dimensional Gaussian distribution functions in the Ramachandran space pertaining to subensembles of polyproline II, beta-strand, right- and left-handed helical, and gamma-turn-like conformations. The intrinsic propensities of eight amino acid residues (x = A, V, F, L, S, E, K, and M) in GxG peptides were determined as mole fractions of these subensembles. Our results show that alanine adopts primarily (approximately 80%) a PPII-like conformation, while valine and phenylalanine were found to sample PPII and beta-strand-like conformations equally. The centers of the respective beta-strand distributions generally do not coincide with canonical values of dihedral angles of residues in parallel or antiparallel beta-strands. In fact, the distributions for most residues found in the beta-region significantly overlap the PPII-region. A comparison with earlier reported results for trivaline reveals that the terminal valines increase the beta-strand propensity of the central valine residue even further. Of the remaining investigated amino acids, methionine preferred PPII the most (0.64), and E, S, L, and K exhibit moderate (0.56-0.45) PPII propensities. Residues V, F, S, E, and L sample, to a significant extent, a region between the canonical PPII and (antiparallel) beta-strand conformations. This region coincides with the sampling reported for L and V using theoretical predictions (Tran et al. Biochemistry 2005, 44, 11369). The distributions of

  16. Two Amino Acid Residues Confer Different Binding Affinities of Abelson Family Kinase Src Homology 2 Domains for Phosphorylated Cortactin*

    PubMed Central

    Gifford, Stacey M.; Liu, Weizhi; Mader, Christopher C.; Halo, Tiffany L.; Machida, Kazuya; Boggon, Titus J.; Koleske, Anthony J.

    2014-01-01

    The closely related Abl family kinases, Arg and Abl, play important non-redundant roles in the regulation of cell morphogenesis and motility. Despite similar N-terminal sequences, Arg and Abl interact with different substrates and binding partners with varying affinities. This selectivity may be due to slight differences in amino acid sequence leading to differential interactions with target proteins. We report that the Arg Src homology (SH) 2 domain binds two specific phosphotyrosines on cortactin, a known Abl/Arg substrate, with over 10-fold higher affinity than the Abl SH2 domain. We show that this significant affinity difference is due to the substitution of arginine 161 and serine 187 in Abl to leucine 207 and threonine 233 in Arg, respectively. We constructed Abl SH2 domains with R161L and S187T mutations alone and in combination and find that these substitutions are sufficient to convert the low affinity Abl SH2 domain to a higher affinity “Arg-like” SH2 domain in binding to a phospho-cortactin peptide. We crystallized the Arg SH2 domain for structural comparison to existing crystal structures of the Abl SH2 domain. We show that these two residues are important determinants of Arg and Abl SH2 domain binding specificity. Finally, we expressed Arg containing an “Abl-like” low affinity mutant Arg SH2 domain (L207R/T233S) and find that this mutant, although properly localized to the cell periphery, does not support wild type levels of cell edge protrusion. Together, these observations indicate that these two amino acid positions confer different binding affinities and cellular functions on the distinct Abl family kinases. PMID:24891505

  17. 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

    2016-08-01

    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

  18. 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. PMID:27631006

  19. Structural insights into the hot spot amino acid residues of mushroom tyrosinase for the bindings of thujaplicins.

    PubMed

    Takahashi, Satoshi; Kamiya, Takanori; Saeki, Kazunori; Nezu, Tomoka; Takeuchi, Shin-Ichiro; Takasawa, Ryoko; Sunaga, Satoshi; Yoshimori, Atsushi; Ebizuka, Shigeo; Abe, Takehiko; Tanuma, Sei-Ichi

    2010-11-15

    Tyrosinase inhibitors are important agents for cosmetic products. We examined here the inhibitory effects of three isomers of thujaplicins (α, β and γ) on mushroom tyrosinase and analyzed their binding modes using a homology model from the crystal structure of Streptomyces castaneoglobisporus tyrosinase (PDB ID: 1wx2). All the thujaplicins were found to be competitive inhibitors and γ-thujaplicin has the most potent inhibitory activity (IC(50)=0.07μM). It is noted that there are good correlations between their observed IC(50) values and their binding free energies calculated by MM-GB/SA. The binding modes of thujaplicins were predicted to be similar to that of Tyr98 of caddie protein (ORF378), which was co-crystallized with S. castaneoglobisporus tyrosinase. Furthermore, free energy decomposition analysis indicated that the potent inhibitory activity of γ-thujaplicin is due to the interactions with His242, Val243 and Pro257 (hot spot amino acid residues) at the active site of tyrosinase. These results provide a novel structural insight into the hot spot of mushroom tyrosinase for the specific binding of γ-thujaplicin.

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

    PubMed

    Wu, Yun; Zheng, Yufei; Tang, Hua

    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. PMID:27631006

  1. 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.

  2. pvSOAR: detecting similar surface patterns of pocket and void surfaces of amino acid residues on proteins.

    PubMed

    Binkowski, T Andrew; Freeman, Patrick; Liang, Jie

    2004-07-01

    Detecting similar protein surfaces provides an important route for discovering unrecognized or novel functional relationship between proteins. The web server pvSOAR (pocket and void Surfaces Of Amino acid Residues) provides an online resource to identify similar protein surface regions. pvSOAR can take a structure either uploaded by a user or obtained from the Protein Data Bank, and identifies similar surface patterns based on geometrically defined pockets and voids. It provides several search modes to compare protein surfaces by similarity in local sequence, local shape and local orientation. Statistically significant search results are reported for visualization and interactive exploration. pvSOAR can be used to predict biological functions of proteins with known three-dimensional structures but unknown biological roles. It can also be used to study functional relationship between proteins and for exploration of the evolutionary origins of structural elements important for protein function. We present an example using pvSOAR to explore the biological roles of a protein whose structure was solved by the structural genomics project. The pvSOAR web server is available at http://pvsoar.bioengr.uic.edu/.

  3. Protective effects of caffeoylquinic acids on the aggregation and neurotoxicity of the 42-residue amyloid β-protein.

    PubMed

    Miyamae, Yusaku; Kurisu, Manami; Murakami, Kazuma; Han, Junkyu; Isoda, Hiroko; Irie, Kazuhiro; Shigemori, Hideyuki

    2012-10-01

    Alzheimer's disease (AD), a neurodegenerative disorder, is characterized by aggregation of 42-mer amyloid β-protein (Aβ42). Aβ42 aggregates through β-sheet formation and induces cytotoxicity against neuronal cells. Aβ42 oligomer, an intermediate of the aggregates, causes memory loss and synaptotoxicity in AD. Inhibition of Aβ42 aggregation by small molecules is thus a promising strategy for the treatment of AD. Caffeoylquinic acid (CQA), a phenylpropanoid found widely in natural sources including foods, shows various biological activities such as anti-oxidative ability. Previously, our group reported that 3,5-di-O-caffeoylquinic acid (3,5-di-CQA) rescued the cognitive impairment in senescence-accelerated-prone mice 8. However, structure-activity relationship of CQA derivatives on the aggregation and neurotoxicity of Aβ42 remains elusive. To evaluate the anti-amyloidogenic property of CQA-related compounds for AD therapy, we examined the effect of CQA and its derivatives on the aggregation and neurotoxicity of Aβ42. In particular, 4,5-di-O-caffeoylquinic acid (4,5-di-CQA) and 3,4,5-tri-O-caffeoylquinic acid (3,4,5-tri-CQA) strongly inhibited the aggregation of Aβ42 in a dose-dependent manner. Structure-activity relationship studies suggested that the caffeoyl group in CQA is essential for the inhibitory activity. These CQAs also suppressed the transformation into β-sheet and cytotoxicity against human neuroblastoma cells of Aβ42. Furthermore, 3,4,5-tri-CQA blocked the formation of Aβ42 oligomer. These results indicate that 3,4,5-tri-CQA could be a potential agent for the prevention of AD.

  4. Cascade dissociations of peptide cation-radicals. Part 1. Scope and effects of amino acid residues in penta-, nona-, and decapeptides.

    PubMed

    Chung, Thomas W; Hui, Renjie; Ledvina, Aaron; Coon, Joshua J; Tureček, Frantisek

    2012-08-01

    Amino acid residue-specific backbone and side-chain dissociations of peptide z ions in MS(3) spectra were elucidated for over 40 pentapeptides with arginine C-terminated sequences of the AAXAR and AAHXR type, nonapeptides of the AAHAAXX"AR and AAHAXAX"AR type, and AAHAAXX"AAR decapeptides. Peptide z(n) ions containing amino acid residues with readily transferrable benzylic or tertiary β-hydrogen atoms (Phe, Tyr, His, Trp, Val) underwent facile backbone cleavages to form dominant z(n-2) or z(n-3) ions. These backbone cleavages are thought to be triggered by a side-chain β-hydrogen atom transfer to the z ion C(α) radical site followed by homolytic dissociation of the adjacent C(α)-CO bond, forming x(n-2) cation-radicals that spontaneously dissociate by loss of HNCO. Amino acid residues that do not have readily transferrable β-hydrogen atoms (Gly, Ala) do not undergo the z(n) → z(n-2) dissociations. The backbone cleavages compete with side-chain dissociations in z ions containing Asp and Asn residues. Side-chain dissociations are thought to be triggered by α-hydrogen atom transfers that activate the C(β)-C(γ) or C(β)-heteroatom bonds for dissociations that dominate the MS(3) spectra of z ions from peptides containing Leu, Cys, Lys, Met, Ser, Arg, Glu, and Gln residues. The Lys, Arg, Gln, and Glu residues also participate in γ-hydrogen atom transfers that trigger other side-chain dissociations. PMID:22669761

  5. Structure-function studies of human deoxyhypusine synthase: identification of amino acid residues critical for the binding of spermidine and NAD.

    PubMed

    Lee, C H; Um, P Y; Park, M H

    2001-05-01

    Deoxyhypusine synthase catalyses the first step in the biosynthesis of hypusine [N(epsilon)-(4-amino-2-hydroxybutyl)lysine]. The crystal structure of human deoxyhypusine synthase in complex with NAD revealed four NAD-binding sites per enzyme tetramer, and led to a prediction of the spermidine-binding pocket. We have replaced each of the seven amino acid residues at the predicted spermidine-binding site, and eleven residues that contact NAD, on an individual basis with alanine. Of the amino acid residues at the spermidine site, substitution of Asp-243, Trp-327, His-288, Asp-316 or Glu-323 with alanine caused an almost complete loss of spermidine binding and enzyme activity; only the mutation Tyr-305-->Ala showed partial binding and activity. His-288-->Ala was also deficient in terms of binding NAD. NAD binding was significantly reduced in all of the NAD-site mutant enzymes, except for Glu-137-->Ala, which showed a normal binding of NAD, but was totally lacking in spermidine binding. Of the NAD-site mutant enzymes, Asp-342-->Ala, Asp-313-->Ala and Asp-238-->Ala displayed the lowest binding of NAD. These enzymes and His-288Ala also showed a reduced binding of spermidine, presumably because spermidine binding is dependent on NAD. These findings permit the positive identification of amino acid residues critical for binding of spermidine and NAD, and provide a new insight into the complex molecular interactions involved in the deoxyhypusine synthase reaction. PMID:11311149

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

    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

  7. 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

  8. Initial interaction of rotavirus strains with N-acetylneuraminic (sialic) acid residues on the cell surface correlates with VP4 genotype, not species of origin.

    PubMed

    Ciarlet, Max; Ludert, Juan E; Iturriza-Gómara, Miren; Liprandi, Ferdinando; Gray, James J; Desselberger, Ulrich; Estes, Mary K

    2002-04-01

    We examined 41 human and animal rotavirus strains representative of all known P genotypes for their dependency on cellular N-acetylneuraminic (sialic) acid (SA) residues for infectivity. Our results showed that all rotaviruses studied, whether of animal or human origin, belonging to P genotypes [1], [2], [3], and [7] depended on SA residues on the cell surface for efficient infectivity but that all human and animal rotavirus strains representative of the remaining known P genotypes were SA independent. The SA residue requirement for efficient infectivity did not change for reassortant rotavirus strains with altered VP4-VP7 combinations. The initial interaction of rotavirus strains with SA residues on the cell surface correlated with VP4 genotype specificity, not with species of origin or VP7 G serotype specificity (P = 0.001; r2 = 1.00, Pearson's correlation coefficient). In addition to being a requirement for infectivity, the presence of SA residues on the cell surface is a requirement for efficient growth in cell culture; recognition of the association of specific P genotypes with the binding of rotavirus to SA residues will facilitate our understanding of the molecular basis of the early events of rotavirus-cell interactions in cell culture models and of pathogenicity in vivo. PMID:11907248

  9. 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

  10. Comparison of molecular dynamics simulation methods for amyloid β(1-42) monomers containing D-aspartic acid residues for predicting retention times in chromatography.

    PubMed

    Oda, Akifumi; Kobayashi, Kana; Takahashi, Ohgi

    2011-11-01

    Molecular dynamics simulations of amyloid β(1-42) containing D-aspartic acid residues were performed using several continuous solvent models to investigate the usefulness of simulation methods for D-amino acid-containing proteins and peptides. Normal molecular dynamics simulations and replica exchange molecular dynamics simulations, which are one of the generalized-ensemble algorithms, were performed. Because the β-structure contents of amyloid β(1-42) peptides obtained by replica exchange molecular dynamics simulations with Onufriev-Bashford-Case generalized Born implicit solvent were qualitatively consistent with experimental data, replica exchange molecular dynamics rather than other methods appeared to be more reasonable for calculations of amyloid β(1-42) containing D-aspartic acid residues. Computational results revealed that peptides with stereoinversion of Asp23 tend to form β-sheet structures by themselves, in contrast to the wild-type peptides that form β-sheet structures only after aggregation. These results are expected to be useful for computational investigations of proteins and peptides such as prediction of retention time of peptides and proteins containing D-aspartic acid residues.

  11. Development of a three-dimensional CysLT1 (LTD4) antagonist model with an incorporated amino acid residue from the receptor.

    PubMed

    Zwaagstra, M E; Schoenmakers, S H; Nederkoorn, P H; Gelens, E; Timmerman, H; Zhang, M Q

    1998-04-23

    This paper describes the molecular modeling of leukotriene CysLT1 (or LTD4) receptor antagonists. Several different structural classes of CysLT1 antagonists were superimposed onto the new and highly rigid CysLT1 antagonist 8-carboxy-3'-[2-(2-quinolinyl)ethenyl]flavone (1, VUF 5017) to generate a common pharmacophoric arrangement. On the basis of known structure-activity relationships of CysLT1 antagonists, the quinoline nitrogen (or a bioisosteric equivalent thereof) and an acidic function were taken as the matching points. In order to optimize the fitting of acidic moieties of all antagonists, an arginine residue from the receptor was proposed as the interaction site for the acidic moieties. Incorporation of this amino acid residue into the model revealed additional interactions between the guanidine group and the nitrogen atoms of quinoline-containing CysLT1 antagonists. In some cases, the arginine may even interact with pi-clouds of phenyl residues of CysLT1 antagonists. The alignment of Montelukast (MK-476) suggests the presence of an additional pocket in the binding site for CysLT1 antagonists. The derived model should be useful for a better understanding of the molecular recognition of the leukotriene CysLT1 receptor.

  12. Glioma residual or recurrence versus radiation necrosis: accuracy of pentavalent technetium-99m-dimercaptosuccinic acid [Tc-99m (V) DMSA] brain SPECT compared to proton magnetic resonance spectroscopy (1H-MRS): initial results.

    PubMed

    Amin, Amr; Moustafa, Hosna; Ahmed, Ebaa; El-Toukhy, Mohamed

    2012-02-01

    We compared pentavalent technetium-99m dimercaptosuccinic acid (Tc-99m (V) DMSA) brain single photon emission computed tomography (SPECT) and proton magnetic resonance spectroscopy ((1)H-MRS) for the detection of residual or recurrent gliomas after surgery and radiotherapy. A total of 24 glioma patients, previously operated upon and treated with radiotherapy, were studied. SPECT was acquired 2-3 h post-administration of 555-740 MBq of Tc-99m (V) DMSA. Lesion to normal (L/N) delayed uptake ratio was calculated as: mean counts of tumor ROI (L)/mean counts of normal mirror symmetric ROI (N). (1)H-MRS was performed using a 1.5-T scanner equipped with a spectroscopy package. SPECT and (1)H-MRS results were compared with pathology or follow-up neuroimaging studies. SPECT and (1)H-MRS showed concordant residue or recurrence in 9/24 (37.5%) patients. Both were true negative in 6/24 (25%) patients. SPECT and (1)H-MRS disagreed in 9 recurrences [7/9 (77.8%) and 2/9 (22.2%) were true positive by SPECT and (1)H-MRS, respectively]. Sensitivity of SPECT and (1)H-MRS in detecting recurrence was 88.8 and 61.1% with accuracies of 91.6 and 70.8%, respectively. A positive association between the delayed L/N ratio and tumor grade was found; the higher the grade, the higher is the L/N ratio (r = 0.62, P = 0.001). Tc-99m (V) DMSA brain SPECT is more accurate compared to (1)H-MRS for the detection of tumor residual tissues or recurrence in glioma patients with previous radiotherapy. It allows early and non-invasive differentiation of residual tumor or recurrence from irradiation necrosis.

  13. Three amino acid residues of an odorant-binding protein are involved in binding odours in Loxostege sticticalis L.

    PubMed

    Yin, J; Zhuang, X; Wang, Q; Cao, Y; Zhang, S; Xiao, C; Li, K

    2015-10-01

    Odorant-binding proteins (OBPs) play an important role in insect olfactory processes and are thought to be responsible for the transport of pheromones and other semiochemicals across the sensillum lymph to the olfactory receptors within the antennal sensilla. As an important general odorant binding protein in the process of olfactory recognition, LstiGOBP1 of Loxostege sticticalis L. has been shown to have good affinity to various plant volatiles. However, the binding specificity of LstiGOBP1 should be further explored in order to better understand the olfactory recognition mechanism of L. sticticalis. In this study, real-time PCR experiments indicated that LstiGOBP1 was expressed primarily in adult antennae. Homology modelling and molecular docking were then conducted on the interactions between LstiGOBP1 and 1-heptanol to understand the interactions between LstiGOBP1 and their ligands. Hydrogen bonds formed by amino acid residues might be crucial for the ligand-binding specificity on molecular docking, a hypothesis that was tested by site-directed mutagenesis. As predicted binding sites for LstiGOBP1, Thr15, Trp43 and Val14 were replaced by alanine to determine the changes in binding affinity. Finally, fluorescence assays revealed that the mutants Thr15 and Trp43 had significantly decreased binding affinity to most odours; in mutants that had two-site mutations, the binding to the six odours that were tested was completely abolished. This result indicates that Thr15 and Trp43 were involved in binding these compounds, possibly by forming multiple hydrogen bonds with the functional groups of the ligands. These results provide new insights into the detailed chemistry of odours' interactions with proteins. PMID:26152502

  14. Effect of lactic acid bacteria inoculant and beet pulp addition on fermentation characteristics and in vitro ruminal digestion of vegetable residue silage.

    PubMed

    Cao, Y; Cai, Y; Takahashi, T; Yoshida, N; Tohno, M; Uegaki, R; Nonaka, K; Terada, F

    2011-08-01

    The objective of this study was to determine the effect of beet pulp (BP) and lactic acid bacteria (LAB) on silage fermentation quality and in vitro ruminal dry matter (DM) digestion of vegetable residues, including white cabbage, Chinese cabbage, red cabbage, and lettuce. Silage was prepared using a small-scale fermentation system, and treatments were designed as control silage without additive or with BP (30% fresh matter basis), LAB inoculant Chikuso-1 (Lactobacillus plantarum, 5mg/kg, fresh matter basis), and BP+LAB. In vitro incubation was performed using rumen fluid mixed with McDougall's artificial saliva (at a ratio of 1:4, vol/vol) at 39°C for 6h to determine the ruminal fermentability of the vegetable residue silages. These vegetable residues contained high levels of crude protein (20.6-22.8% of DM) and moderate levels of neutral detergent fiber (22.7-33.6% of DM). In all silages, the pH sharply decreased and lactic acid increased, and the growth of bacilli, coliform bacteria, molds, and yeasts was inhibited by the low pH at the early stage of ensiling. The silage treated with BP or LAB had a lower pH and a higher lactic acid content than the control silage. After 6h of incubation, all silages had relatively high DM digestibility (38.6-44.9%); in particular, the LAB-inoculated silage had the highest DM digestibility and the lowest methane production. The vegetable residues had high nutritional content and high in vitro DM digestibility. Also, both the addition of a LAB inoculant and moisture adjustment with BP improved the fermentation quality of the vegetable residue silages. In addition, LAB increased DM digestibility and decreased ruminal methane production. PMID:21787927

  15. A Sensitive Gel-based Method Combining Distinct Cyclophellitol-based Probes for the Identification of Acid/Base Residues in Human Retaining β-Glucosidases*

    PubMed Central

    Kallemeijn, Wouter W.; Witte, Martin D.; Voorn-Brouwer, Tineke M.; Walvoort, Marthe T. C.; Li, Kah-Yee; Codée, Jeroen D. C.; van der Marel, Gijsbert A.; Boot, Rolf G.; Overkleeft, Herman S.; Aerts, Johannes M. F. G.

    2014-01-01

    Retaining β-exoglucosidases operate by a mechanism in which the key amino acids driving the glycosidic bond hydrolysis act as catalytic acid/base and nucleophile. Recently we designed two distinct classes of fluorescent cyclophellitol-type activity-based probes (ABPs) that exploit this mechanism to covalently modify the nucleophile of retaining β-glucosidases. Whereas β-epoxide ABPs require a protonated acid/base for irreversible inhibition of retaining β-glucosidases, β-aziridine ABPs do not. Here we describe a novel sensitive method to identify both catalytic residues of retaining β-glucosidases by the combined use of cyclophellitol β-epoxide- and β-aziridine ABPs. In this approach putative catalytic residues are first substituted to noncarboxylic amino acids such as glycine or glutamine through site-directed mutagenesis. Next, the acid/base and nucleophile can be identified via classical sodium azide-mediated rescue of mutants thereof. Selective labeling with fluorescent β-aziridine but not β-epoxide ABPs identifies the acid/base residue in mutagenized enzyme, as only the β-aziridine ABP can bind in its absence. The Absence of the nucleophile abolishes any ABP labeling. We validated the method by using the retaining β-glucosidase GBA (CAZy glycosylhydrolase family GH30) and then applied it to non-homologous (putative) retaining β-glucosidases categorized in GH1 and GH116: GBA2, GBA3, and LPH. The described method is highly sensitive, requiring only femtomoles (nanograms) of ABP-labeled enzymes. PMID:25344605

  16. Extracellular acid block and acid-enhanced inactivation of the Ca2+-activated cation channel TRPM5 involve residues in the S3-S4 and S5-S6 extracellular domains.

    PubMed

    Liu, Dan; Zhang, Zheng; Liman, Emily R

    2005-05-27

    TRPM5, a member of the superfamily of transient receptor potential ion channels, is essential for the detection of bitter, sweet, and amino acid tastes. In heterologous cell types it forms a nonselective cation channel that is activated by intracellular Ca(2+). TRPM5 is likely to be part of the taste transduction cascade, and regulators of TRPM5 are likely to affect taste sensation. In this report we show that TRPM5, but not the related channel TRPM4b, is potently blocked by extracellular acidification. External acidification has two effects, a fast reversible block of the current (IC(50) pH = 6.2) and a slower irreversible enhancement of current inactivation. Mutation of a single Glu residue in the S3-S4 linker and a His residue in the pore region each reduced sensitivity of TRPM5 currents to fast acid block (IC(50) pH = 5.8 for both), and the double mutant was nearly insensitive to acidic pH (IC(50) pH = 5.0). Prolonged exposure to acidic pH enhanced inactivation of TRPM5 currents, and mutant channels that were less sensitive to acid block were also less sensitive to acid-enhanced inactivation, suggesting an intimate association between the two processes. These processes are, however, distinct because the pore mutant H896N, which has normal sensitivity to acid block, shows significant recovery from acid-enhanced inactivation. These data show that extracellular acidification acts through specific residues on TRPM5 to block conduction through two distinct but related mechanisms and suggest a possible interaction between extracellular pH and activation and adaptation of bitter, sweet, and amino acid taste transduction.

  17. New Helical Foldamers: Heterogeneous Backbones with 1:2 and 2:1 [alpha]:[superscript beta]-Amino Acid Residue Patterns

    SciTech Connect

    Schmitt, Margaret A.; Choi, SooHyuk; Guzei, Ilia A.; Gellman, Samuel H.

    2008-10-03

    Foldamers, oligomers with strong folding propensities, are subjects of growing interest because such compounds offer unique scaffolds for the development of molecular function. We report two new foldamer classes, oligopeptides with regular 1:2 or 2:1 patterns of {alpha}- and {beta}-amino acid residues. Two distinct helical conformations are detected via 2D NMR in methanol for each backbone. One of the helices for each backbone is characterized via X-ray crystallography.

  18. 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

  19. 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...

  20. 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.

  1. Functional analysis of three amino acid residues of purR repressor, Trpl47, Gln-218 and Gln-292 in Salmonella typhimurium.

    PubMed

    Zhang, H; Wang, A

    2001-04-01

    The amber mutation sites of 6 purR(am) mutants were determined by cloning and DNA sequencing. The results showed that the mutations were distributed at three different sites in PurR coding region, G(721)(-->A), C(933)(-->T) and C(1155)(-->T), which respectively turn Trp-147, Gln-218 and Gln-292 of PurR into TAG terminal codon. To determine the effect of the three amino acid residues on regulatory function of PurR protein 5 different kinds of tRNA suppressor genes, Su3, Su4, Su6, Su7 and Su9 were used for creating the PurR protein variants with single amino acid substitution. The results indicated that Cys, Glu, Gly, His and Arg which substituted Trp-147 respectively all could not recover the regulation function of PurR. It confirmed that Trp-147 is a critical amino acid for the PurR function. Gln-292 substituted respectively by the same amino acids also could not recover the PurR function, demonstrating that Gln-292 is also an important amino acid residue in PurR.

  2. 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

  3. Pinpointing the putative heparin/sialic acid-binding residues in the 'sushi' domain 7 of factor H: a molecular modeling study.

    PubMed

    Ranganathan, S; Male, D A; Ormsby, R J; Giannakis, E; Gordon, D L

    2000-01-01

    Factor H, a secretory glycoprotein comprising 20 short consensus repeat (SCR) or 'sushi' domains of about 60 amino acids each, is a regulator of the complement system. The complement-regulatory functions of factor H are targeted by its binding to polyanions such as heparin/sialic acid, involving SCRs 7 and 20. Recently, the SCR 7 heparin-binding site was shown to be co-localized with the Streptococcus Group A M protein binding site on factor H (T.K. Blackmore et al., Infect. Immun. 66, 1427 (1998)). Using sequence analysis of all heparin-binding domains of factor H and its closest homologues, molecular modeling of SCRs 6 and 7, and surface electrostatic potential studies, the residues implicated in heparin/sialic acid binding to SCR 7 have been localized to four regions of sequence space containing stretches of basic as well as histidine residues. The heparin-binding site is spatially compact and lies near the interface between SCRs 6 and 7, with residues in the interdomain linker playing a significant role.

  4. 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.

  5. Arginine of retinoic acid receptor beta which coordinates with the carboxyl group of retinoic acid functions independent of the amino acid residues responsible for retinoic acid receptor subtype ligand specificity.

    PubMed

    Zhang, Zeng Ping; Hutcheson, Juliet M; Poynton, Helen C; Gabriel, Jerome L; Soprano, Kenneth J; Soprano, Dianne Robert

    2003-01-15

    The biological actions of retinoic acid (RA) are mediated by retinoic acid receptors (RARalpha, RARbeta, and RARgamma) and retinoid X receptors (RXRalpha, RXRbeta, and RXRgamma). Consistent with the X-ray crystal structures of RARalpha and RARgamma, site-directed mutagenesis studies have demonstrated the importance of a conserved Arg residue (alphaArg(276), betaArg(269), and gammaArg(278)) for coordination with the carboxyl group of RA. However, mutation of Arg(269) to Ala in RARbeta causes only a 3- to 6-fold increase in the K(d) for RA and EC(50) in RA-dependent transcriptional transactivation assays while the homologous mutation in either RARalpha or RARgamma causes a 110-fold and a 45-fold increase in EC(50) value, respectively. To further investigate the nature of this difference, we prepared mutant RARs to determine the effect of conversion of betaR269A to a mutant which mimics either RARalpha ligand selectivity (betaA225S/R269A) or RARgamma ligand selectivity (betaI263M/R269A/V338A). Our results demonstrate that in RARbeta mutants that acquire either RARalpha or RARgamma ligand specificity the Arg(269) position responsible for coordination with the carboxyl group of retinoids continued to function like that of RARbeta. Furthermore, three mutant receptors (betaA225S/R269A, betaA225S/F279, and alphaF286A) were found to have a greater than wild-type affinity for the RARalpha-selective ligand Am580. Finally, a homology-based computer model of the ligand binding domain (LBD) of RARbeta and the X-ray crystal structures of the LBD of both RARalpha and RARgamma are used to describe potential mechanisms responsible for the increased affinity of some mutants for Am580 and for the difference in the effect of mutation of Arg(269) in RARbeta compared to its homologous Arg in RARalpha and RARgamma.

  6. [Effect of mutations and modifications of amino acid residues on zinc-induced interaction of the metal-binding domain of β-amyloid with DNA].

    PubMed

    Khmeleva, S A; Mezentsev, Y V; Kozin, S A; Mitkevich, V A; Medvedev, A E; Ivanov, A S; Bodoev, N V; Makarov, A A; Radko, S P

    2015-01-01

    Interaction of intranuclear β-amyloid with DNA is considered to be a plausible mechanism of Alzheimer's disease pathogenesis. The interaction of single- and double-stranded DNA with synthetic peptides was analyzed using surface plasmon resonance. The peptides represent the metal-binding domain of β-amyloid (amino acids 1-16) and its variants with chemical modifications and point substitutions of amino acid residues which are associated with enhanced neurotoxicity of β-amyloid in cell tests. It has been shown that the presence of zinc ions is necessary for the interaction of the peptides with DNA in solution. H6R substitution has remarkably reduced the ability of domain 1-16 to bind DNA. This is in accordance with the supposition that the coordination of a zinc ion by amino acid residues His6, Glu11, His13, and His14 of the β-amyloid metal-binding domain results in the occurrence of an anion-binding site responsible for the interaction of the domain with DNA. Zinc-induced dimerization and oligomerization of domain 1-16 associated with phosphorylation of Ser8 and the presence of unblocked amino- and carboxy-terminal groups have resulted in a decrease of peptide concentrations required for detection of the peptide-DNA interaction. The presence of multiple anion-binding sites on the dimers and oligomers is responsible for the enhancement of the peptide-DNA interaction. A substitution of the negatively charged residue Asp7 for the neutral residue Asn in close proximity to the anion-binding site of the domain 1-16 of Aβ facilitates the electrostatic interaction between this site and phosphates of a polynucleotide chain, which enhances zinc-induced binding to DNA.

  7. 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.

  8. Amino Acid Residues in Transmembrane Domain 10 of Organic Anion Transporting Polypeptide 1B3 are Critical for Cholecystokinin Octapeptide Transport†

    PubMed Central

    Gui, Chunshan; Hagenbuch, Bruno

    2008-01-01

    Human organic anion transporting polypeptides (OATP) 1B1 and 1B3 are multi-specific transporters that mediate uptake of amphipathic organic compounds into hepatocytes. The two OATPs contain twelve transmembrane domains (TMs) and share 80% amino acid sequence identity. Besides common substrates with OATP1B1, OATP1B3 specifically transports cholecystokinin octapeptide (CCK-8). To determine which structural domains/residues are important for the substrate selectivity of OATP1B3, we constructed a series of chimeric proteins between OATP1B3 and 1B1, expressed them in HEK293 cells and determined uptake of CCK-8 along with surface expression of the proteins. Replacing TM10 in OATP1B3 with TM10 of OATP1B1 resulted in dramatically reduced CCK-8 transport, indicating that TM10 is crucial for recognition and/or translocation of CCK-8. Using site-directed mutagenesis, we identified three key residues within TM10, namely Y537, S545 and T550. When we replaced these residues by the corresponding amino acid residues found in OATP1B1, CCK-8 transport was similarly low as for the replacement of the whole TM10. Kinetic experiments showedthat the Km values for CCK-8 transport in the TM10-replacement and triple mutant were only 1.3 and 1.1 μM, respectively as compared to 16.3 μM for wild-type OATP1B3. Similarly, the Vmax values dropped from 495.5 pmol/normalized mg/min for wild-type OATP1B3 to 13.3 and 19.0 for the TM10-replacement and triple mutant, respectively. Molecular modeling indicated that two of the three identified residues might form hydrogen bonds with CCK-8. In conclusion, we have identified three amino acid residues (Y537, S545 and T550) in TM10 of OATP1B3 that are important for CCK-8 transport. PMID:18690707

  9. 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

  10. 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.

  11. The amino acid residues at 102 and 104 in GP5 of porcine reproductive and respiratory syndrome virus regulate viral neutralization susceptibility to the porcine serum neutralizing antibody.

    PubMed

    Fan, Baochao; Liu, Xing; Bai, Juan; Zhang, Tingjie; Zhang, Qiaoya; Jiang, Ping

    2015-06-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is mainly responsible for the heavy economic losses in pig industry in the world. A number of neutralizing epitopes have been identified in the viral structural proteins GP3, GP4, GP5 and M. In this study, the important amino acid (aa) residues of HP-PRRSV strain BB affecting neutralization susceptibility of antibody were examined using resistant strains generated under neutralizing antibody (NAb) pressure in MARC-145 cells, reverse genetic technique and virus neutralization assay. HP-PRRSV strain BB was passaged under the pressure of porcine NAb serum in vitro. A resistant strain BB34s with 102 and 104 aa substitutions in GP5, which have been predicted to be the positive sites for pressure selection (Delisle et al., 2012), was cloned and identified. To determine the effect of the two aa residues on neutralization, eight recombinant PRRSV strains were generated, and neutralization assay results confirmed that the aa residues 102 and 104 in GP5 played an important role in NAbs against HP-PRRSV in MARC-145 cells and porcine alveolar macrophages. Alignment of GP5 sequences revealed that the variant aa residues at 102 and 104 were frequent among type 2 PRRSV strains. It may be helpful for understanding the mechanism regulating the neutralization susceptibility of PRRSV to the NAbs and monitoring the antigen variant strains in the field.

  12. 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.

  13. 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

  14. 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

  15. TRPA1 is activated by direct addition of cysteine residues to the N-hydroxysuccinyl esters of acrylic and cinnamic acids.

    PubMed

    Sadofsky, Laura R; Boa, Andrew N; Maher, Sarah A; Birrell, Mark A; Belvisi, Maria G; Morice, Alyn H

    2011-01-01

    The nociceptor TRPA1 is thought to be activated through covalent modification of specific cysteine residues on the N terminal of the channel. The precise mechanism of covalent modification with unsaturated carbonyl-containing compounds is unclear, therefore by examining a range of compounds which can undergo both conjugate and/or direct addition reactions we sought to further elucidate the mechanism(s) whereby TRPA1 can be activated by covalent modification. Calcium signalling was used to determine the mechanism of activation of TRPA1 expressed in HEK293 cells with a series of related compounds which were capable of either direct and/or conjugate addition processes. These results were confirmed using physiological recordings with isolated vagus nerve preparations. We found negligible channel activation with chemicals which could only react with cysteine residues via conjugate addition such as acrylamide, acrylic acid, and cinnamic acid. Compounds able to react via either conjugate or direct addition, such as acrolein, methyl vinyl ketone, mesityl oxide, acrylic acid NHS ester, cinnamaldehyde and cinnamic acid NHS ester, activated TRPA1 in a concentration dependent manner as did compounds only capable of direct addition, namely propionic acid NHS ester and hydrocinnamic acid NHS ester. These compounds failed to activate TRPV1 expressed in HEK293 cells or mock transfected HEK293 cells. For molecules capable of direct or conjugate additions, the results suggest for the first time that TRPA1 may be activated preferentially by direct addition of the thiol group of TRPA1 cysteines to the agonist carbonyl carbon of α,β-unsaturated carbonyl-containing compounds.

  16. Ab Initio MD Simulations of the Brønsted Acidity of Glutathione in Aqueous Solutions: Predicting pKa Shifts of the Cysteine Residue.

    PubMed

    Tummanapelli, Anil Kumar; Vasudevan, Sukumaran

    2015-12-10

    The tripeptide glutathione (GSH) is one of the most abundant peptides and the major repository for nonprotein sulfur in both animal and plant cells. It plays a critical role in intracellular oxidative stress management by the reversible formation of glutathione disulfide with the thiol-disulfide pair acting as a redox buffer. The state of charge of the ionizable groups of GSH can influence the redox couple, and hence the pKa value of the cysteine residue of GSH is critical to its functioning. Here we report ab initio Car-Parrinello molecular dynamics simulations of glutathione solvated by 200 water molecules, all of which are considered in the simulation. We show that the free-energy landscape for the protonation-deprotonation reaction of the cysteine residue of GSH computed using metadynamics sampling provides accurate estimates of the pKa and correctly predicts the shift in the dissociation constant values as compared with the isolated cysteine amino acid.

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

    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.

  18. 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

  19. 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

  20. 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.

  1. 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

  2. Five glutamic acid residues in the C-terminal domain of the ChlD subunit play a major role in conferring Mg(2+) cooperativity upon magnesium chelatase.

    PubMed

    Brindley, Amanda A; Adams, Nathan B P; Hunter, C Neil; Reid, James D

    2015-11-10

    Magnesium chelatase catalyzes the first committed step in chlorophyll biosynthesis by inserting a Mg(2+) ion into protoporphyrin IX in an ATP-dependent manner. The cyanobacterial (Synechocystis) and higher-plant chelatases exhibit a complex cooperative response to free magnesium, while the chelatases from Thermosynechococcus elongatus and photosynthetic bacteria do not. To investigate the basis for this cooperativity, we constructed a series of chimeric ChlD proteins using N-terminal, central, and C-terminal domains from Synechocystis and Thermosynechococcus. We show that five glutamic acid residues in the C-terminal domain play a major role in this process.

  3. 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

  4. 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

  5. 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/.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. Identification of the roles of individual amino acid residues of the helix E of the major antenna of photosystem II (LHCII) by alanine scanning mutagenesis.

    PubMed

    Liu, Cheng; Rao, Yan; Zhang, Lei; Yang, Chunhong

    2014-10-01

    The functions of the helix E (W97-F105), an amphiphilic lumenal 310 helix of the major antenna of photosystem II (LHCII), are still unidentified. To elucidate the roles of individual amino acid residue of the helix E, alanine scanning mutagenesis has been performed to mutate every residue of this domain to alanine. The influence of every alanine substitution on the structure and function of LHCII has been investigated biochemically and spectroscopically. The results show that all mutations have little impact on the pigment binding and configuration. However, many mutants presented decreased thermo- or photo-stability compared with the wild type, highlighting the significance of this helix to the stability of LHCII. The most critical residue for stability is W97. The mutant W97A yielded very fragile trimeric pigment protein complexes. The structural analysis revealed that the hydrogen bonding and aromatic interactions between W97, F195, F194 and a water molecule contributed greatly to the stability of LHCII. Moreover, Q103A and F105A have been identified to be able to reinforce the tendency of aggregation in vitro. The structural analysis suggested that the enhancement in aggregation formation for Q103A and F105A might be attributed to the changing hydrophobicity of the region.

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. A requirement of hydrophobic and basic amino acid residues for substrate recognition by Ca2+/calmodulin-dependent protein kinase Ia.

    PubMed Central

    Lee, J C; Kwon, Y G; Lawrence, D S; Edelman, A M

    1994-01-01

    The substrate recognition determinants of Ca2+/calmodulin-dependent protein kinase Ia were investigated by using peptide analogues based on the amino acid sequence around Ser-9 of synapsin I. The Km and Vmax for the synthetic peptide Leu-Arg-Arg-Arg-Leu-Ser-Asp-Ala-Asn-Phe are 3.9 microM and 18.5 mumol/(min.mg), respectively. Deletion of Leu at the -5 position lowers the Vmax/Km by 470-fold. The requirement for a hydrophobic residue at -5 was confirmed by the 90- to 2400-fold reduction in Vmax/Km produced by Arg, Ala, or Asp substitutions, but only 2.6-fold decrease after Phe substitution at this position. A hydrophobic residue is similarly required at the +4 position. Deletion of Phe at this position produces a 67-fold reduction, and substitution of Ala for Phe a 43-fold reduction in Vmax/Km. In contrast, substitution with Leu increases Vmax/Km by 1.8-fold. Arg at -3 is also required for recognition as shown by an approximately 240-fold decrease in Vmax/Km after Ala substitution at this position. Positions -2, -4, and +1 appear to play secondary roles in substrate recognition. Arg at -2 and -4 are positive determinants, since Ala substitution at these positions decreases Vmax/Km by 4.7- and 11-fold, respectively. Asp at +1 is a negative influence, since Ala and Leu substitutions at this position increase Vmax/Km by 2.3- and 6.3-fold, respectively. Substitution of Ala for Leu at -1 or Thr for Ser at the 0 position has little effect on phosphorylation kinetics. Thus, Ca2+/calmodulin-dependent protein kinase Ia has the minimal substrate recognition motif of Hyd-Xaa-Arg-Xaa-Xaa-(Ser*/Thr*)-Xaa-Xaa-Xaa-Hyd, where Hyd represents a hydrophobic amino acid residue. PMID:8022798

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. Subdividing repressor function: DNA binding affinity, selectivity, and allostery can be altered by amino acid substitution of nonconserved residues in a LacI/GalR homologue.

    PubMed

    Zhan, Hongli; Taraban, Marc; Trewhella, Jill; Swint-Kruse, Liskin

    2008-08-01

    Many mutations that impact protein function occur at residues that do not directly contact ligand. To understand the functional contributions from the sequence that links the DNA-binding and regulatory domains of the LacI/GalR homologues, we have created a chimeric protein (LLhP), which comprises the LacI DNA-binding domain, the LacI linker, and the PurR regulatory domain. Although DNA binding site residues are identical in LLhP and LacI, thermodynamic measurements of DNA binding affinity show that LLhP does not discriminate between alternative DNA ligands as well as LacI. In addition, small-angle scattering experiments show that LLhP is more compact than LacI. When DNA is released, LacI shows a 20 A increase in length that was previously attributed to unfolding of the linker. This change is not seen in apo-LLhP, even though the linker sequences of the two proteins are identical. Together, results indicate that long-range functional and structural changes are propagated across the interface that forms between the linker and regulatory domain. These changes could be mediated via the side chains of several linker residues that contact the regulatory domains of the naturally occurring proteins, LacI and PurR. Substitution of these residues in LLhP leads to a range of functional effects. Four variants exhibit altered affinity for DNA, with no changes in selectivity or allosteric response. Another two result in proteins that bind operator DNA with very low affinity and no allosteric response, similar to LacI binding nonspecific DNA sequences. Two more substitutions simultaneously diminish affinity, enhance allostery, and profoundly alter DNA ligand selectivity. Thus, positions within the linker can be varied to modulate different aspects of repressor function.

  1. A single amino acid residue determines the ratio of hydrolysis to transglycosylation catalyzed by β-glucosidases.

    PubMed

    Frutuoso, M A; Marana, S R

    2013-01-01

    The propensity to catalysis of transglycosylation of the β-glucosidase Tmβgly is higher than for Sfβgly. Moreover the propensity to catalysis of transglycosylation is directly proportional to the substrate concentration for Tmβgly, whereas for Sfβgly it is constant. For instance, 60% of a Tmβgly sample catalyzes transglycosylation reactions at 40 mM p-nitrophenyl β-glucoside, whereas only 40% is engaged in hydrolysis of this substrate. For Sfβgly the fraction involved in transglycosylation is only 30 %. In addition, 48 % of a Tmβgly sample catalyzes transglycosylation reactions at 8 mM methylumbelliferyl β-glucoside, whereas Sfβgly does not catalyze transglycosylation using this substrate. Interestingly, these Tmβgly properties were grafted into Sfβgly by a single replacement of a residue forming a channel involved in supplying the catalytic water molecules for attack on the covalent intermediate present in the reaction catalyzed by β-glucosidases. Hence a single residue determines the ratio of hydrolysis to transglycosylation reactions catalyzed by these β-glucosidases.

  2. Substitution of a single amino acid residue in the aromatic/arginine selectivity filter alters the transport profiles of tonoplast aquaporin homologs.

    PubMed

    Azad, Abul Kalam; Yoshikawa, Naoki; Ishikawa, Takahiro; Sawa, Yoshihiro; Shibata, Hitoshi

    2012-01-01

    Aquaporins are integral membrane proteins that facilitate the transport of water and some small solutes across cellular membranes. X-ray crystallography of aquaporins indicates that four amino acids constitute an aromatic/arginine (ar/R) pore constriction known as the selectivity filter. On the basis of these four amino acids, tonoplast aquaporins called tonoplast intrinsic proteins (TIPs) are divided into three groups in Arabidopsis. Herein, we describe the characterization of two group I TIP1s (TgTIP1;1 and TgTIP1;2) from tulip (Tulipa gesneriana). TgTIP1;1 and TgTIP1;2 have a novel isoleucine in loop E (LE2 position) of the ar/R filter; the residue at LE2 is a valine in all group I TIPs from model plants. The homologs showed mercury-sensitive water channel activity in a fast kinetics swelling assay upon heterologous expression in Pichia pastoris. Heterologous expression of both homologs promoted the growth of P. pastoris on ammonium or urea as sole sources of nitrogen and decreased growth and survival in the presence of H(2)O(2). TgTIP1;1- and TgTIP1;2-mediated H(2)O(2) conductance was demonstrated further by a fluorescence assay. Substitutions in the ar/R selectivity filter of TgTIP1;1 showed that mutants that mimicked the ar/R constriction of group I TIPs could conduct the same substrates that were transported by wild-type TgTIP1;1. In contrast, mutants that mimicked group II TIPs showed no evidence of urea or H(2)O(2) conductance. These results suggest that the amino acid residue at LE2 position is critical for the transport selectivity of the TIP homologs and group I TIPs might have a broader spectrum of substrate selectivity than group II TIPs.

  3. Intrinsic contributions of polar amino acid residues toward thermal stability of an ABC–ATPase of mesophilic origin

    PubMed Central

    Sarin, Jyoti; Raghava, Gajendra P.S.; Chakraborti, Pradip K.

    2003-01-01

    The nucleotide-binding subunit of phosphate-specific transporter (PstB) from mesophilic bacterium, Mycobacterium tuberculosis, is a unique ATP-binding cassette (ABC) ATPase because of its unusual ability to hydrolyze ATP at high temperature. In an attempt to define the basis of thermostability, we took a theoretical approach and compared amino acid composition of this protein to that of other PstBs from available bacterial genomes. Interestingly, based on the content of polar amino acids, this protein clustered with the thermophiles. PMID:12931011

  4. 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)

  5. GC-based detection of aldononitrile acetate derivatized glucosamine and muramic acid for microbial residue determination in soil.

    PubMed

    Liang, Chao; Read, Harry W; Balser, Teri C

    2012-05-19

    Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis. The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage. Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives. In

  6. Affinity purification and characterisation of zinc chelating peptides from rapeseed protein hydrolysates: possible contribution of characteristic amino acid residues.

    PubMed

    Xie, Ningning; Huang, Jingjing; Li, Bo; Cheng, Jianghua; Wang, Zhuochen; Yin, Junfeng; Yan, Xiaoming

    2015-04-15

    Zinc is an essential trace element for human growth and development. In this work, zinc-chelating peptides from rapeseed protein hydrolysates produced with alcalase were investigated by affinity chromatography with immobilized zinc and Sephadex G-25 gel filtration. Four small peptides, namely, Ala-Arg, Asn-Ser-Met (NSM), Gly-Lys-Arg, and Glu-Pro-Ser-His, were obtained and identified by reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry. The zinc-chelating ability of the four peptides was further validated by inductively coupled plasma atomic emission spectrometry (ICP-AES). NSM was found to exhibit the highest zinc-chelating rate, which was better than that of reduced glutathione. We speculated that the Asn residue at the amino-terminus might facilitate this zinc-chelating ability. Therefore, utilizing small peptides from rapeseed protein as novel carriers for zinc supplement was feasible.

  7. Catalytically Important Residues of E6AP Ubiquitin Ligase Identified Using Acid-Cleavable Photo-Cross-Linkers.

    PubMed

    Krist, David T; Statsyuk, Alexander V

    2015-07-28

    Inactivation of the E6AP E3 ubiquitin ligase (UBE3A gene) causes Angelman syndrome, while aberrant degradation of p53 by E6AP is implicated in cervical cancers. Herein, we describe the development of photo-cross-linkers to discover catalytic residues of E6AP. Using these cross-linkers, we identified covalent modifications of the E6AP catalytic cysteine and two lysines: Lys(847) and Lys(799). Lys(847) is required for the formation of Lys(48)-linked polyubiquitin chains, while the K799A E6AP mutant was more active at producing Lys(48)-linked polyubiquitin chains. Thus, opposing roles of Lys(799) and Lys(847) pave the path forward to pharmacological inhibitors or activators of E6AP for therapeutic purposes. PMID:26161728

  8. 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

  9. Residual Chemoresponsiveness to Acids in the Superior Laryngeal Nerve in “Taste-Blind” (P2X2/P2X3 Double-KO) Mice

    PubMed Central

    Ohkuri, Tadahiro; Horio, Nao; Stratford, Jennifer M.; Finger, Thomas E.; Ninomiya, Yuzo

    2012-01-01

    Mice lacking both the P2X2 and the P2X3 purinergic receptors (P2X-dblKO) exhibit loss of responses to all taste qualities in the taste nerves innervating the tongue. Similarly, these mice exhibit a near total loss of taste-related behaviors in brief access tests except for a near-normal avoidance of acidic stimuli. This persistent avoidance of acids despite the loss of gustatory neural responses to sour was postulated to be due to continued responsiveness of the superior laryngeal (SL) nerve. However, chemoresponses of the larynx are attributable both to taste buds and to free nerve endings. In order to test whether the SL nerve of P2X-dblKO mice remains responsive to acids but not to other tastants, we recorded responses from the SL nerve in wild-type (WT) and P2X-dblKO mice. WT mice showed substantial SL responses to monosodium glutamate, sucrose, urea, and denatonium—all of which were essentially absent in P2X-dblKO animals. In contrast, the SL nerve of P2X-dblKO mice exhibited near-normal responses to citric acid (50 mM) although responsiveness of both the chorda tympani and the glossopharyngeal nerves to this stimulus were absent or greatly reduced. These results are consistent with the hypothesis that the residual avoidance of acidic solutions by P2X-dblKO mice may be attributable to the direct chemosensitivity of nerve fibers innervating the laryngeal epithelium and not to taste. PMID:22362867

  10. 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.

  11. Ethanol production from residual wood chips of cellulose industry: acid pretreatment investigation, hemicellulosic hydrolysate fermentation, and remaining solid fraction fermentation by SSF process.

    PubMed

    Silva, Neumara Luci Conceição; Betancur, Gabriel Jaime Vargas; Vasquez, Mariana Peñuela; Gomes, Edelvio de Barros; Pereira, Nei

    2011-04-01

    Current research indicates the ethanol fuel production from lignocellulosic materials, such as residual wood chips from the cellulose industry, as new emerging technology. This work aimed at evaluating the ethanol production from hemicellulose of eucalyptus chips by diluted acid pretreatment and the subsequent fermentation of the generated hydrolysate by a flocculating strain of Pichia stipitis. The remaining solid fraction generated after pretreatment was subjected to enzymatic hydrolysis, which was carried out simultaneously with glucose fermentation [saccharification and fermentation (SSF) process] using a strain of Saccharomyces cerevisiae. The acid pretreatment was evaluated using a central composite design for sulfuric acid concentration (1.0-4.0 v/v) and solid to liquid ratio (1:2-1:4, grams to milliliter) as independent variables. A maximum xylose concentration of 50 g/L was obtained in the hemicellulosic hydrolysate. The fermentation of hemicellulosic hydrolysate and the SSF process were performed in bioreactors and the final ethanol concentrations of 15.3 g/L and 28.7 g/L were obtained, respectively.

  12. Identification of amino acid residues photolabeled with 2-azido(alpha-/sup 32/P)adenosine diphosphate in the beta subunit of beef heart mitochondrial F1-ATPase

    SciTech Connect

    Garin, J.; Boulay, F.; Issartel, J.P.; Lunardi, J.; Vignais, P.V.

    1986-07-29

    When beef heart mitochondrial F1-ATPase is photoirradiated in the presence of 2-azido(alpha-/sup 32/P)adenosine diphosphate, the beta subunit of the enzyme is preferentially photolabeled (Dalbon, P., Boulay, F., and Vignais, P. V. (1985) FEBS Lett. 180, 212-218). The site of photolabeling of the beta subunit has been explored. After cyanogen bromide cleavage of the photolabeled beta subunit, only the peptide fragment extending from Gln-293 to Met-358 was found to be labeled. This peptide was isolated and digested by trypsin or Staphylococcus aureus V8 protease. Digestion by trypsin yielded four peptides, one of which spanned residues Ala-338-Arg-356 and contained all the bound radioactivity. When trypsin was replaced by V8 protease, a single peptide spanning residues Leu-342-Met-358 was labeled. Edman degradation of the two labeled peptides showed that radioactivity was localized on the following four amino acids: Leu-342, Ile-344, Tyr-345, and Pro-346.

  13. 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.

  14. 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.

  15. Relationship between enantioselectivity of alternative molecularly imprinted polymeric membranes and species of amino acid residues composing chiral recognition sites.

    PubMed

    Yoshikawa, M; Kondo, Y; Morita, Y

    2001-01-01

    Molecularly imprinted polymeric membranes with tetrapeptide residue H-Asp(OcHex)-Asp(OcHex)-Asp(OcHex)-Asp(OcHex)-CH2- (DDDD) or H-Glu(OBzl)-Glu(OBzl)-Glu(OBzl)-Glu(OBzl)-CH2- (EEEE) were prepared during membrane preparation (casting) processing in the presence of print molecules. The Boc-L-Trp imprinted polymeric membranes thus obtained showed adsorption selectivity toward Ac-L-Trp from its racemic mixtures. From adsorption isotherms of Ac-Trp, the chiral recognition site, that had been formed by the presence of print molecules in the membrane preparation process, exclusively recognized Ac-L-Trp that possessed the same configuration of the print molecule. The affinity constants between chiral recognition sites in the membrane and Ac-L-Trp was determined to be 1.00 x 10(4) mol(-1) dm3 and 1.08 x 10(4) mol(-1) dm3 for the DDDD and EEEE membranes, respectively. Enantioselective electrodialysis could be attained by applying an optimum potential difference to give permselectivity, with a value close to its adsorption selectivity.

  16. Impact of pesticides residue and heavy metals on lipids and fatty acids composition of some seafoods of Red Sea (KSA).

    PubMed

    Al-Malki, Abdulrahman L; Moselhy, Said S

    2011-10-01

    Seafood is considered by nutritionists to be a high source of omega 3 long-chain polyunsaturated fatty acids (n-3 LC-PUFAs) which are involved in prevention of cardiovascular diseases. The aim of the present study is firstly to detect the presence of organochlorine pesticides and heavy metals in some fresh seafood of Red Sea commonly used in KSA in relation to its contents. It was found that the fatty acids 18:2n-6, 18:3n-6, 18:3n-9 was statistically significantly higher in fresh samples than frozen (p < 0.05), smoked (p < 0.01) and canned fishes (p < 0.05), respectively. Total lipids and cholesterol content of canned and smoked fishes were significantly higher than fresh and frozen samples (p < 0.01, <0.05 and p < 0.01, <0.05). Vitamin D was found to be lower in frozen samples than fresh, smoked and canned (p < 0.05 for each). While vitamin A was higher in fresh and smoked as compared with frozen and canned samples. No organochlorine tested were detected in either fresh or processed samples (smoked, frozen and canned). Among the heavy metals, the highest lead level was detected in fresh and smoked samples as compared with frozen and canned samples. No correlation was found between organochlorine compounds in fish and the corresponding levels in fatty acids. We concluded that, all fishes are safe for uses but it is preferable to use fresh samples due to its high content of PUFA and low lead level. PMID:21247990

  17. Specificity for a CCR5 Inhibitor Is Conferred by a Single Amino Acid Residue: ROLE OF ILE198.

    PubMed

    Lau, Gloria; Labrecque, Jean; Metz, Markus; Vaz, Roy; Fricker, Simon P

    2015-04-24

    The chemokine receptors CCR5 and CCR2b share 89% amino acid homology. CCR5 is a co-receptor for HIV and CCR5 antagonists have been investigated as inhibitors of HIV infection. We describe the use of two CCR5 antagonists, Schering-C (SCH-C), which is specific for CCR5, and TAK-779, a dual inhibitor of CCR5 and CCR2b, to probe the CCR5 inhibitor binding site using CCR5/CCR2b chimeric receptors. Compound inhibition in the different chimeras was assessed by inhibition of chemokine-induced calcium flux. SCH-C inhibited RANTES (regulated on activation, normal T cell expressed and secreted) (CCL5)-mediated calcium flux on CCR5 with an IC50 of 22.8 nM but was inactive against monocyte chemoattractant protein-1 (CCL2)-mediated calcium flux on CCR2b. However, SCH-C inhibited CCL2-induced calcium flux against a CCR5/CCR2b chimera consisting of transmembrane domains IV-VI of CCR5 with an IC50 of 55 nM. A sequence comparison of CCR5 and CCR2b identified a divergent amino acid sequence located at the junction of transmembrane domain V and second extracellular loop. Transfer of the CCR5 sequence KNFQTLKIV into CCR2b conferred SCH-C inhibition (IC50 of 122 nM) into the predominantly CCR2b chimera. Furthermore, a single substitution, R206I, conferred partial but significant inhibition (IC50 of 1023 nM) by SCH-C. These results show that a limited amino acid sequence is responsible for SCH-C specificity to CCR5, and we propose a model showing the interaction with CCR5 Ile(198).

  18. 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.

  19. 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.%).

  20. 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

  1. Chronic alcohol consumption augments loss of sialic acid residues and alters erythrocyte membrane charge in type II diabetic patients.

    PubMed

    Degirmenci, Serkan; Akalin, Aysen; Kartkaya, Kazim; Kanbak, Güngör

    2008-01-01

    In this study, the effects of alcohol consumption on erythrocyte membrane properties in type 2 diabetic patients were investigated. Therefore, we measured total and lipid-bound sialic acid (LSA) levels, sialidase activities, and erythrocyte membrane negative charge. Three groups, including control group (n = 20), alcohol-consuming diabetic patients group (n = 14), and diabetic patients without alcohol consumption group (n = 42), were created. Plasma total sialic acid (TSA) levels of the alcohol-consuming diabetic group were elevated as compared to the healthy control and diabetic group (p < 0.001 and p < 0.01, respectively). TSA levels of the diabetic group were significantly elevated as compared to the healthy control group (p > 0.001). Plasma LSA levels of the alcohol-consuming diabetic group were higher than that in the healthy control and diabetic group (p < 0.05 and p < 0.05, respectively). LSA levels of the diabetic group were found to be high as compared to the healthy control group (p < 0.05). Plasma sialidase activities of the alcohol-consuming diabetic group and diabetic group were significantly elevated as compared to the healthy control group (p < 0.05 and p < 0.05, respectively). Sialidase activities of the alcohol-consuming diabetic group were elevated as compared to the diabetic group, but this was not statistically significant (p > 0.05). Erythrocyte membrane negativity levels of the alcohol-consuming diabetic group and diabetic group were significantly decreased (p < 0.001 and p < 0.001, respectively) as compared to the healthy control group. Erythrocyte membrane negativity levels of the alcohol-consuming diabetic group were decreased as compared to the diabetic group, but this was not statistically significant (p > 0.05). In conclusion, our results indicate that chronic alcohol consumption may augment membrane alterations in type 2 diabetic patients.

  2. 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

  3. 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.

  4. 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

  5. Sequence of the cDNA and 5'-flanking region for human acid alpha-glucosidase, detection of an intron in the 5' untranslated leader sequence, definition of 18-bp polymorphisms, and differences with previous cDNA and amino acid sequences.

    PubMed

    Martiniuk, F; Mehler, M; Tzall, S; Meredith, G; Hirschhorn, R

    1990-03-01

    Acid maltase or acid alpha-glucosidase (GAA) is a lysosomal enzyme that hydrolyzes glycogen to glucose and is deficient in glycogen storage disease type II. Previously, we isolated a partial cDNA (1.9 kb) for human GAA; we have now used this cDNA to isolate and determine sequence in longer cDNAs from four additional independent cDNA libraries. Primer extension studies indicated that the mRNA extended approximately 200 bp 5' of the cDNA sequence obtained. Therefore, we isolated a genomic fragment containing 5' cDNA sequences that overlapped the previous cDNA sequence and extended an additional 24 bp to an initiation codon within a Kozak consensus sequence. The sequence of the genomic clone revealed an intron-exon junction 32 bp 5' to the ATG, indicating that the 5' leader sequence was interrupted by an intron. The remaining 186 bp of 5' untranslated sequence was identified approximately 3 kb upstream. The promoter region upstream from the start site of transcription was GC rich and contained areas of homology to Sp1 binding sites but no identifiable CAAT or TATA box. The combined data gave a nucleotide sequence of 2,856 bp for the coding region from the ATG to a stop codon, predicting a protein of 952 amino acids. The 3' untranslated region contained 555 bp with a polyadenylation signal at 3,385 bp followed by 16 bp prior to a poly(A) tail. This sequence of the GAA coding region differs from that reported by Hoefsloot et al. (1988) in three areas that change a total of 42 amino acids. Direct determination of the amino acid sequence in one of these areas confirmed the nucleotide sequence reported here but also disagreed with the directly determined amino acid sequence reported by Hoefsloot et al. (1988). At two other areas, changes in base pairs predicted new restriction sites that were identified in cDNAs from several independent libraries. The amino acid changes in all three ares increased the homology to rabbit-human isomaltase. Therefore, we believe that our

  6. On the activation of bovine plasma factor XIII. Amino acid sequence of the peptide released by thrombin and the terminal residues of the subunit polypeptides.

    PubMed

    Nakamura, S; Iwanaga, S; Suzuki, T

    1975-12-01

    A blood coagulation factor, Factor XIII, was highly purified from bovine fresh plasma by a method similar to those used for human plasma Factor XIII. The isolated Factor XIII consisted of two subunit polypeptides, a and b chains, with molecular weights of 79,000 +/- 2,000 and 75,000 +/- 2,000, respectively. In the conversion of Factor XIII to the active enzyme, Factor XIIIa, by bovine thrombin [EC 3.4.21.5], a peptide was liberated. This peptide, designated tentatively as "activation peptide," was isolated by gel-filtration on a Sephadex G-75 column. It contained a total of 37 amino acid residues with a masked N-terminal residue and C-terminal arginine. The whole amino acid sequence of "Activation peptide" was established by the dansyl-Edman method and standard enzymatic techniques, and the masked N-terminal residue was identified as N-acetylserine by using a rat liver acylamino acid-releasing enzyme. This enzyme specifically cleaved the N-acetylserylglutamyl peptide bond serine and the remaining peptide, which was now reactive to 1-dimethylamino-naphthalene-5-sulfonyl chloride. A comparison of the sequences of human and bovine "Activation peptide" revealed five amino acids replacements, Ser-3 to Thr; Gly-5 to Arg; Ile-14 to Val; Thr-18 to Asn, and Pro-26 to Leu. Another difference was the deletion of Leu-34 in the human peptide. Adsorption chromatography on a hydroxylapatite column in the presence of 0.1% sodium dodecyl sulfate was developed as a preparative procedure for the resolution of the two subunit polypeptides, a or a' chain and b chain, constituting the protein molecule of Factor XIII or Factor XIIIa. End group analyses on the isolated pure chains revealed that the structural change of Factor XIII during activation with thrombin occurs only in the N-terminal portion of the a chain, not in the N-terminal end of the b chain or in the C-terminal ends of the a and b chains. From these results, it was concluded that the activation of bovine plasma Factor XIII

  7. Physico-chemical assessment of a fixated flue-gas desulfurization sludge cap emplaced along with other coal-combustion residues to abate acid mine drainage.

    PubMed

    Naylor, Shawn; Branam, Tracy D; Olyphant, Greg A

    2012-05-01

    Long term monitoring of the physical and chemical effects of using coal-combustion residues (CCRs), in particular fixated flue gas desulfurization (FGD) sludge, as a major component in the reclamation of a pyritic refuse deposit was undertaken to determine the beneficial and detrimental consequences of placing these controversial materials in an unrestricted environment. Monitoring wells, neutron probe access tubes, and weirs were installed before and after reclamation to observe hydrologic conditions and determine how the use of FGD sludge as a recharge barrier was affecting hydrochemical response to ambient weather conditions. Data were collected for six months prior to reclamation and then for an additional 13 years (more intensively during the first 5 years). Statistical analyses of water levels in the pyritic refuse deposit indicate a shift from precipitation- to barometric-controlled fluctuations. These findings, along with minimal variability in soil moisture within the CCR cap and transient perching of groundwater above the cap, are evidence that recharge of the refuse aquifer has been minimized. Statistically significant improvements in the quality of groundwater on-site and surface water leaving the site include long-term declines in acidity, As, and Fe concentrations within the refuse aquifer, attributed to a decrease in recharge of oxygenated water as supported by an analysis of calculated mineral saturation indices. Long-term declines in acidity and associated trace metals discharging from the site are attributed to the post-reclamation loss of sulfate salts brought to the surface by capillary forces. The results of this study indicate that strategic usage of CCRs in reclamation programs can produce beneficial effects, including acid drainage reductions, that are beyond those achieved using traditional reclamation approaches such as the utilization of mine spoil as capping and fill material.

  8. Rapid and simple solid-phase esterification of sialic acid residues for quantitative glycomics by mass spectrometry.

    PubMed

    Miura, Yoshiaki; Shinohara, Yasuro; Furukawa, Jun-ichi; Nagahori, Noriko; Nishimura, Shin-Ichiro

    2007-01-01

    A rapid and quantitative method for solid-phase methyl esterification of carboxy groups of various sialylated oligosaccharides has been established. The method employed a triazene derivative, 3-methyl-1-p-tolyltriazene, for facile derivatization of oligosaccharides immobilized onto general solid supports such as Affi-Gel Hz and gold colloidal nanoparticles in a multiwell plate. The workflow protocol was optimized for the solid-phase processing of captured sialylated/unsialylated oligosaccharides separated from crude sample mixtures by chemical ligation. From tryptic and/or PNGase F-digest mixtures of glycoproteins, purification by chemoselective immobilization, esterification and recovery were achieved in the same well of the filter plate within three hours when used in conjunction with "glycoblotting technology" (S.-I. Nishimura, K. Niikura, M. Kurogochi, T. Matsushita, M. Fumoto, H. Hinou, R. Kamitani, H. Nakagawa, K. Deguchi, N. Miura, K. Monde, H. Kondo, High-throughput protein glycomics: Combined use of chemoselective glycoblotting and MALDI-TOF/TOF mass spectrometry: Angew. Chem. 2005, 117, 93-98; Angew. Chem. Int. Ed. 2005, 44, 91-96). The recovered materials were directly applicable to subsequent characterization by mass spectrometric techniques such as MALDI-TOF for large-scale glycomics of both neutral and sialylated oligosaccharides. On-bead/on-gold nanoparticle derivatization of glycans containing sialic acids allowed rapid and quantitative glycoform profiling by MALDI-TOF MS with reflector and positive ion mode. In addition to its simplicity and speed, the method eliminates the use of unfavorable halogenated solvents such as chloroform and dichloromethane or volatile solvents such as diethyl ether and hexane, resulting in a practical and green chemical method for automated robotic adaptation.

  9. Transporter-associated currents in the gamma-aminobutyric acid transporter GAT-1 are conditionally impaired by mutations of a conserved glycine residue.

    PubMed

    Zhou, Yonggang; Kanner, Baruch I

    2005-05-27

    To determine whether glycine residues play a role in the conformational changes during neurotransmitter transport, we have analyzed site-directed mutants of the gamma-aminobutyric acid (GABA) transporter GAT-1 in a domain containing three consecutive glycines conserved throughout the sodium- and chloride-dependent neurotransmitter transporter family. Only cysteine replacement of glycine 80 resulted in the complete loss of [(3)H]GABA uptake, but oocytes expressing this mutant exhibited the sodium-dependent transient currents thought to reflect a charge-moving conformational change. When sodium was removed and subsequently added back, the transients by G80C did not recover, as opposed to wild type, where recovery was almost complete. Remarkably, the transients by G80C could be restored after exposure of the oocytes to either GABA or a depolarizing pre-pulse. These treatments also resulted in a full recovery of the transients by the wild type. Whereas in wild type lithium leak currents are observed after prior sodium depletion, this was not the case for the glycine 80 mutants unless GABA was added or the oocytes were subjected to a depolarizing pre-pulse. Thus, glycine 80 appears essential for conformational transitions in GAT-1. When this residue is mutated, removal of sodium results in "freezing" the transporter in one conformation from which it can only exit by compensatory changes induced by GABA or depolarization. Our results can be explained by a model invoking two outward-facing states of the empty transporter and a defective transition between these states in the glycine 80 mutants.

  10. 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.

  11. 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

  12. Topoisomerase I-mediated DNA cleavage as a guide to the development of antitumor agents derived from the marine alkaloid lamellarin D: triester derivatives incorporating amino acid residues.

    PubMed

    Tardy, Christelle; Facompré, Michaël; Laine, William; Baldeyrou, Brigitte; García-Gravalos, Dolores; Francesch, Andrés; Mateo, Cristina; Pastor, Alfredo; Jiménez, José A; Manzanares, Ignacio; Cuevas, Carmen; Bailly, Christian

    2004-04-01

    The marine alkaloid lamellarin D (LAM-D) has been recently characterized as a potent poison of human topoisomerase I endowed with remarkable cytotoxic activities against tumor cells. We report here the first structure-activity relationship study in the LAM-D series. Two groups of triester compounds incorporating various substituents on the three phenolic OH at positions 8, 14 and 20 of 6H-[1]benzopyrano[4',3':4,5]pyrrolo[2,1-a]isoquinolin-6-one pentacyclic planar chromophore typical of the parent alkaloid were tested as topoisomerase I inhibitors. The non-amino compounds in group A showed no activity against topoisomerase I and were essentially non cytotoxic. In sharp contrast, compounds in group B incorporating amino acid residues strongly promoted DNA cleavage by human topoisomerase I. LAM-D derivatives tri-substituted with leucine, valine, proline, phenylalanine or alanine residues, or a related amino side chain, stabilize topoisomerase I-DNA complexes. The DNA cleavage sites detected at T downward arrow G or C downward arrow G dinucleotides with these molecules were identical to that of LAM-D but slightly different from those seen with camptothecin which stimulates topoisomerase I-mediated cleavage at T downward arrow G only. In the DNA relaxation and cleavage assays, the corresponding Boc-protected compounds and the analogues of the non-planar LAM-501 derivative lacking the 5-6 double bond in the quinoline B-ring showed no effect on topoisomerase I and were considerably less cytotoxic than the corresponding cationic compounds in the LAM-D series. The presence of positive charges on the molecules enhances DNA interaction but melting temperature studies indicate that DNA binding is not correlated with topoisomerase I inhibition or cytotoxicity. Cell growth inhibition by the 41 lamellarin derivatives was evaluated with a panel of tumor cells lines. With prostate (DU-145 and LN-CaP), ovarian (IGROV and IGROV-ET resistant to ecteinascidin-743) and colon (LoVo and

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

    PubMed

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

    1994-02-18

    Various beta-mutants were investigated either as subunits or as substrates for casein kinase 2 (CK-2), in the absence of presence of polylysine. A total of 21 beta-mutants were characterized for their susceptibility to autophosphorylation, by combining them in equimolar amounts with the recombinant alpha-subunit. Six mutants, i.e. beta A5,6, beta A59-61,63,64, beta A55,57, beta 55-57, beta delta 171-215, and beta delta 150-215 exhibited a > 70% reduction in autophosphorylation. This strongly suggests that in addition to amino acid residues 5,6, distant amino acid residues within the sequence 55-64 are also involved in the process of autophosphorylation, possibly by means of a loop formation. The results obtained with the COOH-terminal-deleted mutants support the view that reconstitution of a functional holoenzyme must occur to allow efficient autophosphorylation. Polylysine prevents the autophosphorylation of beta wt (86% inhibition) inducing a parallel increase of the alpha-subunit autophosphorylation. The autophosphorylation of all mutants, with the exception of beta A55-57 and beta A59-61,63,64, is also inhibited by polylysine (>64%). The alpha-subunit autophosphorylation is increased with all mutants reconstituting a tetrameric holoenzyme. Only with the three largest COOH-terminal deletion mutants beta delta 150-215, beta delta 171-215, and beta delta 181-215 is no significant alpha-subunit autophosphorylation observed. The phosphorylation of the beta-subunit mutants added in large molar excess to CK-2 holoenzyme (either native or recombinant) is also severely impaired by Ala for Glu/Asp substitutions at position 5,6 and in the 55-64 region and by the deletion of the COOH-terminal segments 150-215 and 171-215. Such a phosphorylation is inhibited by polylysine, with the exception of mutants beta delta 171-215 and beta delta 150-215, whose phosphorylation is conversely stimulated by polylysine. The decreased phosphorylation efficiency of those mutants that are

  14. 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

  15. Factor D of the alternative pathway of human complement. Purification, alignment and N-terminal amino acid sequences of the major cyanogen bromide fragments, and localization of the serine residue at the active site.

    PubMed Central

    Johnson, D M; Gagnon, J; Reid, K B

    1980-01-01

    The serine esterase factor D of the complement system was purified from outdated human plasma with a yield of 20% of the initial haemolytic activity found in serum. This represented an approx. 60 000-fold purification. The final product was homogeneous as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with an apparent mol.wt. of 24 000), its migration as a single component in a variety of fractionation procedures based on size and charge, and its N-terminal amino-acid-sequence analysis. The N-terminal amino acid sequence of the first 36 residues of the intact molecule was found to be homologous with the N-terminal amino acid sequences of the catalytic chains of other serine esterases. Factor D showed an especially strong homology (greater than 60% identity) with rat 'group-specific protease' [Woodbury, Katunuma, Kobayashi, Titani, & Neurath (1978) Biochemistry 17, 811-819] over the first 16 amino acid residues. This similarity is of interest since it is considered that both enzymes may be synthesized in their active, rather than zymogen, forms. The three major CNBr fragments of factor D, which had apparent mol.wts. of 15 800, 6600 and 1700, were purified and then aligned by N-terminal amino acid sequence analysis and amino acid analysis. By using factor D labelled with di-[1,3-14C]isopropylphosphofluoridate it was shown that the CNBr fragment of apparent mol.wt. 6600, which is located in the C-terminal region of factor D, contained the active serine residue. The amino acid sequence around this residue was determined. Images Fig. 1. Fig. 2. PMID:6821372

  16. No Previous Public Services Required

    ERIC Educational Resources Information Center

    Taylor, Kelley R.

    2009-01-01

    In 2007, the Supreme Court heard a case that involved the question of whether a school district could be required to reimburse parents who unilaterally placed their child in private school when the child had not previously received special education and related services in a public institution ("Board of Education v. Tom F."). The Court's 4-4…

  17. [Determination of the residues of 3-methyl-quinoxaline-2-carboxylic acid and quinoxaline-2-carboxylic acid in animal origin foods by high performance liquid chromatography-tandem mass spectrometry].

    PubMed

    Zheng, Ling; Wu, Yujie; Li, Yong; Li, Lihua

    2012-07-01

    A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/ MS) method was developed for the simultaneous quantitative determination of 3-methyl-quinoxaline-2-carboxylic acid (MQCA) and quinoxaline-2-carboxylic acid (QCA) as the marker residues for carbadox (CBX) and olaquindox (OLA), respectively, in the muscles and livers of porcine and chicken and in the muscles of fish and shrimp. The MQCA and QCA were deproteinated with 5% metaphosphoric acid in 10% methanol followed by liquid-liquid extraction. Further clean-up was performed by solid phase extraction (SPE) through mixed mode anion-exchange columns (Oasis MAX SPE). The separation of the compounds was carried on a Waters Xterra MS C18 column (150 mm x 2.1 mm, 5 microm) by a gradient elution using methanol and 0.2% formic acid as mobile phases. The analytes were detected by tandem mass spectrometry in multiple reaction monitoring (MRM) mode with positive electrospray ionization. The MQCA and QCA were quantified by internal standard method. The linear ranges were 1.0-20.0 microg/L and the correlation coefficients were not less than 0.9996. The average recoveries and relative standard deviations ranged from 62.4%-118% and 1.48%-28.1% respectively at the spiked levels of 0.1, 0.2 and 1.0 microg/kg for the both markers. The limit of quantitation (LOQ) was 0.1 microg/kg. The method is sensitive, accurate and suitable for the determination and confirmation of MQCA and QCA in animal origin foods.

  18. Structural and energetic analysis to provide insight residues of CYP2C9, 2C11 and 2E1 involved in valproic acid dehydrogenation selectivity.

    PubMed

    Bello, Martiniano; Mendieta-Wejebe, Jessica E; Correa-Basurto, José

    2014-07-15

    Docking and molecular dynamics (MD) simulation have been two computational techniques used to gain insight about the substrate orientation within protein active sites, allowing to identify potential residues involved in the binding and catalytic mechanisms. In this study, both methods were combined to predict the regioselectivity in the binding mode of valproic acid (VPA) on three cytochrome P-450 (CYP) isoforms CYP2C9, CYP2C11, and CYP2E1, which are involved in the biotransformation of VPA yielding reactive hepatotoxic intermediate 2-n-propyl-4-pentenoic acid (4nVPA). There are experimental data about hydrogen atom abstraction of the C4-position of VPA to yield 4nVPA, however, there are not structural evidence about the binding mode of VPA and 4nVPA on CYPs. Therefore, the complexes between these CYP isoforms and VPA or 4nVPA were studied to explore their differences in binding and energetic stabilization. Docking results showed that VPA and 4nVPA are coupled into CYPs binding site in a similar conformation, but it does not explain the VPA hydrogen atom abstraction. On the other hand, MD simulations showed a set of energetic states that reorient VPA at the first ns, then making it susceptible to a dehydrogenation reaction. For 4nVPA, multiple binding modes were observed in which the different states could favor either undergo other reaction mechanism or ligand expulsion from the binding site. Otherwise, the energetic and entropic contribution point out a similar behavior for the three CYP complexes, showing as expected a more energetically favorable binding free energy for the complexes between CYPs and VPA than with 4nVPA.

  19. Six Amino Acid Residues in a 1200 Å2 Interface Mediate Binding of Factor VIII to an IgG4κ Inhibitory Antibody

    PubMed Central

    Lin, Jasper C.; Ettinger, Ruth A.; Schuman, Jason T.; Zhang, Ai-Hong; Wamiq-Adhami, Muhammad; Nguyen, Phuong-Cac T.; Nakaya-Fletcher, Shelley M.; Puranik, Komal; Thompson, Arthur R.; Pratt, Kathleen P.

    2015-01-01

    The development of neutralizing anti-factor VIII (FVIII) antibodies complicates the treatment of many hemophilia A patients. The C-terminal C2 domain is a particularly antigenic FVIII region. A crystal structure of recombinant FVIII-C2 bound to an Fab fragment of the patient-derived monoclonal antibody BO2C11, which recognizes an immunodominant inhibitor epitope on FVIII and blocks its ability to bind von Willebrand factor (VWF) and phospholipids, revealed that 15 amino acids in FVIII contact this antibody. Forty-three recombinant FVIII-C2 proteins, each with a surface-exposed side chain mutated to alanine or another residue, were generated, and surface plasmon resonance studies were carried out to evaluate effects of these substitutions on BO2C11/FVIII-C2 binding affinity. Thermodynamic analysis of experiments carried out at three temperatures indicated that one beta hairpin turn at the antigen-antibody interface (FVIII-F2196, N2198, M2199 and F2200) plus two non-contiguous arginines (FVIII-R2215 and R2220), contributed appreciably to the affinity. B-domain-deleted (BDD) FVIII-F2196A, FVIII-F2196K and FVIII-M2199A were generated and characterized. Their pro-coagulant activities and binding to VWF were similar to those of WT-BDD-FVIII, and FVIII-F2196K avoided neutralization by BO2C11 and murine inhibitory mAb 1B5. This study suggests specific sites for amino acid substitutions to rationally design FVIII variants capable of evading immunodominant neutralizing anti-FVIII antibodies. PMID:25615825

  20. 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

  1. 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

    Han, Ruizhi; Liu, Long; Shin, Hyun-Dong; Chen, Rachel R; Li, Jianghua; Du, Guocheng; Chen, Jian

    2013-12-01

    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.

  2. 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

  3. 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.

  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. Serine 157, a retinoic acid receptor alpha residue phosphorylated by protein kinase C in vitro, is involved in RXR.RARalpha heterodimerization and transcriptional activity.

    PubMed

    Delmotte, M H; Tahayato, A; Formstecher, P; Lefebvre, P

    1999-12-31

    Retinoic acid (RA) regulation of cellular proliferation and differentiation is mediated, at least in part, through two related nuclear receptors, RAR and RXR. RA-induced modulation of gene expression leads generally to cellular differentiation, whereas stimulation of the protein kinase C (PKC) signaling pathway is associated with cellular proliferation. Pursuant to our discovery that prolonged activation of PKCs induced a strong decrease in RA responsiveness of a retinoid-inducible reporter gene, we have further investigated the connections between these two signaling pathways. We demonstrate that PKC isoforms alpha and gamma are able to phosphorylate human RARalpha (hRARalpha) in vitro on a single serine residue located in the extended DNA binding domain (T box). The introduction of a negative charge at this position (serine 157) strongly decreased hRARalpha transcriptional activity, whereas a similar mutation at other PKC consensus phosphorylation sites had no effect. The effect on transcriptional activation was correlated with a decrease in the capacity of hRARalpha to heterodimerize with hRXRalpha. Thus hRARalpha is a direct target for PKCalpha and gamma, which may control retinoid receptor transcriptional activities during cellular proliferation and differentiation.

  6. 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

  7. Probing the S-adenosylmethionine-binding site of rat guanidinoacetate methyltransferase. Effect of site-directed mutagenesis of residues that are conserved across mammalian non-nucleic acid methyltransferases.

    PubMed Central

    Hamahata, A; Takata, Y; Gomi, T; Fujioka, M

    1996-01-01

    Most mammalian non-nucleic acid methyltransferases share three sequence motifs. To gain insight into the S-adenosyl-methionine (AdoMet)-binding site of guanidinoacetate methyltransferase, we mutated several conserved residues that are found in or near motifs I and II. Conversion of either of two glycine residues of motif I (Gly67 and Gly69) to an alanine resulted in an inactive enzyme. These enzymes, although having UV absorption, fluorescence and far-UV CD spectra virtually identical with those of the wild-type enzyme, seem to be conformationally different from the wild-type enzyme as judged by near-UV CD spectra and the extent of urea denaturation, and are apparently not capable of binding AdoMet. Mutation of Tyr136 of motif II to a valine resulted in a decrease in Kcat/Km values for substrates. Changing this residue to a phenylalanine caused only a minor change in Kcat/Km for AdoMet. This suggests that the aromatic side chain stabilizes the binding of AdoMet. Mutagenic changes of Glu89, which is the residue corresponding to the conserved acidic residue on the C-terminal side of motif I, indicated its contribution to AdoMet binding. These results are consistent with the idea that both motifs I and II are crucial in forming the AdoMet binding site of guanidinoacetate methyltransferase. PMID:8694756

  8. Amino acid residues interacting with both the bound quinone and coenzyme, pyrroloquinoline quinone, in Escherichia coli membrane-bound glucose dehydrogenase.

    PubMed

    Mustafa, Golam; Ishikawa, Yoshinori; Kobayashi, Kazuo; Migita, Catharina T; Elias, M D; Nakamura, Satsuki; Tagawa, Seiichi; Yamada, Mamoru

    2008-08-01

    The Escherichia coli membrane-bound glucose dehydrogenase (mGDH) as the primary component of the respiratory chain possesses a tightly bound ubiquinone (UQ) flanking pyrroloquinoline quinone (PQQ) as a coenzyme. Several mutants for Asp-354, Asp-466, and Lys-493, located close to PQQ, that were constructed by site-specific mutagenesis were characterized by enzymatic, pulse radiolysis, and EPR analyses. These mutants retained almost no dehydrogenase activity or ability of PQQ reduction. CD and high pressure liquid chromatography analyses revealed that K493A, D466N, and D466E mutants showed no significant difference in molecular structure from that of the wild-type mGDH but showed remarkably reduced content of bound UQ. A radiolytically generated hydrated electron (e(aq)(-)) reacted with the bound UQ of the wild enzyme and K493R mutant to form a UQ neutral semiquinone with an absorption maximum at 420 nm. Subsequently, intramolecular electron transfer from the bound UQ semiquinone to PQQ occurred. In K493R, the rate of UQ to PQQ electron transfer is about 4-fold slower than that of the wild enzyme. With D354N and D466N mutants, on the other hand, transient species with an absorption maximum at 440 nm, a characteristic of the formation of a UQ anion radical, appeared in the reaction of e(aq)(-), although the subsequent intramolecular electron transfer was hardly affected. This indicates that D354N and D466N are prevented from protonation of the UQ semiquinone radical. Moreover, EPR spectra showed that mutations on Asp-466 or Lys-493 residues changed the semiquinone state of bound UQ. Taken together, we reported here for the first time the existence of a semiquinone radical of bound UQ in purified mGDH and the difference in protonation of ubisemiquinone radical because of mutations in two different amino acid residues, located around PQQ. Furthermore, based on the present results and the spatial arrangement around PQQ, Asp-466 and Lys-493 are suggested to interact both

  9. 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

  10. 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

  11. 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)

  12. Interconversion of ketoprofen recognition in firefly luciferase-catalyzed enantioselective thioesterification reaction using from Pylocoeria miyako (PmL) and Hotaria parvura (HpL) just by mutating two amino acid residues.

    PubMed

    Kato, Dai-ichiro; Hiraishi, Yoshihiro; Maenaka, Mika; Yokoyama, Keisuke; Niwa, Kazuki; Ohmiya, Yoshihiro; Takeo, Masahiro; Negoro, Seiji

    2013-11-01

    We identified the critical amino acid residues for substrate recognition using two firefly luciferases from Pylocoeria miyako (PmL) and Hotaria parvura (HpL), as these two luciferase enzymes exhibit different activities toward ketoprofen. Specifically, PmL can catalyze the apparent enantioselective thioesterification reaction, while HpL cannot. By comparing the amino acid sequences around the active site, we identified two residues (I350 and M397 in PmL and F351 and S398 in HpL) that were different between the two enzymes, and the replacement of these amino acids resulted in changing the ketoprofen recognition pattern. The inactive HpL was converted to the active enzyme toward ketoprofen and vice versa for PmL. These residues also affected the enantioselectivity toward ketoprofen; however, the bioluminescent color was not affected. In addition, using molecular dynamics calculations, the replacement of these two amino acids induced changes in the state of hydrogen bonding between ketoprofen and the S349 side chain through the active site water. As S349 is not considered to influence color tuning, these changes specifically caused the differences in ketoprofen recognition in the enzyme.

  13. 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

  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. 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.

  16. 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

  17. Immunopathogenic and antibacterial effects of H3N2 influenza A virus PB1-F2 map to amino acid residues 62, 75, 79, and 82.

    PubMed

    Alymova, Irina V; Green, Amanda M; van de Velde, Nicholas; McAuley, Julie L; Boyd, Kelli L; Ghoneim, Hazem E; McCullers, Jonathan A

    2011-12-01

    The influenza A virus protein PB1-F2 has been linked to the pathogenesis of both primary viral and secondary bacterial infections. H3N2 viruses have historically expressed full-length PB1-F2 proteins with either proinflammatory (e.g., from influenza A/Hong Kong/1/1968 virus) or noninflammatory (e.g., from influenza A/Wuhan/359/1995 virus) properties. Using synthetic peptides derived from the active C-terminal portion of the PB1-F2 protein from those two viruses, we mapped the proinflammatory domain to amino acid residues L62, R75, R79, and L82 and then determined the role of that domain in H3N2 influenza virus pathogenicity. PB1-F2-derived peptides containing that proinflammatory motif caused significant morbidity, mortality, and pulmonary inflammation in mice, manifesting as increased acute lung injury and the presence of proinflammatory cytokines and inflammatory cells in the lungs compared to peptides lacking this motif, and better supported bacterial infection with Streptococcus pneumoniae. Infections of mice with an otherwise isogenic virus engineered to contain this proinflammatory sequence in PB1-F2 demonstrated increased morbidity resulting from primary viral infections and enhanced development of secondary bacterial pneumonia. The presence of the PB1-F2 noninflammatory (P62, H75, Q79, and S82) sequence in the wild-type virus mediated an antibacterial effect. These data suggest that loss of the inflammatory PB1-F2 phenotype that supports bacterial superinfection during adaptation of H3N2 viruses to humans, coupled with acquisition of antibacterial activity, contributes to the relatively diminished frequency of severe infections seen with seasonal H3N2 influenza viruses in recent decades compared to their first 2 decades of circulation.

  18. 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.

  19. Functional consequences of sulfhydryl modification of the γ-aminobutyric acid transporter 1 at a single solvent-exposed cysteine residue.

    PubMed

    Omoto, Jaison J; Maestas, Matthew J; Rahnama-Vaghef, Ali; Choi, Ye E; Salto, Gerardo; Sanchez, Rachel V; Anderson, Cynthia M; Eskandari, Sepehr

    2012-12-01

    The aims of this study were to optimize the experimental conditions for labeling extracellularly oriented, solvent-exposed cysteine residues of γ-aminobutyric acid transporter 1 (GAT1) with the membrane-impermeant sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) and to characterize the functional and pharmacological consequences of labeling on transporter steady-state and presteady-state kinetic properties. We expressed human GAT1 in Xenopus laevis oocytes and used radiotracer and electrophysiological methods to assay transporter function before and after sulfhydryl modification with MTSET. In the presence of NaCl, transporter exposure to MTSET (1-2.5 mM for 5-20 min) led to partial inhibition of GAT1-mediated transport, and this loss of function was completely reversed by the reducing reagent dithiothreitol. MTSET treatment had no functional effect on the mutant GAT1 C74A, whereas the membrane-permeant reagents N-ethylmaleimide and tetramethylrhodamine-6-maleimide inhibited GABA transport mediated by GAT1 C74A. Ion replacement experiments indicated that MTSET labeling of GAT1 could be driven to completion when valproate replaced chloride in the labeling buffer, suggesting that valproate induces a GAT1 conformation that significantly increases C74 accessibility to the extracellular fluid. Following partial inhibition by MTSET, there was a proportional reduction in both the presteady-state and steady-state macroscopic signals, and the functional and pharmacological properties of the remaining signals were indistinguishable from those of unlabeled GAT1. Therefore, covalent modification of GAT1 at C74 results in completely nonfunctional as well as electrically silent transporters.

  20. Short-chain fatty acids produced in vitro from fibre residues obtained from mixed diets containing different breads and in human faeces during the ingestion of the diets.

    PubMed

    Wisker, E; Daniel, M; Rave, G; Feldheim, W

    2000-07-01

    It was studied whether the type of bread (i.e. a low-fibre wheat-rye mixed bread and coarse or fine wholemeal rye bread) either as part of a diet or alone, had an influence on the short-chain fatty acids (SCFA) produced during in vitro fermentation. Fermentation substrates were dietary fibre residues obtained from diets and breads. In addition, it was investigated whether the faecal SCFA pattern in the inoculum donors, who ingested the experimental diets, could be predicted by in vitro fermentation. Yields of SCFA in vitro were 0.51-0.62 g/g fermented polysaccharide. In vitro, the molar ratios of butyrate were higher for the two high-fibre diets containing coarse or fine wholemeal bread than for the low fibre diet containing wheat-rye mixed bread; the difference was significant for the coarse (P < 0.01), but not for the fine bread diet (P = 0.0678). The coarse wholemeal bread alone produced a higher molar ratio of butyrate than the fine wholemeal bread (P < 0.05) and the wheat-rye mixed bread (P < 0.01). Ingestion by the inoculum donors of the diets containing wholemeal bread led to higher faecal butyrate ratios (molar ratios: coarse bread diet 19.6, fine bread diet 17.7) compared with the wheat-rye mixed bread-containing diet (14.9), but the differences between the diets were not significant. For the diets investigated, there were no significant differences between faecal and in vitro SCFA patterns.

  1. Solution synthesis and biological activity of human pleiotrophin, a novel heparin-binding neurotrophic factor consisting of 136 amino acid residues with five disulfide bonds.

    PubMed

    Inui, T; Nakao, M; Nishio, H; Nishiuchi, Y; Kojima, S; Muramatsu, T; Kimura, T

    2000-05-01

    Human pleiotrophin (hPTN), a novel heparin-binding neurotrophic factor consisting of 136 amino acid residues with five intramolecular disulfide bonds, was synthesized by solution procedure in order to demonstrate the utility of our strategy using our newly developed solvent system, a mixture of trifluoroethanol (TFE) and dichloromethane (DCM) or chloroform (CHL). The final protected peptide was synthesized by coupling two larger protected intermediates, Boc-(1-64)-OH and H-(65-136)-OBzl, in CHL/TFE (3:1; v/v) using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) in the presence of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt). After removal of all protecting groups using the HF procedure followed by treatment with Hg(OAc)2, the fully deprotected peptide was subjected to an oxidative folding reaction. The product was confirmed as having the correct disulfide structure by examining the cystine peptides obtained by enzymatic digestions, and as possessing the same biological activities as those of the natural product. The N- and C-terminal half domains (1-64 and 65-136) were also synthesized, and measurement of their biological activities indicated that the C-terminal half domain displays almost all the activities of the full-length molecule, whereas the N-terminal half domain shows almost no activity. From these results, we were able to confirm that the C-terminal half domain is responsible for the expression of biological activities in the same manner as human midkine (hMK), another heparin-binding neurotrophic growth factor.

  2. Variation of physicochemical properties of drinking water treatment residuals and Phoslock(®) induced by fulvic acid adsorption: Implication for lake restoration.

    PubMed

    Wang, Changhui; Jiang, He-Long; Xu, Huacheng; Yin, Hongbin

    2016-01-01

    The use of phosphorus (P) inactivating agents to reduce internal P loading from sediment for lake restoration has attracted increasing attention. Reasonably, the physicochemical properties of P inactivating agents may vary with the interference of various environmental factors, leading to the change of control effectiveness and risks. In this study, the effect of fulvic acid (FA) adsorption on the properties of two agents, drinking water treatment residuals (DWTRs) and Phoslock®, was investigated. The results showed that after adsorption, there was little change for the main structures of DWTRs and Phoslock®, but the thermostability of Phoslock®, as well as the particle size and settleability of the two agents decreased. The specific surface area and pore volume of DWTRs also decreased, while those of Phoslock® increased. Further analysis indicated that aluminum and iron in DWTRs were stable during FA adsorption, but a substantial increase of lanthanum release from Phoslock® was observed, in particular at first (P < 0.01). Moreover, the P immobilization capability of DWTRs had little change after FA adsorption, while the capability of Phoslock® after FA adsorption decreased in solutions (P < 0.001) and sediments (P < 0.1); interestingly, from the view of engineering application, the performance of Phoslock® was not substantially affected. Overall, each P inactivating agent had its own particular responses of the physicochemical properties to environment factors, and detailed investigations on the applicability of each agent were essential before practical application.

  3. Two-dimensional magnetization exchange spectroscopy of Anabaena 7120 ferredoxin. Nuclear Overhauser effect and electron self-exchange cross peaks from amino acid residues surrounding the Fe-2S* cluster

    SciTech Connect

    Skjeldal, L.; Westler, W.M.; Oh, Byungha; Krezel, A.M.; Markley, J.L.; Holden, H.M.; Jacobson, B.L.; Rayment, I. Univ. of Wisconsin, Madison )

    1991-07-30

    Hyperfine {sup 1}H NMR signals of the 2 Fe-2S* vegetative ferredoxin from Anabaena 7,120 have been studied by two-dimensional (2D) magnetization exchange spectroscopy. The rapid longitudinal relaxation rates of these signals required the use of very short nuclear Overhauser effect (NOE) mixing times (0.5-20 ms). The resulting pattern of NOE cross-relaxation peaks when combined with previous 1D NOE results led to elucidation of the carbon-bound proton spin systems from each of the four cysteines ligated to the 2Fe-2S* cluster in the reduced ferredoxin. Additional NOE cross peaks were observed that provide information about other amino acid residues that interact with the iron-sulfur cluster. NOE cross peaks were assigned tentatively to Leu{sup 27}, Arg{sup 42}, and Ala{sup 43} on the basis of the X-ray coordinates of oxidized Anabaena 7,120 ferredoxin. Three chemical exchange cross peaks were detected in magnetization exchange spectra of half-reduced ferredoxin and assigned to the {sup 1}H{sup {alpha}} protons of Cys{sup 49} and Cys{sup 79} (both of whose sulfur atoms are ligated to Fe(III)) and Arg{sup 42} (whose amide nitrogen is hydrogen-bonded to one of the inorganic sulfurs of the 2Fe-2S* cluster). The chemical exchange cross peaks provide a means of extending assignments in the spectrum of reduced ferredoxin to assignments in the spectrum of the oxidized protein. The results suggest that 2D magnetization exchange spectroscopy employing short mixing times will be useful for the assignment and characterization of hyperfine {sup 1}H peaks in a variety of paramagnetic proteins.

  4. 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.

  5. An acidic amino acid transmembrane helix 10 residue conserved in the neurotransmitter:sodium:symporters is essential for the formation of the extracellular gate of the γ-aminobutyric acid (GABA) transporter GAT-1.

    PubMed

    Ben-Yona, Assaf; Kanner, Baruch I

    2012-03-01

    GAT-1 mediates transport of GABA together with sodium and chloride in an electrogenic process enabling efficient GABAergic transmission. Biochemical and modeling studies based on the structure of the bacterial homologue LeuT are consistent with a mechanism whereby the binding pocket is alternately accessible to either side of the membrane and which predicts that the extracellular part of transmembrane domain 10 (TM10) exhibits aqueous accessibility in the outward-facing conformation only. In this study we have engineered cysteine residues in the extracellular half of TM10 of GAT-1 and probed their state-dependent accessibility to sulfhydryl reagents. In three out of four of the accessible cysteine mutants, the inhibition of transport by a membrane impermeant sulfhydryl reagent was diminished under conditions expected to increase the proportion of inward-facing transporters, such as the presence of GABA together with the cotransported ions. A conserved TM10 aspartate residue, whose LeuT counterpart participates in a "thin" extracellular gate, was found to be essential for transport and only the D451E mutant exhibited residual transport activity. D451E exhibited robust sodium-dependent transient currents with a voltage-dependence indicative of an increased apparent affinity for sodium. Moreover the accessibility of an endogenous cysteine to a membrane impermeant sulfhydryl reagent was enhanced by the D451E mutation, suggesting that sodium binding promotes an outward-facing conformation of the transporter. Our results support the idea that TM10 of GAT-1 lines an accessibility pathway from the extracellular space into the binding pocket and plays a role in the opening and closing of the extracellular transporter gate.

  6. 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

  7. The evolutionary differentiation of two histone H2A.Z variants in chordates (H2A.Z-1 and H2A.Z-2) is mediated by a stepwise mutation process that affects three amino acid residues

    PubMed Central

    Eirín-López, José M; González-Romero, Rodrigo; Dryhurst, Deanna; Ishibashi, Toyotaka; Ausió, Juan

    2009-01-01

    Background The histone H2A family encompasses the greatest number of core histone variants of which the replacement variant H2A.Z is currently one of the most heavily studied. No clear mechanism for the functional variability that H2A.Z imparts to chromatin has yet been proposed. While most of the past studies have referred to H2A.Z generically as a single protein, in vertebrates it is a mixture of two protein forms H2A.Z-1 (previously H2A.Z) and H2A.Z-2 (previously H2A.F/Z or H2A.V) that differ by three amino acids. Results We have performed an extensive study on the long-term evolution of H2A.Z across metazoans with special emphasis on the possible selective mechanisms responsible for the differentiation between H2A.Z-1 and H2A.Z-2. Our results reveal a common origin of both forms early in chordate evolution. The evolutionary process responsible for the differentiation involves refined stepwise mutation change within the codons of the three differential residues. This eventually led to differences in the intensity of the selective constraints acting upon the different H2A.Z forms in vertebrates. Conclusion The results presented in this work definitively reveal that the existence of H2A.Z-1 and H2A.Z-2 is not a whim of random genetic drift. Our analyses demonstrate that H2A.Z-2 is not only subject to a strong purifying selection but it is significantly more evolutionarily constrained than H2A.Z-1. Whether or not the evolutionary drift between H2A.Z-1 and H2A.Z-2 has resulted in a functional diversification of these proteins awaits further research. Nevertheless, the present work suggests that in the process of their differently constrained evolutionary pathways, these two forms may have acquired new or complementary functions. PMID:19193230

  8. 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

  9. 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.

  10. 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

  11. Specificity of the deoxyhypusine hydroxylase-eukaryotic translation initiation factor (eIF5A) interaction: identification of amino acid residues of the enzyme required for binding of its substrate, deoxyhypusine-containing eIF5A.

    PubMed

    Kang, Kee Ryeon; Kim, Yeon Sook; Wolff, Edith C; Park, Myung Hee

    2007-03-16

    Deoxyhypusine hydroxylase (DOHH) is a novel metalloenzyme that catalyzes the final step of the post-translational synthesis of hypusine (Nepsilon-(4-amino-2-hydroxybutyl)lysine) in the eukaryotic translation initiation factor 5A (eIF5A). Hypusine synthesis is unique in that it occurs in only one protein, denoting the strict specificity of the modification enzymes toward the substrate protein. The specificity of the interaction between eIF5A and DOHH was investigated using human eIF5A (eIF5A-1 isoform) and human recombinant DOHH. DOHH displayed a strong preference for binding the deoxyhypusine-containing form of eIF5A, over the eIF5A precursor or the hypusine-containing eIF5A, indicating a role for the deoxyhypusine residue in binding. In addition to the deoxyhypusine residue, a large portion of the eIF5A polypeptide (>20-90 amino acids) is required for effective modification by DOHH. We have identified the amino acid residues of DOHH that are critical for substrate binding by alanine substitution of 36 conserved amino acid residues. Of these, alanine substitution at Glu57, Glu90, Glu208, Glu241, Gly63, or Gly214 caused a severe impairment in eIF5A(Dhp) binding, with a complete loss of binding and activity in the E57A and E208A mutant enzymes. Only aspartate substitution mutants, E57D or E208D, retained partial activity and substrate binding, whereas alanine, glutamine, or asparagine mutants did not. These findings support a proposed model of DOHH-eIF5A binding in which the amino group(s) of the deoxyhypusine side chain of the substrate is primarily anchored by gamma-carboxyl groups of Glu57 and Glu208 at the DOHH active site. PMID:17213197

  12. 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.

  13. Key amino acid residues involved in multi-point binding interactions between brazzein, a sweet protein, and the T1R2-T1R3 human sweet receptor.

    PubMed

    Assadi-Porter, Fariba M; Maillet, Emeline L; Radek, James T; Quijada, Jeniffer; Markley, John L; Max, Marianna

    2010-05-14

    The sweet protein brazzein [recombinant protein with sequence identical with the native protein lacking the N-terminal pyroglutamate (the numbering system used has Asp2 as the N-terminal residue)] activates the human sweet receptor, a heterodimeric G-protein-coupled receptor composed of subunits Taste type 1 Receptor 2 (T1R2) and Taste type 1 Receptor 3 (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 in vitro assay. We mutated surface residues of brazzein at three putative interaction sites: site 1 (Loop43), site 2 (N- and C-termini 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 Loop9-19 (site 3). Results from mutagenesis and chimeras of the receptor indicated that brazzein interacts with both T1R2 and T1R3 and that the Venus flytrap 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 brazzein response. The exception, hT1R2 (human T1R2 subunit of the sweet receptor):R217A/hT1R3 (human T1R3 subunit of the sweet receptor), 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

  14. 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.

  15. Evidence that three histidine residues of a base non-specific and adenylic acid preferential ribonuclease from Rhizopus niveus are involved in the catalytic function.

    PubMed

    Ohgi, K; Horiuchi, H; Watanabe, H; Iwama, M; Takagi, M; Irie, M

    1992-07-01

    In order to study the structure-function relationship of an RNase T2 family enzyme, RNase Rh, from Rhizopus niveus, we investigated the roles of three histidine residues by means of site-specific mutagenesis. One of the three histidine residues of RNase RNAP Rh produced in Saccharomyces cerevisiae by recombinant DNA technology was substituted to a phenylalanine or alanine residue. A Phe or Ala mutant enzyme at His46 or His109 showed less than 0.03%, but a mutant enzyme at His104 showed 0.54% of the enzymatic activity of the wild-type enzyme with RNA as a substrate. Similar results were obtained, when ApU was used as a substrate. The binding constant of a Phe mutant enzyme at His46 or His109 towards 2'-AMP decreased twofold, but that at His104 decreased more markedly. Therefore, we assumed that these three histidine residues are components of the active site of RNase Rh, that His104 contributes to some extent to the binding and less to the catalysis, and that the other two histidine residues and one carboxyl group not yet identified are probably involved in the catalysis. We assigned the C-2 proton resonances of His46, His104, and His109 by comparison of the 1H-NMR spectra of the three mutant enzymes containing Phe in place of His with that of the native enzyme, and also determined the individual pKa values for His46 and His104 to be 6.70 and 5.94. His109 was not titrated in a regular way, but the apparent pKa value was estimated to be around 6.3. The fact that addition of 2'-AMP caused a greater effect on the chemical shift of His104 in the 1NMR spectra as compared with those of the other histidine residues, may support the idea described above on the role of His104.

  16. Murine elongation factor 1 alpha (EF-1 alpha) is posttranslationally modified by novel amide-linked ethanolamine-phosphoglycerol moieties. Addition of ethanolamine-phosphoglycerol to specific glutamic acid residues on EF-1 alpha

    SciTech Connect

    Whiteheart, S.W.; Shenbagamurthi, P.; Chen, L.; Cotter, R.J.; Hart, G.W. )

    1989-08-25

    Elongation Factor 1 alpha (EF-1 alpha), an important eukaryotic translation factor, transports charged aminoacyl-tRNA from the cytosol to the ribosomes during poly-peptide synthesis. Metabolic radiolabeling with ({sup 3}H) ethanolamine shows that, in all cells examined, EF-1 alpha is the major radiolabeled protein. Radiolabeled EF-1 alpha has an apparent Mr = 53,000 and a basic isoelectric point. It is cytosolic and does not contain N-linked oligosaccharides. Trypsin digestion of murine EF-1 alpha generated two major ({sup 3}H)ethanolamine-labeled peptides. Three peptides were sequenced and were identical to two distinct regions of the human EF-1 alpha protein. Blank sequencing cycles coinciding with glutamic acid in the human cDNA-derived sequence were also found to release ({sup 3}H)ethanolamine, and compositional analysis of these peptides confirmed the presence of glutamic acid. Dansylation analysis demonstrates that the amine group of the ethanolamine is blocked. These results indicate that EF-1 alpha is posttranslationally modified by the covalent attachment of ethanolamine via an amide bond to at least two specific glutamic acid residues (Glu-301 and Glu-374). The hydroxyl group of the attached ethanolamine was shown by mass spectrometry and compositional analysis, to be further modified by the addition of a phosphoglycerol unit. This novel posttranslational modification may represent an important alteration of EF-1 alpha, comparable to the regulatory effects of posttranslational methylation of EF-1 alpha lysine residues.

  17. Correlation of loss of activity of human aldehyde dehydrogenase with reaction of bromoacetophenone with glutamic acid-268 and cysteine-302 residues. Partial-sites reactivity of aldehyde dehydrogenase.

    PubMed Central

    Abriola, D P; MacKerell, A D; Pietruszko, R

    1990-01-01

    Bromoacetophenone (2-bromo-1-phenylethanone) has been characterized as an affinity reagent for human aldehyde dehydrogenase (EC 1.2.1.3) [MacKerell, MacWright & Pietruszko (1986) Biochemistry 25, 5182-5189], and has been shown to react specifically with the Glu-268 residue [Abriola, Fields, Stein, MacKerell & Pietruszko (1987) Biochemistry 26, 5679-5684] with an apparent inactivation stoichiometry of two molecules of bromoacetophenone per molecule of enzyme. The specificity of bromoacetophenone for reaction with Glu-268, however, is not absolute, owing to the extreme reactivity of this reagent. When bromo[14C]acetophenone was used to label the human cytoplasmic E1 isoenzyme radioactively and tryptic fragmentation was carried out, peptides besides that containing Glu-268 were found to have reacted with reagent. These peptides were purified by h.p.l.c. and analysed by sequencing and scintillation counting to quantify radioactive label in the material from each cycle of sequencing. Reaction of bromoacetophenone with the aldehyde dehydrogenase molecule during enzyme activity loss occurs with two residues, Glu-268 and Cys-302. The activity loss, however, appears to be proportional to incorporation of label at Glu-268. The large part of incorporation of label at Cys-302 occurs after the activity loss is essentially complete. With both Glu-268 and Cys-302, however, the incorporation of label stops after one molecule of bromoacetophenone has reacted with each residue. Reaction with other residues continues after activity loss is complete. PMID:1968743

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

    SciTech Connect

    Vlahos, C.J.; Dekker, E.E.

    1987-05-01

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

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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

  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%.

  5. Three amino acid residues in the envelope of human immunodeficiency virus type 1 CRF07_BC regulate viral neutralization susceptibility to the human monoclonal neutralizing antibody IgG1b12.

    PubMed

    Nie, Jianhui; Zhao, Juan; Chen, Qingqing; Huang, Weijin; Wang, Youchun

    2014-10-01

    The CD4 binding site (CD4bs) of envelope glycoprotein (Env) is an important conserved target for anti-human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12 (b12) could recognize conformational epitopes that overlap the CD4bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4bs epitopes.

  6. Fatty acid profile in milk from goats, Capra aegagrus hircus, exposed to perchlorate and its relationship with perchlorate residues in human milk.

    PubMed

    Cheng, Qiuqiong; Smith, Ernest E; Kirk, Andrea B; Liu, Fujun; Boylan, Lee Mallory; McCarty, Michael E; Hart, Sybil; Dong, Linxia; Cobb, George P; Jackson, W Andrew; Anderson, Todd A

    2007-10-01

    Polyunsaturated fatty acids (PUFA) in milk are vital for normal growth and development of infant mammals. Changes in fatty acid composition were observed in milk fat from goats dosed with perchlorate (0.1 and 1 mg/kg body weight/day) for 31 days, but the effect was not persistent. Adaptation may be induced in these goats to compensate for the perchlorate effect. In an analysis of fatty acid composition in human milk samples, a weak negative correlation was observed between perchlorate concentrations and total PUFA in 38 human milk samples.

  7. 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

  8. Residue determination of plant activator benzo-1,2,3-thiadiazole-7-carboxylic acid 2-benzoyloxyethyl ester in water, soil, and vegetable by gas chromatography with tandem mass spectrometry.

    PubMed

    Shuying, Li; Yihu, Wang; Qianqian, Sun; Wenjun, Gui; Guonian, Zhu

    2015-06-01

    This study aimed to develop a rapid, specific and sensitive method for the residue determination of benzo-1,2,3-thiadiazole-7-carboxylic acid 2-benzoyloxyethyl ester in soil, vegetable, and water by using gas chromatography with tandem mass spectrometry. During the extraction procedure, modified quick, easy, cheap, effective, rugged, and safe method using acetonitrile was compared with classical oscillating extraction using ethyl acetate and acetone, respectively. Before injection, a solid-phase extraction cartridge of Bond Elut Florisil was used for sample clean-up. The method was fully validated and showed satisfactory linearity (r(2) > 0.99) over the range to be assayed (10-1000 ng/mL), with the limits of detection ranging from 0.092-0.229 ng/mL and the limits of quantification ranging from 0.307-0.763 ng/mL. Recovery values at the spiked concentrations of 10, 100 and 1000 ng/g varied from 85.9-109.3%, 81.0-108.2%, 74.2-113.4% for water, soil and vegetable, respectively, with the maximum relative standard deviations of 12.2%. Results indicated that the established modified quick, easy, cheap, effective, rugged, and safe method coupled to gas chromatography with tandem mass spectrometry was promising for the residue monitoring of benzo-1,2,3-thiadiazole-7-carboxylic acid 2-benzoyloxyethyl ester in the environment and vegetable products.

  9. 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

  10. 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

  11. Lysyl-tRNA synthetase from Bacillus stearothermophilus: the Trp314 residue is shielded in a non-polar environment and is responsible for the fluorescence changes observed in the amino acid activation reaction.

    PubMed

    Takita, Teisuke; Nakagoshi, Makoto; Inouye, Kuniyo; Tonomura, Ben'ichiro

    2003-01-24

    Three Trp variants of lysyl-tRNA synthetase from Bacillus stearothermophilus, in which either one or both of the two Trp residues within the enzyme (Trp314 and Trp332) were substituted by a Phe residue, were produced by site-directed mutagenesis without appreciable loss of catalytic activity. The following two phenomena were observed with W332F and with the wild-type enzyme, but not with W314F: (1) the addition of L-lysine alone decreased the protein fluorescence of the enzyme, but the addition of ATP alone did not; (2) the subsequent addition of ATP after the addition of excess L-lysine restored the fluorescence to its original level. Fluorometry under various conditions and UV-absorption spectroscopy revealed that Trp314, which was about 20A away from the lysine binding site and was shielded in a non-polar environment, was solely responsible for the fluorescence changes of the enzyme in the L-lysine activation reaction. Furthermore, the microenvironmental conditions around the residue were made more polar upon the binding of L-lysine, though its contact with the solvent was still restricted. It was suggested that Trp314 was located in a less polar environment than was Trp332, after comparison of the wavelengths at the peaks of fluorescence emission and of the relative fluorescence quantum yields. Trp332 was thought, based on the fluorescence quenching by some perturbants and the chemical modification with N-bromosuccinimide, to be on the surface of the enzyme, whereas Trp314 was buried inside. The UV absorption difference spectra induced by the L-lysine binding indicated that the state of Trp314, including its electrostatic environment, changed during the process, but Trp332 did not change. The increased fluorescence from Trp314 at acidic pH compared with that at neutral pH suggests that carboxylate(s) are in close proximity to the Trp314 residue. PMID:12507472

  12. Maximum Allowed Solvent Accessibilites of Residues in Proteins

    PubMed Central

    Tien, Matthew Z.; Meyer, Austin G.; Sydykova, Dariya K.; Spielman, Stephanie J.; Wilke, Claus O.

    2013-01-01

    The relative solvent accessibility (RSA) of a residue in a protein measures the extent of burial or exposure of that residue in the 3D structure. RSA is frequently used to describe a protein's biophysical or evolutionary properties. To calculate RSA, a residue's solvent accessibility (ASA) needs to be normalized by a suitable reference value for the given amino acid; several normalization scales have previously been proposed. However, these scales do not provide tight upper bounds on ASA values frequently observed in empirical crystal structures. Instead, they underestimate the largest allowed ASA values, by up to 20%. As a result, many empirical crystal structures contain residues that seem to have RSA values in excess of one. Here, we derive a new normalization scale that does provide a tight upper bound on observed ASA values. We pursue two complementary strategies, one based on extensive analysis of empirical structures and one based on systematic enumeration of biophysically allowed tripeptides. Both approaches yield congruent results that consistently exceed published values. We conclude that previously published ASA normalization values were too small, primarily because the conformations that maximize ASA had not been correctly identified. As an application of our results, we show that empirically derived hydrophobicity scales are sensitive to accurate RSA calculation, and we derive new hydrophobicity scales that show increased correlation with experimentally measured scales. PMID:24278298

  13. Disinfection of wastewater by hydrogen peroxide or peracetic acid: development of procedures for measurement of residual disinfectant and application to a physicochemically treated municipal effluent.

    PubMed

    Wagner, Monika; Brumelis, Daina; Gehr, Ronald

    2002-01-01

    The Montreal Urban Community Wastewater Treatment Plant (MUCWTP) located in Montreal. Quebec, Canada, uses physicochemical treatment processes prior to discharging wastewater into the St. Lawrence River via an outfall tunnel of 2 hours retention time. Although chlorination facilities exist, they are not being used, and the MUCWTP is seeking alternative methods for disinfection to achieve a 2- to 3-log fecal coliform reduction. Liquid chemical disinfectants were attractive options because of their low capital costs. This led to an investigation of the feasibility of using hydrogen peroxide or peracetic acid. A method for measuring peroxycompounds (hydrogen peroxide or peracetic acid plus hydrogen peroxide) was developed using the peroxidase-based oxidation of 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfuric acid) diammonium salt (ABTS) with hydrogen peroxide. The validity of the method was confirmed using effluent from the MUCWTP. Recovery was higher than 90% for peracetic acid levels as low as 1.0 mg/L. Quenching of hydrogen peroxide was achieved with 50-mg/L catalase; quenching of peracetic acid was achieved with 100 mg/L of sodium thiosulfate, followed by 50 mg/L of catalase. Batch disinfection tests were conducted on MUCWTP effluent. Hydrogen peroxide and peracetic acid in wastewater over time could be modeled as a second-order decay, with the decay "constant" being a function of the initial concentration of peroxycompounds. This function was the same for both hydrogen peroxide and peracetic acid, possibly indicating similar decomposition pathways in wastewater matrices. Disinfection was modeled using a modified Hom equation. Required doses of hydrogen peroxide to reach the target fecal coliform levels ranged from 106 to 285 mg/L, with the higher doses occurring when ferric chloride instead of alum was used as the coagulant. Hence, hydrogen peroxide was infeasible as a disinfectant for this application. On the other hand, the peracetic acid dose needed to

  14. 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

  15. 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.

  16. Jamaicensamide A, a Peptide Containing β-Amino-α-keto and Thiazole-Homologated η-Amino Acid Residues from the Sponge Plakina jamaicensis.

    PubMed

    Jamison, Matthew T; Molinski, Tadeusz F

    2016-09-23

    A new cyclic peptide, jamaicensamide A, composed of six amino acids, including a thiazole-homologated amino acid, was isolated from the Bahamian sponge Plakina jamaicensis, along with known compounds bitungolide A and franklinolide A. The structure of the title peptide was solved by integrated analysis of MS, 1D and 2D NMR data, oxidation-hydrolyses to α-amino acids, and their stereodetermination by Marfey's method. The close structural resemblance of Western Atlantic-derived jamaicensamide A to known Western Pacific-derived peptides of lithistid sponges in the genus Theonella and Discodermia suggests a common origin: the symbiotic bacterium Entotheonella sp., a so-called "talented producer" responsible for biosynthesis of most Theonella-associated peptides. Similar natural products from sponges of disparate genera evince the likelihood that these invertebrates harbor the same or a very similar symbiont. PMID:27547840

  17. 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 Central

    Ng, Ivan H. W.; Chan, Kitti W. K.; Zhao, Yongqian; Ooi, Eng Eong; Lescar, Julien; Jans, David A.; Forwood, Jade K.

    2016-01-01

    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

  18. 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

  19. 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 ...

  20. 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.

  1. Srchi24, A Chitinase Homolog Lacking an Essential Glutamic Acid Residue for Hydrolytic Activity, Is Induced during Nodule Development on Sesbania rostrata1

    PubMed Central

    Goormachtig, Sofie; Van de Velde, Willem; Lievens, Sam; Verplancke, Christa; Herman, Sylvia; De Keyser, Annick; Holsters, Marcelle

    2001-01-01

    The interaction between the tropical legume Sesbania rostrata and the bacterium Azorhizobium caulinodans results in the formation of nodules on both stem and roots. Stem nodulation was used as a model system to isolate early markers by differential display. One of them, Srchi24 is a novel early nodulin whose transcript level increased already 4 h after inoculation. This enhancement depended on Nod factor-producing bacteria. Srchi24 transcript levels were induced also by exogenous cytokinins. In situ hybridization and immunolocalization experiments showed that Srchi24 transcripts and proteins were present in the outermost cortical cell layers of the developing nodules. Sequence analyses revealed that Srchi24 is similar to class III chitinases, but lacks an important catalytic glutamate residue. A fusion between a maltose-binding protein and Srchi24 had no detectable hydrolytic activity. A function in nodulation is proposed for the Srchi24 protein. PMID:11553736

  2. Augmentation of the Lipopolysaccharide-Neutralizing Activities of Human Cathelicidin CAP18/LL-37-Derived Antimicrobial Peptides by Replacement with Hydrophobic and Cationic Amino Acid Residues

    PubMed Central

    Nagaoka, Isao; Hirota, Satoko; Niyonsaba, François; Hirata, Michimasa; Adachi, Yoshiyuki; Tamura, Hiroshi; Tanaka, Shigenori; Heumann, Didier

    2002-01-01

    Mammalian myeloid and epithelial cells express various peptide antibiotics (such as defensins and cathelicidins) that contribute to the innate host defense against invading microorganisms. Among these peptides, human cathelicidin CAP18/LL-37 (L1 to S37) possesses not only potent antibacterial activity against gram-positive and gram-negative bacteria but also the ability to bind to gram-negative lipopolysaccharide (LPS) and neutralize its biological activities. In this study, to develop peptide derivatives with improved LPS-neutralizing activities, we utilized an 18-mer peptide (K15 to V32) of LL-37 as a template and evaluated the activities of modified peptides by using the CD14+ murine macrophage cell line RAW 264.7 and the murine endotoxin shock model. By replacement of E16 and K25 with two L residues, the hydrophobicity of the peptide (18-mer LL) was increased, and by further replacement of Q22, D26, and N30 with three K residues, the cationicity of the peptide (18-mer LLKKK) was enhanced. Among peptide derivatives, 18-mer LLKKK displayed the most powerful LPS-neutralizing activity: it was most potent at binding to LPS, inhibiting the interaction between LPS and LPS-binding protein, and attaching to the CD14 molecule, thereby suppressing the binding of LPS to CD14+ cells and attenuating production of tumor necrosis factor alpha (TNF-α) by these cells. Furthermore, in the murine endotoxin shock model, 18-mer LLKKK most effectively suppressed LPS-induced TNF-α production and protected mice from lethal endotoxin shock. Together, these observations indicate that the LPS-neutralizing activities of the amphipathic human CAP18/LL-37-derived 18-mer peptide can be augmented by modifying its hydrophobicity and cationicity, and that 18-mer LLKKK is the most potent of the peptide derivatives, with therapeutic potential for gram-negative bacterial endotoxin shock. PMID:12204946

  3. Prediction of binding modes between protein L-isoaspartyl (D-aspartyl) O-methyltransferase and peptide substrates including isomerized aspartic acid residues using in silico analytic methods for the substrate screening.

    PubMed

    Oda, Akifumi; Noji, Ikuhiko; Fukuyoshi, Shuichi; Takahashi, Ohgi

    2015-12-10

    Because the aspartic acid (Asp) residues in proteins are occasionally isomerized in the human body, not only l-α-Asp but also l-β-Asp, D-α-Asp and D-β-Asp are found in human proteins. In these isomerized aspartic acids, the proportion of D-β-Asp is the largest and the proportions of l-β-Asp and D-α-Asp found in human proteins are comparatively small. To explain the proportions of aspartic acid isomers, the possibility of an enzyme able to repair l-β-Asp and D-α-Asp is frequently considered. The protein L-isoaspartyl (D-aspartyl) O-methyltransferase (PIMT) is considered one of the possible repair enzymes for l-β-Asp and D-α-Asp. Human PIMT is an enzyme that recognizes both l-β-Asp and D-α-Asp, and catalyzes the methylation of their side chains. In this study, the binding modes between PIMT and peptide substrates containing l-β-Asp or D-α-Asp residues were investigated using computational protein-ligand docking and molecular dynamics simulations. The results indicate that carboxyl groups of both l-β-Asp and D-α-Asp were recognized in similar modes by PIMT and that the C-terminal regions of substrate peptides were located in similar positions on PIMT for both the l-β-Asp and D-α-Asp peptides. In contrast, for peptides containing l-α-Asp or D-β-Asp residues, which are not substrates of PIMT, the computationally constructed binding modes between PIMT and peptides greatly differed from those between PIMT and substrates. In the nonsubstrate peptides, not inter- but intra-molecular hydrogen bonds were observed, and the conformations of peptides were more rigid than those of substrates. Thus, the in silico analytical methods were able to distinguish substrates from nonsubstrates and the computational methods are expected to complement experimental analytical methods.

  4. Simultaneous determination of trifloxystrobin and trifloxystrobin acid residue in rice and soil by a modified quick, easy, cheap, effective, rugged, and safe method using ultra high performance liquid chromatography with tandem mass spectrometry.

    PubMed

    Chen, Xixi; Xu, Jun; Liu, Xingang; Tao, Yan; Pan, Xinglu; Zheng, Yongquan; Dong, Fengshou

    2014-07-01

    A sensitive analytical method for the simultaneous determination of trifloxystrobin and its metabolite trifloxystrobin acid in rice including straw, bran, brown rice and soil was developed by using ultra high performance liquid chromatography coupled with tandem mass spectrometry. The fungicide trifloxystrobin and its metabolite trifloxystrobin acid were extracted using acetonitrile with 1% formic acid v/v and subsequently cleaned up by primary secondary amine, octadecylsilane or graphitized carbon black prior to ultra high performance liquid chromatography coupled with tandem mass spectrometry. The determination of two target compounds was achieved in less than 3 min using an electrospray ionization source in positive mode. The limits of detection were below 0.22 μg/kg and the limits of quantification did not exceed 0.74 μg/kg in all matrices, which were much lower than the maximum residue levels established by the Codex Alimentarius Commission. The overall average recoveries in four matrix at three levels (0.1, 1.0 and 5.0 mg/kg) ranged from 74.2 to 107.4% with a relative standard deviations of less than 7.8% (n = 5) for both analytes. The method was demonstrated to be convenient and reliable for the routine monitoring of trifloxystrobin and its metabolite. The developed method was validated and applied for the analysis of degradation study samples.

  5. Role of two amino acid residues' insertion on thermal stability of thermophilic α-amylase AMY121 from a deep sea bacterium Bacillus sp. SCSIO 15121.

    PubMed

    Li, Lizhen; Yang, Jian; Li, Jie; Long, Lijuan; Xiao, Yunzhu; Tian, Xinpeng; Wang, Fazuo; Zhang, Si

    2015-05-01

    α-Amylases from Bacillus licheniformis (BLA) and Bacillus amyloliquefaciens (BAA) are both important industrial enzymes with high similarity in structure but significant differences in thermostability. The mechanisms underlying this discrepancy are still poorly understood. Here, we investigated the role of two amino acids' insertion on the thermostability of these two group amylases. A newly obtained thermophilic amylase AMY121 was found much closer to BLA in both primary structure and enzymological properties. Two amino acids' insertion widespread among BAA group α-amylases was identified as one of the key factors leading to the thermostability differences, since thermostability of insertion mutants (AMY121-EG and AMY121-AA) from AMY121 significantly decreased, while that of deletion mutant from BAA increased. Moreover, we proposed that conformational disturbance caused by insertion mutation might weaken the calcium-binding affinity and consequently decrease the enzyme thermostability.

  6. Redox-linked proton translocation in the b-c1 complex from beef-heart mitochondria reconstituted into phospholipid vesicles. Studies with chemical modifiers of amino acid residues.

    PubMed

    Lorusso, M; Gatti, D; Boffoli, D; Bellomo, E; Papa, S

    1983-12-15

    Possible involvement of polypeptides of b-c1 complex of beef-heart mitochondria in its redox and protonmotive activity has been investigated, by means of chemical modification of amino acid residues in the soluble as well as in the phospholipid-reconstituted b-c1 complex. Treatment of the enzyme with tetranitromethane (C(NO2)4) or with ethoxyformic anhydride (EFA), that modify reversibly tyrosyl and hystidyl residues respectively, resulted in a marked inhibition of electron transport from reduced quinols to cytochrome c. This was accompanied, in b-c1 reconstituted into phospholipid vesicles, by a parallel inhibition of respiratory-linked proton translocation; the H+/e- stoichiometry remained unchanged. Treatment of b-c1 complex with DCCD, that specifically modifies carboxylic groups of glutammic or aspartic residues caused a marked depression of proton translocation in b-c1 vesicles, under conditions where the rate of electron flow in the coupled state, was enhanced. As a consequence the H+/e- stoichiometry was lowered. SDS gel electrophoresis and [14C]DCCD-labelling of the polypeptides of the b-c1 complex showed a major binding of 14C-DCCD to the 8-kDa subunit of the complex and possible cross-linking, induced by DCCD treatment, of polypeptide(s) in the 8-kDa band and the 12-kDa band, with the Fe-s protein of the complex, with the appearance of a new polypeptide band with an apparent molecular mass of about 40 kDa. Involvement of polypeptides of low molecular mass, for which no functional role was so far described, and possibly of the Fe-S protein in the redox-linked proton translocation in b-c1 complex is suggested.

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. Origin of D-amino acids detected in the acid hydrolysates of purified Escherichia coli β-galactosidase.

    PubMed

    Miyamoto, Tetsuya; Sekine, Masae; Ogawa, Tetsuhiro; Hidaka, Makoto; Homma, Hiroshi; Masaki, Haruhiko

    2015-12-10

    In previous report, we detected D-amino acids in the acid hydrolysates of purified recombinant β-galactosidase. Here, we employed a deuterium-hydrogen exchange method to discriminate innate D-amino acids from those generated during hydrolytic incubation. After hydrolysis of β-galactosidase in DCl/D2O, amino acids were derivatized with NBD-F and separated on a reverse-phase column, followed by liquid chromatography-tandem mass spectrometry equipped with a chiral column. Our results show an absence of innate D-amino acid residues in the protein and suggest that the protein undergoes isomerization during a very early stage of hydrolytic incubation.

  13. 5 CFR 534.204 - Previous authorizations.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 5 Administrative Personnel 1 2013-01-01 2013-01-01 false Previous authorizations. 534.204 Section 534.204 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS PAY UNDER OTHER SYSTEMS Student-Employees in Government Hospitals § 534.204 Previous authorizations. The provisions of this subpart do not terminate...

  14. 5 CFR 534.204 - Previous authorizations.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 5 Administrative Personnel 1 2014-01-01 2014-01-01 false Previous authorizations. 534.204 Section 534.204 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS PAY UNDER OTHER SYSTEMS Student-Employees in Government Hospitals § 534.204 Previous authorizations. The provisions of this subpart do not terminate...

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. A phosphoethanolamine transferase specific for the outer 3-deoxy-D-manno-octulosonic acid residue of Escherichia coli lipopolysaccharide. Identification of the eptB gene and Ca2+ hypersensitivity of an eptB deletion mutant.

    PubMed

    Reynolds, C Michael; Kalb, Suzanne R; Cotter, Robert J; Raetz, Christian R H

    2005-06-01

    Addition of a phosphoethanolamine (pEtN) moiety to the outer 3-deoxy-D-manno-octulosonic acid (Kdo) residue of lipopolysaccharide (LPS) in WBB06, a heptose-deficient Escherichia coli mutant, occurs when cells are grown in 5-50 mM CaCl2 (Kanipes, M. I., Lin, S., Cotter, R. J., and Raetz, C. R. H. (2001) J. Biol. Chem. 276, 1156-1163). A Ca2+-induced, membrane-bound enzyme was responsible for the transfer of the pEtN unit to the Kdo domain. We now report the identification of the gene encoding the pEtN transferase. E. coli yhjW was cloned and overexpressed, because it is homologous to a putative pEtN transferase implicated in the modification of the beta-chain heptose residue of Neisseria meningitidis lipo-oligosaccharide (Mackinnon, F. G., Cox, A. D., Plested, J. S., Tang, C. M., Makepeace, K., Coull, P. A., Wright, J. C., Chalmers, R., Hood, D. W., Richards, J. C., and Moxon, E. R. (2002) Mol. Microbiol. 43, 931-943). In vitro assays with Kdo2-4'-[32P]lipid A as the acceptor showed that YhjW (renamed EptB) utilizes phosphatidylethanolamine in the presence of Ca2+ to transfer the pEtN group. Stoichiometric amounts of diacylglycerol were generated during the EptB-catalyzed transfer of pEtN to Kdo2-lipid A. EptB is an inner membrane protein of 574 amino acid residues with five predicted trans-membrane segments within its N-terminal region. An in-frame replacement of eptB with a kanamycin resistance cassette rendered E. coli WBB06 (but not wild-type W3110) hypersensitive to CaCl2 at 5 mM or higher. Ca2+ hypersensitivity was suppressed by excess Mg2+ in the medium or by restoring the LPS core of WBB06. The latter was achieved by reintroducing the waaC and waaF genes, which encode LPS heptosyl transferases I and II, respectively. Our data demonstrate that pEtN modification of the outer Kdo protected cells containing heptose-deficient LPS from damage by high concentrations of Ca2+. Based on its sequence similarity to EptA(PmrC), we propose that the active site of Ept

  20. Seminal role of deletion of amino acid residues in H1-S2 and S-loop regions in eukaryotic β-tubulin investigated from docking and dynamics perspective.

    PubMed

    Selvaa Kumar, C; Gadewal, Nikhil; Mohammed, Sudheer Mm

    2015-08-01

    Tubulin is the fundamental unit of microtubules. It is reported to effect different functions like cell division, chromosomal segregation, motility and intracellular transportation. α- and β-tubulin associate laterally and longitudinally to form protofilaments. Both the subunits are structurally identical to each other except for the deletions reported in H1-S2 and S loop regions in eukaryotic β-tubulin. These deletions mimic the ancestral tubulin protein named Latest Common FtsZ-Tubulin Ancestor (LCFTA) with a shorter S-loop region resulting in weak dimerization. However, in eukaryotic beta tubulin, the significance of this shorter region remains elusive till date. The main objective of this study was to model variants of beta tubulin (βmut1, βmut2 and βmut3) with inserts that lengthened the loop, and to compare them with the native α- and β-subunits to understand their biological significance. Further, one more mutant was modeled with the intention of understanding the counter effect of additional deletion of amino acid residues from both H1-S2 and S-loop regions; this mutant was designated as βmut4. Our study confirms that the insertion of amino acid residues considerably increases the protein-protein interactions in βmut1-βmut1, βmut2-βmut2 and βmut3-βmut3 compared to their native β-subunit. Similarly, the binding affinity of GTP also increases in βmut2 and βmut3 as compared to the wild type. However, these deletions result in decreased protein-protein and ligand interactions in wild beta tubulin and βmut4, as compared to βmut1, βmut2,and βmut3. Therefore, we conclude here that residual inserts in the H1-S2 and S loop sub segments bring about conformational changes in regions critically involved in lateral interactions and in the nucleotide binding site, thus altering the binding affinities between the dimers and the ligands. Regarding the biological importance of such deletions in wild beta tubulin, these deletions result in flexible M

  1. Antioxidant properties of roasted coffee residues.

    PubMed

    Yen, Wen-Jye; Wang, Bor-Sen; Chang, Lee-Wen; Duh, Pin-Der

    2005-04-01

    The antioxidant activity of roasted coffee residues was evaluated. Extraction with four solvents (water, methanol, ethanol, and n-hexane) showed that water extracts of roasted coffee residues (WERCR) produced higher yields and gave better protection for lipid peroxidation. WERCR showed a remarkable protective effect on oxidative damage of protein. In addition, WERCR showed scavenging of free radicals as well as the reducing ability and to bind ferrous ions, indicating that WERCR acts as both primary and secondary antioxidants. The HPLC analyses showed that phenolic acids (chlorogenic acid and caffeic acid) and nonphenolic compounds [caffeine, trigonelline, nicotinic acid, and 5-(hydroxymethyl)furfuraldehyde] remained in roasted coffee residues. These compounds showed a protective effect on a liposome model system. The concentrations of flavonoids and polyphenolic compounds in roasted coffee residues were 8,400 and 20,400 ppm, respectively. In addition, the Maillard reaction products (MRPs) remaining in roasted coffee residues were believed to show antioxidant activity. These data indicate that roasted coffee residues have excellent potential for use as a natural antioxidant source because the antioxidant compounds remained in roasted coffee residues. PMID:15796608

  2. 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.

  3. Amino Acid Residues Critical for the Specificity for Betaine Aldehyde of the Plant ALDH10 Isoenzyme Involved in the Synthesis of Glycine Betaine1[W][OA

    PubMed Central

    Díaz-Sánchez, Ángel G.; González-Segura, Lilian; Mújica-Jiménez, Carlos; Rudiño-Piñera, Enrique; Montiel, Carmina; Martínez-Castilla, León P.; Muñoz-Clares, Rosario A.

    2012-01-01

    Plant Aldehyde Dehydrogenase10 (ALDH10) enzymes catalyze the oxidation of ω-primary or ω-quaternary aminoaldehydes, but, intriguingly, only some of them, such as the spinach (Spinacia oleracea) betaine aldehyde dehydrogenase (SoBADH), efficiently oxidize betaine aldehyde (BAL) forming the osmoprotectant glycine betaine (GB), which confers tolerance to osmotic stress. The crystal structure of SoBADH reported here shows tyrosine (Tyr)-160, tryptophan (Trp)-167, Trp-285, and Trp-456 in an arrangement suitable for cation-π interactions with the trimethylammonium group of BAL. Mutation of these residues to alanine (Ala) resulted in significant Km(BAL) increases and Vmax/Km(BAL) decreases, particularly in the Y160A mutant. Tyr-160 and Trp-456, strictly conserved in plant ALDH10s, form a pocket where the bulky trimethylammonium group binds. This space is reduced in ALDH10s with low BADH activity, because an isoleucine (Ile) pushes the Trp against the Tyr. Those with high BADH activity instead have Ala (Ala-441 in SoBADH) or cysteine, which allow enough room for binding of BAL. Accordingly, the mutation A441I decreased the Vmax/Km(BAL) of SoBADH approximately 200 times, while the mutation A441C had no effect. The kinetics with other ω-aminoaldehydes were not affected in the A441I or A441C mutant, demonstrating that the existence of an Ile in the second sphere of interaction of the aldehyde is critical for discriminating against BAL in some plant ALDH10s. A survey of the known sequences indicates that plants have two ALDH10 isoenzymes: those known to be GB accumulators have a high-BAL-affinity isoenzyme with Ala or cysteine in this critical position, while non GB accumulators have low-BAL-affinity isoenzymes containing Ile. Therefore, BADH activity appears to restrict GB synthesis in non-GB-accumulator plants. PMID:22345508

  4. 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...

  5. 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...

  6. 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...

  7. 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...

  8. Efficient decoration of nanoparticles intended for intracellular drug targeting with targeting residues, as revealed by a new indirect analytical approach.

    PubMed

    Kaplun, Veronika; Stepensky, David

    2014-08-01

    In our previous studies, we developed a nanodrug delivery system (nano-DDS) based on poly(lactic-co-glycolic acid) PLGA nanoparticles encapsulating antigenic peptide and fluorescent marker and 3-stage approach for its decoration with peptide targeting residues. The objectives of this study were (a) to develop methods for quantitative analysis of efficiency of individual conjugation steps and (b) to determine, based on these methods, the efficiency of our 3-stage approach of nano-DDS decoration. We prepared antigenic peptide-loaded PLGA-based nano-DDSs and sequentially decorated them with specific residues using carbodiimide and Click (azide-alkyne Huisgen cycloaddition using copper(I) catalysis) reactions. The extent of cargo encapsulation and release kinetics