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Sample records for acid residues important

  1. 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.

  2. 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

  3. 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.

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

    DOE PAGESBeta

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

    2015-02-27

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

  5. 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

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

    PubMed Central

    2015-01-01

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

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

    PubMed

    Cooper, Gareth R; Moir, Anne

    2011-05-01

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

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

    PubMed Central

    Dangel, Andrew W.; Luther, Amanda

    2014-01-01

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

  9. 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

  10. 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.

  11. 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.

  12. 40 CFR 180.632 - Fenazaquin; import tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Fenazaquin; import tolerances for residues. 180.632 Section 180.632 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... § 180.632 Fenazaquin; import tolerances for residues. (a) General. Import tolerances are established...

  13. 40 CFR 180.632 - Fenazaquin; import tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Fenazaquin; import tolerances for residues. 180.632 Section 180.632 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... § 180.632 Fenazaquin; import tolerances for residues. (a) General. Import tolerances are established...

  14. 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.

  15. Pesticide residues in imported, organic, and "suspect" fruits and vegetables.

    PubMed

    Winter, Carl K

    2012-05-01

    Consumers are frequently urged to avoid imported foods as well as specific fruits and vegetables due to health concerns from pesticide residues and are often encouraged to choose organic fruits and vegetables rather than conventional forms. Studies have demonstrated that while organic fruits and vegetables have lower levels of pesticide residues than do conventional fruits and vegetables, pesticide residues are still frequently detected on organic fruits and vegetables; typical dietary consumer exposure to pesticide residues from conventional fruits and vegetables does not appear to be of health significance. Similarly, research does not demonstrate that imported fruits and vegetables pose greater risks from pesticide residues than do domestic fruits and vegetables or that specific fruits and vegetables singled out as being the most highly contaminated by pesticides should be avoided in their conventional forms. PMID:22335627

  16. 40 CFR 180.180 - Orthoarsenic acid; tolerance for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

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

  17. 40 CFR 180.180 - Orthoarsenic acid; tolerance for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

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

  18. 40 CFR 180.180 - Orthoarsenic acid; tolerance for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

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

  19. Rapid nuclear import of short nucleic acids.

    PubMed

    Kitagawa, Mai; Okamoto, Akimitsu

    2016-10-01

    Exogenous short-chain nucleic acids undergo rapid import into the nucleus. Fluorescence-labeled dT1-13 DNA microinjected into the cytoplasm domain of a HeLa cell was rapidly imported into the nucleus domain within 1min. This is much more rapid than what has been observed for intracellular diffusion of small molecules. In contrast, import of longer nucleic acids with a length of over 30nt into the nucleus was suppressed. PMID:27597250

  20. 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.

  1. 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.

  2. 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.

  3. firestar--advances in the prediction of functionally important residues.

    PubMed

    Lopez, Gonzalo; Maietta, Paolo; Rodriguez, Jose Manuel; Valencia, Alfonso; Tress, Michael L

    2011-07-01

    firestar is a server for predicting catalytic and ligand-binding residues in protein sequences. Here, we present the important developments since the first release of firestar. Previous versions of the server required human interpretation of the results; the server is now fully automatized. firestar has been implemented as a web service and can now be run in high-throughput mode. Prediction coverage has been greatly improved with the extension of the FireDB database and the addition of alignments generated by HHsearch. Ligands in FireDB are now classified for biological relevance. Many of the changes have been motivated by the critical assessment of techniques for protein structure prediction (CASP) ligand-binding prediction experiment, which provided us with a framework to test the performance of firestar. URL: http://firedb.bioinfo.cnio.es/Php/FireStar.php. PMID:21672959

  4. 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

  5. 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.

  6. 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

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

    PubMed

    Fischer, Klaus; Bipp, Hans-Peter

    2005-05-01

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

  8. 40 CFR 180.550 - Arsanilic acid [(4-aminophenyl) arsonic acid]; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

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

  9. Identification of functionally important negatively charged residues in the carboxy end of mouse hepatitis coronavirus A59 nucleocapsid protein.

    PubMed

    Verma, Sandhya; Bednar, Valerie; Blount, Andrew; Hogue, Brenda G

    2006-05-01

    The coronavirus nucleocapsid (N) protein is a multifunctional viral gene product that encapsidates the RNA genome and also plays some as yet not fully defined role in viral RNA replication and/or transcription. A number of conserved negatively charged amino acids are located within domain III in the carboxy end of all coronavirus N proteins. Previous studies suggested that the negatively charged residues are involved in virus assembly by mediating interaction between the membrane (M) protein carboxy tail and nucleocapsids. To determine the importance of these negatively charged residues, a series of alanine and other charged-residue substitutions were introduced in place of those in the N gene within a mouse hepatitis coronavirus A59 infectious clone. Aspartic acid residues 440 and 441 were identified as functionally important. Viruses could not be isolated when both residues were replaced by positively charged amino acids. When either amino acid was replaced by a positively charged residue or both were changed to alanine, viruses were recovered that contained second-site changes within N, but not in the M or envelope protein. The compensatory role of the new changes was confirmed by the construction of new viruses. A few viruses were recovered that retained the D441-to-arginine change and no compensatory changes. These viruses exhibited a small-plaque phenotype and produced significantly less virus. Overall, results from our analysis of a large panel of plaque-purified recovered viruses indicate that the negatively charged residues at positions 440 and 441 are key residues that appear to be involved in virus assembly. PMID:16611893

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

    PubMed

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

    2015-11-01

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

  11. 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

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

    PubMed Central

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

    1994-01-01

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

  13. 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

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

    PubMed

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

    2011-11-01

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

  15. 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.

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

    PubMed

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

    2001-03-01

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

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

    PubMed

    Lavrukhin, O V; Lloyd, R S

    2000-12-12

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

  18. 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.

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

    PubMed

    Archimbault, P; Ambroggi, G; Nicolas, S

    1988-01-01

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

  20. STUDY TO IDENTIFY IMPORTANT PARAMETERS FOR CHARACTERIZING PESTICIDE RESIDUE TRANSFER EFFICIENCIES

    EPA Science Inventory

    To reduce the uncertainty associated with current estimates of children's exposure to pesticides by dermal contact and non-dietary ingestion, residue transfer data are required. Prior to conducting exhaustive studies, a screening study to identify the important parameters for...

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

    PubMed

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

    2016-07-19

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

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

    PubMed

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

    2014-09-01

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

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

    PubMed Central

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

    1971-01-01

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

  4. 7 CFR 29.429 - Disposition of imported tobacco exceeding pesticide residue standards.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Disposition of imported tobacco exceeding pesticide... Disposition of imported tobacco exceeding pesticide residue standards. Within 10 days of the receipt of test results from pesticide test samples, the Director shall notify the importer or entity responsible for...

  5. 7 CFR 29.429 - Disposition of imported tobacco exceeding pesticide residue standards.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Disposition of imported tobacco exceeding pesticide... Disposition of imported tobacco exceeding pesticide residue standards. Within 10 days of the receipt of test results from pesticide test samples, the Director shall notify the importer or entity responsible for...

  6. 7 CFR 29.429 - Disposition of imported tobacco exceeding pesticide residue standards.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Disposition of imported tobacco exceeding pesticide... Disposition of imported tobacco exceeding pesticide residue standards. Within 10 days of the receipt of test results from pesticide test samples, the Director shall notify the importer or entity responsible for...

  7. 7 CFR 29.429 - Disposition of imported tobacco exceeding pesticide residue standards.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Disposition of imported tobacco exceeding pesticide... Disposition of imported tobacco exceeding pesticide residue standards. Within 10 days of the receipt of test results from pesticide test samples, the Director shall notify the importer or entity responsible for...

  8. 7 CFR 29.429 - Disposition of imported tobacco exceeding pesticide residue standards.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Disposition of imported tobacco exceeding pesticide... Disposition of imported tobacco exceeding pesticide residue standards. Within 10 days of the receipt of test results from pesticide test samples, the Director shall notify the importer or entity responsible for...

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

    SciTech Connect

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

    2002-05-01

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

  10. Identification and characterization of important residues in the catalytic mechanism of CMP-Neu5Ac synthetase from Neisseria meningitidis.

    PubMed

    Horsfall, Louise E; Nelson, Adam; Berry, Alan

    2010-07-01

    Sialylated oligosaccharides, present on mammalian outer-cell surfaces, play vital roles in cellular interactions and some bacteria are able to mimic these structures to evade their host's immune system. It would be of great benefit to the study of infectious and autoimmune diseases and cancers, to understand the pathway of sialylation in detail to enable the design and production of inhibitors and mimetics. Sialylation occurs in two stages, the first to activate sialic acid and the second to transfer it to the target molecule. The activation step is catalysed by the enzyme CMP-Neu5Ac synthetase (CNS). Here we used crystal structures of CNS and similar enzymes to predict residues of importance in the CNS from Neisseria meningitidis. Nine residues were mutated to alanine, and the steady-state enzyme kinetic parameters were measured using a continuous assay to detect one of the products of the reaction, pyrophosphate. Mutations that caused the greatest loss in activity included K142A, D211A, D209A and a series of mutations at residue Q104, highlighted from sequence-alignment studies of related enzymes, demonstrating significant roles for these residues in the catalytic mechanism of CNS. The mutations of D211A and D209A provide strong evidence for a previously proposed metal-binding site in the enzyme, and the results of our mutations at residue Q104 lead us to include this residue in the metal-binding site of an intermediate complex. This suggests that, like the sugar-activating lipopolysaccharide-synthesizing CMP-2-keto-3-deoxy-manno-octonic acid synthetase enzyme KdsB, CNS recruits two Mg(2+) ions during the catalytic cycle. PMID:20491913

  11. Interconversion of biologically important carboxylic acids by radiation

    NASA Technical Reports Server (NTRS)

    Negron-Mendoza, A.; Ponnamperuma, C.

    1978-01-01

    The interconversion of a group of biologically important polycarboxylic acids (acetic, fumaric, malic, malonic, succinic, citric, isocitric, tricarballylic) under gamma-ray or ultraviolet radiation was investigated. The formation of high molecular weight compounds was observed in all cases. Succinic acid was formed in almost all radiolysis experiments. Citric, malonic, and succinic acids appeared to be relatively insensitive to radiation. Interconversion of the polycarboxylic acids studied may have occurred under the effect of radiation in the prebiotic earth.

  12. 40 CFR 180.325 - 2-(m-Chlorophenoxy) propionic acid; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

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

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

    Code of Federal Regulations, 2010 CFR

    2010-07-01

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

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

    Code of Federal Regulations, 2011 CFR

    2011-07-01

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

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

    Code of Federal Regulations, 2013 CFR

    2013-07-01

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

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

    Code of Federal Regulations, 2014 CFR

    2014-07-01

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

  17. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

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

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

    PubMed

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

    2006-01-01

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

  19. 40 CFR 180.318 - 4-(2-Methyl-4-chlorophenoxy) butyric acid; tolerance for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

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

  20. Alanine-Scanning Mutational Analysis of Durancin GL Reveals Residues Important for Its Antimicrobial Activity.

    PubMed

    Ju, Xingrong; Chen, Xinquan; Du, Lihui; Wu, Xueyou; Liu, Fang; Yuan, Jian

    2015-07-22

    Durancin GL is a novel class IIa bacteriocin with 43 residues produced by Enterococcus durans 41D. This bacteriocin demonstrates narrow inhibition spectrum and potent antimicrobial activity against several Listeria monocytogenes strains, including nisin-resistant L. monocytogenes NR30. A systematic alanine-scanning mutational analysis with site-directed mutagenesis was performed to analyze durancin GL residues important for antimicrobial activity and specificity. Results showed that three mutations lost their antimicrobial activity, ten mutations demonstrated a decreased effect on the activity, and seven mutations exhibited relatively high activity. With regard to inhibitory spectrum, four mutants demonstrated a narrower antimicrobial spectrum than wild-type durancin GL. Another four mutants displayed a broader target cell spectrum and increased potency relative to wild-type durancin GL. These findings broaden our understanding of durancin GL residues important for its antimicrobial activity and contribute to future rational design of variants with increased potency. PMID:26168032

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

    PubMed Central

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

    2005-01-01

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

  2. 40 CFR 180.155 - 1-Naphthaleneacetic acid; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

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

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

    PubMed

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

    2012-01-01

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

  4. 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.

  5. Importance of residual stresses in the Brillouin gain spectrum of single mode optical fibers.

    PubMed

    Mamdem, Y Sikali; Burov, E; de Montmorillon, L-A; Jaouën, Y; Moreau, G; Gabet, R; Taillade, F

    2012-01-16

    Residual stresses inside optical fibers can impact significantly on Brillouin spectrum properties. We have analyzed the importance of internal stresses on the Brillouin Gain Spectrum (BGS) for a conventional G.652 fiber and compared modeling results to measurements. Then the residual internal stresses have been investigated for a set of trench-assisted fibers: fibers are coming from a single preform with different draw tensions. Numerical modeling based on measured internal stresses profiles are compared with corresponding BGS experimental results. Clearly, Brillouin spectrum is shifted linearly versus draw tension with a coefficient of -20MHz/100g and its linewidth increases. PMID:22274523

  6. 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

  7. 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

  8. 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...

  9. Importance of Basic Residues in Binding of Rous Sarcoma Virus Nucleocapsid to the RNA Packaging Signal

    PubMed Central

    Lee, Eun-gyung; Alidina, Annie; May, Cynthia; Linial, Maxine L.

    2003-01-01

    In the context of the Rous sarcoma virus Gag polyprotein, only the nucleocapsid (NC) domain is required to mediate the specificity of genomic RNA packaging. We have previously showed that the Saccharomyces cerevisiae three-hybrid system provides a rapid genetic assay to analyze the RNA and protein components of the avian retroviral RNA-Gag interactions necessary for specific encapsidation. In this study, using both site-directed mutagenesis and in vivo random screening in the yeast three-hybrid binding assay, we have examined the amino acids in NC required for genomic RNA binding. We found that we could delete either of the two Cys-His boxes without greatly abrogating either RNA binding or packaging, although the two Cys-His boxes are likely to be required for efficient viral assembly and release. In contrast, substitutions for the Zn-coordinating residues within the boxes did prevent RNA binding, suggesting changes in the overall conformation of the protein. In the basic region between the two Cys-His boxes, three positively charged residues, as well as basic residues flanking the two boxes, were necessary for both binding and packaging. Our results suggest that the stretches of positively charged residues within NC that need to be in a proper conformation appear to be responsible for selective recognition and binding to the packaging signal (Ψ)-containing RNAs. PMID:12525635

  10. Importance of basic residues in binding of rous sarcoma virus nucleocapsid to the RNA packaging signal.

    PubMed

    Lee, Eun-gyung; Alidina, Annie; May, Cynthia; Linial, Maxine L

    2003-02-01

    In the context of the Rous sarcoma virus Gag polyprotein, only the nucleocapsid (NC) domain is required to mediate the specificity of genomic RNA packaging. We have previously showed that the Saccharomyces cerevisiae three-hybrid system provides a rapid genetic assay to analyze the RNA and protein components of the avian retroviral RNA-Gag interactions necessary for specific encapsidation. In this study, using both site-directed mutagenesis and in vivo random screening in the yeast three-hybrid binding assay, we have examined the amino acids in NC required for genomic RNA binding. We found that we could delete either of the two Cys-His boxes without greatly abrogating either RNA binding or packaging, although the two Cys-His boxes are likely to be required for efficient viral assembly and release. In contrast, substitutions for the Zn-coordinating residues within the boxes did prevent RNA binding, suggesting changes in the overall conformation of the protein. In the basic region between the two Cys-His boxes, three positively charged residues, as well as basic residues flanking the two boxes, were necessary for both binding and packaging. Our results suggest that the stretches of positively charged residues within NC that need to be in a proper conformation appear to be responsible for selective recognition and binding to the packaging signal (Psi)-containing RNAs. PMID:12525635

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

    PubMed Central

    2015-01-01

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

  12. Identification of functionally important residues in the silkmoth pheromone biosynthesis-activating neuropeptide receptor, an insect ortholog of the vertebrate Neuromedin U Receptor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The biosynthesis of sex pheromone components in many lepidopteran insects is regulated by interactions between pheromone biosynthesis-activating neuropeptide (PBAN) and the PBAN receptor (PBANR), a class-A G-protein-coupled receptor (GPCR). To identify functionally important amino acid residues in t...

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

    PubMed Central

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

    2001-01-01

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

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

    PubMed

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

    2001-01-01

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

  15. Oleanolic acid and related derivatives as medicinally important compounds.

    PubMed

    Sultana, Nighat; Ata, Athar

    2008-12-01

    Oleanolic acid has been isolated from chloroform extract of Olea ferruginea Royle after removal of organic bases and free acids. The literature survey revealed it to be biologically very important. In this review the biological significance of oleanolic acid and its derivatives has been discussed. The aim of this review is to update current knowledge on oleanolic acid and its natural and semisynthetic analogs, focussing on its cytotoxic, antitumer, antioxidant, anti-inflamatory, anti-HIV, acetyl cholinesterase, alpha-glucosidase, antimicrobial, hepatoprotective, anti-inflammatory, antipruritic, spasmolytic activity, anti-angiogenic, antiallergic, antiviral and immunomodulatory activities. We present in this review, for the first time, a compilation of the most relevant scientific papers and technical reports of the chemical, pre-clinical and clinical research on the properties of oleanolic acid and its derivatives. PMID:18618318

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

    PubMed Central

    Conrad, H E

    1980-01-01

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

  17. 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

  18. Importance of Nucleic Acid Recognition in Inflammation and Autoimmunity.

    PubMed

    Barrat, Franck J; Elkon, Keith B; Fitzgerald, Katherine A

    2016-01-01

    An important concept in immunology is the classification of immune responses as either innate or adaptive, based on whether the antigen receptors are encoded in the germline or generated somatically by gene rearrangement. The innate immune system is an ancient mode of immunity, and by being a first layer in our defense against infectious agents, it is essential for our ability to develop rapid and sustained responses to pathogens. We discuss the importance of nucleic acid recognition by the innate immune system to mounting an appropriate immune response to pathogens and also how inflammation driven by uncontrolled recognition of self-nucleic acids can lead to autoimmune diseases. We also summarize current efforts to either harness the immune system using agonists of nucleic acid-specific innate sensors or, on the contrary, by using inhibitors in autoimmune situations. PMID:26526766

  19. [Survey of pesticide residues in imported frozen vegetables and fruits (1989.4~2008.3)].

    PubMed

    Kobayashi, Maki; Ohtsuka, Kenji; Tamura, Yasuhiro; Tomizawa, Sanae; Kamijo, Kyoko; Iwakoshi, Keiko; Kageyama, Yuriko; Nagayama, Toshihiro; Takano, Ichiro

    2011-01-01

    A survey of pesticide residues in 595 imported frozen products on the Tokyo market from April 1989 to March 2008 was carried out. Forty three kinds of pesticides, including organophosphorus, organochlorine, carbamate, pyrethroid and others, were detected between levels of trace (below 0.01 ppm) and 4.6 ppm from 162 samples. Chlorpyrifos, cypermethrin and omethoate were frequently detected in green vegetables (komatsuna leaf and spinach), cypermethrin and methamidophos were detected in pods and seeds (green soybean and string pea), chlorpropham (CIPC) was detected in potato, and captan and carbaryl were detected in berries (blueberry, raspberry and strawberry). The hydrophilic pesticide methamidophos was detected in flesh of lychee. Residue levels of these pesticides were calculated as between less than 0.5% and 30% of their ADI values according to the daily intake of frozen products. Therefore, these frozen products should be safe when they were eaten in customary amounts. PMID:21515967

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

    SciTech Connect

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

    1990-05-01

    Teichuronic acid-peptidoglycan complex isolated from Micrococcus luteus cells by lysozyme digestion in osmotically stabilized medium was treated with mild acid to cleave the linkage joining teichuronic acid to peptidoglycan. This labile linkage was shown to be the phosphodiester which joins N-acetylglucosamine, the residue located at the reducing end of the teichuronic acid, through its anomeric hydroxyl group to a 6-phosphomuramic acid, a residue of the glycan strand of peptidoglycan. {sup 31}P nuclear magnetic resonance spectroscopy of the lysozyme digest of cell walls demonstrated the presence of a phosphodiester which was converted to a phosphomonoester by the conditions which released teichuronic acid from cell walls. Reduction of acid-liberated reducing end groups by NaB{sup 3}H{sub 4} followed by complete acid hydrolysis yielded ({sup 3}H) glucosaminitol from the true reducing end residue of teichuronic acid and ({sup 3}H)glucitol from the sites of fragmentation of teichuronic acid. The amount of N-acetylglucosamine detected was approximately stoichiometric with the amount of phosphate in the complex. Partial fragmentation of teichuronic acid provides an explanation of the previous erroneous identification of the reducing end residue.

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

    PubMed Central

    Neuberger, A.; Ratcliffe, Wendy A.

    1972-01-01

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

  2. 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

  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, 2012 CFR

    2012-07-01

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

  5. 40 CFR 180.155 - 1-Naphthaleneacetic acid; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

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

  6. 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...

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

    PubMed

    Liou, H L; Storch, J

    2001-05-29

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

  8. The Structure of a Plant Tyrosinase from Walnut Leaves Reveals the Importance of "Substrate-Guiding Residues" for Enzymatic Specificity.

    PubMed

    Bijelic, Aleksandar; Pretzler, Matthias; Molitor, Christian; Zekiri, Florime; Rompel, Annette

    2015-12-01

    Tyrosinases and catechol oxidases are members of the class of type III copper enzymes. While tyrosinases accept both mono- and o-diphenols as substrates, only the latter substrate is converted by catechol oxidases. Researchers have been working for decades to elucidate the monophenolase/diphenolase specificity on a structural level and have introduced an early hypothesis that states that the reason for the lack of monophenolase activity in catechol oxidases may be its structurally restricted active site. However, recent structural and biochemical studies of this enzyme class have raised doubts about this theory. Herein, the first crystal structure of a plant tyrosinase (from Juglans regia) is presented. The structure reveals that the distinction between mono- and diphenolase activity does not depend on the degree of restriction of the active site, and thus a more important role for amino acid residues located at the entrance to and in the second shell of the active site is proposed. PMID:26473311

  9. Life cycle assessment and residue leaching: the importance of parameter, scenario and leaching data selection.

    PubMed

    Allegrini, E; Butera, S; Kosson, D S; Van Zomeren, A; Van der Sloot, H A; Astrup, T F

    2015-04-01

    Residues from industrial processes and waste management systems (WMSs) have been increasingly reutilised, leading to landfilling rate reductions and the optimisation of mineral resource utilisation in society. Life cycle assessment (LCA) is a holistic methodology allowing for the analysis of systems and products and can be applied to waste management systems to identify environmental benefits and critical aspects thereof. From an LCA perspective, residue utilisation provides benefits such as avoiding the production and depletion of primary materials, but it can lead to environmental burdens, due to the potential leaching of toxic substances. In waste LCA studies where residue utilisation is included, leaching has generally been neglected. In this study, municipal solid waste incineration bottom ash (MSWI BA) was used as a case study into three LCA scenarios having different system boundaries. The importance of data quality and parameter selection in the overall LCA results was evaluated, and an innovative method to assess metal transport into the environment was applied, in order to determine emissions to the soil and water compartments for use in an LCA. It was found that toxic impacts as a result of leaching were dominant in systems including only MSWI BA utilisation, while leaching appeared negligible in larger scenarios including the entire waste system. However, leaching could not be disregarded a priori, due to large uncertainties characterising other activities in the scenario (e.g. electricity production). Based on the analysis of relevant parameters relative to leaching, and on general results of the study, recommendations are provided regarding the use of leaching data in LCA studies. PMID:25573739

  10. Life cycle assessment and residue leaching: The importance of parameter, scenario and leaching data selection

    SciTech Connect

    Allegrini, E.; Butera, S.; Kosson, D.S.; Van Zomeren, A.; Van der Sloot, H.A.; Astrup, T.F.

    2015-04-15

    Highlights: • Relevance of metal leaching in waste management system LCAs was assessed. • Toxic impacts from leaching could not be disregarded. • Uncertainty of toxicity, due to background activities, determines LCA outcomes. • Parameters such as pH and L/S affect LCA results. • Data modelling consistency and coverage within an LCA are crucial. - Abstract: Residues from industrial processes and waste management systems (WMSs) have been increasingly reutilised, leading to landfilling rate reductions and the optimisation of mineral resource utilisation in society. Life cycle assessment (LCA) is a holistic methodology allowing for the analysis of systems and products and can be applied to waste management systems to identify environmental benefits and critical aspects thereof. From an LCA perspective, residue utilisation provides benefits such as avoiding the production and depletion of primary materials, but it can lead to environmental burdens, due to the potential leaching of toxic substances. In waste LCA studies where residue utilisation is included, leaching has generally been neglected. In this study, municipal solid waste incineration bottom ash (MSWI BA) was used as a case study into three LCA scenarios having different system boundaries. The importance of data quality and parameter selection in the overall LCA results was evaluated, and an innovative method to assess metal transport into the environment was applied, in order to determine emissions to the soil and water compartments for use in an LCA. It was found that toxic impacts as a result of leaching were dominant in systems including only MSWI BA utilisation, while leaching appeared negligible in larger scenarios including the entire waste system. However, leaching could not be disregarded a priori, due to large uncertainties characterising other activities in the scenario (e.g. electricity production). Based on the analysis of relevant parameters relative to leaching, and on general results

  11. 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

  12. Identification of specific calcitonin-like receptor residues important for calcitonin gene-related peptide high affinity binding

    PubMed Central

    Banerjee, Sugato; Evanson, Janel; Harris, Erik; Lowe, Stephen L; Thomasson, Kathryn A; Porter, James E

    2006-01-01

    Background Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide whose biological activity has potential therapeutic value for many vascular related diseases. CGRP is a 37 amino acid neuropeptide that signals through a G protein-coupled receptor belonging to the secretin receptor family. Previous studies on the calcitonin-like receptor (CLR), which requires co-expression of the receptor-activity-modifying protein-1 (RAMP1) to function as a CGRP receptor, have shown an 18 amino acid N-terminus sequence important for binding CGRP. Moreover, several investigations have recognized the C-terminal amidated phenylalanine (F37) of CGRP as essential for docking to the mature receptor. Therefore, we hypothesize that hydrophobic amino acids within the previously characterized 18 amino acid CLR N-terminus domain are important binding contacts for the C-terminal phenylalaninamide of CGRP. Results Two leucine residues within this previously characterized CLR N-terminus domain, when mutated to alanine and expressed on HEK293T cells stably transfected with RAMP1, demonstrated a significantly decreased binding affinity for CGRP compared to wild type receptor. Additional decreases in binding affinity for CGRP were not found when both leucine mutations were expressed in the same CLR construct. Decreased binding characteristic of these leucine mutant receptors was observed for all CGRP ligands tested that contained the necessary amidated phenylalanine at their C-terminus. However, there was no difference in the potency of CGRP to increase cAMP production by these leucine mutant receptors when compared to wild type CLR, consistent with the notion that the neuropeptide C-terminal F37 is important for docking but not activation of the receptor. This observation was conserved when modified CGRP ligands lacking the amidated F37 demonstrated similar potencies to generate cAMP at both wild type and mutant CLRs. Furthermore, these modified CGRP ligands displayed a significant

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

    PubMed

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

    2007-01-01

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

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

    PubMed

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

    2016-01-01

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

  15. Understanding the determinants of substrate specificity in IMP family metallo-β-lactamases: The importance of residue 262

    PubMed Central

    Pegg, Kevin M; Liu, Eleanor M; George, Alex C; LaCuran, Alecander E; Bethel, Christopher R; Bonomo, Robert A; Oelschlaeger, Peter

    2014-01-01

    In Gram-negative bacteria, resistance to β-lactam antibacterials is largely due to β-lactamases and is a growing public health threat. One of the most concerning β-lactamases to evolve in bacteria are the Class B enzymes, the metallo-β-lactamases (MBLs). To date, penams and cephems resistant to hydrolysis by MBLs have not yet been found. As a result of this broad substrate specificity, a better understanding of the role of catalytically important amino acids in MBLs is necessary to design novel β-lactams and inhibitors. Two MBLs, the wild type IMP-1 with serine at position 262, and an engineered variant with valine at the same position (IMP-1-S262V), were previously found to exhibit very different substrate spectra. These findings compelled us to investigate the impact of a threonine at position 262 (IMP-1-S262T) on the substrate spectrum. Here, we explore MBL sequence-structure-activity relationships by predicting and experimentally validating the effect of the S262T substitution in IMP-1. Using site-directed mutagenesis, threonine was introduced at position 262, and the IMP-1-S262T enzyme, as well as the other two enzymes IMP-1 and IMP-1-S262V, were purified and kinetic constants were determined against a range of β-lactam antibacterials. Catalytic efficiencies (kcat/KM) obtained with IMP-1-S262T and minimum inhibitory concentrations (MICs) observed with bacterial cells expressing the protein were intermediate or comparable to the corresponding values with IMP-1 and IMP-1-S262V, validating the role of this residue in catalysis. Our results reveal the important role of IMP residue 262 in β-lactam turnover and support this approach to predict activities of certain novel MBL variants. PMID:25131397

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

    PubMed Central

    Barnhart, Benjamin J.

    1965-01-01

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

  17. 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.

  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. Mutation of the important Tyr-33 residue of chicken avidin: functional and structural consequences.

    PubMed Central

    Marttila, Ari T; Hytönen, Vesa P; Laitinen, Olli H; Bayer, Edward A; Wilchek, Meir; Kulomaa, Markku S

    2003-01-01

    The strong interaction between avidin and biotin is so tight (dissociation constant 10(-15) M) that conditions usually sufficient for protein denaturing fail to dislodge biotin from the avidin-biotin complex. This kind of irreversible binding hinders the use of avidin in applications such as affinity purification or protein immobilization. To address this concern, we have constructed a series of mutants of the strategically positioned Tyr-33 in order to study the role of this residue in biotin binding, and to create avidin variants with more reversible ligand-binding properties. Unexpectedly, an avidin mutant in which Tyr-33 was replaced with phenylalanine (Avm-Y33F) displayed similar biotin-binding characteristics to the native avidin, indicating that the hydrogen bond formed between the hydroxy group of Tyr-33 and the carbonyl oxygen of biotin is not as important for the tight binding of biotin as previously suggested. In terms of the reversibility of biotin binding, Avm-Y33H was the most successful substitution constructed in this study. Interestingly, the binding of this mutant exhibited clear pH-dependence, since at neutral pH it bound to the biotin surface in an irreversible fashion, whereas, at pH 9, 50% of the bound protein could be released with free biotin. Furthermore, although Tyr-33 is located relatively distant from the monomer-monomer interfaces, the mutagenesis of this residue also weakened the quaternary structure of avidin, indicating that the high ligand binding and the high stability of avidin have evolved together and it is difficult to modify one without affecting the other. PMID:12358604

  20. 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

  1. Pore-exposed tyrosine residues of P-glycoprotein are important hydrogen-bonding partners for drugs.

    PubMed

    Dönmez Cakil, Yaprak; Khunweeraphong, Narakorn; Parveen, Zahida; Schmid, Diethart; Artaker, Matthias; Ecker, Gerhard F; Sitte, Harald H; Pusch, Oliver; Stockner, Thomas; Chiba, Peter

    2014-03-01

    The multispecific efflux transporter, P-glycoprotein, plays an important role in drug disposition. Substrate translocation occurs along the interface of its transmembrane domains. The rotational C2 symmetry of ATP-binding cassette transporters implies the existence of two symmetry-related sets of substrate-interacting amino acids. These sets are identical in homodimeric transporters, and remain evolutionary related in full transporters, such as P-glycoprotein, in which substrates bind preferentially, but nonexclusively, to one of two binding sites. We explored the role of pore-exposed tyrosines for hydrogen-bonding interactions with propafenone type ligands in their preferred binding site 2. Tyrosine 953 is shown to form hydrogen bonds not only with propafenone analogs, but also with the preferred site 1 substrate rhodamine123. Furthermore, an accessory role of tyrosine 950 for binding of selected propafenone analogs is demonstrated. The present study demonstrates the importance of domain interface tyrosine residues for interaction of small molecules with P-glycoprotein. PMID:24366667

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

    PubMed

    Sherbakov, D Yu; Triboy, T I

    2007-12-01

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

  3. Threonine 89 Is an Important Residue of Profilin-1 That Is Phosphorylatable by Protein Kinase A

    PubMed Central

    Zeng, Xuemei; Yates, Nathan; Shroff, Sanjeev G.; Koes, David R.; Roy, Partha

    2016-01-01

    Objective Dynamic regulation of actin cytoskeleton is at the heart of all actin-based cellular events. In this study, we sought to identify novel post-translational modifications of Profilin-1 (Pfn1), an important regulator of actin polymerization in cells. Methodology We performed in vitro protein kinase assay followed by mass-spectrometry to identify Protein Kinase A (PKA) phosphorylation sites of Pfn1. By two-dimensional gel electrophoresis (2D-GE) analysis, we further examined the changes in the isoelectric profile of ectopically expressed Pfn1 in HEK-293 cells in response to forskolin (FSK), an activator of cAMP/PKA pathway. Finally, we combined molecular dynamics simulations (MDS), GST pull-down assay and F-actin analyses of mammalian cells expressing site-specific phosphomimetic variants of Pfn1 to predict the potential consequences of phosphorylation of Pfn1. Results and Significance We identified several PKA phosphorylation sites of Pfn1 including Threonine 89 (T89), a novel site. Consistent with PKA’s ability to phosphorylate Pfn1 in vitro, FSK stimulation increased the pool of the most negatively charged form of Pfn1 in HEK-293 cells which can be attenuated by PKA inhibitor H89. MDS predicted that T89 phosphorylation destabilizes an intramolecular interaction of Pfn1, potentially increasing its affinity for actin. The T89D phosphomimetic mutation of Pfn1 elicits several changes that are hallmarks of proteins folded into alternative three-dimensional conformations including detergent insolubility, protein aggregation and accelerated proteolysis, suggesting that T89 is a structurally important residue of Pfn1. Expression of T89D-Pfn1 induces actin:T89D-Pfn1 co-clusters and dramatically reduces overall actin polymerization in cells, indicating an actin-sequestering action of T89D-Pfn1. Finally, rendering T89 non-phosphorylatable causes a positive charge shift in the isoelectric profile of Pfn1 in a 2D gel electrophoresis analysis of cell extracts, a

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

    PubMed

    Miao, Zhichao; Westhof, Eric

    2015-06-23

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

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

    PubMed Central

    Miao, Zhichao; Westhof, Eric

    2015-01-01

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

  6. Delineation of structural domains and identification of functionally important residues in DNA repair enzyme exonuclease VII

    PubMed Central

    Poleszak, Katarzyna; Kaminska, Katarzyna H.; Dunin-Horkawicz, Stanislaw; Lupas, Andrei; Skowronek, Krzysztof J.; Bujnicki, Janusz M.

    2012-01-01

    Exonuclease VII (ExoVII) is a bacterial nuclease involved in DNA repair and recombination that hydrolyses single-stranded DNA. ExoVII is composed of two subunits: large XseA and small XseB. Thus far, little was known about the molecular structure of ExoVII, the interactions between XseA and XseB, the architecture of the nuclease active site or its mechanism of action. We used bioinformatics methods to predict the structure of XseA, which revealed four domains: an N-terminal OB-fold domain, a middle putatively catalytic domain, a coiled-coil domain and a short C-terminal segment. By series of deletion and site-directed mutagenesis experiments on XseA from Escherichia coli, we determined that the OB-fold domain is responsible for DNA binding, the coiled-coil domain is involved in binding multiple copies of the XseB subunit and residues D155, R205, H238 and D241 of the middle domain are important for the catalytic activity but not for DNA binding. Altogether, we propose a model of sequence–structure–function relationships in ExoVII. PMID:22718974

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

    PubMed

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

    2014-10-01

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

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

    PubMed

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

    2016-04-01

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

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

    PubMed

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

    2015-04-01

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

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

    PubMed

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

    2016-06-01

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

  11. 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.)

  12. A Coevolutionary Residue Network at the Site of a Functionally Important Conformational Change in a Phosphohexomutase Enzyme Family

    PubMed Central

    Furdui, Cristina; Beamer, Lesa J.

    2012-01-01

    Coevolution analyses identify residues that co-vary with each other during evolution, revealing sequence relationships unobservable from traditional multiple sequence alignments. Here we describe a coevolutionary analysis of phosphomannomutase/phosphoglucomutase (PMM/PGM), a widespread and diverse enzyme family involved in carbohydrate biosynthesis. Mutual information and graph theory were utilized to identify a network of highly connected residues with high significance. An examination of the most tightly connected regions of the coevolutionary network reveals that most of the involved residues are localized near an interdomain interface of this enzyme, known to be the site of a functionally important conformational change. The roles of four interface residues found in this network were examined via site-directed mutagenesis and kinetic characterization. For three of these residues, mutation to alanine reduces enzyme specificity to ∼10% or less of wild-type, while the other has ∼45% activity of wild-type enzyme. An additional mutant of an interface residue that is not densely connected in the coevolutionary network was also characterized, and shows no change in activity relative to wild-type enzyme. The results of these studies are interpreted in the context of structural and functional data on PMM/PGM. Together, they demonstrate that a network of coevolving residues links the highly conserved active site with the interdomain conformational change necessary for the multi-step catalytic reaction. This work adds to our understanding of the functional roles of coevolving residue networks, and has implications for the definition of catalytically important residues. PMID:22685552

  13. Identification of quantitative trait loci(QTL) controlling important fatty acids in peanut (Arachis hypogaea L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fatty acids play important role in controlling oil quality of peanut. In addition to the major fatty acids, oleic acid (C18:1) and linoleic acid (C18:2) accounting for about 80%, there are several minor fatty acids accounting for about 20% in peanut oil, such as palmitic acid (PA, C16:0), stearic (S...

  14. The second-sphere residue T263 is important for the function and catalytic activity of PTP1B via interaction with the WPD-loop.

    PubMed

    Xiao, Peng; Wang, Xiao; Wang, Hong-Mei; Fu, Xiao-Lei; Cui, Fu-ai; Yu, Xiao; Wen, Shi-shuai; Bi, Wen-Xiang; Sun, Jin-Peng

    2014-12-01

    Protein tyrosine phosphatases have diverse substrate specificities and intrinsic activities that lay the foundations for the fine-tuning of a phosphorylation network to precisely regulate cellular signal transduction. All classical PTPs share common catalytic mechanisms, and the important catalytic residues in the first sphere of their active sites have been well characterized. However, little attention has been paid to the second-sphere residues that are potentially important in defining the intrinsic activity and substrate specificity of PTPs. Here, we find that a conserved second-sphere residue, Thr263, located in the surface Q-loop is important for both the function and activity of PTPs. Using PTP1B as a study model, we found that mutations of Thr263 impaired the negative regulation role of PTP1B in insulin signaling. A detailed mechanistic study utilizing steady-state kinetics, Brønsted analysis and pH dependence in the presence of pNPP or phosphopeptide substrates revealed that Thr263 is required for the stabilization of the leaving group during catalysis. Further crystallographic studies and structural comparison revealed that Thr263 regulates the general acid function through modulation of the WPD-loop by the T263:F182/Y/H interaction pair, which is conserved in 26 out of 32 classical PTPs. In addition, the hydrophobic interaction between Thr263 and Arg1159 of the insulin receptor contributes to the substrate specificity of PTP1B. Taken together, our findings demonstrate the general role of the second-sphere residue Thr263 in PTP catalysis. Our findings suggest that the second sphere residues of PTP active site may play important roles in PTP-mediated function in both normal and diseased states. PMID:25450460

  15. 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

  16. 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

  17. 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

  18. Structure of a Bacterial ABC Transporter Involved in the Import of an Acidic Polysaccharide Alginate.

    PubMed

    Maruyama, Yukie; Itoh, Takafumi; Kaneko, Ai; Nishitani, Yu; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

    2015-09-01

    The acidic polysaccharide alginate represents a promising marine biomass for the microbial production of biofuels, although the molecular and structural characteristics of alginate transporters remain to be clarified. In Sphingomonas sp. A1, the ATP-binding cassette transporter AlgM1M2SS is responsible for the import of alginate across the cytoplasmic membrane. Here, we present the substrate-transport characteristics and quaternary structure of AlgM1M2SS. The addition of poly- or oligoalginate enhanced the ATPase activity of reconstituted AlgM1M2SS coupled with one of the periplasmic solute-binding proteins, AlgQ1 or AlgQ2. External fluorescence-labeled oligoalginates were specifically imported into AlgM1M2SS-containing proteoliposomes in the presence of AlgQ2, ATP, and Mg(2+). The crystal structure of AlgQ2-bound AlgM1M2SS adopts an inward-facing conformation. The interaction between AlgQ2 and AlgM1M2SS induces the formation of an alginate-binding tunnel-like structure accessible to the solvent. The translocation route inside the transmembrane domains contains charged residues suitable for the import of acidic saccharides. PMID:26235029

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

    NASA Astrophysics Data System (ADS)

    Takahashi, Ohgi; Kirikoshi, Ryota

    2014-01-01

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

  20. 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

  1. Site-directed mutagenesis of the human DNA repair enzyme HAP1: identification of residues important for AP endonuclease and RNase H activity.

    PubMed

    Barzilay, G; Walker, L J; Robson, C N; Hickson, I D

    1995-05-11

    HAP1 protein, the major apurinic/apyrimidinic (AP) endonuclease in human cells, is a member of a homologous family of multifunctional DNA repair enzymes including the Escherichia coli exonuclease III and Drosophila Rrp1 proteins. The most extensively characterised member of this family, exonuclease III, exhibits both DNA- and RNA-specific nuclease activities. Here, we show that the RNase H activity characteristic of exonuclease III has been conserved in the human homologue, although the products resulting from RNA cleavage are dissimilar. To identify residues important for enzymatic activity, five mutant HAP1 proteins containing single amino acid substitutions were purified and analysed in vitro. The substitutions were made at sites of conserved amino acids and targeted either acidic or histidine residues because of their known participation in the active sites of hydrolytic nucleases. One of the mutant proteins (replacement of Asp-219 by alanine) showed a markedly reduced enzymatic activity, consistent with a greatly diminished capacity to bind DNA and RNA. In contrast, replacement of Asp-90, Asp-308 or Glu-96 by alanine led to a reduction in enzymatic activity without significantly compromising nucleic acid binding. Replacement of His-255 by alanine led to only a very small reduction in enzymatic activity. Our data are consistent with the presence of a single catalytic active site for the DNA- and RNA-specific nuclease activities of the HAP1 protein. PMID:7784208

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

    PubMed Central

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

    2013-01-01

    The binding of ATP to trimeric P2X receptors (P2XR) causes an enlargement of the receptor extracellular vestibule, leading to opening of the cation-selective transmembrane pore, but specific roles of vestibule amino acid residues in receptor activation have not been evaluated systematically. In this study, alanine or cysteine scanning mutagenesis of V47–V61 and F324–N338 sequences of rat P2X4R revealed that V49, Y54, Q55, F324, and G325 mutants were poorly responsive to ATP and trafficking was only affected by the V49 mutation. The Y54F and Y54W mutations, but not the Y54L mutation, rescued receptor function, suggesting that an aromatic residue is important at this position. Furthermore, the Y54A and Y54C receptor function was partially rescued by ivermectin, a positive allosteric modulator of P2X4R, suggesting a rightward shift in the potency of ATP to activate P2X4R. The Q55T, Q55N, Q55E, and Q55K mutations resulted in non-responsive receptors and only the Q55E mutant was ivermectin-sensitive. The F324L, F324Y, and F324W mutations also rescued receptor function partially or completely, ivermectin action on channel gating was preserved in all mutants, and changes in ATP responsiveness correlated with the hydrophobicity and side chain volume of the substituent. The G325P mutant had a normal response to ATP, suggesting that G325 is a flexible hinge. A topological analysis revealed that the G325 and F324 residues disrupt a β-sheet upon ATP binding. These results indicate multiple roles of the extracellular vestibule amino acid residues in the P2X4R function: the V49 residue is important for receptor trafficking to plasma membrane, the Y54 and Q55 residues play a critical role in channel gating and the F324 and G325 residues are critical for vestibule widening. PMID:23555667

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

    PubMed

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

    1990-02-22

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

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

    PubMed Central

    Tibbs, J.

    1966-01-01

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

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

    PubMed

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

    2016-05-28

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

  6. The importance of crop residue on soil aggregation and soil organic matter components

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Above- and below-ground plant residues are the soil’s main sources of organic materials that bind soil particles together into aggregates and increase soil carbon storage. Serving to stabilize soil particles, soil organic matter assists in supplying plant available nutrients, increases water holding...

  7. 77 FR 3653 - Import Tolerances for Residues of Unapproved New Animal Drugs in Food

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-25

    ... the Federal Register of August 10, 2001 (66 FR 42167), the Agency published an advance notice of... Effects Abroad of Major Federal Actions,'' of January 4, 1979 (44 FR 1957, January 9, 1979); and 21 CFR 25... Tolerances for Residues of Unapproved New Animal Drugs in Food AGENCY: Food and Drug Administration,...

  8. Proline residues in two tightly coupled helices of the sulphate transporter, SHST1, are important for sulphate transport.

    PubMed Central

    Shelden, M C; Loughlin, P; Tierney, M L; Howitt, S M

    2001-01-01

    The sulphate transporter SHST1, from Stylosanthes hamata, features three tightly coupled transmembrane helices which include proline residues that are conserved in most related transporters. We used site-directed mutagenesis and expression of the mutant transporters in yeast to test whether these proline residues are important for function. Four proline residues were replaced by both alanine and leucine. Only one of these proline residues, Pro-144, was essential for sulphate transport. However, mutation of either Pro-133 or Pro-160 resulted in a severe decrease in sulphate transport activity; this was due more to a decrease in transport activity than to a decrease in the amount of mutant SHST1 in the plasma membrane. These results suggest that all three proline residues are important for transport, and that the conformation of the three tightly coupled helices may play a critical role in sulphate transport. We also show that SHST1 undergoes a post-translational modification that is required for trafficking to the plasma membrane. PMID:11368789

  9. Multiple active site residues are important for photochemical efficiency in the light-activated enzyme protochlorophyllide oxidoreductase (POR).

    PubMed

    Menon, Binuraj R K; Hardman, Samantha J O; Scrutton, Nigel S; Heyes, Derren J

    2016-08-01

    Protochlorophyllide oxidoreductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide), an essential, regulatory step in chlorophyll biosynthesis. The unique requirement of the enzyme for light has provided the opportunity to investigate how light energy can be harnessed to power biological catalysis and enzyme dynamics. Excited state interactions between the Pchlide molecule and the protein are known to drive the subsequent reaction chemistry. However, the structural features of POR and active site residues that are important for photochemistry and catalysis are currently unknown, because there is no crystal structure for POR. Here, we have used static and time-resolved spectroscopic measurements of a number of active site variants to study the role of a number of residues, which are located in the proposed NADPH/Pchlide binding site based on previous homology models, in the reaction mechanism of POR. Our findings, which are interpreted in the context of a new improved structural model, have identified several residues that are predicted to interact with the coenzyme or substrate. Several of the POR variants have a profound effect on the photochemistry, suggesting that multiple residues are important in stabilizing the excited state required for catalysis. Our work offers insight into how the POR active site geometry is finely tuned by multiple active site residues to support enzyme-mediated photochemistry and reduction of Pchlide, both of which are crucial to the existence of life on Earth. PMID:27285815

  10. Field evaluations of residual pesticide applications and misting system on militarily relevant materials against medically important mosquitoes in Thailand

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A key strategy to reduce insect-borne disease is to reduce contact between disease vectors and hosts. In the current study, residual pesticide application and misting system were applied on militarily relevant materials and evaluated against medically important mosquitoes. Field evaluations were car...

  11. 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

  12. 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

  13. 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

  14. Residues of a proposed gate region in type I ATP-binding cassette import systems are crucial for function as revealed by mutational analysis.

    PubMed

    Weidlich, Daniela; Wiesemann, Nicole; Heuveling, Johanna; Wardelmann, Kristina; Landmesser, Heidi; Sani, Katayoun Behnam; Worth, Catherine L; Preissner, Robert; Schneider, Erwin

    2013-09-01

    The type I ATP-binding cassette (ABC) importer for positively charged amino acids of the thermophilic bacterium Geobacillus stearothermophilus consists of the extracellular solute binding protein, ArtJ, and a homodimer each of the transmembrane subunit, ArtM, and the nucleotide-binding and -hydrolyzing subunit, ArtP. We have investigated the functional consequences of mutations affecting conserved residues from two peptide regions in ArtM, recently proposed to form a 'gate' by which access of a substrate to the translocation path is controlled (Hollenstein et al., 2007 [14]). Transporter variants were reconstituted into proteoliposomes and assayed for ArtJ/arginine-stimulated ATPase activity. Replacement of residues from region 1 (Arg-63, Pro-66) caused no or only moderate reduction in ATPase activity. In contrast, mutating residues from gate region 2 (Lys-159, Leu-163) resulted in a substantial increase in ATPase activity which, however, as demonstrated for variants ArtM(K159I) and ArtM(K159E), is not coupled to transport. Replacing homologous residues in the closely related histidine transporter of Salmonella enterica serovar Typhimurium (HisJ-QMP2) caused different phenotypes. Mutation to isoleucine of HisQ(K163) or HisM(H172), both homologous to ArtM(K159), abolished ATPase activity. The mutations most likely caused a structural change as revealed by limited proteolysis. In contrast, substantial, albeit reduced, enzymatic activity was observed with variants of HisQ(L167→G) or HisM(L176→G), both homologous to ArtM(L163). Our study provides the first experimental evidence in favor of a crucial role of residues from the proposed gate region in type I ABC importer function. PMID:23747295

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

    SciTech Connect

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

    1980-02-01

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

  16. Identification of catalytically important residues in the active site of Escherichia coli transaldolase.

    PubMed

    Schörken, U; Thorell, S; Schürmann, M; Jia, J; Sprenger, G A; Schneider, G

    2001-04-01

    The roles of invariant residues at the active site of transaldolase B from Escherichia coli have been probed by site-directed mutagenesis. The mutant enzymes D17A, N35A, E96A, T156A, and S176A were purified from a talB-deficient host and analyzed with respect to their 3D structure and kinetic behavior. X-ray analysis showed that side chain replacement did not induce unanticipated structural changes in the mutant enzymes. Three mutations, N35A, E96A, and T156A resulted mainly in an effect on apparent kcat, with little changes in apparent Km values for the substrates. Residues N35 and T156 are involved in the positioning of a catalytic water molecule at the active site and the side chain of E96 participates in concert with this water molecule in proton transfer during catalysis. Substitution of Ser176 by alanine resulted in a mutant enzyme with 2.5% residual activity. The apparent Km value for the donor substrate, fructose 6-phosphate, was increased nearly fivefold while the apparent Km value for the acceptor substrate, erythrose 4-phosphate remained unchanged, consistent with a function for S176 in the binding of the C1 hydroxyl group of the donor substrate. The mutant D17A showed a 300-fold decrease in kcat, and a fivefold increase in the apparent Km value for the acceptor substrate erythrose 4-phosphate, suggesting a role of this residue in carbon-carbon bond cleavage and stabilization of the carbanion/enamine intermediate. PMID:11298760

  17. Evidence that Highly Conserved Residues of Transmembrane Segment 6 of Escherichia coli MntH Are Important for Transport Activity

    PubMed Central

    Haemig, Heather A.H.; Moen, Patrick J.; Brooker, Robert J.

    2010-01-01

    Nramp (natural resistance-associated macrophage protein) family members have been characterized in mammals, yeast, and bacteria as divalent metal ion/H+ symporters. In previous work, a bioinformatic approach was used for the identification of residues that are conserved within the Nramp family [ref.1. Haemig, H.A. and R.J. Brooker, (2004) J Membr. Biol, 201(2): 97-107]. Based on site-directed mutagenesis of highly conserved negatively charged residues, a model was proposed for the metal binding site of the E.coli homolog, MntH. In this study, we have focused on the highly conserved residues, including two histidines, of transmembrane segment 6 (TMS-6). Multiple mutants were made at the eight conserved sites (i.e., Gly-205, Ala-206, Met-209, Pro-210, His-211, Leu-215, His-216, and Ser-217) in TMS-6 of E. coli MntH. Double mutants involving His-211 and His-216 were also made. The results indicate the side chain volume of these residues is critically important for function. In most cases, only substitutions that are closest in side chain volume still permit transport. In addition, the Km for metal binding is largely unaffected by mutations in TMS-6, whereas Vmax values were decreased in all mutants characterized kinetically. Thus, these residues do not appear to play a role in metal binding. Instead, they may comprise an important face on TMS-6 that is critical for protein conformational changes during transport. Also, in contrast to other studies, our data do not strongly indicate that the conserved histidine residues play a role in pH regulation of metal transport. PMID:20441230

  18. Identification of functionally important residues of the silkmoth pheromone biosynthesis-activating neuropeptide receptor, an insect ortholog of the vertebrate neuromedin U receptor.

    PubMed

    Kawai, Takeshi; Katayama, Yukie; Guo, Linjun; Liu, Desheng; Suzuki, Tatsuya; Hayakawa, Kou; Lee, Jae Min; Nagamine, Toshihiro; Hull, J Joe; Matsumoto, Shogo; Nagasawa, Hiromichi; Tanokura, Masaru; Nagata, Koji

    2014-07-01

    The biosynthesis of sex pheromone components in many lepidopteran insects is regulated by the interaction between pheromone biosynthesis-activating neuropeptide (PBAN) and the PBAN receptor (PBANR), a class A G-protein-coupled receptor. To identify functionally important amino acid residues in the silkmoth PBANR, a series of 27 alanine substitutions was generated using a PBANR chimera C-terminally fused with enhanced GFP. The PBANR mutants were expressed in Sf9 insect cells, and their ability to bind and be activated by a core PBAN fragment (C10PBAN(R2K)) was monitored. Among the 27 mutants, 23 localized to the cell surface of transfected Sf9 cells, whereas the other four remained intracellular. Reduced binding relative to wild type was observed with 17 mutants, and decreased Ca(2+) mobilization responses were observed with 12 mutants. Ala substitution of Glu-95, Glu-120, Asn-124, Val-195, Phe-276, Trp-280, Phe-283, Arg-287, Tyr-307, Thr-311, and Phe-319 affected both binding and Ca(2+) mobilization. The most pronounced effects were observed with the E120A mutation. A molecular model of PBANR indicated that the functionally important PBANR residues map to the 2nd, 3rd, 6th, and 7th transmembrane helices, implying that the same general region of class A G-protein-coupled receptors recognizes both peptidic and nonpeptidic ligands. Docking simulations suggest similar ligand-receptor recognition interactions for PBAN-PBANR and the orthologous vertebrate pair, neuromedin U (NMU) and NMU receptor (NMUR). The simulations highlight the importance of two glutamate residues, Glu-95 and Glu-120, in silkmoth PBANR and Glu-117 and Glu-142 in human NMUR1, in the recognition of the most functionally critical region of the ligands, the C-terminal residue and amide. PMID:24847080

  19. Identification of Functionally Important Residues of the Silkmoth Pheromone Biosynthesis-activating Neuropeptide Receptor, an Insect Ortholog of the Vertebrate Neuromedin U Receptor*

    PubMed Central

    Kawai, Takeshi; Katayama, Yukie; Guo, Linjun; Liu, Desheng; Suzuki, Tatsuya; Hayakawa, Kou; Lee, Jae Min; Nagamine, Toshihiro; Hull, J. Joe; Matsumoto, Shogo; Nagasawa, Hiromichi; Tanokura, Masaru; Nagata, Koji

    2014-01-01

    The biosynthesis of sex pheromone components in many lepidopteran insects is regulated by the interaction between pheromone biosynthesis-activating neuropeptide (PBAN) and the PBAN receptor (PBANR), a class A G-protein-coupled receptor. To identify functionally important amino acid residues in the silkmoth PBANR, a series of 27 alanine substitutions was generated using a PBANR chimera C-terminally fused with enhanced GFP. The PBANR mutants were expressed in Sf9 insect cells, and their ability to bind and be activated by a core PBAN fragment (C10PBANR2K) was monitored. Among the 27 mutants, 23 localized to the cell surface of transfected Sf9 cells, whereas the other four remained intracellular. Reduced binding relative to wild type was observed with 17 mutants, and decreased Ca2+ mobilization responses were observed with 12 mutants. Ala substitution of Glu-95, Glu-120, Asn-124, Val-195, Phe-276, Trp-280, Phe-283, Arg-287, Tyr-307, Thr-311, and Phe-319 affected both binding and Ca2+ mobilization. The most pronounced effects were observed with the E120A mutation. A molecular model of PBANR indicated that the functionally important PBANR residues map to the 2nd, 3rd, 6th, and 7th transmembrane helices, implying that the same general region of class A G-protein-coupled receptors recognizes both peptidic and nonpeptidic ligands. Docking simulations suggest similar ligand-receptor recognition interactions for PBAN-PBANR and the orthologous vertebrate pair, neuromedin U (NMU) and NMU receptor (NMUR). The simulations highlight the importance of two glutamate residues, Glu-95 and Glu-120, in silkmoth PBANR and Glu-117 and Glu-142 in human NMUR1, in the recognition of the most functionally critical region of the ligands, the C-terminal residue and amide. PMID:24847080

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

    PubMed

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

    2006-01-27

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

  1. The Importance of Residual Effects When Choosing a Hypnotic: The Unique Profile of Zaleplon.

    PubMed

    Zammit, Gary K.; Kramer, Jeffrey A.

    2001-04-01

    BACKGROUND: Insomnia is a prevalent medical disorder that has significant effects on occupational performance, health, and quality of life. Insomnia places an enormous burden on society through increased visits to physicians, loss of productivity in the workplace, and an increased rate of accidents. An estimated sum of $100 million is spent each year on direct treatment of unresolved insomnia. Physicians need to initiate early effective treatment to prevent development of chronic insomnia and its associated morbidity. Institution of good sleep hygiene practices may be useful in some patients but may not be adequate for resolution of all sleep problems. Behavioral treatments, while effective and durable, are time consuming and not widely utilized in clinical practice. Pharmacotherapy includes benzodiazepine hypnotics, but concerns regarding adverse effects (e.g., residual sedation) prompted the search for safer options. DATA SOURCES: Published and presented studies containing clinical data on zaleplon, a new nonbenzodiazepine sleep medication, were identified via MEDLINE, Current Contents (ISI database), bibliographic reviews, and consultation with sleep specialists. RESULTS: Zaleplon effectively shortens sleep onset time and improves the quality of sleep in patients with insomnia. Whether administered at bedtime or later at night, zaleplon is devoid of residual sedative effects that impair next-day functioning. Follow-up studies evaluating the long-term efficacy and safety of zaleplon showed that decreased time to sleep onset was maintained during therapy lasting up to 52 weeks, without a withdrawal syndrome after discontinuation. CONCLUSION: Insomnia is recurrent and unpredictable in nature. Despite the long-term morbidity of this sleep disorder, research evidence and practice guidelines have not explored long-term use of hypnotics. Many patients could benefit from long-term drug therapy with a sleep medication that is devoid of residual effects and can be taken

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

    PubMed

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

    1995-09-19

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

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

    PubMed

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

    2016-09-01

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

  4. 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.

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

    PubMed

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

    1997-01-01

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

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

    PubMed Central

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

    1992-01-01

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

  9. 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.

  10. Identification of BECN1 and ATG14 Coiled-Coil Interface Residues That Are Important for Starvation-Induced Autophagy.

    PubMed

    Mei, Yang; Su, Minfei; Sanishvili, Ruslan; Chakravarthy, Srinivas; Colbert, Christopher L; Sinha, Sangita C

    2016-08-01

    Autophagy, an essential eukaryotic homeostasis pathway, allows the sequestration of unwanted, damaged, or harmful cytoplasmic components in vesicles called autophagosomes, permitting subsequent lysosomal degradation and nutrient recycling. Autophagosome nucleation is mediated by class III phosphatidylinositol-3-kinase complexes that include two key autophagy proteins, BECN1/Beclin 1 and ATG14/BARKOR, which form parallel heterodimers via their coiled-coil domains (CCDs). Here we present the 1.46 Å X-ray crystal structure of the antiparallel, human BECN1 CCD homodimer, which represents BECN1 oligomerization outside the autophagosome nucleation complex. We use circular dichroism and small-angle X-ray scattering (SAXS) to show that the ATG14 CCD is significantly disordered but becomes more helical in the BECN1:ATG14 heterodimer, although it is less well-folded than the BECN1 CCD homodimer. SAXS also indicates that the BECN1:ATG14 heterodimer is more curved than other BECN1-containing CCD dimers, which has important implications for the structure of the autophagosome nucleation complex. A model of the BECN1:ATG14 CCD heterodimer that agrees well with the SAXS data shows that BECN1 residues at the homodimer interface are also responsible for heterodimerization, allowing us to identify ATG14 interface residues. Finally, we verify the role of BECN1 and ATG14 interface residues in binding by assessing the impact of point mutations of these residues on co-immunoprecipitation of the partner and demonstrate that these mutations abrogate starvation-induced upregulation of autophagy but do not impact basal autophagy. Thus, this research provides insights into structures of the BECN1 CCD homodimer and the BECN1:ATG14 CCD heterodimer and identifies interface residues that are important for BECN1:ATG14 heterodimerization and for autophagy. PMID:27383850

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

    PubMed

    Fan, Hua; Wang, Chaozhan; Wei, Yinmao

    2015-12-01

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

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

    PubMed

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

    2013-06-01

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

  13. 75 FR 1773 - Notice of Receipt of a Pesticide Petition Filed for Residues of Polymeric Polyhydroxy Acid in or...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-13

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

  14. 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

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

    SciTech Connect

    Schellenberg, K.A.; Shaeffer, J.

    1986-05-01

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

  16. 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

  17. Identification of residues important for substrate uptake in a glucose transporter from the filamentous fungus Trichoderma reesei

    PubMed Central

    Zhang, Weixin; Cao, Yanli; Gong, Jing; Bao, Xiaoming; Chen, Guanjun; Liu, Weifeng

    2015-01-01

    The glucose transporter is an important player in cell metabolism that mediates the intracellular uptake of glucose. Here, we characterized the glucose transporter Stp1 from the filamentous fungus Trichoderma reesei. The individual substitution of several conserved residues for Ala in Stp1 corresponding to those interacting with D-glucose in the xylose/H+ symporter XylE inflicted contrasting effects on its ability to support the growth of an hxt-null yeast on glucose. The targeted change of Phe 50, proximal to the substrate-binding site, was also found to exert a profound effect on the activity of Stp1. In contrast with the charged residues, the substitution of Phe 50 with either the hydrophilic residues Asn and Gln or the small residues Gly and Ala significantly enhanced the transport of glucose and its fluorescent analogue, 2-NBDG. On the other hand, a variant with the three substitutions I115F, F199I and P214L displayed remarkably improved activity on glucose and 2-NBDG transport. Further analysis indicated that the combined mutations of Ile 115 and Pro 214, positioned on the lateral surface of the Stp1 N-domain, fully accounted for the enhanced transport activity. These results provide insight into the structural basis for glucose uptake in fungal sugar transporters. PMID:26345619

  18. D-Aspartic acid: an endogenous amino acid with an important neuroendocrine role.

    PubMed

    D'Aniello, Antimo

    2007-02-01

    D-Aspartic acid (d-Asp), an endogenous amino acid present in vertebrates and invertebrates, plays an important role in the neuroendocrine system, as well as in the development of the nervous system. During the embryonic stage of birds and the early postnatal life of mammals, a transient high concentration of d-Asp takes place in the brain and in the retina. d-Asp also acts as a neurotransmitter/neuromodulator. Indeed, this amino acid has been detected in synaptosomes and in synaptic vesicles, where it is released after chemical (K(+) ion, ionomycin) or electric stimuli. Furthermore, d-Asp increases cAMP in neuronal cells and is transported from the synaptic clefts to presynaptic nerve cells through a specific transporter. In the endocrine system, instead, d-Asp is involved in the regulation of hormone synthesis and release. For example, in the rat hypothalamus, it enhances gonadotropin-releasing hormone (GnRH) release and induces oxytocin and vasopressin mRNA synthesis. In the pituitary gland, it stimulates the secretion of the following hormones: prolactin (PRL), luteinizing hormone (LH), and growth hormone (GH) In the testes, it is present in Leydig cells and is involved in testosterone and progesterone release. Thus, a hypothalamus-pituitary-gonads pathway, in which d-Asp is involved, has been formulated. In conclusion, the present work is a summary of previous and current research done on the role of d-Asp in the nervous and endocrine systems of invertebrates and vertebrates, including mammals. PMID:17118457

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

    PubMed

    Michel, K; Atkinson, P W

    2003-07-01

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

  20. 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.

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

    SciTech Connect

    Xu,G.; Chance, M.

    2005-01-01

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

  2. 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

  3. Prediction of enzyme function based on 3D templates of evolutionarily important amino acids

    PubMed Central

    Kristensen, David M; Ward, R Matthew; Lisewski, Andreas Martin; Erdin, Serkan; Chen, Brian Y; Fofanov, Viacheslav Y; Kimmel, Marek; Kavraki, Lydia E; Lichtarge, Olivier

    2008-01-01

    Background Structural genomics projects such as the Protein Structure Initiative (PSI) yield many new structures, but often these have no known molecular functions. One approach to recover this information is to use 3D templates – structure-function motifs that consist of a few functionally critical amino acids and may suggest functional similarity when geometrically matched to other structures. Since experimentally determined functional sites are not common enough to define 3D templates on a large scale, this work tests a computational strategy to select relevant residues for 3D templates. Results Based on evolutionary information and heuristics, an Evolutionary Trace Annotation (ETA) pipeline built templates for 98 enzymes, half taken from the PSI, and sought matches in a non-redundant structure database. On average each template matched 2.7 distinct proteins, of which 2.0 share the first three Enzyme Commission digits as the template's enzyme of origin. In many cases (61%) a single most likely function could be predicted as the annotation with the most matches, and in these cases such a plurality vote identified the correct function with 87% accuracy. ETA was also found to be complementary to sequence homology-based annotations. When matches are required to both geometrically match the 3D template and to be sequence homologs found by BLAST or PSI-BLAST, the annotation accuracy is greater than either method alone, especially in the region of lower sequence identity where homology-based annotations are least reliable. Conclusion These data suggest that knowledge of evolutionarily important residues improves functional annotation among distant enzyme homologs. Since, unlike other 3D template approaches, the ETA method bypasses the need for experimental knowledge of the catalytic mechanism, it should prove a useful, large scale, and general adjunct to combine with other methods to decipher protein function in the structural proteome. PMID:18190718

  4. 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

  5. Invariant Gly Residue Is Important for α-Defensin Folding, Dimerization, and Function

    PubMed Central

    Zhao, Le; Ericksen, Bryan; Wu, Xueji; Zhan, Changyou; Yuan, Weirong; Li, Xu; Pazgier, Marzena; Lu, Wuyuan

    2012-01-01

    The human α-defensins (HNP) are synthesized in vivo as inactive prodefensins, and contain a conserved glycine, Gly17, which is part of a β-bulge structure. It had previously been shown that the glycine main chain torsion angles are in a d-configuration, and that d-amino acids but not l-alanine could be substituted at that position to yield correctly folded peptides without the help of a prodomain. In this study, the glycine to l-alanine mutant defensin was synthesized in the form of a prodefensin using native chemical ligation. The ligation product folded correctly and yielded an active peptide upon CNBr cleavage. The l-Ala17-HNP1 crystal structure depicted a β-bulge identical to wild-type HNP1. However, dimerization was perturbed, causing one monomer to tilt with respect to the other in a dimerization model. Inhibitory activity against the anthrax lethal factor showed a 2-fold reduction relative to wild-type HNP1 as measured by the inhibitory concentration IC50. Self-association was slightly reduced, as detected by surface plasmon resonance measurements. According to the results of the virtual colony count assay, the antibacterial activity against Escherichia coli, Staphylococcus aureus, and Bacillus cereus exhibited a less than 2-fold reduction in virtual lethal dose values. Prodefensins with two other l-amino acid substitutions, Arg and Phe, at the same position did not fold, indicating that only small side chains are tolerable. These results further elucidate the factors governing the region of the β-bulge structure that includes Gly17, illuminating why glycine is conserved in all mammalian α-defensins. PMID:22496447

  6. 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

  7. Metabolic engineering of Pichia pastoris to produce ricinoleic acid, a hydroxy fatty acid of industrial importance.

    PubMed

    Meesapyodsuk, Dauenpen; Chen, Yan; Ng, Siew Hon; Chen, Jianan; Qiu, Xiao

    2015-11-01

    Ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) has many specialized uses in bioproduct industries, while castor bean is currently the only commercial source for the fatty acid. This report describes metabolic engineering of a microbial system (Pichia pastoris) to produce ricinoleic acid using a "push" (synthesis) and "pull" (assembly) strategy. CpFAH, a fatty acid hydroxylase from Claviceps purpurea, was used for synthesis of ricinoleic acid, and CpDGAT1, a diacylglycerol acyl transferase for the triacylglycerol synthesis from the same species, was used for assembly of the fatty acid. Coexpression of CpFAH and CpDGAT1 produced higher lipid contents and ricinoleic acid levels than expression of CpFAH alone. Coexpression in a mutant haploid strain defective in the Δ12 desaturase activity resulted in a higher level of ricinoleic acid than that in the diploid strain. Intriguingly, the ricinoleic acid produced was mainly distributed in the neutral lipid fractions, particularly the free fatty acid form, but with little in the polar lipids. This work demonstrates the effectiveness of the metabolic engineering strategy and excellent capacity of the microbial system for production of ricinoleic acid as an alternative to plant sources for industrial uses. PMID:26323290

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-04-01

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

  10. 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

  11. 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

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

    PubMed

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

    2009-10-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  14. 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

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

    PubMed

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

    2009-04-30

    Cd-Ni filtercakes are produced continuously at the third purification step in the electrolytic production of zinc in the National Iranian Lead and Zinc Company (NILZ) in northwestern Iran. In this research, the dissolution kinetics of cadmium from Cd-Ni residues produced in NILZ plant has been investigated. Hence, the effects of temperature, sulfuric acid concentration, particle size and stirring speed on the kinetics of cadmium dissolution in sulfuric acid were studied. The dissolution kinetics at 25-55 degrees C and tacid concentration, solid/liquid ratio and particle size were also achieved. The rate of reaction at first 5 min based on diffusion-controlled process can be expressed by a semi-empirical equation as:It was determined that the dissolution rate increased with increasing sulfuric acid concentration and decreasing particle size. PMID:18755541

  16. Toxicity of formic acid against red imported fire ants, Solenopsis invicta Buren

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND: Formic acid is a common defensive chemical of formicine ants. Ants often compete with other ants for resources. However, the toxicity of formic acid to any ant species has not been well understood. This study examined the toxicity of formic acid against the red imported fire ants, Sole...

  17. Identification of insulin domains important for binding to and degradation by endosomal acidic insulinase.

    PubMed

    Authier, F; Danielsen, G M; Kouach, M; Briand, G; Chauvet, G

    2001-01-01

    The endosomal compartment of hepatic parenchymal cells contains an acidic endopeptidase, endosomal acidic insulinase (EAI), which hydrolyzes internalized insulin at a limited number of sites. Although the positions of these cleavages are partially known, the residues of insulin important in its binding to and proteolysis by EAI have not been defined. To this end, we have studied the degradation over time of native human insulin and three insulin-analog peptides using a soluble endosomal extract from rat liver parenchyma followed by purification of the products by HPLC and determination of their structure by mass spectrometry. We found variable rates of ligand processing, i.e. high ([Asp(B10)]- and [Glu(A13),Glu(B10)]-insulin), moderate (insulin) and low (the H2-analog). On the basis of IC(50) values, competition studies revealed that human and mutant insulins display nearly equivalent affinity for the EAI. Proteolysis of human and mutant insulins by EAI resulted in eight cleavages in the B-chain which occurred in the central region (Glu(B13)-Leu(B17)) and at the C-terminus (Arg(B22)-Thr(B27)), the latter region comprising the initial cleavages at Phe(B24)-Phe(B25) (major pathway) and Phe(B25)-Tyr(B26) (minor pathway) bonds. Except for the [Glu(A13),Glu(B10)]-insulin mutant, only one cleavage on the A-chain was observed at residues Gln(A15)-Leu(A16). Analysis of the nine cleavage sites showed a preference for hydrophobic and aromatic amino acid residues on both the carboxyl and amino sides of a cleaved peptide bond. Using the B-chain alone as a substrate resulted in a 30-fold increase in affinity for EAI and a 6-fold increase in the rate of hydrolysis compared with native insulin. A similar role for the C-terminal region of the B-chain of insulin in the high-affinity recognition of EAI was supported by the use of the corresponding B(22)-B(30) peptide, which displayed an increase in EAI affinity similar to the entire B-chain vs. wild-type insulin. Thus, we have

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

    PubMed

    Nikita V, Dovidchenko; Oxana V, Galzitskaya

    2008-01-01

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

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

    PubMed Central

    Dovidchenko, Nikita V; Galzitskaya, Oxana V

    2008-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2016-09-01

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

  2. Enhancement of neutrophil-mediated killing of Plasmodium falciparum asexual blood forms by fatty acids: importance of fatty acid structure.

    PubMed Central

    Kumaratilake, L M; Ferrante, A; Robinson, B S; Jaeger, T; Poulos, A

    1997-01-01

    Effects of fatty acids on human neutrophil-mediated killing of Plasmodium falciparum asexual blood forms were investigated by using a quantitative radiometric assay. The results showed that the antiparasitic activity of neutrophils can be greatly increased (>threefold) by short-term treatment with fatty acids with 20 to 24 carbon atoms and at least three double bonds. In particular, the n-3 polyenoic fatty acids, eicosapentaenoic and docosahexaenoic acids, and the n-6 fatty acid, arachidonic acid, significantly enhanced neutrophil antiparasitic activity. This effect was >1.5-fold higher than that induced by an optical concentration of the known agonist cytokine tumor necrosis factor alpha (TNF-alpha). At suboptimal concentrations, the combination of arachidonic acid and TNF-alpha caused a synergistic increase in neutrophil-mediated parasite killing. The fatty acid-induced effect was independent of the availability of serum opsonins but dependent on the structure of the fatty acids. The length of the carbon chain, degree of unsaturation, and availability of a free carboxyl group were important determinants of fatty acid activity. The fatty acids which increased neutrophil-mediated killing primed the enhanced superoxide radical generation of neutrophils in response to P. falciparum as detected by chemiluminescence. Scavengers of oxygen radicals significantly reduced the fatty acid-enhanced parasite killing, but cyclooxygenase and lipoxygenase inhibitors had no effect. These findings have identified a new class of immunoenhancers that could be exploited to increase resistance against Plasmodium species. PMID:9317021

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

    PubMed

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

    2013-06-19

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

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

    NASA Astrophysics Data System (ADS)

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

    2007-02-01

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

  5. C-Terminal Domain Residues Important for Secretion and Attachment of RgpB in Porphyromonas gingivalis▿

    PubMed Central

    Slakeski, Nada; Seers, Christine A.; Ng, Kaiting; Moore, Caroline; Cleal, Steven M.; Veith, Paul D.; Lo, Alvin W.; Reynolds, Eric C.

    2011-01-01

    Porphyromonas gingivalis, a periodontal pathogen, expresses a group of surface proteins with a common C-terminal domain (CTD) that are exported by a novel secretion system to the surface, where they are covalently attached. Using RgpB as a model CTD protein, we have produced a series of site-directed mutations in the CTD sequence at conserved residues and at residues that may be modified and, hence, surface attached. The mutant RgpB proteins were expressed in a P. gingivalis host lacking functional RgpB and RgpA Arg-specific proteases. The RgpB mutants produced were Y674F, Y674F Y718F, T675Q S679Q T682Q T684Q, T693Q, F695A, D696A, N698A, G699P, G716P, T724Q, T728Q T730Q, and K732Q and a protein with a deletion of residues 692 to 702 (Δ692-702). The mutants were characterized for cell-associated Arg-specific protease activity and for cellular distribution using anti-Rgp antibodies and Western blotting of culture fractions. All the mutants exhibited cell-associated Arg-specific activity similar to that of the positive control except for the D696A and Δ692-702 mutants. For all mutants, except D696A and Δ692-702, the RgpB proteins were found modified and attached to the cell surface, which was the same profile found in the positive-control strain. Only trace amounts of the precursor form of the Δ692-702 mutant were detected in the outer membrane, with none detected in the periplasm or culture fluid although cell transcript levels were normal. The results suggest that residues 692 to 702 of the CTD, in particular, residue D696, have an important role in the attachment of RgpB at the cell surface and that without attachment secretion does not occur. PMID:20971915

  6. Reciprocal mutations of neuropeptide Y receptor Y2 in human and chicken identify amino acids important for antagonist binding.

    PubMed

    Berglund, Magnus M; Fredriksson, Robert; Salaneck, Erik; Larhammar, Dan

    2002-05-01

    The neuropeptide Y (NPY) receptor Y2 antagonist BIIE0246 has sub-nanomolar affinity for the human Y2 (hY2) receptor but binds very poorly to chicken Y2 (chY2) with micromolar affinity. Sequence comparisons identified several amino acids for investigation by mutagenesis. Reciprocal mutagenesis between hY2 and chY2 revealed that three of these, individually and in combination, are important for BIIE0246 binding, namely positions Gln(135) in transmembrane (TM) 3, Leu(227) in TM5, and Leu(284) in TM6. Mutagenesis of hY2 to the corresponding amino in chY2 (generating hY2[Q135H,L227Q,L284F]) made the affinity of BIIE0246 as low as for chY2. Introduction into chY2 of the three human residues resulted in antagonist affinity almost as high as for hY2. To distinguish between direct and indirect effects, each of the three residues in hY2 was replaced with alanine. BIIE0246 bound with 28-fold lower affinity to hY2[L227A], suggesting the Leu(227) interacts directly with the antagonist. The other two alanine mutants bound with unaltered affinity, suggesting that the corresponding chY2 residues abolish binding through steric hindrance or charge repulsion. Thus, three amino acid residues can in an additive manner completely account for the difference in antagonist binding between the hY2 and chY2 receptors. These results will be useful for construction of three-dimensional models of the widely divergent NPY receptor subtypes. PMID:11997008

  7. 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

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

    PubMed

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

    2015-11-01

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

  9. 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

  10. 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

  11. 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

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

    PubMed

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

    1995-03-10

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

  13. 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

  14. 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

  15. 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

  16. Critical Assessment of the Important Residues Involved in the Dimerization and Catalysis of MERS Coronavirus Main Protease

    PubMed Central

    Ho, Bo-Lin; Cheng, Shu-Chun; Shi, Lin; Wang, Ting-Yun; Ho, Kuan-I; Chou, Chi-Yuan

    2015-01-01

    Background A highly pathogenic human coronavirus (CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and other places in Saudi Arabia, and has quickly spread to European and Asian countries since September 2012. Up to the 1st October 2015 it has infected at least 1593 people with a global fatality rate of about 35%. Studies to understand the virus are necessary and urgent. In the present study, MERS-CoV main protease (Mpro) is expressed; the dimerization of the protein and its relationship to catalysis are investigated. Methods and Results The crystal structure of MERS-CoV Mpro indicates that it shares a similar scaffold to that of other coronaviral Mpro and consists of chymotrypsin-like domains I and II and a helical domain III of five helices. Analytical ultracentrifugation analysis demonstrated that MERS-CoV Mpro undergoes a monomer to dimer conversion in the presence of a peptide substrate. Glu169 is a key residue and plays a dual role in both dimerization and catalysis. The mutagenesis of other residues found on the dimerization interface indicate that dimerization of MERS-CoV Mpro is required for its catalytic activity. One mutation, M298R, resulted in a stable dimer with a higher level of proteolytic activity than the wild-type enzyme. Conclusions MERS-CoV Mpro shows substrate-induced dimerization and potent proteolytic activity. A critical assessment of the residues important to these processes provides insights into the correlation between dimerization and catalysis within the coronaviral Mpro family. PMID:26658006

  17. 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

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

    PubMed Central

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

    2014-01-01

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

  19. 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).

  20. Amino Acid Compositions of 27 Food Fishes and Their Importance in Clinical Nutrition

    PubMed Central

    Mahanty, Arabinda; Sankar, T. V.; Chakraborty, Kajal; Rangasamy, Anandan; Paul, Baidyanath; Sarma, Debajit; Mathew, Suseela; Asha, Kurukkan Kunnath; Behera, Bijay; Aftabuddin, Md.; Debnath, Dipesh; Vijayagopal, P.; Sridhar, N.; Akhtar, M. S.; Sahi, Neetu; Mitra, Tandrima; Banerjee, Sudeshna; Das, Debajeet; Das, Pushpita; Vijayan, K. K.; Laxmanan, P. T.; Sharma, A. P.

    2014-01-01

    Proteins and amino acids are important biomolecules which regulate key metabolic pathways and serve as precursors for synthesis of biologically important substances; moreover, amino acids are building blocks of proteins. Fish is an important dietary source of quality animal proteins and amino acids and play important role in human nutrition. In the present investigation, crude protein content and amino acid compositions of important food fishes from different habitats have been studied. Crude protein content was determined by Kjeldahl method and amino acid composition was analyzed by high performance liquid chromatography and information on 27 food fishes was generated. The analysis showed that the cold water species are rich in lysine and aspartic acid, marine fishes in leucine, small indigenous fishes in histidine, and the carps and catfishes in glutamic acid and glycine. The enriched nutrition knowledge base would enhance the utility of fish as a source of quality animal proteins and amino acids and aid in their inclusion in dietary counseling and patient guidance for specific nutritional needs. PMID:25379285

  1. Cloning and characterization of heterologous transporters in Saccharomyces cerevisiae and identification of important amino acids for xylose utilization.

    PubMed

    Wang, Chengqiang; Bao, Xiaoming; Li, Yanwei; Jiao, Chunlei; Hou, Jin; Zhang, Qingzhu; Zhang, Weixin; Liu, Weifeng; Shen, Yu

    2015-07-01

    Efficient and specific transporters may enhance pentose uptake and metabolism by Saccharomyces cerevisiae. Eight heterologous sugar transporters were characterized in S. cerevisiae. The transporter Mgt05196p from Meyerozyma guilliermondii showed the highest xylose transport activity among them. Several key amino acid residues of Mgt05196p were suggested by structural and sequence analysis and characterized by site-directed mutagenesis. A conserved aromatic residue-rich motif (YFFYY, position 332-336) in the seventh trans-membrane span plays an important role in D-xylose transport activity. The phenyl ring of the residue at position 336 may take the function to prevent D-xylose from escaping during uptake. F432A and N360S mutations enhanced the D-xylose transport activities of Mgt05196p. Furthermore, mutant N360F specifically transported D-xylose without any glucose-inhibition, high lighting its potential application in constructing glucose-xylose co-fermentation strains for biomass refining. PMID:25944766

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

    PubMed

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

    2016-08-01

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

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

    PubMed

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

    2013-04-01

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

  4. 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

  5. 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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-05-01

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

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    1996-01-30

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

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

    PubMed

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

    2016-01-01

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

  11. Local water dynamics around antifreeze protein residues in the presence of osmolytes: the importance of hydroxyl and disaccharide groups.

    PubMed

    Krishnamoorthy, Anand Narayanan; Holm, Christian; Smiatek, Jens

    2014-10-01

    Antifreeze proteins (AFP) and antifreeze glycoproteins (AFGP) are synthesized by various organisms to enable their cells to survive low temperature environments like in the polar regions. The presence of antifreeze proteins leads to a temperature difference between the melting and freezing point of the solution known as thermal hysteresis. It is nowadays common knowledge that the antifreeze activity of AFPs is mainly determined by a short-range effect which includes a direct binding to the ice phase. Recently, experimental findings also revealed a long-range effect which implies a significant retardation of the water dynamics to facilitate the ice-binding process specifically for AFGPs. The aim of this work is to examine the dynamics of water molecules around different antifreeze protein residues by using atomistic molecular dynamics simulations. A prototype of AFP from antarctic notothenioids with the main subunit alanine-alanine-threonine (AAT) and a mutant (polyalanine) together with the residues of an antifreeze glycoprotein (AFGP) were simulated and compared with respect to their influence on the local water shell. The analysis of the water hydrogen bond characteristics and the dipolar relaxation times reveals a strong retardation effect of the water dynamics around the AFGP prototype. Our numerical results reveal the significant importance of polar units like threonine and disaccharides for the direct binding of water molecules in terms of hydrogen bonds and a significant retardation of water dynamics. In addition, a considerable change of the hydration dynamics is additionally observed in the presence of osmolytes like urea and hydroxyectoine. Our findings indicate that this effect is even more pronounced in the presence of kosmotropic osmolytes. PMID:25207443

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

    PubMed

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

    2004-02-27

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

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

    PubMed

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

    2005-10-15

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

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

    PubMed

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

    2014-04-01

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

  15. 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

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

    PubMed

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

    2015-06-15

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

  17. 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

  18. Importance of the proline-rich multimerization domain on the oligomerization and nucleic acid binding properties of HIV-1 Vif.

    PubMed

    Bernacchi, Serena; Mercenne, Gaëlle; Tournaire, Clémence; Marquet, Roland; Paillart, Jean-Christophe

    2011-03-01

    The HIV-1 viral infectivity factor (Vif) is required for productive infection of non-permissive cells, including most natural HIV-1 targets, where it counteracts the antiviral activities of the cellular cytosine deaminases APOBEC-3G (A3G) and A3F. Vif is a multimeric protein and the conserved proline-rich domain (161)PPLP(164) regulating Vif oligomerization is crucial for its function and viral infectivity. Here, we expressed and purified wild-type Vif and a mutant protein in which alanines were substituted for the proline residues of the (161)PPLP(164) domain. Using dynamic light scattering, circular dichroism and fluorescence spectroscopy, we established the impact of these mutations on Vif oligomerization, secondary structure content and nucleic acids binding properties. In vitro, wild-type Vif formed oligomers of five to nine proteins, while Vif AALA formed dimers and/or trimers. Up to 40% of the unbound wild-type Vif protein appeared to be unfolded, but binding to the HIV-1 TAR apical loop promoted formation of β-sheets. Interestingly, alanine substitutions did not significantly affect the secondary structure of Vif, but they diminished its binding affinity and specificity for nucleic acids. Dynamic light scattering showed that Vif oligomerization, and interaction with folding-promoting nucleic acids, favor formation of high molecular mass complexes. These properties could be important for Vif functions involving RNAs. PMID:21076154

  19. Importance of the proline-rich multimerization domain on the oligomerization and nucleic acid binding properties of HIV-1 Vif

    PubMed Central

    Bernacchi, Serena; Mercenne, Gaëlle; Tournaire, Clémence; Marquet, Roland; Paillart, Jean-Christophe

    2011-01-01

    The HIV-1 viral infectivity factor (Vif) is required for productive infection of non-permissive cells, including most natural HIV-1 targets, where it counteracts the antiviral activities of the cellular cytosine deaminases APOBEC-3G (A3G) and A3F. Vif is a multimeric protein and the conserved proline-rich domain 161PPLP164 regulating Vif oligomerization is crucial for its function and viral infectivity. Here, we expressed and purified wild-type Vif and a mutant protein in which alanines were substituted for the proline residues of the 161PPLP164 domain. Using dynamic light scattering, circular dichroism and fluorescence spectroscopy, we established the impact of these mutations on Vif oligomerization, secondary structure content and nucleic acids binding properties. In vitro, wild-type Vif formed oligomers of five to nine proteins, while Vif AALA formed dimers and/or trimers. Up to 40% of the unbound wild-type Vif protein appeared to be unfolded, but binding to the HIV-1 TAR apical loop promoted formation of β-sheets. Interestingly, alanine substitutions did not significantly affect the secondary structure of Vif, but they diminished its binding affinity and specificity for nucleic acids. Dynamic light scattering showed that Vif oligomerization, and interaction with folding-promoting nucleic acids, favor formation of high molecular mass complexes. These properties could be important for Vif functions involving RNAs. PMID:21076154

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

    PubMed

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

    2015-11-15

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  4. 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.

  5. Histone Sprocket Arginine Residues Are Important for Gene Expression, DNA Repair, and Cell Viability in Saccharomyces cerevisiae.

    PubMed

    Hodges, Amelia J; Gallegos, Isaura J; Laughery, Marian F; Meas, Rithy; Tran, Linh; Wyrick, John J

    2015-07-01

    A critical feature of the intermolecular contacts that bind DNA to the histone octamer is the series of histone arginine residues that insert into the DNA minor groove at each superhelical location where the minor groove faces the histone octamer. One of these "sprocket" arginine residues, histone H4 R45, significantly affects chromatin structure in vivo and is lethal when mutated to alanine or cysteine in Saccharomyces cerevisiae (budding yeast). However, the roles of the remaining sprocket arginine residues (H3 R63, H3 R83, H2A R43, H2B R36, H2A R78, H3 R49) in chromatin structure and other cellular processes have not been well characterized. We have genetically characterized mutations in each of these histone residues when introduced either singly or in combination to yeast cells. We find that pairs of arginine residues that bind DNA adjacent to the DNA exit/entry sites in the nucleosome are lethal in yeast when mutated in combination and cause a defect in histone occupancy. Furthermore, mutations in individual residues compromise repair of UV-induced DNA lesions and affect gene expression and cryptic transcription. This study reveals simple rules for how the location and structural mode of DNA binding influence the biological function of each histone sprocket arginine residue. PMID:25971662

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

    PubMed

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

    2014-03-18

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    SciTech Connect

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

    1985-01-01

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

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. [The important role of vitamins in the over-production of pyruvic acid].

    PubMed

    Li, Y; Chen, J; Lun, S; Rui, X

    2000-10-01

    The effect of nicotinic acid, thiamine, pyridoxine, biotin and riboflavin on the production of pyruvic acid by Torulopsis glabrata WSH-IP303 with glucose as carbon source and NH4Cl as sole nitrogen source was investigated. By using orthogonal experiment method, thiamine was confirmed to be the most important factor affecting the production of pyruvic acid. Based on a certain concentration range of thiamine (0.01-0.015 mg/L), glucose consumption rate can be enhanced by increasing the concentration of nicotinic acid. When the concentration of nicotinic acid, thiamine, pyridoxine, biotin and riboflavin were 8, 0.015, 0.4, 0.04 and 0.1 mg/L, respectively, the concentration and yield to glucose of pyruvic acid reached 52.4 g/L and 0.525 g/g at 48 h in flask culture, respectively. Batch culture was conducted in a 2.5 L fermentor with initial glucose concentration of 120 g/L. By adopting the optimal concentration combination of vitamins, the concentration and yield to glucose of pyruvic acid reached 69.4 g/L and 0.593 g/g at 57.5 h, which were increased by 32.4% and 13% than the best results in flask culture, respectively. PMID:12548766

  15. Repellent Effect of Formic Acid Against the Red Imported Fire Ant (Hymenoptera: Formicidae): A Field Study.

    PubMed

    Wang, Cai; Henderson, Gregg

    2016-04-01

    Previous studies showed that the formic acid secreted by tawny crazy ants not only has fumigation toxicity to the red imported fire ant, Solenopsis invicta Buren (Chen et al. 2013), but also can detoxify fire ant venom (LeBrun et al. 2014). These lead us to a field study to determine if low concentrations of formic acid might be useful in repelling S. invicta. Filter paper discs treated with 1.3% or 5% formic acid (v: v) or distilled water (control) were placed on each of the 46 S. invicta mounds and a disturbance was created. For a minute or less, there were significantly more defending ants on the control discs than that on the paper discs treated with formic acid. After food was added and for the next 40 min, there were significantly more foraging ants on the control discs compared to the treated discs. At 50 min into the test, the number of foraging ants on the control and 1.3% formic acid-treated discs was similar, but both were significantly higher than that on the 5% formic acid-treated discs. In addition, the active foraging (≥10 ants stayed on or around the food) and burying behavior (soil particles were deposited around the food) continued to be inhibited by 5% formic acid. The potential application and ecological significant of this repellent effect is discussed. PMID:26700488

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

    PubMed Central

    Elce, John S.

    1980-01-01

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

  17. 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

  18. Identification of lysine 346 as a functionally important residue for pyridoxal 5'-phosphate binding and catalysis in lysine 2, 3-aminomutase from Bacillus subtilis.

    PubMed

    Chen, D; Frey, P A

    2001-01-16

    Lysine 2,3-aminomutase (LAM) catalyzes the interconversion of L-lysine and L-beta-lysine. The enzyme contains pyridoxal 5'-phosphate (PLP) and a [4Fe-4S] center and requires S-adenosylmethionine (SAM) for activity. The hydrogen transfer is mediated by the 5'-deoxyadenosyl radical generated in a reaction of the iron-sulfur cluster with SAM. PLP facilitates the radical rearrangement by forming a lysine-PLP aldimine, in which the imine group participates in the isomerization mechanism. We here report the identification of lysine 346 as important for PLP binding and catalysis. Reduction of LAM with NaBH(4) rapidly inactivated the enzyme with concomitant UV/visible spectrum changes characteristic of reduction of an aldimine formed between PLP and lysine. Following reduction with NaBH(4) and proteolysis with trypsin, a single phosphopyridoxyl peptide of 36 amino acid residues was identified by reverse-phase liquid chromatography/mass spectrometry (LC/MS). The purified phosphopyridoxyl peptide exhibited an absorption band at 325 nm, and its identity was further confirmed by tandem mass spectrometry (MS/MS) sequencing. The bound PLP is linked to lysine 346 in a PGGGGK (PLP) structure. The sequence of this binding motif is conserved in LAMs from Bacillus and Clostridium and other homologous proteins but is distinct from the PLP-binding motifs found in other PLP enzymes. The function of lysine 346 was further studied by site-directed mutagenesis. The purified K346Q mutant was inactive, and its content of PLP was only approximately 15% of that of the wild-type enzyme. The data indicate that the formation of the aldimine linkage between lysine 346 and PLP is important for LAM catalysis. Sequences similar to the PLP-binding motifs in other enzymes were also present in LAM. However, lysine residues within these motifs neither are the PLP-binding sites in LAM nor are directly involved in LAM catalysis. This study represents the first comprehensive investigation of PLP binding in

  19. 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.

  20. 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

  1. 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

  2. Assessment of oil content and fatty acid composition variability in two economically important Hibiscus species.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Hibiscus genus encompasses more than 300 species, but kenaf (H. cannabinus L.) and roselle (H. sabdariffa L.) are the two most economically important species within the genus. Seeds from these two Hibiscus species contain a relatively high amount of oil with two unusual fatty acids: dihydrosterc...

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

    PubMed

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

    2013-10-01

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

  4. H2rs: Deducing evolutionary and functionally important residue positions by means of an entropy and similarity based analysis of multiple sequence alignments

    PubMed Central

    2014-01-01

    Background The identification of functionally important residue positions is an important task of computational biology. Methods of correlation analysis allow for the identification of pairs of residue positions, whose occupancy is mutually dependent due to constraints imposed by protein structure or function. A common measure assessing these dependencies is the mutual information, which is based on Shannon’s information theory that utilizes probabilities only. Consequently, such approaches do not consider the similarity of residue pairs, which may degrade the algorithm’s performance. One typical algorithm is H2r, which characterizes each individual residue position k by the conn(k)-value, which is the number of significantly correlated pairs it belongs to. Results To improve specificity of H2r, we developed a revised algorithm, named H2rs, which is based on the von Neumann entropy (vNE). To compute the corresponding mutual information, a matrix A is required, which assesses the similarity of residue pairs. We determined A by deducing substitution frequencies from contacting residue pairs observed in the homologs of 35 809 proteins, whose structure is known. In analogy to H2r, the enhanced algorithm computes a normalized conn(k)-value. Within the framework of H2rs, only statistically significant vNE values were considered. To decide on significance, the algorithm calculates a p-value by performing a randomization test for each individual pair of residue positions. The analysis of a large in silico testbed demonstrated that specificity and precision were higher for H2rs than for H2r and two other methods of correlation analysis. The gain in prediction quality is further confirmed by a detailed assessment of five well-studied enzymes. The outcome of H2rs and of a method that predicts contacting residue positions (PSICOV) overlapped only marginally. H2rs can be downloaded from http://www-bioinf.uni-regensburg.de. Conclusions Considering substitution frequencies

  5. Partitioning the Relative Importance of Phylogeny and Environmental Conditions on Phytoplankton Fatty Acids.

    PubMed

    Galloway, Aaron W E; Winder, Monika

    2015-01-01

    Essential fatty acids (EFA), which are primarily generated by phytoplankton, limit growth and reproduction in diverse heterotrophs. The biochemical composition of phytoplankton is well-known to be governed both by phylogeny and environmental conditions. Nutrients, light, salinity, and temperature all affect both phytoplankton growth and fatty acid composition. However, the relative importance of taxonomy and environment on algal fatty acid content has yet to be comparatively quantified, thus inhibiting predictions of changes to phytoplankton food quality in response to global environmental change. We compiled 1145 published marine and freshwater phytoplankton fatty acid profiles, consisting of 208 species from six major taxonomic groups, cultured in a wide range of environmental conditions, and used a multivariate distance-based linear model to quantify the total variation explained by each variable. Our results show that taxonomic group accounts for 3-4 times more variation in phytoplankton fatty acids than the most important growth condition variables. The results underscore that environmental conditions clearly affect phytoplankton fatty acid profiles, but also show that conditions account for relatively low variation compared to phylogeny. This suggests that the underlying mechanism determining basal food quality in aquatic habitats is primarily phytoplankton community composition, and allows for prediction of environmental-scale EFA dynamics based on phytoplankton community data. We used the compiled dataset to calculate seasonal dynamics of long-chain EFA (LCEFA; ≥C20 ɷ-3 and ɷ-6 polyunsaturated fatty acid) concentrations and ɷ-3:ɷ-6 EFA ratios in Lake Washington using a multi-decadal phytoplankton community time series. These analyses quantify temporal dynamics of algal-derived LCEFA and food quality in a freshwater ecosystem that has undergone large community changes as a result of shifting resource management practices, highlighting diatoms

  6. Partitioning the Relative Importance of Phylogeny and Environmental Conditions on Phytoplankton Fatty Acids

    PubMed Central

    Galloway, Aaron W. E.; Winder, Monika

    2015-01-01

    Essential fatty acids (EFA), which are primarily generated by phytoplankton, limit growth and reproduction in diverse heterotrophs. The biochemical composition of phytoplankton is well-known to be governed both by phylogeny and environmental conditions. Nutrients, light, salinity, and temperature all affect both phytoplankton growth and fatty acid composition. However, the relative importance of taxonomy and environment on algal fatty acid content has yet to be comparatively quantified, thus inhibiting predictions of changes to phytoplankton food quality in response to global environmental change. We compiled 1145 published marine and freshwater phytoplankton fatty acid profiles, consisting of 208 species from six major taxonomic groups, cultured in a wide range of environmental conditions, and used a multivariate distance-based linear model to quantify the total variation explained by each variable. Our results show that taxonomic group accounts for 3-4 times more variation in phytoplankton fatty acids than the most important growth condition variables. The results underscore that environmental conditions clearly affect phytoplankton fatty acid profiles, but also show that conditions account for relatively low variation compared to phylogeny. This suggests that the underlying mechanism determining basal food quality in aquatic habitats is primarily phytoplankton community composition, and allows for prediction of environmental-scale EFA dynamics based on phytoplankton community data. We used the compiled dataset to calculate seasonal dynamics of long-chain EFA (LCEFA; ≥C20 ɷ-3 and ɷ-6 polyunsaturated fatty acid) concentrations and ɷ-3:ɷ-6 EFA ratios in Lake Washington using a multi-decadal phytoplankton community time series. These analyses quantify temporal dynamics of algal-derived LCEFA and food quality in a freshwater ecosystem that has undergone large community changes as a result of shifting resource management practices, highlighting diatoms

  7. 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

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

    PubMed

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

    2015-05-01

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

  9. 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

  10. Importance of minimal residual viremia for relapse prediction in patients with chronic hepatitis C genotype 1 infection.

    PubMed

    Wiegand, Johannes; Neumann, Konrad; Böhm, Stephan; Weich, Viola; Teuber, Gerlinde; Klinker, Hartwig; Möller, Bernd; Rasenack, Jens; Hinrichsen, Holger; Gerlach, Tilman; Spengler, Ulrich; Buggisch, Peter; Sarrazin, Christoph; Berg, Thomas

    2011-12-01

    This study demonstrates that a more precise prediction of the individual relapse risk in chronic hepatitis C virus genotype 1 infection can be obtained by kinetics of minimal residual viremia at weeks 4, 8, and 12 in combination with levels of baseline viremia. These data may also help to further individualize new protease inhibitor-based triple therapy regimens. PMID:22021919

  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. Minimal promoter systems reveal the importance of conserved residues in the B-finger of human transcription factor IIB.

    PubMed

    Thompson, Nancy E; Glaser, Bryan T; Foley, Katherine M; Burton, Zachary F; Burgess, Richard R

    2009-09-11

    The "B-finger" of transcription factor IIB (TFIIB) is highly conserved and believed to play a role in the initiation process. We performed alanine substitutions across the B-finger of human TFIIB, made change-of-charge mutations in selected residues, and substituted the B-finger sequence from other organisms. Mutant proteins were examined in two minimal promoter systems (containing only RNA polymerase II, TATA-binding protein, and TFIIB) and in a complex system, using TFIIB-immunodepleted HeLa cell nuclear extract (NE). Mutations in conserved residues located on the sides of the B-finger had the greatest effect on activity in both minimal promoter systems, with mutations in residues Glu-51 and Arg-66 eliminating activity. The double change-of-charge mutant (E51R:R66E) did not show activity in either minimal promoter system. Mutations in the nonconserved residues at the tip of the B-finger did not significantly affect activity. However, all of the mutations in the B-finger showed at least 25% activity in the HeLa cell NE. Chimeric proteins, containing B-finger sequences from species with conserved residues on the side of the B-finger, showed wild-type activity in a minimal promoter system and in the HeLa cell NE. However, chimeric proteins whose sequence showed divergence on the sides of the B-finger had reduced activity. Transcription factor IIF (TFIIF) partially restored activity of the inactive mutants in the minimal promoter system, suggesting that TFIIF in HeLa cell NE helps to rescue the inactive mutations by interacting with either the B-finger or another component of the initiation complex that is influenced by the B-finger. PMID:19590095

  14. Medicinal importance of gallic acid and its ester derivatives: a patent review.

    PubMed

    Choubey, Sneha; Varughese, Lesley Rachel; Kumar, Vinod; Beniwal, Vikas

    2015-01-01

    Gallic acid and its derivatives have a large number of applications in various fields of science. In nature, these compounds are widely distributed in plants and fruits, and thus they are being used as food stuffs, preservatives, etc. directly or indirectly by human community. They have also been implicated as anticarcinogenic, antimicrobial, antimutagenic, antiangiogenic and anti-inflammatory agents besides their use in treating critical diseases like depression, cancer, microbial infections, lipid-related diseases, etc. Herein, an attempt has been made to summarize the important uses of gallic acid derivatives which have extensively been disclosed particularly in various patents. This review would certainly create a great interest of the scientific community toward the developments and uses of gallic acid based compounds in the future. PMID:26174568

  15. Nucleic acid import into mitochondria: New insights into the translocation pathways.

    PubMed

    Weber-Lotfi, Frédérique; Koulintchenko, Milana V; Ibrahim, Noha; Hammann, Philippe; Mileshina, Daria V; Konstantinov, Yuri M; Dietrich, André

    2015-12-01

    Mitochondria have retained indispensable but limited genetic information and they import both proteins and nucleic acids from the cytosol. RNA import is essential for gene expression and regulation, whereas competence for DNA uptake is likely to contribute to organellar genome dynamics and evolution. Contrary to protein import mechanisms, the way nucleic acids cross the mitochondrial membranes remains poorly understood. Using proteomic, genetic and biochemical approaches with both plant and yeast organelles, we develop here a model for DNA uptake into mitochondria. The first step includes the voltage-dependent anion channel and an outer membrane-located precursor fraction of a protein normally located in the inner membrane. To proceed, the DNA is then potentially recruited in the intermembrane space by an accessible subunit of one of the respiratory chain complexes. Final translocation through the inner membrane remains the most versatile but points to the components considered to make the mitochondrial permeability transition pore. Depending on the size, DNA and RNA cooperate or compete for mitochondrial uptake, which shows that they share import mechanisms. On the other hand, our results imply the existence of more than one route for nucleic acid translocation into mitochondria. PMID:26376423

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

    PubMed

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

    2014-10-01

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

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

    PubMed

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

    2014-04-30

    The impacts of various shear forces on floc sizes and structures in humic acid coagulations by polyaluminum chloride (PACl) and nano-Al13 were comparatively studied in this paper. The dynamic floc size was monitored by use of a laser diffraction particle sizing device. The floc structure was evaluated in terms of fractal dimension, analyzed by small-angle laser light scattering (SALLS). The effect of increased shear rate on residual Al of the coagulation effluents was then analyzed on the basis of different floc characteristics generated under various shear conditions. The results showed that floc size decreased with the increasing shear rate for both Al13 and PACl. Besides, floc strength and re-formation ability were also weakened by the enhanced shear force. Al13 resulted in small, strong and better recoverable flocs than PACl and moreover, in the shear range of 100-300 revolution per minute (rpm) (G=40.7-178.3s(-1)), the characteristics of HA-Al13 flocs displayed smaller scale changes than those of HA-PACl flocs. The results of residual Al measurements proved that with shear increased, the residual Al increased continuously but Al13 presented less sensitivity to the varying shear forces. PACl contributed higher residual Al than Al13 under the same shear condition. PMID:24583809

  18. An evaluation of the importance of gastric acid secretion in the absorption of dietary calcium.

    PubMed Central

    Bo-Linn, G W; Davis, G R; Buddrus, D J; Morawski, S G; Santa Ana, C; Fordtran, J S

    1984-01-01

    Since calcium solubility is a prerequisite to calcium absorption, and since solubility of calcium is highly pH-dependent, it has been generally assumed that gastric acid secretion and gastric acidity play an important role in the intestinal absorption of calcium from ingested food or calcium salts such as CaCO3. To evaluate this hypothesis, we developed a method wherein net gastrointestinal absorption of calcium can be measured after ingestion of a single meal. A large dose of cimetidine, which markedly reduced gastric acid secretion, had no effect on calcium absorption in normal subjects, and an achlorhydric patient with pernicious anemia absorbed calcium normally. This was true regardless of the major source of dietary calcium (i.e., milk, insoluble calcium carbonate, or soluble calcium citrate). Moreover, calcium absorption after CaCO3 ingestion was the same when intragastric contents were maintained at pH 7.4 (by in vivo titration) as when intragastric pH was 3.0. On the basis of these results, we conclude that gastric acid secretion and gastric acidity do not normally play a role in the absorption of dietary calcium. Other possible mechanisms by which the gastrointestinal tract might solubilize ingested calcium complexes and salts are discussed. Images PMID:6707197

  19. Positive charges on lysine residues of the extrinsic 18 kDa protein are important to its electrostatic interaction with spinach photosystem II membranes.

    PubMed

    Gao, Jin-Peng; Yong, Zhen-Hua; Zhang, Feng; Ruan, Kang-Cheng; Xu, Chun-He; Chen, Gen-Yun

    2005-11-01

    To determine the contribution of charged amino acids to binding with the photosystem II complex (PSII), the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with N-succinimidyl propionate (NSP) or glycine methyl ester (GME) in the presence of a water-soluble carbodiimide, respectively. Based on isoelectric point shift, 4-10 and 10-14 amino groups were modified in the presence of 2 and 4 mM NSP, respectively. Similarly, 3-4 carboxyl groups were modified by reaction with 100 mM GME. Neutralization of negatively charged carboxyl groups with GME did not alter the binding activity of the extrinsic 18 kDa protein. However, the NSP-modified 18 kDa protein, in which the positively charged amino groups had been modified to uncharged methyl esters, failed to bind with the PSII membrane in the presence of the extrinsic 23 kDa protein. This defect can not be attributed to structural or conformational alterations imposed by chemical modification, as the fluorescence and circular dichroism spectra among native, GME- and NSP-modified extrinsic 18 kDa proteins were similar. Thus, we have concluded that the positive charges of lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSII membranes in the presence of the extrinsic 23 kDa protein. Furthermore, it was found that the negative charges of carboxyl groups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSII membranes. PMID:16270152

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

    NASA Astrophysics Data System (ADS)

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

    2008-12-01

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

  1. Relative importance of nitrite oxidation by hypochlorous acid under chloramination conditions.

    PubMed

    Wahman, David G; Speitel, Gerald E

    2012-06-01

    Nitrification can occur in water distribution systems where chloramines are used as the disinfectant. The resulting product, nitrite, can be oxidized by monochloramine and hypochlorous acid (HOCl), potentially leading to rapid monochloramine loss. This research characterizes the importance of the HOCl reaction, which has typically been ignored because of HOCl's low concentration. Also, the general acid-assisted rate constants for carbonic acid and bicarbonate ion were estimated for the monochloramine reaction. The nitrite oxidation reactions were incorporated into a widely accepted chloramine autodecomposition model, providing a comprehensive model that was implemented in AQUASIM. Batch kinetic experiments were conducted to evaluate the significance of the HOCl reaction and to estimate carbonate buffer rate constants for the monochloramine reaction. The experimental data and model simulations indicated that HOCl may be responsible for up to 60% of the nitrite oxidation, and that the relative importance of the HOCl reaction for typical chloramination conditions peaks between pH 7.5 and 8.5, generally increasing with (1) decreasing nitrite concentration, (2) increasing chlorine to nitrogen mass ratio, and (3) decreasing monochloramine concentration. Therefore, nitrite's reaction with HOCl may be important during chloramination and should be included in water quality models to simulate nitrite and monochloramine's fate. PMID:22571335

  2. 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

  3. C5a RECEPTOR (C5aR) CHIMERAS REVEAL THE IMPORTANCE OF LIPID-FACING RESIDUES IN TRANSPORT COMPETENCE

    PubMed Central

    Klco, Jeffery M.; Sen, Saurabh; Hansen, Jakob L.; Lyngsø, Christina; Nikiforovich, Gregory V.; Sheikh, Soren P.; Baranski, Thomas J.

    2009-01-01

    Residues that mediate helix-helix interactions within the seven transmembranes (TM) of G protein-coupled receptors are important for receptor biogenesis and the receptor switch mechanism. In contrast, the residues directly contacting the lipid bilayer have only recently garnered attention as potential receptor dimerization interfaces. In this study we sought to determine the contributions of these lipid-facing residues to receptor function and oligomerization by systemically generating chimeric C5a receptors in which the entire lipid-exposed surface of a single TM helix was exchanged with the cognate residues from the angiotensin AT1 receptor. Disulfide-trapping and BRET studies demonstrated robust homodimerization of both C5a receptor and AT1R, but no evidence for heterodimerization. Despite relatively conservative substitutions, the lipid-facing chimeras (TM 1, 2, 4, 5, 6, or 7) were retained in the ER/cis-Golgi network. With the exception of the TM7 chimera that did not bind ligand, the lipid-facing chimeras bound ligand with low affinity, but similar to wild-type C5a receptors trapped in the ER with Brefeldin A. These results suggest that the chimeric receptors were properly folded; moreover, native C5a receptors are not fully competent to bind ligand when present in the ER. BRET oligomerization studies demonstrated energy transfer between the wild-type C5aR and the lipid-facing chimeras suggesting that the lipid-facing residues within a single TM segment are not essential for oligomerization. These studies highlight the importance of the lipid-facing residues in C5a receptor for transport competence. PMID:19459935

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2011-09-15

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

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

    PubMed Central

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

    2015-01-01

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

  7. 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.

  8. Crystal Structure of a Four-Layer Aggregate of Engineered TMV CP Implies the Importance of Terminal Residues for Oligomer Assembly

    PubMed Central

    Li, Xiangyang; Song, Baoan; Chen, Xi; Wang, Zhenchao; Zeng, Mengjiao; Yu, Dandan; Hu, Deyu; Chen, Zhuo; Jin, Linhong; Yang, Song; Yang, Caiguang; Chen, Baoen

    2013-01-01

    Background Crystal structures of the tobacco mosaic virus (TMV) coat protein (CP) in its helical and disk conformations have previously been determined at the atomic level. For the helical structure, interactions of proteins and nucleic acids in the main chains were clearly observed; however, the conformation of residues at the C-terminus was flexible and disordered. For the four-layer aggregate disk structure, interactions of the main chain residues could only be observed through water–mediated hydrogen bonding with protein residues. In this study, the effects of the C-terminal peptides on the interactions of TMV CP were investigated by crystal structure determination. Methodology/Principal Findings The crystal structure of a genetically engineered TMV CP was resolved at 3.06 Å. For the genetically engineered TMV CP, a six-histidine (His) tag was introduced at the N-terminus, and the C-terminal residues 155 to 158 were truncated (N-His-TMV CP19). Overall, N-His-TMV CP19 protein self-assembled into the four-layer aggregate form. The conformations of residues Gln36, Thr59, Asp115 and Arg134 were carefully analyzed in the high radius and low radius regions of N-His-TMV CP19, which were found to be significantly different from those observed previously for the helical and four-layer aggregate forms. In addition, the aggregation of the N-His-TMV CP19 layers was found to primarily be mediated through direct hydrogen-bonding. Notably, this engineered protein also can package RNA effectively and assemble into an infectious virus particle. Conclusion The terminal sequence of amino acids influences the conformation and interactions of the four-layer aggregate. Direct protein–protein interactions are observed in the major overlap region when residues Gly155 to Thr158 at the C-terminus are truncated. This engineered TMV CP is reassembled by direct protein–protein interaction and maintains the normal function of the four-layer aggregate of TMV CP in the presence of RNA

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

    PubMed

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

    2016-08-01

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

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

    PubMed

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

    2015-06-15

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

  11. Inhibition of nocturnal acidity is important but not essential for duodenal ulcer healing.

    PubMed Central

    Bianchi Porro, G; Parente, F; Sangaletti, O

    1990-01-01

    We have determined the relative importance of day and night time gastric acid inhibition for duodenal ulcer healing by comparing the anti-ulcer efficacy of a single morning with that of a single bedtime dose of ranitidine. One hundred and thirty patients with active duodenal ulcer were randomly assigned to a double-blind therapy with ranitidine 300 mg at 8 am or the same dose at 10 pm for up to eight weeks. The antisecretory effects of these regimens were also assessed by 24 h intragastric pH monitoring in 18 of these patients. At four weeks ulcers had healed in 41/61 (67%) of patients taking the morning dose and in 47/63 (75%) of those receiving the nocturnal dose (95% CI for the difference -0.09 +0.25; p ns). At eight weeks, the corresponding healing rates were 82% and 85.5%, respectively (95% CI for the difference -0.11 +0.17; p ns). Both treatments were significantly superior to placebo in raising 24 h intragastric pH, although the effects of the morning dose were of shorter duration than those of the nocturnal dose. These findings suggest that suppression of nocturnal acidity is important but not essential to promote healing of duodenal ulcers; a prolonged period of acid inhibition during the day (as obtained with a single large morning dose of H2-blockers) may be equally effective. PMID:2186980

  12. Effect of D to E mutation of the RGD motif in rhodostomin on its activity, structure, and dynamics: importance of the interactions between the D residue and integrin.

    PubMed

    Chen, Chiu-Yueh; Shiu, Jia-Hau; Hsieh, Yao-Husn; Liu, Yu-Chen; Chen, Yen-Chin; Chen, Yi-Chun; Jeng, Wen-Yih; Tang, Ming-Jer; Lo, Szecheng J; Chuang, Woei-Jer

    2009-09-01

    Rhodostomin (Rho) is a snake venom protein containing an RGD motif that specifically inhibits the integrin-binding function. Rho produced in Pichia pastoris inhibits platelet aggregation with a K(I) of 78 nM as potent as native Rho. In contrast, its D51E mutant inhibits platelet aggregation with a K(I) of 49 muM. Structural analysis of Rho and its D51E mutant showed that they have the same tertiary fold with three two-stranded antiparallel beta-sheets. There are no structural backbone differences between the RG[D/E] loop which extends outward from the protein core and the RG[D/E] sequence at its apex in a four-residue RG[D/E]M type I turn. Two minor differences between Rho and its D51E mutant were only found from their backbone dynamics and 3D structures. The R(2) value of E51 is 13% higher than that of the D51 residue. A difference in the charge separation of 1.76 A was found between the sidechains of positive (R49) and negative residues (D51 or E51).The docking of Rho into integrin alphavbeta3 showed that the backbone amide and carbonyl groups of the D51 residue of Rho were formed hydrogen bonds with the integrin residues R216 and R214, respectively. In contrast, these hydrogen bonds were absent in the D51E mutant-integrin complex. Our findings suggest that the interactions between both the sidechain and backbone of the D residue of RGD-containing ligands and integrin are important for their binding. PMID:19280603

  13. Effect of P to A Mutation of the N-Terminal Residue Adjacent to the Rgd Motif on Rhodostomin: Importance of Dynamics in Integrin Recognition

    PubMed Central

    Chen, Yi-Chun; Chang, Yao-Tsung; Chang, Yung-Sheng; Huang, Chun-Hao; Chuang, Woei-Jer

    2012-01-01

    Rhodostomin (Rho) is an RGD protein that specifically inhibits integrins. We found that Rho mutants with the P48A mutation 4.4–11.5 times more actively inhibited integrin α5β1. Structural analysis showed that they have a similar 3D conformation for the RGD loop. Docking analysis also showed no difference between their interactions with integrin α5β1. However, the backbone dynamics of RGD residues were different. The values of the R2 relaxation parameter for Rho residues R49 and D51 were 39% and 54% higher than those of the P48A mutant, which caused differences in S2, Rex, and τe. The S2 values of the P48A mutant residues R49, G50, and D51 were 29%, 14%, and 28% lower than those of Rho. The Rex values of Rho residues R49 and D51 were 0.91 s−1 and 1.42 s−1; however, no Rex was found for those of the P48A mutant. The τe values of Rho residues R49 and D51 were 9.5 and 5.1 times lower than those of P48A mutant. Mutational study showed that integrin α5β1 prefers its ligands to contain (G/A)RGD but not PRGD sequences for binding. These results demonstrate that the N-terminal proline residue adjacent to the RGD motif affect its function and dynamics, which suggests that the dynamic properties of the RGD motif may be important in Rho's interaction with integrin α5β1. PMID:22238583

  14. 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.

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

    PubMed Central

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

    2012-01-01

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

  16. 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.

  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. 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.

  19. 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.

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

    PubMed

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

    2016-03-01

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

  1. Identification of amino acids important for the catalytic activity of the collagen glucosyltransferase associated with the multifunctional lysyl hydroxylase 3 (LH3).

    PubMed

    Wang, Chunguang; Risteli, Maija; Heikkinen, Jari; Hussa, Anna-Kaisa; Uitto, Lahja; Myllyla, Raili

    2002-05-24

    Collagen glucosyltransferase (GGT) activity has recently been shown to be associated with human lysyl hydroxylase (LH) isoform 3 (LH3) (Heikkinen, J., Risteli, M., Wang, C., Latvala, J., Rossi, M., Valtavaara, M., Myllylä, R. (2000) J. Biol. Chem. 275, 36158-36163). The LH and GGT activities of the multifunctional LH3 protein modify lysyl residues in collagens posttranslationally to form hydroxylysyl and glucosylgalactosyl hydroxylysyl residues respectively. We now report that in the nematode, Caenorhabditis elegans, where only one ortholog is found for lysyl hydroxylase, the LH and GGT activities are also associated with the same gene product. The aim of the present studies is the identification of amino acids important for the catalytic activity of GGT. Our data indicate that the GGT active site is separate from the carboxyl-terminal LH active site of human LH3, the amino acids important for the GGT activity being located at the amino-terminal part of the molecule. Site-directed mutagenesis of a conserved cysteine at position 144 to isoleucine and a leucine at position 208 to isoleucine caused a marked reduction in GGT activity. These amino acids were conserved in C. elegans LH and mammalian LH3, but not in LH1 or LH2, which lack GGT activity. The data also reveal a DXD-like motif in LH3 characteristic of many glycosyltransferases and the mutagenesis of aspartates of this motif eliminated the GGT activity. Reduction in GGT activity was not accompanied by a change in the LH activity of the molecule. Thus GGT activity can be manipulated independently of LH activity in LH3. These data provide the information needed to design knock-out studies for investigation of the function of glucosylgalactosyl hydroxylysyl residues of collagens in vivo. PMID:11896059

  2. Metabolic engineering of lactic acid bacteria for the production of industrially important compounds

    PubMed Central

    Papagianni, Maria

    2012-01-01

    Lactic acid bacteria (LAB) are receiving increased attention for use as cell factories for the production of metabolites with wide use by the food and pharmaceutical industries. The availability of efficient tools for genetic modification of LAB during the past decade permitted the application of metabolic engineering strategies at the levels of both the primary and the more complex secondary metabolism. The recent developments in the area with a focus on the production of industrially important metabolites will be discussed in this review. PMID:24688663

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

    PubMed

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

    2015-10-21

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

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

    PubMed

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

    2004-12-01

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

  5. The importance of residues 195-206 of human blood clotting factor VII in the interaction of factor VII with tissue factor

    SciTech Connect

    Wildgoose, P.; Kisiel, W.; Kazim, A.L. )

    1990-09-01

    Previous studies indicated that human and bovine factor VII exhibit 71% amino acid sequence identity. In the present study, competition binding experiments revealed that the interaction of human factor VII with cell-surface human tissue factor was not inhibited by 100-fold molar excess of bovine factor VII. This finding indicated that bovine and human factor VII are not structurally homologous in the region(s) where human factor VII interacts with human tissue factor. On this premise, the authors synthesized three peptides corresponding to regions of human factor VII that exhibited marked structural dissimilarity to bovine factor VII; these regions of dissimilarity included residues 195-206, 263-274, and 314-326. Peptide 195-206 inhibited the interaction of factor VII with cell-surface tissue factor and the activation of factor X by a complex of factor VIIa and tissue factor half-maximally at concentrations of 1-2 mM. A structurally rearranged form of peptide 195-206 containing an aspartimide residue inhibited these reactions half-maximally at concentrations of 250-300 {mu}M. In contrast, neither peptide 263-274 nor peptide 314-326, at 2 mM concentration, significantly affected either factor VIIa interaction with tissue factor or factor VIIa-mediated activation of factor X. The data provide presumptive evidence that residues 195-206 of human factor VII are involved in the interaction of human factor VII with the extracellular domain of human tissue factor apoprotein.

  6. Structural analysis of a penicillin V acylase from Pectobacterium atrosepticum confirms the importance of two Trp residues for activity and specificity.

    PubMed

    Avinash, Vellore Sunder; Panigrahi, Priyabrata; Chand, Deepak; Pundle, Archana; Suresh, Cheravakattu Gopalan; Ramasamy, Sureshkumar

    2016-02-01

    Penicillin V acylases (PVA) catalyze the deacylation of the beta-lactam antibiotic phenoxymethylpenicillin (Pen V). They are members of the Ntn hydrolase family and possess an N-terminal cysteine as the main catalytic nucleophile residue. They form the evolutionarily related cholylglycine hydrolase (CGH) group which includes bile salt hydrolases (BSH) responsible for bile deconjugation. Even though a few PVA and BSH structures have been reported, no structure of a functional PVA from Gram-negative bacteria is available. Here, we report the crystal structure of a highly active PVA from Gram-negative Pectobacterium atrosepticum (PaPVA) at 2.5Å resolution. Structural comparison with PVAs from Gram-positive bacteria revealed that PaPVA had a distinctive tetrameric structure and active site organization. In addition, mutagenesis of key active site residues and biochemical characterization of the resultant variants elucidated the role of these residues in substrate binding and catalysis. The importance of residue Trp23 and Trp87 side chains in binding and correct positioning of Pen V by PVAs was confirmed using mutagenesis and substrate docking with a 15ns molecular dynamics simulation. These results establish the unique nature of Gram-negative CGHs and necessitate further research about their substrate spectrum. PMID:26707624

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

    PubMed

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

    2008-03-12

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

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

    PubMed

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

    2015-12-01

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

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

    PubMed

    Sellami, Sameh; Cherif, Marwa; Jamoussi, Kaïs

    2016-06-01

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

  10. Mineralization of naphtenic acids with thermally-activated persulfate: The important role of oxygen.

    PubMed

    Xu, Xiyan; Pliego, Gema; Zazo, Juan A; Casas, Jose A; Rodriguez, Juan J

    2016-11-15

    This study reports on the mineralization of model naphtenic acids (NAs) in aqueous solution by catalyst-free thermally-activated persulfate (PS) oxidation. These species are found to be pollutants in oil sands process-affected waters. The NAs tested include saturated-ring (cyclohexanecarboxylic and cyclohexanebutyric acids) and aromatic (2-naphthoic and 1,2,3,4-tetrahydro-2-naphthoic acids) structures, at 50mgL(-1)starting concentration. The effect of PS dose within a wide range (10-100% of the theoretical stoichiometric) and working temperature (40-97°C) was investigated. At 80°C and intitial pH=8 complete mineralization of the four NAs was achieved with 40-60% of the stoichiometric PS dose. This is explained because of the important contribution of oxygen, which was experimentally verified and was found to be more effective toward the NAs with a single cyclohexane ring than for the bicyclic aromatic-ring-bearing ones. The effect of chloride and bicarbonate was also checked. The former showed negative effect on the degradation rate of NAs whereas it was negligible or even positive for bicarbonate. The rate of mineralization was well described by simple pseudo-first order kinetics with values of the rate constants normalized to the PS dose within the range of 0.062-0.099h(-1). Apparent activation energy values between 93.7-105.3kJmol(-1) were obtained. PMID:27442986

  11. Quantitative importance of the 25-hydroxylation pathway for bile acid biosynthesis in the rat

    SciTech Connect

    Duane, W.C.; Bjoerkhem, I.H.; Hamilton, J.N.; Mueller, S.M.

    1988-05-01

    During biosynthesis of bile acid, carbons 25-26-27 are removed from the cholesterol side chain. Side-chain oxidation begins either with hydroxylation at the 26-position, in which case the three-carbon fragment is released as propionic acid, or with hydroxylation at the 25-position, in which case the three-carbon fragment is released as acetone. In the present study, we have quantitated the relative importance of these two pathways in vivo by measuring production of (14C) acetone from (14C)-26-cholesterol. Four days after intraperitoneal injection of 20 to 40 muCi (14C)-26-cholesterol and 1 day after beginning a constant intravenous infusion of unlabeled acetone at 25 mumoles per kg per min, 6 male and 2 female Sprague-Dawley rats underwent breath collections. Expired acetone was trapped and purified as the 2,4-dinitrophenylhydrazine derivative. 14CO2 was trapped quantitatively using phenethylamine. Specific activity of breath acetone was multiplied times the acetone infusion rate to calculate production of (14C)acetone. (14C) Acetone production averaged 1.7% of total release of 14C from (14C)-26-cholesterol, estimated by 14CO2 output. The method was validated by showing that (14C) acetone production from (14C)isopropanol averaged 111% of the (14C)isopropanol infusion rate. We conclude that, in the normal rat, the 25-hydroxylation pathway accounts for less than 2% of bile acid synthesis.

  12. 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

  13. Importance of Fatty Acid Compositions in Patients with Peripheral Arterial Disease

    PubMed Central

    Shiba, Yuji; Motoki, Hirohiko; Takeuchi, Takahiro; Okada, Ayako; Tomita, Takeshi; Miyashita, Yusuke; Koyama, Jun; Ikeda, Uichi

    2014-01-01

    Objective Importance of fatty acid components and imbalances has emerged in coronary heart disease. In this study, we analyzed fatty acids and ankle-brachial index (ABI) in a Japanese cohort. Methods Peripheral arterial disease (PAD) was diagnosed in 101 patients by ABI ≤0.90 and/or by angiography. Traditional cardiovascular risk factors and components of serum fatty acids were examined in all patients (mean age 73.2±0.9 years; 81 males), and compared with those in 373 age- and sex-matched control subjects with no evidence of PAD. Results The presence of PAD (mean ABI: 0.71±0.02) was independently associated with low levels of gamma-linolenic acid (GLA) (OR: 0.90; 95% CI: 0.85–0.96; P = 0.002), eicosapentaenoic acid∶arachidonic acid (EPA∶AA) ratio (OR: 0.38; 95% CI: 0.17–0.86; P = 0.021), and estimated glomerular filtration rate (OR: 0.97; 95% CI: 0.96–0.98; P<0.0001), and with a high hemoglobin A1c level (OR: 1.34; 95% CI: 1.06–1.69; P = 0.013). Individuals with lower levels of GLA (≤7.95 µg/mL) and a lower EPA∶AA ratio (≤0.55) had the lowest ABI (0.96±0.02, N = 90), while the highest ABI (1.12±0.01, N = 78) was observed in individuals with higher values of both GLA and EPA∶AA ratio (P<0.0001). Conclusion A low level of GLA and a low EPA∶AA ratio are independently associated with the presence of PAD. Specific fatty acid abnormalities and imbalances could lead to new strategies for risk stratification and prevention in PAD patients. PMID:25191963

  14. The Importance of Nitrous Acid (hono) For Tropospheric Daytime Chemistry In Urban Areas

    NASA Astrophysics Data System (ADS)

    Trick, S.; Geyer, A.; Platt, U.; Bruno, P.; Febo, A.; Acker, K.; Wieprecht, W.

    Nitrous acid (HONO) has been first detected as a typical product of the polluted urban atmosphere about 20 years ago. At this time OH radicals were believed to be formed dominantly by ozone photolysis followed by the reaction with water vapor. Influences of the photolysis of nitrous acid and aldehydes as well as the ozonolysis of alkenes were suggested to be of minor importance for a long time. This was item reasoned by measurements of ozone levels reaching its maximum at noontime, when HONO was found to be below the detection limit at the same time. Here we present the data set and interpretation of an intensive field campaign in May and June 2001 in Rome, Italy during various situations (rainy and clear sky days and photochemical smog events). HONO, NO2, O3, HCHO, NO, SO2, CO, photoly- sis frequencies, aerosol amount and distribution, meteorological data and radon were reported in the city of Rome, at a background station in Rome and in the plume of Rome. During the campaign high HONO daytime levels averaging 200 ppt clear above the detection limits have been measured by three different and independent measurement techniques: Differential Optical Absorption Spectroscopy, Wetted Ef- fluent Diffusion Denuder and High Performance Liquid Chromatography. These mea- surements and first modeling studies give strong evidence that HONO is the most important OH source in urban atmosphere throughout the day. Especially early in the morning, when ozone is still around the detection limit, HONO photolysis was found to initialize photochemical smog events in Rome.

  15. Importance of Branched-Chain Amino Acid Utilization in Francisella Intracellular Adaptation

    PubMed Central

    Gesbert, Gael; Ramond, Elodie; Tros, Fabiola; Dairou, Julien; Frapy, Eric; Barel, Monique

    2014-01-01

    Intracellular bacterial pathogens have adapted their metabolism to optimally utilize the nutrients available in infected host cells. We recently reported the identification of an asparagine transporter required specifically for cytosolic multiplication of Francisella. In the present work, we characterized a new member of the major super family (MSF) of transporters, involved in isoleucine uptake. We show that this transporter (here designated IleP) plays a critical role in intracellular metabolic adaptation of Francisella. Inactivation of IleP severely impaired intracellular F. tularensis subsp. novicida multiplication in all cell types tested and reduced bacterial virulence in the mouse model. To further establish the importance of the ileP gene in F. tularensis pathogenesis, we constructed a chromosomal deletion mutant of ileP (ΔFTL_1803) in the F. tularensis subsp. holarctica live vaccine strain (LVS). Inactivation of IleP in the F. tularensis LVS provoked comparable intracellular growth defects, confirming the critical role of this transporter in isoleucine uptake. The data presented establish, for the first time, the importance of isoleucine utilization for efficient phagosomal escape and cytosolic multiplication of Francisella and suggest that virulent F. tularensis subspecies have lost their branched-chain amino acid biosynthetic pathways and rely exclusively on dedicated uptake systems. This loss of function is likely to reflect an evolution toward a predominantly intracellular life style of the pathogen. Amino acid transporters should be thus considered major players in the adaptation of intracellular pathogens. PMID:25332124

  16. 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.

  17. 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.

  18. Engineering a thermostable fungal GH10 xylanase, importance of N-terminal amino acids.

    PubMed

    Song, Letian; Tsang, Adrian; Sylvestre, Michel

    2015-06-01

    Xylanases are used in many industrial processes including pulp bleaching, baking, detergent, and the hydrolysis of plant cell wall in biofuels production. In this work we have evolved a single domain GH10 xylanase, Xyn10A_ASPNG, from Aspergillus niger to improve its thermostability. We introduced a rational approach involving as the first step a computational analysis to guide the design of a mutagenesis library in targeted regions which identified thermal important residues that were subsequently randomly mutagenized through rounds of iterative saturation mutagenesis (ISM). Focusing on five residues, four rounds of ISM had generated a quintuple mutant 4S1 (R25W/V29A/I31L/L43F/T58I) which exhibited thermal inactivation half-life (t1/2 ) at 60°C that was prolonged by 30 folds in comparison with wild-type enzyme. Whereas the wild-type enzyme retained 0.2% of its initial activity after a heat treatment of 10 min at 60°C and was completely inactivated after 2 min at 65°C, 4S1 mutant retained 30% of its initial activity after 15 min heating at 65°C. Furthermore, the mutant melting temperature (Tm ) increased by 17.4°C compared to the wild type. Each of the five mutations in 4S1 was found to contribute to thermoresistance, but the dramatic improvement of enzyme thermoresistance of 4S1 was attributed to the synergistic effects of the five mutations. Comparison of biochemical data and model structure between 4S1 and the wild-type enzyme suggested that the N-terminal coil of the enzyme is important in stabilizing GH10 xylanase structure. Based on model structure analyses, we propose that enforced hydrophobic interactions within N-terminal elements and between N- and C-terminal ends are responsible for the improved thermostability of Xyn10A_ASPNG. PMID:25640404

  19. 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

  20. 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

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

    SciTech Connect

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

    1987-02-10

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

  2. New functions of the chloroplast Preprotein and Amino acid Transporter (PRAT) family members in protein import.

    PubMed

    Rossig, Claudia; Reinbothe, Christiane; Gray, John; Valdes, Oscar; von Wettstein, Diter; Reinbothe, Steffen

    2014-01-01

    Plant cells contain distinct compartments such as the nucleus, the endomembrane system comprising the endoplasmic reticulum and Golgi apparatus, peroxisomes, vacuoles, as well as mitochondria and chloroplasts. All of these compartments are surrounded by 1 or 2 limiting membranes and need to import proteins from the cytosol. Previous work led to the conclusion that mitochondria and chloroplasts use structurally different protein import machineries in their outer and inner membranes for the uptake of cytosolic precursor proteins. Our most recent data show that there is some unexpected overlap. Three members of the family of preprotein and amino acid transporters, PRAT, were identified in chloroplasts that mediate the uptake of transit sequence-less proteins into the inner plastid envelope membrane. By analogy, mitochondria contain with TIM22 a related PRAT protein that is involved in the import of transit sequence-less proteins into the inner mitochondrial membrane. Both mitochondria and chloroplasts thus make use of similar import mechanisms to deliver some of their proteins to their final place. Because single homologs of HP20- and HP30-like proteins are present in algae such as Chlamydomonas, Ostreococcus, and Volvox, which diverged from land plants approximately 1 billion years ago, it is likely that the discovered PRAT-mediated mechanism of protein translocation evolved concomitantly with the secondary endosymbiotic event that gave rise to green plants. PMID:24476934

  3. Proximate and fatty acid composition of some commercially important fish species from the Sinop region of the Black Sea.

    PubMed

    Kocatepe, Demet; Turan, Hülya

    2012-06-01

    The proximate and fatty acid compositions of the commercially important fish species (Engraulis encrasicolus, Alosa alosa, Belone belone, Scorpaena porcus, Pomatomus saltatrix, Mullus barbatus) from the Sinop region of the Black Sea were examined. The fat contents ranged from 1.26% (for scorpion fish) to 18.12% (for shad). The protein contents were min 14.54% (for red mullet) and maximum 20.26% (for belone). The fatty acid compositions of the fish ranged from 27.83 to 35.91% for saturated fatty acids, 19.50-33.80% for monounsaturated fatty acids and 15.25-40.02% for polyunsaturated fatty acids. Among the saturated fatty acids, palmitic acid (16:0) (17.75-22.20%) was the dominant fatty acid for all the fish species. As a second saturated fatty acid, myristic acid (14:0) was observed in four of the fish species and its content ranged from 4.72 to 7.31%. Whereas, for the other two fish species, the second saturated fatty acid was stearic acid (18:0) ranging between 4.54 and 10.64%. Among the monounsaturated fatty acids, those occurring in the highest proportions were oleic acid (18:1n-9c) (11.67-22.45%) and palmitoleic acid (16:1) (4.50-9.40%). Docosahexaenoic acid (22:6n-3) (5.41-28.52%), eicosapentaenoic acid (20:5n-3) (4.68-11.06) and linoleic acid (18:2n-6) (1.38-3.49%) were dominant polyunsaturated fatty acids, respectively. All the species, in particular the belone, the anchovy and the shad had high levels of the n-3 series. PMID:22322400

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

    PubMed

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

    1994-06-01

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

  5. 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

  6. 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...

  7. 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...

  8. 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

  9. Identification of N-terminal receptor activity-modifying protein residues important for calcitonin gene-related peptide, adrenomedullin, and amylin receptor function.

    PubMed

    Qi, Tao; Christopoulos, George; Bailey, Richard J; Christopoulos, Arthur; Sexton, Patrick M; Hay, Debbie L

    2008-10-01

    Calcitonin-family receptors comprise calcitonin receptor-like receptor (CL) or calcitonin receptor and receptor activity-modifying protein (RAMP) pairings. Calcitonin gene-related peptide (CGRP) receptors are CL/RAMP1, whereas adrenomedullin (AM) receptors are CL/RAMP2 (AM1 receptor) or CL/RAMP3 (AM2 receptor). Amylin (Amy) receptors are RAMP hetero-oligomers with the calcitonin receptor (AMY1, AMY2, and AMY3, respectively). How RAMPs change G protein-coupled receptor pharmacology is not fully understood. We exploited sequence differences between RAMP1 and RAMP3 to identify individual residues capable of altering receptor pharmacology. Alignment of human RAMPs revealed eight residues that are conserved in RAMP2 and RAMP3 but are different in RAMP1. We hypothesized that residues in RAMP2 and RAMP3, but not RAMP1, are responsible for making CL/RAMP2 and CL/RAMP3 AM receptors. Using site-directed mutagenesis, we introduced individual RAMP3 residues into RAMP1 and vice versa in these eight positions. Mutant or wild-type RAMPs were transfected into Cos7 cells with CL or the insert-negative form of the calcitonin receptor [CT(a)]. Agonist-stimulated cAMP production and cell-surface expression of constructs were measured. Position 74 in RAMP1 and RAMP3 was critical for determining AM potency and affinity, and Phe93 in RAMP1 was an important contributor to alphaCGRP potency at CGRP receptors. Mutant RAMP/CT(a) receptor complexes displayed different phenotypes. It is noteworthy that RAMP1 S103N and W74E mutations led to enhanced rAmy potency, probably related to increased cell-surface expression of these complexes. This differs from the effect on CL-based receptors where expression was unchanged. Targeted substitution has emphasized the importance of position 74 in RAMP1/RAMP3 as a key determinant of AM pharmacology. PMID:18593822

  10. Methyl groups of thymine bases are important for nucleic acid recognition by DtxR.

    PubMed

    Chen, C S; White, A; Love, J; Murphy, J R; Ringe, D

    2000-08-29

    The expression of diphtheria toxin is controlled by the diphtheria toxin repressor (DtxR). Under conditions of high iron concentration, DtxR binds the tox operator to inhibit transcription. To study how DNA binding specificity is achieved by this repressor, we solved the crystal structure of the nickel(II) activated DtxR(C102D) mutant complexed with a 43mer DNA duplex containing the DtxR consensus binding sequence. Structural analysis of this complex and comparison with a previously determined DtxR(C102D)-Ni(II)-tox operator ternary complex revealed unusual van der Waals interactions between Ser37/Pro39 of the repressor helix-turn-helix (HTH) motif and the methyl groups of specific thymine bases in the consensus binding sequence. Gel mobility shift assays utilizing deoxyuridine modified duplex DNA probes proved the importance of these interactions: the four methyl groups shown to interact with Ser37/Pro39 in the crystal structure contribute a total of 3.4 kcal/mol to binding energy. Thus, in addition to making base-specific hydrogen-bonding interactions to the DNA through its Gln43 residue, DtxR also recognizes methyl groups at certain positions in the DNA sequence with its Ser37 and Pro39 side chains, to achieve binding specificity toward its cognate operator sequences. PMID:10956029

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2003-03-15

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

  14. Water-soluble LYNX1 Residues Important for Interaction with Muscle-type and/or Neuronal Nicotinic Receptors*

    PubMed Central

    Lyukmanova, Ekaterina N.; Shulepko, Mikhail A.; Buldakova, Svetlana L.; Kasheverov, Igor E.; Shenkarev, Zakhar O.; Reshetnikov, Roman V.; Filkin, Sergey Y.; Kudryavtsev, Denis S.; Ojomoko, Lucy O.; Kryukova, Elena V.; Dolgikh, Dmitry A.; Kirpichnikov, Mikhail P.; Bregestovski, Piotr D.; Tsetlin, Victor I.

    2013-01-01

    Human LYNX1, belonging to the Ly6/neurotoxin family of three-finger proteins, is membrane-tethered with a glycosylphosphatidylinositol anchor and modulates the activity of nicotinic acetylcholine receptors (nAChR). Recent preparation of LYNX1 as an individual protein in the form of water-soluble domain lacking glycosylphosphatidylinositol anchor (ws-LYNX1; Lyukmanova, E. N., Shenkarev, Z. O., Shulepko, M. A., Mineev, K. S., D'Hoedt, D., Kasheverov, I. E., Filkin, S. Y., Krivolapova, A. P., Janickova, H., Dolezal, V., Dolgikh, D. A., Arseniev, A. S., Bertrand, D., Tsetlin, V. I., and Kirpichnikov, M. P. (2011) NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1. J. Biol. Chem. 286, 10618–10627) revealed the attachment at the agonist-binding site in the acetylcholine-binding protein (AChBP) and muscle nAChR but outside it, in the neuronal nAChRs. Here, we obtained a series of ws-LYNX1 mutants (T35A, P36A, T37A, R38A, K40A, Y54A, Y57A, K59A) and examined by radioligand analysis or patch clamp technique their interaction with the AChBP, Torpedo californica nAChR and chimeric receptor composed of the α7 nAChR extracellular ligand-binding domain and the transmembrane domain of α1 glycine receptor (α7-GlyR). Against AChBP, there was either no change in activity (T35A, T37A), slight decrease (K40A, K59A), and even enhancement for the rest mutants (most pronounced for P36A and R38A). With both receptors, many mutants lost inhibitory activity, but the increased inhibition was observed for P36A at α7-GlyR. Thus, there are subtype-specific and common ws-LYNX1 residues recognizing distinct targets. Because ws-LYNX1 was inactive against glycine receptor, its “non-classical” binding sites on α7 nAChR should be within the extracellular domain. Micromolar affinities and fast washout rates measured for ws-LYNX1 and its mutants are in contrast to nanomolar affinities and irreversibility of binding for α-bungarotoxin and

  15. 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

  16. Reciprocal regulation of amino acid import and epigenetic state through Lat1 and EZH2

    PubMed Central

    Dann, Stephen G; Ryskin, Michael; Barsotti, Anthony M; Golas, Jonathon; Shi, Celine; Miranda, Miriam; Hosselet, Christine; Lemon, Luanna; Lucas, Judy; Karnoub, Maha; Wang, Fang; Myers, Jeremy S; Garza, Scott J; Follettie, Maximillian T; Geles, Kenneth G; Klippel, Anke; Rollins, Robert A; Fantin, Valeria R

    2015-01-01

    Lat1 (SLC7A5) is an amino acid transporter often required for tumor cell import of essential amino acids (AA) including Methionine (Met). Met is the obligate precursor of S-adenosylmethionine (SAM), the methyl donor utilized by all methyltransferases including the polycomb repressor complex (PRC2)-specific EZH2. Cell populations sorted for surface Lat1 exhibit activated EZH2, enrichment for Met-cycle intermediates, and aggressive tumor growth in mice. In agreement, EZH2 and Lat1 expression are co-regulated in models of cancer cell differentiation and co-expression is observed at the invasive front of human lung tumors. EZH2 knockdown or small-molecule inhibition leads to de-repression of RXRα resulting in reduced Lat1 expression. Our results describe a Lat1-EZH2 positive feedback loop illustrated by AA depletion or Lat1 knockdown resulting in SAM reduction and concomitant reduction in EZH2 activity. shRNA-mediated knockdown of Lat1 results in tumor growth inhibition and points to Lat1 as a potential therapeutic target. PMID:25979827

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

    PubMed Central

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

    2004-01-01

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

  18. Metabolites derived from omega-3 polyunsaturated fatty acids are important for cardioprotection.

    PubMed

    Gilbert, Kim; Malick, Mandy; Madingou, Ness; Touchette, Charles; Bourque-Riel, Valérie; Tomaro, Leandro; Rousseau, Guy

    2015-12-15

    Although controversial, some data suggest that omega-3 polyunsaturated fatty acids (PUFA) are beneficial to cardiovascular diseases, and could reduce infarct size. In parallel, we have reported that the administration of Resolvin D1 (RvD1), a metabolite of docosahexaenoic acid, an omega-3 PUFA, can reduce infarct size. The present study was designed to determine if the inhibition of two important enzymes involved in the formation of RvD1 from omega-3 PUFA could reduce the cardioprotective effect of omega-3 PUFA. Sprague-Dawley rats were fed with a diet rich in omega-3 PUFA during 10 days before myocardial infarction (MI). Two days before MI, rats received a daily dose of Meloxicam, an inhibitor of cyclooxygenase-2, PD146176, an inhibitor of 15-lipoxygenase, both inhibitors or vehicle. MI was induced by the occlusion of the left coronary artery for 40min followed by reperfusion. Infarct size and neutrophil accumulation were evaluated after 24h of reperfusion while caspase-3, -8 and Akt activities were assessed at 30min of reperfusion. Rats receiving inhibitors, alone or in combination, showed a larger infarct size than those receiving omega-3 PUFA alone. Caspase-3 and -8 activities are higher in ischemic areas with inhibitors while Akt activity is diminished in groups treated with inhibitors. Moreover, the study showed that RvD1 restores cardioprotection when added to the inhibitors. Results from this study indicate that the inhibition of the metabolism of Omega-3 PUFA attenuate their cardioprotective properties. Then, resolvins seem to be an important mediator in the cardioprotection conferred by omega-3 PUFA in our experimental model of MI. PMID:26550951

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

    PubMed

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

    2007-07-01

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

  20. 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.

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

    PubMed Central

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

    2011-01-01

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

  2. SARS-coronavirus spike S2 domain flanked by cysteine residues C822 and C833 is important for activation of membrane fusion

    SciTech Connect

    Madu, Ikenna G.; Belouzard, Sandrine; Whittaker, Gary R.

    2009-10-25

    The S2 domain of the coronavirus spike (S) protein is known to be responsible for mediating membrane fusion. In addition to a well-recognized cleavage site at the S1-S2 boundary, a second proteolytic cleavage site has been identified in the severe acute respiratory syndrome coronavirus (SARS-CoV) S2 domain (R797). C-terminal to this S2 cleavage site is a conserved region flanked by cysteine residues C822 and C833. Here, we investigated the importance of this well conserved region for SARS-CoV S-mediated fusion activation. We show that the residues between C822-C833 are well conserved across all coronaviruses. Mutagenic analysis of SARS-CoV S, combined with cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for the core-conserved residues C822, D830, L831, and C833. Based on available predictive models, we propose that the conserved domain flanked by cysteines 822 and 833 forms a loop structure that interacts with components of the SARS-CoV S trimer to control the activation of membrane fusion.

  3. 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

  4. 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.

  5. Conservation Weighting Functions Enable Covariance Analyses to Detect Functionally Important Amino Acids

    PubMed Central

    Colwell, Lucy J.; Brenner, Michael P.; Murray, Andrew W.

    2014-01-01

    The explosive growth in the number of protein sequences gives rise to the possibility of using the natural variation in sequences of homologous proteins to find residues that control different protein phenotypes. Because in many cases different phenotypes are each controlled by a group of residues, the mutations that separate one version of a phenotype from another will be correlated. Here we incorporate biological knowledge about protein phenotypes and their variability in the sequence alignment of interest into algorithms that detect correlated mutations, improving their ability to detect the residues that control those phenotypes. We demonstrate the power of this approach using simulations and recent experimental data. Applying these principles to the protein families encoded by Dscam and Protocadherin allows us to make testable predictions about the residues that dictate the specificity of molecular interactions. PMID:25379728

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

    SciTech Connect

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

    1993-03-01

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

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

    PubMed

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

    2008-09-01

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

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

    PubMed

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

    2011-08-01

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

  9. The behavior and importance of lactic acid complexation in Talspeak extraction systems

    SciTech Connect

    Grimes, Travis S.; Nilsson, Mikael; Nash, Kenneth L.

    2008-07-01

    Advanced partitioning of spent nuclear fuel in the UREX +la process relies on the TALSPEAK process for separation of fission-product lanthanides from trivalent actinides. The classic TALSPEAK utilizes an aqueous medium of both lactic acid and diethylenetriaminepentaacetic acid and the extraction reagent di(2-ethylhexyl)phosphoric acid in an aromatic diluent. In this study, the specific role of lactic acid and the complexes involved in the extraction of the trivalent actinides and lanthanides have been investigated using {sup 14}C-labeled lactic acid. Our results show that lactic acid partitions between the phases in a complex fashion. (authors)

  10. 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.

  11. 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.

  12. The importance of the excitatory amino acid transporter 3 (EAAT3).

    PubMed

    Bjørn-Yoshimoto, Walden E; Underhill, Suzanne M

    2016-09-01

    The neuronal excitatory amino acid transporter 3 (EAAT3) is fairly ubiquitously expressed in the brain, though it does not necessarily maintain the same function everywhere. It is important in maintaining low local concentrations of glutamate, where its predominant post-synaptic localization can buffer nearby glutamate receptors and modulate excitatory neurotransmission and synaptic plasticity. It is also the main neuronal cysteine uptake system acting as the rate-limiting factor for the synthesis of glutathione, a potent antioxidant, in EAAT3 expressing neurons, while on GABAergic neurons, it is important in supplying glutamate as a precursor for GABA synthesis. Several diseases implicate EAAT3, and modulation of this transporter could prove a useful therapeutic approach. Regulation of EAAT3 could be targeted at several points for functional modulation, including the level of transcription, trafficking and direct pharmacological modulation, and indeed, compounds and experimental treatments have been identified that regulate EAAT3 function at different stages, which together with observations of EAAT3 regulation in patients is giving us insight into the endogenous function of this transporter, as well as the consequences of altered function. This review summarizes work done on elucidating the role and regulation of EAAT3. PMID:27233497

  13. /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.

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

    PubMed

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

    2010-01-12

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

    2013-01-01

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

  17. The importance of amino acid interactions in the crystallization of hydroxyapatite

    PubMed Central

    Jahromi, M. Tavafoghi; Yao, G.; Cerruti, M.

    2013-01-01

    Non-collagenous proteins (NCPs) inhibit hydroxyapatite (HA; Ca5(PO4)3OH) formation in living organisms by binding to nascent nuclei of HA and preventing their further growth. Polar and charged amino acids (AAs) are highly expressed in NCPs, and the negatively charged ones, such as glutamic acid (Glu) and phosphoserine (P-Ser) seem to be mainly responsible for the inhibitory effect of NCPs. Despite the recognized importance of these AAs on the behaviour of NCPs, their specific effect on HA crystallization is still unclear, and controversial results have been reported concerning the efficacy of HA inhibition of positively versus negatively charged AAs. We focused on a positively charged (arginine, Arg) and a negatively charged (Glu) AA, and their combination in the same solution. We studied their inhibitory effect on HA nucleation and growth at physiological temperature and pH and we determined the mechanism by which they can affect HA crystallization. Our results showed a strong inhibitory effect of Arg on HA nucleation; however, Glu was more effective in inhibiting HA crystal growth during the growth stage. The combination of Glu and Arg was less effective in controlling HA nucleation, but it inhibited HA crystal growth. We attributed these differences to the stability of complexes formed between AAs and calcium and phosphate ions at the nucleation stage, and in bonding strength of AAs to HA crystal faces during the growth stage. The AAs also influenced the morphology of synthesized HA. Presence of either Arg or Glu resulted in the formation of spherulites consisting of preferentially oriented nanoplatelets orientation. This was attributed to kinetic factors favoring growth front nucleation (GFN) mechanism. PMID:23269851

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

    PubMed Central

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

    2012-01-01

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

  19. 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.

  20. Tricarboxylic acid cycle intermediate pool size: functional importance for oxidative metabolism in exercising human skeletal muscle.

    PubMed

    Bowtell, Joanna L; Marwood, Simon; Bruce, Mark; Constantin-Teodosiu, Dumitru; Greenhaff, Paul L

    2007-01-01

    The tricarboxylic acid (TCA) cycle is the major final common pathway for oxidation of carbohydrates, lipids and some amino acids, which produces reducing equivalents in the form of nicotinamide adenine dinucleotide and flavin adenine dinucleotide that result in production of large amounts of adenosine triphosphate (ATP) via oxidative phosphorylation. Although regulated primarily by the products of ATP hydrolysis, in particular adenosine diphosphate, the rate of delivery of reducing equivalents to the electron transport chain is also a potential regulatory step of oxidative phosphorylation. The TCA cycle is responsible for the generation of approximately 67% of all reducing equivalents per molecule of glucose, hence factors that influence TCA cycle flux will be of critical importance for oxidative phosphorylation. TCA cycle flux is dependent upon the supply of acetyl units, activation of the three non-equilibrium reactions within the TCA cycle, and it has been suggested that an increase in the total concentration of the TCA cycle intermediates (TCAi) is also necessary to augment and maintain TCA cycle flux during exercise. This article reviews the evidence of the functional importance of the TCAi pool size for oxidative metabolism in exercising human skeletal muscle. In parallel with increased oxidative metabolism and TCA cycle flux during exercise, there is an exercise intensity-dependent 4- to 5-fold increase in the concentration of the TCAi. TCAi concentration reaches a peak after 10-15 minutes of exercise, and thereafter tends to decline. This seems to support the suggestion that the concentration of TCAi may be of functional importance for oxidative phosphorylation. However, researchers have been able to induce dissociations between TCAi pool size and oxidative energy provision using a variety of nutritional, pharmacological and exercise interventions. Brief periods of endurance training (5 days or 7 weeks) have been found to result in reduced TCAi pool

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

    PubMed

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

    2016-07-15

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

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

    PubMed

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

    2015-11-01

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

  3. Diminishing of aggregation for bovine liver catalase through acidic residues modification.

    PubMed

    Hashemnia, S; Moosavi-Movahedi, A A; Ghourchian, H; Ahmad, F; Hakimelahi, G H; Saboury, A A

    2006-12-15

    The tendency of proteins to aggregate is an important problem in biotechnology and the pharmaceutical industry. Because proteins in the aggregated state generally do not have the same biological activity as proteins in the native state. In order to prevent aggregation, it is essential to know the effective parameters in anti-aggregation mechanism. Using a chemical protein modification approach, UV-vis and fluorescence spectroscopies and circular dichroism spectropolarimetry, this study investigates the parameters involved in anti-aggregation mechanism of bovine liver catalase. Our findings clearly indicate that the modified bovine liver catalase provides better protection than the native enzyme against thermal aggregation. It seems that a decrease in hydrophobicity resulting in chemical modification plays an important role in preventing aggregation. PMID:16828155

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

    PubMed

    Alvarez, Patricia; Blanco, Clara; Granda, Marcos

    2007-06-01

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

  5. Prionic Acid: An Effective Sex Attractant for an Important Pest of Sugarcane, Dorysthenes granulosus (Coleoptera: Cerambycidae: Prioninae).

    PubMed

    Wickham, Jacob D; Lu, Wen; Jin, Tao; Peng, Zhengqiang; Guo, Dongfeng; Millar, Jocelyn G; Hanks, Lawrence M; Chen, Yi

    2016-02-01

    Male Dorysthenes granulosus (Thomson, 1860) (Coleoptera: Cerambycidae: Prioninae) were caught in traps baited with racemic 3,5-dimethyldodecanoic acid (prionic acid) during field screening trials in China that tested known cerambycid pheromones. This species is an important pest of sugarcane (Saccharum officinarum L.). In follow-up dose-response trials, plastic sachets loaded with 1 or 0.1 mg of prionic acid were equally attractive to male beetles, whereas lower doses were no better than controls. Two commercial prionic acid lures also were attractive, suggesting that traps baited with prionic acid can be rapidly incorporated into integrated pest management programs targeting this major pest. It is likely that this compound is a major component of the female-produced sex pheromone of D. granulosus because this species is in the same subfamily as Prionus californicus Motschulsky, 1845, the species from which prionic acid was originally identified. PMID:26362990

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

    PubMed Central

    Saito, Kazuki; Ito, Koichi

    2015-01-01

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

  7. Arabidopsis abscisic acid receptors play an important role in disease resistance.

    PubMed

    Lim, Chae Woo; Lee, Sung Chul

    2015-06-01

    Stomata are natural pores of plants and constitute the entry points for water during transpiration. However, they also facilitate the ingress of potentially harmful bacterial pathogens. The phytohormone abscisic acid (ABA) plays a pivotal role in protecting plants against biotic stress, by regulating stomatal closure. In the present study, we investigated the mechanism whereby ABA influences plant defense responses to Pseudomonas syringae pv. tomato (Pst) DC3000, which is a virulent bacterial pathogen of Arabidopsis, at the pre-invasive stage. We found that overexpression of two ABA receptors, namely, RCAR4/PYL10-OX and RCAR5/PYL11-OX (hereafter referred to as RCARs), resulted in ABA-hypersensitive phenotypes being exhibited during the seed germination and seedling growth stages. Sensitivity to ABA enhanced the resistance of RCAR4-OX and RCAR5-OX plants to Pst DC3000, through promoting stomatal closure leading to the development of resistance to this bacterial pathogen. Protein phosphatase HAB1 is an important component that is responsible for ABA signaling and which interacts with ABA receptors. We found that hab1 mutants exhibited enhanced resistance to Pst DC3000; moreover, similar to RCAR4-OX and RCAR5-OX plants, this enhanced resistance was correlated with stomatal closure. Taken together, our findings demonstrate that alteration of RCAR4- or RCAR5-HAB1 mediated ABA signaling influences resistance to bacterial pathogens via stomatal regulation. PMID:25969135

  8. Importance of ALDH1A enzymes in determining human testicular retinoic acid concentrations

    PubMed Central

    Arnold, Samuel L.; Kent, Travis; Hogarth, Cathryn A.; Schlatt, Stefan; Prasad, Bhagwat; Haenisch, Michael; Walsh, Thomas; Muller, Charles H.; Griswold, Michael D.; Amory, John K.; Isoherranen, Nina

    2015-01-01

    Retinoic acid (RA), the active metabolite of vitamin A, is required for spermatogenesis and many other biological processes. RA formation requires irreversible oxidation of retinal to RA by aldehyde dehydrogenase enzymes of the 1A family (ALDH1A). While ALDH1A1, ALDH1A2, and ALDH1A3 all form RA, the expression pattern and relative contribution of these enzymes to RA formation in the testis is unknown. In this study, novel methods to measure ALDH1A protein levels and intrinsic RA formation were used to accurately predict RA formation velocities in individual human testis samples and an association between RA formation and intratesticular RA concentrations was observed. The distinct localization of ALDH1A in the testis suggests a specific role for each enzyme in controlling RA formation. ALDH1A1 was found in Sertoli cells, while only ALDH1A2 was found in spermatogonia, spermatids, and spermatocytes. In the absence of cellular retinol binding protein (CRBP)1, ALDH1A1 was predicted to be the main contributor to intratesticular RA formation, but when CRBP1 was present, ALDH1A2 was predicted to be equally important in RA formation as ALDH1A1. This study provides a comprehensive novel methodology to evaluate RA homeostasis in human tissues and provides insight to how the individual ALDH1A enzymes mediate RA concentrations in specific cell types. PMID:25502770

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

    PubMed

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

    2006-02-01

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

  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. Functional chondroitin sulfate from Enteroctopus dofleini containing a 3-O-sulfo glucuronic acid residue.

    PubMed

    Higashi, Kyohei; Okamoto, Yusuke; Mukuno, Ann; Wakai, Jun; Hosoyama, Saori; Linhardt, Robert J; Toida, Toshihiko

    2015-12-10

    There are several reports that chondroitin sulfate containing K-type units [GlcA (3S)-GalNAc (4S)] exhibiting similar levels of neurite outgrowth promoting activities as CS having high amounts of B-, D- and E-type disulfated disaccharides. Although CS containing K-type units possess important biological activities, there are only few sources, such as king crab cartilage, squid cartilage or sea cucumber. In this study, CS containing 13.9% of K-type units was found in octopus (Enteroctopus dofleini) cartilage using different substrate specificities of chondroitinases. The 2D NMR spectra showed cross-peaks assigned to protons on sugar ring of GlcA (3S), demonstrating the presence of K-type units in octopus CS. Furthermore, proportion of fucosylated disaccharide units in octopus CS was very low. Octopus CS showed high affinity for growth factors and stimulated neurite outgrowth of hippocampal neurons, similar to the activity of squid CS-E. These results strongly suggest that octopus cartilage is a rich source of CS-K and has important biological activities. PMID:26428158

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  14. Identification of arginine 331 as an important active site residue in the class II fructose-1,6-bisphosphate aldolase of Escherichia coli.

    PubMed Central

    Qamar, S.; Marsh, K.; Berry, A.

    1996-01-01

    Treatment of the Class II fructose-1,6-bisphosphate aldolase of Escherichia coli with the arginine-specific alpha-dicarbonyl reagents, butanedione or phenylglyoxal, results in inactivation of the enzyme. The enzyme is protected from inactivation by the substrate, fructose 1,6-bisphosphate, or by inorganic phosphate. Modification with [7-14C] phenylglyoxal in the absence of substrate demonstrates that enzyme activity is abolished by the incorporation of approximately 2 moles of reagent per mole of enzyme. Sequence alignment of the eight known Class II FBP-aldolases shows that only one arginine residue is conserved in all the known sequences. This residue, Arg-331, was mutated to either alanine or glutamic acid. The mutant enzymes were much less susceptible to inactivation by phenylglyoxal. Measurement of the steady-state kinetic parameters revealed that mutation of Arg-331 dramatically increased the K(m) for fructose 1,6-bisphosphate. Comparatively small differences in the inhibitor constant Ki for dihydroxyacetone phosphate or its analogue, 2-phosphoglycolate, were found between the wild-type and mutant enzymes. In contrast, the mutation caused large changes in the kinetic parameters when glyceraldehyde 3-phosphate was used as an inhibitor. Kinetic analysis of the oxidation of the carbanionic aldolase-substrate intermediate of the reaction by hexacyanoferrate (III) revealed that the K(m) for dihydroxyacetone phosphate was again unaffected, whereas that for fructose 1,6-bisphosphate was dramatically increased. Taken together, these results show that Arg-331 is critically involved in the binding of fructose bisphosphate by the enzyme and demonstrate that it interacts with the C-6 phosphate group of the substrate. PMID:8771208

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

    PubMed Central

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

    1996-01-01

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

  16. The importance of glutamate, glycine, and {gamma}-aminobutyric acid transport and regulation in manganese, mercury and lead neurotoxicity

    SciTech Connect

    Fitsanakis, Vanessa A.; Aschner, Michael . E-mail: michael.aschner@vanderbilt.edu

    2005-05-01

    Historically, amino acids were studied in the context of their importance in protein synthesis. In the 1950s, the focus of research shifted as amino acids were recognized as putative neurotransmitters. Today, many amino acids are considered important neurochemicals. Although many amino acids play a role in neurotransmission, glutamate (Glu), glycine (Gly), and {gamma}-aminobutyric acid (GABA) are among the more prevalent and better understood. Glu, the major excitatory neurotransmitter, and Gly and GABA, the major inhibitory neurotransmitters, in the central nervous system, are known to be tightly regulated. Prolonged exposure to environmental toxicants, such as manganese (Mn), mercury (Hg), or lead (Pb), however, can lead to dysregulation of these neurochemicals and subsequent neurotoxicity. While the ability of these metals to disrupt the regulation of Glu, Gly and GABA have been studied, few articles have examined the collective role of these amino acids in the respective metal's mechanism of toxicity. For each of the neurotransmitters above, we will provide a brief synopsis of their regulatory function, including the importance of transport and re-uptake in maintaining their optimal function. Additionally, the review will address the hypothesis that aberrant homeostasis of any of these amino acids, or a combination of the three, plays a role in the neurotoxicity of Mn, Hg, or Pb.

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

    PubMed

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

    2011-01-01

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

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

    PubMed

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

    2016-01-01

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

  19. Importance of tetrahydroiso alpha-acids to the microbiological stability of beer.

    PubMed

    Caballero, Isabel; Agut, Montserrat; Armentia, Alicia; Blanco, Carlos A

    2009-01-01

    While beer provides a very stable microbiological environment, a few niche microorganisms are capable of growth in malt, wort, and beer. The production of off-flavors and development of turbidity in the packaged product are due to the growth and metabolic activity of wild yeast, certain lactic acid bacteria (LAB) and anaerobic Gram-negative bacteria. Beer also contains bitter hop compounds, which are toxic to Gram-positive and Gram-negative bacteria, and contribute to preventing the spoilage of this beverage. In the boiling process, the hop alpha-acids (humulones) are isomerized into iso alpha-acids. These products are responsible for the bitter taste of beer, but they also play an essential role in enhancing foam stability. Antibacterial activity of iso alpha-acids and their hydrogenated derivates (rhoiso alpha-acids and tetrahydroiso alpha-acids) in MRS broth and beer have been evaluated against different LAB (Lactobacillus and Pediococcus) for the determination of their beer-stabilizing capabilities. Besides this, we have determined the minimum inhibitory concentration and the bacteriostatic effect of each compound against Pediococcus. We found that tetrahydroiso alpha-acids (added directly to beer during production processes) are the compounds that present the greatest antibacterial activity against the main agents implicated in beer spoilage. PMID:19714985

  20. Selective protection and relative importance of the carboxylic acid groups of zaragozic acid A for squalene synthase inhibition.

    PubMed

    Biftu, T; Acton, J J; Berger, G D; Bergstrom, J D; Dufresne, C; Kurtz, M M; Marquis, R W; Parsons, W H; Rew, D R; Wilson, K E

    1994-02-01

    Chemistry that allows selective modification of the carboxylic acid groups of the squalene synthase inhibitor zaragozic acid A (1) was developed and applied to the synthesis of compounds modified at the 3-,4-,5-,3,4-,3,5-, and 4,5-positions. A key step in this procedure is the selective debenzylation by transfer hydrogenolysis in the presence of other olefinic groups. These compounds were tested in the rat squalene synthase assay and in vivo mouse model. Modification at C3 retains significant enzyme potency and enhances oral activity, indicating that C3 is not essential for squalene synthase activity. Modification at C4 and C5 results in significant loss in enzyme activity. In contrast, substitution at C3 or C4 enhances in vivo activity. Furthermore, disubstitution at the C3 and C4 positions results in additive in vivo potency. PMID:8308869

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

    PubMed Central

    Friedman, P A; Shia, M A

    1977-01-01

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

  2. Monoclonal antibodies identify residues 199-216 of the integrin alpha2 vWFA domain as a functionally important region within alpha2beta1.

    PubMed Central

    Tuckwell, D S; Smith, L; Korda, M; Askari, J A; Santoso, S; Barnes, M J; Farndale, R W; Humphries, M J

    2000-01-01

    Integrin alpha2beta1 is the major receptor for collagens in the human body, and the collagen-binding site on the alpha2 subunit von Willebrand factor A-type domain (vWFA domain) is now well defined. However, the biologically important conformational changes that are associated with collagen binding, and the means by which the vWFA domain is integrated into the whole integrin are not completely understood. We have raised monoclonal antibodies against recombinant alpha2 vWFA domain for use as probes of function. Three antibodies, JA202, JA215 and JA218, inhibited binding to collagen, collagen I C-propeptide and E-cadherin, demonstrating that their function is important for structurally diverse alpha2beta1 ligands. Cross-blocking studies grouped the epitopes into two clusters: (I) JA202, the inhibitory antibody, Gi9, and a non-inhibitory antibody, JA208; (II) JA215 and JA218. Both clusters were sensitive to events at the collagen binding site, as binding of Gi9, JA202, JA215 and JA218 were inhibited by collagen peptide, JA208 binding was enhanced by collagen peptide, and binding of JA202 was decreased after mutagenesis of the cation-binding residue Thr(221) to alanine. Binding of cluster I antibodies was inhibited by the anti-functional anti-beta1 antibody Mab13, and binding of Gi9 and JA218 to alpha2beta1 was inhibited by substituting Mn(2+) for Mg(2+), demonstrating that these antibodies were sensitive to changes initiated outside the vWFA domain. Mapping of epitopes showed that JA202 and Gi9 bound between residues 212-216, while JA208 bound between residues 199-216. We have therefore identified two epitope clusters with novel properties; i.e. they are intimately associated with the collagen-binding site, responsive to conformational changes at the collagen-binding site and sensitive to events initiated outside the vWFA domain. PMID:10947963

  3. 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

  4. Comparative Analysis of Lipid Content and Fatty Acid Composition of Commercially Important Fish and Shellfish from Sri Lanka and Japan.

    PubMed

    Devadason, Chandravathany; Jayasinghe, Chamila; Sivakanesan, Ramiah; Senarath, Samanthika; Beppu, Fumiaki; Gotoh, Naohiro

    2016-01-01

    Sri Lanka is surrounded by the Indian Ocean, allowing plenty of fishes to be caught. Moreover, these fishes represent one of the undocumented fish resources in the world and their detailed lipid profiles have not been previously examined. In this study, the lipid content and fatty acid composition of 50 commercially important fishes from the Indian Ocean (Sri Lanka) and the Pacific Ocean (Japan) were compared. The total lipid content and fatty acid composition, including eicosapentaenoic acid (C20:5n-3, EPA) and docosahexaenoic acid (C22:6n-3, DHA), differed significantly among species. Fish from the Pacific Ocean had higher proportions of fatty acids, including EPA and DHA. Herrings and mackerels from both oceanic areas demonstrated high levels of EPA and DHA, and n-3/n-6 ratio. Brackish and freshwater fishes from both groups showed low levels of PUFAs. Fish from the Indian Ocean were high in n-6 fatty acids. Monounsaturated fatty acid levels were high in omnivorous fish from the Pacific Ocean, and saturated fatty acid levels were high in fish from the Indian Ocean. The results of this study will be of value in determining the dietary usefulness of fish caught in Sri Lanka. PMID:27373421

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

    SciTech Connect

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

    2010-11-22

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

  6. Steroidogenesis in MA-10 Mouse Leydig Cells Is Altered via Fatty Acid Import into the Mitochondria1

    PubMed Central

    Rone, Malena B.; Midzak, Andrew S.; Martinez-Arguelles, Daniel B.; Fan, Jinjiang; Ye, Xiaoying; Blonder, Josip; Papadopoulos, Vassilios

    2014-01-01

    ABSTRACT Mitochondria are home to many cellular processes, including oxidative phosphorylation and fatty acid metabolism, and in steroid-synthesizing cells, they are involved in cholesterol import and metabolism, which is the initiating step in steroidogenesis. The formation of macromolecular protein complexes aids in the regulation and efficiency of these mitochondrial functions, though because of their dynamic nature, they are hard to identify. To overcome this problem, we used Blue-Native PAGE with whole-gel mass spectrometry on isolated mitochondria from control and hormone-treated MA-10 mouse tumor Leydig cells. The presence of multiple mitochondrial protein complexes was shown. Although these were qualitatively similar under control and human chorionic gonadotropin (hCG)-stimulated conditions, quantitative differences in the components of the complexes emerged after hCG treatment. A prominent decrease was observed with proteins involved in fatty acid import into the mitochondria, implying that mitochondrial beta-oxidation is not essential for steroidogenesis. To confirm this observation, we inhibited fatty acid import utilizing the CPT1a inhibitor etomoxir, resulting in increased steroid production. Conversely, stimulation of mitochondrial beta-oxidation with metformin resulted in a dose-dependent reduction in steroidogenesis. These changes were accompanied by changes in mitochondrial respiration and in the lactic acid formed during glycolysis. Taken together, these results suggest that upon hormonal stimulation, mitochondria efficiently import cholesterol for steroid production at the expense of other lipids necessary for energy production, specifically fatty acids required for beta-oxidation. PMID:25210128

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

    PubMed

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

    2002-07-31

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

  8. 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

  9. [Importance of docosahexaenoic acid (DHA): Functions and recommendations for its ingestion in infants].

    PubMed

    Gil-Campos, M; Dalmau Serra, J

    2010-09-01

    Docosahexaenoic acid (DHA) is a n-3 polyunsaturated fatty acid with multiple functions, although these are still under study. The development of visual and neurological functions have been demonstrated in premature infants and neonates, however, its effects are still being studied in certain chronic neurological diseases, and inflammatory and metabolic disorders. The DHA requirements are not fixed but recommendations must be based on an intake similar to the composition of breastfeeding, and in older children and during pregnancy and lactation, to ensure consumption of oily fish at least twice a week. It is essential to recognise the need for supplementation of this fatty acid in some diseases that require restricted diet, and in metabolic alterations resulting in a deficiency, but also know the scientific evidence on the effects produced in different situations. This review updates this information to propose an adequate intake of DHA at different ages and in different diseases. PMID:20570579

  10. Folic acid and human reproduction—ten important issues for clinicians

    PubMed Central

    Dunlap, Beth; Shelke, Kantha; Salem, Shala A.; Keith, Louis G.

    2011-01-01

    This article presents data on the current best evidence-based clinical practices and controversies surrounding folic acid supplementation/fortification for the prevention of neural tube defects (NTDs) during early pregnancy. Formatted as a series of ten clinical questions, answers and extensive discussion are provided for each point. We assess the history and evidence behind supplementation and fortification, racial/ethnic disparities in NTDs on a global scale, and present information on risk factors for NTDs other than dietary folic acid deficiency. Also discussed are public health challenges, including disparities in NTD rates, population-wide monitoring of NTDs, and tracking safety data in the post-fortification era. Emerging data are also reviewed regarding the role folic acid may play in malignant processes, cardiovascular disease, male fertility, and other medical conditions. PMID:21991291

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

    PubMed

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

    2009-12-25

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

  12. Nucleic acid probes as a diagnostic method for tick-borne hemoparasites of veterinary importance.

    PubMed

    Figueroa, J V; Buening, G M

    1995-03-01

    An increased number of articles on the use of nucleic acid-based hybridization techniques for diagnostic purposes have been recently published. This article reviews nucleic acid-based hybridization as an assay to detect hemoparasite infections of economic relevance in veterinary medicine. By using recombinant DNA techniques, selected clones containing inserts of Anaplasma, Babesia, Cowdria or Theileria genomic DNA sequences have been obtained, and they are now available to be utilized as specific, highly sensitive DNA or RNA probes to detect the presence of the hemoparasite DNA in an infected animal. Either in an isotopic or non-isotopic detection system, probes have allowed scientists to test for--originally in samples collected from experimentally infected animals and later in samples collected in the field--the presence of hemoparasites during the prepatent, patent, convalescent, and chronic periods of the infection in the host. Nucleic acid probes have given researchers the opportunity to carry out genomic analysis of parasite DNA to differentiate hemoparasite species and to identify genetically distinct populations among and within isolates, strains and clonal populations. Prevalence of parasite infection in the tick vector can now be accomplished more specifically with the nucleic acid probes. Lately, with the advent of the polymerase chain reaction technique, small numbers of hemoparasites can be positively identified in the vertebrate host and tick vector. These techniques can be used to assess the veterinary epidemiological situation in a particular geographical region for the planning of control measures. PMID:7597795

  13. Reduction of ferrylmyoglobin by theanine and green tea catechins. Importance of specific Acid catalysis.

    PubMed

    Yin, Jie; Andersen, Mogens L; Skibsted, Leif H

    2013-03-27

    Reduction of the hypervalent heme pigment ferrylmyoglobin by green tea catechins in aqueous solution of pH = 7.5 was investigated by stopped-flow spectroscopy. Reduction by the gallic acid esters epigallocatechin gallate (EGCG, k2 = 1460 L mol(-1) s(-1), 25.0 °C, 0.16 ionic strength) and epicatechin gallate (ECG, 1410 L mol(-1) s(-1)) was found faster than for epicatechin (EC, 300 L mol(-1) s(-1)) and epigallocatechin (EGC, 200 L mol(-1) s(-1)), even though the gallate ion (G, 330 L mol(-1) s(-1)) is similar in rate to EC. The rate for reduction by EC, EGC, ECG, EGCG, and G shows no correlation with their oxidation potentials or phenolic hydrogen-oxygen bond dissociation energy, but with the pKa of the most acidic phenol group. Theanine, with an acidity similar to that of EC, reduces ferrylmyoglobin with a similar rate (200 L mol(-1) s(-1)), in support of general acid catalysis with an initial proton transfer prior to electron transfer. PMID:23461366

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

    PubMed

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

    2008-02-01

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

  15. The importance of the ratio of omega-6/omega-3 essential fatty acids.

    PubMed

    Simopoulos, A P

    2002-10-01

    Several sources of information suggest that human beings evolved on a diet with a ratio of omega-6 to omega-3 essential fatty acids (EFA) of approximately 1 whereas in Western diets the ratio is 15/1-16.7/1. Western diets are deficient in omega-3 fatty acids, and have excessive amounts of omega-6 fatty acids compared with the diet on which human beings evolved and their genetic patterns were established. Excessive amounts of omega-6 polyunsaturated fatty acids (PUFA) and a very high omega-6/omega-3 ratio, as is found in today's Western diets, promote the pathogenesis of many diseases, including cardiovascular disease, cancer, and inflammatory and autoimmune diseases, whereas increased levels of omega-3 PUFA (a low omega-6/omega-3 ratio) exert suppressive effects. In the secondary prevention of cardiovascular disease, a ratio of 4/1 was associated with a 70% decrease in total mortality. A ratio of 2.5/1 reduced rectal cell proliferation in patients with colorectal cancer, whereas a ratio of 4/1 with the same amount of omega-3 PUFA had no effect. The lower omega-6/omega-3 ratio in women with breast cancer was associated with decreased risk. A ratio of 2-3/1 suppressed inflammation in patients with rheumatoid arthritis, and a ratio of 5/1 had a beneficial effect on patients with asthma, whereas a ratio of 10/1 had adverse consequences. These studies indicate that the optimal ratio may vary with the disease under consideration. This is consistent with the fact that chronic diseases are multigenic and multifactorial. Therefore, it is quite possible that the therapeutic dose of omega-3 fatty acids will depend on the degree of severity of disease resulting from the genetic predisposition. A lower ratio of omega-6/omega-3 fatty acids is more desirable in reducing the risk of many of the chronic diseases of high prevalence in Western societies, as well as in the developing countries, that are being exported to the rest of the world. PMID:12442909

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

    PubMed

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

    2004-04-16

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

  17. Hypoxia optimises tumour growth by controlling nutrient import and acidic metabolite export.

    PubMed

    Parks, Scott K; Cormerais, Yann; Marchiq, Ibtissam; Pouyssegur, Jacques

    2016-01-01

    In their quest for survival and successful growth, cancer cells optimise their cellular processes to enable them to outcompete normal cells in their microenvironment. In essence cancer cells: (i) enhance uptake of nutrients/metabolites, (ii) utilise nutrients more efficiently via metabolic alterations and (iii) deal with the metabolic waste products in a way that furthers their progression while hampering the survival of normal tissue. Hypoxia Inducible Factors (HIFs) act as essential drivers of these adaptations via the promotion of numerous membrane proteins including glucose transporters (GLUTs), monocarboxylate transporters (MCTs), amino-acid transporters (LAT1, xCT), and acid-base regulating carbonic anhydrases (CAs). In addition to a competitive growth advantage for tumour cells, these HIF-regulated proteins are implicated in metastasis, cancer 'stemness' and the immune response. Current research indicates that combined targeting of these HIF-regulated membrane proteins in tumour cells will provide promising therapeutic strategies in the future. PMID:26724171

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

    PubMed

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

    2007-04-19

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

  19. Evaluation of the Impact of Ruminant Trans Fatty Acids on Human Health: Important Aspects to Consider.

    PubMed

    Kuhnt, Katrin; Degen, Christian; Jahreis, Gerhard

    2016-09-01

    The definition and evaluation of trans fatty acids (TFA) with regard to foodstuffs and health hazard are not consistent. Based on the current situation, the term should be restricted only to TFA with isolated double bonds in trans-configuration. Conjugated linoleic acids (CLA) should be separately assessed. Ideally, the origin of the consumed fat should be declared, i.e., ruminant TFA (R-TFA) and industrial TFA (non-ruminant; I-TFA). In ruminant fat, more than 50% of R-TFA consists of vaccenic acid (C18:1 t11). In addition, natural CLA, i.e., c9,t11 CLA is also present. Both are elevated in products from organic farming. In contrast to elaidic acid (t9) and t10, which occur mainly in partially hydrogenated industrial fat, t11 is partially metabolized into c9,t11 CLA via Δ9-desaturation. This is the major metabolic criterion used to differentiate between t11 and other trans C18:1. t11 indicates health beneficial effects in several studies. Moreover, CLA in milk fat is associated with the prevention of allergy and asthma. An analysis of the few studies relating to R-TFA alone makes clear that no convincing adverse physiological effect can be attributed to R-TFA. Only extremely high R-TFA intakes cause negative change in blood lipids. In conclusion, in most European countries, the intake of R-TFA is assessed as being low to moderate. Restriction of R-TFA would unjustifiably represent a disadvantage for organic farming of milk. PMID:25746671

  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. PMID:27098519

  1. 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...

  2. 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

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

    PubMed

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

    1996-08-30

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

  4. A method to assess the ranking importance of uncertainties of residual and dissolution trapping of CO2 on a large-scale storage site

    NASA Astrophysics Data System (ADS)

    Audigane, P.; Rohmer, J.; Manceau, J. C.

    2014-12-01

    The long term fate of mobile CO2 remaining after the injection period is a crucial issue for regulators and operators. There are needs to evaluate properly the amount of gas free to migrate and to estimate the fluid movements at long time scales. Often the difficulty is to manage the computational time to assess the large time and dimension scale of the problem. The second limitation is the large level of uncertainty associated to the computation prediction. A variance-based global sensitivity analysis is proposed to assess the importance ranking of uncertainty sources, with regards to the behavior of the mobile CO2 during the post-injection period. We consider three output parameters which characterize the location and the quantity of mobile CO2, considering residual and dissolution trapping. To circumvent both (i) the large number of computationally intensive reservoir-scale flow simulations and (ii) the different nature of uncertainties whether linked to parameters (continuous variables) or to modeling assumptions (scenario-like variables) we propose to use advanced meta-modeling techniques of ACOSSO-type. The feasibility of the approach is demonstrated using a potential site for CO2 storage in the Paris basin (France), for which the amount, nature and quality of the data available at disposal and the associated uncertainties can be seen as representative to those of a storage project at the post-screening stage. A special attention has been paid to confront the results of the sensitivity analysis with the physical interpretation of the processes.

  5. The valine and lysine residues in the conserved FxVTxK motif are important for the function of phylogenetically distant plant cellulose synthases.

    PubMed

    Slabaugh, Erin; Scavuzzo-Duggan, Tess; Chaves, Arielle; Wilson, Liza; Wilson, Carmen; Davis, Jonathan K; Cosgrove, Daniel J; Anderson, Charles T; Roberts, Alison W; Haigler, Candace H

    2016-05-01

    Cellulose synthases (CESAs) synthesize the β-1,4-glucan chains that coalesce to form cellulose microfibrils in plant cell walls. In addition to a large cytosolic (catalytic) domain, CESAs have eight predicted transmembrane helices (TMHs). However, analogous to the structure of BcsA, a bacterial CESA, predicted TMH5 in CESA may instead be an interfacial helix. This would place the conserved FxVTxK motif in the plant cell cytosol where it could function as a substrate-gating loop as occurs in BcsA. To define the functional importance of the CESA region containing FxVTxK, we tested five parallel mutations in Arabidopsis thaliana CESA1 and Physcomitrella patens CESA5 in complementation assays of the relevant cesa mutants. In both organisms, the substitution of the valine or lysine residues in FxVTxK severely affected CESA function. In Arabidopsis roots, both changes were correlated with lower cellulose anisotropy, as revealed by Pontamine Fast Scarlet. Analysis of hypocotyl inner cell wall layers by atomic force microscopy showed that two altered versions of Atcesa1 could rescue cell wall phenotypes observed in the mutant background line. Overall, the data show that the FxVTxK motif is functionally important in two phylogenetically distant plant CESAs. The results show that Physcomitrella provides an efficient model for assessing the effects of engineered CESA mutations affecting primary cell wall synthesis and that diverse testing systems can lead to nuanced insights into CESA structure-function relationships. Although CESA membrane topology needs to be experimentally determined, the results support the possibility that the FxVTxK region functions similarly in CESA and BcsA. PMID:26646446

  6. Residues in the alpha helix 7 of the bacterial maltose binding protein which are important in interactions with the Mal FGK2 complex.

    PubMed

    Szmelcman, S; Sassoon, N; Hofnung, M

    1997-03-01

    The periplasmic maltose binding protein, MalE, is a major element in maltose transport and in chemotaxis towards this sugar. Previous genetic analysis of the MalE protein revealed functional domains involved in transport and chemotactic functions. Among them the surface located alpha helix 7, which is part of the C-lobe, one of the two lobes forming the three dimensional structure of MalE. Small deletions in this region abolished maltose transport, although maintaining wild-type affinity and specificity as well as a normal chemoreceptor function. It was suggested that alpha helix 7 may be implicated in interactions between the maltose binding protein and the membrane-bound protein complex (Duplay P, Szmelcman S. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. II. Chemotaxis towards maltose. J Mol Biol 194:675-678: Duplay P, Szmelcman S, Bedouelle H, Hofnung M. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. I: Transport of maltose. J Mol Biol 194:663-673). In this study, we submitted a region of 14 residues--Asp 207 to Gly 220--encompassing alpha helix 7, to genetic analysis by oligonucleotide mediated random mutagenesis. Out of 127 identified mutations, twelve single and five double mutants with normal affinities towards maltose were selected for further investigation. Two types of mutations were characterized, silent mutations that did not affect maltose transport and mutations that heavily impaired transport kinetics, even thought the maltose binding capacity of the mutant proteins remained normal. Three substitutions at Tyr 210 (Y210S, Y210L, Y210N) drastically reduced maltose transport. One substitution at Ala 213 (A213I) and one substitution at Glu 214 (E214K) also impaired transport. These three identified residues, Tyr 210, Ala 213, and Glu 214, which are constituents of alpha helix 7, therefore seem to play some important role in maltose

  7. The importance of acid-base management for cardiac and cerebral preservation during open heart operations.

    PubMed

    Swan, H

    1984-04-01

    The basic physiologic characteristics of acid-base equilibria during hypothermia were briefly reviewed. By graphic analysis, four possible clinical strategies for managing the acid-base status of the patient undergoing H-CPB were documented. The effect of hemodilution on buffer capacity was charted in a manner applicable to common current operative procedures. During hypothermia for cardiac operations as presently conducted, the perfusionist is in control of the temperature of the body and the perfusion preservation of the body and brain; the surgeon must assume responsibility for preservation of the heart. The literature pertinent to the relationship of the acid-base state to the functions and structural preservation of the heart and brain during the conditions of cooling to and rewarming from deep hypothermia associated with cardiopulmonary bypass, aortic cross clamping, cardioplegia and total circulatory arrest have been reviewed. The evidence is overwhelming that myocardial anoxia caused by aortic occlusion or total circulatory arrest at any temperature to 15 degrees C. result in progressive acidosis which, of itself, is myotoxic. In contrast, alkalinity is ionotropic. Myocardial ischemia, in both adults and infants, should be prevented and treated by alkaline perfusion cooling and by frequent coronary perfusion of a cardiopreservative solution which is extremely cold (4 to 8 degrees C.), oxygenated, has a pH of 7.8, slightly hyperosmolar and which has a hematocrit of 20 per cent (imidazole, erythrocytes and plasma protein colloid), a cardioplegic ionic pattern and energy substrates. Reperfusion of the heart should begin at a 37 pH of 7.8. Evidence is strong that the use of CO2 added to any gas mixture is harmful. It increases myocardial acidosis; it does not increase cerebral blood flow during hypothermia. Protection of the unperfused brain of an infant should emphasize prevention of circulatory arrest prolonged to more than 40 minutes. Temporary reperfusion

  8. Identification of two amino acids within E2 important for the pathogenicity of chimeric classical swine fever virus.

    PubMed

    Wu, Rui; Li, Ling; Zhao, Yu; Tu, Jun; Pan, Zishu

    2016-01-01

    Our previous study demonstrated that a chimeric classical swine fever virus (CSFV) vSM/CE2 containing the E2 gene of the vaccine C-strain on the genetic background of the virulent CSFV strain Shimen (vSM) was attenuated in swine but reversed to virulence after serial passages in PK15 cells. To investigate the molecular basis of the pathogenicity, the genome of the 11th passage vSM/CE2 variant (vSM/CE2-p11) was sequenced, and two amino acid mutations, T745I and M979K, within E2 of vSM/CE2-p11 were observed. Based on reverse genetic manipulation of the chimeric cDNA clone pSM/CE2, the mutated viruses vSM/CE2/T745I, vSMCE2/M979K and vSM/CE2/T745I;M979K were rescued. The data from infection of pigs demonstrated that the M979K amino acid substitution was responsible for pathogenicity. Studies in vitro indicated that T745I and M979K increased infectious virus production and replication. Our results indicated that two residues located at sites 745 and 979 within E2 play a key role in determining the replication in vitro and pathogenicity in vivo of chimeric CSFV vSM/CE2. PMID:26454191

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

    PubMed Central

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

    1996-01-01

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

  10. Relative Rates of Amino Acid Import via the ABC Transporter GlnPQ Determine the Growth Performance of Lactococcus lactis

    PubMed Central

    Fulyani, Faizah; Schuurman-Wolters, Gea K.; Slotboom, Dirk-Jan

    2015-01-01

    ABSTRACT The GlnPQ transporter from Lactococcus lactis has the remarkable feature of having two substrate-binding domains (SBDs) fused to the N terminus of the transmembrane domain (TMD), and thus four SBDs are present in the homodimeric complex. Although X-ray structures and ligand binding data are available for both SBDs, little is known of how different amino acids compete with each other for transport via GlnPQ. Here we show GlnPQ has a broader substrate specificity than previously thought, with the ability to take up asparagine, glutamine, and glutamic acid, albeit via different routes and with different affinities. Asparagine and glutamine compete with each other at the level of binding to SBD1 and SBD2 (with differences in dissociation constant), but at the same time SBD1 and SBD2 compete with each other at the level of interaction with the translocator domain (with differences in affinity constant and rate of transport). Although glutamine transport via SBD1 is outcompeted by physiological concentrations of asparagine, SBD2 ensures high rates of import of the essential amino acid glutamine. Taken together, this study demonstrates that even in the presence of competing asparagine concentrations, GlnPQ has a high capacity to transport glutamine, which matches the high needs of the cell for glutamine and glutamate. IMPORTANCE GlnPQ is an ATP-binding cassette (ABC) transporter for glutamine, glutamic acid, and asparagine. The system is essential in various Gram-positive bacteria, including L. lactis and several pathogens. Here we show how the amino acids compete with each other for binding to the multiple SBDs of GlnPQ and how these SBDs compete with each other for substrate delivery to the transporter. Overall, our results show that GlnPQ has evolved to transport diverse substrates via different paths and to optimally acquire the abundant and essential amino acid glutamine. PMID:26553850

  11. In-frame amber stop codon replacement mutagenesis for the directed evolution of proteins containing non-canonical amino acids: identification of residues open to bio-orthogonal modification.

    PubMed

    Arpino, James A J; Baldwin, Amy J; McGarrity, Adam R; Tippmann, Eric M; Jones, D Dafydd

    2015-01-01

    Expanded genetic code approaches are a powerful means to add new and useful chemistry to proteins at defined residues positions. One such use is the introduction of non-biological reactive chemical handles for site-specific biocompatible orthogonal conjugation of proteins. Due to our currently limited information on the impact of non-canonical amino acids (nAAs) on the protein structure-function relationship, rational protein engineering is a "hit and miss" approach to selecting suitable sites. Furthermore, dogma suggests surface exposed native residues should be the primary focus for introducing new conjugation chemistry. Here we describe a directed evolution approach to introduce and select for in-frame codon replacement to facilitate engineering proteins with nAAs. To demonstrate the approach, the commonly reprogrammed amber stop codon (TAG) was randomly introduced in-frame in two different proteins: the bionanotechnologically important cyt b(562) and therapeutic protein KGF. The target protein is linked at the gene level to sfGFP via a TEV protease site. In absence of a nAA, an in-frame TAG will terminate translation resulting in a non-fluorescent cell phenotype. In the presence of a nAA, TAG will encode for nAA incorporation so instilling a green fluorescence phenotype on E. coli. The presence of endogenously expressed TEV proteases separates in vivo target protein from its fusion to sfGFP if expressed as a soluble fusion product. Using this approach, we incorporated an azide reactive handle and identified residue positions amenable to conjugation with a fluorescence dye via strain-promoted azide-alkyne cycloaddition (SPAAC). Interestingly, best positions for efficient conjugation via SPAAC were residues whose native side chain were buried through analysis of their determined 3D structures and thus may not have been chosen through rational protein engineering. Molecular modeling suggests these buried native residues could become partially exposed on

  12. 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

  13. 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

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

    PubMed

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

    2016-01-13

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

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

    SciTech Connect

    Lin, Z.; Qvarfort, U.

    1996-12-31

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

  16. 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

  17. Structure-Function of CD36 and Importance of Fatty Acid Signal Transduction in Fat Metabolism

    PubMed Central

    Pepino, Marta Yanina; Kuda, Ondrej; Samovski, Dmitri; Abumrad, Nada A

    2015-01-01

    CD36 is a scavenger receptor that functions in high affinity tissue uptake of long chain fatty acids (FA) and contributes under excessive fat supply to lipid accumulation and metabolic dysfunction. This review describes recent evidence regarding the CD36 FA binding site and a potential mechanism for FA transfer. It also presents the view that CD36 and FA signaling coordinate fat utilization based on newly identified CD36 actions that involve oral fat perception, intestinal fat absorption, secretion of the peptides cholecystokinin and secretin, regulation of hepatic lipoprotein output, activation of beta oxidation by muscle and regulation of the production of the FA derived bioactive eicosanoids. Thus abnormalities of fat metabolism and the associated pathology might involve dysfunction of CD36-mediated signal transduction in addition to the changes of FA uptake. PMID:24850384

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

    PubMed

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

    2010-06-15

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

  19. Relative Importance of Nitrite Oxidation by Hypochlorous Acid under Chloramination Conditions

    EPA Science Inventory

    The importance of nitrite’s oxidation by tree chlorine under chloramination conditions was evaluated using batch kinetic experiments and a chloramine model implemented into the computer program AWUASIM. The experimental data was best represented with the inclusion of a reaction b...

  20. Acid-fast lipids are important structural components of oocyst walls of Cryptosporidium, Toxoplasma, and Eimeria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Coccidia are protozoan parasites that cause significant human disease and are of major agricultural importance. Cryptosporidium spp.cause diarrhea in humans and animals, while congenital Toxoplasma infections causes blindness and death. Eimeria kills chickens, so all poultry feed contain antibioti...

  1. 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

  2. Redox regulation of protein tyrosine phosphatase 1B (PTP1B): Importance of steric and electronic effects on the unusual cyclization of the sulfenic acid intermediate to a sulfenyl amide

    NASA Astrophysics Data System (ADS)

    Sarma, Bani Kanta

    2013-09-01

    The redox regulation of protein tyrosine phosphatase 1B (PTP1B) via the unusual transformation of its sulfenic acid (PTP1B-SOH) to a cyclic sulfenyl amide intermediate is studied by using small molecule chemical models. These studies suggest that the sulfenic acids derived from the H2O2-mediated reactions o-amido thiophenols do not efficiently cyclize to sulfenyl amides and the sulfenic acids produced in situ can be trapped by using methyl iodide. Theoretical calculations suggest that the most stable conformer of such sulfenic acids are stabilized by nO → σ*S-OH orbital interactions, which force the -OH group to adopt a position trans to the S⋯O interaction, leading to an almost linear arrangement of the O⋯S-O moiety and this may be the reason for the slow cyclization of such sulfenic acids to their corresponding sulfenyl amides. On the other hand, additional substituents at the 6-position of o-amido phenylsulfenic acids that can induce steric environment and alter the electronic properties around the sulfenic acid moiety by S⋯N or S⋯O nonbonded interactions destabilize the sulfenic acids by inducing strain in the molecule. This may lead to efficient the cyclization of such sulfenic acids. This model study suggests that the amino acid residues in the close proximity of the sulfenic acid moiety in PTP1B may play an important role in the cyclization of PTP1B-SOH to produce the corresponding sulfenyl amide.

  3. The Importance of Sediment Sulfate Reduction to the Sulfate Budget of an Impoundment Receiving Acid Mine Drainage

    NASA Astrophysics Data System (ADS)

    Herlihy, Alan T.; Mills, Aaron L.; Hornberger, George M.; Bruckner, Amy E.

    1987-02-01

    Alkalinity generation by bacterial sulfate reduction (SR) has been shown to be an important neutralizing agent for acid mine drainage and acid precipitation in lakes and reservoirs. In order to quantify the importance of SR in an acidified system, a sulfate influx-efflux budget was constructed for Lake Anna, an impoundment in central Virginia that receives acid mine drainage. For the 1983 and 1984 water years, 48% (namely, 8.0 × 105 kg) of the sulfate entering the impoundment was removed from the water column within the first 2 km of the arm of the lake receiving the pollution. SR rates measured using 35S-labeled sulfate were extrapolated across the surface area of this arm of the lake; this calculated amount of sulfate removed was equal to 200% of the sulfate removed from the lake as calculated in the budget. The calculated alkalinity generated by this sulfate removal was more than twice that necessary to account for the observed pH increase in the impoundment. The magnitude of the sulfate removal and alkalinity generation demonstrates the quantitative importance of SR as an ecosystem level buffering mechanism.

  4. 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

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

    PubMed

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

    2016-01-01

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

  6. 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

  7. 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

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

    PubMed

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

    2015-04-01

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

  9. Key importance of compression properties in the biophysical characteristics of hyaluronic acid soft-tissue fillers.

    PubMed

    Gavard Molliard, Samuel; Albert, Séverine; Mondon, Karine

    2016-08-01

    Hyaluronic acid (HA) soft-tissue fillers are the most popular degradable injectable products used for correcting skin depressions and restoring facial volume loss. From a rheological perspective, HA fillers are commonly characterised through their viscoelastic properties under shear-stress. However, despite the continuous mechanical pressure that the skin applies on the fillers, compression properties in static and dynamic modes are rarely considered. In this article, three different rheological tests (shear-stress test and compression tests in static and dynamic mode) were carried out on nine CE-marked cross-linked HA fillers. Corresponding shear-stress (G', tanδ) and compression (E', tanδc, normal force FN) parameters were measured. We show here that the tested products behave differently under shear-stress and under compression even though they are used for the same indications. G' showed the expected influence on the tissue volumising capacity, and the same influence was also observed for the compression parameters E'. In conclusion, HA soft-tissue fillers exhibit widely different biophysical characteristics and many variables contribute to their overall performance. The elastic modulus G' is not the only critical parameter to consider amongst the rheological properties: the compression parameters E' and FN also provide key information, which should be taken into account for a better prediction of clinical outcomes, especially for predicting the volumising capacity and probably the ability to stimulate collagen production by fibroblasts. PMID:27093589

  10. Fatty acid metabolism pathway play an important role in carcinogenesis of human colorectal cancers by Microarray-Bioinformatics analysis.

    PubMed

    Yeh, Ching-Sheng; Wang, Jaw-Yuan; Cheng, Tian-Lu; Juan, Chin-Hung; Wu, Chan-Han; Lin, Shiu-Ru

    2006-02-28

    The present study systematically explored metabolic pathways and altered expressions of genes speculatively participating in colorectal carcinogenesis by using a Microarray-Bioinformatic analysis methods. The results revealed that 157 genes were up-regulated and 281 genes were down-regulated in colorectal cancer (CRC). Gene Ontology (GO) and relevant bioinformatics tools indicated that the functional category to which 438 genes (12%; 438/3800) of the most frequent alteration belonged was metabolism. The analysis of 10 colorectal cancer tissue specimens demonstrated that genes involved in fatty acid metabolic pathways had high rates of overexpression. In addition, we stimulated CRL-1790 cell line with linoleic acid (a polyunsaturated fatty acid) for 12, 24, 48 and 72 h. Cell proliferation was elevated by 5, 25, 28 and 31% (P<0.05), respectively. Further analyses revealed that the genes increasingly expressed in the cell line included enoyl-Coenzyme A, hydratase/3-hydroxyacyl Coenzyme A dehydrogenase (EHHADH), enoyl Coenzyme A hydratase, short chain, 1, mitochondrial (ECHS1); glutaryl-Coenzyme A dehydrogenase (GCDH), acyl-Coenzyme A oxidase 2, branched chain (ACOX2); acyl-Coenzyme A dehydrogenase, C-2 to C-3 short chain precursor (ACADS); carnitine palmitoyltransferase 1B (CPT1B), acyl-CoA synthetase long-chain family member 5 (ACSL5), and cytochrome P450, family 4, subfamily A, and polypeptide 11 (CYP4A11) genes. This indicated that the stimulating effect of linoleic acid on cell proliferation was due to interference with the metabolic pathway of fatty acid metabolism. In conclusion, genes with altered expression levels in CRC were mainly associated with fatty acid metabolic pathways speculated to have an important role linked to carcinogenesis. PMID:15885896

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

    PubMed

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

    2010-01-13

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

  12. Chiral separation of the clinically important compounds fucose and pipecolic acid using CE: determination of the most effective chiral selector.

    PubMed

    Hadjistasi, Christoforos A; Stavrou, Ioannis J; Stefan-Van Staden, Raluca-Ioana; Aboul-Enein, Hassan Y; Kapnissi-Christodoulou, Constantina P

    2013-09-01

    In this study, simple electrophoretic methods were developed for the chiral separation of the clinically important compounds fucose and pipecolic acid. In recent years, these analytes, and particularly their individual enantiomers, have attracted considerable attention due to their role in biological functions and disorders. The detectability and sensitivity of pipecolic acid and fucose were improved by reacting them with fluorenylmethyloxycarbonyl chloride (FMOC-Cl) and 5-amino-2-naphthalene-sulfonic acid (ANSA), respectively. The enantioseparation conditions were optimized by initially investigating the type of the chiral selector. Different chiral selectors, such as polymeric surfactants and cyclodextrins, were used and the most effective ones were determined with regard to resolution and analysis time. A 10-mM β-cyclodextrin was able to separate the enantiomers of ANSA-DL-fucose and the polymeric surfactant poly(sodium N-undecanoyl-LL-leucine-valinate) was able to separate the enantiomers of FMOC-DL-pipecolic acid, with resolution values of 3.45 and 2.78, respectively. Additional parameters, such as the concentration and the pH of the background electrolyte (BGE), the concentration of the chiral selector, and the addition of modifiers were examined in order to optimize the separations. The addition of the chiral ionic liquid D-alanine tert-butyl ester lactate into the BGE was also investigated, for the first time, in order to improve resolution of the enantiomers. PMID:23757267

  13. Methionine deficiency leads to hepatic fat accretion via impairment of fatty acid import by carnitine palmitoyltransferase I.

    PubMed

    Kikusato, M; Sudo, S; Toyomizu, M

    2015-04-01

    1. To clarify the underlying mechanism of hepatic fat accretion due to methionine (Met) deficiency in broiler chickens, the present study investigated the effect of Met deficiency on the hepatic carnitine palmitoyltransferase (CPT) system, which imports fatty acids into mitochondria. 2. Fifteen-d-old male meat-type chickens were fed on either a control diet (containing 0.52 g/100 g Met) or a Met-deficient diet (containing 0.27 g Met/100 g). After a 10-d feeding period, the birds were killed by decapitation and their livers excised to determine hepatic CPT1 and CPT2 mRNA levels and for the related hepatic fatty acid-supported mitochondrial respiration to be measured. 3. Met deficiency decreased body weight gain and feed efficiency and increased hepatic lipid content compared to the control group. Whereas the hepatic CPT2 mRNA level in the Met-deficient group remained unchanged compared to that of the control group, the CPT1 mRNA level was decreased in the Met-deficient group and CPT1-dependent hepatic mitochondrial respiration was impaired. 4. Our results suggest that the hepatic lipid accretion that occurs in response to Met deficiency might be attributable to the impairment of CPT1-mediated fatty acid import into mitochondria. PMID:25561085

  14. Identification of amino acids important for binding of Clostridium perfringens epsilon toxin to host cells and to HAVCR1

    PubMed Central

    Ivie, Susan E.; McClain, Mark S.

    2012-01-01

    Clostridium perfringens epsilon toxin belongs to the aerolysin-like family of pore-forming toxins and is one of the most potent bacterial toxins known. The epsilon toxin causes fatal enterotoxemia in sheep, goats, and possibly humans. Evidence indicates that the toxin binds to protein receptors including hepatitis A virus cellular receptor 1 (HAVCR1), but the region of the toxin responsible for cell binding has not been identified. In the present study, we identify amino acids within the epsilon toxin important for this cell interaction. Site-specific mutagenesis was used to investigate the role of a surface-accessible cluster of aromatic amino acids, and purified mutant proteins were tested in a series of cell-culture assays to assess cytotoxic activity and cell binding. When added to cells, four mutant proteins (Etx-Y29E, Etx-Y30E, Etx-Y36E and Etx-Y196E) were severely impaired in their ability to not only kill host cells, but also in their ability to permeabilize the plasma membrane. Circular dichroism spectroscopy and thermal stability studies revealed that the wild-type and mutant proteins were similarly folded. Additional experiments revealed that these mutant proteins were defective in binding to host cells and to HAVCR1. These data indicate that an amino acid motif including Y29, Y30, Y36, and Y196 is important for the ability of epsilon toxin to interact with cells and HAVCR1. PMID:22938730

  15. Identification of amino acids important for binding of Clostridium perfringens epsilon toxin to host cells and to HAVCR1.

    PubMed

    Ivie, Susan E; McClain, Mark S

    2012-09-25

    Clostridium perfringens epsilon toxin belongs to the aerolysin-like family of pore-forming toxins and is one of the most potent bacterial toxins known. The epsilon toxin causes fatal enterotoxemia in sheep, goats, and possibly humans. Evidence indicates that the toxin binds to protein receptors including hepatitis A virus cellular receptor 1 (HAVCR1), but the region of the toxin responsible for cell binding has not been identified. In the present study, we identify amino acids within the epsilon toxin important for this cell interaction. Site-specific mutagenesis was used to investigate the role of a surface-accessible cluster of aromatic amino acids, and purified mutant proteins were tested in a series of cell-culture assays to assess cytotoxic activity and cell binding. When added to cells, four mutant proteins (Etx-Y29E, Etx-Y30E, Etx-Y36E and Etx-Y196E) were severely impaired in their ability to not only kill host cells, but also in their ability to permeabilize the plasma membrane. Circular dichroism spectroscopy and thermal stability studies revealed that the wild-type and mutant proteins were similarly folded. Additional experiments revealed that these mutant proteins were defective in binding to host cells and to HAVCR1. These data indicate that an amino acid motif including Y29, Y30, Y36, and Y196 is important for the ability of epsilon toxin to interact with cells and HAVCR1. PMID:22938730

  16. 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

  17. 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

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

    PubMed

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

    1998-10-01

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

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

    PubMed Central

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

    2003-01-01

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

  20. Importance of relative humidity in the oxidative ageing of organic aerosols: case study of the ozonolysis of maleic acid aerosol

    NASA Astrophysics Data System (ADS)

    Gallimore, P. J.; Achakulwisut, P.; Pope, F. D.; Davies, J. F.; Spring, D. R.; Kalberer, M.

    2011-12-01

    Many important atmospheric aerosol processes depend on the chemical composition of the aerosol, e.g. water uptake and particle cloud interactions. Atmospheric ageing processes, such as oxidation reactions, significantly and continuously change the chemical composition of aerosol particles throughout their lifetime. These ageing processes are often poorly understood. In this study we utilize an aerosol flow tube set up and an ultra-high resolution mass spectrometer to explore the effect of relative humidity (RH) in the range of <5-90% on the ozonolysis of maleic acid aerosol which is employed as model organic aerosol system. Due to the slow reaction kinetics relatively high ozone concentrations of 160-200 ppm were used to achieve an appreciable degree of oxidation of maleic acid. The effect of oxidative ageing on the hygroscopicity of maleic acid particles is also investigated using an electrodynamic balance and thermodynamic modelling. RH has a profound effect on the oxidation of maleic acid particles. Very little oxidation is observed at RH < 50% and the only observed reaction products are glyoxylic acid and formic acid. In comparison, when RH > 50% there are about 15 oxidation products identified. This increased oxidation was observed even when the particles were exposed to high humidities long after a low RH ozonolysis reaction. This result might have negative implications for the use of water as an extraction solvent for the analysis of oxidized organic aerosols. These humidity-dependent differences in the composition of the ozonolyzed aerosol demonstrate that water is both a key reactant in the oxidation scheme and a determinant of particle phase and hence diffusivity. The measured chemical composition of the processed aerosol is used to model the hygroscopic growth, which compares favourably with water uptake results from the electrodynamic balance measurements. A reaction mechanism is presented which takes into account the RH dependent observations. This

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

    PubMed

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

    2006-08-24

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

  2. Comparison of two cochlear implantation techniques and their effects on the preservation of residual hearing. Is the surgical approach of any importance?

    PubMed

    Postelmans, J T F; Stokroos, R J; van Spronsen, E; Grolman, W; Tange, R A; Maré, M J; Dreschler, Wouter Albert

    2014-05-01

    The goal of this work was to review the pre-and postsurgical auditory thresholds of two surgical implantation techniques, namely the mastoidectomy with posterior tympanotomy approach (MPTA) and suprameatal approach (SMA), to determine whether there is a difference in the degree of preservation of residual hearing. In a series of 430 consecutive implanted patients 227 patients had measurable pre-operative hearing thresholds at 250, 500, and 1,000 Hz. These patients were divided into two groups according to the surgical technique that was used for implantation. The SMA approach was followed for 84 patients in Amsterdam, whereas the MPTA technique was adhered to 143 patients in Maastricht. The outcome variables of interest were alteration of pre-and postoperative auditory thresholds after cochlear implantation. Complete or partial preservation of residual hearing was obtained in 21.4 and 21.7% in the SMA and MPTA group, respectively. No statistical differences could be found between the SMA and MPTA group (p = 0.96; Chi-square test). The SMA technique is correlated with a similar degree of hearing loss after cochlear implantation compared to the MPTA technique. However, both techniques were not able to conserve a measurable amount of hearing in patients with a substantial degree of residual hearing. Therefore, both surgical techniques need to be refined for patients in which residual acoustical hearing is pursued. PMID:23632865

  3. A Vibrio cholerae Classical TcpA Amino Acid Sequence Induces Protective Antibody That Binds an Area Hypothesized To Be Important for Toxin-Coregulated Pilus Structure

    PubMed Central

    Taylor, Ronald K.; Kirn, Thomas J.; Meeks, Michael D.; Wade, Terri K.; Wade, William F.

    2004-01-01

    Vibrio cholerae is a gram-negative bacterium that has been associated with cholera pandemics since the early 1800s. Whole-cell, killed, and live-attenuated oral cholera vaccines are in use. We and others have focused on the development of a subunit cholera vaccine that features standardized epitopes from various V. cholerae macromolecules that are known to induce protective antibody responses. TcpA protein is assembled into toxin-coregulated pilus (TCP), a type IVb pilus required for V. cholerae colonization, and thus is a strong candidate for a cholera subunit vaccine. Polypeptides (24 to 26 amino acids) in TcpA that can induce protective antibody responses have been reported, but further characterization of their amino acid targets relative to tertiary or quaternary TCP structures has not been done. We report a refinement of the TcpA sequences that can induce protective antibody. One sequence, TcpA 15 (residues 170 to 183), induces antibodies that bind linear TcpA in a Western blot as well as weakly bind soluble TcpA in solution. These antibodies bind assembled pili at high density and provide 80 to 100% protection in the infant mouse protection assay. This is in sharp contrast to other anti-TcpA peptide sera (TcpA 11, TcpA 13, and TcpA 17) that bind very strongly in Western blot and solution assays yet do not provide protection or effectively bind TCP, as evidenced by immunoelectron microscopy. The sequences of TcpA 15 that induce protective antibody were localized on a model of assembled TCP. These sequences are centered on a site that is predicted to be important for TCP structure. PMID:15385509

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

    PubMed

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

    1977-06-15

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

  5. Fatty Acid Composition and Levels of Selected Polyunsaturated Fatty Acids in Four Commercial Important Freshwater Fish Species from Lake Victoria, Tanzania

    PubMed Central

    Robert, Agnes; Mfilinge, Prosper; Limbu, Samwel M.; Mwita, Chacha J.

    2014-01-01

    Fatty acids (FAs) particularly ω3 and ω6 polyunsaturated fatty acids (PUFAs) play important role in human health. This study aimed to investigate the composition and levels of selected ω3 PUFAs in four commercial fish species, Nile perch (Lates niloticus), Nile tilapia (Oreochromis niloticus), Tilapia zillii, and dagaa (Rastrineobola argentea) from Mwanza Gulf in Lake Victoria. The results indicated that 36 types of FAs with different saturation levels were detected. These FAs were dominated by docosahexaenoic (DHA), eicosapentaenoic (EPA), docosapentaenoic (DPA), and eicosatetraenoic acids. O. niloticus had the highest composition of FAs (34) compared to L. niloticus (27), T. zillii (26), and R. argentea (21). The levels of EPA differed significantly among the four commercial fish species (F = 6.19,  P = 0.001). The highest EPA levels were found in R. argentea followed by L. niloticus and O. niloticus and the lowest in T. zillii. The DPA levels showed no significant difference among the four fish species studied (F = 0.652,  P = 0.583). The study concluded that all four commercial species collected from Mwanza Gulf are good for human health, but R. argentea is the best for consumption because it contains higher levels of ω3 FAs, mainly EPA. PMID:25610654

  6. 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

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

    SciTech Connect

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

    1999-03-01

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

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

    PubMed

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

    2011-05-01

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

  9. Acid rock drainage and rock weathering in Antarctica: important sources for iron cycling in the Southern Ocean.

    PubMed

    Dold, B; Gonzalez-Toril, E; Aguilera, A; Lopez-Pamo, E; Cisternas, M E; Bucchi, F; Amils, R

    2013-06-18

    Here we describe biogeochemical processes that lead to the generation of acid rock drainage (ARD) and rock weathering on the Antarctic landmass and describe why they are important sources of iron into the Antarctic Ocean. During three expeditions, 2009-2011, we examined three sites on the South Shetland Islands in Antarctica. Two of them displayed intensive sulfide mineralization and generated acidic (pH 3.2-4.5), iron-rich drainage waters (up to 1.78 mM Fe), which infiltrated as groundwater (as Fe(2+)) and as superficial runoff (as Fe(3+)) into the sea, the latter with the formation of schwertmannite in the sea-ice. The formation of ARD in the Antarctic was catalyzed by acid mine drainage microorganisms found in cold climates, including Acidithiobacillus ferrivorans and Thiobacillus plumbophilus. The dissolved iron (DFe) flux from rock weathering (nonmineralized control site) was calculated to be 0.45 × 10(9) g DFe yr(-1) for the nowadays 5468 km of ice-free Antarctic rock coastline which is of the same order of magnitude as glacial or aeolian input to the Southern Ocean. Additionally, the two ARD sites alone liberate 0.026 and 0.057 × 10(9) g DFe yr(-1) as point sources to the sea. The increased iron input correlates with increased phytoplankton production close to the source. This might even be enhanced in the future by a global warming scenario, and could be a process counterbalancing global warming. PMID:23682976

  10. Elevated levels of perfluoroalkyl acids in family members of occupationally exposed workers: the importance of dust transfer

    NASA Astrophysics Data System (ADS)

    Fu, Jianjie; Gao, Yan; Wang, Thanh; Liang, Yong; Zhang, Aiqian; Wang, Yawei; Jiang, Guibin

    2015-03-01

    The exposure pathways of perfluoroalkyl acids (PFAAs) to humans are still not clear because of the complex living environment, and few studies have simultaneously investigated the bioaccumulative behaviour of different PFAAs in humans. In this study, serum, dust, duplicate diet, and other matrices were collected around a manufacturing plant in China, and homologous series of PFAAs were analysed. PFAA levels in dust and serum of local residents in this area were considerably higher than those in non-polluted area. Although dietary intake was the major exposure pathway in the present study, dust ingestion played an important role in this case. Serum PFAAs in local residents was significantly correlated with dust PFAAs levels in their living or working microenvironment. Serum PFAAs and dust PFAAs were significantly higher in family members of occupational workers (FM) than in ordinary residents (OR) (p < 0.01). After a careful analysis of the PFAAs exposure pathway, a potential pathway in addition to direct dust ingestion was suggested: PFAAs might transferred from occupational worker's clothes to dinners via cooking processes. The bioaccumulative potential of PFHxS and PFOS were higher than other PFAAs, which suggested a substantial difference between the bioaccumulative ability of perfluorinated sulfonic acids and perfluorinated carboxylic acids.

  11. 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

  12. 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.

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

    PubMed

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

    2012-05-15

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

  14. 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

  15. The fatty acid beta-oxidation pathway is important for decidualization of endometrial stromal cells in both humans and mice.

    PubMed

    Tsai, Jui-He; Chi, Maggie M-Y; Schulte, Maureen B; Moley, Kelle H

    2014-02-01

    Embryo implantation and development requires the endometrial stromal cells (ESCs) to undergo decidualization. This differentiation process requires glucose utilization, and blockade of the pentose phosphate pathway inhibits decidualization of ESCs both in vitro and in vivo. Glucose and fatty acids are energy substrates for many cell types, and fatty acid beta-oxidation is critical for embryo implantation. Here, we investigated whether beta-oxidation is required for decidualization of ESCs. As assessed by marker gene expression, decidualization of human primary ESCs was blocked by reducing activity of carnitine calmitoyltransferase I, the rate-limiting enzyme in beta-oxidation, either by short hairpin RNA-mediated silencing or by treatment with the inhibitor etomoxir. Ranolazine (RAN), a partial beta-oxidation inhibitor, blocked early decidualization of a human ESC line. However, decidualization resumed after several days, most likely due to a compensatory up-regulation of GLUT1 expression and an increase in glucose metabolism. Simultaneous inhibition of the beta-oxidation pathway with RAN and the pentose phosphate pathway with glucosamine (GlcN) impaired in vitro decidualization of human ESCs more strongly than inhibition of either pathway alone. These findings were confirmed in murine ESCs in vitro, and exposure to RAN plus GlcN inhibited decidualization in vivo in a deciduoma model. Finally, intrauterine implantation of time-release RAN and GlcN pellets reduced pup number. Importantly, pup number returned to normal after the end of the pellet-active period. This work indicates that both fatty acids and glucose metabolism pathways are important for ESC decidualization, and suggests novel pathways to target for the design of future nonhormonal contraceptives. PMID:24403548

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-11-01

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

  18. 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

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

    PubMed Central

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

    2003-01-01

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

  20. 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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-08-01

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

  3. 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

  4. 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

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

    PubMed

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

    2011-07-15

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

  6. Mfsd2a Is a Transporter for the Essential ω-3 Fatty Acid Docosahexaenoic Acid (DHA) in Eye and Is Important for Photoreceptor Cell Development.

    PubMed

    Wong, Bernice H; Chan, Jia Pei; Cazenave-Gassiot, Amaury; Poh, Rebecca W; Foo, Juat Chin; Galam, Dwight L A; Ghosh, Sujoy; Nguyen, Long N; Barathi, Veluchamy A; Yeo, Sia W; Luu, Chi D; Wenk, Markus R; Silver, David L

    2016-05-13

    Eye photoreceptor membrane discs in outer rod segments are highly enriched in the visual pigment rhodopsin and the ω-3 fatty acid docosahexaenoic acid (DHA). The eye acquires DHA from blood, but transporters for DHA uptake across the blood-retinal barrier or retinal pigment epithelium have not been identified. Mfsd2a is a newly described sodium-dependent lysophosphatidylcholine (LPC) symporter expressed at the blood-brain barrier that transports LPCs containing DHA and other long-chain fatty acids. LPC transport via Mfsd2a has been shown to be necessary for human brain growth. Here we demonstrate that Mfsd2a is highly expressed in retinal pigment epithelium in embryonic eye, before the development of photoreceptors, and is the primary site of Mfsd2a expression in the eye. Eyes from whole body Mfsd2a-deficient (KO) mice, but not endothelium-specific Mfsd2a-deficient mice, were DHA-deficient and had significantly reduced LPC/DHA transport in vivo Fluorescein angiography indicated normal blood-retinal barrier function. Histological and electron microscopic analysis indicated that Mfsd2a KO mice exhibited a specific reduction in outer rod segment length, disorganized outer rod segment discs, and mislocalization of and reduction in rhodopsin early in postnatal development without loss of photoreceptors. Minor photoreceptor cell loss occurred in adult Mfsd2a KO mice, but electroretinography indicated visual function was normal. The developing eyes of Mfsd2a KO mice had activated microglia and up-regulation of lipogenic and cholesterogenic genes, likely adaptations to loss of LPC transport. These findings identify LPC transport via Mfsd2a as an important pathway for DHA uptake in eye and for development of photoreceptor membrane discs. PMID:27008858

  7. Structural and Functional Importance of Transmembrane Domain 3 (TM3) in the Aspartate:Alanine Antiporter AspT: Topology and Function of the Residues of TM3 and Oligomerization of AspT▿

    PubMed Central

    Nanatani, Kei; Maloney, Peter C.; Abe, Keietsu

    2009-01-01

    AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a

  8. Structural and functional importance of transmembrane domain 3 (TM3) in the aspartate:alanine antiporter AspT: topology and function of the residues of TM3 and oligomerization of AspT.

    PubMed

    Nanatani, Kei; Maloney, Peter C; Abe, Keietsu

    2009-04-01

    AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a

  9. The basic leucine zipper transcription factor ABSCISIC ACID RESPONSE ELEMENT-BINDING FACTOR2 is an important transcriptional regulator of abscisic acid-dependent grape berry ripening processes.

    PubMed

    Nicolas, Philippe; Lecourieux, David; Kappel, Christian; Cluzet, Stéphanie; Cramer, Grant; Delrot, Serge; Lecourieux, Fatma

    2014-01-01

    In grape (Vitis vinifera), abscisic acid (ABA) accumulates during fruit ripening and is thought to play a pivotal role in this process, but the molecular basis of this control is poorly understood. This work characterizes ABSCISIC ACID RESPONSE ELEMENT-BINDING FACTOR2 (VvABF2), a grape basic leucine zipper transcription factor belonging to a phylogenetic subgroup previously shown to be involved in ABA and abiotic stress signaling in other plant species. VvABF2 transcripts mainly accumulated in the berry, from the onset of ripening to the harvesting stage, and were up-regulated by ABA. Microarray analysis of transgenic grape cells overexpressing VvABF2 showed that this transcription factor up-regulates and/or modifies existing networks related to ABA responses. In addition, grape cells overexpressing VvABF2 exhibited enhanced responses to ABA treatment compared with control cells. Among the VvABF2-mediated responses highlighted in this study, the synthesis of phenolic compounds and cell wall softening were the most strongly affected. VvABF2 overexpression strongly increased the accumulation of stilbenes that play a role in plant defense and human health (resveratrol and piceid). In addition, the firmness of fruits from tomato (Solanum lycopersicum) plants overexpressing VvABF2 was strongly reduced. These data indicate that VvABF2 is an important transcriptional regulator of ABA-dependent grape berry ripening. PMID:24276949

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

    PubMed Central

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

    2003-01-01

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

  11. Amino acids flanking the central core of Cu,Zn superoxide dismutase are important in retaining enzyme activity after autoclaving.

    PubMed

    Kumar, Arun; Randhawa, Vinay; Acharya, Vishal; Singh, Kashmir; Kumar, Sanjay

    2016-01-01

    Enzymes are known to be denatured upon boiling, although Cu,Zn superoxide dismutase of Potentilla atrosanguinea (Pot-SOD) retains significant catalytic activity even after autoclaving (heating at 121 °C at a pressure of 1.1 kg per square cm for 20 min). The polypeptide backbone of Pot-SOD consists of 152 amino acids with a central core spanning His45 to Cys145 that is involved in coordination of Cu(2+) and Zn(2+) ions. While the central core is essential for imparting catalytic activity and structural stability to the enzyme, the role of sequences flanking the central core was not understood. Experiments with deletion mutants showed that the amino acid sequences flanking the central core were important in retaining activity of Pot-SOD after autoclaving. Molecular dynamics simulations demonstrated the unfavorable structure of mutants due to increased size of binding pocket and enhanced negative charge on the electrostatic surface, resulting in unavailability of the substrate superoxide radical ([Formula: see text]) to the catalytic pocket. Deletion caused destabilization of structural elements and reduced solvent accessibility that further produced unfavorable structural geometry of the protein. PMID:25990646

  12. The Phosphatidic Acid Binding Site of the Arabidopsis Trigalactosyldiacylglycerol 4 (TGD4) Protein Required for Lipid Import into Chloroplasts*

    PubMed Central

    Wang, Zhen; Anderson, Nicholas Scott; Benning, Christoph

    2013-01-01

    Chloroplast membrane lipid synthesis relies on the import of glycerolipids from the ER. The TGD (TriGalactosylDiacylglycerol) proteins are required for this lipid transfer process. The TGD1, -2, and -3 proteins form a putative ABC (ATP-binding cassette) transporter transporting ER-derived lipids through the inner envelope membrane of the chloroplast, while TGD4 binds phosphatidic acid (PtdOH) and resides in the outer chloroplast envelope. We identified two sequences in TGD4, amino acids 1–80 and 110–145, which are necessary and sufficient for PtdOH binding. Deletion of both sequences abolished PtdOH binding activity. We also found that TGD4 from 18:3 plants bound specifically and with increased affinity PtdOH. TGD4 did not interact with other proteins and formed a homodimer both in vitro and in vivo. Our results suggest that TGD4 is an integral dimeric β-barrel lipid transfer protein that binds PtdOH with its N terminus and contains dimerization domains at its C terminus. PMID:23297418

  13. 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...

  14. 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...

  15. 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)

  16. Conserved Ser residues in the basic region of the bZIP-type transcription factor HBP-1a(17): importance in DNA binding and possible targets for phosphorylation.

    PubMed

    Meshi, T; Moda, I; Minami, M; Okanami, M; Iwabuchi, M

    1998-01-01

    HBP-1a(17) is representative of a group of plant bZIP-type transcription factors which includes HBP-1a proteins and G-box-binding factors. We found kinase activity in wheat nuclear extract that phosphorylated HBP-1a(17). Experiments using recombinant HBP-1a(17) derivatives as substrates revealed that all three of the Ser residues in the basic region, Ser-261, Ser-265, and Ser-269, were phosphorylated in a Ca(2+)-stimulated manner. DNA-binding analysis of mutants with a Ser-to-Glu change, prepared to mimic the phosphorylated proteins, indicated that introduction of a negative charge at position 265 or 269 prevents HBP-1a(17) from binding DNA not only in the homodimer of mutants but also in heterodimers with a wild-type protein. It is therefore suggested that the phosphorylation regulates the function of HBP-1a(17) at least at the level of DNA binding. Since Ser-265 and Ser-269 are highly conserved among the plant bZIP-type factors known to date, a common Ca(2+)-mediated regulatory mechanism may exert an effect on the bZIP-type factors through phosphorylation of these conserved Ser residues. PMID:9484468

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

    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

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

    PubMed

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

    2016-05-01

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

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

    PubMed

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

    2016-08-16

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

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

    PubMed

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

    2016-05-11

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

  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. An amino acid substitution in the pyruvate dehydrogenase E1{alpha} gene, affecting mitochondrial import of the precursor protein

    SciTech Connect

    Takakubo, F.; Thorburn, D.R.; Dahl, H.H.M.

    1995-10-01

    A mutation in the mitochondrial targeting sequence was characterized in a male patient with X chromosome-linked pyruvate dehydrogenase E1{alpha} deficiency. The mutation was a base substitution of G by C at nucleotide 134 in the mitochondrial targeting sequence of the PDHA1 gene, resulting in an arginine-to-proline substitution at codon 10 (R10P). Pyruvate dehydrogenase activity in cultured skin fibroblasts was 28% of the control value, and immunoblot analysis revealed a decreased level of pyruvate dehydrogenase E1{alpha}immunoreactivity. Chimeric constructs in which the normal and mutant pyruvate dehydrogenase E1{alpha} targeting sequences were attached to the mitochondrial matrix protein ornithine transcarbamylase were synthesized in a cell free translation system, and mitochondrial import of normal and mutant proteins was compared in vitro. The results show that ornithine transcarbamylase targeted by the mutant pyruvate dehydrogenase E1{alpha} sequence was translocated into the mitochondrial matrix at a reduced rate, suggesting that defective import is responsible for the reduced pyruvate dehydrogenase level in mitochondria. The mutation was also present in an affected brother and the mildly affected mother. The clinical presentations of this X chromosome-linked disorder in affected family members are discussed. To our knowledge, this is the first report of an amino acid substitution in a mitochondrial targeting sequence resulting in a human genetic disease. 58 refs., 5 figs., 1 tab.

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

    PubMed Central

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

    1983-01-01

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

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    1999-02-01

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

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

    PubMed

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

    2015-06-28

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

  7. Increase on the Initial Soluble Heme Levels in Acidic Conditions Is an Important Mechanism for Spontaneous Heme Crystallization In Vitro

    PubMed Central

    Egan, Timothy J.; Wright, David W.; Oliveira, Marcus F.

    2010-01-01

    Background Hemozoin (Hz) is a heme crystal that represents a vital pathway for heme disposal in several blood-feeding organisms. Recent evidence demonstrated that β-hematin (βH) (the synthetic counterpart of Hz) formation occurs under physiological conditions near synthetic or biological hydrophilic-hydrophobic interfaces. This seems to require a heme dimer acting as a precursor of Hz crystals that would be formed spontaneously in the absence of the competing water molecules bound to the heme iron. Here, we aimed to investigate the role of medium polarity on spontaneous βH formation in vitro. Methodology/Principal Findings We assessed the effect of water content on spontaneous βH formation by using the aprotic solvent dimethylsulfoxide (DMSO) and a series of polyethyleneglycols (PEGs). We observed that both DMSO and PEGs (3.350, 6.000, 8.000, and 22.000) increased the levels of soluble heme under acidic conditions. These compounds were able to stimulate the production of βH crystals in the absence of any biological sample. Interestingly, the effects of DMSO and PEGs on βH formation were positively correlated with their capacity to promote previous heme solubilization in acidic conditions. Curiously, a short chain polyethyleneglycol (PEG 300) caused a significant reduction in both soluble heme levels and βH formation. Finally, both heme solubilization and βH formation strongly correlated with reduced medium water activity provided by increased DMSO concentrations. Conclusions The data presented here support the notion that reduction of the water activity is an important mechanism to support spontaneous heme crystallization, which depends on the previous increase of soluble heme levels. PMID:20856937

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

    PubMed

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

    2016-08-01

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2012-12-01

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

  11. TolC is important for bacterial survival and oxidative stress response in Salmonella enterica serovar Choleraesuis in an acidic environment.

    PubMed

    Lee, Jen-Jie; Wu, Ying-Chen; Kuo, Chih-Jung; Hsuan, Shih-Ling; Chen, Ter-Hsin

    2016-09-25

    The outer membrane protein TolC, which is one of the key components of several multidrug efflux pumps, is thought to be involved in various independent systems in Enterobacteriaceae. Since the acidic environment of the stomach is an important protection barrier against foodborne pathogen infections in hosts, we evaluated whether TolC played a role in the acid tolerance of Salmonella enterica serovar Choleraesuis. Comparison of the acid tolerance of the tolC mutant and the parental wild-type strain showed that the absence of TolC limits the ability of Salmonella to sustain life under extreme acidic conditions. Additionally, the mutant exhibited morphological changes during growth in an acidic medium, leading to the conflicting results of cell viability measured by spectrophotometry and colony-forming unit counting. Reverse-transcriptional-PCR analysis indicated that acid-related molecules, apparatus, or enzymes and oxidation-induced factors were significantly affected by the acidic environment in the null-tolC mutant. The elongated cellular morphology was restored by adding antioxidants to the culture medium. Furthermore, we found that increased cellular antioxidative activity provides an overlapping protection against acid killing, demonstrating the complexity of the bacterial acid stress response. Our findings reinforce the multifunctional characteristics of TolC in acid tolerance or oxidative stress resistance and support the correlative protection mechanism between oxygen- and acid-mediated stress responses in Salmonella enterica serovar Choleraesuis. PMID:27599929

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

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

  13. An important role of a pyrethroid-sensing residue F1519 in the action of the N-alkylamide insecticide BTG 502 on the cockroach sodium channel

    PubMed Central

    Du, Yuzhe; Khambay, Bhupinder

    2011-01-01

    Deltamethrin, a pyrethroid insecticide, and BTG 502, an alkylamide insecticide, target voltage-gated sodium channels. Deltamethrin binds to a unique receptor site and causes prolonged opening of sodium channels by inhibiting deactivation and inactivation. Previous 22Na+ influx and receptor binding assays using mouse brain synaptoneurosomes showed that BTG 502 antagonized the binding and action of batrachotoxin (BTX), a site 2 sodium channel neurotoxin. However, the effect of BTG 502 has not been examined directly on sodium channels expressed in Xenopus oocytes. In this study, we examined the effect of BTG 502 on wild-type and mutant cockroach sodium channels expressed in Xenopus oocytes. Toxin competition experiments confirmed that BTG 502 antagonizes the action of BTX and possibly shares a common receptor site with BTX. However, unlike BTX which causes persistent activation of sodium channels, BTG 502 reduces the amplitude of peak sodium current. A previous study showed that BTG 502 was more toxic to pyrethroid-resistant house flies possessing a super-kdr (knockdown resistance) mechanism than to pyrethroid-susceptible house flies. However, we found that the cockroach sodium channels carrying the equivalent super-kdr mutations (M918T and L1014F) were not more sensitive to BTG 502 than the wild-type channel. Instead, a kdr mutation, F1519I, which reduces pyrethroid binding, abolished the action of BTG 502. These results provide evidence the actions of alkylamide and pyrethroid insecticides require a common sodium channel residue. PMID:21426938

  14. Design and Exploration of Novel Boronic Acid Inhibitors Reveals Important Interactions with a Clavulanic Acid-Resistant Sulfhydryl-Variable (SHV) β-Lactamase

    PubMed Central

    Winkler, Marisa L.; Rodkey, Elizabeth A.; Taracila, Magdalena A.; Drawz, Sarah M.; Bethel, Christopher R.; Papp-Wallace, Krisztina M.; Smith, Kerri M.; Xu, Yan; Dwulit-Smith, Jeffrey R.; Romagnoli, Chiara; Caselli, Emilia; Prati, Fabio; van den Akker, Focco; Bonomo, Robert A.

    2014-01-01

    Inhibitor resistant (IR) class A β-lactamases pose a significant threat to many current antibiotic combinations. The K234R substitution in the SHV β-lactamase, from Klebsiella pneumoniae, results in resistance to ampicillin/clavulanate. After site-saturation mutagenesis of Lys-234 in SHV, microbiological and biochemical characterization of the resulting β-lactamases revealed that only –Arg conferred resistance to ampicillin/clavulanate. X-ray crystallography revealed two conformations of Arg-234 and Ser-130 in SHV K234R. The movement of Ser-130 is the principal cause of the observed clavulanate resistance. A panel of boronic acid inhibitors was designed and tested against SHV-1 and SHV K234R. A chiral ampicillin analogue was discovered to have a 2.4 ± 0.2 nM Ki for SHV K234R; the chiral ampicillin analogue formed a more complex hydrogen-bonding network in SHV K234R vs SHV-1. Consideration of the spatial position of Ser-130 and Lys-234 and this hydrogen-bonding network will be important in the design of novel antibiotics targeting IR β-lactamases. PMID:23252553

  15. Transcriptional profiling of canola developing embryo and identification of the important roles of BnDof5.6 in embryo development and fatty acids synthesis.

    PubMed

    Deng, Wei; Yan, Fang; Zhang, Xiaolan; Tang, Yuwei; Yuan, Yujin

    2015-08-01

    Canola is an important vegetable oil crop globally, and the understanding of the molecular mechanism underlying fatty acids biosynthesis during seed embryo development is an important research goal. Here we report the transcriptional profiling analysis of d