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Sample records for acid sequencing mass

  1. Design, synthesis, and characterization of a protein sequencing reagent yielding amino acid derivatives with enhanced detectability by mass spectrometry.

    PubMed Central

    Aebersold, R.; Bures, E. J.; Namchuk, M.; Goghari, M. H.; Shushan, B.; Covey, T. C.

    1992-01-01

    We report the design, chemical synthesis, and structural and functional characterization of a novel reagent for protein sequence analysis by the Edman degradation, yielding amino acid derivatives rapidly detectable at high sensitivity by ion-evaporation mass spectrometry. We demonstrate that the reagent 3-[4'(ethylene-N,N,N-trimethylamino)phenyl]-2-isothiocyanate is chemically stable and shows coupling and cyclization/cleavage yields comparable to phenylisothiocyanate, the standard reagent in chemical sequence analysis, under conditions typically encountered in manual or automated sequence analysis. Amino acid derivatives generated with this reagent were detectable by ion-evaporation mass spectrometry at the subfemtomole sensitivity level at a pace of one sample per minute. Furthermore, derivatives were identified by their mass, thus permitting the rapid and highly sensitive determination of the molecular nature of modified amino acids. Derivatives of amino acids with acidic, basic, polar, or hydrophobic side chains were reproducibly detectable at comparable sensitivities. The polar nature of the reagent required covalent immobilization of polypeptides prior to automated sequence analysis. This reagent, used in automated sequence analysis, has the potential for overcoming the limitations in sensitivity, speed, and the ability to characterize modified amino acid residues inherent in the chemical sequencing methods that are currently used. PMID:1304351

  2. Peptide Mass Fingerprinting and N-Terminal Amino Acid Sequencing of Glycosylated Cysteine Protease of Euphorbia nivulia Buch.-Ham.

    PubMed Central

    Badgujar, Shamkant B.; Mahajan, Raghunath T.

    2013-01-01

    A new cysteine protease named Nivulian-II has been purified from the latex of Euphorbia nivulia Buch.-Ham. The apparent molecular mass of Nivulian-II is 43670.846 Da (MALDI TOF/MS). Peptide mass fingerprint analysis revealed peptide matches to Maturase K (Q52ZV1_9MAGN) of Banksia quercifolia. The N-terminal sequence (DFPPNTCCCICC) showed partial homology with those of other cysteine proteinases of biological origin. This is the first paper to characterize a Nivulian-II of E. nivulia latex with respect to amino acid sequencing. PMID:23476742

  3. Microwave-assisted acid and base hydrolysis of intact proteins containing disulfide bonds for protein sequence analysis by mass spectrometry.

    PubMed

    Reiz, Bela; Li, Liang

    2010-09-01

    Controlled hydrolysis of proteins to generate peptide ladders combined with mass spectrometric analysis of the resultant peptides can be used for protein sequencing. In this paper, two methods of improving the microwave-assisted protein hydrolysis process are described to enable rapid sequencing of proteins containing disulfide bonds and increase sequence coverage, respectively. It was demonstrated that proteins containing disulfide bonds could be sequenced by MS analysis by first performing hydrolysis for less than 2 min, followed by 1 h of reduction to release the peptides originally linked by disulfide bonds. It was shown that a strong base could be used as a catalyst for microwave-assisted protein hydrolysis, producing complementary sequence information to that generated by microwave-assisted acid hydrolysis. However, using either acid or base hydrolysis, amide bond breakages in small regions of the polypeptide chains of the model proteins (e.g., cytochrome c and lysozyme) were not detected. Dynamic light scattering measurement of the proteins solubilized in an acid or base indicated that protein-protein interaction or aggregation was not the cause of the failure to hydrolyze certain amide bonds. It was speculated that there were some unknown local structures that might play a role in preventing an acid or base from reacting with the peptide bonds therein.

  4. Rapid discrimination of Bacillus anthracis from other members of the B. cereus group by mass and sequence of "intact" small acid soluble proteins (SASPs) using mass spectrometry.

    PubMed

    Castanha, Elisangela R; Fox, Alvin; Fox, Karen F

    2006-11-01

    The intentional contamination of buildings, e.g. anthrax in the bioterrorism attacks of 2001, demonstrated that the population can be affected rapidly and lethally if the appropriate treatment is not provided at the right time. Molecular approaches, primarily involving PCR, have proved useful in characterizing "white powders" used in these attacks as well as isolated organisms. However there is a need for a simpler approach, which does not involve temperamental reagents (e.g. enzymes and primers) which could potentially be used by first responders. It is demonstrated here that small acid-soluble proteins (SASPs), located in the core region of Bacillus spores, are reliable biomarkers for identification. The general strategy used in this study was to measure the molecular weight (MW) of an intact SASP by electrospray ionization mass spectrometry (ESI MS) followed by generation of sequence-specific information by ESI MS/MS (tandem mass spectrometry). A prominent SASP of mass 6679 was present in all B. anthracis strains. For B. cereus and B. thuringiensis strains the SASP had a mass of 6712. This represents a two amino acid substitution (serine to alanine; phenylalanine to tyrosine). The only SASP present in the B. anthracis genome consistent with this sequence is encoded by the gene ssB. This protein has a predicted mass of 6810, presumably post-translational processing leads to loss of methionine (mass 131) generating a SASP of mass 6679. This study showed that intact SASPs can be used as a biomarker for identification of B. anthracis; the protocol is simple and rapid. Extrapolation of this approach might prove important for real-time biodetection.

  5. "De-novo" amino acid sequence elucidation of protein G'e by combined "Top-Down" and "Bottom-Up" mass spectrometry

    NASA Astrophysics Data System (ADS)

    Yefremova, Yelena; Al-Majdoub, Mahmoud; Opuni, Kwabena F. M.; Koy, Cornelia; Cui, Weidong; Yan, Yuetian; Gross, Michael L.; Glocker, Michael O.

    2015-03-01

    Mass spectrometric de-novo sequencing was applied to review the amino acid sequence of a commercially available recombinant protein Ǵ with great scientific and economic importance. Substantial deviations to the published amino acid sequence (Uniprot Q54181) were found by the presence of 46 additional amino acids at the N-terminus, including a so-called "His-tag" as well as an N-terminal partial α- N-gluconoylation and α- N-phosphogluconoylation, respectively. The unexpected amino acid sequence of the commercial protein G' comprised 241 amino acids and resulted in a molecular mass of 25,998.9 ± 0.2 Da for the unmodified protein. Due to the higher mass that is caused by its extended amino acid sequence compared with the original protein G' (185 amino acids), we named this protein "protein G'e." By means of mass spectrometric peptide mapping, the suggested amino acid sequence, as well as the N-terminal partial α- N-gluconoylations, was confirmed with 100% sequence coverage. After the protein G'e sequence was determined, we were able to determine the expression vector pET-28b from Novagen with the Xho I restriction enzyme cleavage site as the best option that was used for cloning and expressing the recombinant protein G'e in E. coli. A dissociation constant ( K d ) value of 9.4 nM for protein G'e was determined thermophoretically, showing that the N-terminal flanking sequence extension did not cause significant changes in the binding affinity to immunoglobulins.

  6. The amino acid sequences of eleven tryptic peptides of papaya mosaic virus protein by electron ionization mass spectrometry.

    PubMed

    Parente, A; Short, M N; Self, R; Parsley, K R

    1982-04-01

    Eleven of the fourteen tryptic peptides of papaya mosaic virus protein have been sequenced by electron ionization mass spectrometry using chemical and enzymic hydrolyses and mixture analysis as required. Mid-chain cleavages of N-C bonds produced secondary ion series which allowed up to 16 residues to be sequenced without further hydrolysis. Mixture analysis on hydrolysis products enabled a 24 residue tryptic peptide to be sequenced from the data recorded in a single mass spectrum.

  7. Negative Ion In-Source Decay Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Sequencing Acidic Peptides

    NASA Astrophysics Data System (ADS)

    McMillen, Chelsea L.; Wright, Patience M.; Cassady, Carolyn J.

    2016-05-01

    Matrix-assisted laser desorption/ionization (MALDI) in-source decay was studied in the negative ion mode on deprotonated peptides to determine its usefulness for obtaining extensive sequence information for acidic peptides. Eight biological acidic peptides, ranging in size from 11 to 33 residues, were studied by negative ion mode ISD (nISD). The matrices 2,5-dihydroxybenzoic acid, 2-aminobenzoic acid, 2-aminobenzamide, 1,5-diaminonaphthalene, 5-amino-1-naphthol, 3-aminoquinoline, and 9-aminoacridine were used with each peptide. Optimal fragmentation was produced with 1,5-diaminonphthalene (DAN), and extensive sequence informative fragmentation was observed for every peptide except hirudin(54-65). Cleavage at the N-Cα bond of the peptide backbone, producing c' and z' ions, was dominant for all peptides. Cleavage of the N-Cα bond N-terminal to proline residues was not observed. The formation of c and z ions is also found in electron transfer dissociation (ETD), electron capture dissociation (ECD), and positive ion mode ISD, which are considered to be radical-driven techniques. Oxidized insulin chain A, which has four highly acidic oxidized cysteine residues, had less extensive fragmentation. This peptide also exhibited the only charged localized fragmentation, with more pronounced product ion formation adjacent to the highly acidic residues. In addition, spectra were obtained by positive ion mode ISD for each protonated peptide; more sequence informative fragmentation was observed via nISD for all peptides. Three of the peptides studied had no product ion formation in ISD, but extensive sequence informative fragmentation was found in their nISD spectra. The results of this study indicate that nISD can be used to readily obtain sequence information for acidic peptides.

  8. Effects of Acidic Peptide Size and Sequence on Trivalent Praseodymium Adduction and Electron Transfer Dissociation Mass Spectrometry.

    PubMed

    Commodore, Juliette J; Cassady, Carolyn J

    2017-02-07

    Using the lanthanide ion praseodymium, Pr(III), metallated ion formation and electron transfer dissociation (ETD) were studied for 25 biological and model acidic peptides. For chain lengths of seven or more residues, even highly acidic peptides that can be difficult to protonate by electrospray ionization will metallate and undergo abundant ETD fragmentation. Peptides composed of predominantly acidic residues form only the deprotonated ion, [M + Pr - H](2+) ; this ion yields near complete ETD sequence coverage for larger peptides. Peptides with a mixture of acidic and neutral residues, generate [M + Pr](3+) , which cleaves between every residue for many peptides. Acidic peptides that contain at least one residue with a basic side chain also produce the protonated ion, [M + Pr + H](4+) ; this ion undergoes the most extensive sequence coverage by ETD. Primarily metallated and non-metallated c- and z-ions form for all peptides investigated. Metal adducted product ions are only present when at least half of the peptide sequence can be incorporated into the ion; this suggests that the metal ion simultaneously attaches to more than one acidic site. The only site consistently lacking dissociation is at the N-terminal side of a proline residue. Increasing peptide chain length generates more backbone cleavage for metal-peptide complexes with the same charge state. For acidic peptides with the same length, increasing the precursor ion charge state from 2+ to 3+ also leads to more cleavage. The results of this study indicate that highly acidic peptides can be sequenced by ETD of complexes formed with Pr(III).

  9. RoboOligo: software for mass spectrometry data to support manual and de novo sequencing of post-transcriptionally modified ribonucleic acids.

    PubMed

    Sample, Paul J; Gaston, Kirk W; Alfonzo, Juan D; Limbach, Patrick A

    2015-05-26

    Ribosomal ribonucleic acid (RNA), transfer RNA and other biological or synthetic RNA polymers can contain nucleotides that have been modified by the addition of chemical groups. Traditional Sanger sequencing methods cannot establish the chemical nature and sequence of these modified-nucleotide containing oligomers. Mass spectrometry (MS) has become the conventional approach for determining the nucleotide composition, modification status and sequence of modified RNAs. Modified RNAs are analyzed by MS using collision-induced dissociation tandem mass spectrometry (CID MS/MS), which produces a complex dataset of oligomeric fragments that must be interpreted to identify and place modified nucleosides within the RNA sequence. Here we report the development of RoboOligo, an interactive software program for the robust analysis of data generated by CID MS/MS of RNA oligomers. There are three main functions of RoboOligo: (i) automated de novo sequencing via the local search paradigm. (ii) Manual sequencing with real-time spectrum labeling and cumulative intensity scoring. (iii) A hybrid approach, coined 'variable sequencing', which combines the user intuition of manual sequencing with the high-throughput sampling of automated de novo sequencing.

  10. Characterization of nucleic acids by tandem mass spectrometry - The second decade (2004-2013): From DNA to RNA and modified sequences.

    PubMed

    Schürch, Stefan

    2016-07-01

    Nucleic acids play key roles in the storage and processing of genetic information, as well as in the regulation of cellular processes. Consequently, they represent attractive targets for drugs against gene-related diseases. On the other hand, synthetic oligonucleotide analogues have found application as chemotherapeutic agents targeting cellular DNA and RNA. The development of effective nucleic acid-based chemotherapeutic strategies requires adequate analytical techniques capable of providing detailed information about the nucleotide sequences, the presence of structural modifications, the formation of higher-order structures, as well as the interaction of nucleic acids with other cellular components and chemotherapeutic agents. Due to the impressive technical and methodological developments of the past years, tandem mass spectrometry has evolved to one of the most powerful tools supporting research related to nucleic acids. This review covers the literature of the past decade devoted to the tandem mass spectrometric investigation of nucleic acids, with the main focus on the fundamental mechanistic aspects governing the gas-phase dissociation of DNA, RNA, modified oligonucleotide analogues, and their adducts with metal ions. Additionally, recent findings on the elucidation of nucleic acid higher-order structures by tandem mass spectrometry are reviewed. © 2014 Wiley Periodicals, Inc., Mass Spec Rev 35:483-523, 2016.

  11. Purification, characterization and amino-acid sequence analysis of a thermostable, low molecular mass endo-beta-1,4-glucanase from blue mussel, Mytilus edulis.

    PubMed

    Xu, B; Hellman, U; Ersson, B; Janson, J C

    2000-08-01

    A cellulase (endo-beta-1,4-D-glucanase, EC 3.2.1.4) from blue mussel (Mytilus edulis) was purified to homogeneity using a combination of acid precipitation, heat precipitation, immobilized metal ion affinity chromatography, size-exclusion chromatography and ion-exchange chromatography. Purity was analyzed by SDS/PAGE, IEF and RP-HPLC. The cellulase (endoglucanase) was characterized with regard to enzymatic properties, isoelectric point, molecular mass and amino-acid sequence. It is a single polypeptide chain of 181 amino acids cross-linked with six disulfide bridges. Its molecular mass, as measured by MALDI-MS, is 19 702 Da; a value of 19 710.57 Da was calculated from amino-acid composition. The isoelectric point of the enzyme was estimated by isoelectric focusing in a polyacrylamide gel to a value of 7.6. According to amino-acid composition, the theoretical pI is 7.011. The effect of temperature on the endoglucanase activity, with carboxymethyl cellulose and amorphous cellulose as substrates, respectively, was studied at pH 5.5 and displayed an unusually broad optimum activity temperature range between 30 and 50 degrees C. Another unusual feature is that the enzyme retains 55-60% of its maximum activity at 0 degrees C. The enzyme readily degrades amorphous cellulose and carboxymethyl cellulose but displays no hydrolytic activity towards crystalline cellulose (Avicel) and shows no cross-specificity for xylan; there is no binding to Avicel. The enzyme can withstand 10 min at 100 degrees C without irreversible loss of enzymatic activity. Amino-acid sequence-based classification has revealed that the enzyme belongs to the glycoside hydrolase family 45, subfamily 2 (B. Henrissat, Centre de Recherches sur les Macromolecules Végétales, CNRS, Joseph Fourier Université, Grenoble, France, personal communication).

  12. Composition for nucleic acid sequencing

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2008-08-26

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  13. In-gel microwave-assisted acid hydrolysis of proteins combined with liquid chromatography tandem mass spectrometry for mapping protein sequences.

    PubMed

    Sun, Difei; Wang, Nan; Li, Liang

    2014-01-07

    We report an enabling method for mapping the protein sequence with high sequence coverage. This method combines the high separation power of gel electrophoresis for protein separation with the high sequence coverage capability of microwave-assisted acid hydrolysis (MAAH) mass spectrometry (MS). In-gel MAAH using 25% trifluoroacetic acid was developed and optimized for degrading the gel-separated protein into small peptides suitable for tandem MS sequencing. For bovine serum albumin (BSA) (∼67 kDa), with 4 μg of protein loading onto a gel for separation, followed by excising the protein gel band for in-gel MAAH and then injecting ∼2 μg of the resultant peptides into a liquid chromatography quadrupole time-of-flight mass spectrometer for analysis, 689 ± 54 (n = 3) unique peptides were identified with a protein sequence coverage of 99 ± 1%. Both the number of peptides detected and sequence coverage decreased as the sample amount decreased, mainly due to background interference: 316 ± 59 peptides and 94 ± 3% coverage for 2 μg loading, 136 ± 19 and 76 ± 5% for 1 μg loading, and 30 ± 2 and 32 ± 2% for 0.5 μg loading. To demonstrate the general applicability of the method, 10 gel bands from gel electrophoresis of an albumin-depleted human plasma sample were excised for in-gel MAAH LC-MS analysis. In total, 19 relatively high abundance proteins with molecular weights ranging from ∼8 to ∼160 kD could be mapped with coverage of 100% for six proteins (MW 8759 to 68 425 Da), 96-98% for five proteins (MW 11 458 to 36 431 Da), 92% for three proteins (MW 15 971 to 36 431 Da), 80-87% for four proteins (MW 42 287 to 162 134 Da), and 56% for one protein (MW 51 358 Da). Finally, to demonstrate the applicability of the method for more detailed analysis of complex protein mixtures, two-dimensional (2D) gel electrophoresis was combined with in-gel MAAH, affinity purification, and LC-MS/MS to characterize six bovine alpha-S1-casein phosphoprotein

  14. Determination of the complete amino acid sequence for the coat protein of brome mosaic virus by time-of-flight mass spectrometry. Evidence for mutations associated with change of propagation host.

    PubMed

    She, Y M; Haber, S; Seifers, D L; Loboda, A; Chernushevich, I; Perreault, H; Ens, W; Standing, K G

    2001-06-08

    Time-of-flight mass spectrometry (TOFMS) has been applied to determine the complete coat protein amino acid sequences of a number of distinct brome mosaic virus (BMV) isolates. Ionization was carried out by both electrospray ionization and matrix-assisted laser desorption/ionization (MALDI). After determining overall coat protein masses, the proteins were digested with trypsin or Lys-C proteinases, and the digestion products were analyzed in a MALDI QqTOF mass spectrometer. The N terminus of the coat protein was found to be acetylated in each BMV isolate analyzed. In one isolate (BMV-Valverde), the amino acid sequence was identical to that predicted from the cDNA sequence of the "type" isolate, but deviations from the predicted amino acid sequence were observed for all the other isolates analyzed. When isolates were propagated in different host taxa, modified coat protein sequences were observed in some cases, along with the original sequence. Sequencing by TOFMS may therefore provide a basis for monitoring the effects of host passaging on a virus at the molecular level. Such TOFMS-based analyses assess the complete profiles of coat protein sequences actually present in infected tissues. They are therefore not subject to the selection biases inherent in deducing such sequences from reverse-transcribed viral RNA and cloning the resulting cDNA.

  15. High speed nucleic acid sequencing

    SciTech Connect

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2011-05-17

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid. Each type of labeled nucleotide comprises an acceptor fluorophore attached to a phosphate portion of the nucleotide such that the fluorophore is removed upon incorporation into a growing strand. Fluorescent signal is emitted via fluorescent resonance energy transfer between the donor fluorophore and the acceptor fluorophore as each nucleotide is incorporated into the growing strand. The sequence is deduced by identifying which base is being incorporated into the growing strand.

  16. PhTX-II a Basic Myotoxic Phospholipase A2 from Porthidium hyoprora Snake Venom, Pharmacological Characterization and Amino Acid Sequence by Mass Spectrometry

    PubMed Central

    Huancahuire-Vega, Salomón; Ponce-Soto, Luis Alberto; Marangoni, Sergio

    2014-01-01

    A monomeric basic PLA2 (PhTX-II) of 14149.08 Da molecular weight was purified to homogeneity from Porthidium hyoprora venom. Amino acid sequence by in tandem mass spectrometry revealed that PhTX-II belongs to Asp49 PLA2 enzyme class and displays conserved domains as the catalytic network, Ca2+-binding loop and the hydrophobic channel of access to the catalytic site, reflected in the high catalytic activity displayed by the enzyme. Moreover, PhTX-II PLA2 showed an allosteric behavior and its enzymatic activity was dependent on Ca2+. Examination of PhTX-II PLA2 by CD spectroscopy indicated a high content of alpha-helical structures, similar to the known structure of secreted phospholipase IIA group suggesting a similar folding. PhTX-II PLA2 causes neuromuscular blockade in avian neuromuscular preparations with a significant direct action on skeletal muscle function, as well as, induced local edema and myotoxicity, in mice. The treatment of PhTX-II by BPB resulted in complete loss of their catalytic activity that was accompanied by loss of their edematogenic effect. On the other hand, enzymatic activity of PhTX-II contributes to this neuromuscular blockade and local myotoxicity is dependent not only on enzymatic activity. These results show that PhTX-II is a myotoxic Asp49 PLA2 that contributes with toxic actions caused by P. hyoprora venom. PMID:25365526

  17. Collision-induced release, ion mobility separation, and amino acid sequence analysis of subunits from mass-selected noncovalent protein complexes.

    PubMed

    Rathore, Deepali; Dodds, Eric D

    2014-09-01

    In recent years, mass spectrometry has become a valuable tool for detecting and characterizing protein-protein interactions and for measuring the masses and subunit stoichiometries of noncovalent protein complexes. The gas-phase dissociation of noncovalent protein assemblies via tandem mass spectrometry can be useful in confirming subunit masses and stoichiometries; however, dissociation experiments that are able to yield subunit sequence information must usually be conducted separately. Here, we furnish proof of concept for a method that allows subunit sequence information to be directly obtained from a protein aggregate in a single gas-phase analysis. The experiments were carried out using a quadrupole time-of-flight mass spectrometer equipped with a traveling-wave ion mobility separator. This instrument configuration allows for a noncovalent protein assembly to be quadrupole selected, then subjected to two successive rounds of collision-induced dissociation with an intervening stage of ion mobility separation. This approach was applied to four model proteins as their corresponding homodimers: glucagon, ubiquitin, cytochrome c, and β-lactoglobulin. In each case, b- and y-type fragment ions were obtained upon further collisional activation of the collisionally-released subunits, resulting in up to 50% sequence coverage. Owing to the incorporation of an ion mobility separation, these results also suggest the intriguing possibility of measuring complex mass, complex collisional cross section, subunit masses, subunit collisional cross sections, and sequence information for the subunits in a single gas-phase experiment. Overall, these findings represent a significant contribution towards the realization of protein interactomic analyses, which begin with native complexes and directly yield subunit identities.

  18. Collision-Induced Release, Ion Mobility Separation, and Amino Acid Sequence Analysis of Subunits from Mass-Selected Noncovalent Protein Complexes

    NASA Astrophysics Data System (ADS)

    Rathore, Deepali; Dodds, Eric D.

    2014-09-01

    In recent years, mass spectrometry has become a valuable tool for detecting and characterizing protein-protein interactions and for measuring the masses and subunit stoichiometries of noncovalent protein complexes. The gas-phase dissociation of noncovalent protein assemblies via tandem mass spectrometry can be useful in confirming subunit masses and stoichiometries; however, dissociation experiments that are able to yield subunit sequence information must usually be conducted separately. Here, we furnish proof of concept for a method that allows subunit sequence information to be directly obtained from a protein aggregate in a single gas-phase analysis. The experiments were carried out using a quadrupole time-of-flight mass spectrometer equipped with a traveling-wave ion mobility separator. This instrument configuration allows for a noncovalent protein assembly to be quadrupole selected, then subjected to two successive rounds of collision-induced dissociation with an intervening stage of ion mobility separation. This approach was applied to four model proteins as their corresponding homodimers: glucagon, ubiquitin, cytochrome c, and β-lactoglobulin. In each case, b- and y-type fragment ions were obtained upon further collisional activation of the collisionally-released subunits, resulting in up to 50% sequence coverage. Owing to the incorporation of an ion mobility separation, these results also suggest the intriguing possibility of measuring complex mass, complex collisional cross section, subunit masses, subunit collisional cross sections, and sequence information for the subunits in a single gas-phase experiment. Overall, these findings represent a significant contribution towards the realization of protein interactomic analyses, which begin with native complexes and directly yield subunit identities.

  19. Chip-based sequencing nucleic acids

    DOEpatents

    Beer, Neil Reginald

    2014-08-26

    A system for fast DNA sequencing by amplification of genetic material within microreactors, denaturing, demulsifying, and then sequencing the material, while retaining it in a PCR/sequencing zone by a magnetic field. One embodiment includes sequencing nucleic acids on a microchip that includes a microchannel flow channel in the microchip. The nucleic acids are isolated and hybridized to magnetic nanoparticles or to magnetic polystyrene-coated beads. Microreactor droplets are formed in the microchannel flow channel. The microreactor droplets containing the nucleic acids and the magnetic nanoparticles are retained in a magnetic trap in the microchannel flow channel and sequenced.

  20. The H-Index of `An Approach to Correlate Tandem Mass Spectral Data of Peptides with Amino Acid Sequences in a Protein Database'

    NASA Astrophysics Data System (ADS)

    Washburn, Michael P.

    2015-11-01

    Over 20 years ago a remarkable paper was published in the Journal of American Society for Mass Spectrometry. This paper from Jimmy Eng, Ashley McCormack, and John Yates described the use of protein databases to drive the interpretation of tandem mass spectra of peptides. This paper now has over 3660 citations and continues to average more than 260 per year over the last decade. This is an amazing scientific achievement. The reason for this is the paper was a cutting edge development at the moment in time when genomes of organisms were being sequenced, protein and peptide mass spectrometry was growing into the field of proteomics, and the power of computing was growing quickly in accordance with Moore's law. This work by the Yates lab grew in importance as genomics, proteomics, and computation all advanced and eventually resulted in the widely used SEQUEST algorithm and platform for the analysis of tandem mass spectrometry data. This commentary provides an analysis of the impact of this paper by analyzing the citations it has generated and the impact of these citing papers.

  1. Protein Sequencing with Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Ziady, Assem G.; Kinter, Michael

    The recent introduction of electrospray ionization techniques that are suitable for peptides and whole proteins has allowed for the design of mass spectrometric protocols that provide accurate sequence information for proteins. The advantages gained by these approaches over traditional Edman Degradation sequencing include faster analysis and femtomole, sometimes attomole, sensitivity. The ability to efficiently identify proteins has allowed investigators to conduct studies on their differential expression or modification in response to various treatments or disease states. In this chapter, we discuss the use of electrospray tandem mass spectrometry, a technique whereby protein-derived peptides are subjected to fragmentation in the gas phase, revealing sequence information for the protein. This powerful technique has been instrumental for the study of proteins and markers associated with various disorders, including heart disease, cancer, and cystic fibrosis. We use the study of protein expression in cystic fibrosis as an example.

  2. Solid phase sequencing of double-stranded nucleic acids

    DOEpatents

    Fu, Dong-Jing; Cantor, Charles R.; Koster, Hubert; Smith, Cassandra L.

    2002-01-01

    This invention relates to methods for detecting and sequencing of target double-stranded nucleic acid sequences, to nucleic acid probes and arrays of probes useful in these methods, and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probe comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Nucleic acids whose sequences can be determined include nucleic acids in biological samples such as patient biopsies and environmental samples. Probes may be fixed to a solid support such as a hybridization chip to facilitate automated determination of molecular weights and identification of the target sequence.

  3. Distinguishing Proteins From Arbitrary Amino Acid Sequences

    PubMed Central

    Yau, Stephen S.-T.; Mao, Wei-Guang; Benson, Max; He, Rong Lucy

    2015-01-01

    What kinds of amino acid sequences could possibly be protein sequences? From all existing databases that we can find, known proteins are only a small fraction of all possible combinations of amino acids. Beginning with Sanger's first detailed determination of a protein sequence in 1952, previous studies have focused on describing the structure of existing protein sequences in order to construct the protein universe. No one, however, has developed a criteria for determining whether an arbitrary amino acid sequence can be a protein. Here we show that when the collection of arbitrary amino acid sequences is viewed in an appropriate geometric context, the protein sequences cluster together. This leads to a new computational test, described here, that has proved to be remarkably accurate at determining whether an arbitrary amino acid sequence can be a protein. Even more, if the results of this test indicate that the sequence can be a protein, and it is indeed a protein sequence, then its identity as a protein sequence is uniquely defined. We anticipate our computational test will be useful for those who are attempting to complete the job of discovering all proteins, or constructing the protein universe. PMID:25609314

  4. The complete amino acid sequence of prochymosin.

    PubMed Central

    Foltmann, B; Pedersen, V B; Jacobsen, H; Kauffman, D; Wybrandt, G

    1977-01-01

    The total sequence of 365 amino acid residues in bovine prochymosin is presented. Alignment with the amino acid sequence of porcine pepsinogen shows that 204 amino acid residues are common to the two zymogens. Further comparison and alignment with the amino acid sequence of penicillopepsin shows that 66 residues are located at identical positions in all three proteases. The three enzymes belong to a large group of proteases with two aspartate residues in the active center. This group forms a family derived from one common ancestor. PMID:329280

  5. Method for sequencing nucleic acid molecules

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2006-05-30

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  6. Method for sequencing nucleic acid molecules

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2006-06-06

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  7. Peptide nucleic acid probes with charged photocleavable mass markers

    PubMed Central

    Ball, Rachel J; Green, Philip S; Gale, Nittaya; Langley, G John

    2010-01-01

    Halogen-labelled peptide organic acid (HPOA) monomers have been synthesised and incorporated into sequence-specific peptide nucleic acid (PNA) probes. Three different types of probe have been prepared; the unmodified PNA probe, the PNA probe with a mass marker, and the PNA probe with photocleavable mass marker. All three types of probe have been used in model studies to develop a mass spectrometry-based hybridisation assay for detection of point mutations in DNA. PMID:21687524

  8. Phenolic acid esterases, coding sequences and methods

    DOEpatents

    Blum, David L.; Kataeva, Irina; Li, Xin-Liang; Ljungdahl, Lars G.

    2002-01-01

    Described herein are four phenolic acid esterases, three of which correspond to domains of previously unknown function within bacterial xylanases, from XynY and XynZ of Clostridium thermocellum and from a xylanase of Ruminococcus. The fourth specifically exemplified xylanase is a protein encoded within the genome of Orpinomyces PC-2. The amino acids of these polypeptides and nucleotide sequences encoding them are provided. Recombinant host cells, expression vectors and methods for the recombinant production of phenolic acid esterases are also provided.

  9. Method for identifying and quantifying nucleic acid sequence aberrations

    DOEpatents

    Lucas, J.N.; Straume, T.; Bogen, K.T.

    1998-07-21

    A method is disclosed for detecting nucleic acid sequence aberrations by detecting nucleic acid sequences having both a first and a second nucleic acid sequence type, the presence of the first and second sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. The method uses a first hybridization probe which includes a nucleic acid sequence that is complementary to a first sequence type and a first complexing agent capable of attaching to a second complexing agent and a second hybridization probe which includes a nucleic acid sequence that selectively hybridizes to the second nucleic acid sequence type over the first sequence type and includes a detectable marker for detecting the second hybridization probe. 11 figs.

  10. Method for identifying and quantifying nucleic acid sequence aberrations

    DOEpatents

    Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

    1998-01-01

    A method for detecting nucleic acid sequence aberrations by detecting nucleic acid sequences having both a first and a second nucleic acid sequence type, the presence of the first and second sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. The method uses a first hybridization probe which includes a nucleic acid sequence that is complementary to a first sequence type and a first complexing agent capable of attaching to a second complexing agent and a second hybridization probe which includes a nucleic acid sequence that selectively hybridizes to the second nucleic acid sequence type over the first sequence type and includes a detectable marker for detecting the second hybridization probe.

  11. DeNovoID: a web-based tool for identifying peptides from sequence and mass tags deduced from de novo peptide sequencing by mass spectroscopy.

    PubMed

    Halligan, Brian D; Ruotti, Victor; Twigger, Simon N; Greene, Andrew S

    2005-07-01

    One of the core activities of high-throughput proteomics is the identification of peptides from mass spectra. Some peptides can be identified using spectral matching programs like Sequest or Mascot, but many spectra do not produce high quality database matches. De novo peptide sequencing is an approach to determine partial peptide sequences for some of the unidentified spectra. A drawback of de novo peptide sequencing is that it produces a series of ordered and disordered sequence tags and mass tags rather than a complete, non-degenerate peptide amino acid sequence. This incomplete data is difficult to use in conventional search programs such as BLAST or FASTA. DeNovoID is a program that has been specifically designed to use degenerate amino acid sequence and mass data derived from MS experiments to search a peptide database. Since the algorithm employed depends on the amino acid composition of the peptide and not its sequence, DeNovoID does not have to consider all possible sequences, but rather a smaller number of compositions consistent with a spectrum. DeNovoID also uses a geometric indexing scheme that reduces the number of calculations required to determine the best peptide match in the database. DeNovoID is available at http://proteomics.mcw.edu/denovoid.

  12. Methods for analyzing nucleic acid sequences

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2011-05-17

    The present invention is directed to a method of sequencing a target nucleic acid. The method provides a complex comprising a polymerase enzyme, a target nucleic acid molecule, and a primer, wherein the complex is immobilized on a support Fluorescent label is attached to a terminal phosphate group of the nucleotide or nucleotide analog. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The time duration of the signal from labeled nucleotides or nucleotide analogs that become incorporated is distinguished from freely diffusing labels by a longer retention in the observation volume for the nucleotides or nucleotide analogs that become incorporated than for the freely diffusing labels.

  13. Determination of the sequences of protein-derived peptides and peptide mixtures by mass spectrometry

    PubMed Central

    Morris, Howard R.; Williams, Dudley H.; Ambler, Richard P.

    1971-01-01

    Micro-quantities of protein-derived peptides have been converted into N-acetylated permethyl derivatives, and their sequences determined by low-resolution mass spectrometry without prior knowledge of their amino acid compositions or lengths. A new strategy is suggested for the mass spectrometric sequencing of oligopeptides or proteins, involving gel filtration of protein hydrolysates and subsequent sequence analysis of peptide mixtures. Finally, results are given that demonstrate for the first time the use of mass spectrometry for the analysis of a protein-derived peptide mixture, again without prior knowledge of the protein or components within the mixture. PMID:5158904

  14. 77 FR 65537 - Requirements for Patent Applications Containing Nucleotide Sequence and/or Amino Acid Sequence...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-29

    ... Amino Acid Sequence Disclosures ACTION: Proposed collection; comment request. SUMMARY: The United States....'' SUPPLEMENTARY INFORMATION: I. Abstract Patent applications that contain nucleotide and/or amino acid...

  15. The Star-Forming Main Sequence at Low Galaxy Mass

    NASA Astrophysics Data System (ADS)

    Stierwalt, Sabrina; Johnson, Kelsey E.; Patton, David R.; Besla, Gurtina; Kallivayalil, Nitya; Liss, Sandra; Pearson, Sarah; Privon, George C.; Putman, Mary E.

    2017-01-01

    We present an investigation of the star-forming main sequence at the low mass end. The relation between galaxy stellar mass and star formation rate has been well-studied in the recent literature for a range of redshifts and galaxy type, but almost all of these studies are limited to galaxies with stellar masses above the dwarf galaxy range ( 109 Msun ). Our work, based on the panchromatic TiNy Titans survey of interacting dwarf galaxies, shows that dwarf galaxies extend the well-established main sequence at z=0 down to lower masses. Furthermore, like their more massive counterparts, dwarf mergers appear on an elevated main sequence with higher star formation rates for a given stellar mass. Finally we show that star formation is enhanced to a greater extent in low mass galaxy mergers than for higher mass systems.

  16. Full validation of therapeutic antibody sequences by middle-up mass measurements and middle-down protein sequencing.

    PubMed

    Resemann, Anja; Jabs, Wolfgang; Wiechmann, Anja; Wagner, Elsa; Colas, Olivier; Evers, Waltraud; Belau, Eckhard; Vorwerg, Lars; Evans, Catherine; Beck, Alain; Suckau, Detlev

    2016-01-01

    The regulatory bodies request full sequence data assessment both for innovator and biosimilar monoclonal antibodies (mAbs). Full sequence coverage is typically used to verify the integrity of the analytical data obtained following the combination of multiple LC-MS/MS datasets from orthogonal protease digests (so called "bottom-up" approaches). Top-down or middle-down mass spectrometric approaches have the potential to minimize artifacts, reduce overall analysis time and provide orthogonality to this traditional approach. In this work we report a new combined approach involving middle-up LC-QTOF and middle-down LC-MALDI in-source decay (ISD) mass spectrometry. This was applied to cetuximab, panitumumab and natalizumab, selected as representative US Food and Drug Administration- and European Medicines Agency-approved mAbs. The goal was to unambiguously confirm their reference sequences and examine the general applicability of this approach. Furthermore, a new measure for assessing the integrity and validity of results from middle-down approaches is introduced - the "Sequence Validation Percentage." Full sequence data assessment of the 3 antibodies was achieved enabling all 3 sequences to be fully validated by a combination of middle-up molecular weight determination and middle-down protein sequencing. Three errors in the reference amino acid sequence of natalizumab, causing a cumulative mass shift of only -2 Da in the natalizumab Fd domain, were corrected as a result of this work.

  17. Full validation of therapeutic antibody sequences by middle-up mass measurements and middle-down protein sequencing

    PubMed Central

    Resemann, Anja; Jabs, Wolfgang; Wiechmann, Anja; Wagner, Elsa; Colas, Olivier; Evers, Waltraud; Belau, Eckhard; Vorwerg, Lars; Evans, Catherine; Beck, Alain; Suckau, Detlev

    2016-01-01

    ABSTRACT The regulatory bodies request full sequence data assessment both for innovator and biosimilar monoclonal antibodies (mAbs). Full sequence coverage is typically used to verify the integrity of the analytical data obtained following the combination of multiple LC-MS/MS datasets from orthogonal protease digests (so called “bottom-up” approaches). Top-down or middle-down mass spectrometric approaches have the potential to minimize artifacts, reduce overall analysis time and provide orthogonality to this traditional approach. In this work we report a new combined approach involving middle-up LC-QTOF and middle-down LC-MALDI in-source decay (ISD) mass spectrometry. This was applied to cetuximab, panitumumab and natalizumab, selected as representative US Food and Drug Administration- and European Medicines Agency-approved mAbs. The goal was to unambiguously confirm their reference sequences and examine the general applicability of this approach. Furthermore, a new measure for assessing the integrity and validity of results from middle-down approaches is introduced – the “Sequence Validation Percentage.” Full sequence data assessment of the 3 antibodies was achieved enabling all 3 sequences to be fully validated by a combination of middle-up molecular weight determination and middle-down protein sequencing. Three errors in the reference amino acid sequence of natalizumab, causing a cumulative mass shift of only −2 Da in the natalizumab Fd domain, were corrected as a result of this work. PMID:26760197

  18. Detection of nucleic acid sequences by invader-directed cleavage

    DOEpatents

    Brow, Mary Ann D.; Hall, Jeff Steven Grotelueschen; Lyamichev, Victor; Olive, David Michael; Prudent, James Robert

    1999-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The 5' nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based by charge.

  19. Aspects of oligonucleotide and peptide sequencing with MALDI and electrospray mass spectrometry.

    PubMed

    Owens, D R; Bothner, B; Phung, Q; Harris, K; Siuzdak, G

    1998-09-01

    Biopolymer sequencing with mass spectrometry has become increasingly important and accessible with the development of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). Here we examine the use of sequential digestion for the rapid identification of proteolytic fragments, in turn highlighting the general utility of enzymatic MALDI ladder sequencing and ESI tandem mass spectrometry. Analyses were performed on oligonucleotides ranging in size from 2 to 50 residues, on peptides ranging in size from 7 to 44 residues and on viral coat proteins. MALDI ladder sequencing using exonuclease digestion generated a uniform distribution of ions and provided complete sequence information on the oligonucleotides 2-30 nucleic acid residues long. Only partial sequence information was obtained on the longer oligonucleotides. C-terminal peptide ladder sequencing typically provided information from 4 to 7 amino acids into the peptide. Sequential digestion, or endoprotease followed by exoprotease exposure, was also successfully applied to a trypsin digest of viral proteins. Analysis of ladder sequenced peptides by LCMS generated less information than in the MALDI-MS analysis and ESI-MS2 normally provided partial sequence information on both the small oligonucleotides and peptides. In general, MALDI ladder sequencing offered information on a broader mass range of biopolymers than ESI-MS2 and was relatively straightforward to interpret, especially for oligonucleotides.

  20. Laser mass spectrometry for DNA sequencing, disease diagnosis, and fingerprinting

    SciTech Connect

    Winston Chen, C.H.; Taranenko, N.I.; Zhu, Y.F.; Chung, C.N.; Allman, S.L.

    1997-03-01

    Since laser mass spectrometry has the potential for achieving very fast DNA analysis, the authors recently applied it to DNA sequencing, DNA typing for fingerprinting, and DNA screening for disease diagnosis. Two different approaches for sequencing DNA have been successfully demonstrated. One is to sequence DNA with DNA ladders produced from Snager`s enzymatic method. The other is to do direct sequencing without DNA ladders. The need for quick DNA typing for identification purposes is critical for forensic application. The preliminary results indicate laser mass spectrometry can possibly be used for rapid DNA fingerprinting applications at a much lower cost than gel electrophoresis. Population screening for certain genetic disease can be a very efficient step to reducing medical costs through prevention. Since laser mass spectrometry can provide very fast DNA analysis, the authors applied laser mass spectrometry to disease diagnosis. Clinical samples with both base deletion and point mutation have been tested with complete success.

  1. Laser mass spectrometry for DNA sequencing, disease diagnosis, and fingerprinting

    NASA Astrophysics Data System (ADS)

    Chen, C. H. Winston; Taranenko, N. I.; Zhu, Y. F.; Chung, C. N.; Allman, S. L.

    1997-05-01

    Since laser mass spectrometry has the potential for achieving very fast DNA analysis, we recently applied it to DNA sequencing, DNA typing for fingerprinting, and DNA screening for disease diagnosis. Two different approaches for sequencing DNA have been successfully demonstrated. One is to sequence DNA with DNA ladders produced from Sanger's enzymatic method. The other is to do direct sequencing without DNA ladders. The need for quick DNA typing for identification purposes is critical for forensic application. Our preliminary results indicate laser mass spectrometry can possible be used for rapid DNA fingerprinting applications at a much lower cost than gel electrophoresis. Population screening for certain genetic disease can be a very efficient step to reducing medical costs through prevention. Since laser mass spectrometry can provide very fast DNA analysis, we applied laser mass spectrometry to disease diagnosis. Clinical samples with both base deletion and point mutation have been tested with complete success.

  2. Los Alamos sequence analysis package for nucleic acids and proteins.

    PubMed Central

    Kanehisa, M I

    1982-01-01

    An interactive system for computer analysis of nucleic acid and protein sequences has been developed for the Los Alamos DNA Sequence Database. It provides a convenient way to search or verify various sequence features, e.g., restriction enzyme sites, protein coding frames, and properties of coded proteins. Further, the comprehensive analysis package on a large-scale database can be used for comparative studies on sequence and structural homologies in order to find unnoted information stored in nucleic acid sequences. PMID:6174934

  3. Hybridization and sequencing of nucleic acids using base pair mismatches

    DOEpatents

    Fodor, Stephen P. A.; Lipshutz, Robert J.; Huang, Xiaohua

    2001-01-01

    Devices and techniques for hybridization of nucleic acids and for determining the sequence of nucleic acids. Arrays of nucleic acids are formed by techniques, preferably high resolution, light-directed techniques. Positions of hybridization of a target nucleic acid are determined by, e.g., epifluorescence microscopy. Devices and techniques are proposed to determine the sequence of a target nucleic acid more efficiently and more quickly through such synthesis and detection techniques.

  4. Isolation and characterization of a resistant core peptide of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF); confirmation of the GM-CSF amino acid sequence by mass spectrometry.

    PubMed Central

    Tsarbopoulos, A.; Pramanik, B. N.; Labdon, J. E.; Reichert, P.; Gitlin, G.; Patel, S.; Sardana, V.; Nagabhushan, T. L.; Trotta, P. P.

    1993-01-01

    A trypsin-resistant core peptide of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was isolated and analyzed by high-energy Cs+ liquid secondary-ion (LSI) mass spectrometric analysis. This analysis provided successful detection of the high-mass disulfide-linked core peptide as well as information confirming the existence of disulfide pairing. Similarly, LSI mass spectrometric analysis of the peptide fragments isolated chromatographically from a Staphylococcus aureus V8 protease digest of rhGM-CSF provided rapid confirmation of the cDNA-derived sequence and determination of the existing disulfide bonds between cysteine residues 54-96 and 88-121. Electrospray ionization mass spectrometry was employed to measure the molecular weight of the intact protein and to determine the number of the disulfide bonds in the protein molecule by comparative analysis of the protein before and after reduction with beta-mercaptoethanol. PMID:8268804

  5. Nucleic acid sequence detection using multiplexed oligonucleotide PCR

    DOEpatents

    Nolan, John P.; White, P. Scott

    2006-12-26

    Methods for rapidly detecting single or multiple sequence alleles in a sample nucleic acid are described. Provided are all of the oligonucleotide pairs capable of annealing specifically to a target allele and discriminating among possible sequences thereof, and ligating to each other to form an oligonucleotide complex when a particular sequence feature is present (or, alternatively, absent) in the sample nucleic acid. The design of each oligonucleotide pair permits the subsequent high-level PCR amplification of a specific amplicon when the oligonucleotide complex is formed, but not when the oligonucleotide complex is not formed. The presence or absence of the specific amplicon is used to detect the allele. Detection of the specific amplicon may be achieved using a variety of methods well known in the art, including without limitation, oligonucleotide capture onto DNA chips or microarrays, oligonucleotide capture onto beads or microspheres, electrophoresis, and mass spectrometry. Various labels and address-capture tags may be employed in the amplicon detection step of multiplexed assays, as further described herein.

  6. NCBI Mass Sequence Downloader-Large dataset downloading made easy

    NASA Astrophysics Data System (ADS)

    Pina-Martins, F.; Paulo, O. S.

    Sequence databases, such as NCBI, are a very important resource in many areas of science. Downloading small amounts of sequences to local storage can easily be performed using any recent web browser, but downloading tens of thousands of sequences is not as simple. NCBI Mass Sequence Downloader is an open source program aimed at simplifying obtaining large amounts of sequence data from NCBI databases to local storage. It is written in python (can be run under both python 2 and python 3), and uses PyQt5 for the GUI. The program can be run in either graphical or command line mode. Source code is licensed under the GPLv3, and is supported on Linux, Windows and Mac OSX. Available at https://github.com/ElsevierSoftwareX/SOFTX-D-15-00072.git, https://github.com/StuntsPT/NCBI_Mass_Downloader

  7. Laser desorption mass spectrometry for DNA analysis and sequencing

    SciTech Connect

    Chen, C.H.; Taranenko, N.I.; Tang, K.; Allman, S.L.

    1995-03-01

    Laser desorption mass spectrometry has been considered as a potential new method for fast DNA sequencing. Our approach is to use matrix-assisted laser desorption to produce parent ions of DNA segments and a time-of-flight mass spectrometer to identify the sizes of DNA segments. Thus, the approach is similar to gel electrophoresis sequencing using Sanger`s enzymatic method. However, gel, radioactive tagging, and dye labeling are not required. In addition, the sequencing process can possibly be finished within a few hundred microseconds instead of hours and days. In order to use mass spectrometry for fast DNA sequencing, the following three criteria need to be satisfied. They are (1) detection of large DNA segments, (2) sensitivity reaching the femtomole region, and (3) mass resolution good enough to separate DNA segments of a single nucleotide difference. It has been very difficult to detect large DNA segments by mass spectrometry before due to the fragile chemical properties of DNA and low detection sensitivity of DNA ions. We discovered several new matrices to increase the production of DNA ions. By innovative design of a mass spectrometer, we can increase the ion energy up to 45 KeV to enhance the detection sensitivity. Recently, we succeeded in detecting a DNA segment with 500 nucleotides. The sensitivity was 100 femtomole. Thus, we have fulfilled two key criteria for using mass spectrometry for fast DNA sequencing. The major effort in the near future is to improve the resolution. Different approaches are being pursued. When high resolution of mass spectrometry can be achieved and automation of sample preparation is developed, the sequencing speed to reach 500 megabases per year can be feasible.

  8. Laser Desorption Mass Spectrometry for DNA Sequencing and Analysis

    NASA Astrophysics Data System (ADS)

    Chen, C. H. Winston; Taranenko, N. I.; Golovlev, V. V.; Isola, N. R.; Allman, S. L.

    1998-03-01

    Rapid DNA sequencing and/or analysis is critically important for biomedical research. In the past, gel electrophoresis has been the primary tool to achieve DNA analysis and sequencing. However, gel electrophoresis is a time-consuming and labor-extensive process. Recently, we have developed and used laser desorption mass spectrometry (LDMS) to achieve sequencing of ss-DNA longer than 100 nucleotides. With LDMS, we succeeded in sequencing DNA in seconds instead of hours or days required by gel electrophoresis. In addition to sequencing, we also applied LDMS for the detection of DNA probes for hybridization LDMS was also used to detect short tandem repeats for forensic applications. Clinical applications for disease diagnosis such as cystic fibrosis caused by base deletion and point mutation have also been demonstrated. Experimental details will be presented in the meeting. abstract.

  9. Methods and compositions for efficient nucleic acid sequencing

    DOEpatents

    Drmanac, Radoje

    2006-07-04

    Disclosed are novel methods and compositions for rapid and highly efficient nucleic acid sequencing based upon hybridization with two sets of small oligonucleotide probes of known sequences. Extremely large nucleic acid molecules, including chromosomes and non-amplified RNA, may be sequenced without prior cloning or subcloning steps. The methods of the invention also solve various current problems associated with sequencing technology such as, for example, high noise to signal ratios and difficult discrimination, attaching many nucleic acid fragments to a surface, preparing many, longer or more complex probes and labelling more species.

  10. Methods and compositions for efficient nucleic acid sequencing

    DOEpatents

    Drmanac, Radoje

    2002-01-01

    Disclosed are novel methods and compositions for rapid and highly efficient nucleic acid sequencing based upon hybridization with two sets of small oligonucleotide probes of known sequences. Extremely large nucleic acid molecules, including chromosomes and non-amplified RNA, may be sequenced without prior cloning or subcloning steps. The methods of the invention also solve various current problems associated with sequencing technology such as, for example, high noise to signal ratios and difficult discrimination, attaching many nucleic acid fragments to a surface, preparing many, longer or more complex probes and labelling more species.

  11. Kit for detecting nucleic acid sequences using competitive hybridization probes

    DOEpatents

    Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

    2001-01-01

    A kit is provided for detecting a target nucleic acid sequence in a sample, the kit comprising: a first hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a first portion of the target sequence, the first hybridization probe including a first complexing agent for forming a binding pair with a second complexing agent; and a second hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a second portion of the target sequence to which the first hybridization probe does not selectively hybridize, the second hybridization probe including a detectable marker; a third hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a first portion of the target sequence, the third hybridization probe including the same detectable marker as the second hybridization probe; and a fourth hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a second portion of the target sequence to which the third hybridization probe does not selectively hybridize, the fourth hybridization probe including the first complexing agent for forming a binding pair with the second complexing agent; wherein the first and second hybridization probes are capable of simultaneously hybridizing to the target sequence and the third and fourth hybridization probes are capable of simultaneously hybridizing to the target sequence, the detectable marker is not present on the first or fourth hybridization probes and the first, second, third, and fourth hybridization probes each include a competitive nucleic acid sequence which is sufficiently complementary to a third portion of the target sequence that the competitive sequences of the first, second, third, and fourth hybridization probes compete with each other to hybridize to the third portion of the

  12. Analysis of cloned cDNA and genomic sequences for phytochrome: complete amino acid sequences for two gene products expressed in etiolated Avena.

    PubMed Central

    Hershey, H P; Barker, R F; Idler, K B; Lissemore, J L; Quail, P H

    1985-01-01

    Cloned cDNA and genomic sequences have been analyzed to deduce the amino acid sequence of phytochrome from etiolated Avena. Restriction endonuclease site polymorphism between clones indicates that at least four phytochrome genes are expressed in this tissue. Sequence analysis of two complete and one partial coding region shows approximately 98% homology at both the nucleotide and amino acid levels, with the majority of amino acid changes being conservative. High sequence homology is also found in the 5'-untranslated region but significant divergence occurs in the 3'-untranslated region. The phytochrome polypeptides are 1128 amino acid residues long corresponding to a molecular mass of 125 kdaltons. The known protein sequence at the chromophore attachment site occurs only once in the polypeptide, establishing that phytochrome has a single chromophore per monomer covalently linked to Cys-321. Computer analyses of the amino acid sequences have provided predictions regarding a number of structural features of the phytochrome molecule. PMID:3001642

  13. Analysis and Annotation of Nucleic Acid Sequence

    SciTech Connect

    States, David J.

    2004-07-28

    The aims of this project were to develop improved methods for computational genome annotation and to apply these methods to improve the annotation of genomic sequence data with a specific focus on human genome sequencing. The project resulted in a substantial body of published work. Notable contributions of this project were the identification of basecalling and lane tracking as error processes in genome sequencing and contributions to improved methods for these steps in genome sequencing. This technology improved the accuracy and throughput of genome sequence analysis. Probabilistic methods for physical map construction were developed. Improved methods for sequence alignment, alternative splicing analysis, promoter identification and NF kappa B response gene prediction were also developed.

  14. Sequencing of Oligourea Foldamers by Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Bathany, Katell; Owens, Neil W.; Guichard, Gilles; Schmitter, Jean-Marie

    2013-03-01

    This study is focused on sequence analysis of peptidomimetic helical oligoureas by means of tandem mass spectrometry, to build a basis for de novo sequencing for future high-throughput combinatorial library screening of oligourea foldamers. After the evaluation of MS/MS spectra obtained for model compounds with either MALDI or ESI sources, we found that the MALDI-TOF-TOF instrument gave more satisfactory results. MS/MS spectra of oligoureas generated by decay of singly charged precursor ions show major ion series corresponding to fragmentation across both CO-NH and N'H-CO urea bonds. Oligourea backbones fragment to produce a pattern of a, x, b, and y type fragment ions. De novo decoding of spectral information is facilitated by the occurrence of low mass reporter ions, representative of constitutive monomers, in an analogous manner to the use of immonium ions for peptide sequencing.

  15. Dipeptide Sequence Determination: Analyzing Phenylthiohydantoin Amino Acids by HPLC

    NASA Astrophysics Data System (ADS)

    Barton, Janice S.; Tang, Chung-Fei; Reed, Steven S.

    2000-02-01

    Amino acid composition and sequence determination, important techniques for characterizing peptides and proteins, are essential for predicting conformation and studying sequence alignment. This experiment presents improved, fundamental methods of sequence analysis for an upper-division biochemistry laboratory. Working in pairs, students use the Edman reagent to prepare phenylthiohydantoin derivatives of amino acids for determination of the sequence of an unknown dipeptide. With a single HPLC technique, students identify both the N-terminal amino acid and the composition of the dipeptide. This method yields good precision of retention times and allows use of a broad range of amino acids as components of the dipeptide. Students learn fundamental principles and techniques of sequence analysis and HPLC.

  16. Applications of mass spectrometry to DNA fingerprinting and DNA sequencing

    SciTech Connect

    Jacobson, K.B.; Buchanan, M.V.; Chen, C.H.; Doktycz, M.J.; McLuckey, S.A. ); Arlinghaus, H.F. )

    1993-01-01

    DNA fingerprinting and sequencing rely on polyacrylamide gel electrophoresis to determine the sizes of the DNA fragments. Innovative altematives to polyacrylamide gel electrophoresis are under investigation for characterization of such fingerprinting and sequencing. One method uses stable isotopes of tin and other elements to label the DNAwhereas other procedures do not require labels. The detectors in each case are mass spectrometers that detect either the stable isotopes or the DNA fragments themselves. If successful, these methods will speed up the rate of DNA analysis by one or two orders of magnitude.

  17. Applications of mass spectrometry to DNA fingerprinting and DNA sequencing

    SciTech Connect

    Jacobson, K.B.; Buchanan, M.V.; Chen, C.H.; Doktycz, M.J.; McLuckey, S.A.; Arlinghaus, H.F.

    1993-06-01

    DNA fingerprinting and sequencing rely on polyacrylamide gel electrophoresis to determine the sizes of the DNA fragments. Innovative altematives to polyacrylamide gel electrophoresis are under investigation for characterization of such fingerprinting and sequencing. One method uses stable isotopes of tin and other elements to label the DNAwhereas other procedures do not require labels. The detectors in each case are mass spectrometers that detect either the stable isotopes or the DNA fragments themselves. If successful, these methods will speed up the rate of DNA analysis by one or two orders of magnitude.

  18. Amino acid sequence of mouse submaxillary gland renin.

    PubMed Central

    Misono, K S; Chang, J J; Inagami, T

    1982-01-01

    The complete amino acid sequences of the heavy chain and light chain of mouse submaxillary gland renin have been determined. The heavy chain consists of 288 amino acid residues having a Mr of 31,036 calculated from the sequence. The light chain contains 48 amino acid residues with a Mr of 5,458. The sequence of the heavy chain was determined by automated Edman degradations of the cyanogen bromide peptides and tryptic peptides generated after citraconylation, as well as other peptides generated therefrom. The sequence of the light chain was derived from sequence analyses of the peptides generated by cyanogen bromide cleavage or by digestion with Staphylococcus aureus protease. The sequences in the active site regions in renin containing two catalytically essential aspartyl residues 32 and 215 were found identical with those in pepsin, chymosin, and penicillopepsin. Comparison of the amino acid sequence of renin with that of porcine pepsin indicated a 42% sequence identity of the heavy chain with the amino-terminal and middle regions and a 46% identity of the light chain with the carboxyl-terminal region of the porcine pepsin sequence. Residues identical in renin and pepsin are distributed throughout the length of the molecules, suggesting a similarity in their overall structures. PMID:6812055

  19. Sequence microheterogeneity of parvalbumin pI 5.0 of pike: a mass spectrometric study.

    PubMed

    Permyakov, Sergei E; Karnoup, Anton S; Bakunts, Anush G; Permyakov, Eugene A

    2009-01-01

    Parvalbumin (PA) is a muscle and neuronal calcium-binding protein, the major fish and frog allergen. Its characteristic feature is the presence of multiple isoforms with significantly different amino acid sequences. Here we show that the major isoform of northern pike muscle PA (pI 5.0, alpha-PA) exhibits microheterogeneity of amino acid sequence. ESI Q-TOF mass-spectrometry (MS) analysis of alpha-PA sample showed the presence of two components with mass difference of 71 Da. Analysis of tryptic and endoproteinase Asp-N digests of alpha-PA by MALDI-TOF MS revealed peptides, corresponding to two different amino acid sequences. The sequence differences between variant proteins are limited to AB-domain and include substitutions K27A and L31K, and an extra Leu residue between K11 and K12. Since the affected residues comprise a cluster on the surface of PA, an involvement of the identified region into target recognition is suggested. The substitutions at positions 27 and 31 are located in the region of previously identified epitopes of parvalbumin relevant for PA-specific IgE and IgG binding, which suggests different immunoactivities of the variants. The found microheterogeneity of PA is suggested to be of importance for physiological adaptation of the propulsive musculature to developmental and/or environmental requirements and may contribute to PA allergenicity.

  20. Purification, characterization and partial amino acid sequence of glycogen synthase from Saccharomyces cerevisiae.

    PubMed Central

    Carabaza, A; Arino, J; Fox, J W; Villar-Palasi, C; Guinovart, J J

    1990-01-01

    Glycogen synthase from Saccharomyces cerevisiae was purified to homogeneity. The enzyme showed a subunit molecular mass of 80 kDa. The holoenzyme appears to be a tetramer. Antibodies developed against purified yeast glycogen synthase inactivated the enzyme in yeast extracts and allowed the detection of the protein in Western blots. Amino acid analysis showed that the enzyme is very rich in glutamate and/or glutamine residues. The N-terminal sequence (11 amino acid residues) was determined. In addition, selected tryptic-digest peptides were purified by reverse-phase h.p.l.c. and submitted to gas-phase sequencing. Up to eight sequences (79 amino acid residues) could be aligned with the human muscle enzyme sequence. Levels of identity range between 37 and 100%, indicating that, although human and yeast glycogen synthases probably share some conserved regions, significant differences in their primary structure should be expected. Images Fig. 1. Fig. 2. Fig. 3. PMID:2114092

  1. Single-chain structure of human ceruloplasmin: the complete amino acid sequence of the whole molecule.

    PubMed Central

    Takahashi, N; Ortel, T L; Putnam, F W

    1984-01-01

    We have determined the amino acid sequence of the amino-terminal 67,000-dalton (67-kDa) fragment of human ceruloplasmin and have established overlapping sequences between the 67-kDa and 50-kDa fragments and between the 50-kDa and 19-kDa fragments. The 67-kDa fragment contains 480 amino acid residues and three glucosamine oligosaccharides. These results together with our previous sequence data for the 50-kDa and 19-kDa fragments complete the amino acid sequence of human ceruloplasmin. The polypeptide chain has a total of 1,046 amino acid residues (Mr 120,085) and has attachment sites for four glucosamine oligosaccharides; together these account for the total molecular mass of human ceruloplasmin (132 kDa). The sequence analysis of the peptides overlapping the fragments showed that one additional amino acid, arginine, is present between the 67-kDa and 50-kDa fragments, and another, lysine, is between the 50-kDa and 19-kDa fragments. Only two apparent sites of amino acid interchange have been identified in the polypeptide chain. Both involve a single-point interchange of glycine and lysine that would result in a difference in charge. The results of the complete sequence analysis verified that human ceruloplasmin is composed of a single polypeptide chain and that the subunit-like fragments are produced by proteolytic cleavage during purification (and possibly also in vivo). PMID:6582496

  2. Amino Acid Sequence of Human Cholinesterase

    DTIC Science & Technology

    1985-10-01

    liquid chromatography (HPLC). Activity testing of the aged, DFP-labeled cholinesterase showed that 99.8% of the active sites had been labeled, since...acids were quantitated by ninhydrin at the AAA Labs, or by derivatization with phenylisothiocyanate at the University of Michigan. The latter method

  3. Cystatin. Amino acid sequence and possible secondary structure.

    PubMed Central

    Schwabe, C; Anastasi, A; Crow, H; McDonald, J K; Barrett, A J

    1984-01-01

    The amino acid sequence of cystatin, the protein from chicken egg-white that is a tight-binding inhibitor of many cysteine proteinases, is reported. Cystatin is composed of 116 amino acid residues, and the Mr is calculated to be 13 143. No striking similarity to any other known sequence has been detected. The results of computer analysis of the sequence and c.d. spectrometry indicate that the secondary structure includes relatively little alpha-helix (about 20%) and that the remainder is mainly beta-structure. PMID:6712597

  4. Subset of Kappa and Lambda Germline Sequences Result in Light Chains with a Higher Molecular Mass Phenotype.

    PubMed

    Barnidge, David R; Lundström, Susanna L; Zhang, Bo; Dasari, Surendra; Murray, David L; Zubarev, Roman A

    2015-12-04

    In our previous work, we showed that electrospray ionization of intact polyclonal kappa and lambda light chains isolated from normal serum generates two distinct, Gaussian-shaped, molecular mass distributions representing the light-chain repertoire. During the analysis of a large (>100) patient sample set, we noticed a low-intensity molecular mass distribution with a mean of approximately 24 250 Da, roughly 800 Da higher than the mean of the typical kappa molecular-mass distribution mean of 23 450 Da. We also observed distinct clones in this region that did not appear to contain any typical post-translational modifications that would account for such a large mass shift. To determine the origin of the high molecular mass clones, we performed de novo bottom-up mass spectrometry on a purified IgM monoclonal light chain that had a calculated molecular mass of 24 275.03 Da. The entire sequence of the monoclonal light chain was determined using multienzyme digestion and de novo sequence-alignment software and was found to belong to the germline allele IGKV2-30. The alignment of kappa germline sequences revealed ten IGKV2 and one IGKV4 sequences that contained additional amino acids in their CDR1 region, creating the high-molecular-mass phenotype. We also performed an alignment of lambda germline sequences, which showed additional amino acids in the CDR2 region, and the FR3 region of functional germline sequences that result in a high-molecular-mass phenotype. The work presented here illustrates the ability of mass spectrometry to provide information on the diversity of light-chain molecular mass phenotypes in circulation, which reflects the germline sequences selected by the immunoglobulin-secreting B-cell population.

  5. Mouse Vk gene classification by nucleic acid sequence similarity.

    PubMed

    Strohal, R; Helmberg, A; Kroemer, G; Kofler, R

    1989-01-01

    Analyses of immunoglobulin (Ig) variable (V) region gene usage in the immune response, estimates of V gene germline complexity, and other nucleic acid hybridization-based studies depend on the extent to which such genes are related (i.e., sequence similarity) and their organization in gene families. While mouse Igh heavy chain V region (VH) gene families are relatively well-established, a corresponding systematic classification of Igk light chain V region (Vk) genes has not been reported. The present analysis, in the course of which we reviewed the known extent of the Vk germline gene repertoire and Vk gene usage in a variety of responses to foreign and self antigens, provides a classification of mouse Vk genes in gene families composed of members with greater than 80% overall nucleic acid sequence similarity. This classification differed in several aspects from that of VH genes: only some Vk gene families were as clearly separated (by greater than 25% sequence dissimilarity) as typical VH gene families; most Vk gene families were closely related and, in several instances, members from different families were very similar (greater than 80%) over large sequence portions; frequently, classification by nucleic acid sequence similarity diverged from existing classifications based on amino-terminal protein sequence similarity. Our data have implications for Vk gene analyses by nucleic acid hybridization and describe potentially important differences in sequence organization between VH and Vk genes.

  6. Main sequence mass loss and the ages of stars

    NASA Technical Reports Server (NTRS)

    Willson, L. A.

    1989-01-01

    The potentially observable consequences of the pulsation/rotation-induced mass loss from main-sequence A and F stars proposed by Willson et al. (1987) are discussed, reviewing the results of recent investigations. Particular attention is given to (1) evidence for a deficiency in A stars and an excess of F and G stars, as predicted by the theory, (2) cluster HR diagrams and age estimates, and (3) modifications to standard models of solar-system evolution. It is pointed out that the time scales and mass-loss rates required to explain the observed properties of clusters and field stars in this theory are the same as those needed to account for the early development of the solar system.

  7. Amino acid sequence of myoglobin from emu (Dromaius novaehollandiae) skeletal muscle.

    PubMed

    Suman, S P; Joseph, P; Li, S; Beach, C M; Fontaine, M; Steinke, L

    2010-11-01

    The objective of the present study was to characterize the primary structure of emu myoglobin (Mb). Emu Mb was isolated from Iliofibularis muscle employing gel-filtration chromatography. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry was employed to determine the exact molecular mass of emu Mb in comparison with horse Mb, and Edman degradation was utilized to characterize the amino acid sequence. The molecular mass of emu Mb was 17,380 Da and was close to those reported for ratite and poultry myoglobins. Similar to myoglobins from meat-producing livestock and birds, emu Mb has 153 amino acids. Emu Mb contains 9 histidines. Proximal and distal histidines, responsible for coordinating oxygen-binding property of Mb, are conserved in emu. Emu Mb shared more than 90% homology with ratite and chicken myoglobins, whereas it demonstrated only less than 70% sequence similarity with ruminant myoglobins.

  8. Amino acid sequence repertoire of the bacterial proteome and the occurrence of untranslatable sequences

    PubMed Central

    Navon, Sharon Penias; Kornberg, Guy; Chen, Jin; Schwartzman, Tali; Tsai, Albert; Puglisi, Elisabetta Viani; Puglisi, Joseph D.; Adir, Noam

    2016-01-01

    Bioinformatic analysis of Escherichia coli proteomes revealed that all possible amino acid triplet sequences occur at their expected frequencies, with four exceptions. Two of the four underrepresented sequences (URSs) were shown to interfere with translation in vivo and in vitro. Enlarging the URS by a single amino acid resulted in increased translational inhibition. Single-molecule methods revealed stalling of translation at the entrance of the peptide exit tunnel of the ribosome, adjacent to ribosomal nucleotides A2062 and U2585. Interaction with these same ribosomal residues is involved in regulation of translation by longer, naturally occurring protein sequences. The E. coli exit tunnel has evidently evolved to minimize interaction with the exit tunnel and maximize the sequence diversity of the proteome, although allowing some interactions for regulatory purposes. Bioinformatic analysis of the human proteome revealed no underrepresented triplet sequences, possibly reflecting an absence of regulation by interaction with the exit tunnel. PMID:27307442

  9. Amino acid sequence repertoire of the bacterial proteome and the occurrence of untranslatable sequences.

    PubMed

    Navon, Sharon Penias; Kornberg, Guy; Chen, Jin; Schwartzman, Tali; Tsai, Albert; Puglisi, Elisabetta Viani; Puglisi, Joseph D; Adir, Noam

    2016-06-28

    Bioinformatic analysis of Escherichia coli proteomes revealed that all possible amino acid triplet sequences occur at their expected frequencies, with four exceptions. Two of the four underrepresented sequences (URSs) were shown to interfere with translation in vivo and in vitro. Enlarging the URS by a single amino acid resulted in increased translational inhibition. Single-molecule methods revealed stalling of translation at the entrance of the peptide exit tunnel of the ribosome, adjacent to ribosomal nucleotides A2062 and U2585. Interaction with these same ribosomal residues is involved in regulation of translation by longer, naturally occurring protein sequences. The E. coli exit tunnel has evidently evolved to minimize interaction with the exit tunnel and maximize the sequence diversity of the proteome, although allowing some interactions for regulatory purposes. Bioinformatic analysis of the human proteome revealed no underrepresented triplet sequences, possibly reflecting an absence of regulation by interaction with the exit tunnel.

  10. Amino acid sequences of proteins from Leptospira serovar pomona.

    PubMed

    Alves, S F; Lefebvre, R B; Probert, W

    2000-01-01

    This report describes a partial amino acid sequences from three putative outer envelope proteins from Leptospira serovar pomona. In order to obtain internal fragments for protein sequencing, enzymatic and chemical digestion was performed. The enzyme clostripain was used to digest the proteins 32 and 45 kDa. In situ digestion of 40 kDa molecular weight protein was accomplished using cyanogen bromide. The 32 kDa protein generated two fragments, one of 21 kDa and another of 10 kDa that yielded five residues. A fragment of 24 kDa that yielded nineteen residues of amino acids was obtained from 45 kDa protein. A fragment with a molecular weight of 20 kDa, yielding a twenty amino acids sequence from the 40 kDa protein.

  11. LESSONS IN DE NOVO PEPTIDE SEQUENCING BY TANDEM MASS SPECTROMETRY

    PubMed Central

    Medzihradszky, Katalin F.; Chalkley, Robert J.

    2015-01-01

    Mass spectrometry has become the method of choice for the qualitative and quantitative characterization of protein mixtures isolated from all kinds of living organisms. The raw data in these studies are MS/MS spectra, usually of peptides produced by proteolytic digestion of a protein. These spectra are “translated” into peptide sequences, normally with the help of various search engines. Data acquisition and interpretation have both been automated, and most researchers look only at the summary of the identifications without ever viewing the underlying raw data used for assignments. Automated analysis of data is essential due to the volume produced. However, being familiar with the finer intricacies of peptide fragmentation processes, and experiencing the difficulties of manual data interpretation allow a researcher to be able to more critically evaluate key results, particularly because there are many known rules of peptide fragmentation that are not incorporated into search engine scoring. Since the most commonly used MS/MS activation method is collision-induced dissociation (CID), in this article we present a brief review of the history of peptide CID analysis. Next, we provide a detailed tutorial on how to determine peptide sequences from CID data. Although the focus of the tutorial is de novo sequencing, the lessons learned and resources supplied are useful for data interpretation in general. PMID:25667941

  12. Extensive amino acid sequence homologies between animal lectins

    SciTech Connect

    Paroutaud, P.; Levi, G.; Teichberg, V.I.; Strosberg, A.D.

    1987-09-01

    The authors have established the amino acid sequence of the ..beta..-D-galactoside binding lectin from the electric eel and the sequences of several peptides from a similar lectin isolated from human placenta. These sequences were compared with the published sequences of peptides derived from the ..beta..-D-galactoside binding lectin from human lung and with sequences deduced from cDNAs assigned to the ..beta..-D-galactoside binding lectins from chicken embryo skin and human hepatomas. Significant homologies were observed. One of the highly conserved regions that contains a tryptophan residue and two glutamic acid resides is probably part of the ..beta..-D-galactoside binding site, which, on the basis of spectroscopic studies of the electric eel lectin, is expected to contain such residues. The similarity of the hydropathy profiles and the predicted secondary structure of the lectins from chicken skin and electric eel, in spite of differences in their amino acid sequences, strongly suggests that these proteins have maintained structural homologies during evolution and together with the other ..beta..-D-galactoside binding lectins were derived form a common ancestor gene.

  13. Amino acid sequence of porcine spleen cathepsin D.

    PubMed Central

    Shewale, J G; Tang, J

    1984-01-01

    The amino acid sequence of porcine spleen cathepsin D heavy chain has been determined and, hence, the complete structure of this enzyme is now known. The sequence of heavy chain was constructed by aligning the structures of peptides generated by cyanogen bromide, trypsin, and endo-proteinase Lys C cleavages. The structure of the light chain has been published previously. The cathepsin D molecule contains 339 amino acid residues in two polypeptide chains: a 97-residue light chain and a 242-residue heavy chain, with a combined Mr of 36,779 (without carbohydrate). There are two carbohydrate units linked to asparagine residues 70 and 192. The disulfide bond arrangement in cathepsin D is probably similar to that of pepsin, because the positions of six half-cystine residues are conserved. The active site aspartyl residues, corresponding to aspartic acid-32 and -215 of pepsin, are located at residues 33 and 224 in the cathepsin D molecule. The amino acid sequence around these aspartyl residues is strongly conserved. Cathepsin D shows a strong homology with other acid proteases. When the sequence of cathepsin D, renin, and pepsin are aligned, 32.7% of the residues are identical. The homology is observed throughout the length of the molecules, indicating that three-dimensional structures of all three molecules are similar. PMID:6587385

  14. GenoMass software: a tool based on electrospray ionization tandem mass spectrometry for characterization and sequencing of oligonucleotide adducts

    PubMed Central

    Sharma, Vaneet K; Glick, James; Liao, Qing; Shen, Chang; Vouros, Paul

    2012-01-01

    The analysis of DNA adducts is of importance in understanding DNA damage, and in the last few years mass spectrometry (MS) has emerged as the most comprehensive and versatile tool for routine characterization of modified oligonucleotides. The structural analysis of modified oligonucleotides, although routinely analyzed using mass spectrometry, is followed by a large amount of data, and a significant challenge is to locate the exact position of the adduct by computational spectral interpretation, which still is a bottleneck. In this report, we present an additional feature of the in-house developed GenoMass software, which determines the exact location of an adduct in modified oligonucleotides by connecting tandem mass spectrometry (MS/MS) to a combinatorial isomer library generated in silico for nucleic acids. The performance of this MS/MS approach using GenoMass software was evaluated by MS/MS data interpretation for an unadducted and its corresponding N-acetylaminofluorene (AAF) adducted 17-mer (5′OH-CCT ACC CCT TCC TTG TA-3′OH) oligonucleotide. Further computational screening of this AAF adducted 17-mer oligonucleotide (5′OH-CCT ACC CCT TCC TTG TA-3′OH) from a complex oligonucleotide mixture was performed using GenoMass. Finally, GenoMass was also used to identify the positional isomers of the AAF adducted 15-mer oligonucleotide (5′OH-ATGAACCGGAGGCCC-3′OH). GenoMass is a simple, fast, data interpretation software that uses an in silico constructed library to relate the MS/MS sequencing approach to identify the exact location of adduct on oligonucleotides. PMID:22689626

  15. [Measurement of the amino acid sequence for the fusion protein FP3 with LC-MS/MS].

    PubMed

    Li, Xiang; Gao, Xiang-Dong; Tao, Lei; Pei, De-Ning; Guo, Ying; Rao, Chun-Ming; Wang, Jun-Zhi

    2012-02-01

    The amino acid sequence of the fusion protein FP3 was measured by two types of LC-MS/MS and its primary structure was confirmed. After reduction and alkylation, the protein was digested with trypsin and glycosyl groups in glycopeptide were removed by PNGase F. The mixed peptides were separated by LC, then Q-TOF and Ion trap tandem mass spectrometry were used to measure b, y fragment ions of each peptide to analyze the amino acid sequence of fusion protein FP3. Seventy-six percent of full amino acid sequence of the fusion protein FP3 was measured by LC-ESI-Q-TOF with the remaining 24% completed by LC-ESI-Trap. As LC-MS and tandem mass spectrometry are rapid, sensitive, accurate to measure the protein amino acid sequence, they are important approach to structure analysis and identification of recombinant protein.

  16. Active site amino acid sequence of human factor D.

    PubMed

    Davis, A E

    1980-08-01

    Factor D was isolated from human plasma by chromatography on CM-Sephadex C50, Sephadex G-75, and hydroxylapatite. Digestion of reduced, S-carboxymethylated factor D with cyanogen bromide resulted in three peptides which were isolated by chromatography on Sephadex G-75 (superfine) equilibrated in 20% formic acid. NH2-Terminal sequences were determined by automated Edman degradation with a Beckman 890C sequencer using a 0.1 M Quadrol program. The smallest peptide (CNBr III) consisted of the NH2-terminal 14 amino acids. The other two peptides had molecular weights of 17,000 (CNBr I) and 7000 (CNBr II). Overlap of the NH2-terminal sequence of factor D with the NH2-terminal sequence of CNBr I established the order of the peptides. The NH2-terminal 53 residues of factor D are somewhat more homologous with the group-specific protease of rat intestine than with other serine proteases. The NH2-terminal sequence of CNBr II revealed the active site serine of factor D. The typical serine protease active site sequence (Gly-Asp-Ser-Gly-Gly-Pro was found at residues 12-17. The region surrounding the active site serine does not appear to be more highly homologous with any one of the other serine proteases. The structural data obtained point out the similarities between factor D and the other proteases. However, complete definition of the degree of relationship between factor D and other proteases will require determination of the remainder of the primary structure.

  17. The amino acid sequence of iguana (Iguana iguana) pancreatic ribonuclease.

    PubMed

    Zhao, W; Beintema, J J; Hofsteenge, J

    1994-01-15

    The pyrimidine-specific ribonuclease superfamily constitutes a group of homologous proteins so far found only in higher vertebrates. Four separate families are found in mammals, which have resulted from gene duplications in mammalian ancestors. To learn more about the evolutionary history of this superfamily, the primary structure and other characteristics of the pancreatic enzyme from iguana (Iguana iguana), a herbivorous lizard species belonging to the reptiles, have been determined. The polypeptide chain consists of 119 amino acid residues. The positions of insertions and deletions in the sequence are identical to those in the enzyme from snapping turtle. However, the two enzymes differ at 54% of the amino acid positions. Iguana ribonuclease contains no carbohydrate, although the enzyme possesses three recognition sites for carbohydrate attachment, and has a high number of acidic residues in a localized part of the sequence.

  18. The amino acid sequence of Lady Amherst's pheasant (Chrysolophus amherstiae) and golden pheasant (Chrysolophus pictus) egg-white lysozymes.

    PubMed

    Araki, T; Kuramoto, M; Torikata, T

    1990-09-01

    The amino acids of Lady Amherst's pheasant and golden pheasant egg-white lysozymes have been sequenced. The carboxymethylated lysozymes were digested with trypsin followed by sequencing of the tryptic peptides. Lady Amherst's pheasant lysozyme proved to consist of 129 amino acid residues, and a relative molecular mass of 14,423 Da was calculated. This lysozyme had 6 amino acids substitutions when compared with hen egg-white lysozyme: Phe3 to Tyr, His15 to Leu, Gln41 to His, Asn77 to His, Gln 121 to Asn, and a newly found substitution of Ile124 to Thr. The amino acid sequence of golden pheasant lysozyme was identical to that of Lady Amherst's phesant lysozyme. The phylogenetic tree constructured by the comparison of amino acid sequences of phasianoid birds lysozymes revealed a minimum genetic distance between these pheasants and the turkey-peafowl group.

  19. Trichomonas vaginalis acidic phospholipase A2: isolation and partial amino acid sequence.

    PubMed

    Escobedo-Guajardo, Brenda L; González-Salazar, Francisco; Palacios-Corona, Rebeca; Torres de la Cruz, Víctor M; Morales-Vallarta, Mario; Mata-Cárdenas, Benito D; Garza-González, Jesús N; Rivera-Silva, Gerardo; Vargas-Villarreal, Javier

    2013-12-01

    Sexually transmitted diseases are a major cause of acute disease worldwide, and trichomoniasis is the most common and curable disease, generating more than 170 million cases annually worldwide. Trichomonas vaginalis is the causal agent of trichomoniasis and has the ability to destroy in vitro cell monolayers of the vaginal mucosa, where the phospholipases A2 (PLA2) have been reported as potential virulence factors. These enzymes have been partially characterized from the subcellular fraction S30 of pathogenic T. vaginalis strains. The main objective of this study was to purify a phospholipase A2 from T. vaginalis, make a partial characterization, obtain a partial amino acid sequence, and determine its enzymatic participation as hemolytic factor causing lysis of erythrocytes. Trichomonas S30, RF30 and UFF30 sub-fractions from GT-15 strain have the capacity to hydrolyze [2-(14)C-PA]-PC at pH 6.0. Proteins from the UFF30 sub-fraction were separated by affinity chromatography into two eluted fractions with detectable PLA A2 activity. The EDTA-eluted fraction was analyzed by HPLC using on-line HPLC-tandem mass spectrometry and two protein peaks were observed at 8.2 and 13 kDa. Peptide sequences were identified from the proteins present in the eluted EDTA UFF30 fraction; bioinformatic analysis using Protein Link Global Server charged with T. vaginalis protein database suggests that eluted peptides correspond a putative ubiquitin protein in the 8.2 kDa fraction and a phospholipase preserved in the 13 kDa fraction. The EDTA-eluted fraction hydrolyzed [2-(14)C-PA]-PC lyses erythrocytes from Sprague-Dawley in a time and dose-dependent manner. The acidic hemolytic activity decreased by 84% with the addition of 100 μM of Rosenthal's inhibitor.

  20. Amino acid sequence of bovine heart coupling factor 6.

    PubMed Central

    Fang, J K; Jacobs, J W; Kanner, B I; Racker, E; Bradshaw, R A

    1984-01-01

    The amino acid sequence of bovine heart mitochondrial coupling factor 6 (F6) has been determined by automated Edman degradation of the whole protein and derived peptides. Preparations based on heat precipitation and ethanol extraction showed allotypic variation at three positions while material further purified by HPLC yielded only one sequence that also differed by a Phe-Thr replacement at residue 62. The mature protein contains 76 amino acids with a calculated molecular weight of 9006 and a pI of approximately equal to 5, in good agreement with experimentally measured values. The charged amino acids are mainly clustered at the termini and in one section in the middle; these three polar segments are separated by two segments relatively rich in nonpolar residues. Chou-Fasman analysis suggests three stretches of alpha-helix coinciding (or within) the high-charge-density sequences with a single beta-turn at the first polar-nonpolar junction. Comparison of the F6 sequence with those of other proteins did not reveal any homologous structures. PMID:6149548

  1. Amino acid sequence and comparative antigenicity of chicken metallothionein.

    PubMed Central

    McCormick, C C; Fullmer, C S; Garvey, J S

    1988-01-01

    The complete amino acid sequence of metallothionein (MT) from chicken liver is reported. The primary structure was determined by automated sequence analysis of peptides produced by limited acid hydrolysis and by trypsin digestion. The comparative antigenicity of chicken MT was determined by radioimmunoassay using rabbit anti-rat MT polyclonal antibody. Chicken MT consists of 63 amino acids as compared to 61 found in MTs from mammals. One insertion (and two substitutions) occurs in the amino-terminal region, a region considered invariant among mammalian MTs. Eighteen of the 20 cysteines in chicken MT were aligned with cysteines from other mammalian sequences. Two cysteines near the carboxyl terminus are shifted by one residue due to the insertion of proline in that region. Overall, the chicken protein showed approximately equal to 68% sequence identity in a comparison with various mammalian MTs. The affinity of the polyclonal antibody for chicken MT was decreased by 2 orders of magnitude in comparison to that of a mammalian MT (rat MT isoforms). This reduced affinity is attributed to major substitutions in chicken MT in the regions of the principal determinants of mammalian MTs. Theoretical analysis of the primary structure predicted the secondary structure to consist of reverse turns and random coils with no stable beta or helix conformations. There is no evidence that chicken MT differs functionally from mammalian MTs. PMID:2448773

  2. Sequences Of Amino Acids For Human Serum Albumin

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C.

    1992-01-01

    Sequences of amino acids defined for use in making polypeptides one-third to one-sixth as large as parent human serum albumin molecule. Smaller, chemically stable peptides have diverse applications including service as artificial human serum and as active components of biosensors and chromatographic matrices. In applications involving production of artificial sera from new sequences, little or no concern about viral contaminants. Smaller genetically engineered polypeptides more easily expressed and produced in large quantities, making commercial isolation and production more feasible and profitable.

  3. False sugar sequence ions in electrospray tandem mass spectrometry of underivatized sialyl-Lewis-type oligosaccharides

    NASA Astrophysics Data System (ADS)

    Ernst, Beat; Müller, Dieter R.; Richter, Wilhelm J.

    1997-01-01

    Formation of "false" sugar sequence ions from branched tetrasaccharides of the sialyl-Lewis-type by migration of fucose towards sialic acid residues is shown to occur in [M + H]+ and [M + NH4]+ ions produced by electrospray ionization and subjected to low energy collision induced dissociation (CID). For the verification of their composition and sequence, such irregular ions were produced in the orifice region of the ion source, mass selected in Q1, and subjected to a second CID step in Q2 of a triple quadrupole analyser. When produced and analysed in the same "double CID" fashion, the branched B3 ions still containing all four sugar subunits show such migration to only a minor extent. The analysis of Bn fragment ions with high numbers for n may thus have advantages over the analysis of M-like species

  4. Sequencing Lys-N Proteolytic Peptides by ESI and MALDI Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Dupré, Mathieu; Cantel, Sonia; Verdié, Pascal; Martinez, Jean; Enjalbal, Christine

    2011-02-01

    In this study, we explored the MS/MS behavior of various synthetic peptides that possess a lysine residue at the N-terminal position. These peptides were designed to mimic peptides produced upon proteolysis by the Lys-N enzyme, a metalloendopeptidase issued from a Japanese fungus Grifola frondosa that was recently investigated in proteomic studies as an alternative to trypsin digestion, as a specific cleavage at the amide X-Lys chain is obtained that provides N-terminal lysine peptide fragments. In contrast to tryptic peptides exhibiting a lysine or arginine residue solely at the C-terminal position, and are thus devoid of such basic amino acids within the sequence, these Lys-N proteolytic peptides can contain the highly basic arginine residue anywhere within the peptide chain. The fragmentation patterns of such sequences with the ESI-QqTOF and MALDI-TOF/TOF mass spectrometers commonly used in proteomic bottom-up experiments were investigated.

  5. Nanopores and nucleic acids: prospects for ultrarapid sequencing

    NASA Technical Reports Server (NTRS)

    Deamer, D. W.; Akeson, M.

    2000-01-01

    DNA and RNA molecules can be detected as they are driven through a nanopore by an applied electric field at rates ranging from several hundred microseconds to a few milliseconds per molecule. The nanopore can rapidly discriminate between pyrimidine and purine segments along a single-stranded nucleic acid molecule. Nanopore detection and characterization of single molecules represents a new method for directly reading information encoded in linear polymers. If single-nucleotide resolution can be achieved, it is possible that nucleic acid sequences can be determined at rates exceeding a thousand bases per second.

  6. De Novo Sequencing of Peptides from Top-Down Tandem Mass Spectra

    SciTech Connect

    Vyatkina, Kira; Wu, Si; Dekker, Lennard J. M.; VanDuijn, Martijn M.; Liu, Xiaowen; Tolić, Nikola; Dvorkin, Mikhail; Alexandrova, Sonya; Luider, Theo M.; Paša-Tolić, Ljiljana; Pevzner, Pavel A.

    2015-11-06

    De novo sequencing of proteins and peptides is one of the most important problems in mass spectrometry-driven proteomics. A variety of methods have been developed to accomplish this task from a set of bottom-up tandem (MS/MS) mass spectra. However, a more recently emerged top-down technology, now gaining more and more popularity, opens new perspectives for protein analysis and characterization, implying a need in efficient algorithms for processing this kind of MS/MS data. Here we describe a method that allows to retrieve from a set of top-down MS/MS spectra long and accurate sequence fragments of the proteins contained in a sample. To this end, we outline a strategy for generating high-quality sequence tags from top-down spectra, and introduce the concept of a T-Bruijn graph by adapting to the case of tags the notion of an A-Bruijn graph widely used in genomics. The output of the proposed approach represents the set of amino acid strings spelled out by optimal paths in the connected components of a T-Bruijn graph. We illustrate its performance on top-down datasets acquired from carbonic anhydrase 2 (CAH2) and the Fab region of alemtuzumab.

  7. Complete amino acid sequence of ananain and a comparison with stem bromelain and other plant cysteine proteases.

    PubMed Central

    Lee, K L; Albee, K L; Bernasconi, R J; Edmunds, T

    1997-01-01

    The amino acid sequences of ananain (EC3.4.22.31) and stem bromelain (3.4.22.32), two cysteine proteases from pineapple stem, are similar yet ananain and stem bromelain possess distinct specificities towards synthetic peptide substrates and different reactivities towards the cysteine protease inhibitors E-64 and chicken egg white cystatin. We present here the complete amino acid sequence of ananain and compare it with the reported sequences of pineapple stem bromelain, papain and chymopapain from papaya and actinidin from kiwifruit. Ananain is comprised of 216 residues with a theoretical mass of 23464 Da. This primary structure includes a sequence insert between residues 170 and 174 not present in stem bromelain or papain and a hydrophobic series of amino acids adjacent to His-157. It is possible that these sequence differences contribute to the different substrate and inhibitor specificities exhibited by ananain and stem bromelain. PMID:9355753

  8. Stereochemical Sequence Ion Selectivity: Proline versus Pipecolic-acid-containing Protonated Peptides

    NASA Astrophysics Data System (ADS)

    Abutokaikah, Maha T.; Guan, Shanshan; Bythell, Benjamin J.

    2017-01-01

    Substitution of proline by pipecolic acid, the six-membered ring congener of proline, results in vastly different tandem mass spectra. The well-known proline effect is eliminated and amide bond cleavage C-terminal to pipecolic acid dominates instead. Why do these two ostensibly similar residues produce dramatically differing spectra? Recent evidence indicates that the proton affinities of these residues are similar, so are unlikely to explain the result [Raulfs et al., J. Am. Soc. Mass Spectrom. 25, 1705-1715 (2014)]. An additional hypothesis based on increased flexibility was also advocated. Here, we provide a computational investigation of the "pipecolic acid effect," to test this and other hypotheses to determine if theory can shed additional light on this fascinating result. Our calculations provide evidence for both the increased flexibility of pipecolic-acid-containing peptides, and structural changes in the transition structures necessary to produce the sequence ions. The most striking computational finding is inversion of the stereochemistry of the transition structures leading to "proline effect"-type amide bond fragmentation between the proline/pipecolic acid-congeners: R (proline) to S (pipecolic acid). Additionally, our calculations predict substantial stabilization of the amide bond cleavage barriers for the pipecolic acid congeners by reduction in deleterious steric interactions and provide evidence for the importance of experimental energy regime in rationalizing the spectra.

  9. The complementary deoxyribonucleic acid sequence of guinea pig endometrial prorelaxin.

    PubMed

    Lee, Y A; Bryant-Greenwood, G D; Mandel, M; Greenwood, F C

    1992-03-01

    The nucleotide sequence of the relaxin gene transcript in the endometrium of the late pregnant guinea pig has been determined. The strategy used was a combination of polymerase chain reaction (PCR) with primers designed from the mRNA sequence of porcine preprorelaxin, rapid amplification of cDNA ends-PCR, and blunt end cloning in M13 mp18. With heterologous primers, a 226-basepair (bp) segment of the guinea pig relaxin gene sequence was obtained and was used to design a guinea pig-specific primer for use with the rapid amplification of cDNA ends-PCR method. The latter allowed completion of the sequence of 336 bp, with a 96-bp overlap. The sequence obtained shows greater homology at both the nucleotide and amino acid levels with porcine and human relaxins H1 and H2 than with rat relaxin, supporting the thesis that the guinea pig is not a rodent. The transcription of the guinea pig endometrial relaxin gene during pregnancy was confirmed by Northern analysis of guinea pig endometrial tissues with a species-specific cDNA probe. The endometrial relaxin gene is transcribed during pregnancy, but not in lactation, consistent with the observed immunostaining for relaxin.

  10. Quantum-Sequencing: Biophysics of quantum tunneling through nucleic acids

    NASA Astrophysics Data System (ADS)

    Casamada Ribot, Josep; Chatterjee, Anushree; Nagpal, Prashant

    2014-03-01

    Tunneling microscopy and spectroscopy has extensively been used in physical surface sciences to study quantum tunneling to measure electronic local density of states of nanomaterials and to characterize adsorbed species. Quantum-Sequencing (Q-Seq) is a new method based on tunneling microscopy for electronic sequencing of single molecule of nucleic acids. A major goal of third-generation sequencing technologies is to develop a fast, reliable, enzyme-free single-molecule sequencing method. Here, we present the unique ``electronic fingerprints'' for all nucleotides on DNA and RNA using Q-Seq along their intrinsic biophysical parameters. We have analyzed tunneling spectra for the nucleotides at different pH conditions and analyzed the HOMO, LUMO and energy gap for all of them. In addition we show a number of biophysical parameters to further characterize all nucleobases (electron and hole transition voltage and energy barriers). These results highlight the robustness of Q-Seq as a technique for next-generation sequencing.

  11. Molecular cloning and amino acid sequence of human 5-lipoxygenase

    SciTech Connect

    Matsumoto, T.; Funk, C.D.; Radmark, O.; Hoeoeg, J.O.; Joernvall, H.; Samuelsson, B.

    1988-01-01

    5-Lipoxygenase (EC 1.13.11.34), a Ca/sup 2 +/- and ATP-requiring enzyme, catalyzes the first two steps in the biosynthesis of the peptidoleukotrienes and the chemotactic factor leukotriene B/sub 4/. A cDNA clone corresponding to 5-lipoxygenase was isolated from a human lung lambda gt11 expression library by immunoscreening with a polyclonal antibody. Additional clones from a human placenta lambda gt11 cDNA library were obtained by plaque hybridization with the /sup 32/P-labeled lung cDNA clone. Sequence data obtained from several overlapping clones indicate that the composite DNAs contain the complete coding region for the enzyme. From the deduced primary structure, 5-lipoxygenase encodes a 673 amino acid protein with a calculated molecular weight of 77,839. Direct analysis of the native protein and its proteolytic fragments confirmed the deduced composition, the amino-terminal amino acid sequence, and the structure of many internal segments. 5-Lipoxygenase has no apparent sequence homology with leukotriene A/sub 4/ hydrolase or Ca/sup 2 +/-binding proteins. RNA blot analysis indicated substantial amounts of an mRNA species of approx. = 2700 nucleotides in leukocytes, lung, and placenta.

  12. The amino acid sequence of chymopapain from Carica papaya.

    PubMed Central

    Watson, D C; Yaguchi, M; Lynn, K R

    1990-01-01

    Chymopapain is a polypeptide of 218 amino acid residues. It has considerable structural similarity with papain and papaya proteinase omega, including conservation of the catalytic site and of the disulphide bonding. Chymopapain is like papaya proteinase omega in carrying four extra residues between papain positions 168 and 169, but differs from both papaya proteinases in the composition of its S2 subsite, as well as in having a second thiol group, Cys-117. Some evidence for the amino acid sequence of chymopapain has been deposited as Supplementary Publication SUP 50153 (12 pages) at the British Library Document Supply Centre, Boston Spa., Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1990) 265, 5. The information comprises Supplement Tables 1-4, which contain, in order, amino acid compositions of peptides from tryptic, peptic, CNBr and mild acid cleavages, Supplement Fig. 1, showing re-fractionation of selected peaks from Fig. 2 of the main paper. Supplement Fig. 2, showing cation-exchange chromatography of the earliest-eluted peak of Fig. 3 of the main paper, Supplement Fig. 3, showing reverse-phase h.p.l.c. of the later-eluted peak from Fig. 3 of the main paper, and Supplement Fig. 4, showing the separation of peptides after mild acid hydrolysis of CNBr-cleavage fragment CB3. PMID:2106878

  13. Complete amino acid sequence of branched-chain amino acid aminotransferase (transaminase B) of Salmonella typhimurium, identification of the coenzyme-binding site and sequence comparison analysis

    SciTech Connect

    Feild, M.J.

    1988-01-01

    The complete amino acid sequence of the subunit of branched-chain amino acid aminotransferase of Salmonella typhimurium was determined by automated Edman degradation of peptide fragments generated by chemical and enzymatic digestion of S-carboxymethylated and S-pyridylethylated transaminase B. Peptide fragments of transaminase B were generated by treatment of the enzyme with trypsin, Staphylococcus aureus V8 protease, endoproteinase Lys-C, and cyanogen bromide. Protocols were developed for separation of the peptide fragments by reverse-phase high performance liquid chromatography (HPLC), ion-exchange HPLC, and SDS-urea gel electrophoresis. The enzyme subunit contains 308 amino acid residues and has a molecular weight of 33,920 daltons. The coenzyme-binding site was determined by treatment of the enzyme, containing bound pyridoxal 5-phosphate, with tritiated sodium borohydride prior to trypsin digestion. Monitoring radioactivity incorporation and peptide map comparisons with an apoenzyme tryptic digest, allowed identification of the pyridoxylated-peptide which was isolated by reverse-phase HPLC and sequenced. The coenzyme-binding site is a lysyl residue at position 159. Some peptides were further characterized by fast atom bombardment mass spectrometry.

  14. Amino acid sequence of band-3 protein from rainbow trout erythrocytes derived from cDNA.

    PubMed Central

    Hübner, S; Michel, F; Rudloff, V; Appelhans, H

    1992-01-01

    In this report we present the first complete band-3 cDNA sequence of a poikilothermic lower vertebrate. The primary structure of the anion-exchange protein band 3 (AE1) from rainbow trout erythrocytes was determined by nucleotide sequencing of cDNA clones. The overlapping clones have a total length of 3827 bp with a 5'-terminal untranslated region of 150 bp, a 2754 bp open reading frame and a 3'-untranslated region of 924 bp. Band-3 protein from trout erythrocytes consists of 918 amino acid residues with a calculated molecular mass of 101 827 Da. Comparison of its amino acid sequence revealed a 60-65% identity within the transmembrane spanning sequence of band-3 proteins published so far. An additional insertion of 24 amino acid residues within the membrane-associated domain of trout band-3 protein was identified, which until now was thought to be a general feature only of mammalian band-3-related proteins. PMID:1637296

  15. The amino acid sequence of rabbit cardiac troponin I.

    PubMed Central

    Grand, R J; Wilkinson, J M

    1976-01-01

    The complete amino acid sequence of troponin I from rabbit cardiac muscle was determined by the isolation of four unique CNBr fragments, together with overlapping tryptic peptides containing radioactive methionine residues. Overlap data for residues 35-36, 93-94 and 140-145 are incomplete, the sequence at these positions being based on homology with the sequence of the fast-skeletal-muscle protein. Cardiac troponin I is a single polypeptide chain of 206 residues with mol.wt. 23550 and an extinction coefficient, E 1%,1cm/280, of 4.37. The protein has a net positive charge of 14 and is thus somewhat more basic than troponin I from fast-skeletal muscle. Comparison of the sequences of troponin I from cardiac and fast skeletal muscle show that the cardiac protein has 26 extra residues at the N-terminus which account for the larger size of the protein. In the remainder of sequence there is a considerable degree of homology, this being greater in the C-terminal two-thirds of the molecule. The region in the cardiac protein corresponding to the peptide with inhibitory activity from the fast-skeletal-muscle protein is very similar and it seems unlikely that this is the cause of the difference in inhibitory activity between the two proteins. The region responsible for binding troponin C, however, possesses a lower degree of homology. Detailed evidence on which the sequence is based has been deposited as Supplementary Publication SUP 50072 (20 pages), at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7QB, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1976) 153, 5. PMID:1008822

  16. The complete amino acid sequence of a trypsin inhibitor from Bauhinia variegata var. candida seeds.

    PubMed

    Di Ciero, L; Oliva, M L; Torquato, R; Köhler, P; Weder, J K; Camillo Novello, J; Sampaio, C A; Oliveira, B; Marangoni, S

    1998-11-01

    Trypsin inhibitors of two varieties of Bauhinia variegata seeds have been isolated and characterized. Bauhinia variegata candida trypsin inhibitor (BvcTI) and B. variegata lilac trypsin inhibitor (BvlTI) are proteins with Mr of about 20,000 without free sulfhydryl groups. Amino acid analysis shows a high content of aspartic acid, glutamic acid, serine, and glycine, and a low content of histidine, tyrosine, methionine, and lysine in both inhibitors. Isoelectric focusing for both varieties detected three isoforms (pI 4.85, 5.00, and 5.15), which were resolved by HPLC procedure. The trypsin inhibitors show Ki values of 6.9 and 1.2 nM for BvcTI and BvlTI, respectively. The N-terminal sequences of the three trypsin inhibitor isoforms from both varieties of Bauhinia variegata and the complete amino acid sequence of B. variegata var. candida L. trypsin inhibitor isoform 3 (BvcTI-3) are presented. The sequences have been determined by automated Edman degradation of the reduced and carboxymethylated proteins of the peptides resulting from Staphylococcus aureus protease and trypsin digestion. BvcTI-3 is composed of 167 residues and has a calculated molecular mass of 18,529. Homology studies with other trypsin inhibitors show that BvcTI-3 belongs to the Kunitz family. The putative active site encompasses Arg (63)-Ile (64).

  17. Multiple site-selective insertions of non-canonical amino acids into sequence-repetitive polypeptides

    PubMed Central

    Wu, I-Lin; Patterson, Melissa A.; Carpenter Desai, Holly E.; Mehl, Ryan A.; Giorgi, Gianluca

    2013-01-01

    A simple and efficient method is described for introduction of non-canonical amino acids at multiple, structurally defined sites within recombinant polypeptide sequences. E. coli MRA30, a bacterial host strain with attenuated activity for release factor 1 (RF1), is assessed for its ability to support the incorporation of a diverse range of non-canonical amino acids in response to multiple encoded amber (TAG) codons within genetic templates derived from superfolder GFP and an elastin-mimetic protein polymer. Suppression efficiency and isolated protein yield were observed to depend on the identity of the orthogonal aminoacyl-tRNA synthetase/tRNACUA pair and the non-canonical amino acid substrate. This approach afforded elastin-mimetic protein polymers containing non-canonical amino acid derivatives at up to twenty-two positions within the repeat sequence with high levels of substitution. The identity and position of the variant residues was confirmed by mass spectrometric analysis of the full-length polypeptides and proteolytic cleavage fragments resulting from thermolysin digestion. The accumulated data suggest that this multi-site suppression approach permits the preparation of protein-based materials in which novel chemical functionality can be introduced at precisely defined positions within the polypeptide sequence. PMID:23625817

  18. De Novo Sequencing of Top-Down Tandem Mass Spectra: A Next Step towards Retrieving a Complete Protein Sequence

    PubMed Central

    Vyatkina, Kira

    2017-01-01

    De novo sequencing of tandem (MS/MS) mass spectra represents the only way to determine the sequence of proteins from organisms with unknown genomes, or the ones not directly inscribed in a genome—such as antibodies, or novel splice variants. Top-down mass spectrometry provides new opportunities for analyzing such proteins; however, retrieving a complete protein sequence from top-down MS/MS spectra still remains a distant goal. In this paper, we review the state-of-the-art on this subject, and enhance our previously developed Twister algorithm for de novo sequencing of peptides from top-down MS/MS spectra to derive longer sequence fragments of a target protein. PMID:28248257

  19. Amino acid sequence of a mouse immunoglobulin mu chain.

    PubMed Central

    Kehry, M; Sibley, C; Fuhrman, J; Schilling, J; Hood, L E

    1979-01-01

    The complete amino acid sequence of the mouse mu chain from the BALB/c myeloma tumor MOPC 104E is reported. The C mu region contains four consecutive homology regions of approximately 110 residues and a COOH-terminal region of 19 residues. A comparison of this mu chain from mouse with a complete mu sequence from human (Ou) and a partial mu chain sequence from dog (Moo) reveals a striking gradient of increasing homology from the NH2-terminal to the COOH-terminal portion of these mu chains, with the former being the least and the latter the most highly conserved. Four of the five sites of carbohydrate attachment appear to be at identical residue positions when the constant regions of the mouse and human mu chains are compared. The mu chain of MOPC 104E has a carbohydrate moiety attached in the second hypervariable region. This is particularly interesting in view of the fact that MOPC 104E binds alpha-(1 leads to 3)-dextran, a simple carbohydrate. The structural and functional constraints imposed by these comparative sequence analyses are discussed. PMID:111247

  20. Ultrasensitive nucleic acid sequence detection by single-molecule electrophoresis

    SciTech Connect

    Castro, A; Shera, E.B.

    1996-09-01

    This is the final report of a one-year laboratory-directed research and development project at Los Alamos National Laboratory. There has been considerable interest in the development of very sensitive clinical diagnostic techniques over the last few years. Many pathogenic agents are often present in extremely small concentrations in clinical samples, especially at the initial stages of infection, making their detection very difficult. This project sought to develop a new technique for the detection and accurate quantification of specific bacterial and viral nucleic acid sequences in clinical samples. The scheme involved the use of novel hybridization probes for the detection of nucleic acids combined with our recently developed technique of single-molecule electrophoresis. This project is directly relevant to the DOE`s Defense Programs strategic directions in the area of biological warfare counter-proliferation.

  1. Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry.

    PubMed

    Asara, John M; Schweitzer, Mary H; Freimark, Lisa M; Phillips, Matthew; Cantley, Lewis C

    2007-04-13

    Fossilized bones from extinct taxa harbor the potential for obtaining protein or DNA sequences that could reveal evolutionary links to extant species. We used mass spectrometry to obtain protein sequences from bones of a 160,000- to 600,000-year-old extinct mastodon (Mammut americanum) and a 68-million-year-old dinosaur (Tyrannosaurus rex). The presence of T. rex sequences indicates that their peptide bonds were remarkably stable. Mass spectrometry can thus be used to determine unique sequences from ancient organisms from peptide fragmentation patterns, a valuable tool to study the evolution and adaptation of ancient taxa from which genomic sequences are unlikely to be obtained.

  2. Mass spectrometry of polycyclic tetracarboxylic ('ARN') acids and tetramethyl esters.

    PubMed

    Sutton, Paul A; Smith, Benjamin E; Rowland, Steven J

    2010-11-15

    Polycyclic C(80) tetracarboxylic (so-called 'ARN') acids are found as calcium salts in deposits which form in certain oilfield pipelines and equipment. Characterisation of these acids is important for improving the prediction and hence avoidance or minimisation of oilfield deposition problems. Although several of the acids have been isolated and characterised (as regioisomeric mixtures) by nuclear magnetic resonance spectroscopy, mass spectrometric methods are likely to be much more useful for the routine analysis of oils and deposits containing the acids. A publication summarising the mass spectra of the purified acids and major derivatives might thus be a very useful source of reference for scientists and technologists studying these unusual compounds. We now report the characterisation of several of the purified acids and of the tetramethyl esters by electrospray ionisation mass spectrometry (ESI-MS) in both positive ion and negative ion modes, by multistage ESI-MS with a suggested rationalisation of the ions produced, by positive ion atmospheric solids analysis probe (ASAP) atmospheric pressure chemical ionisation (APCI), and by positive ion electron ionisation (EI)-MS. Tentative identifications of C(80) acyclic, mono-, bi- and tricylic tetraacids and the δ(13)C isotope values of a mixture of the semi-pure acids determined by MS are also reported for the first time.

  3. Nucleic acid (cDNA) and amino acid sequences of alpha-type gliadins from wheat (Triticum aestivum).

    PubMed Central

    Kasarda, D D; Okita, T W; Bernardin, J E; Baecker, P A; Nimmo, C C; Lew, E J; Dietler, M D; Greene, F C

    1984-01-01

    The complete amino acid sequence for an alpha-type gliadin protein of wheat (Triticum aestivum Linnaeus) endosperm has been derived from a cloned cDNA sequence. An additional cDNA clone that corresponds to about 75% of a similar alpha-type gliadin has been sequenced and shows some important differences. About 97% of the composite sequence of A-gliadin (an alpha-type gliadin fraction) has also been obtained by direct amino acid sequencing. This sequence shows a high degree of similarity with amino acid sequences derived from both cDNA clones and is virtually identical to one of them. On the basis of sequence information, after loss of the signal sequence, the mature alpha-type gliadins may be divided into five different domains, two of which may have evolved from an ancestral gliadin gene, whereas the remaining three contain repeating sequences that may have developed independently. Images PMID:6589619

  4. A case study of de novo sequence analysis of N-sulfonated peptides by MALDI TOF/TOF mass spectrometry.

    PubMed

    Samyn, Bart; Debyser, Griet; Sergeant, Kjell; Devreese, Bart; Van Beeumen, Jozef

    2004-12-01

    The simplicity and sensitivity of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry have increased its application in recent years. The most common method of "peptide mass fingerprint" analysis often does not provide robust identification. Additional sequence information, obtained by post-source decay or collision induced dissociation, provides additional constraints for database searches. However, de novo sequencing by mass spectrometry is not yet common practice, most likely because of the difficulties associated with the interpretation of high and low energy CID spectra. Success with this type of sequencing requires full sequence coverage and demands better quality spectra than those typically used for data base searching. In this report we show that full-length de novo sequencing is possible using MALDI TOF/TOF analysis. The interpretation of MS/MS data is facilitated by N-terminal sulfonation after protection of lysine side chains (Keough et al., Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7131-7136). Reliable de novo sequence analysis has been obtained using sub-picomol quantities of peptides and peptide sequences of up to 16 amino acid residues in length have been determined. The simple, predictable fragmentation pattern allows routine de novo interpretation, either manually or using software. Characterization of the complete primary structure of a peptide is often hindered because of differences in fragmentation efficiencies and in specific fragmentation patterns for different peptides. These differences are controlled by various structural parameters including the nature of the residues present. The influence of the presence of internal Pro, acidic and basic residues on the TOF/TOF fragmentation pattern will be discussed, both for underivatized and guanidinated/sulfonated peptides.

  5. A method for high-sensitivity peptide sequencing using postsource decay matrix-assisted laser desorption ionization mass spectrometry

    PubMed Central

    Keough, T.; Youngquist, R. S.; Lacey, M. P.

    1999-01-01

    A method has been developed for de novo peptide sequencing using matrix-assisted laser desorption ionization mass spectrometry. This method will facilitate biological studies that require rapid determination of peptide or protein sequences, e.g., determination of posttranslational modifications, identification of active compounds isolated from combinatorial peptide libraries, and the selective identification of proteins as part of proteome studies. The method involves fast, one-step addition of a sulfonic acid group to the N terminus of tryptic peptides followed by acquisition of postsource decay (PSD) fragment ion spectra. The derivatives are designed to promote efficient charge site-initiated fragmentation of the backbone amide bonds and to selectively enhance the detection of a single fragment ion series that contains the C terminus of the molecule (y-ions). The overall method has been applied to pmol quantities of peptides. The resulting PSD fragment ion spectra often exhibit uninterrupted sequences of 20 or more amino acid residues. However, fragmentation efficiency decreases considerably at amide bonds on the C-terminal side of Pro. The spectra are simple enough that de novo sequence tagging is routine. The technique has been successfully applied to peptide mixtures, to high-mass peptides (up to 3,600 Da) and to the unambiguous identification of proteins isolated from two-dimensional gel electrophoresis. The PSD spectra of these derivatized peptides often allow far more selective protein sequence database searches than those obtained from the spectra of native peptides. PMID:10377380

  6. Structural gene and complete amino acid sequence of Vibrio alginolyticus collagenase.

    PubMed Central

    Takeuchi, H; Shibano, Y; Morihara, K; Fukushima, J; Inami, S; Keil, B; Gilles, A M; Kawamoto, S; Okuda, K

    1992-01-01

    The DNA encoding the collagenase of Vibrio alginolyticus was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, bacteria carrying the gene exhibited both collagenase antigen and collagenase activity. The open reading frame from the ATG initiation codon was 2442 bp in length for the collagenase structural gene. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature collagenase consists of 739 amino acids with an Mr of 81875. The amino acid sequences of 20 polypeptide fragments were completely identical with the deduced amino acid sequences of the collagenase gene. The amino acid composition predicted from the DNA sequence was similar to the chemically determined composition of purified collagenase reported previously. The analyses of both the DNA and amino acid sequences of the collagenase gene were rigorously performed, but we could not detect any significant sequence similarity to other collagenases. Images Fig. 2. PMID:1311172

  7. Effects of main-sequence mass loss on the turnoff ages of population 1 clusters

    NASA Astrophysics Data System (ADS)

    Guzik, Joyce A.

    Willson, Bowen, and Struck-Marcell have proposed that stars of spectral types A through early-G lose a significant portion of their mass during the early main-sequence phase. The proposed mass loss is driven by pulsation, and facilitated by rapid rotation. One implication of this hypothesis is that the main-sequence turnoff is an invalid indicator of cluster age, as present turnoff stars may have had higher projenitor masses; hence clusters appear older than they actually are. This paper presents examples of cluster HR diagrams synthesized with mass-losing stars of solar metallicity, initial masses 1-2 M, and exponentially-decreasing mass-loss rates with e-folding times 1 to 2 Gyr. The increases in apparent turnoff age of Pop. I clusters, and the potential of the hypothesis to account for blue stragglers as normal stars that have not lost mass (or lost mass more slowly) are discussed.

  8. RECONCILING THE OBSERVED STAR-FORMING SEQUENCE WITH THE OBSERVED STELLAR MASS FUNCTION

    SciTech Connect

    Leja, Joel; Van Dokkum, Pieter G.; Franx, Marijn; Whitaker, Katherine E.

    2015-01-10

    We examine the connection between the observed star-forming sequence (SFR ∝ M {sup α}) and the observed evolution of the stellar mass function in the range 0.2 < z < 2.5. We find that the star-forming sequence cannot have a slope α ≲ 0.9 at all masses and redshifts because this would result in a much higher number density at 10 < log (M/M {sub ☉}) < 11 by z = 1 than is observed. We show that a transition in the slope of the star-forming sequence, such that α = 1 at log (M/M {sub ☉}) < 10.5 and α = 0.7-0.13z (Whitaker et al.) at log (M/M {sub ☉}) > 10.5, greatly improves agreement with the evolution of the stellar mass function. We then derive a star-forming sequence that reproduces the evolution of the mass function by design. This star-forming sequence is also well described by a broken power law, with a shallow slope at high masses and a steep slope at low masses. At z = 2, it is offset by ∼0.3 dex from the observed star-forming sequence, consistent with the mild disagreement between the cosmic star formation rate (SFR) and recent observations of the growth of the stellar mass density. It is unclear whether this problem stems from errors in stellar mass estimates, errors in SFRs, or other effects. We show that a mass-dependent slope is also seen in other self-consistent models of galaxy evolution, including semianalytical, hydrodynamical, and abundance-matching models. As part of the analysis, we demonstrate that neither mergers nor hidden low-mass quiescent galaxies are likely to reconcile the evolution of the mass function and the star-forming sequence. These results are supported by observations from Whitaker et al.

  9. Nucleic acid (cDNA) and amino acid sequences of the maize endosperm protein glutelin-2.

    PubMed Central

    Prat, S; Cortadas, J; Puigdomènech, P; Palau, J

    1985-01-01

    The cDNA coding for a glutelin-2 protein from maize endosperm has been cloned and the complete amino acid sequence of the protein derived for the first time. An immature maize endosperm cDNA bank was screened for the expression of a beta-lactamase:glutelin-2 (G2) fusion polypeptide by using antibodies against the purified 28 kd G2 protein. A clone corresponding to the 28 kd G2 protein was sequenced and the primary structure of this protein was derived. Five regions can be defined in the protein sequence: an 11 residue N-terminal part, a repeated region formed by eight units of the sequence Pro-Pro-Pro-Val-His-Leu, an alternating Pro-X stretch 21 residues long, a Cys rich domain and a C-terminal part rich in Gln. The protein sequence is preceded by 19 residues which have the characteristics of the signal peptide found in secreted proteins. Unlike zeins, the main maize storage proteins, 28 kd glutelin-2 has several homologous sequences in common with other cereal storage proteins. Images PMID:3839076

  10. Spermatogenesis of the lizard Lacerta vivipara: histological studies and amino acid sequence of a protamine lacertine 1.

    PubMed

    Martinage, A; Depeiges, A; Wouters, D; Morel, L; Sautière, P

    1996-06-01

    The lizard Lacerta vivipara is a seasonal breeder with a well characterized reproductive cycle. An histological study of the lizard testis has been performed at different stages of spermatogenesis and the nuclear basic proteins content was assessed by electrophoretical analysis. Two protamines, lacertines 1 and 2, are present in spermatozoa in April and May. We have isolated lacertine1 and characterized a protamine with a mass of 4,963.7 Da. Amino acid sequence of this protamine (41 residues) was established from data provided by automated Edman degradation. It is characterized by a basic amino acid stretch in the N- and C-terminal regions and by a central part which only consists of 3 different intermingled amino acids. This protamine presents 62% homology with scylliorhinine Z3 from dog-fish Scylliorhinus caniculus and 58% homology with quail protamine. The reported lizard protamine sequence is the first reptilian protamine sequence available so far.

  11. Probing Protein Structure by Amino Acid-Specific Covalent Labeling and Mass Spectrometry

    PubMed Central

    Mendoza, Vanessa Leah; Vachet, Richard W.

    2009-01-01

    For many years, amino acid-specific covalent labeling has been a valuable tool to study protein structure and protein interactions, especially for systems that are difficult to study by other means. These covalent labeling methods typically map protein structure and interactions by measuring the differential reactivity of amino acid side chains. The reactivity of amino acids in proteins generally depends on the accessibility of the side chain to the reagent, the inherent reactivity of the label and the reactivity of the amino acid side chain. Peptide mass mapping with ESI- or MALDI-MS and peptide sequencing with tandem MS are typically employed to identify modification sites to provide site-specific structural information. In this review, we describe the reagents that are most commonly used in these residue-specific modification reactions, details about the proper use of these covalent labeling reagents, and information about the specific biochemical problems that have been addressed with covalent labeling strategies. PMID:19016300

  12. 37 CFR 1.821 - Nucleotide and/or amino acid sequence disclosures in patent applications.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2010-07-01 2010-07-01 false Nucleotide and/or amino acid... Biotechnology Invention Disclosures Application Disclosures Containing Nucleotide And/or Amino Acid Sequences § 1.821 Nucleotide and/or amino acid sequence disclosures in patent applications. (a) Nucleotide...

  13. 37 CFR 1.821 - Nucleotide and/or amino acid sequence disclosures in patent applications.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2012-07-01 2012-07-01 false Nucleotide and/or amino acid... Biotechnology Invention Disclosures Application Disclosures Containing Nucleotide And/or Amino Acid Sequences § 1.821 Nucleotide and/or amino acid sequence disclosures in patent applications. (a) Nucleotide...

  14. 37 CFR 1.821 - Nucleotide and/or amino acid sequence disclosures in patent applications.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2014-07-01 2014-07-01 false Nucleotide and/or amino acid... Biotechnology Invention Disclosures Application Disclosures Containing Nucleotide And/or Amino Acid Sequences § 1.821 Nucleotide and/or amino acid sequence disclosures in patent applications. (a) Nucleotide...

  15. 37 CFR 1.821 - Nucleotide and/or amino acid sequence disclosures in patent applications.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2011-07-01 2011-07-01 false Nucleotide and/or amino acid... Biotechnology Invention Disclosures Application Disclosures Containing Nucleotide And/or Amino Acid Sequences § 1.821 Nucleotide and/or amino acid sequence disclosures in patent applications. (a) Nucleotide...

  16. 37 CFR 1.821 - Nucleotide and/or amino acid sequence disclosures in patent applications.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2013-07-01 2013-07-01 false Nucleotide and/or amino acid... Biotechnology Invention Disclosures Application Disclosures Containing Nucleotide And/or Amino Acid Sequences § 1.821 Nucleotide and/or amino acid sequence disclosures in patent applications. (a) Nucleotide...

  17. A Quantitative Tool to Distinguish Isobaric Leucine and Isoleucine Residues for Mass Spectrometry-Based De Novo Monoclonal Antibody Sequencing

    NASA Astrophysics Data System (ADS)

    Poston, Chloe N.; Higgs, Richard E.; You, Jinsam; Gelfanova, Valentina; Hale, John E.; Knierman, Michael D.; Siegel, Robert; Gutierrez, Jesus A.

    2014-07-01

    De novo sequencing by mass spectrometry (MS) allows for the determination of the complete amino acid (AA) sequence of a given protein based on the mass difference of detected ions from MS/MS fragmentation spectra. The technique relies on obtaining specific masses that can be attributed to characteristic theoretical masses of AAs. A major limitation of de novo sequencing by MS is the inability to distinguish between the isobaric residues leucine (Leu) and isoleucine (Ile). Incorrect identification of Ile as Leu or vice versa often results in loss of activity in recombinant antibodies. This functional ambiguity is commonly resolved with costly and time-consuming AA mutation and peptide sequencing experiments. Here, we describe a set of orthogonal biochemical protocols, which experimentally determine the identity of Ile or Leu residues in monoclonal antibodies (mAb) based on the selectivity that leucine aminopeptidase shows for n-terminal Leu residues and the cleavage preference for Leu by chymotrypsin. The resulting observations are combined with germline frequencies and incorporated into a logistic regression model, called Predictor for Xle Sites (PXleS) to provide a statistical likelihood for the identity of Leu at an ambiguous site. We demonstrate that PXleS can generate a probability for an Xle site in mAbs with 96% accuracy. The implementation of PXleS precludes the expression of several possible sequences and, therefore, reduces the overall time and resources required to go from spectra generation to a biologically active sequence for a mAb when an Ile or Leu residue is in question.

  18. Human liver apolipoprotein B-100 cDNA: complete nucleic acid and derived amino acid sequence.

    PubMed Central

    Law, S W; Grant, S M; Higuchi, K; Hospattankar, A; Lackner, K; Lee, N; Brewer, H B

    1986-01-01

    Human apolipoprotein B-100 (apoB-100), the ligand on low density lipoproteins that interacts with the low density lipoprotein receptor and initiates receptor-mediated endocytosis and low density lipoprotein catabolism, has been cloned, and the complete nucleic acid and derived amino acid sequences have been determined. ApoB-100 cDNAs were isolated from normal human liver cDNA libraries utilizing immunoscreening as well as filter hybridization with radiolabeled apoB-100 oligodeoxynucleotides. The apoB-100 mRNA is 14.1 kilobases long encoding a mature apoB-100 protein of 4536 amino acids with a calculated amino acid molecular weight of 512,723. ApoB-100 contains 20 potential glycosylation sites, and 12 of a total of 25 cysteine residues are located in the amino-terminal region of the apolipoprotein providing a potential globular structure of the amino terminus of the protein. ApoB-100 contains relatively few regions of amphipathic helices, but compared to other human apolipoproteins it is enriched in beta-structure. The delineation of the entire human apoB-100 sequence will now permit a detailed analysis of the conformation of the protein, the low density lipoprotein receptor binding domain(s), and the structural relationship between apoB-100 and apoB-48 and will provide the basis for the study of genetic defects in apoB-100 in patients with dyslipoproteinemias. PMID:3464946

  19. Computer selection of oligonucleotide probes from amino acid sequences for use in gene library screening.

    PubMed

    Yang, J H; Ye, J H; Wallace, D C

    1984-01-11

    We present a computer program, FINPROBE, which utilizes known amino acid sequence data to deduce minimum redundancy oligonucleotide probes for use in screening cDNA or genomic libraries or in primer extension. The user enters the amino acid sequence of interest, the desired probe length, the number of probes sought, and the constraints on oligonucleotide synthesis. The computer generates a table of possible probes listed in increasing order of redundancy and provides the location of each probe in the protein and mRNA coding sequence. Activation of a next function provides the amino acid and mRNA sequences of each probe of interest as well as the complementary sequence and the minimum dissociation temperature of the probe. A final routine prints out the amino acid sequence of the protein in parallel with the mRNA sequence listing all possible codons for each amino acid.

  20. Purification, characterization, gene cloning and nucleotide sequencing of D: -stereospecific amino acid amidase from soil bacterium: Delftia acidovorans.

    PubMed

    Hongpattarakere, Tipparat; Komeda, Hidenobu; Asano, Yasuhisa

    2005-12-01

    The D-amino acid amidase-producing bacterium was isolated from soil samples using an enrichment culture technique in medium broth containing D-phenylalanine amide as a sole source of nitrogen. The strain exhibiting the strongest activity was identified as Delftia acidovorans strain 16. This strain produced intracellular D-amino acid amidase constitutively. The enzyme was purified about 380-fold to homogeneity and its molecular mass was estimated to be about 50 kDa, on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was active preferentially toward D-amino acid amides rather than their L-counterparts. It exhibited strong amino acid amidase activity toward aromatic amino acid amides including D-phenylalanine amide, D-tryptophan amide and D-tyrosine amide, yet it was not specifically active toward low-molecular-weight D-amino acid amides such as D-alanine amide, L-alanine amide and L-serine amide. Moreover, it was not specifically active toward oligopeptides. The enzyme showed maximum activity at 40 degrees C and pH 8.5 and appeared to be very stable, with 92.5% remaining activity after the reaction was performed at 45 degrees C for 30 min. However, it was mostly inactivated in the presence of phenylmethanesulfonyl fluoride or Cd2+, Ag+, Zn2+, Hg2+ and As3+ . The NH2 terminal and internal amino acid sequences of the enzyme were determined; and the gene was cloned and sequenced. The enzyme gene damA encodes a 466-amino-acid protein (molecular mass 49,860.46 Da); and the deduced amino acid sequence exhibits homology to the D-amino acid amidase from Variovorax paradoxus (67.9% identity), the amidotransferase A subunit from Burkholderia fungorum (50% identity) and other enantioselective amidases.

  1. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry: a powerful tool for the mass and sequence analysis of natural and modified oligonucleotides.

    PubMed Central

    Pieles, U; Zürcher, W; Schär, M; Moser, H E

    1993-01-01

    We report the analysis and characterization of natural and modified oligonucleotides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The present technology was highly improved for this class of compounds by using a new matrix, 2,4,6-trihydroxy acetophenone, together with di- and triammonium salts of organic or inorganic acids to suppress peak broadening due to multiple ion adducts. This methodology can be used in combination with time dependent degradation of oligonucleotides by exonucleases as powerful tool to determine sequence compositions. PMID:8341593

  2. Medical Sequencing at the extremes of Human Body Mass

    SciTech Connect

    Ahituv, Nadav; Kavaslar, Nihan; Schackwitz, Wendy; Ustaszewski,Anna; Martin, Joes; Hebert, Sybil; Doelle, Heather; Ersoy, Baran; Kryukov, Gregory; Schmidt, Steffen; Yosef, Nir; Ruppin, Eytan; Sharan,Roded; Vaisse, Christian; Sunyaev, Shamil; Dent, Robert; Cohen, Jonathan; McPherson, Ruth; Pennacchio, Len A.

    2006-09-01

    Body weight is a quantitative trait with significantheritability in humans. To identify potential genetic contributors tothis phenotype, we resequenced the coding exons and splice junctions of58 genes in 379 obese and 378 lean individuals. Our 96Mb survey included21 genes associated with monogenic forms of obesity in humans or mice, aswell as 37 genes that function in body weight-related pathways. We foundthat the monogenic obesity-associated gene group was enriched for rarenonsynonymous variants unique to the obese (n=46) versus lean (n=26)populations. Computational analysis further predicted a significantlygreater fraction of deleterious variants within the obese cohort.Consistent with the complex inheritance of body weight, we did notobserve obvious familial segregation in the majority of the 28 availablekindreds. Taken together, these data suggest that multiple rare alleleswith variable penetrance contribute to obesity in the population andprovide a deep medical sequencing based approach to detectthem.

  3. Mass balance of perfluoroalkyl acids in the Baltic Sea.

    PubMed

    Filipovic, Marko; Berger, Urs; McLachlan, Michael S

    2013-05-07

    A mass balance was assembled for perfluorohexanoic acid (PFHxA), perfluorooctanoic acid (PFOA), perfluorodecanoic acid (PFDA), and perfluorooctanesulfonic acid (PFOS) in the Baltic Sea. Inputs (from riverine discharge, atmospheric deposition, coastal wastewater discharges, and the North Sea) and outputs (to sediment burial, transformation of the chemical, and the North Sea), as well as the inventory in the Baltic Sea, were estimated from recently published monitoring data. Formation of the chemicals in the water column from precursors was not considered. River inflow and atmospheric deposition were the dominant inputs, while wastewater treatment plant (WWTP) effluents made a minor contribution (<5%). A mass balance of the Oder River watershed was assembled to explore the sources of the perfluoroalkyl acids (PFAAs) in the river inflow. It indicated that WWTP effluents made only a moderate contribution to riverine discharge (21% for PFOA, 6% for PFOS), while atmospheric deposition to the watershed was 1-2 orders of magnitude greater than WWTP discharges. The input to the Baltic Sea exceeded the output for all four PFAAs, suggesting that inputs were higher during 2005-2010 than during the previous 20 years despite efforts to reduce emissions of PFAAs. One possible explanation is the retention and delayed release of PFAAs from atmospheric deposition in the soils and groundwater of the watershed.

  4. 37 CFR 1.822 - Symbols and format to be used for nucleotide and/or amino acid sequence data.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... for nucleotide and/or amino acid sequence data. 1.822 Section 1.822 Patents, Trademarks, and... Amino Acid Sequences § 1.822 Symbols and format to be used for nucleotide and/or amino acid sequence data. (a) The symbols and format to be used for nucleotide and/or amino acid sequence data...

  5. 37 CFR 1.822 - Symbols and format to be used for nucleotide and/or amino acid sequence data.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... for nucleotide and/or amino acid sequence data. 1.822 Section 1.822 Patents, Trademarks, and... Amino Acid Sequences § 1.822 Symbols and format to be used for nucleotide and/or amino acid sequence data. (a) The symbols and format to be used for nucleotide and/or amino acid sequence data...

  6. 37 CFR 1.822 - Symbols and format to be used for nucleotide and/or amino acid sequence data.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... for nucleotide and/or amino acid sequence data. 1.822 Section 1.822 Patents, Trademarks, and... Amino Acid Sequences § 1.822 Symbols and format to be used for nucleotide and/or amino acid sequence data. (a) The symbols and format to be used for nucleotide and/or amino acid sequence data...

  7. 37 CFR 1.822 - Symbols and format to be used for nucleotide and/or amino acid sequence data.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... for nucleotide and/or amino acid sequence data. 1.822 Section 1.822 Patents, Trademarks, and... Amino Acid Sequences § 1.822 Symbols and format to be used for nucleotide and/or amino acid sequence data. (a) The symbols and format to be used for nucleotide and/or amino acid sequence data...

  8. 37 CFR 1.822 - Symbols and format to be used for nucleotide and/or amino acid sequence data.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... for nucleotide and/or amino acid sequence data. 1.822 Section 1.822 Patents, Trademarks, and... Amino Acid Sequences § 1.822 Symbols and format to be used for nucleotide and/or amino acid sequence data. (a) The symbols and format to be used for nucleotide and/or amino acid sequence data...

  9. Dust discs around low-mass main-sequence stars

    NASA Technical Reports Server (NTRS)

    Wolstencroft, R. D.; Walker, Helen J.

    1988-01-01

    The current understanding of the formation of circumstellar disks as a natural accompaniment to the process of low-mass star formation is examined. Models of the thermal emission from the dust disks around the prototype stars Alpha Lyr, Alpha PsA, Beta Pic, and Epsilon Eri are discussed, which indicate that the central regions of three of these disks are almost devoid of dust within radii ranging between 17 and 26 AU, with the temperature of the hottest zone lying between about 115 and 210 K. One possible explanation of the dust-free zones is the presence of a planet at the inner boundary of each cloud which sweeps up grains crossing its orbit.

  10. Human retroviruses and AIDS 1996. A compilation and analysis of nucleic acid and amino acid sequences

    SciTech Connect

    Myers, G.; Foley, B.; Korber, B.; Mellors, J.W.; Jeang, K.T.; Wain-Hobson, S.

    1997-04-01

    This compendium and the accompanying floppy diskettes are the result of an effort to compile and rapidly publish all relevant molecular data concerning the human immunodeficiency viruses (HIV) and related retroviruses. The scope of the compendium and database is best summarized by the five parts that it comprises: (1) Nuclear Acid Alignments and Sequences; (2) Amino Acid Alignments; (3) Analysis; (4) Related Sequences; and (5) Database Communications. Information within all the parts is updated throughout the year on the Web site, http://hiv-web.lanl.gov. While this publication could take the form of a review or sequence monograph, it is not so conceived. Instead, the literature from which the database is derived has simply been summarized and some elementary computational analyses have been performed upon the data. Interpretation and commentary have been avoided insofar as possible so that the reader can form his or her own judgments concerning the complex information. In addition to the general descriptions of the parts of the compendium, the user should read the individual introductions for each part.

  11. Tandem Mass Spectrum Sequencing: An Alternative to Database Search Engines in Shotgun Proteomics.

    PubMed

    Muth, Thilo; Rapp, Erdmann; Berven, Frode S; Barsnes, Harald; Vaudel, Marc

    2016-01-01

    Protein identification via database searches has become the gold standard in mass spectrometry based shotgun proteomics. However, as the quality of tandem mass spectra improves, direct mass spectrum sequencing gains interest as a database-independent alternative. In this chapter, the general principle of this so-called de novo sequencing is introduced along with pitfalls and challenges of the technique. The main tools available are presented with a focus on user friendly open source software which can be directly applied in everyday proteomic workflows.

  12. Transcriptome Sequencing in Response to Salicylic Acid in Salvia miltiorrhiza

    PubMed Central

    Zhang, Xiaoru; Dong, Juane; Liu, Hailong; Wang, Jiao; Qi, Yuexin; Liang, Zongsuo

    2016-01-01

    Salvia miltiorrhiza is a traditional Chinese herbal medicine, whose quality and yield are often affected by diseases and environmental stresses during its growing season. Salicylic acid (SA) plays a significant role in plants responding to biotic and abiotic stresses, but the involved regulatory factors and their signaling mechanisms are largely unknown. In order to identify the genes involved in SA signaling, the RNA sequencing (RNA-seq) strategy was employed to evaluate the transcriptional profiles in S. miltiorrhiza cell cultures. A total of 50,778 unigenes were assembled, in which 5,316 unigenes were differentially expressed among 0-, 2-, and 8-h SA induction. The up-regulated genes were mainly involved in stimulus response and multi-organism process. A core set of candidate novel genes coding SA signaling component proteins was identified. Many transcription factors (e.g., WRKY, bHLH and GRAS) and genes involved in hormone signal transduction were differentially expressed in response to SA induction. Detailed analysis revealed that genes associated with defense signaling, such as antioxidant system genes, cytochrome P450s and ATP-binding cassette transporters, were significantly overexpressed, which can be used as genetic tools to investigate disease resistance. Our transcriptome analysis will help understand SA signaling and its mechanism of defense systems in S. miltiorrhiza. PMID:26808150

  13. Human retroviruses and aids, 1992. A compilation and analysis of nucleic acid and amino acid sequences

    SciTech Connect

    Myers, G.; Korber, B.; Berzofsky, J.A.; Pavlakis, G.N.; Smith, R.F.

    1992-10-01

    This compendium and the accompanying floppy diskettes are the result of an effort to compile and rapidly publish all relevant molecular data concerning the human immunodeficiency viruses (HIV) and related retroviruses. The scope of the compendium and database is best summarized by the five parts that it comprises: (1) HIV and SIV Nucleotide Sequences; (H) Amino Acid Sequences; (III) Analyses; (IV) Related Sequences; and (V) Database Communications. information within all the parts is updated at least twice in each year, which accounts for the modes of binding and pagination in the compendium. While this publication could take the form of a review or sequence monograph, it is not so conceived. Instead, the literature from which the database is derived has simply been summarized and some elementary computational analyses have been performed upon the data. Interpretation and commentary have been avoided insofar as possible so that the reader can form his or her own judgments concerning the complex information. In addition to the general descriptions below of the parts of the compendium, the user should read the individual introductions for each part.

  14. Cloning and sequencing of the Bet v 1-homologous allergen Fra a 1 in strawberry (Fragaria ananassa) shows the presence of an intron and little variability in amino acid sequence.

    PubMed

    Musidlowska-Persson, Anna; Alm, Rikard; Emanuelsson, Cecilia

    2007-02-01

    The Fra a 1 allergen in strawberry (Fragaria ananassa) is homologous to the major birch pollen allergen Bet v 1, which has numerous isoforms differing in terms of amino acid sequence and immunological impact. To map the extent of sequence differences in the Fra a 1 allergen, PCR cloning and sequencing was applied. Several genomic sequences of Fra a 1, with a length of either 584, 591 or 594 nucleotides, were obtained from three different strawberry varieties. All contained one intron, with the length of either 101 or 110 nucleotides. By sequencing 30 different clones, eight different DNA sequences were obtained, giving in total five potential Fra a 1 protein isoforms, with high sequence similarity (>97% sequence identity) and only seven positions of amino acid variability, which were largely confirmed by mass spectrometry of expressed proteins. We conclude that the sequence variability in the strawberry allergen Fra a 1 is small, within and between strawberry varieties, and that multiple spots, previously detected in 2DE, are presumably due to differences in post-translational modification rather than differences in amino acid sequence. The most abundant Fra a 1 isoform sequence, recombinantly expressed in Escherichia coli after removal of the intron, was recognized by IgE from strawberry allergic patients. It cross-reacted with antibodies to Bet v 1 and the homologous apple allergen Mal d 1 (61 and 78% sequence identity, respectively), and will be used in further analyses of variation in Fra a 1-expression.

  15. Completion of the amino acid sequence of the alpha 1 chain from type I calf skin collagen. Amino acid sequence of alpha 1(I)B8.

    PubMed Central

    Glanville, R W; Breitkreutz, D; Meitinger, M; Fietzek, P P

    1983-01-01

    The complete amino acid sequence of the 279-residue CNBr peptide CB8 from the alpha 1 chain of type I calf skin collagen is presented. It was determined by sequencing overlapping fragments of CB8 produced by Staphylococcus aureus V8 proteinase, trypsin, Endoproteinase Arg-C and hydroxylamine. Tryptic cleavages were also made specific for lysine by blocking arginine residues with cyclohexane-1,2-dione. This completes the amino acid sequence analysis of the 1054-residues-long alpha (I) chain of calf skin collagen. PMID:6354180

  16. An Integrated Sequence-Structure Database incorporating matching mRNA sequence, amino acid sequence and protein three-dimensional structure data.

    PubMed Central

    Adzhubei, I A; Adzhubei, A A; Neidle, S

    1998-01-01

    We have constructed a non-homologous database, termed the Integrated Sequence-Structure Database (ISSD) which comprises the coding sequences of genes, amino acid sequences of the corresponding proteins, their secondary structure and straight phi,psi angles assignments, and polypeptide backbone coordinates. Each protein entry in the database holds the alignment of nucleotide sequence, amino acid sequence and the PDB three-dimensional structure data. The nucleotide and amino acid sequences for each entry are selected on the basis of exact matches of the source organism and cell environment. The current version 1.0 of ISSD is available on the WWW at http://www.protein.bio.msu.su/issd/ and includes 107 non-homologous mammalian proteins, of which 80 are human proteins. The database has been used by us for the analysis of synonymous codon usage patterns in mRNA sequences showing their correlation with the three-dimensional structure features in the encoded proteins. Possible ISSD applications include optimisation of protein expression, improvement of the protein structure prediction accuracy, and analysis of evolutionary aspects of the nucleotide sequence-protein structure relationship. PMID:9399866

  17. Complete amino acid sequence and structure characterization of the taste-modifying protein, miraculin.

    PubMed

    Theerasilp, S; Hitotsuya, H; Nakajo, S; Nakaya, K; Nakamura, Y; Kurihara, Y

    1989-04-25

    The taste-modifying protein, miraculin, has the unusual property of modifying sour taste into sweet taste. The complete amino acid sequence of miraculin purified from miracle fruits by a newly developed method (Theerasilp, S., and Kurihara, Y. (1988) J. Biol. Chem. 263, 11536-11539) was determined by an automatic Edman degradation method. Miraculin was a single polypeptide with 191 amino acid residues. The calculated molecular weight based on the amino acid sequence and the carbohydrate content (13.9%) was 24,600. Asn-42 and Asn-186 were linked N-glycosidically to carbohydrate chains. High homology was found between the amino acid sequences of miraculin and soybean trypsin inhibitor.

  18. Detection and isolation of nucleic acid sequences using a bifunctional hybridization probe

    DOEpatents

    Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

    2000-01-01

    A method for detecting and isolating a target sequence in a sample of nucleic acids is provided using a bifunctional hybridization probe capable of hybridizing to the target sequence that includes a detectable marker and a first complexing agent capable of forming a binding pair with a second complexing agent. A kit is also provided for detecting a target sequence in a sample of nucleic acids using a bifunctional hybridization probe according to this method.

  19. Proteogenomics: Integrating Next-Generation Sequencing and Mass Spectrometry to Characterize Human Proteomic Variation

    NASA Astrophysics Data System (ADS)

    Sheynkman, Gloria M.; Shortreed, Michael R.; Cesnik, Anthony J.; Smith, Lloyd M.

    2016-06-01

    Mass spectrometry-based proteomics has emerged as the leading method for detection, quantification, and characterization of proteins. Nearly all proteomic workflows rely on proteomic databases to identify peptides and proteins, but these databases typically contain a generic set of proteins that lack variations unique to a given sample, precluding their detection. Fortunately, proteogenomics enables the detection of such proteomic variations and can be defined, broadly, as the use of nucleotide sequences to generate candidate protein sequences for mass spectrometry database searching. Proteogenomics is experiencing heightened significance due to two developments: (a) advances in DNA sequencing technologies that have made complete sequencing of human genomes and transcriptomes routine, and (b) the unveiling of the tremendous complexity of the human proteome as expressed at the levels of genes, cells, tissues, individuals, and populations. We review here the field of human proteogenomics, with an emphasis on its history, current implementations, the types of proteomic variations it reveals, and several important applications.

  20. Proteogenomics: Integrating Next-Generation Sequencing and Mass Spectrometry to Characterize Human Proteomic Variation.

    PubMed

    Sheynkman, Gloria M; Shortreed, Michael R; Cesnik, Anthony J; Smith, Lloyd M

    2016-06-12

    Mass spectrometry-based proteomics has emerged as the leading method for detection, quantification, and characterization of proteins. Nearly all proteomic workflows rely on proteomic databases to identify peptides and proteins, but these databases typically contain a generic set of proteins that lack variations unique to a given sample, precluding their detection. Fortunately, proteogenomics enables the detection of such proteomic variations and can be defined, broadly, as the use of nucleotide sequences to generate candidate protein sequences for mass spectrometry database searching. Proteogenomics is experiencing heightened significance due to two developments: (a) advances in DNA sequencing technologies that have made complete sequencing of human genomes and transcriptomes routine, and (b) the unveiling of the tremendous complexity of the human proteome as expressed at the levels of genes, cells, tissues, individuals, and populations. We review here the field of human proteogenomics, with an emphasis on its history, current implementations, the types of proteomic variations it reveals, and several important applications.

  1. Proteogenomics: Integrating Next-Generation Sequencing and Mass Spectrometry to Characterize Human Proteomic Variation

    PubMed Central

    Sheynkman, Gloria M.; Shortreed, Michael R.; Cesnik, Anthony J.; Smith, Lloyd M.

    2016-01-01

    Mass spectrometry–based proteomics has emerged as the leading method for detection, quantification, and characterization of proteins. Nearly all proteomic workflows rely on proteomic databases to identify peptides and proteins, but these databases typically contain a generic set of proteins that lack variations unique to a given sample, precluding their detection. Fortunately, proteogenomics enables the detection of such proteomic variations and can be defined, broadly, as the use of nucleotide sequences to generate candidate protein sequences for mass spectrometry database searching. Proteogenomics is experiencing heightened significance due to two developments: (a) advances in DNA sequencing technologies that have made complete sequencing of human genomes and transcriptomes routine, and (b) the unveiling of the tremendous complexity of the human proteome as expressed at the levels of genes, cells, tissues, individuals, and populations. We review here the field of human proteogenomics, with an emphasis on its history, current implementations, the types of proteomic variations it reveals, and several important applications. PMID:27049631

  2. Comment on "Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry".

    PubMed

    Buckley, Mike; Walker, Angela; Ho, Simon Y W; Yang, Yue; Smith, Colin; Ashton, Peter; Oates, Jane Thomas; Cappellini, Enrico; Koon, Hannah; Penkman, Kirsty; Elsworth, Ben; Ashford, Dave; Solazzo, Caroline; Andrews, Phillip; Strahler, John; Shapiro, Beth; Ostrom, Peggy; Gandhi, Hasand; Miller, Webb; Raney, Brian; Zylber, Maria Ines; Gilbert, M Thomas P; Prigodich, Richard V; Ryan, Michael; Rijsdijk, Kenneth F; Janoo, Anwar; Collins, Matthew J

    2008-01-04

    We used authentication tests developed for ancient DNA to evaluate claims by Asara et al. (Reports, 13 April 2007, p. 280) of collagen peptide sequences recovered from mastodon and Tyrannosaurus rex fossils. Although the mastodon samples pass these tests, absence of amino acid composition data, lack of evidence for peptide deamidation, and association of alpha1(I) collagen sequences with amphibians rather than birds suggest that T. rex does not.

  3. Molecular cloning and sequencing of a cDNA encoding the thioesterase domain of the rat fatty acid synthetase.

    PubMed

    Naggert, J; Witkowski, A; Mikkelsen, J; Smith, S

    1988-01-25

    A cloned cDNA containing the entire coding sequence for the long-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase I) component as well as the 3'-noncoding region of the fatty acid synthetase has been isolated using an expression vector and domain-specific antibodies. The coding region was assigned to the thioesterase I domain by identification of sequences coding for characterized peptide fragments, amino-terminal analysis of the isolated thioesterase I domain and the presence of the serine esterase active-site sequence motif. The thioesterase I domain is 306 amino acids long with a calculated molecular mass of 33,476 daltons; its DNA is flanked at the 5'-end by a region coding for the acyl carrier protein domain and at the 3'-end by a 1,537-base pairs-long noncoding sequence with a poly(A) tail. The thioesterase I domain exhibits a low, albeit discernible, homology with the discrete medium-chain S-acyl fatty acid synthetase thioester hydrolases (thioesterase II) from rat mammary gland and duck uropygial gland, suggesting a distant but common evolutionary ancestry for these proteins.

  4. Masses of Pre-Main Sequence Binary Stars-Part 2

    NASA Astrophysics Data System (ADS)

    Simon, Michal

    1991-07-01

    There are still no pre-main sequence stars with reliably known masses. This represents a serious gap in our understanding of low-mass star formation. The goal of this long-term program is to measure the masses of pre-main sequence binaries selected from our survey (ref. 3) of the Taurus star forming region by IR lunar occultation and imaging. We propose to use the Fine Guide Sensors in the Transfer Function Mode to determine the apparent orbits of the binaries. Since the distance to the region is known, the apparent orbits will yield the total masses of the binaries. THIS PROPOSAL CONTAINS ONE FOLLOW-UP VISIT TO HV-TAU-C ONLY. THE REST OF THE EXPOSURES ARE IN 3842.

  5. Molecular outflows and mass loss in the pre-main-sequence stars

    NASA Astrophysics Data System (ADS)

    Levreault, Russell M.

    1988-07-01

    Molecular outflows are used here to probe mass loss in premain sequence (PMS) stars. Mass-loss rates are determined for 26 objects ranging in luminosity from four to about 100,000 solar, in mass from 0.5 to 30 solar, and in age from about 10,000 to about a million years. The derived mass-loss rates range from 9 x 10 to the -9th to 9 x 10 to the -4th solar mass/yr, with a typical value of 3 x 10 to the -7th solar mass/yr. PMS objects showing mass loss fall in a clearly demarcated region of the H-R diagram. The mass loss is proportional to bolometric luminosity to the 0.6 power and to stellar mass to the 1.8 power. The implications of these findings for the nature of the PMS mass-loss mechanism, for self-regulated low-mass star formation, and for planetary formation are discussed.

  6. Amino acid sequence of rabbit kidney neutral endopeptidase 24.11 (enkephalinase) deduced from a complementary DNA.

    PubMed Central

    Devault, A; Lazure, C; Nault, C; Le Moual, H; Seidah, N G; Chrétien, M; Kahn, P; Powell, J; Mallet, J; Beaumont, A

    1987-01-01

    Neutral endopeptidase (EC 3.4.24.11) is a major constituent of kidney brush border membranes. It is also present in the brain where it has been shown to be involved in the inactivation of opioid peptides, methionine- and leucine-enkephalins. For this reason this enzyme is often called 'enkephalinase'. In order to characterize the primary structure of the enzyme, oligonucleotide probes were designed from partial amino acid sequences and used to isolate clones from kidney cDNA libraries. Sequencing of the cDNA inserts revealed the complete primary structure of the enzyme. Neutral endopeptidase consists of 750 amino acids. It contains a short N-terminal cytoplasmic domain (27 amino acids), a single membrane-spanning segment (23 amino acids) and an extracellular domain that comprises most of the protein mass. The comparison of the primary structure of neutral endopeptidase with that of thermolysin, a bacterial Zn-metallopeptidase, indicates that most of the amino acid residues involved in Zn coordination and catalytic activity in thermolysin are found within highly honmologous sequences in neutral endopeptidase. Images Fig. 1. Fig. 3. PMID:2440677

  7. Progress in mass spectrometry of nucleic acid constituents: analysis of xenobiotic modifications and measurements at high mass

    NASA Astrophysics Data System (ADS)

    McCloskey, James A.; Crain, Pamela F.

    1992-09-01

    Significant recent progress in the applications of mass spectrometry in nucleic acid chemistry has been realized in two diverse areas. These are the characterization and quantification of xenobiotically modified nucleic acid constituents, and the mass spectrometry of large oligonucleotides, primarily by electrospray and laser desorption methods. Selected examples of the literature in these two fields are reviewed for the period 1988 through mid-1991.

  8. Fragmentation Characteristics of Deprotonated N-linked Glycopeptides: Influences of Amino Acid Composition and Sequence

    NASA Astrophysics Data System (ADS)

    Nishikaze, Takashi; Kawabata, Shin-ichirou; Tanaka, Koichi

    2014-06-01

    Glycopeptide structural analysis using tandem mass spectrometry is becoming a common approach for elucidating site-specific N-glycosylation. The analysis is generally performed in positive-ion mode. Therefore, fragmentation of protonated glycopeptides has been extensively investigated; however, few studies are available on deprotonated glycopeptides, despite the usefulness of negative-ion mode analysis in detecting glycopeptide signals. Here, large sets of glycopeptides derived from well-characterized glycoproteins were investigated to understand the fragmentation behavior of deprotonated N-linked glycopeptides under low-energy collision-induced dissociation (CID) conditions. The fragment ion species were found to be significantly variable depending on their amino acid sequence and could be classified into three types: (i) glycan fragment ions, (ii) glycan-lost fragment ions and their secondary cleavage products, and (iii) fragment ions with intact glycan moiety. The CID spectra of glycopeptides having a short peptide sequence were dominated by type (i) glycan fragments (e.g., 2,4AR, 2,4AR-1, D, and E ions). These fragments define detailed structural features of the glycan moiety such as branching. For glycopeptides with medium or long peptide sequences, the major fragments were type (ii) ions (e.g., [peptide + 0,2X0-H]- and [peptide-NH3-H]-). The appearance of type (iii) ions strongly depended on the peptide sequence, and especially on the presence of Asp, Asn, and Glu. When a glycosylated Asn is located on the C-terminus, an interesting fragment having an Asn residue with intact glycan moiety, [glycan + Asn-36]-, was abundantly formed. Observed fragments are reasonably explained by a combination of existing fragmentation rules suggested for N-glycans and peptides.

  9. The Binary Mass Transfer Origin of the Red Blue Straggler Sequence in M30

    NASA Astrophysics Data System (ADS)

    Xin, Y.; Ferraro, F. R.; Lu, P.; Deng, L.; Lanzoni, B.; Dalessandro, E.; Beccari, G.

    2015-03-01

    Two separated sequences of blue straggler stars (BSSs) have been revealed by Ferraro et al. in the color-magnitude diagram (CMD) of the Milky Way globular cluster M30. Their presence has been suggested to be related to the two BSS formation channels (namely, collisions and mass transfer in close binaries) operating within the same stellar system. The blue sequence was indeed found to be well reproduced by collisional BSS models. In contrast, no specific models for mass-transfer BSSs were available for an old stellar system like M30. Here we present binary evolution models, including case-B mass transfer and binary merging, specifically calculated for this cluster. We discuss in detail the evolutionary track of a 0.9 + 0.5 M ⊙ binary, which spends approximately 4 Gyr in the BSS region of the CMD of a 13 Gyr old cluster. We also run Monte Carlo simulations to study the distribution of mass-transfer BSSs in the CMD and to compare it with the observational data. Our results show that (1) the color and magnitude distribution of synthetic mass-transfer BSSs defines a strip in the CMD that nicely matches the observed red-BSS sequence, thus providing strong support to the mass-transfer origin for these stars; (2) the CMD distribution of synthetic BSSs never attains the observed location of the blue-BSS sequence, thus reinforcing the hypothesis that the latter formed through a different channel (likely collisions); (3) most (~60%) of the synthetic BSSs are produced by mass-transfer models, while the remaining <40% requires the contribution from merger models.

  10. Identification of random nucleic acid sequence aberrations using dual capture probes which hybridize to different chromosome regions

    DOEpatents

    Lucas, J.N.; Straume, T.; Bogen, K.T.

    1998-03-24

    A method is provided for detecting nucleic acid sequence aberrations using two immobilization steps. According to the method, a nucleic acid sequence aberration is detected by detecting nucleic acid sequences having both a first nucleic acid sequence type (e.g., from a first chromosome) and a second nucleic acid sequence type (e.g., from a second chromosome), the presence of the first and the second nucleic acid sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. In the method, immobilization of a first hybridization probe is used to isolate a first set of nucleic acids in the sample which contain the first nucleic acid sequence type. Immobilization of a second hybridization probe is then used to isolate a second set of nucleic acids from within the first set of nucleic acids which contain the second nucleic acid sequence type. The second set of nucleic acids are then detected, their presence indicating the presence of a nucleic acid sequence aberration. 14 figs.

  11. Identification of random nucleic acid sequence aberrations using dual capture probes which hybridize to different chromosome regions

    DOEpatents

    Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

    1998-01-01

    A method is provided for detecting nucleic acid sequence aberrations using two immobilization steps. According to the method, a nucleic acid sequence aberration is detected by detecting nucleic acid sequences having both a first nucleic acid sequence type (e.g., from a first chromosome) and a second nucleic acid sequence type (e.g., from a second chromosome), the presence of the first and the second nucleic acid sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. In the method, immobilization of a first hybridization probe is used to isolate a first set of nucleic acids in the sample which contain the first nucleic acid sequence type. Immobilization of a second hybridization probe is then used to isolate a second set of nucleic acids from within the first set of nucleic acids which contain the second nucleic acid sequence type. The second set of nucleic acids are then detected, their presence indicating the presence of a nucleic acid sequence aberration.

  12. Effects of main-sequence mass loss on the turnoff ages of globular clusters

    SciTech Connect

    Guzik, J.A.

    1989-01-01

    Willson, Bowen, and Struck-Marcell have proposed that globular cluster main-sequence turnoff ages can be reconciled with the lower ages of the Galaxy and universe deduced from other methods by incorporating an epoch of early main-sequence mass-loss by stars of spectral types A through early-F. The proposed mass loss is pulsation-driven, and facilitated by rapid rotation. This paper presents stellar evolution calculations of Pop. II (Z = 0.001) mass-losing stars of initial mass 0.8 to 1.6 M/sub /circle dot//, with exponentially-decreasing mass loss rates of e-folding times 0.5 to 2.0 Gyr, evolving to a final mass of 0.7 M/sub /circle dot//. The calculations indicate that a globular cluster with apparent turnoff age 18 Gyr could have an actual age as low as /approximately/12 Gyr. Observational implications that may help to verify the hypothesis, e.g. low C/N abundance ratios among red giants following first dredge-up, blue stragglers, red giant deficiencies, and signatures in cluster mass/luminosity functions, are also discussed.25 refs., 4 figs., 3 tabs.

  13. The amino acid sequence of protein CM-3 from Dendroaspis polylepis polylepis (black mamba) venom.

    PubMed

    Joubert, F J

    1985-01-01

    Protein CM-3 from Dendroaspis polylepis polylepis venom was purified by gel filtration and ion exchange chromatography. It comprises 65 amino acids including eight half-cystines. The complete amino acid sequence of protein CM-3 has been elucidated. The sequence (residues 1-50) resembles that of the N-terminal sequence of the subunits of a synergistic type protein and residues 51-65 that of the C-terminal sequence of an angusticeps type protein. Mixtures of protein CM-3 and angusticeps type proteins showed no apparent synergistic effect, in that their toxicity in combination was no greater than the sum of their individual toxicities.

  14. The amino acid sequences of the Fd fragments of two human γ heavy chains

    PubMed Central

    Press, E. M.; Hogg, N. M.

    1970-01-01

    The amino acid sequences of the Fd fragments of two human pathological immunoglobulins of the immunoglobulin G1 class are reported. Comparison of the two sequences shows that the heavy-chain variable regions are similar in length to those of the light chains. The existence of heavy chain variable region subgroups is also deduced, from a comparison of these two sequences with those of another γ 1 chain, Eu, a μ chain, Ou, and the partial sequence of a fourth γ 1 chain, Ste. Carbohydrate has been found to be linked to an aspartic acid residue in the variable region of one of the γ 1 chains, Cor. PMID:5449120

  15. Preserving the Sequence of a Biopolymer's Monomers as They Enter an Electrospray Mass Spectrometer

    NASA Astrophysics Data System (ADS)

    Maulbetsch, William; Wiener, Benjamin; Poole, William; Bush, Joseph; Stein, Derek

    2016-11-01

    This paper investigates how faithfully an electrospray mass spectrometer reports the order of monomers of a single biopolymer in the context of two sequencing strategies. We develop a simplified one-dimensional theoretical model of the dynamics of Brownian particles in the Taylor cone of an electropray source, where free monomers drift towards the apex in an elongational force gradient. The likelihood that neighboring particles will invert their order decreases near the apex because the strength of the force gradient increases. Neighboring monomers on a stretched biopolymer should be cleaved by photofragmentation within about 3 nm of the apex if they are to enter the mass spectrometer in sequence with 95% probability under typical experimental conditions. Alternatively, if the monomers are cleaved processively at milliseconds-long intervals by an enzyme, their sequence will be faithfully reported with 95% confidence if the enzyme is within about 117 nm of the apex.

  16. Identification of staphylococcal species based on variations in protein sequences (mass spectrometry) and DNA sequence (sodA microarray).

    PubMed

    Kooken, Jennifer; Fox, Karen; Fox, Alvin; Altomare, Diego; Creek, Kim; Wunschel, David; Pajares-Merino, Sara; Martínez-Ballesteros, Ilargi; Garaizar, Javier; Oyarzabal, Omar; Samadpour, Mansour

    2014-02-01

    This report is among the first using sequence variation in newly discovered protein markers for staphylococcal (or indeed any other bacterial) speciation. Variation, at the DNA sequence level, in the sodA gene (commonly used for staphylococcal speciation) provided excellent correlation. Relatedness among strains was also assessed using protein profiling using microcapillary electrophoresis and pulsed field electrophoresis. A total of 64 strains were analyzed including reference strains representing the 11 staphylococcal species most commonly isolated from man (Staphylococcus aureus and 10 coagulase negative species [CoNS]). Matrix assisted time of flight ionization/ionization mass spectrometry (MALDI TOF MS) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC ESI MS/MS) were used for peptide analysis of proteins isolated from gel bands. Comparison of experimental spectra of unknowns versus spectra of peptides derived from reference strains allowed bacterial identification after MALDI TOF MS analysis. After LC-MS/MS analysis of gel bands bacterial speciation was performed by comparing experimental spectra versus virtual spectra using the software X!Tandem. Finally LC-MS/MS was performed on whole proteomes and data analysis also employing X!tandem. Aconitate hydratase and oxoglutarate dehydrogenase served as marker proteins on focused analysis after gel separation. Alternatively on full proteomics analysis elongation factor Tu generally provided the highest confidence in staphylococcal speciation.

  17. DNA sequencing with capillary electrophoresis and single cell analysis with mass spectrometry

    SciTech Connect

    Fung, N.

    1998-03-27

    Since the first demonstration of the laser in the 1960`s, lasers have found numerous applications in analytical chemistry. In this work, two different applications are described, namely, DNA sequencing with capillary gel electrophoresis and single cell analysis with mass spectrometry. Two projects are described in which high-speed DNA separations with capillary gel electrophoresis were demonstrated. In the third project, flow cytometry and mass spectrometry were coupled via a laser vaporization/ionization interface and individual mammalian cells were analyzed. First, DNA Sanger fragments were separated by capillary gel electrophoresis. A separation speed of 20 basepairs per minute was demonstrated with a mixed poly(ethylene oxide) (PEO) sieving solution. In addition, a new capillary wall treatment protocol was developed in which bare (or uncoated) capillaries can be used in DNA sequencing. Second, a temperature programming scheme was used to separate DNA Sanger fragments. Third, flow cytometry and mass spectrometry were coupled with a laser vaporization/ionization interface.

  18. The Chinese hamster Alu-equivalent sequence: a conserved highly repetitious, interspersed deoxyribonucleic acid sequence in mammals has a structure suggestive of a transposable element.

    PubMed Central

    Haynes, S R; Toomey, T P; Leinwand, L; Jelinek, W R

    1981-01-01

    A consensus sequence has been determined for a major interspersed deoxyribonucleic acid repeat in the genome of Chinese hamster ovary cells (CHO cells). This sequence is extensively homologous to (i) the human Alu sequence (P. L. Deininger et al., J. Mol. Biol., in press), (ii) the mouse B1 interspersed repetitious sequence (Krayev et al., Nucleic Acids Res. 8:1201-1215, 1980) (iii) an interspersed repetitious sequence from African green monkey deoxyribonucleic acid (Dhruva et al., Proc. Natl. Acad. Sci. U.S.A. 77:4514-4518, 1980) and (iv) the CHO and mouse 4.5S ribonucleic acid (this report; F. Harada and N. Kato, Nucleic Acids Res. 8:1273-1285, 1980). Because the CHO consensus sequence shows significant homology to the human Alu sequence it is termed the CHO Alu-equivalent sequence. A conserved structure surrounding CHO Alu-equivalent family members can be recognized. It is similar to that surrounding the human Alu and the mouse B1 sequences, and is represented as follows: direct repeat-CHO-Alu-A-rich sequence-direct repeat. A composite interspersed repetitious sequence has been identified. Its structure is represented as follows: direct repeat-residue 47 to 107 of CHO-Alu-non-Alu repetitious sequence-A-rich sequence-direct repeat. Because the Alu flanking sequences resemble those that flank known transposable elements, we think it likely that the Alu sequence dispersed throughout the mammalian genome by transposition. Images PMID:9279371

  19. Mass spectral studies of the carboxylic acid ionophore antibiotic griseochelin and its derivatives.

    PubMed

    Schade, W; Gräfe, U; Schmidt, J

    1988-04-01

    The electron impact (EI) mass spectra (75 eV) of the new carboxylic acid ionophore griseochelin and some of its derivatives are discussed. The mass spectral fragmentation was studied using exact mass measurements and deuterium labelling. Furthermore, the negative ion mass spectra (2-4 eV) of these compounds are compared with their EI mass spectra.

  20. The amino acid sequence of goat beta-lactoglobulin.

    PubMed

    Préaux, G; Braunitzer, G; Schrank, B; Stangl, A

    1979-11-01

    The isolation of beta-lactoglobulin from milk of the goat is described. The purified protein was checked for purity and has been characterized by its gross composition and end groups. The native or the modified protein was then degraded by tryptic and cyanogen bromide cleavage. The cleavage products were isolated and sequenced in the sequenator using a Quadrol and propyne program. These data provide the complete sequence of beta-lactoglobulin of the goat. The results are discussed and compared particularly with bovine beta-lactoglobulin components AB. Some biological aspects are described.

  1. Layered materials with coexisting acidic and basic sites for catalytic one-pot reaction sequences.

    PubMed

    Motokura, Ken; Tada, Mizuki; Iwasawa, Yasuhiro

    2009-06-17

    Acidic montmorillonite-immobilized primary amines (H-mont-NH(2)) were found to be excellent acid-base bifunctional catalysts for one-pot reaction sequences, which are the first materials with coexisting acid and base sites active for acid-base tamdem reactions. For example, tandem deacetalization-Knoevenagel condensation proceeded successfully with the H-mont-NH(2), affording the corresponding condensation product in a quantitative yield. The acidity of the H-mont-NH(2) was strongly influenced by the preparation solvent, and the base-catalyzed reactions were enhanced by interlayer acid sites.

  2. The amino acid sequence of Ole e I, the major allergen from olive tree (Olea europaea) pollen.

    PubMed

    Villalba, M; Batanero, E; López-Otín, C; Sánchez, L M; Monsalve, R I; González de la Peña, M A; Lahoz, C; Rodríguez, R

    1993-09-15

    The complete primary structure of the major allergen from Olea europaea (olive tree) pollen, Ole e I (IUIS nomenclature), has been determined. The amino acid sequence was established by automated Edman degradation of the reduced and alkylated molecule as well as of selected fragments obtained by proteolytic digestions. Ole e I contains a single polypeptide chain of 145 amino acid residues with a calculated molecular mass of 16331 Da. No free sulfhydryl groups have been detected in the native protein. The molecule contains a putative glycosylation site. A high degree of microheterogeneity has been observed, mainly centered in the first 33% of the molecule. Comparison of Ole e I sequence with protein sequence databases showed no similarity with other known allergens. However, it has a 36% and 38% sequence identity with the putative polypeptide structures, deduced, respectively, from nucleotide sequences of genes isolated from tomato anthers and corn pollen, which have been suggested to be involved in the growing of the pollen tube. Therefore, the olive tree allergen may be a constitutive protein of the pollen involved in reproductive functions.

  3. Synthesis of gamma,delta-unsaturated glycolic acids via sequenced brook and Ireland--claisen rearrangements.

    PubMed

    Schmitt, Daniel C; Johnson, Jeffrey S

    2010-03-05

    Organozinc, -magnesium, and -lithium nucleophiles initiate a Brook/Ireland-Claisen rearrangement sequence of allylic silyl glyoxylates resulting in the formation of gamma,delta-unsaturated alpha-silyloxy acids.

  4. Computer Simulation of the Determination of Amino Acid Sequences in Polypeptides

    ERIC Educational Resources Information Center

    Daubert, Stephen D.; Sontum, Stephen F.

    1977-01-01

    Describes a computer program that generates a random string of amino acids and guides the student in determining the correct sequence of a given protein by using experimental analytic data for that protein. (MLH)

  5. BEYOND THE MAIN SEQUENCE: TESTING THE ACCURACY OF STELLAR MASSES PREDICTED BY THE PARSEC EVOLUTIONARY TRACKS

    SciTech Connect

    Ghezzi, Luan; Johnson, John Asher

    2015-10-20

    Characterizing the physical properties of exoplanets and understanding their formation and orbital evolution requires precise and accurate knowledge of their host stars. Accurately measuring stellar masses is particularly important because they likely influence planet occurrence and the architectures of planetary systems. Single main-sequence stars typically have masses estimated from evolutionary tracks, which generally provide accurate results due to their extensive empirical calibration. However, the validity of this method for subgiants and giants has been called into question by recent studies, with suggestions that the masses of these evolved stars could have been overestimated. We investigate these concerns using a sample of 59 benchmark evolved stars with model-independent masses (from binary systems or asteroseismology) obtained from the literature. We find very good agreement between these benchmark masses and the ones estimated using evolutionary tracks. The average fractional difference in the mass interval ∼0.7–4.5 M{sub ⊙} is consistent with zero (−1.30 ± 2.42%), with no significant trends in the residuals relative to the input parameters. A good agreement between model-dependent and -independent radii (−4.81 ± 1.32%) and surface gravities (0.71 ± 0.51%) is also found. The consistency between independently determined ages for members of binary systems adds further support for the accuracy of the method employed to derive the stellar masses. Taken together, our results indicate that determination of masses of evolved stars using grids of evolutionary tracks is not significantly affected by systematic errors, and is thus valid for estimating the masses of isolated stars beyond the main sequence.

  6. The Solar Neighborhood. XXXVII: The Mass-Luminosity Relation for Main-sequence M Dwarfs

    NASA Astrophysics Data System (ADS)

    Benedict, G. F.; Henry, T. J.; Franz, O. G.; McArthur, B. E.; Wasserman, L. H.; Jao, Wei-Chun; Cargile, P. A.; Dieterich, S. B.; Bradley, A. J.; Nelan, E. P.; Whipple, A. L.

    2016-11-01

    We present a mass-luminosity relation (MLR) for red dwarfs spanning a range of masses from 0.62 {{ M }}⊙ to the end of the stellar main sequence at 0.08 {{ M }}⊙ . The relation is based on 47 stars for which dynamical masses have been determined, primarily using astrometric data from Fine Guidance Sensors (FGS) 3 and 1r, white-light interferometers on the Hubble Space Telescope (HST), and radial velocity data from McDonald Observatory. For our HST/FGS sample of 15 binaries, component mass errors range from 0.4% to 4.0% with a median error of 1.8%. With these and masses from other sources, we construct a V-band MLR for the lower main sequence with 47 stars and a K-band MLR with 45 stars with fit residuals half of those of the V band. We use GJ 831 AB as an example, obtaining an absolute trigonometric parallax, π abs = 125.3 ± 0.3 mas, with orbital elements yielding {{ M }}{{A}}=0.270+/- 0.004 {{ M }}⊙ and {{ M }}{{B}}=0.145+/- 0.002 {{ M }}⊙ . The mass precision rivals that derived for eclipsing binaries. A remaining major task is the interpretation of the intrinsic cosmic scatter in the observed MLR for low-mass stars in terms of physical effects. In the meantime, useful mass values can be estimated from the MLR for the ubiquitous red dwarfs that account for 75% of all stars, with applications ranging from the characterization of exoplanet host stars to the contribution of red dwarfs to the mass of the universe. Based on observations made with the NASA/ESA Hubble Space Telescope, obtained at the Space Telescope Science Institute, which is operated by the Association of Universities for Research in Astronomy, Inc., under NASA contract NAS5-26555.

  7. Genome sequence of the acid-tolerant strain Rhizobium sp. LPU83.

    PubMed

    Wibberg, Daniel; Tejerizo, Gonzalo Torres; Del Papa, María Florencia; Martini, Carla; Pühler, Alfred; Lagares, Antonio; Schlüter, Andreas; Pistorio, Mariano

    2014-04-20

    Rhizobia are important members of the soil microbiome since they enter into nitrogen-fixing symbiosis with different legume host plants. Rhizobium sp. LPU83 is an acid-tolerant Rhizobium strain featuring a broad-host-range. However, it is ineffective in nitrogen fixation. Here, the improved draft genome sequence of this strain is reported. Genome sequence information provides the basis for analysis of its acid tolerance, symbiotic properties and taxonomic classification.

  8. The amino acid sequence of monal pheasant lysozyme and its activity.

    PubMed

    Araki, T; Matsumoto, T; Torikata, T

    1998-10-01

    The amino acid sequence of monal pheasant lysozyme and its activity were analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had one amino acid substitution at position 102 (Arg to Gly) comparing with Indian peafowl lysozyme and four amino acid substitutions at positions 3 (Phe to Tyr), 15 (His to Leu), 41 (Gln to His), and 121 (Gln to His) with chicken lysozyme. Analysis of the time-courses of reaction using N-acetylglucosamine pentamer as a substrate showed a difference of binding free energy change (-0.4 kcal/mol) at subsites A between monal pheasant and Indian peafowl lysozyme. This was assumed to be caused by the amino acid substitution at subsite A with loss of a positive charge at position 102 (Arg102 to Gly).

  9. The naked T Tauri stars - The low-mass pre-main sequence unveiled

    NASA Technical Reports Server (NTRS)

    Walter, Frederick M.

    1987-01-01

    The search for low-mass premain-sequence (PMS) stars associated with X-ray sources in regions of star formation is discussed. The survey to date has revealed at least 30 low-mass PMS stars in the Tau-Aur region, and a comparable number in Oph. These stars are the naked T Tau stars, unveiled versions of the well-known classical T Tau stars. The properties of these newly discovered PMS stars and their relation to the classical T Tau stars are discussed, and it is concluded that the naked T Tau stars are the true low-mass PMS stars, and that the observable characteristics defining the classical T Tau stars are due to the interaction of an underlying, fairly normal star with a dominant circumstellar environment. The impact the naked T Tau stars are likely to have on models of the PMS evolution of low-mass stars is considered.

  10. Multiple Genome Sequences of Important Beer-Spoiling Lactic Acid Bacteria

    PubMed Central

    Geissler, Andreas J.; Vogel, Rudi F.

    2016-01-01

    Seven strains of important beer-spoiling lactic acid bacteria were sequenced using single-molecule real-time sequencing. Complete genomes were obtained for strains of Lactobacillus paracollinoides, Lactobacillus lindneri, and Pediococcus claussenii. The analysis of these genomes emphasizes the role of plasmids as the genomic foundation of beer-spoiling ability. PMID:27795248

  11. Gastropod arginine kinases from Cellana grata and Aplysia kurodai. Isolation and cDNA-derived amino acid sequences.

    PubMed

    Suzuki, T; Inoue, N; Higashi, T; Mizobuchi, R; Sugimura, N; Yokouchi, K; Furukohri, T

    2000-12-01

    Arginine kinase (AK) was isolated from the radular muscle of the gastropod molluscs Cellana grata (subclass Prosobranchia) and Aplysia kurodai (subclass Opisthobranchia), respectively, by ammonium sulfate fractionation, Sephadex G-75 gel filtration and DEAE-ion exchange chromatography. The denatured relative molecular mass values were estimated to be 40 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated enzyme from Aplysia gave a Km value of 0.6 mM for arginine and a Vmax value of 13 micromole Pi min(-1) mg protein(-1) for the forward reaction. These values are comparable to other molluscan AKs. The cDNAs encoding Cellana and Aplysia AKs were amplified by polymerase chain reaction, and the nucleotide sequences of 1,608 and 1,239 bp, respectively, were determined. The open reading frame for Cellana AK is 1044 nucleotides in length and encodes a protein with 347 amino acid residues, and that for A. kurodai is 1077 nucleotides and 354 residues. The cDNA-derived amino acid sequences were validated by chemical sequencing of internal lysyl endopeptidase peptides. The amino acid sequences of Cellana and Aplysia AKs showed the highest percent identity (66-73%) with those of the abalone Nordotis and turbanshell Battilus belonging to the same class Gastropoda. These AK sequences still have a strong homology (63-71%) with that of the chiton Liolophura (class Polyplacophora), which is believed to be one of the most primitive molluscs. On the other hand, these AK sequences are less homologous (55-57%) with that of the clam Pseudocardium (class Bivalvia), suggesting that the biological position of the class Polyplacophora should be reconsidered.

  12. PASTA: Ultra-Large Multiple Sequence Alignment for Nucleotide and Amino-Acid Sequences.

    PubMed

    Mirarab, Siavash; Nguyen, Nam; Guo, Sheng; Wang, Li-San; Kim, Junhyong; Warnow, Tandy

    2015-05-01

    We introduce PASTA, a new multiple sequence alignment algorithm. PASTA uses a new technique to produce an alignment given a guide tree that enables it to be both highly scalable and very accurate. We present a study on biological and simulated data with up to 200,000 sequences, showing that PASTA produces highly accurate alignments, improving on the accuracy and scalability of the leading alignment methods (including SATé). We also show that trees estimated on PASTA alignments are highly accurate--slightly better than SATé trees, but with substantial improvements relative to other methods. Finally, PASTA is faster than SATé, highly parallelizable, and requires relatively little memory.

  13. Bromine isotopic signature facilitates de novo sequencing of peptides in free-radical-initiated peptide sequencing (FRIPS) mass spectrometry.

    PubMed

    Nam, Jungjoo; Kwon, Hyuksu; Jang, Inae; Jeon, Aeran; Moon, Jingyu; Lee, Sun Young; Kang, Dukjin; Han, Sang Yun; Moon, Bongjin; Oh, Han Bin

    2015-02-01

    We recently showed that free-radical-initiated peptide sequencing mass spectrometry (FRIPS MS) assisted by the remarkable thermochemical stability of (2,2,6,6-tetramethyl-piperidin-1-yl)oxyl (TEMPO) is another attractive radical-driven peptide fragmentation MS tool. Facile homolytic cleavage of the bond between the benzylic carbon and the oxygen of the TEMPO moiety in o-TEMPO-Bz-C(O)-peptide and the high reactivity of the benzylic radical species generated in •Bz-C(O)-peptide are key elements leading to extensive radical-driven peptide backbone fragmentation. In the present study, we demonstrate that the incorporation of bromine into the benzene ring, i.e. o-TEMPO-Bz(Br)-C(O)-peptide, allows unambiguous distinction of the N-terminal peptide fragments from the C-terminal fragments through the unique bromine doublet isotopic signature. Furthermore, bromine substitution does not alter the overall radical-driven peptide backbone dissociation pathways of o-TEMPO-Bz-C(O)-peptide. From a practical perspective, the presence of the bromine isotopic signature in the N-terminal peptide fragments in TEMPO-assisted FRIPS MS represents a useful and cost-effective opportunity for de novo peptide sequencing.

  14. SETG: Nucleic Acid Extraction and Sequencing for In Situ Life Detection on Mars

    NASA Astrophysics Data System (ADS)

    Mojarro, A.; Hachey, J.; Tani, J.; Smith, A.; Bhattaru, S. A.; Pontefract, A.; Doebler, R.; Brown, M.; Ruvkun, G.; Zuber, M. T.; Carr, C. E.

    2016-10-01

    We are developing an integrated nucleic acid extraction and sequencing instrument: the Search for Extra-Terrestrial Genomes (SETG) for in situ life detection on Mars. Our goals are to identify related or unrelated nucleic acid-based life on Mars.

  15. Draft Genome Sequence of Cyanobacterium sp. Strain IPPAS B-1200 with a Unique Fatty Acid Composition

    PubMed Central

    Starikov, Alexander Y.; Usserbaeva, Aizhan A.; Sinetova, Maria A.; Sarsekeyeva, Fariza K.; Zayadan, Bolatkhan K.; Ustinova, Vera V.; Kupriyanova, Elena V.; Los, Dmitry A.

    2016-01-01

    Here, we report the draft genome of Cyanobacterium sp. IPPAS strain B-1200, isolated from Lake Balkhash, Kazakhstan, and characterized by the unique fatty acid composition of its membrane lipids, which are enriched with myristic and myristoleic acids. The approximate genome size is 3.4 Mb, and the predicted number of coding sequences is 3,119. PMID:27856596

  16. Sequencing and computational analysis of complete genome sequences of Citrus yellow mosaic badna virus from acid lime and pummelo.

    PubMed

    Borah, Basanta K; Johnson, A M Anthony; Sai Gopal, D V R; Dasgupta, Indranil

    2009-08-01

    Citrus yellow mosaic badna virus (CMBV), a member of the Family Caulimoviridae, Genus Badnavirus, is the causative agent of Citrus mosaic disease in India. Although the virus has been detected in several citrus species, only two full-length genomes, one each from Sweet orange and Rangpur lime, are available in publicly accessible databases. In order to obtain a better understanding of the genetic variability of the virus in other citrus mosaic-affected citrus species, we performed the cloning and sequence analysis of complete genomes of CMBV from two additional citrus species, Acid lime and Pummelo. We show that CMBV genomes from the two hosts share high homology with previously reported CMBV sequences and hence conclude that the new isolates represent variants of the virus present in these species. Based on in silico sequence analysis, we predict the possible function of the protein encoded by one of the five ORFs.

  17. Parvalbumins from coelacanth muscle. III. Amino acid sequence of the major component.

    PubMed

    Jauregui-Adell, J; Pechere, J F

    1978-09-26

    The primary structure of the major parvalbumin (pI = 4.52) from coelacanth muscle (Latimeria chalumnae) has been determined. Sequence analysis of the tryptic peptides, in some cases obtained with beta-trypsin, accounts for the total amino acid content of the protein. Chymotryptic peptides provide appropriate sequence overlaps, to complete the localization of the tryptic peptides. Examination of the amino acid sequence of this protein shows the typical structure of a beta-parvalbumin. Its position in the dendrogram of related calcium-binding proteins corresponds to that usually accepted for crossopterygians.

  18. Determination of Elizabethkingia Diversity by MALDI-TOF Mass Spectrometry and Whole-Genome Sequencing

    PubMed Central

    Gumpert, Heidi; Faurholt, Cecilie Haase; Westh, Henrik

    2017-01-01

    In a hospital-acquired infection with multidrug-resistant Elizabethkingia, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and 16S rRNA gene analysis identified the pathogen as Elizabethkingia miricola. Whole-genome sequencing, genus-level core genome analysis, and in silico DNA-DNA hybridization of 35 Elizabethkingia strains indicated that the species taxonomy should be further explored. PMID:28098550

  19. Uncertainties in the Determination of the Upper Mass Limit for Zero-Age Main Sequence Stars

    NASA Astrophysics Data System (ADS)

    Klapp, J.; Langer, N.; Fricke, K. J.

    1987-05-01

    In a recent investigation Klapp et al. 1987 obtained a critical nass of 440 M@ for the overstability of very massive extreme population I stars at the main sequence. In this work we investigate the dependence of Klapp et al. 1987 results upon the program input physics. We find that stars in the 100 - 500 Me range are marginally stable (or unstable) and that this mass range should be considered as a transition region from stability to overstability of very massive stars.

  20. Amino acid sequence of anionic peroxidase from the windmill palm tree Trachycarpus fortunei.

    PubMed

    Baker, Margaret R; Zhao, Hongwei; Sakharov, Ivan Yu; Li, Qing X

    2014-12-10

    Palm peroxidases are extremely stable and have uncommon substrate specificity. This study was designed to fill in the knowledge gap about the structures of a peroxidase from the windmill palm tree Trachycarpus fortunei. The complete amino acid sequence and partial glycosylation were determined by MALDI-top-down sequencing of native windmill palm tree peroxidase (WPTP), MALDI-TOF/TOF MS/MS of WPTP tryptic peptides, and cDNA sequencing. The propeptide of WPTP contained N- and C-terminal signal sequences which contained 21 and 17 amino acid residues, respectively. Mature WPTP was 306 amino acids in length, and its carbohydrate content ranged from 21% to 29%. Comparison to closely related royal palm tree peroxidase revealed structural features that may explain differences in their substrate specificity. The results can be used to guide engineering of WPTP and its novel applications.

  1. Method and apparatus for enhanced sequencing of complex molecules using surface-induced dissociation in conjunction with mass spectrometric analysis

    DOEpatents

    Laskin, Julia [Richland, WA; Futrell, Jean H [Richland, WA

    2008-04-29

    The invention relates to a method and apparatus for enhanced sequencing of complex molecules using surface-induced dissociation (SID) in conjunction with mass spectrometric analysis. Results demonstrate formation of a wide distribution of structure-specific fragments having wide sequence coverage useful for sequencing and identifying the complex molecules.

  2. The coronal temperatures of low-mass main-sequence stars

    NASA Astrophysics Data System (ADS)

    Johnstone, C. P.; Güdel, M.

    2015-06-01

    Aims: We study the X-ray emission of low-mass main-sequence stars to derive a reliable general scaling law between coronal temperature and the level of X-ray activity. Methods: We collect ROSAT measurements of hardness ratios and X-ray luminosities for a large sample of stars to derive which stellar X-ray emission parameter is most closely correlated with coronal temperature. We calculate average coronal temperatures for a sample of 24 low-mass main-sequence stars with measured emission measure distributions (EMDs) collected from the literature. These EMDs are based on high-resolution X-ray spectra measured by XMM-Newton and Chandra. Results: We confirm that there is one universal scaling relation between coronal average temperature and surface X-ray flux, FX, that applies to all low-mass main-sequence stars. We find that coronal temperature is related to FX by T̅cor = 0.11 FX0.26, where T̅cor is in MK and FX is in erg s-1 cm-2.

  3. TranslatorX: multiple alignment of nucleotide sequences guided by amino acid translations.

    PubMed

    Abascal, Federico; Zardoya, Rafael; Telford, Maximilian J

    2010-07-01

    We present TranslatorX, a web server designed to align protein-coding nucleotide sequences based on their corresponding amino acid translations. Many comparisons between biological sequences (nucleic acids and proteins) involve the construction of multiple alignments. Alignments represent a statement regarding the homology between individual nucleotides or amino acids within homologous genes. As protein-coding DNA sequences evolve as triplets of nucleotides (codons) and it is known that sequence similarity degrades more rapidly at the DNA than at the amino acid level, alignments are generally more accurate when based on amino acids than on their corresponding nucleotides. TranslatorX novelties include: (i) use of all documented genetic codes and the possibility of assigning different genetic codes for each sequence; (ii) a battery of different multiple alignment programs; (iii) translation of ambiguous codons when possible; (iv) an innovative criterion to clean nucleotide alignments with GBlocks based on protein information; and (v) a rich output, including Jalview-powered graphical visualization of the alignments, codon-based alignments coloured according to the corresponding amino acids, measures of compositional bias and first, second and third codon position specific alignments. The TranslatorX server is freely available at http://translatorx.co.uk.

  4. Complete amino acid sequence of an acidic, cardiotoxic phospholipase A2 from the venom of Ophiophagus hannah (King Cobra): a novel cobra venom enzyme with "pancreatic loop".

    PubMed

    Huang, M Z; Gopalakrishnakone, P; Chung, M C; Kini, R M

    1997-02-15

    A phospholipase A2 (OHV A-PLA2) from the venom of Ophiophagus hannah (King cobra) is an acidic protein exhibiting cardiotoxicity, myotoxicity, and antiplatelet activity. The complete amino acid sequence of OHV A-PLA2 has been determined using a combination of Edman degradation and mass spectrometric techniques. OHV A-PLA2 is composed of a single chain of 124 amino acid residues with 14 cysteines and a calculated molecular weight of 13719 Da. It contains the loop of residues (62-66) found in pancreatic PLA2s and hence belongs to class IB enzymes. This pancreatic loop is between two proline residues (Pro 59 and Pro 68) and contains several hydrophilic amino acids (Ser and Asp). This region has high degree of conformational flexibility and is on the surface of the molecule, and hence it may be a potential protein-protein interaction site. A relatively low sequence homology is found between OHV A-PLA2 and other known cardiotoxic PLA2s, and hence a contiguous segment could not be identified as a site responsible for the cardiotoxic activity.

  5. Amino acid sequence of homologous rat atrial peptides: natriuretic activity of native and synthetic forms.

    PubMed Central

    Seidah, N G; Lazure, C; Chrétien, M; Thibault, G; Garcia, R; Cantin, M; Genest, J; Nutt, R F; Brady, S F; Lyle, T A

    1984-01-01

    A substance called atrial natriuretic factor (ANF), localized in secretory granules of atrial cardiocytes, was isolated as four homologous natriuretic peptides from homogenates of rat atria. The complete sequence of the longest form showed that it is composed of 33 amino acids. The three other shorter forms (2-33, 3-33, and 8-33) represent amino-terminally truncated versions of the 33 amino acid parent molecule as shown by analysis of sequence, amino acid composition, or both. The proposed primary structure agrees entirely with the amino acid composition and reveals no significant sequence homology with any known protein or segment of protein. The short form ANF-(8-33) was synthesized by a multi-fragment condensation approach and the synthetic product was shown to exhibit specific activity comparable to that of the natural ANF-(3-33). PMID:6232612

  6. Nucleotide and deduced amino acid sequences of a new subtilisin from an alkaliphilic Bacillus isolate.

    PubMed

    Saeki, Katsuhisa; Magallones, Marietta V; Takimura, Yasushi; Hatada, Yuji; Kobayashi, Tohru; Kawai, Shuji; Ito, Susumu

    2003-10-01

    The gene for a new subtilisin from the alkaliphilic Bacillus sp. KSM-LD1 was cloned and sequenced. The open reading frame of the gene encoded a 97 amino-acid prepro-peptide plus a 307 amino-acid mature enzyme that contained a possible catalytic triad of residues, Asp32, His66, and Ser224. The deduced amino acid sequence of the mature enzyme (LD1) showed approximately 65% identity to those of subtilisins SprC and SprD from alkaliphilic Bacillus sp. LG12. The amino acid sequence identities of LD1 to those of previously reported true subtilisins and high-alkaline proteases were below 60%. LD1 was characteristically stable during incubation with surfactants and chemical oxidants. Interestingly, an oxidizable Met residue is located next to the catalytic Ser224 of the enzyme as in the cases of the oxidation-susceptible subtilisins reported to date.

  7. Shark myelin basic protein: amino acid sequence, secondary structure, and self-association.

    PubMed

    Milne, T J; Atkins, A R; Warren, J A; Auton, W P; Smith, R

    1990-09-01

    Myelin basic protein (MBP) from the Whaler shark (Carcharhinus obscurus) has been purified from acid extracts of a chloroform/methanol pellet from whole brains. The amino acid sequence of the majority of the protein has been determined and compared with the sequences of other MBPs. The shark protein has only 44% homology with the bovine protein, but, in common with other MBPs, it has basic residues distributed throughout the sequence and no extensive segments that are predicted to have an ordered secondary structure in solution. Shark MBP lacks the triproline sequence previously postulated to form a hairpin bend in the molecule. The region containing the putative consensus sequence for encephalitogenicity in the guinea pig contains several substitutions, thus accounting for the lack of activity of the shark protein. Studies of the secondary structure and self-association have shown that shark MBP possesses solution properties similar to those of the bovine protein, despite the extensive differences in primary structure.

  8. Complete cDNA and derived amino acid sequence of human factor V

    SciTech Connect

    Jenny, R.J.; Pittman, D.D.; Toole, J.J.; Kriz, R.W.; Aldape, R.A.; Hewick, R.M.; Kaufman, R.J.; Mann, K.G.

    1987-07-01

    cDNA clones encoding human factor V have been isolated from an oligo(dT)-primed human fetal liver cDNA library prepared with vector Charon 21A. The cDNA sequence of factor V from three overlapping clones includes a 6672-base-pair (bp) coding region, a 90-bp 5' untranslated region, and a 163-bp 3' untranslated region within which is a poly(A)tail. The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. Direct comparison with human factor VIII reveals considerable homology between proteins in amino acid sequence and domain structure: a triplicated A domain and duplicated C domain show approx. 40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share approx. 40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and approx. 9 additional semiconserved repeats of nine amino acids of the form Asp-Leu-Ser-Gln-Thr-Thr/Asn-Leu-Ser-Pro and 2 additional semiconserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues.

  9. Solid phase sequencing of biopolymers

    DOEpatents

    Cantor, Charles; Koster, Hubert

    2010-09-28

    This invention relates to methods for detecting and sequencing target nucleic acid sequences, to mass modified nucleic acid probes and arrays of probes useful in these methods, and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probes comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Nucleic acids whose sequences can be determined include DNA or RNA in biological samples such as patient biopsies and environmental samples. Probes may be fixed to a solid support such as a hybridization chip to facilitate automated molecular weight analysis and identification of the target sequence.

  10. Estimation of submarine mass failure probability from a sequence of deposits with age dates

    USGS Publications Warehouse

    Geist, Eric L.; Chaytor, Jason D.; Parsons, Thomas E.; ten Brink, Uri S.

    2013-01-01

    The empirical probability of submarine mass failure is quantified from a sequence of dated mass-transport deposits. Several different techniques are described to estimate the parameters for a suite of candidate probability models. The techniques, previously developed for analyzing paleoseismic data, include maximum likelihood and Type II (Bayesian) maximum likelihood methods derived from renewal process theory and Monte Carlo methods. The estimated mean return time from these methods, unlike estimates from a simple arithmetic mean of the center age dates and standard likelihood methods, includes the effects of age-dating uncertainty and of open time intervals before the first and after the last event. The likelihood techniques are evaluated using Akaike’s Information Criterion (AIC) and Akaike’s Bayesian Information Criterion (ABIC) to select the optimal model. The techniques are applied to mass transport deposits recorded in two Integrated Ocean Drilling Program (IODP) drill sites located in the Ursa Basin, northern Gulf of Mexico. Dates of the deposits were constrained by regional bio- and magnetostratigraphy from a previous study. Results of the analysis indicate that submarine mass failures in this location occur primarily according to a Poisson process in which failures are independent and return times follow an exponential distribution. However, some of the model results suggest that submarine mass failures may occur quasiperiodically at one of the sites (U1324). The suite of techniques described in this study provides quantitative probability estimates of submarine mass failure occurrence, for any number of deposits and age uncertainty distributions.

  11. An analysis of amino acid sequences surrounding archaeal glycoprotein sequons.

    PubMed

    Abu-Qarn, Mehtap; Eichler, Jerry

    2007-05-01

    Despite having provided the first example of a prokaryal glycoprotein, little is known of the rules governing the N-glycosylation process in Archaea. As in Eukarya and Bacteria, archaeal N-glycosylation takes place at the Asn residues of Asn-X-Ser/Thr sequons. Since not all sequons are utilized, it is clear that other factors, including the context in which a sequon exists, affect glycosylation efficiency. As yet, the contribution to N-glycosylation made by sequon-bordering residues and other related factors in Archaea remains unaddressed. In the following, the surroundings of Asn residues confirmed by experiment as modified were analyzed in an attempt to define sequence rules and requirements for archaeal N-glycosylation.

  12. Characterization of antimicrobial histone sequences and posttranslational modifications by mass spectrometry.

    PubMed

    Ouvry-Patat, Séverine A; Schey, Kevin L

    2007-05-01

    Histones typically play a role in DNA packaging and transcription regulation. These proteins are heavily modified by acetylation, methylation, phosphorylation and/or ubiquitination, and various combinations of these modifications alter histone functions and form the basis of the histone code. Furthermore, histones, including those found in shrimp, have recently been found to possess antimicrobial properties; however, the sequences and posttranslational modifications of shrimp histones are largely unknown. In this study mass spectrometry was used to characterize the primary structure of the shrimp antimicrobial histone. A combination of in-solution digestion and in-gel propionylation/digestion followed by LC-MS-MS and MALDI-TOF-TOF analysis was used. Over 80% of each histone sequence was obtained by in-solution digestion; however, none of the N-terminal domains was sequenced with this method. An in-gel propionylation method was optimized to recover and sequence the extremely hydrophilic histone N-termini. This method was then applied to shrimp hemocyte lysates separated on a 1-D SDS-PAGE gel. Overall, 95% coverage was obtained for the histone sequences as well as the identification of posttranslational sites such as acetylation, methylation and phosphorylation.

  13. Mass spectrometric identification, sequence evolution, and intraspecific variability of dimeric peptides encoded by cockroach akh genes.

    PubMed

    Sturm, Sebastian; Predel, Reinhard

    2015-02-01

    Neuropeptides are structurally the most diverse group of messenger molecules of the nervous system. Regarding neuropeptide identification, distribution, function, and evolution, insects are among the best studied invertebrates. Indeed, more than 100 neuropeptides are known from single species. Most of these peptides can easily be identified by direct tissue or cell profiling using MALDI-TOF MS. In these experiments, protein hormones with extensive post-translational modifications such as inter- and intramolecular disulfides are usually missed. It is evident that an exclusion of these bioactive molecules hinders the utilization of direct profiling methods in comprehensive peptidomic analyses. In the current study, we focus on the detection and structural elucidation of homo- and heterodimeric adipokinetic hormone precursor-related peptides (APRPs) of cockroaches. The physiological relevance of these molecules with highly conserved sequences in insects is still uncertain. Sequence similarities with vertebrate growth hormone-releasing factors have been reported, but remarkably, few data regarding APRP processing exist and these data are restricted to locusts. Here, we elucidated sequences of carbamidomethylated APRP monomers of different cockroaches by means of MALDI-TOF MS(2), and we were able to identify a surprisingly large number of APRP sequences, resulting either from intraspecific amino acid substitutions within the APRP sequences or C-terminal truncated APRPs.

  14. Amino acid sequence and some properties of phytolacain G, a cysteine protease from growing fruit of pokeweed, Phytolacca americana.

    PubMed

    Uchikoba, T; Arima, K; Yonezawa, H; Shimada, M; Kaneda, M

    2000-10-18

    A protease, phytolacain G, has been found to appear on CM-Sepharose ion-exchange chromatography of greenish small-size fruits of pokeweed, Phytolacca americana L, from ca. 2 weeks after flowering, and increases during fruit enlargement. Reddish ripe fruit of the pokeweed contained both phytolacain G and R. The molecular mass of phytolacain G was estimated to be 25.5 kDa by SDS-PAGE. Its amino acid sequence was reconstructed by automated sequence analysis of the peptides obtained after cleavage with Achromobacter protease I, chymotrypsin, and cyanogen bromide. The enzyme is composed of 216 amino acid residues, of which it shares 152 identical amino acid residues (70%) with phytolacain R, 126 (58%) with melain G, 108 (50%) with papain, 106 (49%) with actinidain, and 96 (44%) with stem bromelain. The amino acid residues forming the substrate binding S(2) pocket of papain, Tyr67, Pro68, Trp69, Val133, and Phe207, were predicted to be replaced by Trp, Met, His, Ala, and Ser in phytolacain G, respectively. As a consequence of these substitutions, the S(2) pocket is expected to be less hydrophobic in phytolacain G than in papain.

  15. "Polymeromics": Mass spectrometry based strategies in polymer science toward complete sequencing approaches: a review.

    PubMed

    Altuntaş, Esra; Schubert, Ulrich S

    2014-01-15

    Mass spectrometry (MS) is the most versatile and comprehensive method in "OMICS" sciences (i.e. in proteomics, genomics, metabolomics and lipidomics). The applications of MS and tandem MS (MS/MS or MS(n)) provide sequence information of the full complement of biological samples in order to understand the importance of the sequences on their precise and specific functions. Nowadays, the control of polymer sequences and their accurate characterization is one of the significant challenges of current polymer science. Therefore, a similar approach can be very beneficial for characterizing and understanding the complex structures of synthetic macromolecules. MS-based strategies allow a relatively precise examination of polymeric structures (e.g. their molar mass distributions, monomer units, side chain substituents, end-group functionalities, and copolymer compositions). Moreover, tandem MS offer accurate structural information from intricate macromolecular structures; however, it produces vast amount of data to interpret. In "OMICS" sciences, the software application to interpret the obtained data has developed satisfyingly (e.g. in proteomics), because it is not possible to handle the amount of data acquired via (tandem) MS studies on the biological samples manually. It can be expected that special software tools will improve the interpretation of (tandem) MS output from the investigations of synthetic polymers as well. Eventually, the MS/MS field will also open up for polymer scientists who are not MS-specialists. In this review, we dissect the overall framework of the MS and MS/MS analysis of synthetic polymers into its key components. We discuss the fundamentals of polymer analyses as well as recent advances in the areas of tandem mass spectrometry, software developments, and the overall future perspectives on the way to polymer sequencing, one of the last Holy Grail in polymer science.

  16. Nucleic and Amino Acid Sequences Support Structure-Based Viral Classification

    PubMed Central

    Sinclair, Robert M.; Ravantti, Janne J.

    2017-01-01

    ABSTRACT Viral capsids ensure viral genome integrity by protecting the enclosed nucleic acids. Interactions between the genome and capsid and between individual capsid proteins (i.e., capsid architecture) are intimate and are expected to be characterized by strong evolutionary conservation. For this reason, a capsid structure-based viral classification has been proposed as a way to bring order to the viral universe. The seeming lack of sufficient sequence similarity to reproduce this classification has made it difficult to reject structural convergence as the basis for the classification. We reinvestigate whether the structure-based classification for viral coat proteins making icosahedral virus capsids is in fact supported by previously undetected sequence similarity. Since codon choices can influence nascent protein folding cotranslationally, we searched for both amino acid and nucleotide sequence similarity. To demonstrate the sensitivity of the approach, we identify a candidate gene for the pandoravirus capsid protein. We show that the structure-based classification is strongly supported by amino acid and also nucleotide sequence similarities, suggesting that the similarities are due to common descent. The correspondence between structure-based and sequence-based analyses of the same proteins shown here allow them to be used in future analyses of the relationship between linear sequence information and macromolecular function, as well as between linear sequence and protein folds. IMPORTANCE Viral capsids protect nucleic acid genomes, which in turn encode capsid proteins. This tight coupling of protein shell and nucleic acids, together with strong functional constraints on capsid protein folding and architecture, leads to the hypothesis that capsid protein-coding nucleotide sequences may retain signatures of ancient viral evolution. We have been able to show that this is indeed the case, using the major capsid proteins of viruses forming icosahedral capsids

  17. Classification of mouse VK groups based on the partial amino acid sequence to the first invariant tryptophan: impact of 14 new sequences from IgG myeloma proteins.

    PubMed

    Potter, M; Newell, J B; Rudikoff, S; Haber, E

    1982-12-01

    Fourteen new VK sequences derived from BALB/c IgG myeloma proteins were determined to the first invariant tryptophan (Trp 35). These partial sequences were compared with 65 other published VK sequences using a computer program. The 79 sequences were organized according to the length of the sequence from the amino terminus to the first invariant tryptophan (Trp 35), into seven groups (33, 34, 35, 36, 39, 40 and 41aa). A distance matrix of all 79 sequences was then computed, i.e. the number of amino acid substitutions necessary to convert one sequence to another was determined. From these data a dendrogram was constructed. Most of the VK sequences fell into clusters or closely related groups. The definition of a sequence group is arbitrary but facilitates the classification of VK proteins. We used 12 substitutions as the basis for defining a sequence group based on the known number of substitutions that are found in the VK21 proteins. By this criterion there were 18 groups in the Trp 35 dendrogram. Twelve of the 14 new sequences fell into one of these sequence groups; two formed new sequence groups. Collective amino acid sequencing is still encountering new VK structures indicating more sequences will be required to attain an accurate estimate of the total number of VK groups. Updated dendrograms can be quickly generated to include newly generated sequences.

  18. Detection and isolation of nucleic acid sequences using competitive hybridization probes

    DOEpatents

    Lucas, J.N.; Straume, T.; Bogen, K.T.

    1997-04-01

    A method for detecting a target nucleic acid sequence in a sample is provided using hybridization probes which competitively hybridize to a target nucleic acid. According to the method, a target nucleic acid sequence is hybridized to first and second hybridization probes which are complementary to overlapping portions of the target nucleic acid sequence, the first hybridization probe including a first complexing agent capable of forming a binding pair with a second complexing agent and the second hybridization probe including a detectable marker. The first complexing agent attached to the first hybridization probe is contacted with a second complexing agent, the second complexing agent being attached to a solid support such that when the first and second complexing agents are attached, target nucleic acid sequences hybridized to the first hybridization probe become immobilized on to the solid support. The immobilized target nucleic acids are then separated and detected by detecting the detectable marker attached to the second hybridization probe. A kit for performing the method is also provided. 7 figs.

  19. Detection and isolation of nucleic acid sequences using competitive hybridization probes

    DOEpatents

    Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

    1997-01-01

    A method for detecting a target nucleic acid sequence in a sample is provided using hybridization probes which competitively hybridize to a target nucleic acid. According to the method, a target nucleic acid sequence is hybridized to first and second hybridization probes which are complementary to overlapping portions of the target nucleic acid sequence, the first hybridization probe including a first complexing agent capable of forming a binding pair with a second complexing agent and the second hybridization probe including a detectable marker. The first complexing agent attached to the first hybridization probe is contacted with a second complexing agent, the second complexing agent being attached to a solid support such that when the first and second complexing agents are attached, target nucleic acid sequences hybridized to the first hybridization probe become immobilized on to the solid support. The immobilized target nucleic acids are then separated and detected by detecting the detectable marker attached to the second hybridization probe. A kit for performing the method is also provided.

  20. Amino acid sequence around the active-site serine residue in the acyltransferase domain of goat mammary fatty acid synthetase.

    PubMed Central

    Mikkelsen, J; Højrup, P; Rasmussen, M M; Roepstorff, P; Knudsen, J

    1985-01-01

    Goat mammary fatty acid synthetase was labelled in the acyltransferase domain by formation of O-ester intermediates by incubation with [1-14C]acetyl-CoA and [2-14C]malonyl-CoA. Tryptic-digest and CNBr-cleavage peptides were isolated and purified by high-performance reverse-phase and ion-exchange liquid chromatography. The sequences of the malonyl- and acetyl-labelled peptides were shown to be identical. The results confirm the hypothesis that both acetyl and malonyl groups are transferred to the mammalian fatty acid synthetase complex by the same transferase. The sequence is compared with those of other fatty acid synthetase transferases. PMID:3922356

  1. Extended main sequence turn-offs in low mass intermediate-age clusters

    NASA Astrophysics Data System (ADS)

    Piatti, Andrés E.; Bastian, Nate

    2016-05-01

    We present an imaging analysis of four low mass stellar clusters (≲5000 M⊙) in the outer regions of the LMC in order to shed light on the extended main sequence turn-off (eMSTO) phenomenon observed in high mass clusters. The four clusters have ages between 1-2 Gyr and two of them appear to host eMTSOs. The discovery of eMSTOs in such low mass clusters - more than 5 times less massive than the eMSTO clusters previously studied - suggests that mass is not the controlling factor in whether clusters host eMSTOs. Additionally, the narrow extent of the eMSTO in the two older clusters (~2 Gyr) is in agreement with predictions of the stellar rotation scenario, as lower mass stars are expected to be magnetically braked, meaning that their colour magnitude diagrams should be better reproduced by canonical simple stellar populations. We also performed a structural analysis on all the clusters and found that a large core radius is not a requisite for a cluster to exhibit an eMSTO. Full Table 2, and Tables 3-5 are only available at the CDS via anonymous ftp to http://cdsarc.u-strasbg.fr (http://130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/590/A50

  2. Ligation with nucleic acid sequence-based amplification.

    PubMed

    Ong, Carmichael; Tai, Warren; Sarma, Aartik; Opal, Steven M; Artenstein, Andrew W; Tripathi, Anubhav

    2012-01-01

    This work presents a novel method for detecting nucleic acid targets using a ligation step along with an isothermal, exponential amplification step. We use an engineered ssDNA with two variable regions on the ends, allowing us to design the probe for optimal reaction kinetics and primer binding. This two-part probe is ligated by T4 DNA Ligase only when both parts bind adjacently to the target. The assay demonstrates that the expected 72-nt RNA product appears only when the synthetic target, T4 ligase, and both probe fragments are present during the ligation step. An extraneous 38-nt RNA product also appears due to linear amplification of unligated probe (P3), but its presence does not cause a false-positive result. In addition, 40 mmol/L KCl in the final amplification mix was found to be optimal. It was also found that increasing P5 in excess of P3 helped with ligation and reduced the extraneous 38-nt RNA product. The assay was also tested with a single nucleotide polymorphism target, changing one base at the ligation site. The assay was able to yield a negative signal despite only a single-base change. Finally, using P3 and P5 with longer binding sites results in increased overall sensitivity of the reaction, showing that increasing ligation efficiency can improve the assay overall. We believe that this method can be used effectively for a number of diagnostic assays.

  3. Preparation of protein samples for mass spectrometry and N-terminal sequencing.

    PubMed

    Glenn, Gary

    2014-01-01

    The preparation of protein samples for mass spectrometry and N-terminal sequencing is a key step in successfully identifying proteins. Mass spectrometry is a very sensitive technique, and as such, samples must be prepared carefully since they can be subject to contamination of the sample (e.g., due to incomplete subcellular fractionation or purification of a multiprotein complex), overwhelming of the sample by highly abundant proteins, and contamination from skin or hair (keratin can be a very common hit). One goal of sample preparation for mass spec is to reduce the complexity of the sample - in the example presented here, mitochondria are purified, solubilized, and fractionated by sucrose density gradient sedimentation prior to preparative 1D SDS-PAGE. It is important to verify the purity and integrity of the sample so that you can have confidence in the hits obtained. More protein is needed for N-terminal sequencing and ideally it should be purified to a single band when run on an SDS-polyacrylamide gel. The example presented here involves stably expressing a tagged protein in HEK293 cells and then isolating the protein by affinity purification and SDS-PAGE.

  4. Thin-film technology for direct visual detection of nucleic acid sequences: applications in clinical research.

    PubMed

    Jenison, Robert D; Bucala, Richard; Maul, Diana; Ward, David C

    2006-01-01

    Certain optical conditions permit the unaided eye to detect thickness changes on surfaces on the order of 20 A, which are of similar dimensions to monomolecular interactions between proteins or hybridization of complementary nucleic acid sequences. Such detection exploits specific interference of reflected white light, wherein thickness changes are perceived as surface color changes. This technology, termed thin-film detection, allows for the visualization of subattomole amounts of nucleic acid targets, even in complex clinical samples. Thin-film technology has been applied to a broad range of clinically relevant indications, including the detection of pathogenic bacterial and viral nucleic acid sequences and the discrimination of sequence variations in human genes causally related to susceptibility or severity of disease.

  5. Low-Mass Pre-Main-Sequence Stars in the Magellanic Clouds

    NASA Astrophysics Data System (ADS)

    Gouliermis, Dimitrios A.

    2012-09-01

    The stellar Initial Mass Function (IMF) suggests that stars with sub-solar mass form in very large numbers. Most attractive places for catching low-mass star formation in the act are young stellar clusters and associations, still (half-)embedded in star-forming regions. The low-mass stars in such regions are still in their pre-main-sequence (PMS) evolutionary phase, i.e., they have not started their lives on the main-sequence yet. The peculiar nature of these objects and the contamination of their samples by the fore- and background evolved populations of the Galactic disk impose demanding observational techniques, such as X-ray surveying and optical spectroscopy of large samples for the detection of complete numbers of PMS stars in the Milky Way. The Magellanic Clouds, the metal-poor companion galaxies to our own, demonstrate an exceptional star formation activity. The low extinction and stellar field contamination in star-forming regions of these galaxies imply a more efficient detection of low-mass PMS stars than in the Milky Way, but their distance from us make the application of the above techniques unfeasible. Nonetheless, imaging with the Hubble Space Telescope within the last five years yield the discovery of solar and sub-solar PMS stars in the Magellanic Clouds from photometry alone. Unprecedented numbers of such objects are identified as the low-mass stellar content of star-forming regions in these galaxies, changing completely our picture of young stellar systems outside the Milky Way, and extending the extragalactic stellar IMF below the persisting threshold of a few solar masses. This review presents the recent developments in the investigation of the PMS stellar content of the Magellanic Clouds, with special focus on the limitations by single-epoch photometry that can only be circumvented by the detailed study of the observable behavior of these stars in the color-magnitude diagram. The achieved characterization of the low-mass PMS stars in the

  6. Conservation of Shannon's redundancy for proteins. [information theory applied to amino acid sequences

    NASA Technical Reports Server (NTRS)

    Gatlin, L. L.

    1974-01-01

    Concepts of information theory are applied to examine various proteins in terms of their redundancy in natural originators such as animals and plants. The Monte Carlo method is used to derive information parameters for random protein sequences. Real protein sequence parameters are compared with the standard parameters of protein sequences having a specific length. The tendency of a chain to contain some amino acids more frequently than others and the tendency of a chain to contain certain amino acid pairs more frequently than other pairs are used as randomness measures of individual protein sequences. Non-periodic proteins are generally found to have random Shannon redundancies except in cases of constraints due to short chain length and genetic codes. Redundant characteristics of highly periodic proteins are discussed. A degree of periodicity parameter is derived.

  7. RNA internal standard synthesis by nucleic acid sequence-based amplification for competitive quantitative amplification reactions.

    PubMed

    Lo, Wan-Yu; Baeumner, Antje J

    2007-02-15

    Nucleic acid sequence-based amplification (NASBA) reactions have been demonstrated to successfully synthesize new sequences based on deletion and insertion reactions. Two RNA internal standards were synthesized for use in competitive amplification reactions in which quantitative analysis can be achieved by coamplifying the internal standard with the wild type sample. The sequences were created in two consecutive NASBA reactions using the E. coli clpB mRNA sequence as model analyte. The primer sequences of the wild type sequence were maintained, and a 20-nt-long segment inside the amplicon region was exchanged for a new segment of similar GC content and melting temperature. The new RNA sequence was thus amplifiable using the wild type primers and detectable via a new inserted sequence. In the first reaction, the forwarding primer and an additional 20-nt-long sequence was deleted and replaced by a new 20-nt-long sequence. In the second reaction, a forwarding primer containing as 5' overhang sequence the wild type primer sequence was used. The presence of pure internal standard was verified using electrochemiluminescence and RNA lateral-flow biosensor analysis. Additional sequence deletion in order to shorten the internal standard amplicons and thus generate higher detection signals was found not to be required. Finally, a competitive NASBA reaction between one internal standard and the wild type sequence was carried out proving its functionality. This new rapid construction method via NASBA provides advantages over the traditional techniques since it requires no traditional cloning procedures, no thermocyclers, and can be completed in less than 4 h.

  8. Plasma-desorption mass spectrometry as an aid in protein sequence determination. Application of the method on a cuticular protein from the migratory locust (Locusta migratoria).

    PubMed Central

    Klarskov, K; Højrup, P; Andersen, S O; Roepstorff, P

    1989-01-01

    The complete amino acid sequence of a structural protein, protein 8, isolated from the pharate cuticle of the locust Locusta migratoria was determined. Protein 8 contains 148 amino acid residues and has an Mr of 15,224. By the extensive use of information obtained by plasma-desorption mass spectrometry (p.d.m.s.) it was possible to reduce the need for conventional sequence determination and to improve the reliability of the results. On the basis of the determined Mr of the intact protein all the peptides that constitute the complete sequence could be isolated from a time-course enzymic digestion. The isolated peptides were sequenced by using a combination of Edman degradation and carboxypeptidase digestion monitored by p.d.m.s. The alignment of the peptides was established from the time-course digestion and further verified by a second enzymic digestion. The primary structure of the protein consists of two hydrophilic and two hydrophobic regions. The hydrophobic regions are enriched in alanine, valine and proline and dominated by a repetitive sequence Ala-Ala-Pro-(Ala/Val). The sequence strengthens the view that the cuticle proteins belong to a unique family of structural proteins. PMID:2590176

  9. Relative Stability of Peptide Sequence Ions Generated by Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Bythell, Benjamin J.; Hendrickson, Christopher L.; Marshall, Alan G.

    2012-04-01

    We report the use of unimolecular dissociation by infrared radiation for gaseous multiphoton energy transfer to determine relative activation energy (Ea,laser) for dissociation of peptide sequence ions. The sequence ions of interest are mass-isolated; the entire ion cloud is then irradiated with a continuous wave CO2 laser, and the first order rate constant, kd, is determined for each of a series of laser powers. Provided these conditions are met, a plot of the natural logarithm of kd versus the natural logarithm of laser power yields a straight line, whose slope provides a measure of Ea,laser. This method reproduces the Ea values from blackbody radiative dissociation (BIRD) for the comparatively large, singly and doubly protonated bradykinin ions (nominally y 9 and y 9 2+ ). The comparatively small sequence ion systems produce Ea,laser values that are systematic underestimates of theoretical barriers calculated with density functional theory (DFT). However, the relative Ea,laser values are in qualitative agreement with the mobile proton model and available theory. Additionally, novel protonated cyclic-dipeptide (diketopiperazine) fragmentation reactions are analyzed with DFT. FT-ICR MS provides access to sequence ions generated by electron capture dissociation, infrared multiphoton dissociation, and collisional activation methods (i.e., b n , y m , c n , z m • ions).

  10. Gene analysis using mass spectrometric cleaved amplified polymorphic sequence (MS-CAPS) with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF).

    PubMed

    Kajiwara, Hideyuki

    2015-01-01

    Mass spectrometric cleaved amplified polymorphic sequence (MS-CAPS) is a method for detecting genes using a combination of short PCR and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). MS-CAPS can identify a single nucleotide polymorphism (SNP) in less than one hour and is suitable for plants, animals, bacteria, and food.

  11. A Machine Learning Based Approach to de novo Sequencing of Glycans from Tandem Mass Spectrometry Spectrum.

    PubMed

    Kumozaki, Shotaro; Sato, Kengo; Sakakibara, Yasubumi

    2015-01-01

    Recently, glycomics has been actively studied and various technologies for glycomics have been rapidly developed. Currently, tandem mass spectrometry (MS/MS) is one of the key experimental tools for identification of structures of oligosaccharides. MS/MS can observe MS/MS peaks of fragmented glycan ions including cross-ring ions resulting from internal cleavages, which provide valuable information to infer glycan structures. Thus, the aim of de novo sequencing of glycans is to find the most probable assignments of observed MS/MS peaks to glycan substructures without databases. However, there are few satisfiable algorithms for glycan de novo sequencing from MS/MS spectra. We present a machine learning based approach to de novo sequencing of glycans from MS/MS spectrum. First, we build a suitable model for the fragmentation of glycans including cross-ring ions, and implement a solver that employs Lagrangian relaxation with a dynamic programming technique. Then, to optimize scores for the algorithm, we introduce a machine learning technique called structured support vector machines that enable us to learn parameters including scores for cross-ring ions from training data, i.e., known glycan mass spectra. Furthermore, we implement additional constraints for core structures of well-known glycan types including N-linked glycans and O-linked glycans. This enables us to predict more accurate glycan structures if the glycan type of given spectra is known. Computational experiments show that our algorithm performs accurate de novo sequencing of glycans. The implementation of our algorithm and the datasets are available at http://glyfon.dna.bio.keio.ac.jp/.

  12. Pulsating low-mass white dwarfs in the frame of new evolutionary sequences. I. Adiabatic properties

    NASA Astrophysics Data System (ADS)

    Córsico, A. H.; Althaus, L. G.

    2014-09-01

    Context. Many low-mass white dwarfs with masses M∗/M⊙ ≲ 0.45, including the so-called extremely low-mass white dwarfs (M∗/M⊙ ≲ 0.20 - 0.25), have recently been discovered in the field of our Galaxy through dedicated photometric surveys. The subsequent discovery of pulsations in some of them has opened the unprecedented opportunity of probing the internal structure of these ancient stars. Aims: We present a detailed adiabatic pulsational study of these stars based on full evolutionary sequences derived from binary star evolution computations. The main aim of this study is to provide a detailed theoretical basis of reference for interpreting present and future observations of variable low-mass white dwarfs. Methods: Our pulsational analysis is based on a new set of He-core white-dwarf models with masses ranging from 0.1554 to 0.4352 M⊙ derived by computing the non-conservative evolution of a binary system consisting of an initially 1 M⊙ ZAMS star and a 1.4 M⊙ neutron star. We computed adiabatic radial (ℓ = 0) and non-radial (ℓ = 1,2) p and g modes to assess the dependence of the pulsational properties of these objects on stellar parameters such as the stellar mass and the effective temperature, as well as the effects of element diffusion. Results: We found that for white dwarf models with masses below ~ 0.18 M⊙, g modes mainly probe the core regions and p modes the envelope, therefore pulsations offer the opportunity of constraining both the core and envelope chemical structure of these stars via asteroseismology. For models with M∗ ≳ 0.18 M⊙, on the other hand, g modes are very sensitive to the He/H compositional gradient and therefore can be used as a diagnostic tool for constraining the H envelope thickness. Because both types of objects have not only very distinct evolutionary histories (according to whether the progenitor stars have experienced CNO-flashes or not), but also have strongly different pulsation properties, we propose to

  13. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry

    PubMed Central

    Syka, John E. P.; Coon, Joshua J.; Schroeder, Melanie J.; Shabanowitz, Jeffrey; Hunt, Donald F.

    2004-01-01

    Peptide sequence analysis using a combination of gas-phase ion/ion chemistry and tandem mass spectrometry (MS/MS) is demonstrated. Singly charged anthracene anions transfer an electron to multiply protonated peptides in a radio frequency quadrupole linear ion trap (QLT) and induce fragmentation of the peptide backbone along pathways that are analogous to those observed in electron capture dissociation. Modifications to the QLT that enable this ion/ion chemistry are presented, and automated acquisition of high-quality, single-scan electron transfer dissociation MS/MS spectra of phosphopeptides separated by nanoflow HPLC is described. PMID:15210983

  14. Proceedings of the relevance of mass spectrometry to DNA sequence determination: Research needs for the Human Genome Program

    SciTech Connect

    Edmonds, C.G.; Smith, R.D. ); Smith, L.M. )

    1990-11-01

    A workshop was sponsored for the US Department of Energy (DOE), Office of Health and Environmental Research by Pacific Northwest Laboratory, April 4--5, 1990, in Seattle, Washington, to examine the potential role of mass spectrometry in the joint DOE/National Institutes of Health (NIH) Human Genome Program. The workshop was occasioned by recent developments in mass spectrometry that are providing new levels for selectivity, sensitivity, and, in particular, new methods of ionization appropriate for large biopolymers such as DNA. During discussions, three general mass spectrometric approaches to the determination of DNA sequence were considered: (1) the mass spectrometric detection of isotopic labels from DNA sequencing mixtures separated using gel electrophoresis, (2) the direct mass spectrometric analysis from direct ionization of unfractionated sequencing mixtures where the measured mass of the constituents functions to identify and order the base sequence (replacing separation by gel electrophoresis), and (3) an approach in which a single highly charged molecular ion of a large DNA segment produced is rapidly sequenced in an ion cyclotron resonance ion trap. The consensus of the workshop was that, on the basis of the new developments, mass spectrometry has the potential to provide the substantial increases in sequencing speed required for the Human Genome Program. 66 refs., 3 tabs.

  15. Mass functions for globular cluster main sequences based on CCD photometry and stellar models

    NASA Astrophysics Data System (ADS)

    McClure, Robert D.; Vandenberg, Don A.; Smith, Graeme H.; Fahlman, Gregory G.; Richer, Harvey B.; Hesser, James E.; Harris, William E.; Stetson, Peter B.; Bell, R. A.

    1986-08-01

    Main-sequence luminosity functions constructed from CCD observations of globular clusters reveal a strong trend in slope with metal abundance. Theoretical luminosity functions constructed from VandenBerg and Bell's (1985) isochrones have been fitted to the observations and reveal a trend between x, the power-law index of the mass function, and metal abundance. The most metal-poor clusters require an index of about x = 2.5, whereas the most metal-rich clusters exhibit an index of x of roughly -0.5. The luminosity functions for two sparse clusters, E3 and Pal 5, are distinct from those of the more massive clusters, in that they show a turndown which is possibly a result of mass loss or tidal disruption.

  16. The Impact of Starspots on Mass and Age Estimates for Pre-main Sequence Stars

    NASA Astrophysics Data System (ADS)

    Somers, Garrett; Pinsonneault, Marc H.

    2016-01-01

    We investigate the impact of starspots on the evolution of late-type stars during the pre-main sequence (pre-MS). We find that heavy spot coverage increases the radii of stars by 4-10%, consistent with inflation factors in eclipsing binary systems, and suppresses the rate of pre-MS lithium depletion, leading to a dispersion in zero-age MS Li abundance (comparable to observed spreads) if a range of spot properties exist within clusters from 3-10 Myr. This concordance with data implies that spots induce a range of radii at fixed mass during the pre-MS. These spots decrease the luminosity and T eff of stars, leading to a displacement on the HR diagram. This displacement causes isochrone derived masses and ages to be systematically under-estimated, and can lead to the spurious appearance of an age spread in a co-eval population.

  17. Defining sequence space and reaction products within the cyanuric acid hydrolase (AtzD)/barbiturase protein family.

    PubMed

    Seffernick, Jennifer L; Erickson, Jasmine S; Cameron, Stephan M; Cho, Seunghee; Dodge, Anthony G; Richman, Jack E; Sadowsky, Michael J; Wackett, Lawrence P

    2012-09-01

    Cyanuric acid hydrolases (AtzD) and barbiturases are homologous, found almost exclusively in bacteria, and comprise a rare protein family with no discernible linkage to other protein families or an X-ray structural class. There has been confusion in the literature and in genome projects regarding the reaction products, the assignment of individual sequences as either cyanuric acid hydrolases or barbiturases, and spurious connection of this family to another protein family. The present study has addressed those issues. First, the published enzyme reaction products of cyanuric acid hydrolase are incorrectly identified as biuret and carbon dioxide. The current study employed (13)C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry to show that cyanuric acid hydrolase releases carboxybiuret, which spontaneously decarboxylates to biuret. This is significant because it revealed that homologous cyanuric acid hydrolases and barbiturases catalyze completely analogous reactions. Second, enzymes that had been annotated incorrectly in genome projects have been reassigned here by bioinformatics, gene cloning, and protein characterization studies. Third, the AtzD/barbiturase family has previously been suggested to consist of members of the amidohydrolase superfamily, a large class of metallohydrolases. Bioinformatics and the lack of bound metals both argue against a connection to the amidohydrolase superfamily. Lastly, steady-state kinetic measurements and observations of protein stability suggested that the AtzD/barbiturase family might be an undistinguished protein family that has undergone some resurgence with the recent introduction of industrial s-triazine compounds such as atrazine and melamine into the environment.

  18. Amino acid sequences of two nonspecific lipid-transfer proteins from germinated castor bean.

    PubMed

    Takishima, K; Watanabe, S; Yamada, M; Suga, T; Mamiya, G

    1988-11-01

    The amino acid sequence of two nonspecific lipid-transfer proteins (nsLTP) B and C from germinated castor bean seeds have been determined. Both the proteins consist of 92 residues, as for nsLTP previously reported, and their calculated Mr values are 9847 and 9593 for nsLTP-B and nsLTP-C, respectively. The sequences of nsLTP-B and nsLTP-C, compared to the known sequence of nsLTP-A from the same source, are 68% and 35% similar, respectively. No variation was found at the positions of the cysteine residues, indicating that they might be involved in disulfide bridges.

  19. Automated Glycan Sequencing from Tandem Mass Spectra of N-Linked Glycopeptides.

    PubMed

    Yu, Chuan-Yih; Mayampurath, Anoop; Zhu, Rui; Zacharias, Lauren; Song, Ehwang; Wang, Lei; Mechref, Yehia; Tang, Haixu

    2016-06-07

    Mass spectrometry has become a routine experimental tool for proteomic biomarker analysis of human blood samples, partly due to the large availability of informatics tools. As one of the most common protein post-translational modifications (PTMs) in mammals, protein glycosylation has been observed to alter in multiple human diseases and thus may potentially be candidate markers of disease progression. While mass spectrometry instrumentation has seen advancements in capabilities, discovering glycosylation-related markers using existing software is currently not straightforward. Complete characterization of protein glycosylation requires the identification of intact glycopeptides in samples, including identification of the modification site as well as the structure of the attached glycans. In this paper, we present GlycoSeq, an open-source software tool that implements a heuristic iterated glycan sequencing algorithm coupled with prior knowledge for automated elucidation of the glycan structure within a glycopeptide from its collision-induced dissociation tandem mass spectrum. GlycoSeq employs rules of glycosidic linkage as defined by glycan synthetic pathways to eliminate improbable glycan structures and build reasonable glycan trees. We tested the tool on two sets of tandem mass spectra of N-linked glycopeptides cell lines acquired from breast cancer patients. After employing enzymatic specificity within the N-linked glycan synthetic pathway, the sequencing results of GlycoSeq were highly consistent with the manually curated glycan structures. Hence, GlycoSeq is ready to be used for the characterization of glycan structures in glycopeptides from MS/MS analysis. GlycoSeq is released as open source software at https://github.com/chpaul/GlycoSeq/ .

  20. A classification of glycosyl hydrolases based on amino acid sequence similarities.

    PubMed Central

    Henrissat, B

    1991-01-01

    The amino acid sequences of 301 glycosyl hydrolases and related enzymes have been compared. A total of 291 sequences corresponding to 39 EC entries could be classified into 35 families. Only ten sequences (less than 5% of the sample) could not be assigned to any family. With the sequences available for this analysis, 18 families were found to be monospecific (containing only one EC number) and 17 were found to be polyspecific (containing at least two EC numbers). Implications on the folding characteristics and mechanism of action of these enzymes and on the evolution of carbohydrate metabolism are discussed. With the steady increase in sequence and structural data, it is suggested that the enzyme classification system should perhaps be revised. PMID:1747104

  1. In silico comparative analysis of DNA and amino acid sequences for prion protein gene.

    PubMed

    Kim, Y; Lee, J; Lee, C

    2008-01-01

    Genetic variability might contribute to species specificity of prion diseases in various organisms. In this study, structures of the prion protein gene (PRNP) and its amino acids were compared among species of which sequence data were available. Comparisons of PRNP DNA sequences among 12 species including human, chimpanzee, monkey, bovine, ovine, dog, mouse, rat, wallaby, opossum, chicken and zebrafish allowed us to identify candidate regulatory regions in intron 1 and 3'-untranslated region (UTR) in addition to the coding region. Highly conserved putative binding sites for transcription factors, such as heat shock factor 2 (HSF2) and myocite enhancer factor 2 (MEF2), were discovered in the intron 1. In 3'-UTR, the functional sequence (ATTAAA) for nucleus-specific polyadenylation was found in all the analysed species. The functional sequence (TTTTTAT) for maturation-specific polyadenylation was identically observed only in ovine, and one or two nucleotide mismatches in the other species. A comparison of the amino acid sequences in 53 species revealed a large sequence identity. Especially the octapeptide repeat region was observed in all the species but frog and zebrafish. Functional changes and susceptibility to prion diseases with various isoforms of prion protein could be caused by numeric variability and conformational changes discovered in the repeat sequences.

  2. Complete amino acid sequence of the N-terminal extension of calf skin type III procollagen.

    PubMed Central

    Brandt, A; Glanville, R W; Hörlein, D; Bruckner, P; Timpl, R; Fietzek, P P; Kühn, K

    1984-01-01

    The N-terminal extension peptide of type III procollagen, isolated from foetal-calf skin, contains 130 amino acid residues. To determine its amino acid sequence, the peptide was reduced and carboxymethylated or aminoethylated and fragmented with trypsin, Staphylococcus aureus V8 proteinase and bacterial collagenase. Pyroglutamate aminopeptidase was used to deblock the N-terminal collagenase fragment to enable amino acid sequencing. The type III collagen extension peptide is homologous to that of the alpha 1 chain of type I procollagen with respect to a three-domain structure. The N-terminal 79 amino acids, which contain ten of the 12 cysteine residues, form a compact globular domain. The next 39 amino acids are in a collagenase triplet sequence (Gly- Xaa - Yaa )n with a high hydroxyproline content. Finally, another short non-collagenous domain of 12 amino acids ends at the cleavage site for procollagen aminopeptidase, which cleaves a proline-glutamine bond. In contrast with type I procollagen, the type III procollagen extension peptides contain interchain disulphide bridges located at the C-terminus of the triple-helical domain. PMID:6331392

  3. Protein identification from two-dimensional gel electrophoresis analysis of Klebsiella pneumoniae by combined use of mass spectrometry data and raw genome sequences

    PubMed Central

    Wang, Wei; Sun, Jibin; Nimtz, Manfred; Deckwer, Wolf-Dieter; Zeng, An-Ping

    2003-01-01

    Separation of proteins by two-dimensional gel electrophoresis (2-DE) coupled with identification of proteins through peptide mass fingerprinting (PMF) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is the widely used technique for proteomic analysis. This approach relies, however, on the presence of the proteins studied in public-accessible protein databases or the availability of annotated genome sequences of an organism. In this work, we investigated the reliability of using raw genome sequences for identifying proteins by PMF without the need of additional information such as amino acid sequences. The method is demonstrated for proteomic analysis of Klebsiella pneumoniae grown anaerobically on glycerol. For 197 spots excised from 2-DE gels and submitted for mass spectrometric analysis 164 spots were clearly identified as 122 individual proteins. 95% of the 164 spots can be successfully identified merely by using peptide mass fingerprints and a strain-specific protein database (ProtKpn) constructed from the raw genome sequences of K. pneumoniae. Cross-species protein searching in the public databases mainly resulted in the identification of 57% of the 66 high expressed protein spots in comparison to 97% by using the ProtKpn database. 10 dha regulon related proteins that are essential for the initial enzymatic steps of anaerobic glycerol metabolism were successfully identified using the ProtKpn database, whereas none of them could be identified by cross-species searching. In conclusion, the use of strain-specific protein database constructed from raw genome sequences makes it possible to reliably identify most of the proteins from 2-DE analysis simply through peptide mass fingerprinting. PMID:14653859

  4. Systematic studies of the mass spectrometric properties of alkaline earth metal cationized amino acids and peptides

    NASA Astrophysics Data System (ADS)

    Küjckelmann, Ulrich; Müller, Dietrich; Weber, Carsten

    1997-07-01

    The results of a systematic study of the gas phase interactions of α-amino acids and peptides (4-15 amino acids) with alkaline earth metals, observed with mass spectrometric techniques, are presented. Furthermore, a model for the cationization with calcium at the C-terminal amino acid arginine in rotaviral polypeptides is presented.

  5. Identification of acylglycerols containing dihydroxy fatty acids in castor oil by mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ricinoleate, a monohydroxy fatty acid, in castor oil has many industrial uses. Dihydroxy fatty acids can also be used in industry. The C18 HPLC fractions of castor oil were used for mass spectrometry of lithium addicts to identify the acylglycerols containing dihydroxy fatty acids. Four diacylglycer...

  6. 37 CFR 1.824 - Form and format for nucleotide and/or amino acid sequence submissions in computer readable form.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... nucleotide and/or amino acid sequence submissions in computer readable form. 1.824 Section 1.824 Patents... And/or Amino Acid Sequences § 1.824 Form and format for nucleotide and/or amino acid sequence... readable form may be created by any means, such as word processors, nucleotide/amino acid sequence...

  7. 37 CFR 1.824 - Form and format for nucleotide and/or amino acid sequence submissions in computer readable form.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... nucleotide and/or amino acid sequence submissions in computer readable form. 1.824 Section 1.824 Patents... And/or Amino Acid Sequences § 1.824 Form and format for nucleotide and/or amino acid sequence... readable form may be created by any means, such as word processors, nucleotide/amino acid sequence...

  8. 37 CFR 1.824 - Form and format for nucleotide and/or amino acid sequence submissions in computer readable form.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... nucleotide and/or amino acid sequence submissions in computer readable form. 1.824 Section 1.824 Patents... And/or Amino Acid Sequences § 1.824 Form and format for nucleotide and/or amino acid sequence... readable form may be created by any means, such as word processors, nucleotide/amino acid sequence...

  9. CycloBranch: De Novo Sequencing of Nonribosomal Peptides from Accurate Product Ion Mass Spectra

    NASA Astrophysics Data System (ADS)

    Novák, Jiří; Lemr, Karel; Schug, Kevin A.; Havlíček, Vladimír

    2015-07-01

    Nonribosomal peptides have a wide range of biological and medical applications. Their identification by tandem mass spectrometry remains a challenging task. A new open-source de novo peptide identification engine CycloBranch was developed and successfully applied in identification or detailed characterization of 11 linear, cyclic, branched, and branch-cyclic peptides. CycloBranch is based on annotated building block databases the size of which is defined by the user according to ribosomal or nonribosomal peptide origin. The current number of involved nonisobaric and isobaric building blocks is 287 and 521, respectively. Contrary to all other peptide sequencing tools utilizing either peptide libraries or peptide fragment libraries, CycloBranch represents a true de novo sequencing engine developed for accurate mass spectrometric data. It is a stand-alone and cross-platform application with a graphical and user-friendly interface; it supports mzML, mzXML, mgf, txt, and baf file formats and can be run in parallel on multiple threads. It can be downloaded for free from http://ms.biomed.cas.cz/cyclobranch/, where the User's manual and video tutorials can be found.

  10. The amino acid sequence of cytochromes c-551 from three species of Pseudomonas

    PubMed Central

    Ambler, R. P.; Wynn, Margaret

    1973-01-01

    The amino acid sequences of the cytochromes c-551 from three species of Pseudomonas have been determined. Each resembles the protein from Pseudomonas strain P6009 (now known to be Pseudomonas aeruginosa, not Pseudomonas fluorescens) in containing 82 amino acids in a single peptide chain, with a haem group covalently attached to cysteine residues 12 and 15. In all four sequences 43 residues are identical. Although by bacteriological criteria the organisms are closely related, the differences between pairs of sequences range from 22% to 39%. These values should be compared with the differences in the sequence of mitochondrial cytochrome c between mammals and amphibians (about 18%) or between mammals and insects (about 33%). Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 50015 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 5. PMID:4352718

  11. Draft Genome Sequence of Sorghum Grain Mold Fungus Epicoccum sorghinum, a Producer of Tenuazonic Acid

    PubMed Central

    Oliveira, Rodrigo C.; Davenport, Karen W.; Hovde, Blake; Silva, Danielle; Chain, Patrick S. G.; Correa, Benedito

    2017-01-01

    ABSTRACT The facultative plant pathogen Epicoccum sorghinum is associated with grain mold of sorghum and produces the mycotoxin tenuazonic acid. This fungus can have serious economic impact on sorghum production. Here, we report the draft genome sequence of E. sorghinum (USPMTOX48). PMID:28126937

  12. Snake venom. The amino acid sequence of protein A from Dendroaspis polylepis polylepis (black mamba) venom.

    PubMed

    Joubert, F J; Strydom, D J

    1980-12-01

    Protein A from Dendroaspis polylepis polylepis venom comprises 81 amino acids, including ten half-cystine residues. The complete primary structures of protein A and its variant A' were elucidated. The sequences of proteins A and A', which differ in a single position, show no homology with various neurotoxins and non-neurotoxic proteins and represent a new type of elapid venom protein.

  13. Draft Genome Sequence of Bacillus coagulans NL01, a Wonderful l-Lactic Acid Producer

    PubMed Central

    Zheng, Zhaojuan; Jiang, Ting; Lin, Xi; Zhou, Jie

    2015-01-01

    Here, we report the draft genome sequence of Bacillus coagulans NL01, which could produce high optically pure l-lactic acid using xylose as a sole carbon source. The draft genome is 3,505,081 bp, with 144 contigs. About 3,903 protein-coding genes and 92 rRNAs are predicted from this assembly. PMID:26089419

  14. Angular momentum transport efficiency in post-main sequence low-mass stars

    NASA Astrophysics Data System (ADS)

    Spada, F.; Gellert, M.; Arlt, R.; Deheuvels, S.

    2016-05-01

    Context. Using asteroseismic techniques, it has recently become possible to probe the internal rotation profile of low-mass (≈1.1-1.5 M⊙) subgiant and red giant stars. Under the assumption of local angular momentum conservation, the core contraction and envelope expansion occurring at the end of the main sequence would result in a much larger internal differential rotation than observed. This suggests that angular momentum redistribution must be taking place in the interior of these stars. Aims: We investigate the physical nature of the angular momentum redistribution mechanisms operating in stellar interiors by constraining the efficiency of post-main sequence rotational coupling. Methods: We model the rotational evolution of a 1.25M⊙ star using the Yale Rotational stellar Evolution Code. Our models take into account the magnetic wind braking occurring at the surface of the star and the angular momentum transport in the interior, with an efficiency dependent on the degree of internal differential rotation. Results: We find that models including a dependence of the angular momentum transport efficiency on the radial rotational shear reproduce very well the observations. The best fit of the data is obtained with an angular momentum transport coefficient scaling with the ratio of the rotation rate of the radiative interior over that of the convective envelope of the star as a power law of exponent ≈3. This scaling is consistent with the predictions of recent numerical simulations of the Azimuthal Magneto-Rotational Instability. Conclusions: We show that an angular momentum transport process whose efficiency varies during the stellar evolution through a dependence on the level of internal differential rotation is required to explain the observed post-main sequence rotational evolution of low-mass stars.

  15. De novo sequence analysis and intact mass measurements for characterization of phycocyanin subunit isoforms from the blue-green alga Aphanizomenon flos-aquae.

    PubMed

    Rinalducci, Sara; Roepstorff, Peter; Zolla, Lello

    2009-04-01

    In this work, partial characterization of the primary structure of phycocyanin from the cyanobacterium Aphanizomenon flos-aquae (AFA) was achieved by mass spectrometry de novo sequencing with the aid of chemical derivatization. Combining N-terminal sulfonation of tryptic peptides by 4-sulfophenyl isothiocyanate (SPITC) and MALDI-TOF/TOF analyses, facilitated the acquisition of sequence information for AFA phycocyanin subunits. In fact, SPITC-derivatized peptides underwent facile fragmentation, predominantly resulting in y-series ions in the MS/MS spectra and often exhibiting uninterrupted sequences of 20 or more amino acid residues. This strategy allowed us to carry out peptide fragment fingerprinting and de novo sequencing of several peptides belonging to both alpha- and beta-phycocyanin polypeptides, obtaining a sequence coverage of 67% and 75%, respectively. The presence of different isoforms of phycocyanin subunits was also revealed; subsequently Intact Mass Measurements (IMMs) by both MALDI- and ESI-MS supported the detection of these protein isoforms. Finally, we discuss the evolutionary importance of phycocyanin isoforms in cyanobacteria, suggesting the possible use of the phycocyanin operon for a correct taxonomic identity of this species.

  16. Amino acid sequences of heterotrophic and photosynthetic ferredoxins from the tomato plant (Lycopersicon esculentum Mill.).

    PubMed

    Kamide, K; Sakai, H; Aoki, K; Sanada, Y; Wada, K; Green, L S; Yee, B C; Buchanan, B B

    1995-11-01

    Several forms (isoproteins) of ferredoxin in roots, leaves, and green and red pericarps in tomato plants (Lycopersicon esculentum Mill.) were earlier identified on the basis of N-terminal amino acid sequence and chromatographic behavior (Green et al. 1991). In the present study, a large scale preparation made possible determination of the full length amino acid sequence of the two ferredoxins from leaves. The ferredoxins characteristic of fruit and root were sequenced from the amino terminus to the 30th residue or beyond. The leaf ferredoxins were confirmed to be expressed in pericarp of both green and red fruit. The ferredoxins characteristic of fruit and root appeared to be restricted to those tissue. The results extend earlier findings in demonstrating that ferredoxin occurs in the major organs of the tomato plant where it appears to function irrespective of photosynthetic competence.

  17. Amino acid sequence of myoglobin from white-tailed deer (Odocoileus virginianus).

    PubMed

    Joseph, Poulson; Suman, Surendranath P; Li, Shuting; Fontaine, Michele; Steinke, Laurey

    2012-10-01

    Our objective was to determine the primary structure of white-tailed deer myoglobin (Mb). White-tailed deer Mb was isolated from cardiac muscles employing ammonium sulfate precipitation and gel-filtration chromatography. The amino acid sequence was determined by Edman degradation. Sequence analyses of intact Mb as well as tryptic- and cyanogen bromide-peptides yielded the complete primary structure of white-tailed deer Mb, which shared 100% similarity with red deer Mb. White-tailed deer Mb consists of 153 amino acid residues and shares more than 96% sequence similarity with myoglobins from meat-producing ruminants, such as cattle, buffalo, sheep, and goat. Similar to sheep and goat myoglobins, white-tailed deer Mb contains 12 histidine residues. Proximal (position 93) and distal (position 64) histidine residues responsible for maintaining the stability of heme are conserved in white-tailed deer Mb.

  18. Adiabatic Mass Loss in Binary Stars. II. From Zero-age Main Sequence to the Base of the Giant Branch

    NASA Astrophysics Data System (ADS)

    Ge, Hongwei; Webbink, Ronald F.; Chen, Xuefei; Han, Zhanwen

    2015-10-01

    In the limit of extremely rapid mass transfer, the response of a donor star in an interacting binary becomes asymptotically one of adiabatic expansion. We survey here adiabatic mass loss from Population I stars (Z = 0.02) of mass 0.10 M⊙-100 M⊙ from the zero-age main sequence to the base of the giant branch, or to central hydrogen exhaustion for lower main sequence stars. The logarithmic derivatives of radius with respect to mass along adiabatic mass-loss sequences translate into critical mass ratios for runaway (dynamical timescale) mass transfer, evaluated here under the assumption of conservative mass transfer. For intermediate- and high-mass stars, dynamical mass transfer is preceded by an extended phase of thermal timescale mass transfer as the star is stripped of most of its envelope mass. The critical mass ratio qad (throughout this paper, we follow the convention of defining the binary mass ratio as q ≡ Mdonor/Maccretor) above which this delayed dynamical instability occurs increases with advancing evolutionary age of the donor star, by ever-increasing factors for more massive donors. Most intermediate- or high-mass binaries with nondegenerate accretors probably evolve into contact before manifesting this instability. As they approach the base of the giant branch, however, and begin developing a convective envelope, qad plummets dramatically among intermediate-mass stars, to values of order unity, and a prompt dynamical instability occurs. Among low-mass stars, the prompt instability prevails throughout main sequence evolution, with qad declining with decreasing mass, and asymptotically approaching qad = 2/3, appropriate to a classical isentropic n = 3/2 polytrope. Our calculated qad values agree well with the behavior of time-dependent models by Chen & Han of intermediate-mass stars initiating mass transfer in the Hertzsprung gap. Application of our results to cataclysmic variables, as systems that must be stable against rapid mass transfer, nicely

  19. ADIABATIC MASS LOSS IN BINARY STARS. II. FROM ZERO-AGE MAIN SEQUENCE TO THE BASE OF THE GIANT BRANCH

    SciTech Connect

    Ge, Hongwei; Chen, Xuefei; Han, Zhanwen; Webbink, Ronald F. E-mail: rwebbink@illinois.edu

    2015-10-10

    In the limit of extremely rapid mass transfer, the response of a donor star in an interacting binary becomes asymptotically one of adiabatic expansion. We survey here adiabatic mass loss from Population I stars (Z = 0.02) of mass 0.10 M{sub ⊙}–100 M{sub ⊙} from the zero-age main sequence to the base of the giant branch, or to central hydrogen exhaustion for lower main sequence stars. The logarithmic derivatives of radius with respect to mass along adiabatic mass-loss sequences translate into critical mass ratios for runaway (dynamical timescale) mass transfer, evaluated here under the assumption of conservative mass transfer. For intermediate- and high-mass stars, dynamical mass transfer is preceded by an extended phase of thermal timescale mass transfer as the star is stripped of most of its envelope mass. The critical mass ratio q{sub ad} (throughout this paper, we follow the convention of defining the binary mass ratio as q ≡ M{sub donor}/M{sub accretor}) above which this delayed dynamical instability occurs increases with advancing evolutionary age of the donor star, by ever-increasing factors for more massive donors. Most intermediate- or high-mass binaries with nondegenerate accretors probably evolve into contact before manifesting this instability. As they approach the base of the giant branch, however, and begin developing a convective envelope, q{sub ad} plummets dramatically among intermediate-mass stars, to values of order unity, and a prompt dynamical instability occurs. Among low-mass stars, the prompt instability prevails throughout main sequence evolution, with q{sub ad} declining with decreasing mass, and asymptotically approaching q{sub ad} = 2/3, appropriate to a classical isentropic n = 3/2 polytrope. Our calculated q{sub ad} values agree well with the behavior of time-dependent models by Chen and Han of intermediate-mass stars initiating mass transfer in the Hertzsprung gap. Application of our results to cataclysmic variables, as systems

  20. Nucleotide sequence and the encoded amino acids of human apolipoprotein A-I mRNA.

    PubMed Central

    Law, S W; Brewer, H B

    1984-01-01

    The cDNA clones encoding the precursor form of human liver apolipoprotein A-I (apoA-I), preproapoA-I, have been isolated from a cDNA library. A 17-base synthetic oligonucleotide based on residues 108-113 of apoA-I and a 26-base primer-extended, dideoxynucleotide-terminated cDNA were used as hybridization probes to select for recombinant plasmids bearing the apoA-I sequence. The complete nucleic acid sequence of human liver preproapoA-I has been determined by analysis of the cloned cDNA. The sequence is composed of 801 nucleotides encoding 267 amino acid residues. PreproapoA-I contains an 18-amino-acid prepeptide and a 6-amino-acid propeptide connected to the amino terminus of the 243-amino acid mature apoA-I. Southern blotting analysis of chromosomal DNA obtained from peripheral blood indicated the apoA-I gene is contained in a 2.1-kilobase-pair Pst I fragment and there is no gross difference in structural organization between the normal apoA-I gene and the Tangier disease apoA-I gene. Images PMID:6198645

  1. Mathematical Characterization of Protein Sequences Using Patterns as Chemical Group Combinations of Amino Acids.

    PubMed

    Das, Jayanta Kumar; Das, Provas; Ray, Korak Kumar; Choudhury, Pabitra Pal; Jana, Siddhartha Sankar

    2016-01-01

    Comparison of amino acid sequence similarity is the fundamental concept behind the protein phylogenetic tree formation. By virtue of this method, we can explain the evolutionary relationships, but further explanations are not possible unless sequences are studied through the chemical nature of individual amino acids. Here we develop a new methodology to characterize the protein sequences on the basis of the chemical nature of the amino acids. We design various algorithms for studying the variation of chemical group transitions and various chemical group combinations as patterns in the protein sequences. The amino acid sequence of conventional myosin II head domain of 14 family members are taken to illustrate this new approach. We find two blocks of maximum length 6 aa as 'FPKATD' and 'Y/FTNEKL' without repeating the same chemical nature and one block of maximum length 20 aa with the repetition of chemical nature which are common among all 14 members. We also check commonality with another motor protein sub-family kinesin, KIF1A. Based on our analysis we find a common block of length 8 aa both in myosin II and KIF1A. This motif is located in the neck linker region which could be responsible for the generation of mechanical force, enabling us to find the unique blocks which remain chemically conserved across the family. We also validate our methodology with different protein families such as MYOI, Myosin light chain kinase (MLCK) and Rho-associated protein kinase (ROCK), Na+/K+-ATPase and Ca2+-ATPase. Altogether, our studies provide a new methodology for investigating the conserved amino acids' pattern in different proteins.

  2. Dihydrofolate reductase: low-resolution mass-spectrometric analysis of an elastase digest as a sequencing tool (Short Communication)

    PubMed Central

    Morris, Howard R.; Batley, Karen E.; Harding, Nigel G. L.; Bjur, Richard A.; Dann, John G.; King, Rodney W.

    1974-01-01

    An elastase digest of a protein of unknown structure, dihydrofolate reductase, was studied by mass spectrometry. This soluble digest contained a large number of small peptides in different yields, within the ideal molecular-weight range (200–1200) for mixture-analysis mass spectrometry. Sequences of the major component peptides in the digest are reported. PMID:4207389

  3. The simultaneous quantitation of ten amino acids in soil extracts by mass fragmentography

    NASA Technical Reports Server (NTRS)

    Pereira, W. E.; Hoyano, Y.; Reynolds, W. E.; Summons, R. E.; Duffield, A. M.

    1972-01-01

    A specific and sensitive method for the identification and simultaneous quantitation by mass fragmentography of ten of the amino acids present in soil was developed. The technique uses a computer driven quadrupole mass spectrometer and a commercial preparation of deuterated amino acids is used as internal standards for purposes of quantitation. The results obtained are comparable with those from an amino acid analyzer. In the quadrupole mass spectrometer-computer system up to 25 pre-selected ions may be monitored sequentially. This allows a maximum of 12 different amino acids (one specific ion in each of the undeuterated and deuterated amino acid spectra) to be quantitated. The method is relatively rapid (analysis time of approximately one hour) and is capable of the quantitation of nanogram quantities of amino acids.

  4. Solid phase sequencing of biopolymers

    SciTech Connect

    Cantor, Charles R.; Hubert, Koster

    2014-06-24

    This invention relates to methods for detecting and sequencing target nucleic acid sequences, to mass modified nucleic acid probes and arrays of probes useful in these methods, and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probes comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Probes may be affixed to a solid support such as a hybridization chip to facilitate automated molecular weight analysis and identification of the target sequence.

  5. Software scripts for quality checking of high-throughput nucleic acid sequencers.

    PubMed

    Lazo, G R; Tong, J; Miller, R; Hsia, C; Rausch, C; Kang, Y; Anderson, O D

    2001-06-01

    We have developed a graphical interface to allow the researcher to view and assess the quality of sequencing results using a series of program scripts developed to process data generated by automated sequencers. The scripts are written in Perl programming language and are executable under the cgibin directory of a Web server environment. The scripts direct nucleic acid sequencing trace file data output from automated sequencers to be analyzed by the phred molecular biology program and are displayed as graphical hypertext mark-up language (HTML) pages. The scripts are mainly designed to handle 96-well microtiter dish samples, but the scripts are also able to read data from 384-well microtiter dishes 96 samples at a time. The scripts may be customized for different laboratory environments and computer configurations. Web links to the sources and discussion page are provided.

  6. Amino acid sequence of myoglobin from the chiton Liolophura japonica and a phylogenetic tree for molluscan globins.

    PubMed

    Suzuki, T; Furukohri, T; Okamoto, S

    1993-02-01

    Myoglobin was isolated from the radular muscle of the chiton Liolophura japonica, a primitive archigastropodic mollusc. Liolophura contains three monomeric myoglobins (I, II, and III), and the complete amino acid sequence of myoglobin I has been determined. It is composed of 145 amino acid residues, and the molecular mass was calculated to be 16,070 D. The E7 distal histidine, which is replaced by valine or glutamine in several molluscan globins, is conserved in Liolophura myoglobin. The autoxidation rate at physiological conditions indicated that Liolophura oxymyoglobin is fairly stable when compared with other molluscan myoglobins. The amino acid sequence of Liolophura myoglobin shows low homology (11-21%) with molluscan dimeric myoglobins and hemoglobins, but shows higher homology (26-29%) with monomeric myoglobins from the gastropodic molluscs Aplysia, Dolabella, and Bursatella. A phylogenetic tree was constructed from 19 molluscan globin sequences. The tree separated them into two distinct clusters, a cluster for muscle myoglobins and a cluster for erythrocyte or gill hemoglobins. The myoglobin cluster is divided further into two subclusters, corresponding to monomeric and dimeric myoglobins, respectively. Liolophura myoglobin was placed on the branch of monomeric myoglobin lineage, showing that it diverged earlier from other monomeric myoglobins. The hemoglobin cluster is also divided into two subclusters. One cluster contains homodimeric, heterodimeric, tetrameric, and didomain chains of erythrocyte hemoglobins of the blood clams Anadara, Scapharca, and Barbatia. Of special interest is the other subcluster. It consists of three hemoglobin chains derived from the bacterial symbiontharboring clams Calyptogena and Lucina, in which hemoglobins are supposed to play an important role in maintaining the symbiosis with sulfide bacteria.

  7. Identification of metallothionein subisoforms in HPLC using accurate mass and online sequencing by electrospray hybrid linear ion trap-orbital ion trap mass spectrometry.

    PubMed

    Mounicou, Sandra; Ouerdane, Laurent; L'Azou, Béatrice; Passagne, Isabelle; Ohayon-Courtès, Céline; Szpunar, Joanna; Lobinski, Ryszard

    2010-08-15

    A comprehensive approach to the characterization of metallothionein (MT) isoforms based on microbore HPLC with multimodal detection was developed. MTs were separated as Cd(7) complexes, detected by ICP MS and tentatively identified by molecular mass measured with 1-2 ppm accuracy using Orbital ion trap mass spectrometry. The identification was validated by accurate mass of the corresponding apo-MTs after postcolumn acidification and by their sequences acquired online by higher-energy collision dissociation MS/MS. The detection limits down to 10 fmol and 45 fmol could be obtained by ESI MS for apo- and Cd(7)-isoforms, respectively, and were lower than those obtained by ICP MS (100 fmol). The individual MT isoforms could be sequenced at levels as low as 200 fmol with the sequence coverage exceeding 90%. The approach was successfully applied to the identification of MT isoforms induced in a pig kidney cell line (LLC-PK(1)) exposed to CdS nanoparticles.

  8. Identification of protein SUMOylation sites by mass spectrometry using combined microwave-assisted aspartic acid cleavage and tryptic digestion

    PubMed Central

    Osula, Omoruyi; Swatkoski, Stephen; Cotter, Robert J.

    2012-01-01

    SUMO (Small-Ubiquitin-like MOdifier) is a post-translational modifier of protein substrates at lysine residues that conjugates to proteins in response to various changes in the cell. As a result of SUMO modification, marked changes in transcription regulation, DNA repair, subcellular localization, and mitosis, among other cellular processes, are known to occur. However, while the identification of ubiquitylation sites by mass spectrometry is aided in part by the presence of a small di-amino acid GlyGly “tag” that remains on lysine residues following tryptic digestion, SUMOylation poses a particular challenge as the absence of a basic residue near to the SUMO C-terminus results in a significant 27 or 32 amino acid sequence branch conjugated to the substrate peptide. MS/MS analyses of these branch peptides generally reveal abundant fragment ions resulting from cleavage of the SUMO tail, but which obscure those needed for characterizing the target peptide sequence. Other approaches for identifying SUMO substrates exist and include overexpression of the SUMO isoforms using an N-terminal histidine tag, as well as site-directed mutagenesis of the C-terminal end of the SUMO sequence. Here, we employ combined enzymatic/chemical approaches which serve to shorten the SUMO tag, and thus help to simplify SUMO spectra, making interpretation of mass spectra and location of the SUMOylation site easier. As described in this report, we demonstrate a method for identifying SUMOylation sites using three commercially available SUMO- modified isoforms, and by employing acid-only and acid/trypsin cleavage strategies. These approaches were carried out using MALDI-TOF and LC/MS instrumentation, along with CID and ETD fragmentation. PMID:22576878

  9. Preparation of Nucleic Acid Libraries for Personalized Sequencing Systems Using an Integrated Microfluidic Hub Technology (Seventh Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting 2012)

    ScienceCinema

    Patel, Kamlesh D [Ken; SNL,

    2016-07-12

    Kamlesh (Ken) Patel from Sandia National Laboratories (Livermore, California) presents "Preparation of Nucleic Acid Libraries for Personalized Sequencing Systems Using an Integrated Microfluidic Hub Technology " at the 7th Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting held in June, 2012 in Santa Fe, NM.

  10. Enhanced Digestion Efficiency, Peptide Ionization Efficiency, and Sequence Resolution for Protein Hydrogen/Deuterium Exchange Monitored by FT-ICR Mass Spectrometry

    PubMed Central

    Zhang, Hui-Min; Kazazic, Saša; Schaub, Tanner M.; Tipton, Jeremiah D.; Emmett, Mark R.; Marshall, Alan G.

    2009-01-01

    Solution-phase hydrogen/deuterium exchange (HDX) monitored by high-resolution FT-ICR mass spectrometry offers a rapid method to study protein conformations and protein-protein interactions. Pepsin is usually used to digest proteins in HDX and is known as lack of cleavage specificity. To improve digestion efficiency and specificity, we have optimized digestion conditions and cleavage preferences for pepsin and protease type XIII from Aspergillus saitoi. A dilution series of the proteases was used to determine the digestion efficiency for several test proteins. Protease type XIII prefers to cleave on the C-terminal end of basic amino acids and produced the highest number of fragments and the best sequence coverage compared to pepsin or protease type XVIII from Rhizhopus. Furthermore, protease type XIII exhibited much less self-digestion than pepsin, and thus is superior for HDX experiments. Many highly overlapped segments from protease type XIII and pepsin digestion, combined with high-resolution FT-ICR mass spectrometry, provide high sequence resolution (to as few as one or two amino acids) for the assignment of amide hydrogen exchange rate. Our H/D exchange results correlate well with the secondary and tertiary structure of myoglobin. Such assignments of highly overlapped fragments promise to greatly enhance the accuracy and sequence resolution for determining conformational differences resulting from ligand binding or protein-protein interactions. PMID:19551977

  11. Role of the two-component leader sequence and mature amino acid sequences in extracellular export of endoglucanase EGL from Pseudomonas solanacearum.

    PubMed Central

    Huang, J Z; Schell, M A

    1992-01-01

    The egl gene of Pseudomonas solanacearum encodes a 43-kDa extracellular endoglucanase (mEGL) involved in wilt disease caused by this phytopathogen. Egl is initially translated with a 45-residue, two-part leader sequence. The first 19 residues are apparently removed by signal peptidase II during export of Egl across the inner membrane (IM); the remaining residues of the leader sequence (modified with palmitate) are removed during export across the outer membrane (OM). Localization of Egl-PhoA fusion proteins showed that the first 26 residues of the Egl leader sequence are required and sufficient to direct lipid modification, processing, and export of Egl or PhoA across the IM but not the OM. Fusions of the complete 45-residue leader sequence or of the leader and increasing portions of mEgl sequences to PhoA did not cause its export across the OM. In-frame deletion of portions of mEGL-coding sequences blocked export of the truncated polypeptides across the OM without affecting export across the IM. These results indicate that the first part of the leader sequence functions independently to direct export of Egl across the IM while the second part and sequences and structures in mEGL are involved in export across the OM. Computer analysis of the mEgl amino acid sequence obtained from its nucleotide sequence identified a region of mEGL similar in amino acid sequence to regions in other prokaryotic endoglucanases. Images PMID:1735723

  12. Studies on adenosine triphosphate transphosphorylases. Amino acid sequence of rabbit muscle ATP-AMP transphosphorylase.

    PubMed

    Kuby, S A; Palmieri, R H; Frischat, A; Fischer, A H; Wu, L H; Maland, L; Manship, M

    1984-05-22

    The total amino acid sequence of rabbit muscle adenylate kinase has been determined, and the single polypeptide chain of 194 amino acid residues starts with N-acetylmethionine and ends with leucyllysine at its carboxyl terminus, in agreement with the earlier data on its amino acid composition [Mahowald, T. A., Noltmann, E. A., & Kuby, S. A. (1962) J. Biol. Chem. 237, 1138-1145] and its carboxyl-terminus sequence [Olson, O. E., & Kuby, S. A. (1964) J. Biol. Chem. 239, 460-467]. Elucidation of the primary structure was based on tryptic and chymotryptic cleavages of the performic acid oxidized protein, cyanogen bromide cleavages of the 14C-labeled S-carboxymethylated protein at its five methionine sites (followed by maleylation of peptide fragments), and tryptic cleavages at its 12 arginine sites of the maleylated 14C-labeled S-carboxymethylated protein. Calf muscle myokinase, whose sequence has also been established, differs primarily from the rabbit muscle myokinase's sequence in the following: His-30 is replaced by Gln-30; Lys-56 is replaced by Met-56; Ala-84 and Asp 85 are replaced by Val-84 and Asn-85. A comparison of the four muscle-type adenylate kinases, whose covalent structures have now been determined, viz., rabbit, calf, porcine, and human [for the latter two sequences see Heil, A., Müller, G., Noda, L., Pinder, T., Schirmer, H., Schirmer, I., & Von Zabern, I. (1974) Eur. J. Biochem. 43, 131-144, and Von Zabern, I., Wittmann-Liebold, B., Untucht-Grau, R., Schirmer, R. H., & Pai, E. F. (1976) Eur. J. Biochem. 68, 281-290], demonstrates an extraordinary degree of homology.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Mathematical Characterization of Protein Sequences Using Patterns as Chemical Group Combinations of Amino Acids

    PubMed Central

    Choudhury, Pabitra Pal; Jana, Siddhartha Sankar

    2016-01-01

    Comparison of amino acid sequence similarity is the fundamental concept behind the protein phylogenetic tree formation. By virtue of this method, we can explain the evolutionary relationships, but further explanations are not possible unless sequences are studied through the chemical nature of individual amino acids. Here we develop a new methodology to characterize the protein sequences on the basis of the chemical nature of the amino acids. We design various algorithms for studying the variation of chemical group transitions and various chemical group combinations as patterns in the protein sequences. The amino acid sequence of conventional myosin II head domain of 14 family members are taken to illustrate this new approach. We find two blocks of maximum length 6 aa as ‘FPKATD’ and ‘Y/FTNEKL’ without repeating the same chemical nature and one block of maximum length 20 aa with the repetition of chemical nature which are common among all 14 members. We also check commonality with another motor protein sub-family kinesin, KIF1A. Based on our analysis we find a common block of length 8 aa both in myosin II and KIF1A. This motif is located in the neck linker region which could be responsible for the generation of mechanical force, enabling us to find the unique blocks which remain chemically conserved across the family. We also validate our methodology with different protein families such as MYOI, Myosin light chain kinase (MLCK) and Rho-associated protein kinase (ROCK), Na+/K+-ATPase and Ca2+-ATPase. Altogether, our studies provide a new methodology for investigating the conserved amino acids’ pattern in different proteins. PMID:27930687

  14. Deduced amino acid sequence of human pulmonary surfactant proteolipid: SPL(pVal)

    SciTech Connect

    Whitsett, J.A.; Glasser, S.W.; Korfhagen, T.R.; Weaver, T.E.; Clark, J.; Pilot-Matias, T.; Meuth, J.; Fox, J.L.

    1987-05-01

    Hydrophobic, proteolipid-like protein of Mr 6500 was isolated from ether/ethanol extracts of human, canine and bovine pulmonary surfactant. Amino acid composition of the protein demonstrated a remarkable abundance of hydrophobic residues, particularly valine and leucine. The N-terminal amino acid sequence of the human protein was determined: N-Leu-Ile-Pro-Cys-Cys-Pro-Val-Asn-Leu-Lys-Arg-Leu-Leu-Ile-Val4... An oligonucleotide probe was used to screen an adult human lung cDNA library and resulted in detection of cDNA clones with predicted amino acid sequence with close identity to the N-terminal amino acid sequence of the human peptide. SPL(pVal) was found within the reading frame of a larger peptide. SPL(pVal) results from proteolytic processing of a larger preprotein. Northern blot analysis detected in a single 1.0 kilobase SPL(pVal) RNA which was less abundant in fetal than in adult lung. Mixtures of purified canine and bovine SPL(pVal) and synthetic phospholipids display properties of rapid adsorption and surface tension lowering activity characteristic of surfactant. Human SPL(pVal) is a pulmonary surfactant proteolipid which may therefore be useful in combination with phospholipids and/or other surfactant proteins for the treatment of surfactant deficiency such as hyaline membrane disease in newborn infants.

  15. Use of Composite Protein Database including Search Result Sequences for Mass Spectrometric Analysis of Cell Secretome

    PubMed Central

    Shin, Jihye; Kim, Gamin; Kabir, Mohammad Humayun; Park, Seong Jun; Lee, Seoung Taek; Lee, Cheolju

    2015-01-01

    Mass spectrometric (MS) data of human cell secretomes are usually run through the conventional human database for identification. However, the search may result in false identifications due to contamination of the secretome with fetal bovine serum (FBS) proteins. To overcome this challenge, here we provide a composite protein database including human as well as 199 FBS protein sequences for MS data search of human cell secretomes. Searching against the human-FBS database returned more reliable results with fewer false-positive and false-negative identifications compared to using either a human only database or a human-bovine database. Furthermore, the improved results validated our strategy without complex experiments like SILAC. We expect our strategy to improve the accuracy of human secreted protein identification and to also add value for general use. PMID:25822838

  16. Rapid on-membrane proteolytic cleavage for Edman sequencing and mass spectrometric identification of proteins.

    PubMed

    Pham, Victoria C; Henzel, William J; Lill, Jennie R

    2005-11-01

    A method for the rapid limited enzymatic cleavage of PVDF membrane-immobilized proteins is described. This method allows the fast characterization of PVDF blotted proteins by peptide mass fingerprinting (Henzel, W. J., Billeci, T. M., Stults, J. T., Wong, S. C., Grimley, C., Wantanabe, C., Proc. Natl. Acad. Sci. USA 1993, 90, 5011-5015), LC-MS/MS, or N-terminal sequencing and has been demonstrated on a range of proteins using a full complement of proteolytic enzymes. This technique allows the generation of proteolytic fragments between 5 and 60 min (depending on the enzyme employed), which is significantly faster than previously reported on-membrane digestion methods. To date, this on-membrane rapid digestion protocol has aided in the identification and confirmation of mutation sites in over 200 recombinant proteins.

  17. SUBGROUPS OF AMINO ACID SEQUENCES IN THE VARIABLE REGIONS OF IMMUNOGLOBULIN HEAVY CHAINS*

    PubMed Central

    Cunningham, Bruce A.; Pflumm, Mollie N.; User, Urs Rutisha; Edelman, Gerald M.

    1969-01-01

    The amino acid sequence of the first 133 residues of the heavy (γ) chain from a human γG immunoglobulin (He) has been determined. This γ-chain is identical in Gm type to that of protein Eu, the complete sequence of which has been reported. Comparison of the two sequences substantiates the previous suggestion that there are subgroups of variable regions of heavy chains. The variable region of Eu has been assigned to subgroup I and that of He to subgroup II; on the other hand, the constant regions of the two proteins appear to be identical. Comparison of the sequence of the heavy chain of He with the heavy chain sequences determined in other laboratories suggests that the variable region of subgroup II is at least 118 residues long. The nature and distribution of amino acid variations in this heavy chain subgroup resemble those observed in light chain subgroups. These studies provide evidence that the translocation hypothesis applies to heavy as well as to light chains, viz., genes for variable regions (V) are somatically translocated to genes for constant regions (C) to form complete VC structural genes. Images PMID:5264153

  18. Complete nucleic acid sequence of Penaeus stylirostris densovirus (PstDNV) from India.

    PubMed

    Rai, Praveen; Safeena, Muhammed P; Karunasagar, Iddya; Karunasagar, Indrani

    2011-06-01

    Infectious hypodermal and hematopoietic necrosis virus (IHHNV) of shrimp, recently been classified as Penaeus stylirostris densovirus (PstDNV). The complete nucleic acid sequence of PstDNV from India was obtained by cloning and sequencing of different DNA fragment of the virus. The genome organisation of PstDNV revealed that there were three major coding domains: a left ORF (NS1) of 2001 bp, a mid ORF (NS2) of 1092 bp and a right ORF (VP) of 990 bp. The complete genome and amino acid sequences of three proteins viz., NS1, NS2 and VP were compared with the genomes of the virus reported from Hawaii, China and Mexico and with partial sequence available from isolates from different regions. The phylogenetic analysis of shrimp, insect and vertebrate parvovirus sequences showed that the Indian PstDNV isolate is phylogenetically more closely related to one of the three isolates from Taiwan (AY355307), and two isolates (AY362547 and AY102034) from Thailand.

  19. L-Rhamnose-binding lectin from eggs of the Echinometra lucunter: Amino acid sequence and molecular modeling.

    PubMed

    Carneiro, Rômulo Farias; Teixeira, Claudener Souza; de Melo, Arthur Alves; de Almeida, Alexandra Sampaio; Cavada, Benildo Sousa; de Sousa, Oscarina Viana; da Rocha, Bruno Anderson Matias; Nagano, Celso Shiniti; Sampaio, Alexandre Holanda

    2015-01-01

    An L-rhamnose-binding lectin named ELEL was isolated from eggs of the rock boring sea urchin Echinometra lucunter by affinity chromatography on lactosyl-agarose. ELEL is a homodimer linked by a disulfide bond with subunits of 11 kDa each. The new lectin was inhibited by saccharides possessing the same configuration of hydroxyl groups at C-2 and C-4, such as L-rhamnose, melibiose, galactose and lactose. The amino acid sequence of ELEL was determined by tandem mass spectrometry. The ELEL subunit has 103 amino acids, including nine cysteine residues involved in four conserved intrachain disulfide bonds and one interchain disulfide bond. The full sequence of ELEL presents conserved motifs commonly found in rhamnose-binding lectins, including YGR, DPC and KYL. A three-dimensional model of ELEL was created, and molecular docking revealed favorable binding energies for interactions between ELEL and rhamnose, melibiose and Gb3 (Galα1-4Galβ1-4Glcβ1-Cer). Furthermore, ELEL was able to agglutinate Gram-positive bacterial cells, suggesting its ability to recognize pathogens.

  20. Extending the Substellar Sequence to Planetary Masses with Distances from Spitzer Parallaxes

    NASA Astrophysics Data System (ADS)

    Dupuy, Trent

    A major goal in contemporary astrophysics is to extend the study of stellar atmospheres to the much cooler atmospheres of gas-giant extrasolar planets. One key pathway is the identification of free-floating substellar objects that not only share common temperatures with exoplanets but also common masses and thus surface gravities. In recent years, searches for ever colder freefloating objects have steadily pushed the census to ever lower masses and finally into this longsought planetary-mass regime (5-15 Jupiter masses; Y dwarfs). The empirical backbone of the exploration of these new substellar samples is the accurate measurement of distances via trigonometric parallaxes. Distances allow us to make empirical sequences of absolute magnitude versus color and spectral type to compare to better studied normal brown dwarfs, as well as providing absolute bolometric fluxes and thereby luminosities and temperatures. However, unlike simple photometry or spectroscopy, parallax measurements are time consuming and prone to large systematic errors when attempted by combining data from multiple telescopes and bandpasses. Spitzer/IRAC is poised to deliver the definitive sample of parallax distances for the coldest brown dwarfs. It is the only facility capable of measuring high-precision astrometry efficiently for a large sample of such faint substellar objects, as validated by our past work. Spitzer was never intended to deliver high quality astrometry, but using our custom built astrometric pipeline we have successfully produced the first precise IRAC astrometry and measured distances for a small initial sample of the coldest brown dwarfs (Dupuy & Kraus 2013). We showed that in spite of the seemingly subtle spectral changes from T8 to Y0 (only 2 subtypes), the mean effective temperature plummets by almost a factor of 2, from 700 K for T8 dwarfs to 400 K for Y0 dwarfs. Even in this small sample, we found some objects that seemed to defy the expectation that later spectral type

  1. DNA Cloning of Plasmodium falciparum Circumsporozoite Gene: Amino Acid Sequence of Repetitive Epitope

    NASA Astrophysics Data System (ADS)

    Enea, Vincenzo; Ellis, Joan; Zavala, Fidel; Arnot, David E.; Asavanich, Achara; Masuda, Aoi; Quakyi, Isabella; Nussenzweig, Ruth S.

    1984-08-01

    A clone of complementary DNA encoding the circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum has been isolated by screening an Escherichia coli complementary DNA library with a monoclonal antibody to the CS protein. The DNA sequence of the complementary DNA insert encodes a four-amino acid sequence: proline-asparagine-alanine-asparagine, tandemly repeated 23 times. The CS β -lactamase fusion protein specifically binds monoclonal antibodies to the CS protein and inhibits the binding of these antibodies to native Plasmodium falciparum CS protein. These findings provide a basis for the development of a vaccine against Plasmodium falciparum malaria.

  2. Amino-Acid Sequence of NADP-Specific Glutamate Dehydrogenase of Neurospora crassa

    PubMed Central

    Wootton, John C.; Chambers, Geoffrey K.; Holder, Anthony A.; Baron, Andrew J.; Taylor, John G.; Fincham, John R. S.; Blumenthal, Kenneth M.; Moon, Kenneth; Smith, Emil L.

    1974-01-01

    A tentative primary structure of the NADP-specific glutamate dehydrogenase [L-glutamate: NADP oxidoreductase (deaminating), EC 1.4.1.4] from Neurospora crassa has been determined. The proposed sequence contains 452 amino-acid residues in each of the identical subunits of the hexameric enzyme. Comparison of the sequence with that of the bovine liver enzyme reveals considerable homology in the amino-terminal portion of the chain, including the vicinity of the reactive lysine, with only shorter stretches of homology within the carboxyl-terminal regions. The significance of this distribution of homologous regions is discussed. PMID:4155068

  3. Seq2Logo: a method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion

    PubMed Central

    Thomsen, Martin Christen Frølund; Nielsen, Morten

    2012-01-01

    Seq2Logo is a web-based sequence logo generator. Sequence logos are a graphical representation of the information content stored in a multiple sequence alignment (MSA) and provide a compact and highly intuitive representation of the position-specific amino acid composition of binding motifs, active sites, etc. in biological sequences. Accurate generation of sequence logos is often compromised by sequence redundancy and low number of observations. Moreover, most methods available for sequence logo generation focus on displaying the position-specific enrichment of amino acids, discarding the equally valuable information related to amino acid depletion. Seq2logo aims at resolving these issues allowing the user to include sequence weighting to correct for data redundancy, pseudo counts to correct for low number of observations and different logotype representations each capturing different aspects related to amino acid enrichment and depletion. Besides allowing input in the format of peptides and MSA, Seq2Logo accepts input as Blast sequence profiles, providing easy access for non-expert end-users to characterize and identify functionally conserved/variable amino acids in any given protein of interest. The output from the server is a sequence logo and a PSSM. Seq2Logo is available at http://www.cbs.dtu.dk/biotools/Seq2Logo (14 May 2012, date last accessed). PMID:22638583

  4. Seq2Logo: a method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion.

    PubMed

    Thomsen, Martin Christen Frølund; Nielsen, Morten

    2012-07-01

    Seq2Logo is a web-based sequence logo generator. Sequence logos are a graphical representation of the information content stored in a multiple sequence alignment (MSA) and provide a compact and highly intuitive representation of the position-specific amino acid composition of binding motifs, active sites, etc. in biological sequences. Accurate generation of sequence logos is often compromised by sequence redundancy and low number of observations. Moreover, most methods available for sequence logo generation focus on displaying the position-specific enrichment of amino acids, discarding the equally valuable information related to amino acid depletion. Seq2logo aims at resolving these issues allowing the user to include sequence weighting to correct for data redundancy, pseudo counts to correct for low number of observations and different logotype representations each capturing different aspects related to amino acid enrichment and depletion. Besides allowing input in the format of peptides and MSA, Seq2Logo accepts input as Blast sequence profiles, providing easy access for non-expert end-users to characterize and identify functionally conserved/variable amino acids in any given protein of interest. The output from the server is a sequence logo and a PSSM. Seq2Logo is available at http://www.cbs.dtu.dk/biotools/Seq2Logo (14 May 2012, date last accessed).

  5. Proteomics of Soil and Sediment: Protein Identification by De Novo Sequencing of Mass Spectra Complements Traditional Database Searching

    NASA Astrophysics Data System (ADS)

    Miller, S.; Rizzo, A. I.; Waldbauer, J.

    2014-12-01

    Proteomics has the potential to elucidate the metabolic pathways and taxa responsible for in situ biogeochemical transformations. However, low rates of protein identification from high resolution mass spectra have been a barrier to the development of proteomics in complex environmental samples. Much of the difficulty lies in the computational challenge of linking mass spectra to their corresponding proteins. Traditional database search methods for matching peptide sequences to mass spectra are often inadequate due to the complexity of environmental proteomes and the large database search space, as we demonstrate with soil and sediment proteomes generated via a range of extraction methods. One alternative to traditional database searching is de novo sequencing, which identifies peptide sequences without the need for a database. BLAST can then be used to match de novo sequences to similar genetic sequences. Assigning confidence to putative identifications has been one hurdle for the implementation of de novo sequencing. We found that accurate de novo sequences can be screened by quality score and length. Screening criteria are verified by comparing the results of de novo sequencing and traditional database searching for well-characterized proteomes from simple biological systems. The BLAST hits of screened sequences are interrogated for taxonomic and functional information. We applied de novo sequencing to organic topsoil and marine sediment proteomes. Peak-rich proteomes, which can result from various extraction techniques, yield thousands of high-confidence protein identifications, an improvement over previous proteomic studies of soil and sediment. User-friendly software tools for de novo metaproteomics analysis have been developed. This "De Novo Analysis" Pipeline is also a faster method of data analysis than constructing a tailored sequence database for traditional database searching.

  6. Proteomics of Soil and Sediment: Protein Identification by De Novo Sequencing of Mass Spectra Complements Traditional Database Searching

    NASA Astrophysics Data System (ADS)

    Miller, S.; Rizzo, A. I.; Waldbauer, J.

    2015-12-01

    Proteomics has the potential to elucidate the metabolic pathways and taxa responsible for in situ biogeochemical transformations. However, low rates of protein identification from high resolution mass spectra have been a barrier to the development of proteomics in complex environmental samples. Much of the difficulty lies in the computational challenge of linking mass spectra to their corresponding proteins. Traditional database search methods for matching peptide sequences to mass spectra are often inadequate due to the complexity of environmental proteomes and the large database search space, as we demonstrate with soil and sediment proteomes generated via a range of extraction methods. One alternative to traditional database searching is de novo sequencing, which identifies peptide sequences without the need for a database. BLAST can then be used to match de novo sequences to similar genetic sequences. Assigning confidence to putative identifications has been one hurdle for the implementation of de novo sequencing. We found that accurate de novo sequences can be screened by quality score and length. Screening criteria are verified by comparing the results of de novo sequencing and traditional database searching for well-characterized proteomes from simple biological systems. The BLAST hits of screened sequences are interrogated for taxonomic and functional information. We applied de novo sequencing to organic topsoil and marine sediment proteomes. Peak-rich proteomes, which can result from various extraction techniques, yield thousands of high-confidence protein identifications, an improvement over previous proteomic studies of soil and sediment. User-friendly software tools for de novo metaproteomics analysis have been developed. This "De Novo Analysis" Pipeline is also a faster method of data analysis than constructing a tailored sequence database for traditional database searching.

  7. Isolation, amino acid sequence and biological characterization of an "aspartic-49" phospholipase A₂ from Bothrops (Rhinocerophis) ammodytoides venom.

    PubMed

    Clement, Herlinda; Costa de Oliveira, Vanessa; Zamudio, Fernando Z; Lago, Néstor R; Valdez-Cruz, Norma A; Bérnard Valle, Melisa; Hajos, Silvia E; Alagón, Alejandro; Possani, Lourival D; de Roodt, Adolfo R

    2012-12-01

    A phospholipase enzyme was separated by chromatography from the venom of the snake Bothrops (Rhinocerophis) ammodytoides and characterized. The experimentally determined molecular weight was 13,853.65 Da, and the full primary structure was determined by Edman degradation and mass spectrometry analysis. The enzyme contains 122 amino acids residues closely stabilized by 7 disulfide bridges with an isoelectric point of 6.13. Sequence comparison with other known secretory PLA2 shows that the enzyme isolated belongs to the group II, presenting an aspartic acid residue at position 48 (numbered by convention as Asp49) of the active site, and accordingly displaying enzymatic activity. The enzyme corresponds to 3% of the total mass of the venom. The enzyme is mildly toxic to mice. The intravenous LD₅₀ of this phospholipase in CD-1 mice was around 6 μg/g of mouse body weight (more exactly 117 μg/mouse of 20 g) and the minimal mortal dose (MMD) was estimated to be close to 10 μg/g. In contrast, the LD₅₀ of the venom was circa 2 μg/g mouse body weight. Toxicological analyses of the purified enzyme were performed in vitro and in vivo using experimental animals (mice and rats). The enzyme at high doses caused pulmonary congestion, intraperitoneal bleeding, inhibition of clot retraction and muscle tissue alterations with increasing of creatine kinase levels.

  8. Method for high-volume sequencing of nucleic acids: random and directed priming with libraries of oligonucleotides

    DOEpatents

    Studier, F.W.

    1995-04-18

    Random and directed priming methods for determining nucleotide sequences by enzymatic sequencing techniques, using libraries of primers of lengths 8, 9 or 10 bases, are disclosed. These methods permit direct sequencing of nucleic acids as large as 45,000 base pairs or larger without the necessity for subcloning. Individual primers are used repeatedly to prime sequence reactions in many different nucleic acid molecules. Libraries containing as few as 10,000 octamers, 14,200 nonamers, or 44,000 decamers would have the capacity to determine the sequence of almost any cosmid DNA. Random priming with a fixed set of primers from a smaller library can also be used to initiate the sequencing of individual nucleic acid molecules, with the sequence being completed by directed priming with primers from the library. In contrast to random cloning techniques, a combined random and directed priming strategy is far more efficient. 2 figs.

  9. Method for high-volume sequencing of nucleic acids: random and directed priming with libraries of oligonucleotides

    DOEpatents

    Studier, F. William

    1995-04-18

    Random and directed priming methods for determining nucleotide sequences by enzymatic sequencing techniques, using libraries of primers of lengths 8, 9 or 10 bases, are disclosed. These methods permit direct sequencing of nucleic acids as large as 45,000 base pairs or larger without the necessity for subcloning. Individual primers are used repeatedly to prime sequence reactions in many different nucleic acid molecules. Libraries containing as few as 10,000 octamers, 14,200 nonamers, or 44,000 decamers would have the capacity to determine the sequence of almost any cosmid DNA. Random priming with a fixed set of primers from a smaller library can also be used to initiate the sequencing of individual nucleic acid molecules, with the sequence being completed by directed priming with primers from the library. In contrast to random cloning techniques, a combined random and directed priming strategy is far more efficient.

  10. Sequence-specific thermodynamic properties of nucleic acids influence both transcriptional pausing and backtracking in yeast

    PubMed Central

    2017-01-01

    RNA Polymerase II pauses and backtracks during transcription, with many consequences for gene expression and cellular physiology. Here, we show that the energy required to melt double-stranded nucleic acids in the transcription bubble predicts pausing in Saccharomyces cerevisiae far more accurately than nucleosome roadblocks do. In addition, the same energy difference also determines when the RNA polymerase backtracks instead of continuing to move forward. This data-driven model corroborates—in a genome wide and quantitative manner—previous evidence that sequence-dependent thermodynamic features of nucleic acids influence both transcriptional pausing and backtracking. PMID:28301878

  11. Characterization of N,N-dimethyl amino acids by electrospray ionization-tandem mass spectrometry.

    PubMed

    Naresh Chary, V; Sudarshana Reddy, B; Kumar, Ch Dinesh; Srinivas, R; Prabhakar, S

    2015-05-01

    Methylation is an essential metabolic process for a number of critical reactions in the body. Methyl groups are involved in the healthy function of the body life processes, by conducting methylation process involving specific enzymes. In these processes, various amino acids are methylated, and the occurrence of methylated amino acids in nature is diverse. Nowadays, mass-spectrometric-based identification of small molecules as biomarkers for diseases is a growing research. Although all dimethyl amino acids are metabolically important molecules, mass spectral data are available only for a few of them in the literature. In this study, we report synthesis and characterization of all dimethyl amino acids, by electrospray ionization-tandem mass spectrometry (MS/MS) experiments on protonated molecules. The MS/MS spectra of all the studied dimethyl amino acids showed preliminary loss of H2O + CO to form corresponding immonium ions. The other product ions in the spectra are highly characteristic of the methyl groups on the nitrogen and side chain of the amino acids. The amino acids, which are isomeric and isobaric with the studied dimethyl amino acids, gave distinctive MS/MS spectra. The study also included MS/MS analysis of immonium ions of dimethyl amino acids that provide information on side chain structure, and it is further tested to determine the N-terminal amino acid of the peptides.

  12. Disulfide bond cleavage in TEMPO-free radical initiated peptide sequencing mass spectrometry.

    PubMed

    Lee, Minhee; Lee, Younjin; Kang, Minhyuk; Park, Hyeyeon; Seong, Yeonmi; Sung, Bong June; Moon, Bongjin; Oh, Han Bin

    2011-08-01

    The gas-phase free radical initiated peptide sequencing (FRIPS) fragmentation behavior of o-TEMPO-Bz-conjugated peptides with an intra- and intermolecular disulfide bond was investigated using MS(n) tandem mass spectrometry experiments. Investigated peptides included four peptides with an intramolecular cyclic disulfide bond, Bactenecin (RLCRIVVIRVCR), TGF-α (CHSGYVGVRC), MCH (DFDMLRCMLGRVFRPCWQY) and Adrenomedullin (16-31) (CRFGTCTVQKLAHQIY), and two peptides with an intermolecular disulfide bond. Collisional activation of the benzyl radical conjugated peptide cation, which was generated through the release of a TEMPO radical from o-TEMPO-Bz-conjugated peptides upon initial collisional activation, produced a large number of peptide backbone fragments in which the S-S or C-S bond was readily cleaved. The observed peptide backbone fragments included a-, c-, x- or z-types, which indicates that the radical-driven peptide fragmentation mechanism plays an important role in TEMPO-FRIPS mass spectrometry. FRIPS application of the linearly linked disulfide peptides further showed that the S-S or C-S bond was selectively and preferentially cleaved, followed by peptide backbone dissociations. In the FRIPS mass spectra, the loss of •SH or •SSH was also abundantly found. On the basis of these findings, FRIPS fragmentation pathways for peptides with a disulfide bond are proposed. For the cleavage of the S-S bond, the abstraction of a hydrogen atom at C(β) by the benzyl radical is proposed to be the initial radical abstraction/transfer reaction. On the other hand, H-abstraction at C(α) is suggested to lead to C-S bond cleavage, which yields [ion ± S] fragments or the loss of •SH or •SSH.

  13. Habitable Zones Around Main-Sequence Stars: Dependence on Planetary Mass

    NASA Technical Reports Server (NTRS)

    Kopparapu, Ravi Kumar; Ramirez, Ramses M.; Kotte, James Schottel; Kasting, James F.; Domagal-Goldman, Shawn; Eymet, Vincent

    2014-01-01

    The ongoing discoveries of extra-solar planets are unveiling a wide range of terrestrial mass (size) planets around their host stars. In this Letter, we present estimates of habitable zones (HZs) around stars with stellar effective temperatures in the range 2600 K-7200 K, for planetary masses between 0.1M and 5M. Assuming H2O-(inner HZ) and CO2-(outer HZ) dominated atmospheres, and scaling the background N2 atmospheric pressure with the radius of the planet, our results indicate that larger planets have wider HZs than do smaller ones. Specifically, with the assumption that smaller planets will have less dense atmospheres, the inner edge of the HZ (runaway greenhouse limit) moves outward (approx.10% lower than Earth flux) for low mass planets due to larger greenhouse effect arising from the increased H2O column depth. For larger planets, the H2O column depth is smaller, and higher temperatures are needed before water vapor completely dominates the outgoing long-wave radiation. Hence the inner edge moves inward (approx.7% higher than Earth's flux). The outer HZ changes little due to the competing effects of the greenhouse effect and an increase in albedo. New, three-dimensional climate model results from other groups are also summarized, and we argue that further, independent studies are needed to verify their predictions. Combined with our previous work, the results presented here provide refined estimates of HZs around main-sequence stars and provide a step toward a more comprehensive analysis of HZs.

  14. HABITABLE ZONES AROUND MAIN-SEQUENCE STARS: DEPENDENCE ON PLANETARY MASS

    SciTech Connect

    Kopparapu, Ravi Kumar; Ramirez, Ramses M.; Kasting, James F.; SchottelKotte, James; Domagal-Goldman, Shawn; Eymet, Vincent

    2014-06-01

    The ongoing discoveries of extra-solar planets are unveiling a wide range of terrestrial mass (size) planets around their host stars. In this Letter, we present estimates of habitable zones (HZs) around stars with stellar effective temperatures in the range 2600 K-7200 K, for planetary masses between 0.1 M {sub ⊕} and 5 M {sub ⊕}. Assuming H{sub 2}O-(inner HZ) and CO{sub 2}-(outer HZ) dominated atmospheres, and scaling the background N{sub 2} atmospheric pressure with the radius of the planet, our results indicate that larger planets have wider HZs than do smaller ones. Specifically, with the assumption that smaller planets will have less dense atmospheres, the inner edge of the HZ (runaway greenhouse limit) moves outward (∼10% lower than Earth flux) for low mass planets due to larger greenhouse effect arising from the increased H{sub 2}O column depth. For larger planets, the H{sub 2}O column depth is smaller, and higher temperatures are needed before water vapor completely dominates the outgoing longwave radiation. Hence the inner edge moves inward (∼7% higher than Earth's flux). The outer HZ changes little due to the competing effects of the greenhouse effect and an increase in albedo. New, three-dimensional climate model results from other groups are also summarized, and we argue that further, independent studies are needed to verify their predictions. Combined with our previous work, the results presented here provide refined estimates of HZs around main-sequence stars and provide a step toward a more comprehensive analysis of HZs.

  15. Respiratory syncytial virus fusion glycoprotein: nucleotide sequence of mRNA, identification of cleavage activation site and amino acid sequence of N-terminus of F1 subunit.

    PubMed Central

    Elango, N; Satake, M; Coligan, J E; Norrby, E; Camargo, E; Venkatesan, S

    1985-01-01

    The amino acid sequence of respiratory syncytial virus fusion protein (Fo) was deduced from the sequence of a partial cDNA clone of mRNA and from the 5' mRNA sequence obtained by primer extension and dideoxysequencing. The encoded protein of 574 amino acids is extremely hydrophobic and has a molecular weight of 63371 daltons. The site of proteolytic cleavage within this protein was accurately mapped by determining a partial amino acid sequence of the N-terminus of the larger subunit (F1) purified by radioimmunoprecipitation using monoclonal antibodies. Alignment of the N-terminus of the F1 subunit within the deduced amino acid sequence of Fo permitted us to identify a sequence of lys-lys-arg-lys-arg-arg at the C-terminus of the smaller N-terminal F2 subunit that appears to represent the cleavage/activation domain. Five potential sites of glycosylation, four within the F2 subunit, were also identified. Three extremely hydrophobic domains are present in the protein; a) the N-terminal signal sequence, b) the N-terminus of the F1 subunit that is analogous to the N-terminus of the paramyxovirus F1 subunit and the HA2 subunit of influenza virus hemagglutinin, and c) the putative membrane anchorage domain near the C-terminus of F1. Images PMID:2987829

  16. Analysis of protein function and its prediction from amino acid sequence.

    PubMed

    Clark, Wyatt T; Radivojac, Predrag

    2011-07-01

    Understanding protein function is one of the keys to understanding life at the molecular level. It is also important in the context of human disease because many conditions arise as a consequence of alterations of protein function. The recent availability of relatively inexpensive sequencing technology has resulted in thousands of complete or partially sequenced genomes with millions of functionally uncharacterized proteins. Such a large volume of data, combined with the lack of high-throughput experimental assays to functionally annotate proteins, attributes to the growing importance of automated function prediction. Here, we study proteins annotated by Gene Ontology (GO) terms and estimate the accuracy of functional transfer from protein sequence only. We find that the transfer of GO terms by pairwise sequence alignments is only moderately accurate, showing a surprisingly small influence of sequence identity (SID) in a broad range (30-100%). We developed and evaluated a new predictor of protein function, functional annotator (FANN), from amino acid sequence. The predictor exploits a multioutput neural network framework which is well suited to simultaneously modeling dependencies between functional terms. Experiments provide evidence that FANN-GO (predictor of GO terms; available from http://www.informatics.indiana.edu/predrag) outperforms standard methods such as transfer by global or local SID as well as GOtcha, a method that incorporates the structure of GO.

  17. The Complete Genome Sequence of the Lactic Acid Bacterium Lactococcus lactis ssp. lactis IL1403

    PubMed Central

    Bolotin, Alexander; Wincker, Patrick; Mauger, Stéphane; Jaillon, Olivier; Malarme, Karine; Weissenbach, Jean; Ehrlich, S. Dusko; Sorokin, Alexei

    2001-01-01

    Lactococcus lactis is a nonpathogenic AT-rich gram-positive bacterium closely related to the genus Streptococcus and is the most commonly used cheese starter. It is also the best-characterized lactic acid bacterium. We sequenced the genome of the laboratory strain IL1403, using a novel two-step strategy that comprises diagnostic sequencing of the entire genome and a shotgun polishing step. The genome contains 2,365,589 base pairs and encodes 2310 proteins, including 293 protein-coding genes belonging to six prophages and 43 insertion sequence (IS) elements. Nonrandom distribution of IS elements indicates that the chromosome of the sequenced strain may be a product of recent recombination between two closely related genomes. A complete set of late competence genes is present, indicating the ability of L. lactis to undergo DNA transformation. Genomic sequence revealed new possibilities for fermentation pathways and for aerobic respiration. It also indicated a horizontal transfer of genetic information from Lactococcus to gram-negative enteric bacteria of Salmonella-Escherichia group. [The sequence data described in this paper has been submitted to the GenBank data library under accession no. AE005176.] PMID:11337471

  18. Evolution of phosphagen kinase V. cDNA-derived amino acid sequences of two molluscan arginine kinases from the chiton Liolophura japonica and the turbanshell Battilus cornutus.

    PubMed

    Suzuki, T; Ban, T; Furukohri, T

    1997-06-20

    The cDNAs of arginine kinases from the chiton Liolophura japonica (Polyplacophora) and the turbanshell Battilus cornutus (Gastropoda) were amplified by polymerase chain reaction (PCR), and the complete nucleotide sequences of 1669 and 1624 bp, respectively, were determined. The open reading frame for Liolophura arginine kinase is 1050 nucleotides in length and encodes a protein with 349 amino acid residues, and that for Battilus is 1077 nucleotides and 358 residues. The validity of the cDNA-derived amino acid sequence was supported by chemical sequencing of internal tryptic peptides. The molecular masses were calculated to be 39,057 and 39,795 Da, respectively. The amino acid sequence of Liolophura arginine kinase showed 65-68% identity with those of Battilus and Nordotis (abalone) arginine kinases, and the homology between Battilus and Nordotis was 79%. Molluscan arginine kinases also show lower, but significant homology (38-43%) with rabbit creatine kinase. The sequences of arginine kinases could be used as a molecular clock to elucidate the phylogeny of Mollusca, one of the most diverse animal phyla.

  19. Self-sequencing of amino acids and origins of polyfunctional protocells

    NASA Technical Reports Server (NTRS)

    Fox, S. W.

    1984-01-01

    The role of proteins in the origin of living things is discussed. It has been experimentally established that amino acids can sequence themselves under simulated geological conditions with highly nonrandom products which accordingly contain diverse information. Multiple copies of each type of macromolecule are formed, resulting in greater power for any protoenzymic molecule than would accrue from a single copy of each type. Thermal proteins are readily incorporated into laboratory protocells. The experimental evidence for original polyfunctional protocells is discussed.

  20. Amino acid sequence of atrial natriuretic peptides in human coronary sinus plasma.

    PubMed

    Yandle, T; Crozier, I; Nicholls, G; Espiner, E; Carne, A; Brennan, S

    1987-07-31

    Two atrial natriuretic peptides were purified from pooled human coronary sinus plasma by Sep-Pak extraction, immunoaffinity chromatography and reverse phase HPLC. The amino acid sequences of the two peptides were homologous with 99-126 human atrial natriuretic peptide (hANP) and 106-126 hANP, the latter being most probably linked to 99-105 ANP by the disulphide bond. The molar ratio of the peptides in plasma, as assessed by radioimmunoassay was 10:3.

  1. Amino Acid Sequences Mediating Vascular Cell Adhesion Molecule 1 Binding to Integrin Alpha 4: Homologous DSP Sequence Found for JC Polyoma VP1 Coat Protein

    PubMed Central

    Meyer, Michael Andrew

    2013-01-01

    The JC polyoma viral coat protein VP1 was analyzed for amino acid sequences homologies to the IDSP sequence which mediates binding of VLA-4 (integrin alpha 4) to vascular cell adhesion molecule 1. Although the full sequence was not found, a DSP sequence was located near the critical arginine residue linked to infectivity of the virus and binding to sialic acid containing molecules such as integrins (3). For the JC polyoma virus, a DSP sequence was found at residues 70, 71 and 72 with homology also noted for the mouse polyoma virus and SV40 virus. Three dimensional modeling of the VP1 molecule suggests that the DSP loop has an accessible site for interaction from the external side of the assembled viral capsid pentamer. PMID:24147211

  2. Amino Acid Sequences Mediating Vascular Cell Adhesion Molecule 1 Binding to Integrin Alpha 4: Homologous DSP Sequence Found for JC Polyoma VP1 Coat Protein.

    PubMed

    Meyer, Michael Andrew

    2013-01-01

    The JC polyoma viral coat protein VP1 was analyzed for amino acid sequences homologies to the IDSP sequence which mediates binding of VLA-4 (integrin alpha 4) to vascular cell adhesion molecule 1. Although the full sequence was not found, a DSP sequence was located near the critical arginine residue linked to infectivity of the virus and binding to sialic acid containing molecules such as integrins (3). For the JC polyoma virus, a DSP sequence was found at residues 70, 71 and 72 with homology also noted for the mouse polyoma virus and SV40 virus. Three dimensional modeling of the VP1 molecule suggests that the DSP loop has an accessible site for interaction from the external side of the assembled viral capsid pentamer.

  3. Amino acid sequence similarity between rabies virus glycoprotein and snake venom curaremimetic neurotoxins.

    PubMed

    Lentz, T L; Wilson, P T; Hawrot, E; Speicher, D W

    1984-11-16

    Evidence was presented earlier that a host-cell receptor for the highly neurotropic rabies virus might be the acetylcholine receptor. The amino acid sequence of the glycoprotein of rabies virus was compared by computer analysis with that of snake venom curaremimetic neurotoxins, potent ligands of the acetylcholine receptor. A statistically significant sequence relation was found between a segment of the rabies glycoprotein and the entire sequence of long neurotoxins. The greatest identity occurs with residues considered most important in neurotoxicity, including those interacting with the acetylcholine binding site of the acetylcholine receptor. Because of the similarity between the glycoprotein and the receptor-binding region of the neurotoxins, this region of the viral glycoprotein may function as a recognition site for the acetylcholine receptor. Direct binding of the rabies virus glycoprotein to the acetylcholine receptor could contribute to the neurotropism of this virus.

  4. Partial amino acid sequence of human pancreatic stone protein, a novel pancreatic secretory protein.

    PubMed Central

    Montalto, G; Bonicel, J; Multigner, L; Rovery, M; Sarles, H; De Caro, A

    1986-01-01

    Pancreatic stone protein (PSP) is the major organic component of human pancreatic stones. With the use of monoclonal antibody immunoadsorbents, five immunoreactive forms (PSP-S) with close Mr values (14,000-19,000) were isolated from normal pancreatic juice. By CM-Trisacryl M chromatography the lowest-Mr form (PSP-S1) was separated from the others and some of its molecular characteristics were investigated. The Mr of the PSP-S1 polypeptide chain calculated from the amino acid composition was about 16,100. The N-terminal sequences (40 residues) of PSP and PSP-S1 are identical, which suggests that the peptide backbone is the same for both of these polypeptides. The PSP-S1 sequence was determined up to residue 65 and was found to be different from all other known protein sequences. Images Fig. 1. PMID:3541906

  5. Detection of alkylmethylphosphonic acids on leaf surfaces by static secondary ion mass spectrometry

    SciTech Connect

    Ingram, J.C.; Groenewold, G.S.; Appelhans, A.D.; Delmore, J.E.; Dahl, D.A. )

    1995-01-01

    Detection of environmental degradation products of nerve agents directly from the surface of plant leaves using static secondary ion mass spectrometry (SIMS) is demonstrated. Pinacolylmethylphosphonic acid (PMPA), isopropylmethylphosphonic acid (IMPA), and ethylmethylphosphonic acid (EMPA) were spiked from aqueous solutions onto philodendron leaves prior to analysis by static SIMS. Fragment ions were observed in the anion SIMS spectra from all three compounds at m/z 63, 77, 79, and 95, which are attributed to PO[sub 2][sup [minus

  6. Characterization of the microbial acid mine drainage microbial community using culturing and direct sequencing techniques.

    PubMed

    Auld, Ryan R; Myre, Maxine; Mykytczuk, Nadia C S; Leduc, Leo G; Merritt, Thomas J S

    2013-05-01

    We characterized the bacterial community from an AMD tailings pond using both classical culturing and modern direct sequencing techniques and compared the two methods. Acid mine drainage (AMD) is produced by the environmental and microbial oxidation of minerals dissolved from mining waste. Surprisingly, we know little about the microbial communities associated with AMD, despite the fundamental ecological roles of these organisms and large-scale economic impact of these waste sites. AMD microbial communities have classically been characterized by laboratory culturing-based techniques and more recently by direct sequencing of marker gene sequences, primarily the 16S rRNA gene. In our comparison of the techniques, we find that their results are complementary, overall indicating very similar community structure with similar dominant species, but with each method identifying some species that were missed by the other. We were able to culture the majority of species that our direct sequencing results indicated were present, primarily species within the Acidithiobacillus and Acidiphilium genera, although estimates of relative species abundance were only obtained from direct sequencing. Interestingly, our culture-based methods recovered four species that had been overlooked from our sequencing results because of the rarity of the marker gene sequences, likely members of the rare biosphere. Further, direct sequencing indicated that a single genus, completely missed in our culture-based study, Legionella, was a dominant member of the microbial community. Our results suggest that while either method does a reasonable job of identifying the dominant members of the AMD microbial community, together the methods combine to give a more complete picture of the true diversity of this environment.

  7. [MOLECULAR EVOLUTION OF ION CHANNELS: AMINO ACID SEQUENCES AND 3D STRUCTURES].

    PubMed

    Korkosh, V S; Zhorov, B S; Tikhonov, D B

    2016-01-01

    An integral part of modern evolutionary biology is comparative analysis of structure and function of macromolecules such as proteins. The first and critical step to understand evolution of homologous proteins is their amino acid sequence alignment. However, standard algorithms fop not provide unambiguous sequence alignments for proteins of poor homology. More reliable results can be obtained by comparing experimental 3D structures obtained at atomic resolution, for instance, with the aid of X-ray structural analysis. If such structures are lacking, homology modeling is used, which may take into account indirect experimental data on functional roles of individual amino-acid residues. An important problem is that the sequence alignment, which reflects genetic modifications, does not necessarily correspond to the functional homology. The latter depends on three-dimensional structures which are critical for natural selection. Since alignment techniques relying only on the analysis of primary structures carry no information on the functional properties of proteins, including 3D structures into consideration is very important. Here we consider several examples involving ion channels and demonstrate that alignment of their three-dimensional structures can significantly improve sequence alignments obtained by traditional methods.

  8. The amino acid sequence of the aspartate aminotransferase from baker's yeast (Saccharomyces cerevisiae).

    PubMed Central

    Cronin, V B; Maras, B; Barra, D; Doonan, S

    1991-01-01

    1. The single (cytosolic) aspartate aminotransferase was purified in high yield from baker's yeast (Saccharomyces cerevisiae). 2. Amino-acid-sequence analysis was carried out by digestion of the protein with trypsin and with CNBr; some of the peptides produced were further subdigested with Staphylococcus aureus V8 proteinase or with pepsin. Peptides were sequenced by the dansyl-Edman method and/or by automated gas-phase methods. The amino acid sequence obtained was complete except for a probable gap of two residues as indicated by comparison with the structures of counterpart proteins in other species. 3. The N-terminus of the enzyme is blocked. Fast-atom-bombardment m.s. was used to identify the blocking group as an acetyl one. 4. Alignment of the sequence of the enzyme with those of vertebrate cytosolic and mitochondrial aspartate aminotransferases and with the enzyme from Escherichia coli showed that about 25% of residues are conserved between these distantly related forms. 5. Experimental details and confirmatory data for the results presented here are given in a Supplementary Publication (SUP 50164, 25 pages) that has been deposited at the British Library Document Supply Centre, Boston Spa. Wetherby, West Yorkshire LS23 7 BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273, 5. PMID:1859361

  9. Analysis of amino acid sequence variations and immunoglobulin E-binding epitopes of German cockroach tropomyosin.

    PubMed

    Jeong, Kyoung Yong; Lee, Jongweon; Lee, In-Yong; Ree, Han-Il; Hong, Chein-Soo; Yong, Tai-Soon

    2004-09-01

    The allergenicities of tropomyosins from different organisms have been reported to vary. The cDNA encoding German cockroach tropomyosin (Bla g 7) was isolated, expressed, and characterized previously. In the present study, the amino acid sequence variations in German cockroach tropomyosin were analyzed in order to investigate its influence on allergenicity. We also undertook the identification of immunodominant peptides containing immunoglobulin E (IgE) epitopes which may facilitate the development of diagnostic and immunotherapeutic strategies based on the recombinant proteins. Two-dimensional gel electrophoresis and immunoblot analysis with mouse anti-recombinant German cockroach tropomyosin serum was performed to investigate the isoforms at the protein level. Reverse transcriptase PCR (RT-PCR) was applied to examine the sequence diversity. Eleven different variants of the deduced amino acid sequences were identified by RT-PCR. German cockroach tropomyosin has only minor sequence variations that did not seem to affect its allergenicity significantly. These results support the molecular basis underlying the cross-reactivities of arthropod tropomyosins. Recombinant fragments were also generated by PCR, and IgE-binding epitopes were assessed by enzyme-linked immunosorbent assay. Sera from seven patients revealed heterogeneous IgE-binding responses. This study demonstrates multiple IgE-binding epitope regions in a single molecule, suggesting that full-length tropomyosin should be used for the development of diagnostic and therapeutic reagents.

  10. A comparison of molecular mass determination of hyaluronic acid using SEC/MALLS and sedimentation equilibrium.

    PubMed

    Hokputsa, Sanya; Jumel, Kornelia; Alexander, Catherine; Harding, Stephen E

    2003-08-01

    Hyaluronic acid (HA) is a natural polysaccharide with importance in the pharmaceutical, medical and cosmetic industries. Determining factors in its final applications are its physicochemical properties, particularly molecular mass. A high molecular mass HA was degraded using five different hydroxyl free-radical starting concentrations chemically produced from ascorbic acid and hydrogen peroxide. The aims of the study were to investigate the effect of different hydroxyl free-radical concentrations on the chain length of HA and compare the molecular masses obtained from analytical ultracentrifugation using sedimentation equilibrium experiments and size exclusion chromatography/multi-angle laser light scattering (SEC/MALLS). The results indicated that their molecular masses varied, depending on the degree of hydroxyl free-radical starting concentration. Molecular mass values obtained from sedimentation equilibrium experiments for each sample showed the same trend as those obtained from the SEC/MALLS in the range of molecular masses studied. The molecular masses obtained from sedimentation equilibrium for high molecular mass samples from reciprocal plots of apparent weight average molecular mass against concentration gave values similar to those obtained by SEC/MALLS. In contrast, the molecular mass from conventional plots for high molecular mass samples were much lower than those from SEC/MALLS, even when high ionic strength buffers were used.

  11. Determination of α-keto acids in pork meat and Iberian ham via tandem mass spectrometry.

    PubMed

    Hidalgo, Francisco J; Navarro, José L; Delgado, Rosa M; Zamora, Rosario

    2013-09-01

    An analytical method which offers accurate determination and identification of eight α-keto acids (α-ketoglutaric acid, pyruvic acid, 4-hydroxyphenylpyruvic acid, 3-methyl-2-oxobutyric acid, α-keto-γ-methylthiobutyric acid, 4-methyl-2-oxovaleric acid, 3-methyl-2-oxovaleric acid, and phenylpyruvic acid) in pork meat and Iberian ham samples is reported. The method utilises a highly selective and sensitive method of multiple reaction monitoring (MRM) by mass spectrometry. The analytical method is simple (although the chemical derivatisation of the α-keto acids with dansylhydrazine is required), precise (<18% RSD), accurate (90-110%), sensitive (0.01-0.34 mg/kg of defatted and freeze-dried meat depending on the α-keto acid) and linear (R>0.99) over several orders of magnitude (until 0.01-146.1 mg/kg of defatted and freeze-dried meat depending on the α-keto acid). Using this methodology, α-keto acids were found to be present in pork meat to a low extent, and their concentration increased when they were determined in Iberian ham. This is the first report of the presence of α-keto acids in both pork meats and Iberian hams.

  12. A comparative mass spectrometric study of fatty acids and metals in some seed extracts.

    PubMed

    Suvar, Sonia Niculina; Bleiziffer, R; Podea, P; Iordache, A; Voica, C; Zgavarogea, R; Culea, M

    2016-01-01

    A major cause of cardiovascular diseases and cancer is diet content, so the optimization of micronutrients in food is very important. Omega-3 fatty acids supplementation for patients had beneficial effects on subjective global assessment score and metabolic profiles. Fatty acids content and the metal ions in different seeds (e.g. linseed, poppy, grape, hemp, nuts, pumpkin, sesame, watermelon, chia) recommended as food supplements, purchased on the Romanian market, were compared. Gas chromatography coupled to mass spectrometry (GC-MS) was used as an excellent technique for fatty acids identification and quantitation, and inductively coupled plasma mass spectrometer (ICP-MS) for analytical measurements of metals.

  13. Electrospray ionization mass spectrometric studies of some imidazole amidoximes and nitrolic acids and their esters.

    PubMed

    Oresmaa, Larisa; Aulaskari, Paula; Vainiotalo, Pirjo

    2006-01-01

    The fragmentations of the [M+H]+ ions of imidazole amidoximes, and nitrolic acids and their esters, were studied by collision-induced dissociation experiments and by determining the accurate masses of the product ions on an electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer. The fragmentation pathways of the amidoximes varied with the substituent in the imidazole ring at position 1N, allowing two regioisomers to be distinguished. Nitrolic acids decompose in solution to nitrile oxides, and the studied nitrolic acid behaved in the same way in the gas phase. The esters decompose similarly to their parent compounds.

  14. Studies of the acidic components of the Colorado Green River formation oil shale-Mass spectrometric identification of the methyl esters of extractable acids.

    NASA Technical Reports Server (NTRS)

    Haug, P.; Schnoes, H. K.; Burlingame, A. L.

    1971-01-01

    Study of solvent extractable acidic constituents of oil shale from the Colorado Green River Formation. Identification of individual components is based on gas chromatographic and mass spectrometric data obtained for their respective methyl esters. Normal acids, isoprenoidal acids, alpha, omega-dicarboxylic acids, mono-alpha-methyl dicarboxylic acids and methyl ketoacids were identified. In addition, the presence of monocyclic, benzoic, phenylalkanoic and naphthyl-carboxylic acids, as well as cycloaromatic acids, is demonstrated by partial identification.

  15. Molecular resolution and fragmentation of fulvic acid by electrospray ionization/multistage tandem mass spectrometry

    USGS Publications Warehouse

    Leenheer, J.A.; Rostad, C.E.; Gates, Paul M.; Furlong, E.T.; Ferrer, I.

    2001-01-01

    Molecular weight distributions of fulvic acid from the Suwannee River, Georgia, were investigated by electrospray ionization/quadrupole mass spectrometry (ESI/QMS), and fragmentation pathways of specific fulvic acid masses were investigated by electrospray ionization/ion trap multistage tandem mass spectrometry (ESI/MST/MS). ESI/QMS studies of the free acid form of low molecular weight poly(carboxylic acid) standards in 75% methanol/25% water mobile phase found that negative ion detection gave the optimum generation of parent ions that can be used for molecular weight determinations. However, experiments with poly(acrylic acid) mixtures and specific high molecular weight standards found multiply charged negative ions that gave a low bias to molecular mass distributions. The number of negative charges on a molecule is dependent on the distance between charges. ESI/MST/MS of model compounds found characteristic water loss from alcohol dehydration and anhydride formation, as well as CO2 loss from decarboxylation, and CO loss from ester structures. Application of these fragmentation pathways to specific masses of fulvic acid isolated and fragmented by ESI/MST/MS is indicative of specific structures that can serve as a basis for future structural confirmation after these hypothesized structures are synthesized.

  16. Complete amino acid sequence of a histidine-rich proteolytic fragment of human ceruloplasmin.

    PubMed

    Kingston, I B; Kingston, B L; Putnam, F W

    1979-04-01

    The complete amino acid sequence has been determined for a fragment of human ceruloplasmin [ferroxidase; iron(II):oxygen oxidoreductase, EC 1.16.3.1]. The fragment (designated Cp F5) contains 159 amino acid residues and has a molecular weight of 18,650; it lacks carbohydrate, is rich in histidine, and contains one free cysteine that may be part of a copper-binding site. This fragment is present in most commercial preparations of ceruloplasmin, probably owing to proteolytic degradation, but can also be obtained by limited cleavage of single-chain ceruloplasmin with plasmin. Cp F5 probably is an intact domain attached to the COOH-terminal end of single-chain ceruloplasmin via a labile interdomain peptide bond. A model of the secondary structure predicted by empirical methods suggests that almost one-third of the amino acid residues are distributed in alpha helices, about a third in beta-sheet structure, and the remainder in beta turns and unidentified structures. Computer analysis of the amino acid sequence has not demonstrated a statistically significant relationship between this ceruloplasmin fragment and any other protein, but there is some evidence for an internal duplication.

  17. Processing and amino acid sequence analysis of the mouse mammary tumor virus env gene product.

    PubMed Central

    Arthur, L O; Copeland, T D; Oroszlan, S; Schochetman, G

    1982-01-01

    The envelope proteins of mouse mammary tumor virus (MMTV) are synthesized from a subgenomic 24S mRNA as a 75,000-dalton glycosylated precursor polyprotein which is eventually processed to the mature glycoproteins gp52 and gp36. In vivo synthesis of this env precursor in the presence of the core glycosylation inhibitor tunicamycin yielded a precursor of approximately 61,000 daltons (P61env). However, a 67,000-dalton protein (P67env) was obtained from cell-free translation with the MMTV 24S mRNA as the template. To determine whether the portion of the protein cleaved from P67env to give P61env was removed from the NH2-terminal end of P67env and as such would represent a leader sequence, the NH2-terminal amino acid sequence of the terminal peptide gp52 was determined. Glutamic acid, and not methionine, was found to be the amino-terminal residue of gp52, indicating that the cleaved portion was derived from the NH2-terminal end of P67env. The NH2-terminal amino acid sequences of gp52's from endogenous and exogenous C3H MMTVs were determined though 46 residues and found to be identical. However, amino acid composition and type-specific gp52 radioimmunoassays from MMTVs grown in heterologous cells indicated primary structure differences between gp52's of the two viruses. The nucleic acid sequence of cloned MMTV DNA fragments (J. Majors and H. E. Varmus, personal communication) in conjunction with the NH2-terminal sequence of gp52 allowed localization of the env gene in the MMTV genome. Nucleotides coding for the NH2 terminus of gp52 begin approximately 0.8 kilobase to the 3' side of the single EcoRI cleavage site. Localization of the env gene at that point agrees with the proposed gene order -gag-pol-env- and also allows sufficient coding potential for the glycoprotein precursor without extending into the long terminal repeat. Images PMID:6281457

  18. Effects of coating of dicarboxylic acids on the mass-mobility relationship of soot particles.

    PubMed

    Xue, Huaxin; Khalizov, Alexei F; Wang, Lin; Zheng, Jun; Zhang, Renyi

    2009-04-15

    Atandem differential mobility analyzer (TDMA) and a differential mobility analyzer-aerosol particle mass analyzer (DMA-APM) have been employed to study morphology and hygroscopicity of soot aerosol internally mixed with dicarboxylic acids. The effective densities, fractal dimensions, and dynamic shape factors of soot particles before and after coating with succinic and glutaric acids are determined. Coating of soot with succinic acid results in a significant increase in the particle mobility diameter, mass, and effective density, but these properties recover to their initial values once succinic acid is removed by heating, suggesting that no restructuring of the soot core occurs. This conclusion is also supported from the observation of similar fractal dimensions and dynamic shape factors for fresh and coated/heated soot aggregates. Also, no change is observed when succinic acid-coated aggregates are cycled through elevated relative humidity (5% to 90% to 5% RH) below the succinic acid deliquescence point. When soot is coated with glutaric acid, the particle mass increases, but the mobility diameter shrinks by 10-40%. Cycling the soot aerosol coated with glutaric acid through elevated relative humidity leads to an additional mass increase, indicating that condensed water remains within the coating even at low RH. The fractal dimension of soot particles increases after coating and remains high when glutaric acid is removed by heating. The dynamic shape factor of glutaric acid-coated and heated soot is significantly lower than that of fresh soot, suggesting a significant restructuring of the soot agglomerates by glutaric acid. The results imply that internal mixing of soot aerosol during atmospheric aging leads to changes in hygroscopicity, morphology, and effective density, which likely modify their effects on direct and indirect climate forcing and deposition in the human respiratory system.

  19. Complete Genome Sequence of a thermotolerant sporogenic lactic acid bacterium, Bacillus coagulans strain 36D1

    PubMed Central

    Rhee, Mun Su; Moritz, Brélan E.; Xie, Gary; Glavina del Rio, T.; Dalin, E.; Tice, H.; Bruce, D.; Goodwin, L.; Chertkov, O.; Brettin, T.; Han, C.; Detter, C.; Pitluck, S.; Land, Miriam L.; Patel, Milind; Ou, Mark; Harbrucker, Roberta; Ingram, Lonnie O.; Shanmugam, K. T.

    2011-01-01

    Bacillus coagulans is a ubiquitous soil bacterium that grows at 50-55 °C and pH 5.0 and ferments various sugars that constitute plant biomass to L (+)-lactic acid. The ability of this sporogenic lactic acid bacterium to grow at 50-55 °C and pH 5.0 makes this organism an attractive microbial biocatalyst for production of optically pure lactic acid at industrial scale not only from glucose derived from cellulose but also from xylose, a major constituent of hemicellulose. This bacterium is also considered as a potential probiotic. Complete genome sequence of a representative strain, B. coagulans strain 36D1, is presented and discussed. PMID:22675583

  20. BeadCons: detection of nucleic acid sequences by flow cytometry.

    PubMed

    Horejsh, Douglas; Martini, Federico; Capobianchi, Maria Rosaria

    2005-11-01

    Molecular beacons are single-stranded nucleic acid structures with a terminal fluorophore and a distal, terminal quencher. These molecules are typically used in real-time PCR assays, but have also been conjugated with solid matrices. This unit describes protocols related to molecular beacon-conjugated beads (BeadCons), whose specific hybridization with complementary target sequences can be resolved by cytometry. Assay sensitivity is achieved through the concentration of fluorescence signal on discrete particles. By using molecular beacons with different fluorophores and microspheres of different sizes, it is possible to construct a fluid array system with each bead corresponding to a specific target nucleic acid. Methods are presented for the design, construction, and use of BeadCons for the specific, multiplexed detection of unlabeled nucleic acids in solution. The use of bead-based detection methods will likely lead to the design of new multiplex molecular diagnostic tools.

  1. Measuring nanometer distances in nucleic acids using a sequence-independent nitroxide probe

    PubMed Central

    Qin, Peter Z; Haworth, Ian S; Cai, Qi; Kusnetzow, Ana K; Grant, Gian Paola G; Price, Eric A; Sowa, Glenna Z; Popova, Anna; Herreros, Bruno; He, Honghang

    2008-01-01

    This protocol describes the procedures for measuring nanometer distances in nucleic acids using a nitroxide probe that can be attached to any nucleotide within a given sequence. Two nitroxides are attached to phosphorothioates that are chemically substituted at specific sites of DNA or RNA. Inter-nitroxide distances are measured using a four-pulse double electron–electron resonance technique, and the measured distances are correlated to the parent structures using a Web-accessible computer program. Four to five days are needed for sample labeling, purification and distance measurement. The procedures described herein provide a method for probing global structures and studying conformational changes of nucleic acids and protein/nucleic acid complexes. PMID:17947978

  2. Complete Genome Sequence of a thermotolerant sporogenic lactic acid bacterium, Bacillus coagulans strain 36D1.

    PubMed

    Rhee, Mun Su; Moritz, Brélan E; Xie, Gary; Glavina Del Rio, T; Dalin, E; Tice, H; Bruce, D; Goodwin, L; Chertkov, O; Brettin, T; Han, C; Detter, C; Pitluck, S; Land, Miriam L; Patel, Milind; Ou, Mark; Harbrucker, Roberta; Ingram, Lonnie O; Shanmugam, K T

    2011-12-31

    Bacillus coagulans is a ubiquitous soil bacterium that grows at 50-55 °C and pH 5.0 and ferments various sugars that constitute plant biomass to L (+)-lactic acid. The ability of this sporogenic lactic acid bacterium to grow at 50-55 °C and pH 5.0 makes this organism an attractive microbial biocatalyst for production of optically pure lactic acid at industrial scale not only from glucose derived from cellulose but also from xylose, a major constituent of hemicellulose. This bacterium is also considered as a potential probiotic. Complete genome sequence of a representative strain, B. coagulans strain 36D1, is presented and discussed.

  3. Oxidative degradation of bis (2,4,4-trimethylpentyl) dithiophosphinic acid in nitric acid studied by electrospray ionization mass spectrometry

    SciTech Connect

    G. S. Groenewold; D. R. Peterman

    2012-10-01

    Samples of bis(2,4,4-trimethylpentyl)dithiophosphinic acid (Cyanex-301) were analyzed using direct infusion electrospray ionization mass spectrometry. Positive ion spectra of standard and stereo-pure acids displayed ions typical of the unmodified compound, cationized monomeric and dimeric cluster ion species. In addition, a significant ions 2 u less than the dimeric clusters were seen, that correspond to an oxidatively coupled species designated Cyx2 that is observed as H- or Na-cationized species in the electrospray analyses. Based on uncorrected ion intensities, Cyx2 is estimated to account for about 20% of the total in the standard materials. When samples that were contacted with 3 M HNO3 were analyzed, the positive ion spectrum consisted nearly entirely of ions derived from the oxidatively coupled product, indicating that the acid promotes coupling. The negative ion spectra of the standard acids consisted nearly entirely of the conjugate base that is formed by deprotonation of the acids, and cluster ions containing multiple acid molecules. The negative spectra of the HNO3-contacted samples also contained the conjugate base of the unmodified acid, but also two other species that correspond to the dioxo- and perthio- derivatives. It is concluded that HNO3 contact causes significant oxidation, forming at least three major products, Cyx2, the perthio-acid, and the dioxo-acid.

  4. Luminosity and mass functions of the three main sequences of the globular cluster NGC 2808

    NASA Astrophysics Data System (ADS)

    Milone, A. P.; Piotto, G.; Bedin, L. R.; Cassisi, S.; Anderson, J.; Marino, A. F.; Pietrinferni, A.; Aparicio, A.

    2012-01-01

    High-precision HST photometry has revealed that the globular cluster (GC) NGC 2808 hosts a triple main sequence (MS) corresponding to three stellar populations with different helium abundances. We carried out photometry on ACS/WFC HST images of NGC 2808 with the main purpose of measuring the luminosity function (LF) of stars in the three different MSs, and the binary fraction in the cluster. We used isochrones to transform the observed LFs into mass functions (MFs). We estimate that the fraction of binary systems in NGC 2808 is fbin ≃ 0.05, and find that the three MSs have very similar LFs. The slopes of the corresponding MFs are α = -1.2 ± 0.3 for the red MS, α = -0.9 ± 0.3 for the middle MS, and α = -0.9 ± 0.4 for the blue one, the same, to within the errors. There is marginal evidence of a MF flattening for masses ℳ ≤ 0.6ℳ⊙ for the the reddest (primordial) MS. These results represent the first direct measurement of the present-day MF and LF in distinct stellar populations of a GC, and provide constraints on models of the formation and evolution of multiple generations of stars in these objects. Based on observations with the NASA/ESA Hubble Space Telescope, obtained at the Space Telescope Science Institute, which is operated by AURA, Inc., under NASA contract NAS 5-26555, under the programs GO-9899 and GO-10922.Appendices are available in electronic form at http://www.aanda.org

  5. A 25-Amino Acid Sequence of the Arabidopsis TGD2 Protein Is Sufficient for Specific Binding of Phosphatidic Acid*

    PubMed Central

    Lu, Binbin; Benning, Christoph

    2009-01-01

    Genetic analysis suggests that the TGD2 protein of Arabidopsis is required for the biosynthesis of endoplasmic reticulum derived thylakoid lipids. TGD2 is proposed to be the substrate-binding protein of a presumed lipid transporter consisting of the TGD1 (permease) and TGD3 (ATPase) proteins. The TGD1, -2, and -3 proteins are localized in the inner chloroplast envelope membrane. TGD2 appears to be anchored with an N-terminal membrane-spanning domain into the inner envelope membrane, whereas the C-terminal domain faces the intermembrane space. It was previously shown that the C-terminal domain of TGD2 binds phosphatidic acid (PtdOH). To investigate the PtdOH binding site of TGD2 in detail, the C-terminal domain of the TGD2 sequence lacking the transit peptide and transmembrane sequences was fused to the C terminus of the Discosoma sp. red fluorescent protein (DR). This greatly improved the solubility of the resulting DR-TGD2C fusion protein following production in Escherichia coli. The DR-TGD2C protein bound PtdOH with high specificity, as demonstrated by membrane lipid-protein overlay and liposome association assays. Internal deletion and truncation mutagenesis identified a previously undescribed minimal 25-amino acid fragment in the C-terminal domain of TGD2 that is sufficient for PtdOH binding. Binding characteristics of this 25-mer were distinctly different from those of TGD2C, suggesting that additional sequences of TGD2 providing the proper context for this 25-mer are needed for wild type-like PtdOH binding. PMID:19416982

  6. Exact masses and chemical formulas of individual Suwannee River fulvic acids from ultrahigh resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectra.

    PubMed

    Stenson, Alexandra C; Marshall, Alan G; Cooper, William T

    2003-03-15

    Molecular formulas have been assigned for 4626 individual Suwannee River fulvic acids based on accurate mass measurements from ions generated by electrospray ionization and observed by ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). Formula assignments were possible because of the mass accuracy of FTICR MS at high field (9.4 T) and the regular mass spacing patterns found in fulvic acid mixtures. Sorting the 4626 individually observed ions according to Kendrick mass defect and nominal mass series (z* score) revealed that all could be assigned to 1 of 266 distinct homologous series that differ in oxygen content and double bond equivalence. Tandem mass spectrometry based on infrared multiphoton dissociation identified labile fragments of fulvic acid molecules, whose chemical formulas led to plausible structures consistent with degraded lignin as a source of Suwannee River fulvic acids.

  7. Nucleotide sequence of the luxC gene encoding fatty acid reductase of the lux operon from Photobacterium leiognathi.

    PubMed

    Lin, J W; Chao, Y F; Weng, S F

    1993-02-26

    The nucleotide sequence of the luxC gene (EMBL Accession No. 65156) encoding fatty acid reductase (FAR) of the lux operon from Photobacterium leiognathi PL741 was determined and the encoded amino acid sequence deduced. The fatty acid reductase is a component of the fatty acid reductase complex. The complex is responsible for converting fatty acid to aldehyde which serves as the substrate in the luciferase-catalyzed bioluminescent reaction. The protein comprises 478 amino acid residues and has a calculated M(r) of 53,858. Alignment and comparison of the fatty acid reductase of P. leiognathi with that of Vibrio harveyi B392 and Vibrio fischeri ATCC 7744 shows that there is 70% and 59% amino acid residues identity, respectively.

  8. A theoretical study of acoustic glitches in low-mass main-sequence stars

    SciTech Connect

    Verma, Kuldeep; Antia, H. M.; Basu, Sarbani; Mazumdar, Anwesh E-mail: antia@tifr.res.in E-mail: anwesh@tifr.res.in

    2014-10-20

    There are regions in stars, such as ionization zones and the interface between radiative and convective regions, that cause a localized sharp variation in the sound speed. These are known as 'acoustic glitches'. Acoustic glitches leave their signatures on the oscillation frequencies of stars, and hence these signatures can be used as diagnostics of these regions. In particular, the signatures of these glitches can be used as diagnostics for the position of the second helium ionization zone and that of the base of the envelope convection zone. With the help of stellar models, we study the properties of these acoustic glitches in main-sequence stars. We find that the acoustic glitch due to the helium ionization zone does not correspond to the dip in the adiabatic index Γ{sub 1} caused by the ionization of He II, but to the peak in Γ{sub 1} between the He I and He II ionization zones. We find that it is easiest to study the acoustic glitch that is due to the helium ionization zone in stars with masses in the range 0.9-1.2 M {sub ☉}.

  9. Evaporation and NARS Nitric Acid Mass Balance Summary: 2000--2005

    SciTech Connect

    B.D. Kreutzberg; R.L. Ames; K.M. Hansel

    2005-11-01

    A compilation of the historical nitric acid processing data for the evaporation and nitric acid recycle system (NARS) in TA-55 has provided general acid mass balance trends, as well as the location of missing information in both the evaporation system and NARS data logs. The data were accumulated during the calendar years 2000 to 2005. After making a number of processing assumptions, the empirical system information was used to create an interactive spreadsheet that predicts, with moderate accuracy, some of the various stream variables for the combined evaporation and acid recycle processes. Empirical data and interactive calculations were compared to an Aspen Plus{trademark} simulation of the process.

  10. Nucleotide sequence of the Klebsiella pneumoniae nifD gene and predicted amino acid sequence of the alpha-subunit of nitrogenase MoFe protein.

    PubMed Central

    Ioannidis, I; Buck, M

    1987-01-01

    The nucleotide sequence of the Klebsiella pneumoniae nifD gene is presented and together with the accompanying paper [Holland, Zilberstein, Zamir & Sussman (1987) Biochem. J. 247, 277-285] completes the sequence of the nifHDK genes encoding the nitrogenase polypeptides. The K. pneumoniae nifD gene encodes the 483-amino acid-residue nitrogenase alpha-subunit polypeptide of Mr 54156. The alpha-subunit has five strongly conserved cysteine residues at positions 63, 89, 155, 184 and 275, some occurring in a region showing both primary sequence and potential structural homology to the K. pneumoniae nitrogenase beta-subunit. A comparison with six other alpha-subunit amino acid sequences has been made, which indicates a number of potentially important domains within alpha-subunits. PMID:3322262

  11. Dynamics and mass accommodation of HCl molecules on sulfuric acid-water surfaces.

    PubMed

    Behr, P; Scharfenort, U; Ataya, K; Zellner, R

    2009-09-28

    A molecular beam technique has been used to study the dynamics and mass accommodation of HCl molecules in collision with sulfuric acid-water surfaces. The experiments were performed by directing a nearly mono-energetic beam of HCl molecules onto a continuously renewed liquid film of 54-76 wt% sulfuric acid at temperatures between 213 K and 243 K. Deuterated sulfuric acid was used to separate sticking but non-reactive collisions from those that involved penetration through the phase boundary followed by dissociation and recombination with D+. The results indicate that the mass accommodation of HCl on sulfuric acid-water surfaces decreases sharply with increasing acidity over the concentration range 54-76 wt%. Using the capillary wave theory of mass accommodation this effect is explained by a change of the surface dynamics. Regarding the temperature dependence it is found that the mass accommodation of HCl increases with increasing temperature and is limited by the bulk phase viscosity and driven by the restoring forces of the surface tension. These findings imply that under atmospheric conditions the uptake of HCl from the gas phase depends crucially on the bulk phase parameters of the sulfuric acid aerosol.

  12. Complete amino acid sequence of the A chain of human complement-classical-pathway enzyme C1r.

    PubMed Central

    Arlaud, G J; Willis, A C; Gagnon, J

    1987-01-01

    The amino acid sequence of human C1r A chain was determined, from sequence analysis performed on fragments obtained from C1r autolytic cleavage, cleavage of methionyl bonds, tryptic cleavages at arginine and lysine residues, and cleavages by staphylococcal proteinase. The polypeptide chain has an N-terminal serine residue and contains 446 amino acid residues (Mr 51,200). The sequence data allow chemical characterization of fragments alpha (positions 1-211), beta (positions 212-279) and gamma (positions 280-446) yielded from C1r autolytic cleavage, and identification of the two major cleavage sites generating these fragments. Position 150 of C1r A chain is occupied by a modified amino acid residue that, upon acid hydrolysis, yields erythro-beta-hydroxyaspartic acid, and that is located in a sequence homologous to the beta-hydroxyaspartic acid-containing regions of Factor IX, Factor X, protein C and protein Z. Sequence comparison reveals internal homology between two segments (positions 10-78 and 186-257). Two carbohydrate moieties are attached to the polypeptide chain, both via asparagine residues at positions 108 and 204. Combined with the previously determined sequence of C1r B chain [Arlaud & Gagnon (1983) Biochemistry 22, 1758-1764], these data give the complete sequence of human C1r. PMID:3036070

  13. Nucleotide sequences of the Pseudomonas savastanoi indoleacetic acid genes show homology with Agrobacterium tumefaciens T-DNA

    PubMed Central

    Yamada, Tetsuji; Palm, Curtis J.; Brooks, Bob; Kosuge, Tsune

    1985-01-01

    We report the nucleotide sequences of iaaM and iaaH, the genetic determinants for, respectively, tryptophan 2-monooxygenase and indoleacetamide hydrolase, the enzymes that catalyze the conversion of L-tryptophan to indoleacetic acid in the tumor-forming bacterium Pseudomonas syringae pv. savastanoi. The sequence analysis indicates that the iaaM locus contains an open reading frame encoding 557 amino acids that would comprise a protein with a molecular weight of 61,783; the iaaH locus contains an open reading frame of 455 amino acids that would comprise a protein with a molecular weight of 48,515. Significant amino acid sequence homology was found between the predicted sequence of the tryptophan monooxygenase of P. savastanoi and the deduced product of the T-DNA tms-1 gene of the octopine-type plasmid pTiA6NC from Agrobacterium tumefaciens. Strong homology was found in the 25 amino acid sequence in the putative FAD-binding region of tryptophan monooxygenase. Homology was also found in the amino acid sequences representing the central regions of the putative products of iaaH and tms-2 T-DNA. The results suggest a strong similarity in the pathways for indoleacetic acid synthesis encoded by genes in P. savastanoi and in A. tumefaciens T-DNA. Images PMID:16593610

  14. Prediction of flexible/rigid regions from protein sequences using k-spaced amino acid pairs

    PubMed Central

    Chen, Ke; Kurgan, Lukasz A; Ruan, Jishou

    2007-01-01

    Background Traditionally, it is believed that the native structure of a protein corresponds to a global minimum of its free energy. However, with the growing number of known tertiary (3D) protein structures, researchers have discovered that some proteins can alter their structures in response to a change in their surroundings or with the help of other proteins or ligands. Such structural shifts play a crucial role with respect to the protein function. To this end, we propose a machine learning method for the prediction of the flexible/rigid regions of proteins (referred to as FlexRP); the method is based on a novel sequence representation and feature selection. Knowledge of the flexible/rigid regions may provide insights into the protein folding process and the 3D structure prediction. Results The flexible/rigid regions were defined based on a dataset, which includes protein sequences that have multiple experimental structures, and which was previously used to study the structural conservation of proteins. Sequences drawn from this dataset were represented based on feature sets that were proposed in prior research, such as PSI-BLAST profiles, composition vector and binary sequence encoding, and a newly proposed representation based on frequencies of k-spaced amino acid pairs. These representations were processed by feature selection to reduce the dimensionality. Several machine learning methods for the prediction of flexible/rigid regions and two recently proposed methods for the prediction of conformational changes and unstructured regions were compared with the proposed method. The FlexRP method, which applies Logistic Regression and collocation-based representation with 95 features, obtained 79.5% accuracy. The two runner-up methods, which apply the same sequence representation and Support Vector Machines (SVM) and Naïve Bayes classifiers, obtained 79.2% and 78.4% accuracy, respectively. The remaining considered methods are characterized by accuracies below 70

  15. Nucleic and amino acid sequences relating to a novel transketolase, and methods for the expression thereof

    DOEpatents

    Croteau, Rodney Bruce; Wildung, Mark Raymond; Lange, Bernd Markus; McCaskill, David G.

    2001-01-01

    cDNAs encoding 1-deoxyxylulose-5-phosphate synthase from peppermint (Mentha piperita) have been isolated and sequenced, and the corresponding amino acid sequences have been determined. Accordingly, isolated DNA sequences (SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7) are provided which code for the expression of 1-deoxyxylulose-5-phosphate synthase from plants. In another aspect the present invention provides for isolated, recombinant DXPS proteins, such as the proteins having the sequences set forth in SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8. In other aspects, replicable recombinant cloning vehicles are provided which code for plant 1-deoxyxylulose-5-phosphate synthases, or for a base sequence sufficiently complementary to at least a portion of 1-deoxyxylulose-5-phosphate synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding a plant 1-deoxyxylulose-5-phosphate synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant 1-deoxyxylulose-5-phosphate synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant 1-deoxyxylulose-5-phosphate synthase may be used to obtain expression or enhanced expression of 1-deoxyxylulose-5-phosphate synthase in plants in order to enhance the production of 1-deoxyxylulose-5-phosphate, or its derivatives such as isopentenyl diphosphate (BP), or may be otherwise employed for the regulation or expression of 1-deoxyxylulose-5-phosphate synthase, or the production of its products.

  16. NEGATIVE ION ELECTROSPRAY OF BROMO- AND CHLORACETIC ACIDS AND AN EVALUATION OF EXACT MASS MEASUREMENTS WITH A BENCH-TOP TIME-OF-FLIGHT MASS SPECTROMETER

    EPA Science Inventory

    The negative ion electrospray mass spectra of six bromo- and chloroacetic acids were measured using two different electrospray interfaces and single quadrupole and bench-top time-of-flight mass spectrometers. With each acid at 50 ug/mL in aqueous methanol at pH 10, the anions ob...

  17. Fatty acids of Thespesia populnea: Mass spectrometry of picolinyl esters of cyclopropene fatty acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Thespesia populnea belongs to the plant family of Malvaceae which contain cyclopropane and cyclopropene fatty acids. However, previous literature reports vary regarding the content of these compounds in Thespesia populnea seed oil. In this work, the content of malvalic acid (8,9-methylene-9-heptade...

  18. Gene sequence and predicted amino acid sequence of the motA protein, a membrane-associated protein required for flagellar rotation in Escherichia coli.

    PubMed Central

    Dean, G E; Macnab, R M; Stader, J; Matsumura, P; Burks, C

    1984-01-01

    The motA and motB gene products of Escherichia coli are integral membrane proteins necessary for flagellar rotation. We determined the DNA sequence of the region containing the motA gene and its promoter. Within this sequence, there is an open reading frame of 885 nucleotides, which with high probability (98% confidence level) meets criteria for a coding sequence. The 295-residue amino acid translation product had a molecular weight of 31,974, in good agreement with the value determined experimentally by gel electrophoresis. The amino acid sequence, which was quite hydrophobic, was subjected to a theoretical analysis designed to predict membrane-spanning alpha-helical segments of integral membrane proteins; four such hydrophobic helices were predicted by this treatment. Additional amphipathic helices may also be present. A remarkable feature of the sequence is the existence of two segments of high uncompensated charge density, one positive and the other negative. Possible organization of the protein in the membrane is discussed. Asymmetry in the amino acid composition of translated DNA sequences was used to distinguish between two possible initiation codons. The use of this method as a criterion for authentication of coding regions is described briefly in an Appendix. PMID:6090403

  19. Genome Sequence Analysis of the Naphthenic Acid Degrading and Metal Resistant Bacterium Cupriavidus gilardii CR3

    PubMed Central

    Xiao, Jingfa; Hao, Lirui; Crowley, David E.; Zhang, Zhewen; Yu, Jun; Huang, Ning; Huo, Mingxin; Wu, Jiayan

    2015-01-01

    Cupriavidus sp. are generally heavy metal tolerant bacteria with the ability to degrade a variety of aromatic hydrocarbon compounds, although the degradation pathways and substrate versatilities remain largely unknown. Here we studied the bacterium Cupriavidus gilardii strain CR3, which was isolated from a natural asphalt deposit, and which was shown to utilize naphthenic acids as a sole carbon source. Genome sequencing of C. gilardii CR3 was carried out to elucidate possible mechanisms for the naphthenic acid biodegradation. The genome of C. gilardii CR3 was composed of two circular chromosomes chr1 and chr2 of respectively 3,539,530 bp and 2,039,213 bp in size. The genome for strain CR3 encoded 4,502 putative protein-coding genes, 59 tRNA genes, and many other non-coding genes. Many genes were associated with xenobiotic biodegradation and metal resistance functions. Pathway prediction for degradation of cyclohexanecarboxylic acid, a representative naphthenic acid, suggested that naphthenic acid undergoes initial ring-cleavage, after which the ring fission products can be degraded via several plausible degradation pathways including a mechanism similar to that used for fatty acid oxidation. The final metabolic products of these pathways are unstable or volatile compounds that were not toxic to CR3. Strain CR3 was also shown to have tolerance to at least 10 heavy metals, which was mainly achieved by self-detoxification through ion efflux, metal-complexation and metal-reduction, and a powerful DNA self-repair mechanism. Our genomic analysis suggests that CR3 is well adapted to survive the harsh environment in natural asphalts containing naphthenic acids and high concentrations of heavy metals. PMID:26301592

  20. Genome Sequence Analysis of the Naphthenic Acid Degrading and Metal Resistant Bacterium Cupriavidus gilardii CR3.

    PubMed

    Wang, Xiaoyu; Chen, Meili; Xiao, Jingfa; Hao, Lirui; Crowley, David E; Zhang, Zhewen; Yu, Jun; Huang, Ning; Huo, Mingxin; Wu, Jiayan

    2015-01-01

    Cupriavidus sp. are generally heavy metal tolerant bacteria with the ability to degrade a variety of aromatic hydrocarbon compounds, although the degradation pathways and substrate versatilities remain largely unknown. Here we studied the bacterium Cupriavidus gilardii strain CR3, which was isolated from a natural asphalt deposit, and which was shown to utilize naphthenic acids as a sole carbon source. Genome sequencing of C. gilardii CR3 was carried out to elucidate possible mechanisms for the naphthenic acid biodegradation. The genome of C. gilardii CR3 was composed of two circular chromosomes chr1 and chr2 of respectively 3,539,530 bp and 2,039,213 bp in size. The genome for strain CR3 encoded 4,502 putative protein-coding genes, 59 tRNA genes, and many other non-coding genes. Many genes were associated with xenobiotic biodegradation and metal resistance functions. Pathway prediction for degradation of cyclohexanecarboxylic acid, a representative naphthenic acid, suggested that naphthenic acid undergoes initial ring-cleavage, after which the ring fission products can be degraded via several plausible degradation pathways including a mechanism similar to that used for fatty acid oxidation. The final metabolic products of these pathways are unstable or volatile compounds that were not toxic to CR3. Strain CR3 was also shown to have tolerance to at least 10 heavy metals, which was mainly achieved by self-detoxification through ion efflux, metal-complexation and metal-reduction, and a powerful DNA self-repair mechanism. Our genomic analysis suggests that CR3 is well adapted to survive the harsh environment in natural asphalts containing naphthenic acids and high concentrations of heavy metals.

  1. Formic and Acetic Acid Observations over Colorado by Chemical Ionization Mass Spectrometry and Organic Acids' Role in Air Quality

    NASA Astrophysics Data System (ADS)

    Treadaway, V.; O'Sullivan, D. W.; Heikes, B.; Silwal, I.; McNeill, A.

    2015-12-01

    Formic acid (HFo) and acetic acid (HAc) have both natural and anthropogenic sources and a role in the atmospheric processing of carbon. These organic acids also have an increasing importance in setting the acidity of rain and snow as precipitation nitrate and sulfate concentrations have decreased. Primary emissions for both organic acids include biomass burning, agriculture, and motor vehicle emissions. Secondary production is also a substantial source for both acids especially from biogenic precursors, secondary organic aerosols (SOAs), and photochemical production from volatile organic compounds (VOCs) and oxygenated volatile organic compounds (OVOCs). Chemical transport models underestimate organic acid concentrations and recent research has sought to develop additional production mechanisms. Here we report HFo and HAc measurements during two campaigns over Colorado using the peroxide chemical ionization mass spectrometer (PCIMS). Iodide clusters of both HFo and HAc were recorded at mass-to-charge ratios of 173 and 187, respectively. The PCIMS was flown aboard the NCAR Gulfstream-V platform during the Deep Convective Clouds and Chemistry Experiment (DC3) and aboard the NCAR C-130 during the Front Range Air Pollution and Photochemistry Experiment (FRAPPE). The DC3 observations were made in May and June 2012 extending from the surface to 13 km over the central and eastern United States. FRAPPE observations were made in July and August 2014 from the surface to 7 km over Colorado. DC3 measurements reported here are focused over the Colorado Front Range and complement the FRAPPE observations. DC3 HFo altitude profiles are characterized by a decrease up to 6 km followed by an increase either back to boundary layer mixing ratio values or higher (a "C" shape). Organic acid measurements from both campaigns are interpreted with an emphasis on emission sources (both natural and anthropogenic) over Colorado and in situ photochemical production especially ozone precursors.

  2. The impact of monomer sequence and stereochemistry on the swelling and erosion of biodegradable poly(lactic-co-glycolic acid) matrices.

    PubMed

    Washington, Michael A; Swiner, Devin J; Bell, Kerri R; Fedorchak, Morgan V; Little, Steven R; Meyer, Tara Y

    2017-02-01

    Monomer sequence is demonstrated to be a primary factor in determining the hydrolytic degradation profile of poly(lactic-co-glycolic acid)s (PLGAs). Although many approaches have been used to tune the degradation of PLGAs, little effort has been expended in exploring the sequence-control strategy exploited by nature in biopolymers. Cylindrical matrices and films prepared from a series of sequenced and random PLGAs were subjected to hydrolysis in a pH 7.4 buffer at 37 °C. Swelling ranged from 107% for the random racemic PLGA with a 50:50 ratio of lactic (L) to glycolic (G) units to 6% for the sequenced alternating copolymer poly LG. Erosion followed an inverse trend with the random 50:50 PLGA showing an erosion half-life of 3-4 weeks while poly LG required ca. >10 weeks. Stereosequence was found to play a large role in determining swelling and erosion; stereopure analogs swelled less and were slower to lose mass. Molecular weight loss followed similar trends and increases in dispersity correlated with the onset of significant swelling. The relative proportion of rapidly cleavable G-G linkages relative to G-L/L-G (moderate) and L-L (slow) correlates strongly with the degree of swelling observed and the rate of erosion. The dramatic sequence-dependent variation in swelling, in the absence of a parallel hydrophilicity trend, suggest that osmotic pressure, driven by the differential accumulation of degradation products, plays an important role.

  3. Repeat sequence chromosome specific nucleic acid probes and methods of preparing and using

    DOEpatents

    Weier, Heinz-Ulrich G.; Gray, Joe W.

    1995-01-01

    A primer directed DNA amplification method to isolate efficiently chromosome-specific repeated DNA wherein degenerate oligonucleotide primers are used is disclosed. The probes produced are a heterogeneous mixture that can be used with blocking DNA as a chromosome-specific staining reagent, and/or the elements of the mixture can be screened for high specificity, size and/or high degree of repetition among other parameters. The degenerate primers are sets of primers that vary in sequence but are substantially complementary to highly repeated nucleic acid sequences, preferably clustered within the template DNA, for example, pericentromeric alpha satellite repeat sequences. The template DNA is preferably chromosome-specific. Exemplary primers ard probes are disclosed. The probes of this invention can be used to determine the number of chromosomes of a specific type in metaphase spreads, in germ line and/or somatic cell interphase nuclei, micronuclei and/or in tissue sections. Also provided is a method to select arbitrarily repeat sequence probes that can be screened for chromosome-specificity.

  4. Unconventional amino acid sequence of the sun anemone (Stoichactis helianthus) polypeptide neurotoxin

    SciTech Connect

    Kem, W.; Dunn, B.; Parten, B.; Pennington, M.; Price, D.

    1986-05-01

    A 5000 dalton polypeptide neurotoxin (Sh-NI) purified by G50 Sephadex, P-cellulose, and SP-Sephadex chromatography was homogeneous by isoelectric focusing. Sh-NI was highly toxic to crayfish (LD/sub 50/ 0.6 ..mu..g/kg) but without effect upon mice at 15,000 ..mu..g/kg (i.p. injection). The reduced, /sup 3/H-carboxymethylated toxin and its fragments were subjected to automatic Edman degradation and the resulting PTH-amino acids were identified by HPLC, back hydrolysis, and scintillation counting. Peptides resulting from proteolytic (clostripain, staphylococcal protease) and chemical (tryptophan) cleavage were sequenced. The sequence is: AACKCDDEGPDIRTAPLTGTVDLGSCNAGWEKCASYYTIIADCCRKKK. This sequence differs considerably from the homologous Anemonia and Anthopleura toxins; many of the identical residues (6 half-cystines, G9, P10, R13, G19, G29, W30) are probably critical for folding rather than receptor recognition. However, the Sh-NI sequence closely resembles Radioanthus macrodactylus neurotoxin III and r. paumotensis II. The authors propose that Sh-NI and related Radioanthus toxins act upon a different site on the sodium channel.

  5. Repeat sequence chromosome specific nucleic acid probes and methods of preparing and using

    DOEpatents

    Weier, H.U.G.; Gray, J.W.

    1995-06-27

    A primer directed DNA amplification method to isolate efficiently chromosome-specific repeated DNA wherein degenerate oligonucleotide primers are used is disclosed. The probes produced are a heterogeneous mixture that can be used with blocking DNA as a chromosome-specific staining reagent, and/or the elements of the mixture can be screened for high specificity, size and/or high degree of repetition among other parameters. The degenerate primers are sets of primers that vary in sequence but are substantially complementary to highly repeated nucleic acid sequences, preferably clustered within the template DNA, for example, pericentromeric alpha satellite repeat sequences. The template DNA is preferably chromosome-specific. Exemplary primers and probes are disclosed. The probes of this invention can be used to determine the number of chromosomes of a specific type in metaphase spreads, in germ line and/or somatic cell interphase nuclei, micronuclei and/or in tissue sections. Also provided is a method to select arbitrarily repeat sequence probes that can be screened for chromosome-specificity. 18 figs.

  6. Sequence-defined bioactive macrocycles via an acid-catalysed cascade reaction

    NASA Astrophysics Data System (ADS)

    Porel, Mintu; Thornlow, Dana N.; Phan, Ngoc N.; Alabi, Christopher A.

    2016-06-01

    Synthetic macrocycles derived from sequence-defined oligomers are a unique structural class whose ring size, sequence and structure can be tuned via precise organization of the primary sequence. Similar to peptides and other peptidomimetics, these well-defined synthetic macromolecules become pharmacologically relevant when bioactive side chains are incorporated into their primary sequence. In this article, we report the synthesis of oligothioetheramide (oligoTEA) macrocycles via a one-pot acid-catalysed cascade reaction. The versatility of the cyclization chemistry and modularity of the assembly process was demonstrated via the synthesis of >20 diverse oligoTEA macrocycles. Structural characterization via NMR spectroscopy revealed the presence of conformational isomers, which enabled the determination of local chain dynamics within the macromolecular structure. Finally, we demonstrate the biological activity of oligoTEA macrocycles designed to mimic facially amphiphilic antimicrobial peptides. The preliminary results indicate that macrocyclic oligoTEAs with just two-to-three cationic charge centres can elicit potent antibacterial activity against Gram-positive and Gram-negative bacteria.

  7. Microbial community dynamics in bioaugmented sequencing batch reactors for bromoamine acid removal.

    PubMed

    Qu, Yuanyuan; Zhou, Jiti; Wang, Jing; Fu, Xiang; Xing, Linlin

    2005-05-01

    Sphingomonas xenophaga QYY with the ability to degrade bromoamine acid (BAA) was previously isolated from sludge samples. The enhancement of BAA removal by strain QYY in sequencing batch reactors (SBRs) was investigated in this study. The results showed that augmented SBRs exhibited stronger abilities to degrade BAA than the non-augmented control one. In order to estimate the relationship between community dynamics and function of augmented SBRs, a combined method based on fingerprints (ribosomal intergenic spacer analysis, RISA) and 16S rRNA gene sequencing was used. The results indicated that the microbial community dynamics were substantially changed, and the introduced strain QYY was persistent in the augmented systems. This study suggests that it is feasible and potentially useful to enhance BAA removal using BAA-degrading bacteria, such as S. xenophaga QYY.

  8. NullSeq: A Tool for Generating Random Coding Sequences with Desired Amino Acid and GC Contents

    PubMed Central

    Liu, Sophia S.; Hockenberry, Adam J.; Lancichinetti, Andrea; Jewett, Michael C.

    2016-01-01

    The existence of over- and under-represented sequence motifs in genomes provides evidence of selective evolutionary pressures on biological mechanisms such as transcription, translation, ligand-substrate binding, and host immunity. In order to accurately identify motifs and other genome-scale patterns of interest, it is essential to be able to generate accurate null models that are appropriate for the sequences under study. While many tools have been developed to create random nucleotide sequences, protein coding sequences are subject to a unique set of constraints that complicates the process of generating appropriate null models. There are currently no tools available that allow users to create random coding sequences with specified amino acid composition and GC content for the purpose of hypothesis testing. Using the principle of maximum entropy, we developed a method that generates unbiased random sequences with pre-specified amino acid and GC content, which we have developed into a python package. Our method is the simplest way to obtain maximally unbiased random sequences that are subject to GC usage and primary amino acid sequence constraints. Furthermore, this approach can easily be expanded to create unbiased random sequences that incorporate more complicated constraints such as individual nucleotide usage or even di-nucleotide frequencies. The ability to generate correctly specified null models will allow researchers to accurately identify sequence motifs which will lead to a better understanding of biological processes as well as more effective engineering of biological systems. PMID:27835644

  9. Morphological tranformation of calcite crystal growth by prismatic "acidic" polypeptide sequences.

    SciTech Connect

    Kim, I; Giocondi, J L; Orme, C A; Collino, J; Evans, J S

    2007-02-13

    Many of the interesting mechanical and materials properties of the mollusk shell are thought to stem from the prismatic calcite crystal assemblies within this composite structure. It is now evident that proteins play a major role in the formation of these assemblies. Recently, a superfamily of 7 conserved prismatic layer-specific mollusk shell proteins, Asprich, were sequenced, and the 42 AA C-terminal sequence region of this protein superfamily was found to introduce surface voids or porosities on calcite crystals in vitro. Using AFM imaging techniques, we further investigate the effect that this 42 AA domain (Fragment-2) and its constituent subdomains, DEAD-17 and Acidic-2, have on the morphology and growth kinetics of calcite dislocation hillocks. We find that Fragment-2 adsorbs on terrace surfaces and pins acute steps, accelerates then decelerates the growth of obtuse steps, forms clusters and voids on terrace surfaces, and transforms calcite hillock morphology from a rhombohedral form to a rounded one. These results mirror yet are distinct from some of the earlier findings obtained for nacreous polypeptides. The subdomains Acidic-2 and DEAD-17 were found to accelerate then decelerate obtuse steps and induce oval rather than rounded hillock morphologies. Unlike DEAD-17, Acidic-2 does form clusters on terrace surfaces and exhibits stronger obtuse velocity inhibition effects than either DEAD-17 or Fragment-2. Interestingly, a 1:1 mixture of both subdomains induces an irregular polygonal morphology to hillocks, and exhibits the highest degree of acute step pinning and obtuse step velocity inhibition. This suggests that there is some interplay between subdomains within an intra (Fragment-2) or intermolecular (1:1 mixture) context, and sequence interplay phenomena may be employed by biomineralization proteins to exert net effects on crystal growth and morphology.

  10. Sequence selective recognition of double-stranded RNA using triple helix-forming peptide nucleic acids.

    PubMed

    Zengeya, Thomas; Gupta, Pankaj; Rozners, Eriks

    2014-01-01

    Noncoding RNAs are attractive targets for molecular recognition because of the central role they play in gene expression. Since most noncoding RNAs are in a double-helical conformation, recognition of such structures is a formidable problem. Herein, we describe a method for sequence-selective recognition of biologically relevant double-helical RNA (illustrated on ribosomal A-site RNA) using peptide nucleic acids (PNA) that form a triple helix in the major grove of RNA under physiologically relevant conditions. Protocols for PNA preparation and binding studies using isothermal titration calorimetry are described in detail.

  11. Fast computational methods for predicting protein structure from primary amino acid sequence

    DOEpatents

    Agarwal, Pratul Kumar

    2011-07-19

    The present invention provides a method utilizing primary amino acid sequence of a protein, energy minimization, molecular dynamics and protein vibrational modes to predict three-dimensional structure of a protein. The present invention also determines possible intermediates in the protein folding pathway. The present invention has important applications to the design of novel drugs as well as protein engineering. The present invention predicts the three-dimensional structure of a protein independent of size of the protein, overcoming a significant limitation in the prior art.

  12. Lactobacillus kefiri shows inter-strain variations in the amino acid sequence of the S-layer proteins.

    PubMed

    Malamud, Mariano; Carasi, Paula; Bronsoms, Sílvia; Trejo, Sebastián A; Serradell, María de Los Angeles

    2017-04-01

    The S-layer is a proteinaceous envelope constituted by subunits that self-assemble to form a two-dimensional lattice that covers the surface of different species of Bacteria and Archaea, and it could be involved in cell recognition of microbes among other several distinct functions. In this work, both proteomic and genomic approaches were used to gain knowledge about the sequences of the S-layer protein (SLPs) encoding genes expressed by six aggregative and sixteen non-aggregative strains of potentially probiotic Lactobacillus kefiri. Peptide mass fingerprint (PMF) analysis confirmed the identity of SLPs extracted from L. kefiri, and based on the homology with phylogenetically related species, primers located outside and inside the SLP-genes were employed to amplify genomic DNA. The O-glycosylation site SASSAS was found in all L. kefiri SLPs. Ten strains were selected for sequencing of the complete genes. The total length of the mature proteins varies from 492 to 576 amino acids, and all SLPs have a calculated pI between 9.37 and 9.60. The N-terminal region is relatively conserved and shows a high percentage of positively charged amino acids. Major differences among strains are found in the C-terminal region. Different groups could be distinguished regarding the mature SLPs and the similarities observed in the PMF spectra. Interestingly, SLPs of the aggregative strains are 100% homologous, although these strains were isolated from different kefir grains. This knowledge provides relevant data for better understanding of the mechanisms involved in SLPs functionality and could contribute to the development of products of biotechnological interest from potentially probiotic bacteria.

  13. Mass spectrometric determination of acromelic acid A from a new poisonous mushroom: Clitocybe amoenolens.

    PubMed

    Bessard, J; Saviuc, P; Chane-Yene, Y; Monnet, S; Bessard, G

    2004-11-05

    As Clitocybe acromelalga, the mushroom Clitocybe amoenolens is responsible for erythermalgia. Acromelic acids isolated from C. acromelalga have been suspected to be to some extend the active principles. The objective was to develop a specific and sensitive liquid chromatographic-mass spectrometric method that would allow acromelic acid A identification and quantification in mushrooms. The method involved a single-step methanol-water extraction followed by a selective cleanup of the extract with solid-phase extraction cartridges (strong-anion exchange). The chromatographic separation was achieved on a porous graphitic carbon column with acetonitrile-water-formic acid as mobile phase. Detection was done with a mass analyzer equipped with a TurboIonSpray source, operated in the negative ionization mode. Acromelic acid A concentration was determined in dried mushroom at around 325 ng/mg in C. amoenolens and 283 ng/mg in C. acromelalga.

  14. Fluorescence energy transfer as a probe for nucleic acid structures and sequences.

    PubMed Central

    Mergny, J L; Boutorine, A S; Garestier, T; Belloc, F; Rougée, M; Bulychev, N V; Koshkin, A A; Bourson, J; Lebedev, A V; Valeur, B

    1994-01-01

    The primary or secondary structure of single-stranded nucleic acids has been investigated with fluorescent oligonucleotides, i.e., oligonucleotides covalently linked to a fluorescent dye. Five different chromophores were used: 2-methoxy-6-chloro-9-amino-acridine, coumarin 500, fluorescein, rhodamine and ethidium. The chemical synthesis of derivatized oligonucleotides is described. Hybridization of two fluorescent oligonucleotides to adjacent nucleic acid sequences led to fluorescence excitation energy transfer between the donor and the acceptor dyes. This phenomenon was used to probe primary and secondary structures of DNA fragments and the orientation of oligodeoxynucleotides synthesized with the alpha-anomers of nucleoside units. Fluorescence energy transfer can be used to reveal the formation of hairpin structures and the translocation of genes between two chromosomes. PMID:8152922

  15. Amino acid sequence of two neurotoxins from the venom of the Egyptian black snake (Walterinnesia aegyptia).

    PubMed

    Samejima, Y; Aoki-Tomomatsu, Y; Yanagisawa, M; Mebs, D

    1997-02-01

    The venom of the Egyptian black snake Walterinnesia aegyptia contains at least three toxins, which act postsynaptically to block the neuromuscular transmission of isolated rat phrenic nerve-diaphragm and chicken biventer cervicis muscle. The complete amino acid sequence of the two toxins, W-III and W-IV, consisting of 62 amino acid residues, was elucidated by Edman degradation of fragments obtained after Staphylococcus aureus protease and prolylpeptidase digestion. Although the toxins exhibit close structural homology to other short-chain postsynaptic neurotoxins from Elapidae venoms, toxin IV is unique by having a free SH-group (cysteine) at position 16. In position 35 of W-III, which is located at the tip of the central loop, threonine is replaced by lysine, which may alter the interaction of the toxin with the acetylcholine receptor, since the toxin is seven times less lethal than toxin W-IV.

  16. Fatty acids composition of Caenorhabditis elegans using accurate mass GCMS-QTOF.

    PubMed

    Henry, Parise; Owopetu, Olufunmilayo; Adisa, Demilade; Nguyen, Thao; Anthony, Kevin; Ijoni-Animadu, David; Jamadar, Sakha; Abdel-Rahman, Fawzia; Saleh, Mahmoud A

    2016-08-02

    The free living nematode Caenorhabditis elegans is a proven model organism for lipid metabolism research. Total lipids of C. elegans were extracted using chloroform and methanol in 2:1 ratio (v/v). Fatty acids composition of the extracted total lipids was converted to their corresponding fatty acids methyl esters (FAMEs) and analyzed by gas chromatography/accurate mass quadrupole time of flight mass spectrometry using both electron ionization and chemical ionization techniques. Twenty-eight fatty acids consisting of 12 to 22 carbon atoms were identified, 65% of them were unsaturated. Fatty acids containing 12 to17 carbons were mostly saturated with stearic acid (18:0) as the major constituent. Several branched-chain fatty acids were identified. Methyl-14-methylhexadecanoate (iso- 17:0) was the major identified branched fatty acid. This is the first report to detect the intact molecular parent ions of the identified fatty acids in C. elegans using chemical ionization compared to electron ionization which produced fragmentations of the FAMEs.

  17. [Determination of 13C enrichment in soil amino acid enantiomers by gas chromatogram/mass spectrometry].

    PubMed

    He, Hong-Bo; Zhang, Wei; Ding, Xue-Li; Bai, Zhen; Liu, Ning; Zhang, Xu-Dong

    2008-06-01

    The transformation and renewal of amino acid enantiomers is of significance in indicating the turnover mechanism of soil organic matter. In this paper, a method of gas chromatogram/mass spectrometry combined with U-13 C-glucose incubation was developed to determine the 13C enrichment in soil amino acid enantiomers, which could effectively differentiate the original and the newly synthesized amino acids in soil matrix. The added U-13 C-glucose was utilized rapidly to structure the amino acid carbon skeleton, and the change of relative abundance of isotope ions could be determined by mass spectrometry. The direct incorporation of U-13 C glucose was estimated by the intensity increase of m/z (F + n) to F (F was parent fragment, and n was the carbon number in the fragment), while the total isotope incorporation from the added 13C could be calculated according to the abundance ratio increment summation from m/z (Fa + 1) through (Fa + T) (Fa was the fragment containing all original skeleton carbons, and T was the carbon number in the amino acid molecule). The 13C enrichment in the target compound was expressed as atom percentage excess (APE), and that of D-amino acid needed to be corrected by the coefficient of hydrolysis-induced racemization. The 13C enrichment reflected the carbon turnover velocity of individual amino acid enantiomers, and was powerful to investigate the dynamics of soil amino acids.

  18. Mass spectral determination of aldehydes, ketones, and carboxylic acids using 1,1-dimethylhydrazine.

    PubMed

    McDaniel, C A; Howard, R W

    1985-03-01

    Analyses of nanogram to milligram quantities of aliphatic aldehydes, fatty acids, and unhindered aliphatic ketones such as those typically found in pheromonal blends have been effected by treating these mixtures with 1,1-dimethylhydrazine. The aldehydes and ketones formN,N-dimethylhydrazones, while the fatty acids form methyl esters. Structural elucidation of the reaction products was achieved using EI and CI gas chromatography-mass spectrometry.

  19. Complete genome sequence of Lactococcus lactis IO-1, a lactic acid bacterium that utilizes xylose and produces high levels of L-lactic acid.

    PubMed

    Kato, Hiroaki; Shiwa, Yuh; Oshima, Kenshiro; Machii, Miki; Araya-Kojima, Tomoko; Zendo, Takeshi; Shimizu-Kadota, Mariko; Hattori, Masahira; Sonomoto, Kenji; Yoshikawa, Hirofumi

    2012-04-01

    We report the complete genome sequence of Lactococcus lactis IO-1 (= JCM7638). It is a nondairy lactic acid bacterium, produces nisin Z, ferments xylose, and produces predominantly L-lactic acid at high xylose concentrations. From ortholog analysis with other five L. lactis strains, IO-1 was identified as L. lactis subsp. lactis.

  20. Complete genome sequence of Bacillus amyloliquefaciens LL3, which exhibits glutamic acid-independent production of poly-γ-glutamic acid.

    PubMed

    Geng, Weitao; Cao, Mingfeng; Song, Cunjiang; Xie, Hui; Liu, Li; Yang, Chao; Feng, Jun; Zhang, Wei; Jin, Yinghong; Du, Yang; Wang, Shufang

    2011-07-01

    Bacillus amyloliquefaciens is one of most prevalent Gram-positive aerobic spore-forming bacteria with the ability to synthesize polysaccharides and polypeptides. Here, we report the complete genome sequence of B. amyloliquefaciens LL3, which was isolated from fermented food and presents the glutamic acid-independent production of poly-γ-glutamic acid.

  1. Formation Sequences of Iron Minerals in the Acidic Alteration Products and Variation of Hydrothermal Fluid Conditions

    NASA Astrophysics Data System (ADS)

    Isobe, H.; Yoshizawa, M.

    2008-12-01

    Iron minerals have important role in environmental issues not only on the Earth but also other terrestrial planets. Iron mineral species related to alteration products of primary minerals with surface or subsurface fluids are characterized by temperature, acidity and redox conditions of the fluids. We can see various iron- bearing alteration products in alteration products around fumaroles in geothermal/volcanic areas. In this study, zonal structures of iron minerals in alteration products of the geothermal area are observed to elucidate temporal and spatial variation of hydrothermal fluids. Alteration of the pyroxene-amphibole andesite of Garan-dake volcano, Oita, Japan occurs by the acidic hydrothermal fluid to form cristobalite leaching out elements other than Si. Hand specimens with unaltered or weakly altered core and cristobalite crust show various sequences of layers. XRD analysis revealed that the alteration degree is represented by abundance of cristobalite. Intermediately altered layers are characterized by occurrence including alunite, pyrite, kaolinite, goethite and hematite. A specimen with reddish brown core surrounded by cristobalite-rich white crust has brown colored layers at the boundary of core and the crust. Reddish core is characterized by occurrence of crystalline hematite by XRD. Another hand specimen has light gray core, which represents reduced conditions, and white cristobalite crust with light brown and reddish brown layers of ferric iron minerals between the core and the crust. On the other hand, hornblende crystals, typical ferrous iron-bearing mineral of the host rock, are well preserved in some samples with strongly decolorized cristobalite-rich groundmass. Hydrothermal alteration experiments of iron-rich basaltic material shows iron mineral species depend on acidity and temperature of the fluid. Oxidation states of the iron-bearing mineral species are strongly influenced by the acidity and redox conditions. Variations of alteration

  2. 16S rRNA Gene Sequencing, Multilocus Sequence Analysis, and Mass Spectrometry Identification of the Proposed New Species “Clostridium neonatale”

    PubMed Central

    Bouvet, Philippe; Ferraris, Laurent; Dauphin, Brunhilde; Popoff, Michel-Robert; Butel, Marie Jose

    2014-01-01

    In 2002, an outbreak of necrotizing enterocolitis in a Canadian neonatal intensive care unit was associated with a proposed novel species of Clostridium, “Clostridium neonatale.” To date, there are no data about the isolation, identification, or clinical significance of this species. Additionally, C. neonatale has not been formally classified as a new species, rendering its identification challenging. Indeed, the C. neonatale 16S rRNA gene sequence shows high similarity to another Clostridium species involved in neonatal necrotizing enterocolitis, Clostridium butyricum. By performing a polyphasic study combining phylogenetic analysis (16S rRNA gene sequencing and multilocus sequence analysis) and phenotypic characterization with mass spectrometry, we demonstrated that C. neonatale is a new species within the Clostridium genus sensu stricto, for which we propose the name Clostridium neonatale sp. nov. Now that the status of C. neonatale has been clarified, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) can be used for better differential identification of C. neonatale and C. butyricum clinical isolates. This is necessary to precisely define the role and clinical significance of C. neonatale, a species that may have been misidentified and underrepresented during previous neonatal necrotizing enterocolitis studies. PMID:25232167

  3. 16S rRNA gene sequencing, multilocus sequence analysis, and mass spectrometry identification of the proposed new species "Clostridium neonatale".

    PubMed

    Bouvet, Philippe; Ferraris, Laurent; Dauphin, Brunhilde; Popoff, Michel-Robert; Butel, Marie Jose; Aires, Julio

    2014-12-01

    In 2002, an outbreak of necrotizing enterocolitis in a Canadian neonatal intensive care unit was associated with a proposed novel species of Clostridium, "Clostridium neonatale." To date, there are no data about the isolation, identification, or clinical significance of this species. Additionally, C. neonatale has not been formally classified as a new species, rendering its identification challenging. Indeed, the C. neonatale 16S rRNA gene sequence shows high similarity to another Clostridium species involved in neonatal necrotizing enterocolitis, Clostridium butyricum. By performing a polyphasic study combining phylogenetic analysis (16S rRNA gene sequencing and multilocus sequence analysis) and phenotypic characterization with mass spectrometry, we demonstrated that C. neonatale is a new species within the Clostridium genus sensu stricto, for which we propose the name Clostridium neonatale sp. nov. Now that the status of C. neonatale has been clarified, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can be used for better differential identification of C. neonatale and C. butyricum clinical isolates. This is necessary to precisely define the role and clinical significance of C. neonatale, a species that may have been misidentified and underrepresented during previous neonatal necrotizing enterocolitis studies.

  4. Complete Genome Sequence of Enterobacter cloacae UW5, a Rhizobacterium Capable of High Levels of Indole-3-Acetic Acid Production.

    PubMed

    Coulson, Thomas J D; Patten, Cheryl L

    2015-08-06

    We report the complete genome sequence of Enterobacter cloacae UW5, an indole-3-acetic acid-producing rhizobacterium originally isolated from the rhizosphere of grass. The 4.9-Mbp genome has a G+C content of 54% and contains 4,496 protein-coding sequences.

  5. Complete Genome Sequence of Enterobacter cloacae UW5, a Rhizobacterium Capable of High Levels of Indole-3-Acetic Acid Production

    PubMed Central

    Coulson, Thomas J. D.

    2015-01-01

    We report the complete genome sequence of Enterobacter cloacae UW5, an indole-3-acetic acid-producing rhizobacterium originally isolated from the rhizosphere of grass. The 4.9-Mbp genome has a G+C content of 54% and contains 4,496 protein-coding sequences. PMID:26251488

  6. Genome Sequence of the Lactic Acid Bacterium Lactococcus lactis subsp. lactis TOMSC161, Isolated from a Nonscalded Curd Pressed Cheese

    PubMed Central

    Velly, H.; Abraham, A.-L.; Loux, V.; Delacroix-Buchet, A.; Fonseca, F.; Bouix, M.

    2014-01-01

    Lactococcus lactis is a lactic acid bacterium used in the production of many fermented foods, such as dairy products. Here, we report the genome sequence of L. lactis subsp. lactis TOMSC161, isolated from nonscalded curd pressed cheese. This genome sequence provides information in relation to dairy environment adaptation. PMID:25377704

  7. Deoxyribonucleic acid sequence of araBAD promoter mutants of Escherichia coli.

    PubMed

    Horwitz, A H; Morandi, C; Wilcox, G

    1980-05-01

    The controlling site region for the araBAD operon is defined, in part, by two classes of cis-acting constitutive mutations. The aralc mutations allow low-level constitutive expression of ara-BAD in the absence of the positive regulatory protein coded for by the araC gene, whereas the araXc mutations allow expression of araBAD in the absence of the cyclic adenosine monophosphate receptor protein. Six independently isolated aralc mutations and three independently isolated araXc mutations were cloned onto the plasmid pBR322 using in vitro recombinant deoxyribonucleic acid techniques and in vivo recombination between plasmid and chromosomal deoxyribonucleic acid. The location of these mutations was determined by deoxyribonucleic acid sequence analysis. All of the aralc mutations occurred at position -35 within the araBAD promoter (+1 = messenger ribonucleic acid start for araBAD) and resulted from an AT leads to GC transition. All of the araXc mutations occurred at position -10 within the araBAD promoter and resulted from a GC leads to AT transition. Models are presented to explain the mode of action of the aralc and araXc mutations.

  8. Loss of Free Fatty Acid Receptor 2 leads to impaired islet mass and beta cell survival

    PubMed Central

    Villa, Stephanie R.; Priyadarshini, Medha; Fuller, Miles H.; Bhardwaj, Tanya; Brodsky, Michael R.; Angueira, Anthony R.; Mosser, Rockann E.; Carboneau, Bethany A.; Tersey, Sarah A.; Mancebo, Helena; Gilchrist, Annette; Mirmira, Raghavendra G.; Gannon, Maureen; Layden, Brian T.

    2016-01-01

    The regulation of pancreatic β cell mass is a critical factor to help maintain normoglycemia during insulin resistance. Nutrient-sensing G protein-coupled receptors (GPCR) contribute to aspects of β cell function, including regulation of β cell mass. Nutrients such as free fatty acids (FFAs) contribute to precise regulation of β cell mass by signaling through cognate GPCRs, and considerable evidence suggests that circulating FFAs promote β cell expansion by direct and indirect mechanisms. Free Fatty Acid Receptor 2 (FFA2) is a β cell-expressed GPCR that is activated by short chain fatty acids, particularly acetate. Recent studies of FFA2 suggest that it may act as a regulator of β cell function. Here, we set out to explore what role FFA2 may play in regulation of β cell mass. Interestingly, Ffar2−/− mice exhibit diminished β cell mass at birth and throughout adulthood, and increased β cell death at adolescent time points, suggesting a role for FFA2 in establishment and maintenance of β cell mass. Additionally, activation of FFA2 with Gαq/11-biased agonists substantially increased β cell proliferation in in vitro and ex vivo proliferation assays. Collectively, these data suggest that FFA2 may be a novel therapeutic target to stimulate β cell growth and proliferation. PMID:27324831

  9. In the TTF-1 homeodomain the contribution of several amino acids to DNA recognition depends on the bound sequence.

    PubMed Central

    Fabbro, D; Tell, G; Leonardi, A; Pellizzari, L; Pucillo, C; Lonigro, R; Formisano, S; Damante, G

    1996-01-01

    The thyroid transcription factor-1 homeodomain (TTF-1HD) shows a peculiar DNA binding specificity, preferentially recognizing sequences containing the 5'-CAAG-3' core motif. Most other homeodomains instead recognize sites containing the 5'-TAAT-3' core motif. Here, we show that TTF-1HD efficiently recognizes another sequence, called D1, devoid of the 5'-CAAG-3' core motif. Different experimental approaches indicate that TTF-1HD contacts the D1 sequence in a manner which is different to that used to interact with sequences containing the 5'-CAAG-3' core motif. The binding activities that mutants of TTF-1HD display with the D1 sequence or with the sequence containing the 5'-CAAG-3' core motif indicate that the role of several DNA-contacting amino acids is different. In particular, during recognition of the D1 sequence, backbone-interacting amino acids not relevant in binding to sequences containing the 5'-CAAG-3' core motif play an important role. In the TTF-1HD, therefore, the contribution of several amino acids to DNA recognition depends on the bound sequence. These data indicate that although a common bonding network exists in all of the HD/DNA complexes, peculiarities important for DNA recognition may occur in single cases. PMID:8811078

  10. The Interior Structure Constants as an Age Diagnostic for Low-mass, Pre-main-sequence Detached Eclipsing Binary Stars

    NASA Astrophysics Data System (ADS)

    Feiden, Gregory A.; Dotter, Aaron

    2013-03-01

    We propose a novel method for determining the ages of low-mass, pre-main-sequence stellar systems using the apsidal motion of low-mass detached eclipsing binaries. The apsidal motion of a binary system with an eccentric orbit provides information regarding the interior structure constants of the individual stars. These constants are related to the normalized stellar interior density distribution and can be extracted from the predictions of stellar evolution models. We demonstrate that low-mass, pre-main-sequence stars undergoing radiative core contraction display rapidly changing interior structure constants (greater than 5% per 10 Myr) that, when combined with observational determinations of the interior structure constants (with 5%-10% precision), allow for a robust age estimate. This age estimate, unlike those based on surface quantities, is largely insensitive to the surface layer where effects of magnetic activity are likely to be most pronounced. On the main sequence, where age sensitivity is minimal, the interior structure constants provide a valuable test of the physics used in stellar structure models of low-mass stars. There are currently no known systems where this technique is applicable. Nevertheless, the emphasis on time domain astronomy with current missions, such as Kepler, and future missions, such as LSST, has the potential to discover systems where the proposed method will be observationally feasible.

  11. Molecular cloning, encoding sequence, and expression of vaccinia virus nucleic acid-dependent nucleoside triphosphatase gene.

    PubMed Central

    Rodriguez, J F; Kahn, J S; Esteban, M

    1986-01-01

    A rabbit poxvirus genomic library contained within the expression vector lambda gt11 was screened with polyclonal antiserum prepared against vaccinia virus nucleic acid-dependent nucleoside triphosphatase (NTPase)-I enzyme. Five positive phage clones containing from 0.72- to 2.5-kilobase-pair (kbp) inserts expressed a beta-galactosidase fusion protein that was reactive by immunoblotting with the NTPase-I antibody. Hybridization analysis allowed the location of this gene within the vaccinia HindIIID restriction fragment. From the known nucleotide sequence of the 16-kbp vaccinia HindIIID fragment, we identified a region that contains a 1896-base open reading frame coding for a 631-amino acid protein. Analysis of the complete sequence revealed a highly basic protein, with hydrophilic COOH and NH2 termini, various hydrophobic domains, and no significant homology to other known proteins. Translational studies demonstrate that NTPase-I belongs to a late class of viral genes. This protein is highly conserved among Orthopoxviruses. Images PMID:3025846

  12. The amino acid sequences and activities of synergistic hemolysins from Staphylococcus cohnii.

    PubMed

    Mak, Pawel; Maszewska, Agnieszka; Rozalska, Malgorzata

    2008-10-01

    Staphylococcus cohnii ssp. cohnii and S. cohnii ssp. urealyticus are a coagulase-negative staphylococci considered for a long time as unable to cause infections. This situation changed recently and pathogenic strains of these bacteria were isolated from hospital environments, patients and medical staff. Most of the isolated strains were resistant to many antibiotics. The present work describes isolation and characterization of several synergistic peptide hemolysins produced by these bacteria and acting as virulence factors responsible for hemolytic and cytotoxic activities. Amino acid sequences of respective hemolysins from S. cohnii ssp. cohnii (named as H1C, H2C and H3C) and S. cohnii ssp. urealyticus (H1U, H2U and H3U) were identical. Peptides H1 and H3 possessed significant amino acid homology to three synergistic hemolysins secreted by Staphylococcus lugdunensis and to putative antibacterial peptide produced by Staphylococcus saprophyticus ssp. saprophyticus. On the other hand, hemolysin H2 had a unique sequence. All isolated peptides lysed red cells from different mammalian species and exerted a cytotoxic effect on human fibroblasts.

  13. Complete amino acid sequence of a Lolium perenne (perennial rye grass) pollen allergen, Lol p II.

    PubMed

    Ansari, A A; Shenbagamurthi, P; Marsh, D G

    1989-07-05

    The complete amino acid sequence of a Lolium perenne (rye grass) pollen allergen, Lol p II was determined by automated Edman degradation of the protein and selected fragments. Cleavage of the protein by enzymatic and chemical techniques established an unambiguous sequence for the protein. Lol p II contains 97 amino acid residues, with a calculated molecular weight of 10,882. The protein lacks cysteine and glutamine and shows no evidence of glycosylation. Theoretical predictions by Fraga's (Fraga, S. (1982) Can. J. Chem. 60, 2606-2610) and Hopp and Woods' (Hopp, T. P., and Woods, K. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 3824-3828) methods indicate the presence of four hydrophilic regions, which may contribute to sequential or parts of conformational B-cell epitopes. Analysis of amphipathic regions by Berzofsky's method indicates the presence of a highly amphipathic region, which may contain, or contribute to, an Ia/T-cell epitope. This latter segment of Lol p II was found to be highly homologous with an antibody-binding segment of the major rye allergen Lol p I and may explain why immune responsiveness to both the allergens is associated with HLA-DR3.

  14. The Sequence-Specific Cellular Uptake of Spherical Nucleic Acid Nanoparticle Conjugates

    PubMed Central

    Narayan, Suguna P.; Choi, Chung Hang J.; Hao, Liangliang; Calabrese, Colin M.; Auyeung, Evelyn; Zhang, Chuan; Goor, Olga J.G.M.

    2015-01-01

    We investigated the sequence-dependent cellular uptake of spherical nucleic acid nanoparticle conjugates (SNAs). This process occurs by interaction with class A scavenger receptors (SR-A) and caveolae-mediated endocytosis. It is known that linear poly(guanine) (poly G) is a natural ligand for SR-A, and it has been proposed that interaction of poly G with SR-A is dependent on the formation of G-quadruplexes. Since G-rich oligonucleotides are known to interact strongly with SR-A, we hypothesized that SNAs with higher G contents would be able to enter cells in larger amounts than SNAs composed of other nucleotides, and as such we measured cellular internalization of SNAs as a function of constituent oligonucleotide sequence. Indeed, SNAs with enriched G content show the highest cellular uptake. Using this hypothesis, we chemically conjugated a small molecule (camptothecin) with SNAs to create drug-SNA conjugates and observed that poly G SNAs deliver the most camptothecin to cells and have the highest cytotoxicity in cancer cells. Our data elucidate important design considerations for enhancing the intracellular delivery of spherical nucleic acids. PMID:26097111

  15. Partial amino acid sequences around sulfhydryl groups of soybean beta-amylase.

    PubMed

    Nomura, K; Mikami, B; Morita, Y

    1987-08-01

    Sulfhydryl (SH) groups of soybean beta-amylase were modified with 5-(iodoaceto-amidoethyl)aminonaphthalene-1-sulfonate (IAEDANS) and the SH-containing peptides exhibiting fluorescence were purified after chymotryptic digestion of the modified enzyme. The sequence analysis of the peptides derived from the modification of all SH groups in the denatured enzyme revealed the existence of six SH groups, in contrast to five reported previously. One of them was found to have extremely low reactivity toward SH-reagents without reduction. In the native state, IAEDANS reacted with 2 mol of SH groups per mol of the enzyme (SH1 and SH2) accompanied with inactivation of the enzyme owing to the modification of SH2 located near the active site of this enzyme. The selective modification of SH2 with IAEDANS was attained after the blocking of SH1 with 5,5'-dithiobis-(2-nitrobenzoic acid). The amino acid sequences of the peptides containing SH1 and SH2 were determined to be Cys-Ala-Asn-Pro-Gln and His-Gln-Cys-Gly-Gly-Asn-Val-Gly-Asp-Ile-Val-Asn-Ile-Pro-Ile-Pro-Gln-Trp, respectively.

  16. Genome Sequence of Lactobacillus rhamnosus Strain CASL, an Efficient l-Lactic Acid Producer from Cheap Substrate Cassava

    PubMed Central

    Yu, Bo; Su, Fei; Wang, Limin; Zhao, Bo; Qin, Jiayang; Ma, Cuiqing; Xu, Ping; Ma, Yanhe

    2011-01-01

    Lactobacillus rhamnosus is a type of probiotic bacteria with industrial potential for l-lactic acid production. We announce the draft genome sequence of L. rhamnosus CASL (2,855,156 bp with a G+C content of 46.6%), which is an efficient producer of l-lactic acid from cheap, nonfood substrate cassava with a high production titer. PMID:22123765

  17. [Nucleotide sequence of genes for alpha- and beta-subunits of luciferase from Photobacterium leiognathi].

    PubMed

    Illarionov, B A; Protopopova, M V; Karginov, V A; Mertvetsov, N P; Gitel'zon, I I

    1988-03-01

    Nucleotide sequence of the Photobacterium leiognathi DNA containing genes of alpha and beta subunits of luciferase has been determined. We also deduced amino acid sequence and molecular mass of luciferase and localized luciferase genes in the sequenced DNA fragment.

  18. Stable Isotope Peptide Mass Spectrometry To Decipher Amino Acid Metabolism in Dehalococcoides Strain CBDB1

    PubMed Central

    Marco-Urrea, Ernest; Seifert, Jana; von Bergen, Martin

    2012-01-01

    Dehalococcoides species are key players in the anaerobic transformation of halogenated solvents at contaminated sites. Here, we analyze isotopologue distributions in amino acid pools from peptides of Dehalococcoides strain CBDB1 after incubation with 13C-labeled acetate or bicarbonate as a carbon source. The resulting data were interpreted with regard to genome annotations to identify amino acid biosynthesis pathways. In addition to using gas chromatography-mass spectrometry (GC-MS) for analyzing derivatized amino acids after protein hydrolysis, we introduce a second, much milder method, in which we directly analyze peptide masses after tryptic digest and peptide fragments by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS). With this method, we identify isotope incorporation patterns for 17 proteinaceous amino acids, including proline, cysteine, lysine, and arginine, which escaped previous analyses in Dehalococcoides. Our results confirmed lysine biosynthesis via the α-aminoadipate pathway, precluding lysine formation from aspartate. Similarly, the isotopologue pattern obtained for arginine provided biochemical evidence of its synthesis from glutamate. Direct peptide MS/MS analysis of the labeling patterns of glutamine and asparagine, which were converted to glutamate and aspartate during protein hydrolysis, gave biochemical evidence of their precursors and confirmed glutamate biosynthesis via a Re-specific citrate synthase. By addition of unlabeled free amino acids to labeled cells, we show that in strain CBDB1 none of the 17 tested amino acids was incorporated into cell mass, indicating that they are all synthesized de novo. Our approach is widely applicable and provides a means to analyze amino acid metabolism by studying specific proteins even in mixed consortia. PMID:22661690

  19. Amino acid sequence of versutoxin, a lethal neurotoxin from the venom of the funnel-web spider Atrax versutus.

    PubMed

    Brown, M R; Sheumack, D D; Tyler, M I; Howden, M E

    1988-03-01

    The complete amino acid sequence of versutoxin, a lethal neurotoxic polypeptide isolated from the venom of male and female funnel-web spiders of the species Atrax versutus, was determined. Sequencing was performed in a gas-phase protein sequencer by automated Edman degradation of the S-carboxymethylated toxin and fragments of it produced by reaction with CNBr. Versutoxin consisted of a single chain of 42 amino acid residues. It was found to have a high proportion of basic residues and of cystine. The primary structure showed marked homology with that of robustoxin, a novel neurotoxin recently isolated from the venom of another funnel-web-spider species, Atrax robustus.

  20. Amino acid sequence of versutoxin, a lethal neurotoxin from the venom of the funnel-web spider Atrax versutus.

    PubMed Central

    Brown, M R; Sheumack, D D; Tyler, M I; Howden, M E

    1988-01-01

    The complete amino acid sequence of versutoxin, a lethal neurotoxic polypeptide isolated from the venom of male and female funnel-web spiders of the species Atrax versutus, was determined. Sequencing was performed in a gas-phase protein sequencer by automated Edman degradation of the S-carboxymethylated toxin and fragments of it produced by reaction with CNBr. Versutoxin consisted of a single chain of 42 amino acid residues. It was found to have a high proportion of basic residues and of cystine. The primary structure showed marked homology with that of robustoxin, a novel neurotoxin recently isolated from the venom of another funnel-web-spider species, Atrax robustus. PMID:3355530

  1. Application of a Mass Spectrometric Approach to Detect the Presence of Fatty Acid Biosynthetic Phosphopeptides.

    PubMed

    Lau, Benjamin Yii Chung; Clerens, Stefan; Morton, James D; Dyer, Jolon M; Deb-Choudhury, Santanu; Ramli, Umi Salamah

    2016-04-01

    The details of plant lipid metabolism are relatively well known but the regulation of fatty acid production at the protein level is still not understood. Hence this study explores the importance of phosphorylation as a mechanism to control the activity of fatty acid biosynthetic enzymes using low and high oleic acid mesocarps of oil palm fruit (Elaeis guineensis variety of Tenera). Adaptation of neutral loss-triggered tandem mass spectrometry and selected reaction monitoring to detect the neutral loss of phosphoric acid successfully found several phosphoamino acid-containing peptides. These peptides corresponded to the peptides from acetyl-CoA carboxylase and 3-enoyl-acyl carrier protein reductase as identified by their precursor ion masses. These findings suggest that these enzymes were phosphorylated at 20th week after anthesis. Phosphorylation could have reduce their activities towards the end of fatty acid biosynthesis at ripening stage. Implication of phosphorylation in the regulation of fatty acid biosynthesis at protein level has never been reported.

  2. Clostridium sticklandii, a specialist in amino acid degradation:revisiting its metabolism through its genome sequence

    PubMed Central

    2010-01-01

    Background Clostridium sticklandii belongs to a cluster of non-pathogenic proteolytic clostridia which utilize amino acids as carbon and energy sources. Isolated by T.C. Stadtman in 1954, it has been generally regarded as a "gold mine" for novel biochemical reactions and is used as a model organism for studying metabolic aspects such as the Stickland reaction, coenzyme-B12- and selenium-dependent reactions of amino acids. With the goal of revisiting its carbon, nitrogen, and energy metabolism, and comparing studies with other clostridia, its genome has been sequenced and analyzed. Results C. sticklandii is one of the best biochemically studied proteolytic clostridial species. Useful additional information has been obtained from the sequencing and annotation of its genome, which is presented in this paper. Besides, experimental procedures reveal that C. sticklandii degrades amino acids in a preferential and sequential way. The organism prefers threonine, arginine, serine, cysteine, proline, and glycine, whereas glutamate, aspartate and alanine are excreted. Energy conservation is primarily obtained by substrate-level phosphorylation in fermentative pathways. The reactions catalyzed by different ferredoxin oxidoreductases and the exergonic NADH-dependent reduction of crotonyl-CoA point to a possible chemiosmotic energy conservation via the Rnf complex. C. sticklandii possesses both the F-type and V-type ATPases. The discovery of an as yet unrecognized selenoprotein in the D-proline reductase operon suggests a more detailed mechanism for NADH-dependent D-proline reduction. A rather unusual metabolic feature is the presence of genes for all the enzymes involved in two different CO2-fixation pathways: C. sticklandii harbours both the glycine synthase/glycine reductase and the Wood-Ljungdahl pathways. This unusual pathway combination has retrospectively been observed in only four other sequenced microorganisms. Conclusions Analysis of the C. sticklandii genome and

  3. Amino acid sequence of neurotoxin III of the scorpion Androctonus austrialis Hector.

    PubMed

    Kopeyan, C; Martinez, G; Rochat, H

    1979-03-01

    The amino acid sequence of neurotoxin III, purified from the venom of the North African scorpion Androctonus australis Hector, has been determined by Edman degradation using a liquid-phase sequencer. Carboxypeptidase A hydrolyses confirmed not only the sequence of the five last residues but also the presence of a free alpha-carboxylic group at the C-terminus. Edman degradation was conducted on one hand with the Quadrol [N,N,N',N'-tetrakis(2-hydroxypropyl)ethylene diamine] program and S-alkylated protein before or after coupling with sulfophenylisothiocynate (the first 34 residues were thus identified), on the other hand on tryptic and chymotryptic peptides with a dimethylbenzylamine program (residues 1--23 and 31--34 were confirmed, the positions of residues 35-64 were established). Neurotoxin III was found to belong to the same group of scorpion toxins active on mammals as neurotoxin I purified from the same venom (50 homologous positions exist in the two proteins).

  4. Isolation and amino acid sequences of squirrel monkey (Saimiri sciurea) insulin and glucagon

    SciTech Connect

    Yu, Jinghua ); Eng, J.; Yalow, R.S. City Univ. of New York, NY )

    1990-12-01

    It was reported two decades ago that insulin was not detectable in the glucose-stimulated state in Saimiri sciurea, the New World squirrel monkey, by a radioimmunoassay system developed with guinea pig anti-pork insulin antibody and labeled park insulin. With the same system, reasonable levels were observed in rhesus monkeys and chimpanzees. This suggested that New World monkeys, like the New World hystricomorph rodents such as the guinea pig and the coypu, might have insulins whose sequences differ markedly from those of Old World mammals. In this report the authors describe the purification and amino acid sequences of squirrel monkey insulin and glucagon. They demonstrate that the substitutions at B29, B27, A2, A4, and A17 of squirrel monkey insulin are identical with those previously found in another New World primate, the owl monkey (Aotus trivirgatus). The immunologic cross-reactivity of this insulin in their immunoassay system is only a few percent of that of human insulin. It appears that the peptides of the New World monkeys have diverged less from those of the Old World mammals than have those of the New World hystricomorph rodents. The striking improvements in peptide purification and sequencing have the potential for adding new information concerning the evolutionary divergence of species.

  5. Purification, amino acid sequence and characterisation of kangaroo IGF-I.

    PubMed

    Yandell, C A; Francis, G L; Wheldrake, J F; Upton, Z

    1998-01-01

    Insulin-like growth factor-I (IGF-I) and IGF-II have been purified to homogeneity from kangaroo (Macropus fuliginosus) serum, thus this represents the first report of the purification, sequencing and characterisation of marsupial IGFs. N-Terminal protein sequencing reveals that there are six amino acid differences between kangaroo and human IGF-I. Kangaroo IGF-II has been partially sequenced and no differences were found between human and kangaroo IGF-II in the 53 residues identified. Thus the IGFs appear to be remarkably structurally conserved during mammalian radiation. In addition, in vitro characterisation of kangaroo IGF-I demonstrated that the functional properties of human, kangaroo and chicken IGF-I are very similar. In an assay measuring the ability of the proteins to stimulate protein synthesis in rat L6 myoblasts, all IGF-I proteins were found to be equally potent. The ability of all three proteins to compete for binding with radiolabelled human IGF-I to type-1 IGF receptors in L6 myoblasts and in Sminthopsis crassicaudata transformed lung fibroblasts, a marsupial cell line, was comparable. Furthermore, kangaroo and human IGF-I react equally in a human IGF-I RIA using a human reference standard, radiolabelled human IGF-I and a polyclonal antibody raised against recombinant human IGF-I. This study indicates that not only is the primary structure of eutherian and metatherian IGF-I conserved, but also the proteins appear to be functionally similar.

  6. Complete Genome Sequence of the Prototype Lactic Acid Bacterium Lactococcus lactis subsp. cremoris MG1363▿

    PubMed Central

    Wegmann, Udo; O'Connell-Motherway, Mary; Zomer, Aldert; Buist, Girbe; Shearman, Claire; Canchaya, Carlos; Ventura, Marco; Goesmann, Alexander; Gasson, Michael J.; Kuipers, Oscar P.; van Sinderen, Douwe; Kok, Jan

    2007-01-01

    Lactococcus lactis is of great importance for the nutrition of hundreds of millions of people worldwide. This paper describes the genome sequence of Lactococcus lactis subsp. cremoris MG1363, the lactococcal strain most intensively studied throughout the world. The 2,529,478-bp genome contains 81 pseudogenes and encodes 2,436 proteins. Of the 530 unique proteins, 47 belong to the COG (clusters of orthologous groups) functional category “carbohydrate metabolism and transport,” by far the largest category of novel proteins in comparison with L. lactis subsp. lactis IL1403. Nearly one-fifth of the 71 insertion elements are concentrated in a specific 56-kb region. This integration hot-spot region carries genes that are typically associated with lactococcal plasmids and a repeat sequence specifically found on plasmids and in the “lateral gene transfer hot spot” in the genome of Streptococcus thermophilus. Although the parent of L. lactis MG1363 was used to demonstrate lysogeny in Lactococcus, L. lactis MG1363 carries four remnant/satellite phages and two apparently complete prophages. The availability of the L. lactis MG1363 genome sequence will reinforce its status as the prototype among lactic acid bacteria through facilitation of further applied and fundamental research. PMID:17307855

  7. Development of microwave-assisted acid hydrolysis of proteins using a commercial microwave reactor and its combination with LC-MS for protein full-sequence analysis.

    PubMed

    Chen, Lu; Wang, Nan; Li, Liang

    2014-11-01

    Microwave-assisted acid hydrolysis (MAAH) can be used to degrade a protein non-specifically into many peptides with overlapping sequences which can be identified by mass spectrometry (MS) to produce a sequence map that covers the full sequence of a protein. The success of this method for protein sequence analysis depends on the proper control of the MAAH process, which is currently done using a household microwave oven. However, to meet the regulatory or good laboratory practice (GLP) requirement in a clinical or pharmaceutical laboratory, using a commercial microwave device is often required. In this paper, we report a method of performing MAAH using a CEM Discover single-mode microwave reactor. It is shown that, using an optimized protocol for MAAH, reproducible results comparable to those obtained using a household microwave oven can be generated using the commercial reactor. To illustrate the potential applications of MAAH MS for characterizing clinically relevant proteins, this method was applied, for the first time, to map the amino acid sequences of normal and sickle-cell human hemoglobin as well as bovine hemoglobin. Full sequence coverage was readily achieved from 294 and 266 unique peptides matched to the alpha and beta subunits of normal hemoglobin, respectively, 334 and 265 unique peptides matched to the alpha and beta submit units of sickle-cell hemoglobin, and 377 and 224 unique peptides matched to the alpha and beta subunits of bovine hemoglobin. This method opens the possibility for any laboratory to use a commercial laboratory equipment to perform MAAH MS for protein full-sequence analysis.

  8. Phosphorylation of serine residues in histidine-tag sequences attached to recombinant protein kinases: a cause of heterogeneity in mass and complications in function.

    PubMed

    Du, Ping; Loulakis, Pat; Luo, Chun; Mistry, Anil; Simons, Samuel P; LeMotte, Peter K; Rajamohan, Francis; Rafidi, Kristina; Coleman, Kevin G; Geoghegan, Kieran F; Xie, Zhi

    2005-12-01

    High-level recombinant expression of protein kinases in eukaryotic cells or Escherichia coli commonly gives products that are phosphorylated by autocatalysis or by the action of endogenous kinases. Here, we report that phosphorylation occurred on serine residues adjacent to hexahistidine affinity tags (His-tags) derived from several commercial expression vectors and fused to overexpressed kinases. The result was observed with a variety of recombinant kinases expressed in either insect cells or E. coli. Multiple phosphorylations of His-tagged full-length Aurora A, a protein serine/threonine kinase, were detected by mass spectrometry when it was expressed in insect cells in the presence of okadaic acid, a protein phosphatase inhibitor. Peptide mapping by liquid chromatography-mass spectrometry detected phosphorylations on all three serine residues in an N-terminal tag, alpha-N-acetyl-MHHHHHHSSGLPRGS. The same sequence was also phosphorylated, but only at a low level, when a His-tagged protein tyrosine kinase, Pyk2 was expressed in insect cells and activated in vitro. When catalytic domains of Aurora A and several other protein serine/threonine kinases were expressed in E. coli, serines in the affinity tag sequence GSSHHHHHHSSGLVPRGS were also variably phosphorylated. His-Aurora A with hyperphosphorylation of the serine residues in the tag aggregated and resisted thrombin-catalyzed removal of the tag. Treatment with alkaline phosphatase partly restored sensitivity to thrombin. The same His-tag sequence was also detected bearing alpha-N-d-gluconoylation in addition to multiple phosphorylations. The results show that histidine-tag sequences can receive complicated posttranslational modification, and that the hyperphosphorylation and resulting heterogeneity of the recombinant fusion proteins can interfere with downstream applications.

  9. The ABRF Edman Sequencing Research Group 2008 Study: Investigation into Homopolymeric Amino Acid N-Terminal Sequence Tags and Their Effects on Automated Edman Degradation

    PubMed Central

    Thoma, R. S.; Smith, J. S.; Sandoval, W.; Leone, J. W.; Hunziker, P.; Hampton, B.; Linse, K. D.; Denslow, N. D.

    2009-01-01

    The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and executes interlaboratory studies investigating the use of automated Edman degradation for protein and peptide analysis. In 2008, the ESRG enlisted the help of core sequencing facilities to investigate the effects of a repeating amino acid tag at the N-terminus of a protein. Commonly, to facilitate protein purification, an affinity tag containing a polyhistidine sequence is conjugated to the N-terminus of the protein. After expression, polyhistidine-tagged protein is readily purified via chelation with an immobilized metal affinity resin. The addition of the polyhistidine tag presents unique challenges for the determination of protein identity using Edman degradation chemistry. Participating laboratories were asked to sequence one protein engineered in three configurations: with an N-terminal polyhistidine tag; with an N-terminal polyalanine tag; or with no tag. Study participants were asked to return a data file containing the uncorrected amino acid picomole yields for the first 17 cycles. Initial and repetitive yield (R.Y.) information and the amount of lag were evaluated. Information about instrumentation and sample treatment was also collected as part of the study. For this study, the majority of participating laboratories successfully called the amino acid sequence for 17 cycles for all three test proteins. In general, laboratories found it more difficult to call the sequence containing the polyhistidine tag. Lag was observed earlier and more consistently with the polyhistidine-tagged protein than the polyalanine-tagged protein. Histidine yields were significantly less than the alanine yields in the tag portion of each analysis. The polyhistidine and polyalanine protein-R.Y. calculations were found to be equivalent. These calculations showed that the nontagged portion from each protein was equivalent. The terminal histidines from the tagged portion of the protein

  10. Constraining the low-mass Slope of the star formation sequence at 0.5 < z < 2.5

    SciTech Connect

    Whitaker, Katherine E.; Henry, Alaina; Rigby, Jane R.; Franx, Marijn; Fumagalli, Mattia; Labbé, Ivo; Leja, Joel; Van Dokkum, Pieter G.; Momcheva, Ivelina G.; Nelson, Erica J.; Skelton, Rosalind E.; Brammer, Gabriel B.

    2014-11-10

    We constrain the slope of the star formation rate (SFR; log Ψ) to stellar mass (log M {sub *}) relation down to log (M {sub *}/M {sub ☉}) = 8.4 (log (M {sub *}/M {sub ☉}) = 9.2) at z = 0.5 (z = 2.5) with a mass-complete sample of 39,106 star-forming galaxies selected from the 3D-HST photometric catalogs, using deep photometry in the CANDELS fields. For the first time, we find that the slope is dependent on stellar mass, such that it is steeper at low masses (log Ψ∝log M {sub *}) than at high masses (log Ψ∝(0.3-0.6)log M {sub *}). These steeper low-mass slopes are found for three different star formation indicators: the combination of the ultraviolet (UV) and infrared (IR), calibrated from a stacking analysis of Spitzer/MIPS 24 μm imaging; β-corrected UV SFRs; and Hα SFRs. The normalization of the sequence evolves differently in distinct mass regimes as well: for galaxies less massive than log (M {sub *}/M {sub ☉}) < 10 the specific SFR (Ψ/M {sub *}) is observed to be roughly self-similar with Ψ/M {sub *}∝(1 + z){sup 1.9}, whereas more massive galaxies show a stronger evolution with Ψ/M {sub *}∝(1 + z){sup 2.2-3.5} for log (M {sub *}/M {sub ☉}) = 10.2-11.2. The fact that we find a steep slope of the star formation sequence for the lower mass galaxies will help reconcile theoretical galaxy formation models with the observations.

  11. Molecular weight determination of methyl esters of mycolic acids using thermospray mass spectrometry.

    PubMed

    Ioneda, T; Beaman, B L

    1992-11-01

    Methyl esters of normal fatty acids, corynomycolate and corynomycolenate were used as model compounds for thermospray mass spectrometric procedures for molecular weight determination of the related nocardial mycolic acids. By using ammonium acetate at the positive ion generator, in both cases, a family of ions was produced. The following members were found and corresponded to the adducts: (1) M + H; M + NH4 and M + H + NH4 for methyl esters of normal fatty acids, whereas M + H, M + 2H and M + H + NH4 were the adducts most frequently observed with methyl corynomycolates. The methyl esters of C40-C48 mycolic acids from Rhodococcus rhodochrous exhibited prominent peaks corresponding to adducts M + H + NH4 whereas those corresponding to M + 2H showed slightly lower intensities. The structure M + H had no significant representatives with this subclass of mycolic acids. A similar pattern was observed with methyl esters of C50-C54 mycolic acids from Nocardia asteroides GUH-2. Ion peaks C50-C54 representing adducts M + 2H and M + H + NH4 prevailed in the mass spectrum. In this case, the intensities of peaks corresponding to M + 2H were slightly higher than those of the M + H + NH4. Essentially three main species of nocardomycolic acids were detected: (1) monounsaturated C50:1, C52:1 and C54:1; (2) diunsaturated C50:2, C52:2 and C54:2 and (3) triunsaturated C52:3 and C54:3 mycolic acids. The most abundant mycolic acid was C52:2 followed in decreasing abundance by C52:1, C54:2, C50:2, C52:3 and C54:3 mycolic acids.

  12. The amino acid sequence around the active-site cysteine and histidine residues, and the buried cysteine residue in ficin.

    PubMed

    Husain, S S; Lowe, G

    1970-04-01

    Ficin that had been prepared from the latex of Ficus glabrata by salt fractionation and chromatography on carboxymethylcellulose was completely and irreversibly inhibited with 1,3-dibromo[2-(14)C]acetone and then treated with N-(4-dimethylamino-3,5-dinitrophenyl)maleimide in 6m-guanidinium chloride. After reduction and carboxymethylation of the labelled protein, it was digested with trypsin and alpha-chymotrypsin. Two radioactive peptides and two coloured peptides were isolated chromatographically and their sequences determined. The radioactive peptides revealed the amino acid sequences around the active-site cysteine and histidine residues and showed a high degree of homology with the omino acid sequence around the active-site cysteine and histidine residues in papain. The coloured peptides allowed the amino acid sequence around the buried cysteine residue in ficin to be determined.

  13. The `heavy' subunit of the photosynthetic reaction centre from Rhodopseudomonas viridis: isolation of the gene, nucleotide and amino acid sequence

    PubMed Central

    Michel, H.; Weyer, K. A.; Gruenberg, H.; Lottspeich, F.

    1985-01-01

    The gene coding for the `heavy' subunit of the photosynthetic reaction centre from Rhodopseudomonas viridis was isolated in an expression vector. Expression of the heavy subunit in Escherichia coli was detected with antibodies raised against crystalline reaction centres. The entire subunit, and not a fusion protein, was expressed in E. coli. The protein coding region of the gene was sequenced and the amino acid sequence derived. Part of the amino acid sequence was confirmed by chemical sequence analysis of the protein. The heavy subunit consists of 258 amino acids and its mol. wt. is 28 345. It possesses one membrane-spanning α-helical segment, as was revealed by the concomitant X-ray structure analysis. ImagesFig. 1.Fig. 2. PMID:16453623

  14. Purification, amino acid sequence and immunological characterization of Ole e 6, a cysteine-enriched allergen from olive tree pollen.

    PubMed

    Batanero, E; Ledesma, A; Villalba, M; Rodríguez, R

    1997-06-30

    The Ole e 6 allergen from olive tree pollen has been isolated by combining gel permeation and reverse-phase chromatographies. It is a single and highly acidic (pI 4.2) polypeptide chain protein. Its NH2-terminal amino acid sequence has been determined by Edman degradation. Total RNA from the olive tree pollen was isolated, and a specific cDNA was amplified by the polymerase chain reaction using a degenerate oligonucleotide primer designed according to the NH2-terminal sequence of the protein. The nucleotide sequencing of the cDNA rendered an open reading frame encoding a 50 amino acid polypeptide chain, in which two sets of the sequential motif Cys-X3-Cys-X3-Cys are present. No sequence similarity has been found between this protein and other previously described polypeptides.

  15. Nucleotide and derived amino acid sequences of the major porin of Comamonas acidovorans and comparison of porin primary structures.

    PubMed Central

    Gerbl-Rieger, S; Peters, J; Kellermann, J; Lottspeich, F; Baumeister, W

    1991-01-01

    The DNA sequence of the gene which codes for the major outer membrane porin (Omp32) of Comamonas acidovorans has been determined. The structural gene encodes a precursor consisting of 351 amino acid residues with a signal peptide of 19 amino acid residues. Comparisons with amino acid sequences of outer membrane proteins and porins from several other members of the class Proteobacteria and of the Chlamydia trachomatis porin and the Neurospora crassa mitochondrial porin revealed a motif of eight regions of local homology. The results of this analysis are discussed with regard to common structural features of porins. PMID:1848840

  16. The evolution of proteins from random amino acid sequences: II. Evidence from the statistical distributions of the lengths of modern protein sequences.

    PubMed

    White, S H

    1994-04-01

    This paper continues an examination of the hypothesis that modern proteins evolved from random heteropeptide sequences. In support of the hypothesis, White and Jacobs (1993, J Mol Evol 36:79-95) have shown that any sequence chosen randomly from a large collection of nonhomologous proteins has a 90% or better chance of having a lengthwise distribution of amino acids that is indistinguishable from the random expectation regardless of amino acid type. The goal of the present study was to investigate the possibility that the random-origin hypothesis could explain the lengths of modern protein sequences without invoking specific mechanisms such as gene duplication or exon splicing. The sets of sequences examined were taken from the 1989 PIR database and consisted of 1,792 "super-family" proteins selected to have little sequence identity, 623 E. coli sequences, and 398 human sequences. The length distributions of the proteins could be described with high significance by either of two closely related probability density functions: The gamma distribution with parameter 2 or the distribution for the sum of two exponential random independent variables. A simple theory for the distributions was developed which assumes that (1) protoprotein sequences had exponentially distributed random independent lengths, (2) the length dependence of protein stability determined which of these protoproteins could fold into compact primitive proteins and thereby attain the potential for biochemical activity, (3) the useful protein sequences were preserved by the primitive genome, and (4) the resulting distribution of sequence lengths is reflected by modern proteins. The theory successfully predicts the two observed distributions which can be distinguished by the functional form of the dependence of protein stability on length. The theory leads to three interesting conclusions. First, it predicts that a tetra-nucleotide was the signal for primitive translation termination. This prediction is

  17. The Effects of Post-Main-Sequence Solar Mass Loss on the Stability of Our Planetary System

    NASA Astrophysics Data System (ADS)

    Duncan, Martin J.; Lissauer, Jack J.

    1998-08-01

    We present the results of extensive long-term integrations of systems of planets with orbits initially identical to subsets of the planets within our Solar System, but with the Sun's mass decreased relative to the masses of the planets. For systems based on the giant planets, we find an approximate power-law correlation between the time elapsed until a pair of planetary orbits cross and the solar-to-planetary-mass ratio, provided that this ratio is ≲0.4 times its current value. However, deviations from this relationship at larger ratios suggest that this correlation may not be useful in predicting the lifetime of the current system. Detailed simulations of the evolution of planetary orbits through the solar mass loss phase at the end of the Sun's main-sequence lifetime suggest that the orbits of those terrestrial planets that survive the Sun's red giant phase are likely to remain stable for (possibly much) longer than a billion years and those of the giant planets are likely to remain stable for (possibly much) more than ten billion years. Pluto is likely to escape from its current 2:3 mean-motion resonance with Neptune within a few billion years beyond the Sun's main sequence lifetime if subject only to gravitational forces; its prognosis is likely to be even poorer when nongravitational forces are included. Implications for the effects of stellar mass loss on the stability of other planetary systems are discussed.

  18. Exercise and amino acid anabolic cell signaling and the regulation of skeletal muscle mass.

    PubMed

    Pasiakos, Stefan M

    2012-07-01

    A series of complex intracellular networks influence the regulation of skeletal muscle protein turnover. In recent years, studies have examined how cellular regulators of muscle protein turnover modulate metabolic mechanisms contributing to the loss, gain, or conservation of skeletal muscle mass. Exercise and amino acids both stimulate anabolic signaling potentially through several intracellular pathways including the mammalian target of rapamycin complex 1 and the mitogen activated protein kinase cell signaling cascades. As novel molecular regulators of muscle integrity continue to be explored, a contemporary analysis of the literature is required to understand the metabolic mechanisms by which contractile forces and amino acids affect cellular process that contribute to long-term adaptations and preservation of muscle mass. This article reviews the literature related to how exercise and amino acid availability affect cellular regulators of skeletal muscle mass, especially highlighting recent investigations that have identified mechanisms by which contractile forces and amino acids modulate muscle health. Furthermore, this review will explore integrated exercise and nutrition strategies that promote the maintenance of muscle health by optimizing exercise, and amino acid-induced cell signaling in aging adults susceptible to muscle loss.

  19. An amino acid sequence motif sufficient for subnuclear localization of an arginine/serine-rich splicing factor.

    PubMed

    Hedley, M L; Amrein, H; Maniatis, T

    1995-12-05

    We have identified an amino acid sequence in the Drosophila Transformer (Tra) protein that is capable of directing a heterologous protein to nuclear speckles, regions of the nucleus previously shown to contain high concentrations of spliceosomal small nuclear RNAs and splicing factors. This sequence contains a nucleoplasmin-like bipartite nuclear localization signal (NLS) and a repeating arginine/serine (RS) dipeptide sequence adjacent to a short stretch of basic amino acids. Sequence comparisons from a number of other splicing factors that colocalize to nuclear speckles reveal the presence of one or more copies of this motif. We propose a two-step subnuclear localization mechanism for splicing factors. The first step is transport across the nuclear envelope via the nucleoplasmin-like NLS, while the second step is association with components in the speckled domain via the RS dipeptide sequence.

  20. Purification and partial amino acid sequence of the chloroplast cytochrome b-559.

    PubMed

    Widger, W R; Cramer, W A; Hermodson, M; Meyer, D; Gullifor, M

    1984-03-25

    The hydrophobic cytochrome b-559, purified from unstacked, ethanol-washed spinach thylakoid membranes, using extraction with 2% Triton X-100 in 4 M urea and three chromatographic steps in the presence of protease inhibitors, has a dominant band on sodium dodecyl sulfate-urea gels corresponding to Mr = 10,000. The yield of this preparation is 30-50% (5-10 mg) starting with 600 mg of chlorophyll. The heme content yields a calculated molecular weight of no more than 17,500/heme, and perhaps somewhat smaller after correction for impurities. The Mr = 10,000 band is stained by the tetramethylbenzidine-H2O2 heme reagent on lithium dodecyl sulfate gels run at 0 degrees C. The Mr = 10,000 protein, further separated by high performance liquid chromatography, contains a unique NH2 terminus that is not blocked, and the amino acid sequence for the first 27 residues is NH2-Ser-Gly-Ser-Thr-Gly-Glu-Arg-Ser-Phe-Ala-Asp-Ile-Ile-Thr-Ser-Ile-Arg-Tyr-Trp -Val-Ile-X-Ser-Ile-Thr-Ile-Pro. . . COOH. Approximately 55% of the amino acids are hydrophobic, based on amino acid analysis of the Mr = 10,000 peptide, which also indicated the presence of at least one histidine. Only one cytochrome b-559 component could be identified, whose yield indicated that it arises from a single b-559 protein in chloroplasts corresponding to the in situ high potential cytochrome of the chloroplast photosystem II.

  1. Distinguishing Aspartic and Isoaspartic Acids in Peptides by Several Mass Spectrometric Fragmentation Methods

    NASA Astrophysics Data System (ADS)

    DeGraan-Weber, Nick; Zhang, Jun; Reilly, James P.

    2016-12-01

    Six ion fragmentation techniques that can distinguish aspartic acid from its isomer, isoaspartic acid, were compared. MALDI post-source decay (PSD), MALDI 157 nm photodissociation, tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) charge tagging in PSD and photodissociation, ESI collision-induced dissociation (CID), electron transfer dissociation (ETD), and free-radical initiated peptide sequencing (FRIPS) with CID were applied to peptides containing either aspartic or isoaspartic acid. Diagnostic ions, such as the y-46 and b+H2O, are present in PSD, photodissociation, and charge tagging. c•+57 and z-57 ions are observed in ETD and FRIPS experiments. For some molecules, aspartic and isoaspartic acid yield ion fragments with significantly different intensities. ETD and charge tagging appear to be most effective at distinguishing these residues.

  2. Reprint of "Identification of staphylococcal species based on variations in protein sequences (mass spectrometry) and DNA sequence (sodA microarray)".

    PubMed

    Kooken, Jennifer; Fox, Karen; Fox, Alvin; Altomare, Diego; Creek, Kim; Wunschel, David; Pajares-Merino, Sara; Martínez-Ballesteros, Ilargi; Garaizar, Javier; Oyarzabal, Omar; Samadpour, Mansour

    2014-01-01

    This report is among the first using sequence variation in newly discovered protein markers for staphylococcal (or indeed any other bacterial) speciation. Variation, at the DNA sequence level, in the sodA gene (commonly used for staphylococcal speciation) provided excellent correlation. Relatedness among strains was also assessed using protein profiling using microcapillary electrophoresis and pulsed field electrophoresis. A total of 64 strains were analyzed including reference strains representing the 11 staphylococcal species most commonly isolated from man (Staphylococcus aureus and 10 coagulase negative species [CoNS]). Matrix assisted time of flight ionization/ionization mass spectrometry (MALDI TOF MS) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC ESI MS/MS) were used for peptide analysis of proteins isolated from gel bands. Comparison of experimental spectra of unknowns versus spectra of peptides derived from reference strains allowed bacterial identification after MALDI TOF MS analysis. After LC-MS/MS analysis of gel bands bacterial speciation was performed by comparing experimental spectra versus virtual spectra using the software X!Tandem. Finally LC-MS/MS was performed on whole proteomes and data analysis also employing X!tandem. Aconitate hydratase and oxoglutarate dehydrogenase served as marker proteins on focused analysis after gel separation. Alternatively on full proteomics analysis elongation factor Tu generally provided the highest confidence in staphylococcal speciation.

  3. THE STAR FORMATION HISTORIES OF RED-SEQUENCE GALAXIES, MASS-TO-LIGHT RATIOS AND THE FUNDAMENTAL PLANE

    SciTech Connect

    Allanson, Steven P.; Hudson, Michael J.; Smith, Russell J.; Lucey, John R.

    2009-09-10

    This paper addresses the challenge of understanding the typical star formation histories of red-sequence galaxies, using linestrength indices and mass-to-light ratios as complementary constraints on their stellar age distribution. We first construct simple parametric models of the star formation history that bracket a range of scenarios, and fit these models to the linestrength indices of low-redshift cluster red-sequence galaxies. For giant galaxies, we confirm the downsizing trend, i.e., the stellar populations are younger, on average, for lower {sigma} galaxies. We find, however, that this trend flattens or reverses at {sigma} {approx}< 70 km s{sup -1}. We then compare predicted stellar mass-to-light ratios with dynamical mass-to-light ratios derived from the fundamental plane (FP), or by the SAURON group. For galaxies with {sigma} {approx} 70 km s{sup -1}, models with a late 'frosting' of young stars and models with exponential star formation histories have stellar mass-to-light ratios that are larger than observed dynamical mass-to-light ratios by factors of 1.7 and 1.4, respectively, and so are rejected. The single stellar population (SSP) model is consistent with the FP, and requires a modest amount of dark matter (between 20% and 30%) to account for the difference between stellar and dynamical mass-to-light ratios. A model in which star formation was 'quenched' at intermediate ages is also consistent with the observations, although in this case less dark matter is required for low mass galaxies. We also find that the contribution of stellar populations to the 'tilt' of the fundamental plane is highly dependent on the assumed star formation history: for the SSP model, the tilt of the FP is driven primarily by stellar-population effects. For a quenched model, two-thirds of the tilt is due to stellar populations and only one-third is due to dark matter or non-homology.

  4. Gas chromatography-mass spectrometry profiles of urinary organic acids in healthy captive cheetahs (Acinonyx jubatus).

    PubMed

    Tordiffe, Adrian Stephen Wolferstan; van Reenen, Mari; Reyers, Fred; Mienie, Lodewyk Jacobus

    2017-04-01

    In captivity, cheetahs (Acinonyx jubatus) frequently suffer from several unusual chronic diseases that rarely occur in their free-ranging counterparts. In order to develop a better understanding of their metabolism and health we documented the urine organic acids of 41 apparently healthy captive cheetahs, in an untargeted metabolomic study, using gas chromatography-mass spectrometry. A total of 339 organic acids were detected and annotated. Phenolic compounds, thought to be produced by the anaerobic fermentation of aromatic amino acids in the distal colon, as well as their corresponding glycine conjugates, were present in high concentrations. The most abundant organic acids in the cheetahs' urine were an as yet unidentified compound and a novel cadaverine metabolite, tentatively identified as N(1),N(5)-dimethylpentane-1,5-diamine. Pantothenic acid and citramalic acid concentrations correlated negatively with age, while glutaric acid concentrations correlated positively with age, suggesting possible dysregulation of coenzyme A metabolism in older cheetahs. This study provides a baseline of urine organic acid reference values in captive cheetahs and suggests important avenues for future research in this species.

  5. OMA and OPA--software-supported mass spectra analysis of native and modified nucleic acids.

    PubMed

    Nyakas, Adrien; Blum, Lorenz C; Stucki, Silvan R; Reymond, Jean-Louis; Schürch, Stefan

    2013-02-01

    The platform-independent software package consisting of the oligonucleotide mass assembler (OMA) and the oligonucleotide peak analyzer (OPA) was created to support the analysis of oligonucleotide mass spectra. It calculates all theoretically possible fragments of a given input sequence and annotates it to an experimental spectrum, thus, saving a large amount of manual processing time. The software performs analysis of precursor and product ion spectra of oligonucleotides and their analogues comprising user-defined modifications of the backbone, the nucleobases, or the sugar moiety, as well as adducts with metal ions or drugs. The ability to expand the library of building blocks and to implement individual structural variations makes it extremely useful for supporting the analysis of therapeutically active compounds. The functionality of the software tool is demonstrated on the examples of a platinated double-stranded oligonucleotide and a modified RNA sequence. Experiments also reveal the unique dissociation behavior of platinated higher-order DNA structures.

  6. OMA and OPA—Software-Supported Mass Spectra Analysis of Native and Modified Nucleic Acids

    NASA Astrophysics Data System (ADS)

    Nyakas, Adrien; Blum, Lorenz C.; Stucki, Silvan R.; Reymond, Jean-Louis; Schürch, Stefan

    2013-02-01

    The platform-independent software package consisting of the oligonucleotide mass assembler (OMA) and the oligonucleotide peak analyzer (OPA) was created to support the analysis of oligonucleotide mass spectra. It calculates all theoretically possible fragments of a given input sequence and annotates it to an experimental spectrum, thus, saving a large amount of manual processing time. The software performs analysis of precursor and product ion spectra of oligonucleotides and their analogues comprising user-defined modifications of the backbone, the nucleobases, or the sugar moiety, as well as adducts with metal ions or drugs. The ability to expand the library of building blocks and to implement individual structural variations makes it extremely useful for supporting the analysis of therapeutically active compounds. The functionality of the software tool is demonstrated on the examples of a platinated double-stranded oligonucleotide and a modified RNA sequence. Experiments also reveal the unique dissociation behavior of platinated higher-order DNA structures.

  7. Alignment of 700 globin sequences: extent of amino acid substitution and its correlation with variation in volume.

    PubMed Central

    Kapp, O. H.; Moens, L.; Vanfleteren, J.; Trotman, C. N.; Suzuki, T.; Vinogradov, S. N.

    1995-01-01

    Seven-hundred globin sequences, including 146 nonvertebrate sequences, were aligned on the basis of conservation of secondary structure and the avoidance of gap penalties. Of the 182 positions needed to accommodate all the globin sequences, only 84 are common to all, including the absolutely conserved PheCD1 and HisF8. The mean number of amino acid substitutions per position ranges from 8 to 13 for all globins and 5 to 9 for internal positions. Although the total sequence volumes have a variation approximately 2-3%, the variation in volume per position ranges from approximately 13% for the internal to approximately 21% for the surface positions. Plausible correlations exist between amino acid substitution and the variation in volume per position for the 84 common and the internal but not the surface positions. The amino acid substitution matrix derived from the 84 common positions was used to evaluate sequence similarity within the globins and between the globins and phycocyanins C and colicins A, via calculation of pairwise similarity scores. The scores for globin-globin comparisons over the 84 common positions overlap the globin-phycocyanin and globin-colicin scores, with the former being intermediate. For the subset of internal positions, overlap is minimal between the three groups of scores. These results imply a continuum of amino acid sequences able to assume the common three-on-three alpha-helical structure and suggest that the determinants of the latter include sites other than those inaccessible to solvent. PMID:8535255

  8. Amino acid substitutions in genetic variants of human serum albumin and in sequences inferred from molecular cloning

    SciTech Connect

    Takahashi, N.; Takahashi, Y.; Blumberg, B.S.; Putnam, F.W.

    1987-07-01

    The structural changes in four genetic variants of human serum albumin were analyzed by tandem high-pressure liquid chromatography (HPLC) of the tryptic peptides, HPLC mapping and isoelectric focusing of the CNBr fragments, and amino acid sequence analysis of the purified peptides. Lysine-372 of normal (common) albumin A was changed to glutamic acid both in albumin Naskapi, a widespread polymorphic variant of North American Indians, and in albumin Mersin found in Eti Turks. The two variants also exhibited anomalous migration in NaDodSO/sub 4//PAGE, which is attributed to a conformational change. The identity of albumins Naskapi and Mersin may have originated through descent from a common mid-Asiatic founder of the two migrating ethnic groups, or it may represent identical but independent mutations of the albumin gene. In albumin Adana, from Eti Turks, the substitution site was not identified but was localized to the region from positions 447 through 548. The substitution of aspartic acid-550 by glycine was found in albumin Mexico-2 from four individuals of the Pima tribe. Although only single-point substitutions have been found in these and in certain other genetic variants of human albumin, five differences exist in the amino acid sequences inferred from cDNA sequences by workers in three other laboratories. However, our results on albumin A and on 14 different genetic variants accord with the amino acid sequence of albumin deduced from the genomic sequence. The apparent amino acid substitutions inferred from comparison of individual cDNA sequences probably reflect artifacts in cloning or in cDNA sequence analysis rather than polymorphism of the coding sections of the albumin gene.

  9. Identification of 19 phthalic acid esters in dairy products by gas chromatography with mass spectrometry.

    PubMed

    Wu, Pinggu; Cai, Chenggang; Yang, Dajin; Wang, Liyuan; Zhou, Yan; Shen, Xianghong; Ma, Bingjie; Tang, Jun

    2015-01-01

    A detection method for 19 kinds of phthalic acid ester compounds analyzed by n-hexane/ether/acetonitrile 1:7:8 v/v/v mixed solvent extraction, quick, easy, cheap, effective, rugged, and safe purification and internal standard method of quantitative gas chromatography with mass spectrometry was established. This method can effectively remove interfering materials, such as lipids, fatty acids, and pigments, from dairy products. The 19 kinds of phthalic acid ester compounds were within a 0.025-0.2 mg/kg range, the recovery rate was 65.2-125.7%, relative standard deviation was 7.9-15.4% (n = 6), and the limit of detection was 0.005-0.02 mg/kg. Concentrations of the 19 kinds of phthalic acid ester compounds ranged between 0.01 and 0.12 mg/kg in ten dairy materials and 20 dairy products. The established method is simple, rapid, accurate, and highly sensitive.

  10. Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus

    PubMed Central

    Abd El Galil, Khaled H.; El Sokkary, M. A.; Kheira, S. M.; Salazar, Andre M.; Yates, Marylynn V.; Chen, Wilfred; Mulchandani, Ashok

    2005-01-01

    A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water. PMID:16269748

  11. Evolutionary connections of biological kingdoms based on protein and nucleic acid sequence evidence

    NASA Technical Reports Server (NTRS)

    Dayhoff, M. O.

    1983-01-01

    Prokaryotic and eukaryotic evolutionary trees are developed from protein and nucleic-acid sequences by the methods of numerical taxonomy. Trees are presented for bacterial ferredoxins, 5S ribosomal RNA, c-type cytochromes , cytochromes c2 and c', and 5.8S ribosomal RNA; the implications for early evolution are discussed; and a composite tree showing the branching of the anaerobes, aerobes, archaebacteria, and eukaryotes is shown. Single lines are found for all oxygen-evolving photosynthetic forms and for the salt-loving and high-temperature forms of archaebacteria. It is argued that the eukaryote mitochondria, chloroplasts, and cytoplasmic host material are descended from free-living prokaryotes that formed symbiotic associations, with more than one symbiotic event involved in the evolution of each organelle.

  12. Sequence-defined shuttles for targeted nucleic acid and protein delivery.

    PubMed

    Röder, Ruth; Wagner, Ernst

    2014-01-01

    Molecular medicine opens into a space of novel specific therapeutic agents: intracellularly active drugs such as peptides, proteins or nucleic acids, which are not able to cross cell membranes and enter the intracellular space on their own. Through the development of cell-targeted shuttles for specific delivery, this restriction in delivery has the potential to be converted into an advantage. On the one hand, due to the multiple extra- and intracellular barriers, such carrier systems need to be multifunctional. On the other hand, they must be precise and reproducibly manufactured due to pharmaceutical reasons. Here we review the design of precise sequence-defined delivery carriers, including solid-phase synthesized peptides and nonpeptidic oligomers, or nucleotide-based carriers such as aptamers and origami nanoboxes.

  13. Identification of amino acid sequences in the polyomavirus capsid proteins that serve as nuclear localization signals

    NASA Technical Reports Server (NTRS)

    Chang, D.; Haynes, J. I. Jr; Brady, J. N.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The molecular mechanism participating in the transport of newly synthesized proteins from the cytoplasm to the nucleus in mammalian cells is poorly understood. Recently, the nuclear localization signal sequences (NLS) of many nuclear proteins have been identified, and most have been found to be composed of a highly basic amino acid stretch. A genetic "subtractive" and a biochemical "additive" approach were used in our studies to identify the NLS's of the polyomavirus structural capsid proteins. An NLS was identified at the N-terminus (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) of the major capsid protein VP1 and at the C-terminus (Glu307 -Glu-Asp-Gly-Pro-Glu-Lys-Lys-Lys-Arg-Arg-Leu318) of the VP2/VP3 minor capsid proteins.

  14. The amino acid sequence of a carbohydrate-containing fragment of hen ovotransferrin.

    PubMed Central

    Kingston, I B; Williams, J

    1975-01-01

    1. Hen ovotransferrin was treated with CNBr and fractionated by gel filtration. 2. After further treatment by reduction and carboxymethylation a carbohydrate-containing fragment of molecular weight 11990 was obtained (fragment BCd). 3. The amino acid sequence of this fragment was determined. It consists of a single chain of 94 residues. 4. The structure of a tryptic glycopeptide derived from whole ovotransferrin permitted a further eight residues to be assigned at the N-terminus of fragment BCd. 5. Heterogeneity was found at two positions. 6. Further evidence has been deposited as Supplementary Publication SUP 50045 (19 pages) at the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1975), 145, 5. PMID:1172663

  15. Reduction in database search space by utilization of amino acid composition information from electron transfer dissociation and higher-energy collisional dissociation mass spectra.

    PubMed

    Hansen, Thomas A; Kryuchkov, Fedor; Kjeldsen, Frank

    2012-08-07

    With high-mass accuracy and consecutively obtained electron transfer dissociation (ETD) and higher-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS), reliable (≥97%) and sensitive fragment ions have been extracted for identification of specific amino acid residues in peptide sequences. The analytical benefit of these specific amino acid composition (AAC) ions is to restrict the database search space and provide identification of peptides with higher confidence and reduced false negative rates. The 6706 uniquely identified peptide sequences determined with a conservative Mascot score of >30 were used to characterize the AAC ions. The loss of amino acid side chains (small neutral losses, SNLs) from the charge reduced peptide radical cations was studied using ETD. Complementary AAC information from HCD spectra was provided by immonium ions. From the ETD/HCD mass spectra, 5162 and 6720 reliable SNLs and immonium ions were successfully extracted, respectively. Automated application of the AAC information during database searching resulted in an average 3.5-fold higher confidence level of peptide identification. In addition, 4% and 28% more peptides were identified above the significance level in a standard and extended search space, respectively.

  16. Phylogenetic analysis of beta-papillomaviruses as inferred from nucleotide and amino acid sequence data.

    PubMed

    Gottschling, Marc; Köhler, Anja; Stockfleth, Eggert; Nindl, Ingo

    2007-01-01

    Human papillomaviruses (HPV) of the beta-group seem to be involved in the pathogenesis of non-melanoma skin cancer. Papillomaviruses are host specific and are considered closely co-evolving with their hosts. Evolutionary incongruence between early genes and late genes has been reported among oncogenic genital alpha-papillomaviruses and considerably challenge phylogenetic reconstructions. We investigated the relationships of 29 beta-HPV (25 types plus four putative new types, subtypes, or variants) as inferred from codon aligned and amino acid sequence data of the genes E1, E2, E6, E7, L1, and L2 using likelihood, distance, and parsimony approaches. An analysis of a L1 fragment included additional nucleotide and amino acid sequences from seven non-human beta-papillomaviruses. Early genes and late genes evolution did not conflict significantly in beta-papillomaviruses based on partition homogeneity tests (p > or = 0.001). As inferred from the complete genome analyses, beta-papillomaviruses were monophyletic and segregated into four highly supported monophyletic assemblages corresponding to the species 1, 2, 3, and fused 4/5. They basically split into the species 1 and the remainder of beta-papillomaviruses, whose species 3, 4, and 5 constituted the sistergroup of species 2. beta-Papillomaviruses have been isolated from humans, apes, and monkeys, and phylogenetic analyses of the L1 fragment showed non-human papillomaviruses highly polyphyletic nesting within the HPV species. Thus, host and virus phylogenies were not congruent in beta-papillomaviruses, and multiple invasions across species borders may contribute (additionally to host-linked evolution) to their diversification.

  17. Identification and sequence determination of recombinant Clostridium perfringens alpha-toxin by use of electrospray ionization mass spectrometry.

    PubMed

    Saito, Hitoshi; Inoue, Masaharu; Tomiki, Masayoshi; Nemoto, Hiroshi; Komoriya, Tomoe; Kimata, Junko; Watanabe, Kunitomo; Kohno, Hideki

    2009-01-01

    Only a few methods exist for simple, sensitive and rapid detection of alpha-toxin in clinical and biological samples. The aim of our study was to establish a procedure for the production of an antibody against a recombinant antigen with confirmed sequence identity. We applied a noble approach based on proteomics using a mass spectrometer for the conclusive identification of the recombinant alpha-toxin that was subsequently used as an antigen. The recombinant alpha-toxin was produced in Escherichia coli. A clinical isolate of Clostridium perfringens GAI 94074 was amplified by polymerase chain reaction (PCR) and subsequently, cloning was performed. Three different fragments were cloned using a pET100/D-TOPO vector. These fragments coded for a ribosome binding site, a signal peptide and the alpha-toxin gene, respectively. Recombinant pET100 plasmids were cloned into TOP 10 cells and the isolated plasmids were transferred into BL21 Star (DE3) cells. Their expression was then induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). Recombinant E. coli transformed with a plasmid encoding the alpha-toxin gene alone produced a biologically inactive protein. On the other hand, E. coli carrying the plasmid encoding the toxin sequence and its native signal peptide sequence, or the toxin sequence along with the ribosome binding sequence and the signal peptide sequence secreted an active alpha-toxin with phospholipase activity. Accordingly, the C. perfringens gene encoding the alpha-toxin protein along with its signal peptide was successfully cloned, expressed, and secreted by E. coli. Furthermore, without consideration of its activity, we used mass spectrometry to confirm that the expressed protein was indeed the alpha-toxin. Thus, the identification of alpha-toxin protein using both the biological activity testing and the mass spectrometry analysis is expected to verify the significant production of C. perfringens antibody. The study for the analysis of recombinant alpha

  18. Amino acid sequence homology between rat and human C-reactive protein.

    PubMed Central

    Taylor, J A; Bruton, C J; Anderson, J K; Mole, J E; De Beer, F C; Baltz, M L; Pepys, M B

    1984-01-01

    The rat serum protein that undergoes Ca2+-dependent binding to pneumococcal C-polysaccharide and to phosphocholine residues, and that is evidently a member of the pentraxin family of proteins by virtue of its appearance under the electron microscope, has been variously designated as rat C-reactive protein (CRP) [de Beer, Baltz, Munn, Feinstein, Taylor, Bruton, Clamp & Pepys (1982) Immunology 45, 55-70], 'phosphoryl choline-binding protein' [Nagpurkar & Mookerjea (1981) J. Biol. Chem. 256, 7440-7448] and rat serum amyloid P component (SAP) [Pontet, D'Asnieres, Gache, Escaig & Engler (1981) Biochim. Biophys. Acta 671, 202-210]. The partial amino acid sequence (45 residues) towards the C-terminus of this protein was determined, and it showed 71.7% identity with the known sequence of human CRP but only 54.3% identity with human SAP. Since human CRP and SAP are themselves approximately 50% homologous, the level of identity between the rat protein and human SAP is evidence only of membership of the pentraxin family. In contrast, the much greater resemblance to human CRP confirms that the rat C-polysaccharide-binding/phosphocholine-binding protein is in fact rat CRP. PMID:6477504

  19. High-throughput sequencing of peptoids and peptide-peptoid hybrids by partial edman degradation and mass spectrometry.

    PubMed

    Thakkar, Amit; Cohen, Allison S; Connolly, Michael D; Zuckermann, Ronald N; Pei, Dehua

    2009-03-09

    A method for the rapid sequence determination of peptoids [oligo(N-substituted glycines)] and peptide-peptoid hybrids selected from one-bead-one-compound combinatorial libraries has been developed. In this method, beads carrying unique peptoid (or peptide-peptoid) sequences were subjected to multiple cycles of partial Edman degradation (PED) by treatment with a 1:3 (mol/mol) mixture of phenyl isothiocyanate (PITC) and 9-fluorenylmethyl chloroformate (Fmoc-Cl) to generate a series of N-terminal truncation products for each resin-bound peptoid. After PED, the Fmoc group was removed from the N-terminus and any reacted side chains via piperidine treatment. The resulting mixture of the full-length peptoid and its truncation products was analyzed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry, to reveal the sequence of the full-length peptoid. With a slight modification, the method was also effective in the sequence determination of peptide-peptoid hybrids. This rapid, high-throughput, sensitive, and inexpensive sequencing method should greatly expand the utility of combinatorial peptoid libraries in biomedical and materials research.

  20. Domoic Acid Poisoning as a Possible Cause of Seasonal Cetacean Mass Stranding Events in Tasmania, Australia.

    PubMed

    Bengtson Nash, S M; Baddock, M C; Takahashi, E; Dawson, A; Cropp, R

    2017-01-01

    The periodic trend to cetacean mass stranding events in the Australian island state of Tasmania remains unexplained. This article introduces the hypothesis that domoic acid poisoning may be a causative agent in these events. The hypothesis arises from the previously evidenced role of aeolian dust as a vector of iron input to the Southern Ocean; the role of iron enrichment in Pseudo-nitzschia bloom proliferation and domoic acid production; and importantly, the characteristic toxicosis of domoic acid poisoning in mammalian subjects leading to spatial navigation deficits. As a pre-requisite for quantitative evaluation, the plausibility of this hypothesis was considered through correlation analyses between historical monthly stranding event numbers, mean monthly chlorophyll concentration and average monthly atmospheric dust loading. Correlation of these variables, which under the domoic acid stranding scenario would be linked, revealed strong agreement (r = 0.80-0.87). We therefore advocate implementation of strategic quantitative investigation of the role of domoic acid in Tasmanian cetacean mass stranding events.

  1. Amino acid sequences of alpha-helical segments from S-carbosymethylkerateine-A. Complete sequence of a type-I segment.

    PubMed Central

    Gough, K H; Inglis, A S; Crewther, W G

    1978-01-01

    The amino acid sequence of a type-I helical segment from the low-sulphur protein (S-carboxymethylkerateine-A) of wool was determined by combining automatic and manual-sequencing data. Whereas in the type-II helical segment most of the cationic groups occur in pairs, 11 of the 22 anionic residues in the sequence of the type-I segment were situated next to a second anionic residue. This suggests possible interactions between type-I and type-II helical segments in alpha-keratin. As observed with the sequence of a type-II helical segment a model constructed on 3.6 residues per turn of helix shows a line of hydrophobic residues along the helix, thereby supporting the physicochemical evidence that the molecule is predominantly helical and forms part of a coiled-coil structure. Examination of the sequence data by predictive methods indicates the possibilty of extensive sections of alpha-helix interspersed with discontinuities. The molecule contains a number of regions with peptide sequences identical with those found by other workers after enzymic digestion of fractions from oxidized wool. Images Fig. 1. PMID:697725

  2. Species Identification of Archaeological Skin Objects from Danish Bogs: Comparison between Mass Spectrometry-Based Peptide Sequencing and Microscopy-Based Methods

    PubMed Central

    Brandt, Luise Ørsted; Schmidt, Anne Lisbeth; Mannering, Ulla; Sarret, Mathilde; Kelstrup, Christian D.; Olsen, Jesper V.; Cappellini, Enrico

    2014-01-01

    Denmark has an extraordinarily large and well-preserved collection of archaeological skin garments found in peat bogs, dated to approximately 920 BC – AD 775. These objects provide not only the possibility to study prehistoric skin costume and technologies, but also to investigate the animal species used for the production of skin garments. Until recently, species identification of archaeological skin was primarily performed by light and scanning electron microscopy or the analysis of ancient DNA. However, the efficacy of these methods can be limited due to the harsh, mostly acidic environment of peat bogs leading to morphological and molecular degradation within the samples. We compared species assignment results of twelve archaeological skin samples from Danish bogs using Mass Spectrometry (MS)-based peptide sequencing, against results obtained using light and scanning electron microscopy. While it was difficult to obtain reliable results using microscopy, MS enabled the identification of several species-diagnostic peptides, mostly from collagen and keratins, allowing confident species discrimination even among taxonomically close organisms, such as sheep and goat. Unlike previous MS-based methods, mostly relying on peptide fingerprinting, the shotgun sequencing approach we describe aims to identify the complete extracted ancient proteome, without preselected specific targets. As an example, we report the identification, in one of the samples, of two peptides uniquely assigned to bovine foetal haemoglobin, indicating the production of skin from a calf slaughtered within the first months of its life. We conclude that MS-based peptide sequencing is a reliable method for species identification of samples from bogs. The mass spectrometry proteomics data were deposited in the ProteomeXchange Consortium with the dataset identifier PXD001029. PMID:25260035

  3. Species identification of archaeological skin objects from Danish bogs: comparison between mass spectrometry-based peptide sequencing and microscopy-based methods.

    PubMed

    Brandt, Luise Ørsted; Schmidt, Anne Lisbeth; Mannering, Ulla; Sarret, Mathilde; Kelstrup, Christian D; Olsen, Jesper V; Cappellini, Enrico

    2014-01-01

    Denmark has an extraordinarily large and well-preserved collection of archaeological skin garments found in peat bogs, dated to approximately 920 BC - AD 775. These objects provide not only the possibility to study prehistoric skin costume and technologies, but also to investigate the animal species used for the production of skin garments. Until recently, species identification of archaeological skin was primarily performed by light and scanning electron microscopy or the analysis of ancient DNA. However, the efficacy of these methods can be limited due to the harsh, mostly acidic environment of peat bogs leading to morphological and molecular degradation within the samples. We compared species assignment results of twelve archaeological skin samples from Danish bogs using Mass Spectrometry (MS)-based peptide sequencing, against results obtained using light and scanning electron microscopy. While it was difficult to obtain reliable results using microscopy, MS enabled the identification of several species-diagnostic peptides, mostly from collagen and keratins, allowing confident species discrimination even among taxonomically close organisms, such as sheep and goat. Unlike previous MS-based methods, mostly relying on peptide fingerprinting, the shotgun sequencing approach we describe aims to identify the complete extracted ancient proteome, without preselected specific targets. As an example, we report the identification, in one of the samples, of two peptides uniquely assigned to bovine foetal haemoglobin, indicating the production of skin from a calf slaughtered within the first months of its life. We conclude that MS-based peptide sequencing is a reliable method for species identification of samples from bogs. The mass spectrometry proteomics data were deposited in the ProteomeXchange Consortium with the dataset identifier PXD001029.

  4. Pyrolysis-gas chromatography-mass spectrometry of a series of bile acid sequestrants.

    PubMed

    Haskins, N J; Eckers, C; Mitchell, R

    1992-09-01

    Pyrolysis of a series of polymers based on polystyrene and used as bile acid sequestrants produced characteristic mixtures of compounds which were analysed by gas chromatography-mass spectrometry. The nature of the substituent groups was clearly apparent while the polymer backbone gave rise to representative styrenes. The reproducibility of the results was examined by experimenting with the temperature of pyrolysis. It was found that at low temperatures very little fragmentation of the polystyrene backbone occurred but the substituents were still released in high yield. The orientation of the various substituted styrenes generated by pyrolysis was confirmed by the use of gas chromatography with infrared and mass spectrometric detection.

  5. Extraction Chromatographic Methods in the Sample Preparation Sequence for Thermal Ionization Mass Spectrometric Analysis of Plutonium Isotopes

    SciTech Connect

    Grate, Jay W.; O'Hara, Matthew J.; Farawila, Anne F.; Douglas, Matthew; Haney, Morgan M.; Peterson, Steve L.; Maiti, Tapas C.; Aardahl, Christopher L.

    2011-10-17

    A sample preparation sequence for actinide isotopic analysis by TIMS is described that includes column-based extraction chromatography as the first separation step, followed by anion exchange column separations. The sequence is designed to include a wet ashing step after the extraction chromatography to prevent any leached extractant or oxalic acid eluent reagents from interfering with subsequent separations, source preparation, or TIMS ionization. TEVA-resin and DGA-resin materials, containing extractants that consist only of C, N, O, and H atoms, were investigated for isolation of plutonium. Radiotracer level studies confirmed expected high yields from column-based separation procedures. Femtogram-level studies were carried out with TIMS detection, using multiple isotopic spikes through the separation sequence. Pu recoveries were 87% and 86% for TEVA- and DGA-resins separations respectively. The Pu recoveries from 400 {mu}L anion-exchange column separations were 89% and 93% for trial sequences incorporating TEVA and DGA-resin. Thus, a prior extraction chromatography step in the sequence did not interfere with the subsequent anion exchange separation when a simple wet ash step was carried out in between these column separations. The average measurement efficiency, for Pu, encompassing the chemical separation recoveries and the TIMS ionization efficiency, was 2.73 {+-} 0.77% (2-sigma) for the DGA-resin trials and 2.67 {+-} 0.54% for the TEVA-resin trials, compared to 3.41% and 2.37% (average 2.89%) for two spikes in the experimental set. These compare with an average measurement efficiency of 2.78 {+-} 1.70%, n = 33 from process benchmark analyses using Pu spikes processed through a sequence of oxalate precipitation, wet ash, iron hydroxide precipitation, and anion exchange column separations. We conclude that extraction chromatography can be a viable separation procedure as part of a multistep sequence for TIMS sample preparation.

  6. The amino acid sequence of the cytochrome c-554(547) from the chemolithotrophic bacterium Thiobacillus neapolitanus.

    PubMed Central

    Ambler, R P; Meyer, T E; Trudinger, P A; Kamen, M D

    1985-01-01

    An amino acid sequence is proposed for the cytochrome c-554(547) from the bacterium Thiobacillus neapolitanus N.C.I.B. 8539). It consists of a polypeptide chain of 91 residues, with a pair of haem-attachment cysteine residues at positions 15 and 18. There is similarity in sequence with each of the halves of the sequence of the dihaem cytochromes c4 and with a cytochrome c-554(548) from a halophilic strain of Paracoccus. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50127 (11 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1985) 225, 5. PMID:2988504

  7. Human Retroviruses and AIDS. A compilation and analysis of nucleic acid and amino acid sequences: I--II; III--V

    SciTech Connect

    Myers, G.; Korber, B.; Wain-Hobson, S.; Smith, R.F.; Pavlakis, G.N.

    1993-12-31

    This compendium and the accompanying floppy diskettes are the result of an effort to compile and rapidly publish all relevant molecular data concerning the human immunodeficiency viruses (HIV) and related retroviruses. The scope of the compendium and database is best summarized by the five parts that it comprises: (I) HIV and SIV Nucleotide Sequences; (II) Amino Acid Sequences; (III) Analyses; (IV) Related Sequences; and (V) Database Communications. Information within all the parts is updated at least twice in each year, which accounts for the modes of binding and pagination in the compendium.

  8. Identification of Novel Perfluoroalkyl Ether Carboxylic Acids (PFECAs) and Sulfonic Acids (PFESAs) in Natural Waters Using Accurate Mass Time-of-Flight Mass Spectrometry (TOFMS).

    PubMed

    Strynar, Mark; Dagnino, Sonia; McMahen, Rebecca; Liang, Shuang; Lindstrom, Andrew; Andersen, Erik; McMillan, Larry; Thurman, Michael; Ferrer, Imma; Ball, Carol

    2015-10-06

    Recent scientific scrutiny and concerns over exposure, toxicity, and risk have led to international regulatory efforts resulting in the reduction or elimination of certain perfluorinated compounds from various products and waste streams. Some manufacturers have started producing shorter chain per- and polyfluorinated compounds to try to reduce the potential for bioaccumulation in humans and wildlife. Some of these new compounds contain central ether oxygens or other minor modifications of traditional perfluorinated structures. At present, there has been very limited information published on these "replacement chemistries" in the peer-reviewed literature. In this study we used a time-of-flight mass spectrometry detector (LC-ESI-TOFMS) to identify fluorinated compounds in natural waters collected from locations with historical perfluorinated compound contamination. Our workflow for discovery of chemicals included sequential sampling of surface water for identification of potential sources, nontargeted TOFMS analysis, molecular feature extraction (MFE) of samples, and evaluation of features unique to the sample with source inputs. Specifically, compounds were tentatively identified by (1) accurate mass determination of parent and/or related adducts and fragments from in-source collision-induced dissociation (CID), (2) in-depth evaluation of in-source adducts formed during analysis, and (3) confirmation with authentic standards when available. We observed groups of compounds in homologous series that differed by multiples of CF2 (m/z 49.9968) or CF2O (m/z 65.9917). Compounds in each series were chromatographically separated and had comparable fragments and adducts produced during analysis. We detected 12 novel perfluoroalkyl ether carboxylic and sulfonic acids in surface water in North Carolina, USA using this approach. A key piece of evidence was the discovery of accurate mass in-source n-mer formation (H(+) and Na(+)) differing by m/z 21.9819, corresponding to the

  9. Nucleic acid sequence of an internal image-bearing monoclonal anti-idiotype and its comparison to the sequence of the external antigen.

    PubMed Central

    Bruck, C; Co, M S; Slaoui, M; Gaulton, G N; Smith, T; Fields, B N; Mullins, J I; Greene, M I

    1986-01-01

    The monoclonal anti-idiotypic antibody (mAb2) 87.92.6 directed against the 9B.G5 antibody specific for the virus neutralizing epitope on the mammalian reovirus type 3 hemagglutinin was previously demonstrated to express an internal image of the receptor binding epitope of the reovirus type 3. Furthermore, this mAb2 has autoimmune reactivity to the cell surface receptor of the reovirus. The nucleotide and deduced amino acid sequences of the 87.92.6 mAb2 heavy and light chains are described in this report. The sequence analysis reveals that the same heavy chain variable and joining (VH and JH) gene segments are used by the 87.92.6 anti-idiotypic mAb2 and by the dominant idiotypes of the BALB/c anti-GAT (cGAT) and anti-NP (NPa) responses. [GAT; random polymer that is 60% glutamic acid, 30% alanine, and 10% tyrosine. NP; (4-hydroxy-3-nitrophenyl)-acetyl.] Despite extensive homology at the level of the heavy chain variable regions, the NPa positive BALB/c anti-NP monoclonal antibody 17.2.25 binds neither 9B.G5 nor the cellular receptor for the hemagglutinin. Amino acid sequence comparison between the viral hemagglutinin and the 87.92.6 mAb2 light chain "internal image," reveals an area of significant homology indicating that antigen mimicry by antibodies may be achieved by sharing primary structure. PMID:2428036

  10. Draft Genome Sequence of Escherichia coli O157:H7 ATCC 35150 and a Nalidixic Acid-Resistant Mutant Derivative

    PubMed Central

    Markell, James A.; Koziol, Adam G.

    2015-01-01

    Shiga toxin-producing Escherichia coli strains, occasionally isolated from food, are of public health importance. Here, we report on the 5.30-Mbp draft genome sequence of E. coli O157:H7 EDL931 (strain ATCC 35150) and the 5.32-Mbp draft genome sequence of a nalidixic acid-resistant mutant derivative used as a distinguishable control strain in food-testing laboratories. PMID:26205873

  11. Acid-base-driven matrix-assisted mass spectrometry for targeted metabolomics.

    PubMed

    Shroff, Rohit; Rulísek, Lubomír; Doubsky, Jan; Svatos, Ales

    2009-06-23

    The ability to charge huge biomolecules without breaking them apart has made matrix-assisted laser desorption/ionization (MALDI) mass spectrometry an indispensable tool for biomolecular analysis. Conventional, empirically selected matrices produce abundant matrix ion clusters in the low-mass region (<500 Da), hampering the application of MALDI-MS to metabolomics. An ionization mode of MAILD, a rational protocol for matrix selection based on Brønsted-Lowry acid-base theory and its application to metabolomics, biological screening/profiling/imaging, and clinical diagnostics is illustrated. Numerous metabolites, covering important metabolic pathways (Krebs' cycle, fatty acid and glucosinolate biosynthesis), were detected in extracts, biofluids, and/or in biological tissues (Arabidopsis thaliana, Drosophila melanogaster, Acyrthosiphon pisum, and human blood). This approach moves matrix selection from "black art" to rational design and sets a paradigm for small-molecule analysis via MALDI-MS.

  12. Enhancement of acidic gases in biomass burning impacted air masses over Canada

    NASA Technical Reports Server (NTRS)

    Lefer, B. L.; Talbot, R. W.; Harriss, R. C.; Bradshaw, J. D.; Sandholm, S. T.; Olson, J. O.; Sachse, G. W.; Collins, J.; Shipham, M. A.; Blake, D. R.

    1994-01-01

    Biomass-burning impacted air masses sampled over central and eastern Canada during the summer of 1990 as part of ABLE 3B contained enhanced mixing ratios of gaseous HNO3, HCOOH, CH3COOH, and what appears to be (COOH)2. These aircraft-based samples were collected from a variety of fresh burning plumes and more aged haze layers from different source regions. Values of the enhancement factor, delta X/delta CO, where X represents an acidic gas, for combustion-impacted air masses sampled both near and farther away from the fires, were relatively uniform. However, comparison of carboxylic acid emission ratios measured in laboratory fires to field plume enhancement factors indicates significant in-plume production of HCOOH. Biomass-burning appears to be an important source of HNO3, HCOOH, and CH3COOH to the troposphere over subarctic Canada.

  13. Poly(lactic acid)-Mass production, processing, industrial applications, and end of life.

    PubMed

    Castro-Aguirre, E; Iñiguez-Franco, F; Samsudin, H; Fang, X; Auras, R

    2016-12-15

    Global awareness of material sustainability has increased the demand for bio-based polymers like poly(lactic acid) (PLA), which are seen as a desirable alternative to fossil-based polymers because they have less environmental impact. PLA is an aliphatic polyester, primarily produced by industrial polycondensation of lactic acid and/or ring-opening polymerization of lactide. Melt processing is the main technique used for mass production of PLA products for the medical, textile, plasticulture, and packaging industries. To fulfill additional desirable product properties and extend product use, PLA has been blended with other resins or compounded with different fillers such as fibers, and micro- and nanoparticles. This paper presents a review of the current status of PLA mass production, processing techniques and current applications, and also covers the methods to tailor PLA properties, the main PLA degradation reactions, PLA products' end-of-life scenarios and the environmental footprint of this unique polymer.

  14. DNA sequence analysis by hybridization with oligonucleotide microchips: MALDI mass spectrometry identification of 5mers contiguously stacked to microchip oligonucleotides

    PubMed Central

    Stomakhin, Andrey A.; Vasiliskov, Vadim A.; Timofeev, Edward; Schulga, Dennis; Cotter, Richard J.; Mirzabekov, Andrei D.

    2000-01-01

    Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) has been applied to increase the informational output from DNA sequence analysis. It has been used to analyze DNA by hybridization with microarrays of gel-immobilized oligonucleotides extended with stacked 5mers. In model experiments, a 28 nt long DNA fragment was hybridized with 10 immobilized, overlapping 8mers. Then, in a second round of hybridization DNA–8mer duplexes were hybridized with a mixture of 10 5mers. The stability of the 5mer complex with DNA was increased to raise the melting temperature of the duplex by 10–15°C as a result of stacking interaction with 8mers. Contiguous 13 bp duplexes containing an internal break were formed. MALDI MS identified one or, in some cases, two 5mers contiguously stacked to each DNA–8mer duplex formed on the microchip. Incorporating a mass label into 5mers optimized MALDI MS monitoring. This procedure enabled us to reconstitute the sequence of a model DNA fragment and identify polymorphic nucleotides. The application of MALDI MS identification of contiguously stacked 5mers to increase the length of DNA for sequence analysis is discussed. PMID:10666462

  15. Identification of acylglycerols containing dihydroxy fatty acids in castor (Ricinus communis L.)oil by mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ricinoleate, a monohydroxy fatty acid, in castor oil has many industrial uses. Dihydroxy fatty acids can also be used in industry. The C18 HPLC fractions of castor oil were used for mass spectrometry to identify the acylglycerols containing dihydroxy fatty acids. Four diacylglycerols identified were...

  16. Identification of trihydroxy fatty acids in castor oil and the regiospecific quantification of the triacylglycerols by mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ricinoleate, a monohydroxy fatty acid, has many industrial uses. Trihydroxy fatty acids can also be used in industry. We report here the identification of diacylglycerols and triacylglycerols containing trihydroxy fatty acids in castor oil. The C18 HPLC fractions of castor oil were used for mass spe...

  17. Gas chromatographic/mass spectrometric determination of lysergic acid diethylamide (LSD) in serum samples.

    PubMed

    Musshoff, F; Daldrup, T

    1997-08-04

    A sensitive method for the detection and quantification of lysergic acid diethylamide (LSD) in serum samples is described. After liquid-liquid extraction the trimethylsilyl derivative of LSD is detected by gas chromatography-mass spectrometry. Experiments with spiked samples resulted in a recovery of 76%, the coefficient of variation was 9.3%. Excellent linearity was obtained over the range 0.1-10 ng ml-1. Additionally experiments demonstrating the light sensitivity of LSD are presented together with casuistics.

  18. Identification of acylglycerols containing dihydroxy fatty acids in castor oil by mass spectrometry.

    PubMed

    Lin, Jiann-Tsyh; Arcinas, Arthur; Harden, Leslie A

    2009-04-01

    Ricinoleate, a monohydroxy fatty acid, in castor oil has many industrial uses. Dihydroxy fatty acids can also be used in industry. The C(18) HPLC fractions of castor oil were analyzed by electrospray ionization mass spectrometry of lithium adducts to identify the acylglycerols containing dihydroxy fatty acids and the dihydroxy fatty acids. Four diacylglycerols identified were diOH18:1-diOH18:1, diOH18:2-OH18:1, diOH18:1-OH18:1 and diOH18:0-OH18:1. Eight triacylglycerols identified were diOH18:1-diOH18:1-diOH18:1, diOH18:1-diOH18:1-diOH18:0, diOH18:2-diOH18:1-OH18:1, diOH18:1-diOH18:1-OH18:1, diOH18:1-diOH18:0-OH18:1, diOH18:2-OH18:1-OH18:1, diOH18:1-OH18:1-OH18:1 and diOH18:0-OH18:1-OH18:1. The locations of fatty acids on the glycerol backbone were not determined. The structures of these three newly identified dihydroxy fatty acids were proposed as 11,12-dihydroxy-9-octadecenoic acid, 11,12-dihydroxy-9,13-octadecadienoic acid and 11,12-dihydroxyoctadecanoic acid. These individual acylglycerols were at the levels of about 0.5% or less in castor oil and can be isolated from castor oil or overproduced in a transgenic oil seed plant for future industrial uses.

  19. Analysis of free amino acids in natural waters by liquid chromatography-tandem mass spectrometry.

    PubMed

    How, Zuo Tong; Busetti, Francesco; Linge, Kathryn L; Kristiana, Ina; Joll, Cynthia A; Charrois, Jeffrey W A

    2014-11-28

    This paper reports a new analytical method for the analysis of 18 amino acids in natural waters using solid-phase extraction (SPE) followed by liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) operated in multiple reaction monitoring mode. Two different preconcentration methods, solid-phase extraction and concentration under reduced pressure, were tested in development of this method. Although concentration under reduced pressure provided better recoveries and method limits of detection for amino acids in ultrapure water, SPE was a more suitable extraction method for real samples due to the lower matrix effects for this method. Even though the strong cation exchange resin used in SPE method introduced exogenous matrix interferences into the sample extracts (inorganic salt originating from the acid-base reaction during the elution step), the SPE method still incorporates a broad sample clean-up and minimised endogenous matrix effects by reducing interferences originating from real water samples. The method limits of quantification (MLQ) for the SPE LC-MS/MS method in ultrapure water ranged from 0.1 to 100 μg L(-1) as N for the different amino acids. The MLQs of the early eluting amino acids were limited by the presence of matrix interfering species, such as inorganic salts in natural water samples. The SPE LC-MS/MS method was successfully applied to the analysis of amino acids in 3 different drinking water source waters: the average total free amino acid content in these waters was found to be 19 μg L(-1) as N, while among the 18 amino acids analysed, the most abundant amino acids were found to be tyrosine, leucine and isoleucine.

  20. Determination of ascorbic acid and carotenoids in food commodities by liquid chromatography with mass spectrometry detection.

    PubMed

    Frenich, A Garrido; Torres, M E Hernández; Vega, A Belmonte; Vidal, J L Martínez; Bolaños, P Plaza

    2005-09-21

    Two methods, one to determine ascorbic acid and one to determine lycopene and beta-carotene, in vegetables and fruits by liquid chromatography coupled with mass spectrometry (LC-MS) have been established. The chromatographic separation of the studied compounds and their MS parameters were optimized to improve selectivity and sensitivity. In both methods, separation was carried out with two coupled columns, first a C(18) and then a dC(18), using as mobile phase 70% methanol (0.005% acetic acid) and 30% acetic acid 0.05% for ascorbic acid determination and a mixture of methanol, tetrahydrofuran, and acetonitrile (60:30:10 v/v/v) for carotenoid analysis in isocratic mode. The molecular ion was selected for the quantification in selective ion monitoring (SIM) mode. Ascorbic acid was detected with electrospray ionization probe (ESI) in negative mode, while chemical ionization atmospheric pressure (APCI) in positive mode was used for the target carotenoids. The methodology for ascorbic acid analysis is based on an extraction with polytron using methanol and a mixture of methaphosphoric acid and acetic acid. Extraction of the carotenoids was carried out with tetrahydrofuran/methanol (1:1) (v/v). The proposed methods were applied, after their corresponding validations, to the analysis of four varieties of tomatoes, tomato in tin enriched and dried tomato, and to the analysis of mango and kiwi fruits, to compare the content in these compounds. Moreover, the influence of the process of freezing and the effect that the manipulation/preservation has in the content of ascorbic acid in tomato have also been studied.

  1. Comment on "Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry".

    PubMed

    Pevzner, Pavel A; Kim, Sangtae; Ng, Julio

    2008-08-22

    Asara et al. (Reports, 13 April 2007, p. 280) reported sequencing of Tyrannosaurus rex proteins and used them to establish the evolutionary relationships between birds and dinosaurs. We argue that the reported T. rex peptides may represent statistical artifacts and call for complete data release to enable experimental and computational verification of their findings.

  2. Standard pre-main sequence models of low-mass stars

    SciTech Connect

    Prada Moroni, P. G.; Degl'Innocenti, S.; Tognelli, E.

    2014-05-09

    The main characteristics of standard pre-main sequence (PMS) models are described. A discussion of the uncer-tainties affecting the current generation of PMS evolutionary tracks and isochrones is also provided. In particular, the impact of the uncertainties in the adopted equation of state, radiative opacity, nuclear cross sections, and initial chemical abundances are analysed.

  3. The peculiar structural features of kiwi fruit pectin methylesterase: amino acid sequence, oligosaccharides structure, and modeling of the interaction with its natural proteinaceous inhibitor.

    PubMed

    Ciardiello, M Antonietta; D'Avino, Rossana; Amoresano, Angela; Tuppo, Lisa; Carpentieri, Andrea; Carratore, Vito; Tamburrini, Maurizio; Giovane, Alfonso; Pucci, Piero; Camardella, Laura

    2008-04-01

    Pectin methylesterase (PME) from kiwi fruit (Actinidia deliciosa) is a glycoprotein, showing an apparent molecular mass of 50 kDa upon size exclusion chromatography and SDS-PAGE. The primary structure, elucidated by direct sequencing of the protein, comprises 321 amino acid residues providing a molecular mass of 35 kDa. The protein has an acetylated Thr residue at the amino terminus and five N-glycosylation consensus sequences, four of which are actually glycosylated. A careful investigation of the oligosaccharide structures demonstrated that PME glycans belong to complex type oligosaccharides essentially consisting of xylosylated polyfucosylated biantennary structures. Alignment with known mature plant PME sequences indicates that the postulated active site residues are conserved. Kiwi PME activity is inhibited following the interaction with the proteinaceous inhibitor PMEI, isolated from the same source. Gel-filtration experiments show that kiwi PME/PMEI complex is stable in a large pH range and dissociates only at pH 10.0. Modeling of the interaction with the inhibitor was performed by using the crystal structure of the complex between kiwi PMEI and tomato PME as a template. The model shows that the binding site is the same reported for tomato PME. However, additional salt link interactions are found to connect the external loops of kiwi PME to PMEI. This finding may explain the higher pH stability of the complex formed by the two kiwi proteins respect to that formed by PMEI and tomato PME.

  4. Enzymatic properties and nucleotide and amino acid sequences of a thermostable beta-agarase from a novel species of deep-sea Microbulbifer.

    PubMed

    Ohta, Y; Hatada, Y; Nogi, Y; Miyazaki, M; Li, Z; Akita, M; Hidaka, Y; Goda, S; Ito, S; Horikoshi, K

    2004-05-01

    An agar-degrading bacterium, strain JAMB-A7, was isolated from the sediment in Sagami Bay, Japan, at a depth of 1,174 m and identified as a novel species of the genus Microbulbifer. The gene for a novel beta-agarase from the isolate was cloned and sequenced. It encodes a protein of 441 amino acids with a calculated molecular mass of 48,989 Da. The deduced amino acid sequence showed similarity to those of known beta-agarases in glycoside hydrolase family 16, with only 34-55% identity. A sequence similar to a carbohydrate-binding module was found in the C-terminal region of the enzyme. The recombinant agarase was hyper-produced extracellularly using Bacillus subtilis as the host, and the enzyme purified to homogeneity had a specific activity of 398 U (mg protein)(-1) at pH 7.0 and 50 degrees C. It was thermostable, with a half-life of 502 min at 50 degrees C. The optimal pH and temperature for activity were around 7 and 50 degrees C, respectively. The pattern of agarose hydrolysis showed that the enzyme was an endo-type beta-agarase, and the final main product was neoagarotetraose. The activity was not inhibited by NaCl, EDTA, and various surfactants at high concentrations. In particular, sodium dodecyl sulfate had no inhibitory effect up to 2%.

  5. EVOLUTION OF VERY MASSIVE POPULATION III STARS WITH MASS ACCRETION FROM PRE-MAIN SEQUENCE TO COLLAPSE

    SciTech Connect

    Ohkubo, Takuya; Nomoto, Ken'ichi; Umeda, Hideyuki; Yoshida, Naoki; Tsuruta, Sachiko E-mail: umeda@astron.s.u-tokyo.ac.j E-mail: naoki.yoshida@ipmu.j

    2009-12-01

    We calculate the evolution of zero-metallicity Population III (Pop III) stars whose mass grows from the initial mass of approx1 M{sub sun} by accreting the surrounding gases. Our calculations cover whole evolutionary stages from the pre-main sequence, via various nuclear burning stages, through the final core-collapse or pair-creation instability phases. We adopt two different sets of stellar mass accretion rates as our fiducial models. One is derived from a cosmological simulation of the first generation (PopIII.1) stars, and the other is derived from a simulation of the second generation stars that are affected by radiation from PopIII.1 stars. The latter represents one case of PopIII.2 stars. We also adopt additional models that include radiative feedback effects. We show that the final mass of Pop III.1 stars can be as large as approx1000 M {sub sun}, beyond the mass range (140-300 M{sub sun}) for the pair-instability supernovae. Such massive stars undergo core-collapse to form intermediate-mass black holes, which may be the seeds for merger trees to supermassive black holes. On the other hand, Pop III.2 stars become less massive (approx<40-60 M{sub sun}), being in the mass range of ordinary iron core-collapse stars. Such stars explode and eject heavy elements to contribute to chemical enrichment of the early universe as observed in the abundance patterns of extremely metal-poor stars in the Galactic halo. In view of the large range of possible accretion rates, further studies are important to see if these fiducial models are actually the cases.

  6. Nucleotide and deduced amino acid sequences of a subtilisin-like serine protease from a deep-sea bacterium, Alkalimonas collagenimarina AC40(T).

    PubMed

    Kurata, Atsushi; Uchimura, Kohsuke; Shimamura, Shigeru; Kobayashi, Tohru; Horikoshi, Koki

    2007-11-01

    The acpI gene encoding an alkaline protease (AcpI) from a deep-sea bacterium, Alkalimonas collagenimarina AC40(T), was shotgun-cloned and sequenced. It had a 1,617-bp open reading frame encoding a protein of 538 amino acids. Based on analysis of the deduced amino acid sequence, AcpI is a subtilisin-like serine protease belonging to subtilase family A. It consists of a prepropeptide, a catalytic domain, and a prepeptidase C-terminal domain like other serine proteases from the genera Pseudomonas, Shewanella, Alteromonas, and Xanthomonas. Heterologous expression of the acpI gene in Escherichia coli cells yielded a 28-kDa recombinant AcpI (rAcpI), suggesting that both the prepropeptide and prepeptidase C-terminal domains were cleaved off to give the mature form. Analysis of N-terminal and C-terminal amino acid sequences of purified rAcpI showed that the mature enzyme would be composed of 273 amino acids. The optimal pH and temperature for the caseinolytic activity of the purified rAcpI were 9.0-9.5 and 45 degrees C in 100 mM glycine-NaOH buffer. Calcium ions slightly enhanced the enzyme activity and stability. The enzyme favorably hydrolyzed gelatin, collagen, and casein. AcpI from A. collagenimarina AC40(T) was also purified from culture broth, and its molecular mass was around 28 kDa, indicating that the cleavage manner of the enzyme is similar to that in E. coli cells.

  7. New roles for Smad signaling and phosphatidic acid in the regulation of skeletal muscle mass.

    PubMed

    Goodman, Craig A; Hornberger, Troy A

    2014-01-01

    Skeletal muscle is essential for normal bodily function and the loss of skeletal muscle (i.e. muscle atrophy/wasting) can have a major impact on mobility, whole-body metabolism, disease resistance, and quality of life. Thus, there is a clear need for the development of therapies that can prevent the loss, or increase, of skeletal muscle mass. However, in order to develop such therapies, we will first have to develop a thorough understanding of the molecular mechanisms that regulate muscle mass. Fortunately, our knowledge is rapidly advancing, and in this review, we will summarize recent studies that have expanded our understanding of the roles that Smad signaling and the synthesis of phosphatidic acid play in the regulation of skeletal muscle mass.

  8. High sensitivity measurement of amino acid isotope enrichment using liquid chromatography-mass spectrometry.

    PubMed

    van Eijk, Hans M H; Wijnands, Karolina A P; Bessems, Babs A F M; Olde Damink, Steven W; Dejong, Cornelis H C; Poeze, Martijn

    2012-09-15

    Measurement of the incorporation or conversion of infused stable isotope enriched metabolites in vivo such as amino acids plays a key role in metabolic research. Specific routes are frequently probed in knockout mouse models limiting the available amount of sample. Although less precise as compared to combustion-isotope ratio mass spectrometry (C-IRMS), gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS) techniques are therefore often the method of choice to measure isotopic enrichment of target metabolites. However, under conditions of metabolic depletion, the precision of these systems becomes limiting. In this paper, studies were performed to enhance the sensitivity and precision of isotope enrichment measurements using LC-MS. Ion-statistics and resolution were identified as critical factors for this application when using a linear trap mass spectrometer. The combination with an automated pre-column derivatization and a carefully selected solvent mix allowed us to measure isotopic enrichments down to 0.005% at plasma concentrations as low as 5 μmol/l, an improvement by a factor of 100 compared to alternative methods. The resulting method now allowed measurement of the in vivo conversion of the amino acid arginine into citrulline as a marker for the production of nitric oxide in an in vivo murine endotoxemia model with depleted plasma levels of arginine and citrulline.

  9. Amino acid analysis in micrograms of meteorite sample by nanoliquid chromatography-high-resolution mass spectrometry.

    PubMed

    Callahan, Michael P; Martin, Mildred G; Burton, Aaron S; Glavin, Daniel P; Dworkin, Jason P

    2014-03-07

    Amino acids and their enantiomers in a 360 microgram sample of Murchison meteorite were unambiguously identified and quantified using chemical derivatization and nanoliquid chromatography coupled to nanoelectrospray ionization high resolution orbitrap mass spectrometry techniques. The distribution and abundance of amino acids were similar to past studies of Murchison meteorite but the samples used here were three orders of magnitude lower. The analytical method was also highly sensitive, and some amino acid reference standards were successfully detected at a level of ∼200 attomoles (on column). These results may open up the possibility for investigating other less studied, sample-limited extraterrestrial samples (e.g., micrometeorites, interplanetary dust particles, and cometary particles) for biologically-relevant organic molecules.

  10. Water chemical ionization mass spectrometry of aldehydes, ketones esters, and carboxylic acids

    SciTech Connect

    Hawthorne, S.B.; Miller, D.J.

    1986-11-01

    Chemical ionization mass spectrometry (CI) of aliphatic and aromatic carbonyl compounds using water as the reagent gas provides intense pseudomolecular ions and class-specific fragmentation patterns that can be used to identify aliphatic aldehydes, ketones, carboxylic acids, and esters. The length of ester acyl and alkyl groups can easily be determined on the basis of loss of alcohols from the protonated parent. Water CI provides for an approximately 200:1 selectivity of carbonyl species over alkanes. No reagent ions are detected above 55 amu, allowing species as small as acetone, propanal, acetic acid, and methyl formate to be identified. When deuterate water was used as the reagent, only the carboxylic acids and ..beta..-diketones showed significant H/D exchange. The use of water CI to identify carbonyl compounds in a wastewater from the supercritical water extraction of lignite coal, in lemon oil, and in whiskey volatiles is discussed.

  11. Mass spectrometric and Edman sequencing of lipocortin I isolated by two-dimensional SDS/PAGE of human melanoma lysates.

    PubMed Central

    Hall, S C; Smith, D M; Masiarz, F R; Soo, V W; Tran, H M; Epstein, L B; Burlingame, A L

    1993-01-01

    We have integrated preparative two-dimensional polyacrylamide gel electrophoresis with high-performance tandem mass spectrometry and Edman degradation. By using this approach, we have isolated and identified, by partial sequencing, a human melanoma protein (34 kDa, pI 6.4) as lipocortin I. To our knowledge, this protein was not previously known to be associated with melanoma cells. The identity of the protein was confirmed by two-dimensional immunoblot analysis. High-energy collision-induced dissociation analysis revealed the sequence and acetylation of the N-terminal tryptic peptide and an acrylamide-modified cysteine in another tryptic peptide. Thus, knowledge concerning both the primary structure and covalent modifications of proteins isolated from two-dimensional gels can be obtained directly by this approach, which is applicable to a broad range of biological problems. Images Fig. 1 Fig. 5 PMID:8446611

  12. Body mass index, gestational weight gain and fatty acid concentrations during pregnancy: the Generation R Study.

    PubMed

    Vidakovic, Aleksandra Jelena; Jaddoe, Vincent W V; Gishti, Olta; Felix, Janine F; Williams, Michelle A; Hofman, Albert; Demmelmair, Hans; Koletzko, Berthold; Tiemeier, Henning; Gaillard, Romy

    2015-11-01

    Obesity during pregnancy may be correlated with an adverse nutritional status affecting pregnancy and offspring outcomes. We examined the associations of prepregnancy body mass index and gestational weight gain with plasma fatty acid concentrations in mid-pregnancy. This study was embedded in a population-based prospective cohort study among 5636 women. We obtained prepregnancy body mass index and maximum weight gain during pregnancy by questionnaires. We measured concentrations of saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), n-3 polyunsaturated fatty acid (n-3 PUFA) and n-6 polyunsaturated fatty acid (n-6 PUFA) at a median gestational age of 20.5 (95% range 17.1-24.9) weeks. We used multivariate linear regression models. As compared to normal weight women, obese women had higher total SFA concentrations [difference: 0.10 standard deviation (SD) (95% Confidence Interval (CI) 0, 0.19)] and lower total n-3 PUFA concentrations [difference: - 0.11 SD (95% CI - 0.20, - 0.02)]. As compared to women with sufficient gestational weight gain, those with excessive gestational weight gain had higher SFA concentrations [difference: 0.16 SD (95% CI 0.08, 0.25)], MUFA concentrations [difference: 0.16 SD (95% CI 0.08, 0.24)] and n-6 PUFA concentrations [difference: 0.12 SD (95% CI 0.04, 0.21)]. These results were not materially affected by adjustment for maternal characteristics. Our results suggest that obesity and excessive weight gain during pregnancy are associated with an adverse fatty acids profile. Further studies are needed to assess causality and direction of the observed associations.

  13. Effect of dimethylamine on the gas phase sulfuric acid concentration measured by Chemical Ionization Mass Spectrometry.

    PubMed

    Rondo, L; Ehrhart, S; Kürten, A; Adamov, A; Bianchi, F; Breitenlechner, M; Duplissy, J; Franchin, A; Dommen, J; Donahue, N M; Dunne, E M; Flagan, R C; Hakala, J; Hansel, A; Keskinen, H; Kim, J; Jokinen, T; Lehtipalo, K; Leiminger, M; Praplan, A; Riccobono, F; Rissanen, M P; Sarnela, N; Schobesberger, S; Simon, M; Sipilä, M; Smith, J N; Tomé, A; Tröstl, J; Tsagkogeorgas, G; Vaattovaara, P; Winkler, P M; Williamson, C; Wimmer, D; Baltensperger, U; Kirkby, J; Kulmala, M; Petäjä, T; Worsnop, D R; Curtius, J

    2016-03-27

    Sulfuric acid is widely recognized as a very important substance driving atmospheric aerosol nucleation. Based on quantum chemical calculations it has been suggested that the quantitative detection of gas phase sulfuric acid (H2SO4) by use of Chemical Ionization Mass Spectrometry (CIMS) could be biased in the presence of gas phase amines such as dimethylamine (DMA). An experiment (CLOUD7 campaign) was set up at the CLOUD (Cosmics Leaving OUtdoor Droplets) chamber to investigate the quantitative detection of H2SO4 in the presence of dimethylamine by CIMS at atmospherically relevant concentrations. For the first time in the CLOUD experiment, the monomer sulfuric acid concentration was measured by a CIMS and by two CI-APi-TOF (Chemical Ionization-Atmospheric Pressure interface-Time Of Flight) mass spectrometers. In addition, neutral sulfuric acid clusters were measured with the CI-APi-TOFs. The CLOUD7 measurements show that in the presence of dimethylamine (<5 to 70 pptv) the sulfuric acid monomer measured by the CIMS represents only a fraction of the total H2SO4, contained in the monomer and the clusters that is available for particle growth. Although it was found that the addition of dimethylamine dramatically changes the H2SO4 cluster distribution compared to binary (H2SO4-H2O) conditions, the CIMS detection efficiency does not seem to depend substantially on whether an individual H2SO4 monomer is clustered with a DMA molecule. The experimental observations are supported by numerical simulations based on A Self-contained Atmospheric chemistry coDe coupled with a molecular process model (Sulfuric Acid Water NUCleation) operated in the kinetic limit.

  14. Effect of dimethylamine on the gas phase sulfuric acid concentration measured by Chemical Ionization Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Rondo, L.; Ehrhart, S.; Kürten, A.; Adamov, A.; Bianchi, F.; Breitenlechner, M.; Duplissy, J.; Franchin, A.; Dommen, J.; Donahue, N. M.; Dunne, E. M.; Flagan, R. C.; Hakala, J.; Hansel, A.; Keskinen, H.; Kim, J.; Jokinen, T.; Lehtipalo, K.; Leiminger, M.; Praplan, A.; Riccobono, F.; Rissanen, M. P.; Sarnela, N.; Schobesberger, S.; Simon, M.; Sipilä, M.; Smith, J. N.; Tomé, A.; Tröstl, J.; Tsagkogeorgas, G.; Vaattovaara, P.; Winkler, P. M.; Williamson, C.; Wimmer, D.; Baltensperger, U.; Kirkby, J.; Kulmala, M.; Petäjä, T.; Worsnop, D. R.; Curtius, J.

    2016-03-01

    Sulfuric acid is widely recognized as a very important substance driving atmospheric aerosol nucleation. Based on quantum chemical calculations it has been suggested that the quantitative detection of gas phase sulfuric acid (H2SO4) by use of Chemical Ionization Mass Spectrometry (CIMS) could be biased in the presence of gas phase amines such as dimethylamine (DMA). An experiment (CLOUD7 campaign) was set up at the CLOUD (Cosmics Leaving OUtdoor Droplets) chamber to investigate the quantitative detection of H2SO4 in the presence of dimethylamine by CIMS at atmospherically relevant concentrations. For the first time in the CLOUD experiment, the monomer sulfuric acid concentration was measured by a CIMS and by two CI-APi-TOF (Chemical Ionization-Atmospheric Pressure interface-Time Of Flight) mass spectrometers. In addition, neutral sulfuric acid clusters were measured with the CI-APi-TOFs. The CLOUD7 measurements show that in the presence of dimethylamine (<5 to 70 pptv) the sulfuric acid monomer measured by the CIMS represents only a fraction of the total H2SO4, contained in the monomer and the clusters that is available for particle growth. Although it was found that the addition of dimethylamine dramatically changes the H2SO4 cluster distribution compared to binary (H2SO4-H2O) conditions, the CIMS detection efficiency does not seem to depend substantially on whether an individual H2SO4 monomer is clustered with a DMA molecule. The experimental observations are supported by numerical simulations based on A Self-contained Atmospheric chemistry coDe coupled with a molecular process model (Sulfuric Acid Water NUCleation) operated in the kinetic limit.

  15. Effect of dimethylamine on the gas phase sulfuric acid concentration measured by Chemical Ionization Mass Spectrometry

    PubMed Central

    Ehrhart, S.; Kürten, A.; Adamov, A.; Bianchi, F.; Breitenlechner, M.; Duplissy, J.; Franchin, A.; Dommen, J.; Donahue, N. M.; Dunne, E. M.; Flagan, R. C.; Hakala, J.; Hansel, A.; Keskinen, H.; Kim, J.; Jokinen, T.; Lehtipalo, K.; Leiminger, M.; Praplan, A.; Riccobono, F.; Rissanen, M. P.; Sarnela, N.; Schobesberger, S.; Simon, M.; Sipilä, M.; Smith, J. N.; Tomé, A.; Tröstl, J.; Tsagkogeorgas, G.; Vaattovaara, P.; Winkler, P. M.; Williamson, C.; Wimmer, D.; Baltensperger, U.; Kirkby, J.; Kulmala, M.; Petäjä, T.; Worsnop, D. R.; Curtius, J.

    2016-01-01

    Abstract Sulfuric acid is widely recognized as a very important substance driving atmospheric aerosol nucleation. Based on quantum chemical calculations it has been suggested that the quantitative detection of gas phase sulfuric acid (H2SO4) by use of Chemical Ionization Mass Spectrometry (CIMS) could be biased in the presence of gas phase amines such as dimethylamine (DMA). An experiment (CLOUD7 campaign) was set up at the CLOUD (Cosmics Leaving OUtdoor Droplets) chamber to investigate the quantitative detection of H2SO4 in the presence of dimethylamine by CIMS at atmospherically relevant concentrations. For the first time in the CLOUD experiment, the monomer sulfuric acid concentration was measured by a CIMS and by two CI‐APi‐TOF (Chemical Ionization‐Atmospheric Pressure interface‐Time Of Flight) mass spectrometers. In addition, neutral sulfuric acid clusters were measured with the CI‐APi‐TOFs. The CLOUD7 measurements show that in the presence of dimethylamine (<5 to 70 pptv) the sulfuric acid monomer measured by the CIMS represents only a fraction of the total H2SO4, contained in the monomer and the clusters that is available for particle growth. Although it was found that the addition of dimethylamine dramatically changes the H2SO4 cluster distribution compared to binary (H2SO4‐H2O) conditions, the CIMS detection efficiency does not seem to depend substantially on whether an individual H2SO4 monomer is clustered with a DMA molecule. The experimental observations are supported by numerical simulations based on A Self‐contained Atmospheric chemistry coDe coupled with a molecular process model (Sulfuric Acid Water NUCleation) operated in the kinetic limit. PMID:27610289

  16. Mass

    SciTech Connect

    Quigg, Chris

    2007-12-05

    In the classical physics we inherited from Isaac Newton, mass does not arise, it simply is. The mass of a classical object is the sum of the masses of its parts. Albert Einstein showed that the mass of a body is a measure of its energy content, inviting us to consider the origins of mass. The protons we accelerate at Fermilab are prime examples of Einsteinian matter: nearly all of their mass arises from stored energy. Missing mass led to the discovery of the noble gases, and a new form of missing mass leads us to the notion of dark matter. Starting with a brief guided tour of the meanings of mass, the colloquium will explore the multiple origins of mass. We will see how far we have come toward understanding mass, and survey the issues that guide our research today.

  17. Mass loss from pre-main-sequence accretion disks. I - The accelerating wind of FU Orionis

    NASA Technical Reports Server (NTRS)

    Calvet, Nuria; Hartmann, Lee; Kenyon, Scott J.

    1993-01-01

    We present evidence that the wind of the pre-main-sequence object FU Orionis arises from the surface of the luminous accretion disk. A disk wind model calculated assuming radiative equilibrium explains the differential behavior of the observed asymmetric absorption-line profiles. The model predicts that strong lines should be asymmetric and blueshifted, while weak lines should be symmetric and double-peaked due to disk rotation, in agreement with observations. We propose that many blueshifted 'shell' absorption features are not produced in a true shell of material, but rather form in a differentially expanding wind that is rapidly rotating. The inference of rapid rotation supports the proposal that pre-main-sequence disk winds are rotationally driven.

  18. Method for the detection of specific nucleic acid sequences by polymerase nucleotide incorporation

    DOEpatents

    Castro, Alonso

    2004-06-01

    A method for rapid and efficient detection of a target DNA or RNA sequence is provided. A primer having a 3'-hydroxyl group at one end and having a sequence of nucleotides sufficiently homologous with an identifying sequence of nucleotides in the target DNA is selected. The primer is hybridized to the identifying sequence of nucleotides on the DNA or RNA sequence and a reporter molecule is synthesized on the target sequence by progressively binding complementary nucleotides to the primer, where the complementary nucleotides include nucleotides labeled with a fluorophore. Fluorescence emitted by fluorophores on single reporter molecules is detected to identify the target DNA or RNA sequence.

  19. De novo sequencing and characterization of a novel Bowman-Birk inhibitor from Lathyrus sativus L. seeds by electrospray mass spectrometry.

    PubMed

    Tamburino, Rachele; Severino, Valeria; Sandomenico, Annamaria; Ruvo, Menotti; Parente, Augusto; Chambery, Angela; Di Maro, Antimo

    2012-10-30

    Bowman-Birk serine protease inhibitors (BBIs) from legume seeds are small proteins showing a two-head structure with distinct reactive site loops, which inhibit two molecules of the same enzyme or two different proteases. Purification and characterization of new BBIs is of broad interest for understanding the basic molecular mechanisms underlying natural defence against the action of proteolytic enzymes. In this study, two novel acidic BBIs (LSI-1a and LSI-2a) were isolated from L. sativus seeds using classical biochemical techniques and characterized for their inhibitory activity. In addition, the N-terminal sequencing of LSI-1a was performed by Edman degradation up to residue 10 and the complete primary structure of the most abundant form (LSI-2a) was determined by using a combination of mass spectrometry approaches, including MALDI-TOF MS, tandem MS and Electron Transfer Dissociation coupled with Proton Transfer Reaction (ETD/PTR) top-down sequencing of N- and C-termini. Furthermore, the LSI-2a dimerization surface has also been investigated by a combination of gel filtration, electrophoretic techniques and homology modelling. Knowing the structure of small proteins inhibiting proteolytic enzymes is of general importance for understanding the defence mechanisms against degradation for their use in biological applications as well as for designing artificial inhibitors.

  20. Identification of tropomyosins as major allergens in antarctic krill and mantis shrimp and their amino acid sequence characteristics.

    PubMed

    Motoyama, Kanna; Suma, Yota; Ishizaki, Shoichiro; Nagashima, Yuji; Lu, Ying; Ushio, Hideki; Shiomi, Kazuo

    2008-01-01

    Tropomyosin represents a major allergen of decapod crustaceans such as shrimps and crabs, and its highly conserved amino acid sequence (>90% identity) is a molecular basis of the immunoglobulin E (IgE) cross-reactivity among decapods. At present, however, little information is available about allergens in edible crustaceans other than decapods. In this study, the major allergen in two species of edible crustaceans, Antarctic krill Euphausia superba and mantis shrimp Oratosquilla oratoria that are taxonomically distinct from decapods, was demonstrated to be tropomyosin by IgE-immunoblotting using patient sera. The cross-reactivity of the tropomyosins from both species with decapod tropomyosins was also confirmed by inhibition IgE immunoblotting. Sequences of the tropomyosins from both species were determined by complementary deoxyribonucleic acid cloning. The mantis shrimp tropomyosin has high sequence identity (>90% identity) with decapod tropomyosins, especially with fast-type tropomyosins. On the other hand, the Antarctic krill tropomyosin is characterized by diverse alterations in region 13-42, the amino acid sequence of which is highly conserved for decapod tropomyosins, and hence, it shares somewhat lower sequence identity (82.4-89.8% identity) with decapod tropomyosins than the mantis shrimp tropomyosin. Quantification by enzyme-linked immunosorbent assay revealed that Antarctic krill contains tropomyosin at almost the same level as decapods, suggesting that its allergenicity is equivalent to decapods. However, mantis shrimp was assumed to be substantially not allergenic because of the extremely low content of tropomyosin.

  1. Investigating Mass Transport Limitations on Xylan Hydrolysis During Dilute Acid Pretreatment of Poplar

    SciTech Connect

    Mittal, Ashutosh; Pilath, Heid M.; Parent, Yves; Chatterjee, Siddharth G.; Donohoe, Bryon S.; Yarbrough, John M.; Himmel, Michael E.; Nimlos, Mark R.; Johnson, David K.

    2014-04-28

    Mass transport limitations could be an impediment to achieving high sugar yields during biomass pretreatment and thus be a critical factor in the economics of biofuels production. The objective of this work was to study the mass transfer restrictions imposed by the structure of biomass on the hydrolysis of xylan during dilute acid pretreatment of biomass. Mass transfer effects were studied by pretreating poplar wood at particle sizes ranging from 10 micrometers to 10 mm. This work showed a significant reduction in the rate of xylan hydrolysis in poplar when compared to the intrinsic rate of hydrolysis for isolated xylan that is possible in the absence of mass transfer. In poplar samples we observed no significant difference in the rates of xylan hydrolysis over more than two orders of magnitude in particle size. It appears that no additional mass transport restrictions are introduced by increasing particle size from 10 micrometers to 10 mm. This work suggests that the rates of xylan hydrolysis in biomass particles are limited primarily by the diffusion of hydrolysis products out of plant cell walls. A mathematical description is presented to describe the kinetics of xylan hydrolysis that includes transport of the hydrolysis products through biomass into the bulk solution. The modeling results show that the effective diffusion coefficient of the hydrolysis products in the cell wall is several orders of magnitude smaller than typical values in other applications signifying the role of plant cell walls in offering resistance to diffusion of the hydrolysis products.

  2. 2,5-Dihydroxybenzoic acid solution in MALDI-MS: ageing and use for mass calibration.

    PubMed

    Teearu, Anu; Vahur, Signe; Haljasorg, Uku; Leito, Ivo; Haljasorg, Tõiv; Toom, Lauri

    2014-10-01

    2,5-Dihydroxybenzoic acid (DHB) is one of the most widely used and studied matrix compounds in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. However, the influence of ageing of the DHB solution on the MALDI mass spectra has not been yet systematically studied. In this work, the possible changes occurring in the acidified acetonitrile/water solution of the MALDI matrix compound DHB during 1-year usage period have been monitored with MALDI-Fourier transform ion cyclotron resonance mass spectrometer (MALDI-FT-ICR-MS) and attenuated total reflectance Fourier transform infrared (ATR-FT-IR) spectroscopy. No significant ageing products have been detected. The ability of the aged DHB solution to act as a MALDI matrix was tested with two materials widely used in art and conservation - bone glue (a proteinaceous material) and shellac resin (a resinous material) - and good results were obtained. A number of peaks in the mass spectra measured from the DHB solution were identified, which can be used for internal calibration of the mass axis.

  3. Human parainfluenza type 3 virus hemagglutinin-neuraminidase glycoprotein: nucleotide sequence of mRNA and limited amino acid sequence of the purified protein.

    PubMed Central

    Elango, N; Coligan, J E; Jambou, R C; Venkatesan, S

    1986-01-01

    The nucleotide sequence of mRNA for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 3 virus obtained from the corresponding cDNA clone had a single long open reading frame encoding a putative protein of 64,254 daltons consisting of 572 amino acids. The deduced protein sequence was confirmed by limited N-terminal amino acid microsequencing of CNBr cleavage fragments of native HN that was purified by immunoprecipitation. The HN protein is moderately hydrophobic and has four potential sites (Asn-X-Ser/Thr) of N-glycosylation in the C-terminal half of the molecule. It is devoid of both the N-terminal signal sequence and the C-terminal membrane anchorage domain characteristic of the hemagglutinin of influenza virus and the fusion (F0) protein of the paramyxoviruses. Instead, it has a single prominent hydrophobic region capable of membrane insertion beginning at 32 residues from the N terminus. This N-terminal membrane insertion is similar to that of influenza virus neuraminidase and the recently reported structures of HN proteins of Sendai virus and simian virus 5. Images PMID:3003381

  4. Simultaneous determination of acidic pesticides in vegetables and fruits by liquid chromatography--tandem mass spectrometry.

    PubMed

    Shida, Shizuka S; Nemoto, Satoru; Matsuda, Rieko

    2015-01-01

    A sensitive and efficient method has been developed for the simultaneous determination of 73 multi-class acidic pesticides, such as phenoxy acid and sulfonylurea herbicides, in vegetables and fruits. The sample preparation procedure was carefully optimized for the efficient removal of co-extracted matrix components. The method involves extraction of acidic pesticides with acetonitrile containing hydrochloric acid, removal of water from crude extract by salting out, and sequential cleanup by octadecylsilyl silica gel and silica gel columns. For samples containing high amounts of pigments, such as spinach, additional cleanup using a graphitized carbon column was performed prior to liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Recovery tests were performed for five times for each sample of cabbage, spinach, potato, eggplant, orange, and apple fortified at 0.01 mg kg-1. Out of the 73 tested pesticides, 70 for cabbage, 67 for spinach, 69 for potato, 67 for eggplant, 64 for orange, and 70 for apple were within the range of 70-120%, with relative standard deviations below 25%. Nitenpyram and pyrasulfotole showed low recoveries for all the samples tested, probably due to low recoveries from silica gel column. The developed method effectively removed co-extracted matrix components and was highly selective, with no interfering peaks found in the chromatograms of blank samples. The overall results indicate that the developed method is suitable for the quantitative analysis of acidic pesticide residues in vegetables and fruits.

  5. Fish protein hydrolysate elevates plasma bile acids and reduces visceral adipose tissue mass in rats.

    PubMed

    Liaset, Bjørn; Madsen, Lise; Hao, Qin; Criales, Gabriel; Mellgren, Gunnar; Marschall, Hanns-Ulrich; Hallenborg, Philip; Espe, Marit; Frøyland, Livar; Kristiansen, Karsten

    2009-04-01

    Conjugation of bile acids (BAs) to the amino acids taurine or glycine increases their solubility and promotes liver BA secretion. Supplementing diets with taurine or glycine modulates BA metabolism and enhances fecal BA excretion in rats. However, it is still unclear whether dietary proteins varying in taurine and glycine contents alter BA metabolism, and thereby modulate the recently discovered systemic effects of BAs. Here we show that rats fed a diet containing saithe fish protein hydrolysate (saithe FPH), rich in taurine and glycine, for 26 days had markedly elevated fasting plasma BA levels relative to rats fed soy protein or casein. Concomitantly, the saithe FPH fed rats had reduced liver lipids and fasting plasma TAG levels. Furthermore, visceral adipose tissue mass was reduced and expression of genes involved in fatty acid oxidation and energy expenditure was induced in perirenal/retroperitoneal adipose tissues of rats fed saithe FPH. Our results provide the first evidence that dietary protein sources with different amino acid compositions can modulate the level of plasma bile acids and our data suggest potential novel mechanisms by which dietary protein sources can affect energy metabolism.

  6. The Quenched Mass Portion of Star-forming Galaxies and the Origin of the Star Formation Sequence Slope

    NASA Astrophysics Data System (ADS)

    Pan, Zhizheng; Zheng, Xianzhong; Kong, Xu

    2017-01-01

    Observationally, a massive disk galaxy can harbor a bulge component that is comparably inactive as a quiescent galaxy. It has been speculated that the quenched component contained in star-forming galaxies (SFGs) is the reason why the star formation main sequence (MS) has a shallow slope at high masses. In this paper, we present a toy model to quantify the quenched mass portion of SFGs (fQ) at fixed stellar mass (M*) and to reconcile the MS slopes in both the low- and the high-mass regimes. In this model, each SFG is composed of a star-forming plus a quenched component. The mass of the star-forming component (MSF) correlates with the star formation rate (SFR) following a relation SFR \\propto {M}{SF}{α {SF}}, where αSF ∼ 1.0. The quenched component contributes to the stellar mass but not to the SFR. It is thus possible to quantify fQ based on the departure of the observed MS slope α from αSF. Adopting the redshift-dependent MS slope reported by Whitaker et al., we explore the evolution of the {f}{{Q}}{--}{M}* relations over z = [0.5, 2.5]. We find that Milky Way-like SFGs (with {M}* ≈ {10}10.7 {M}ȯ ) typically have an fQ = 30%–40% at z ∼ 2.25, whereas this value rapidly rises up to 70%–80% at z ∼ 0.75. The origin of an α ∼ 1.0 MS slope seen in the low-mass regime is also discussed. We argue for a scenario in which the majority of low-mass SFGs stay in a “steady-stage” star formation phase. In this phase, the SFR is mainly regulated by stellar feedback and not significantly influenced by the quenching mechanisms, thus remaining roughly constant over cosmic time. This scenario successfully produces an α ∼ 1.0 MS slope, as well as the observed MS evolution from z = 2.5 to z = 0 at low masses.

  7. Sequence dependent N-terminal rearrangement and degradation of peptide nucleic acid (PNA) in aqueous solution

    NASA Technical Reports Server (NTRS)

    Eriksson, M.; Christensen, L.; Schmidt, J.; Haaima, G.; Orgel, L.; Nielsen, P. E.

    1998-01-01

    The stability of the PNA (peptide nucleic acid) thymine monomer inverted question markN-[2-(thymin-1-ylacetyl)]-N-(2-aminoaminoethyl)glycine inverted question mark and those of various PNA oligomers (5-8-mers) have been measured at room temperature (20 degrees C) as a function of pH. The thymine monomer undergoes N-acyl transfer rearrangement with a half-life of 34 days at pH 11 as analyzed by 1H NMR; and two reactions, the N-acyl transfer and a sequential degradation, are found by HPLC analysis to occur at measurable rates for the oligomers at pH 9 or above. Dependent on the amino-terminal sequence, half-lives of 350 h to 163 days were found at pH 9. At pH 12 the half-lives ranged from 1.5 h to 21 days. The results are discussed in terms of PNA as a gene therapeutic drug as well as a possible prebiotic genetic material.

  8. Solubility Challenges in High Concentration Monoclonal Antibody Formulations: Relationship with Amino Acid Sequence and Intermolecular Interactions.

    PubMed

    Pindrus, Mariya; Shire, Steven J; Kelley, Robert F; Demeule, Barthélemy; Wong, Rita; Xu, Yiren; Yadav, Sandeep

    2015-11-02

    The purpose of this work was to elucidate the molecular interactions leading to monoclonal antibody self-association and precipitation and utilize biophysical measurements to predict solubility behavior at high protein concentration. Two monoclonal antibodies (mAb-G and mAb-R) binding to overlapping epitopes were investigated. Precipitation of mAb-G solutions was most prominent at high ionic strength conditions and demonstrated strong dependence on ionic strength, as well as slight dependence on solution pH. At similar conditions no precipitation was observed for mAb-R solutions. Intermolecular interactions (interaction parameter, kD) related well with high concentration solubility behavior of both antibodies. Upon increasing buffer ionic strength, interactions of mAb-R tended to weaken, while those of mAb-G became more attractive. To investigate the role of amino acid sequence on precipitation behavior, mutants were designed by substituting the CDR of mAb-R into the mAb-G framework (GM-1) or deleting two hydrophobic residues in the CDR of mAb-G (GM-2). No precipitation was observed at high ionic strength for either mutant. The molecular interactions of mutants were similar in magnitude to those of mAb-R. The results suggest that presence of hydrophobic groups in the CDR of mAb-G may be responsible for compromising its solubility at high ionic strength conditions since deleting these residues mitigated the solubility issue.

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

    PubMed Central

    Schwartz, Russell; Istrail, Sorin; King, Jonathan

    2001-01-01

    Patterns of hydrophobic and hydrophilic residues play a major role in protein folding and function. Long, predominantly hydrophobic strings of 20–22 amino acids each are associated with transmembrane helices and have been used to identify such sequences. Much less attention has been paid to hydrophobic sequences within globular proteins. In prior work on computer simulations of the competition between on-pathway folding and off-pathway aggregate formation, we found that long sequences of consecutive hydrophobic residues promoted aggregation within the model, even controlling for overall hydrophobic content. We report here on an analysis of the frequencies of different lengths of contiguous blocks of hydrophobic residues in a database of amino acid sequences of proteins of known structure. Sequences of three or more consecutive hydrophobic residues are found to be significantly less common in actual globular proteins than would be predicted if residues were selected independently. The result may reflect selection against long blocks of hydrophobic residues within globular proteins relative to what would be expected if residue hydrophobicities were independent of those of nearby residues in the sequence. PMID:11316883

  10. Negative ion electrospray of bromo- and chloroacetic acids and an evaluation of exact mass measurements with a bench-top time-of-flight mass spectrometer

    PubMed

    Debre; Budde; Song

    2000-09-01

    The negative ion electrospray mass spectra of six bromo- and chloroacetic acids were measured using two different electrospray interfaces and single quadrupole and bench-top time-of-flight mass spectrometers. With each acid at 50 microg/mL in aqueous methanol at pH 10, the anions observed included deprotonated molecules, adducts, and fragment ions. With each acid at 100 ng/mL in aqueous acetonitrile at pH 10, mainly deprotonated molecules are observed. The exact m/z measuring capability of the time-of-flight mass spectrometer was evaluated to assess the potential for the determination of the individual acids in mixtures without an on-line separation. Mean measurement errors were nearly always less than +/- 9 ppm and the majority were less than +/- 5 ppm. Potential interferences by substances having similar exact masses and the ability to form anions in aqueous solutions were evaluated. The estimated detection limits of the five regulated haloacetic acids in drinking water, without a sample preconcentration step, are in the range of 24-86 ng/mL, which is within about a factor of 10 of the levels required for routine monitoring of the acids. Actual drinking water samples were not analyzed pending the development of slightly more sensitive techniques and quantitative analytical procedures.

  11. High speed DNA sequence analysis by matrix-assisted laser desorption mass spectrometry. Final report for period February 15, 1991 - February 14, 2001

    SciTech Connect

    Smith, Lloyd M.

    2001-04-17

    This grant had as its focus (i) to develop chemistry and enzymology to permit the enzymatic synthesis of 2' fluoro modified Sanger sequencing reactions, which would be resistant to fragmentation during MALDI process, (ii) to develop rapid MALDI analyses of DNA sequence polymorphisms using Peptide Nucleic Acid (PNA) DNA analogs in conjunction with solid phase chemistry (iii) to study the fundamental mechanisms occurring in the MALDI analysis of nucleic acids.

  12. Profiling of soil fatty acids using comprehensive two-dimensional gas chromatography with mass spectrometry detection.

    PubMed

    Zeng, Annie Xu; Chin, Sung-Tong; Patti, Antonio; Marriott, Philip J

    2013-11-22

    Profiling of phospholipid fatty acids (PLFA) represents a challenging goal for distinguishing the diversity of microbial communities and biomass in the complex and heterogeneous soil ecosystem. Comprehensive two-dimensional gas chromatography (GC×GC) coupled with simultaneous flame ionisation and mass spectrometry detection was applied as a culture-independent method for PLFA profiling of microbial classification in forest soil. A number of column sets were evaluated for the GC×GC separation of fatty acid methyl esters (FAME). Due to better isomeric separation and compound patterns on the 2D contour plot, an apolar-polar column combination was selected for soil microbial PLFA characterisation. A comprehensive view of PLFA composition with carbon chain length varying from 12 to 20 was observed in forest soil samples, with the commonly reported bacterial FAME of iso-/anteiso-, methyl-branched-, cyclopropyl-, and hydroxyl-substituted FA identified by their mass spectral and retention time according to authentic standards. Notably, some uncommon oxygenated FAME were found in high abundance and were further characterised by GC×GC coupled with high resolution mass spectrometry. This tentatively revealed geometric pairs of methyl 9,10-epoxyoctadecanoate isomers.

  13. Tandem mass spectrometry of poly(methacrylic Acid) oligomers produced by negative mode electrospray ionization.

    PubMed

    Giordanengo, Rémi; Viel, Stéphane; Allard-Breton, Béatrice; Thévand, André; Charles, Laurence

    2009-01-01

    Dissociation of small poly(methyl acrylic acid) (PMAA) anions produced by electrospray was characterized by tandem mass spectrometry. Upon collisional activation, singly, and doubly deprotonated PMAA oligomers were shown to fragment via two major reactions, dehydration and decarboxylation. The elimination of a water molecule would occur between two consecutive acid groups in a charged-remote mechanism, giving rise to cyclic anhydrides, and was shown to proceed as many times as pairs of neutral pendant groups were available. As a result, the number of dehydration steps, together with the abundance of the fragment ions produced after the release of all water molecules, revealed the polymerization degree of the molecule in the particular case of doubly charged oligomers. For singly deprotonated molecules, the exact number of MAA units could be reached from the number of carbon dioxide molecules successively eliminated from the fully dehydrated precursor ions. In contrast to dehydration, decarboxylation reactions would proceed via a charge-induced mechanism. The proposed dissociation mechanisms are consistent with results commonly reported in thermal degradation studies of poly(acrylic acid) resins and were supported by accurate mass measurements. These fragmentation rules were successfully applied to characterize a polymeric impurity detected in the tested PMAA sample.

  14. Mass spectrometric and theoretical studies on the decarboxylation of the anionic lithium complexes of the doubly deprotonated dicarboxylic acids

    NASA Astrophysics Data System (ADS)

    Zhang, Xiang

    2012-05-01

    On the basis of the tandem mass spectrometry (MS/MS) technique and density functional theory (DFT) calculations, we have studied the decarboxylation reactions of several anionic lithium complexes of the doubly deprotonated dicarboxylic acids, which include succinic acid, L-malic acid, L-mercaptosuccinic acid, L-aspartic acid and oxaloacetic acid, etc. Tandem mass spectrometry experiments indicate that the decarboxylation reactions of these complexes in the gas phase can all take place. DFT calculations show that the α-substituted groups in the dicarboxylic acids, such as sbnd OH, sbnd NH2 and sbnd SH can advance the decarboxylation of the corresponding anionic lithium complexes. Meanwhile, the decarboxylation generally happens at the carboxylate group that is away from the substituent. This opinion is also supported by the bond angle analyses of the carboxylate groups.

  15. [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.

  16. Toward a Chemical Evolutionary Sequence in High-Mass Star Formation

    NASA Astrophysics Data System (ADS)

    Gerner, Thomas; Beuther, Henrik; Semenov, Dmitry; Linz, Hendrik; Vasyunina, Tatiana; Henning, Thomas

    Understanding the chemical evolution of young (high-mass) star-forming regions is a central topic in star formation research. The chemistry plays two main roles here: to study the evolution from simple to complex molecules, and to investigate the underlying physical processes. With these aims in mind, we observed a diverse sample of 60 high-mass star-forming regions in different evolutionary stages. In the early phase, quiescent Infrared Dark Clouds (IRDCs), consisting of cold and dense gas and dust, and emitting mainly at (sub-)millimeter wavelength, are formed. In the next phase, the so called High Mass Protostellar Objects (HMPOs) form, which host a central, likely still accreting protostar and already show emission at mid-infrared wavelengths. In the Hot Molecular Core phase (HMC) the central source heats up the surrounding environment, evaporating molecular-rich ices, which gives rise to a rich chemistry leading to complex molecules such as long carbon chains. Finally the UV-radiation from the embedded protostars ionizes the gas around and forms an Ultra Compact HII (UCHII) region. In these objects many of the previously formed complex molecules are not longer detected as they got destroyed by the ionizing radiation. For our observations, we used the IRAM 30m telescope with the total bandpass of 16 GHz and good spectral resolution (˜0.3/0.7 km/s at 1/3 mm). We derived their large-scale chemical abundances, assuming LTE and optically thin emission. To set these results into context, we model the chemical evolution in such environments with a state-of-the-art chemical model. This enables us to put constraints on the chemical evolution, the age and parameters such as the temperature and the density of the molecular clouds.

  17. Isolation, characterization, and amino acid sequences of auracyanins, blue copper proteins from the green photosynthetic bacterium Chloroflexus aurantiacus

    NASA Technical Reports Server (NTRS)

    McManus, J. D.; Brune, D. C.; Han, J.; Sanders-Loehr, J.; Meyer, T. E.; Cusanovich, M. A.; Tollin, G.; Blankenship, R. E.

    1992-01-01

    Three small blue copper proteins designated auracyanin A, auracyanin B-1, and auracyanin B-2 have been isolated from the thermophilic green gliding photosynthetic bacterium Chloroflexus aurantiacus. All three auracyanins are peripheral membrane proteins. Auracyanin A was described previously (Trost, J. T., McManus, J. D., Freeman, J. C., Ramakrishna, B. L., and Blankenship, R. E. (1988) Biochemistry 27, 7858-7863) and is not glycosylated. The two B forms are glycoproteins and have almost identical properties to each other, but are distinct from the A form. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis apparent monomer molecular masses are 14 (A), 18 (B-2), and 22 (B-1) kDa. The amino acid sequences of the B forms are presented. All three proteins have similar absorbance, circular dichroism, and resonance Raman spectra, but the electron spin resonance signals are quite different. Laser flash photolysis kinetic analysis of the reactions of the three forms of auracyanin with lumiflavin and flavin mononucleotide semiquinones indicates that the site of electron transfer is negatively charged and has an accessibility similar to that found in other blue copper proteins. Copper analysis indicates that all three proteins contain 1 mol of copper per mol of protein. All three auracyanins exhibit a midpoint redox potential of +240 mV. Light-induced absorbance changes and electron spin resonance signals suggest that auracyanin A may play a role in photosynthetic electron transfer. Kinetic data indicate that all three proteins can donate electrons to cytochrome c-554, the electron donor to the photosynthetic reaction center.

  18. Primary structure of three cationic peptides from porcine neutrophils. Sequence determination by the combined usage of electrospray ionization mass spectrometry and Edman degradation.

    PubMed

    Mirgorodskaya, O A; Shevchenko, A A; Abdalla, K O; Chernushevich, I V; Egorov, T A; Musoliamov, A X; Kokryakov, V N; Shamova, O V

    1993-09-20

    The primary structure of three major cationic peptides from porcine neutrophils has been determined. The sequencing was made by the combined use of electrospray ionization mass spectrometry and Edman degradation. The determined sequences unambiguously show that these peptides can not be considered as defensins.

  19. Complete Genomic Sequence and Mass Spectrometric Analysis of Highly Diverse, Atypical Bacillus thuringiensis phage 0305φ8-36

    PubMed Central

    Thomas, Julie A.; Hardies, Stephen C.; Rolando, Mandy; Hayes, Shirley J.; Lieman, Karen; Carroll, Christopher A.; Weintraub, Susan T.; Serwer, Philip

    2007-01-01

    To investigate the apparent genomic complexity of long-genome bacteriophages, we have sequenced the 218,948-bp genome (6479 bp terminal repeat), and identified the virion proteins (55), of Bacillus thuringiensis bacteriophage 0305φ8-36. Phage 0305φ8-36 is an atypical myovirus with three large curly tail fibers. An accurate mode of DNA pyrosequencing was used to sequence the genome and mass spectrometry was used to accomplish the comprehensive virion protein survey. Advanced informatic techniques were used to identify classical morphogenesis genes. The 0305φ8-36 genes were highly diverged; 19% of 247 closely spaced genes have similarity to proteins with known functions. Genes for virion-associated, apparently fibrous proteins in a new class were found, in addition to strong candidates for the curly fiber genes. Phage 0305φ8-36 has twice the virion protein coding sequence of T4. Based on its genomic isolation, 0305φ8-36 is a resource for future studies of vertical gene transmission. PMID:17673272

  20. Classifying nucleic acid sub-sequences as introns or exons using genetic programming

    SciTech Connect

    Handley, S.

    1995-12-31

    An evolutionary computation technique, genetic programming, created programs that classify messenger RNA sequences into one of two classes: (1) the sequence is expressed as (part of) a protein (an exon), or (2) not expressed as protein (an intron).

  1. 5S ribosomal ribonucleic acid sequences in Bacteroides and Fusobacterium: evolutionary relationships within these genera and among eubacteria in general

    NASA Technical Reports Server (NTRS)

    Van den Eynde, H.; De Baere, R.; Shah, H. N.; Gharbia, S. E.; Fox, G. E.; Michalik, J.; Van de Peer, Y.; De Wachter, R.

    1989-01-01

    The 5S ribosomal ribonucleic acid (rRNA) sequences were determined for Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides capillosus, Bacteroides veroralis, Porphyromonas gingivalis, Anaerorhabdus furcosus, Fusobacterium nucleatum, Fusobacterium mortiferum, and Fusobacterium varium. A dendrogram constructed by a clustering algorithm from these sequences, which were aligned with all other hitherto known eubacterial 5S rRNA sequences, showed differences as well as similarities with respect to results derived from 16S rRNA analyses. In the 5S rRNA dendrogram, Bacteroides clustered together with Cytophaga and Fusobacterium, as in 16S rRNA analyses. Intraphylum relationships deduced from 5S rRNAs suggested that Bacteroides is specifically related to Cytophaga rather than to Fusobacterium, as was suggested by 16S rRNA analyses. Previous taxonomic considerations concerning the genus Bacteroides, based on biochemical and physiological data, were confirmed by the 5S rRNA sequence analysis.

  2. Clickable Nucleic Acids: Sequence-Controlled Periodic Copolymer/Oligomer Synthesis by Orthogonal Thiol-X Reactions.

    PubMed

    Xi, Weixian; Pattanayak, Sankha; Wang, Chen; Fairbanks, Benjamin; Gong, Tao; Wagner, Justine; Kloxin, Christopher J; Bowman, Christopher N

    2015-11-23

    Synthetic polymer approaches generally lack the ability to control the primary sequence, with sequence control referred to as the holy grail. Two click chemistry reactions were now combined to form nucleobase-containing sequence-controlled polymers in simple polymerization reactions. Two distinct approaches are used to form these click nucleic acid (CNA) polymers. These approaches employ thiol-ene and thiol-Michael reactions to form homopolymers of a single nucleobase (e.g., poly(A)n ) or homopolymers of specific repeating nucleobase sequences (e.g., poly(ATC)n). Furthermore, the incorporation of monofunctional thiol-terminated polymers into the polymerization system enables the preparation of multiblock copolymers in a single reaction vessel; the length of the diblock copolymer can be tuned by the stoichiometric ratio and/or the monomer functionality. These polymers are also used for organogel formation where complementary CNA-based polymers form reversible crosslinks.

  3. PHOTOMETRIC DETERMINATION OF THE MASS ACCRETION RATES OF PRE-MAIN-SEQUENCE STARS. IV. RECENT STAR FORMATION IN NGC 602

    SciTech Connect

    De Marchi, Guido; Beccari, Giacomo; Panagia, Nino E-mail: gbeccari@eso.org

    2013-09-20

    We have studied the young stellar populations in NGC 602, in the Small Magellanic Cloud, using a novel method that we have developed to combine Hubble Space Telescope photometry in the V, I, and Hα bands. We have identified about 300 pre-main-sequence (PMS) stars, all of which are still undergoing active mass accretion, and have determined their physical parameters (effective temperature, luminosity, age, mass, and mass accretion rate). Our analysis shows that star formation has been present in this field over the last 60 Myr. In addition, we can recognize at least two clear, distinct, and prominent episodes in the recent past: one about 2 Myr ago, but still ongoing in regions of higher nebulosity, and one (or more) older than 30 Myr, encompassing both stars dispersed in the field and two smaller clusters located about 100'' north of the center of NGC 602. The relative locations of younger and older PMS stars do not imply a causal effect or triggering of one generation on the other. The strength of the two episodes appears to be comparable, but the episodes occurring more than 30 Myr ago might have been even stronger than the current one. We have investigated the evolution of the mass accretion rate, M-dot{sub acc}, as a function of the stellar parameters finding that log M-dot{sub acc}≅-0.6 log t + log m + c, where t is the age of the star, m is its mass, and c is a decreasing function of the metallicity.

  4. Enzymatic properties, crystallization, and deduced amino acid sequence of an alkaline endoglucanase from Bacillus circulans.

    PubMed

    Hakamada, Yoshihiro; Endo, Keiji; Takizawa, Shuichi; Kobayashi, Tohru; Shirai, Tsuyoshi; Yamane, Takashi; Ito, Susumu

    2002-04-15

    A high-isoelectric-point (pI), alkaline endo-1,4-beta-glucanase (Egl-257) of Bacillus circulans KSM-N257 was purified to homogeneity and crystallized. The purified enzyme hydrolyzed carboxymethyl cellulose (CMC) with optima of pH 8.5 and 55 degrees C. The molecular mass was 43 kDa, and the pI was pH 9.3. The structural gene contained a single open reading frame of 1221 bp, corresponding to 407 amino acids (aa), including a 30-aa signal peptide (377 aa and 41,680 Da for the mature enzyme). Egl-257 hydrolyzed lichenan and showed 76.3% aa identity to a lichenase from B. circulans WL-12 belonging to glycosyl hydrolase family 8 but did not hydrolyze laminarin, curdran, and xylan at all. This indicates that Egl-257 is a true endo-1,4-beta-glucanase. However, this enzyme was not active on p-nitrophenyl beta-D-cellotrioside and p-nitrophenyl beta-D-cellotetraoside. It was crystallized by the hanging-drop vapor-diffusion method with phosphate plus CdCl(2) as precipitant. Pyramid-like crystals were formed, and they diffracted X-rays beyond 2.2 A resolution. It belongs to the space group P2(1)2(1)2(1) with unit cell parameters of a=62.5 A, b=71.7 A, and c=88.6 A.

  5. Webinar Presentation: Suspect Screening of Environmental Organic Acids in Human Serum Using High-resolution Mass Spectrometry (HRMS)

    EPA Pesticide Factsheets

    This presentation, Suspect Screening of Environmental Organic Acids in Human Serum Using High-resolution Mass Spectrometry (HRMS), was given at the NIEHS/EPA Children's Centers 2016 Webinar Series: Exposome held on May 11, 2016.

  6. Sequence Comparison and Phylogeny of Nucleotide Sequence of Coat Protein and Nucleic Acid Binding Protein of a Distinct Isolate of Shallot virus X from India.

    PubMed

    Majumder, S; Baranwal, V K

    2011-06-01

    Shallot virus X (ShVX), a type species in the genus Allexivirus of the family Alfaflexiviridae has been associated with shallot plants in India and other shallot growing countries like Russia, Germany, Netherland, and New Zealand. Coat protein (CP) and nucleic acid binding protein (NB) region of the virus was obtained by reverse transcriptase polymerase chain reaction from scales leaves of shallot bulbs. The partial cDNA contained two open reading frames encoding proteins of molecular weights of 28.66 and 14.18 kDa belonging to Flexi_CP super-family and viral NB super-family, respectively. The percent identity and phylogenetic analysis of amino acid sequences of CP and NB region of the virus associated with shallot indicated that it was a distinct isolate of ShVX.

  7. Differentiation of acetic acid bacteria based on sequence analysis of 16S-23S rRNA gene internal transcribed spacer sequences.

    PubMed

    González, Angel; Mas, Albert

    2011-06-30

    The 16S-23S gene internal transcribed spacer sequence of sixty-four strains belonging to different acetic acid bacteria genera were analyzed, and phylogenetic trees were generated for each genera. The topologies of the different trees were in accordance with the 16S rRNA gene trees, although the similarity percentages obtained between the species was shown to be much lower. These values suggest the usefulness of including the 16S-23S gene internal transcribed spacer region as a part of the polyphasic approach required for the further classification of acetic acid bacteria. Furthermore, the region could be a good target for primer and probe design. It has also been validated for use in the identification of unknown samples of this bacterial group from wine vinegar and fruit condiments.

  8. Uncertainty analysis on simple mass balance model to calculate critical loads for soil acidity.

    PubMed

    Li, Harbin; McNulty, Steven G

    2007-10-01

    Simple mass balance equations (SMBE) of critical acid loads (CAL) in forest soil were developed to assess potential risks of air pollutants to ecosystems. However, to apply SMBE reliably at large scales, SMBE must be tested for adequacy and uncertainty. Our goal was to provide a detailed analysis of uncertainty in SMBE so that sound strategies for scaling up CAL estimates to the national scale could be developed. Specifically, we wanted to quantify CAL uncertainty under natural variability in 17 model parameters, and determine their relative contributions in predicting CAL. Results indicated that uncertainty in CAL came primarily from components of base cation weathering (BC(w); 49%) and acid neutralizing capacity (46%), whereas the most critical parameters were BC(w) base rate (62%), soil depth (20%), and soil temperature (11%). Thus, improvements in estimates of these factors are crucial to reducing uncertainty and successfully scaling up SMBE for national assessments of CAL.

  9. Influence of eicosapentaenoic acid supplementation on lean body mass in cancer cachexia.

    PubMed

    Murphy, R A; Yeung, E; Mazurak, V C; Mourtzakis, M

    2011-11-08

    Cancer cachexia is characterised by a progressive loss of muscle, resulting in functional impairment and shorter survival. Eicosapentaenoic acid, an n-3 polyunsaturated fatty acid found in fish, has been studied for its role as an anti-cachexia therapy. Initial results of eicosapentaenoic supplementation in advanced cancer were promising with improvements in lean body mass (LBM), appetite and quality of life. However, subsequent larger phase III clinical trials reported minimal benefits of supplementation. Recently, several studies have used different study designs, which may provide insight on the effectiveness of eicosapentaenoic in cancer cachexia and also on potential sources of divergent results in previous trials. This review examines the potential benefit of eicosapentaenoic supplementation on LBM and discusses limitations with current studies to identify methods which may aid in progressing the research of future clinical trials.

  10. Simultaneous determination of gibberellic acid, indole-3-acetic acid and abscisic acid in wheat extracts by solid-phase extraction and liquid chromatography-electrospray tandem mass spectrometry.

    PubMed

    Hou, Shengjie; Zhu, Jiang; Ding, Mingyu; Lv, Guohua

    2008-08-15

    A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous determination of three representative phytohormones in plant samples: gibberellic acid (GA(3)), indole-3-acetic acid (IAA) and abscisic acid (ABA). A solid-phase extraction (SPE) pretreatment method was used to concentrate and purify the three phytohormones of different groups from plant samples. The separation was carried out on a C(18) reversed-phase column, using methanol/water containing 0.2% formic acid (50:50, v/v) as the isocratic mobile phase at the flow-rate of 1.0 mL min(-1), and the three phytohormones were eluted within 7 min. A linear ion trap mass spectrometer equipped with electrospray ionization source was operated in negative ion mode. Selective reaction monitoring (SRM) was employed for quantitative measurement. The SRM transitions monitored were as 345-->239, 301 for GA(3), 174-->130 for IAA and 263-->153, 219 for ABA. Good linearities were found within the ranges of 5-200 microg mL(-1) for IAA and 0.005-10 microg mL(-1) for ABA and GA(3). Their detection limits based on a signal-to-noise ratio of three were 0.005 microg mL(-1), 2.2 microg mL(-1) and 0.003 microg mL(-1) for GA(3), IAA and ABA, respectively. Good recoveries from 95.5% to 102.4% for the three phytohormones were obtained. The results demonstrated that the SPE-LC-MS/MS method developed is highly effective for analyzing trace amounts of the three phytohormones in plant samples.

  11. Characterization of Hydroxyphthioceranoic and Phthioceranoic Acids by Charge-Switch Derivatization and CID Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Hsu, Fong-Fu

    2016-04-01

    Hydroxyphthioceranoic (HPA) and phthioceranoic (PA) acids are polymethylated long chain fatty acids with and without a hydroxyl group attached to the carbon next to the terminal methyl-branched carbon distal to the carboxylic end of the long-chain fatty acid, respectively. They are the major components of the sulfolipids found in the cell wall of Mycobacterium tuberculosis (M. tuberculosis) strain H37Rv. In this report, I describe CID linear ion-trap MSn mass spectrometric approaches combined with charge-reverse derivatization strategy toward characterization of these complex lipids, which were released from sulfolipids by alkaline hydrolysis and sequentially derivatized to the N-(4-aminomethylphenyl) pyridinium (AMPP) derivatives. This method affords complete characterization of HPA and PA, including the location of the hydroxyl group and the multiple methyl side chains. The study also led to the notion that the hydroxyphthioceranoic acid in sulfolipid consists of two (for hC24) to 12 (for hC52) methyl branches, and among them 2,4,6,8,10,12,14,16-octamethyl-17-hydroxydotriacontanoic acid (hC40) is the most prominent, while phthioceranoic acids are the minor constituents. These results confirm our previous findings that sulfolipid II, a family of homologous 2-stearoyl(palmitoyl)-3,6,6'-tris(hydroxyphthioceranoy1)-trehalose 2'-sulfates is the predominant species, and sulfolipid I, a family of homologous 2-stearoyl(palmitoyl)-3-phthioceranoyl-6,6'-bis(hydroxyphthioceranoy1)-trehalose 2'-sulfates is the minor species in the cell wall of M. tuberculosis.

  12. Calibration and intercomparison of acetic acid measurements using proton transfer reaction mass spectrometry (PTR-MS)

    USGS Publications Warehouse

    Haase, K.B.; Keene, W.C.; Pszenny, A.A.P.; Mayne, H.R.; Talbot, R.W.; Sive, B.C.

    2012-01-01

    Acetic acid is one of the most abundant organic acids in the ambient atmosphere, with maximum mixing ratios reaching into the tens of parts per billion by volume (ppbv) range. The identities and associated magnitudes of the major sources and sinks for acetic acid are poorly characterized, due in part to the limitation in available measurement techniques. This paper demonstrates that Proton Transfer Reaction Mass Spectrometry (PTR-MS) can reliably quantify acetic acid vapor in ambient air. Three different PTR-MS configurations were calibrated at low ppbv mixing ratios using permeation tubes, which yielded calibration factors between 7.0 and 10.9 normalized counts per second per ppbv (ncps ppbv−1) at a drift tube field strength of 132 townsend (Td). Detection limits ranged from 0.06 to 0.32 ppbv with dwell times of 5 s. These calibration factors showed negligible humidity dependence. Using the experimentally determined calibration factors, PTR-MS measurements of acetic acid during the International Consortium for Atmospheric Research on Transport and Transformation (ICARTT) campaign were validated against results obtained using Mist Chambers coupled with Ion Chromatography (MC/IC). An orthogonal least squares linear regression of paired data yielded a slope of 1.14 ± 0.06 (2σ), an intercept of 0.049 ± 20 (2σ) ppbv, and an R2 of 0.78. The median mixing ratio of acetic acid on Appledore Island, ME during the ICARTT campaign was 0.530 ± 0.025 ppbv with a minimum of 0.075 ± 0.004 ppbv, and a maximum of 3.555 ± 0.171 ppbv.

  13. Role of protein and amino acids in promoting lean mass accretion with resistance exercise and attenuating lean mass loss during energy deficit in humans.

    PubMed

    Churchward-Venne, Tyler A; Murphy, Caoileann H; Longland, Thomas M; Phillips, Stuart M

    2013-08-01

    Amino acids are major nutrient regulators of muscle protein turnover. After protein ingestion, hyperaminoacidemia stimulates increased rates of skeletal muscle protein synthesis, suppresses muscle protein breakdown, and promotes net muscle protein accretion for several hours. These acute observations form the basis for strategized protein intake to promote lean mass accretion, or prevent lean mass loss over the long term. However, factors such as protein dose, protein source, and timing of intake are important in mediating the anabolic effects of amino acids on skeletal muscle and must be considered within the context of evaluating the reported efficacy of long-term studies investigating protein supplementation as part of a dietary strategy to promote lean mass accretion and/or prevent lean mass loss. Current research suggests that dietary protein supplementation can augment resistance exercise-mediated gains in skeletal muscle mass and strength and can preserve skeletal muscle mass during periods of diet-induced energy restriction. Perhaps less appreciated, protein supplementation can augment resistance training-mediated gains in skeletal muscle mass even in individuals habitually consuming 'adequate' (i.e., >0.8 g kg⁻¹ day⁻¹) protein. Additionally, overfeeding energy with moderate to high-protein intake (15-25 % protein or 1.8-3.0 g kg⁻¹ day⁻¹) is associated with lean, but not fat mass accretion, when compared to overfeeding energy with low protein intake (5 % protein or ~0.68 g kg⁻¹ day⁻¹). Amino acids represent primary nutrient regulators of skeletal muscle anabolism, capable of enhancing lean mass accretion with resistance exercise and attenuating the loss of lean mass during periods of energy deficit, although factors such as protein dose, protein source, and timing of intake are likely important in mediating these effects.

  14. Analysis of the citric acid cycle intermediates using gas chromatography-mass spectrometry.

    PubMed

    Kombu, Rajan S; Brunengraber, Henri; Puchowicz, Michelle A

    2011-01-01

    Researchers view analysis of the citric acid cycle (CAC) intermediates as a metabolomic approach to identifying unexpected correlations between apparently related and unrelated pathways of metabolism. Relationships of the CAC intermediates, as measured by their concentrations and relative ratios, offer useful information to understanding interrelationships between the CAC and metabolic pathways under various physiological and pathological conditions. This chapter presents a relatively simple method that is sensitive for simultaneously measuring concentrations of CAC intermediates (relative and absolute) and other related intermediates of energy metabolism using gas chromatography-mass spectrometry.

  15. Measurements of mass attenuation coefficient, effective atomic number and electron density of some amino acids

    NASA Astrophysics Data System (ADS)

    Kore, Prashant S.; Pawar, Pravina P.

    2014-05-01

    The mass attenuation coefficients of some amino acids, such as DL-aspartic acid-LR(C4H7NO4), L-glutamine (C4H10N2O3), creatine monohydrate LR(C4H9N3O2H2O), creatinine hydrochloride (C4H7N3O·HCl) L-asparagine monohydrate(C4H9N3O2H2O), L-methionine LR(C5H11NO2S), were measured at 122, 356, 511, 662, 1170, 1275 and 1330 keV photon energies using a well-collimated narrow beam good geometry set-up. The gamma-rays were detected using NaI (Tl) scintillation detection system with a resolution of 0.101785 at 662 keV. The attenuation coefficient data were then used to obtain the effective atomic numbers (Zeff), and effective electron densities (Neff) of amino acids. It was observed that the effective atomic number (Zeff) and effective electron densities (Neff) initially decrease and tend to be almost constant as a function of gamma-ray energy. Zeff and Neff experimental values showed good agreement with the theoretical values with less than 1% error for amino acids.

  16. Electrospray ionization mass spectrometric observation of ligand exchange of zinc pyrithione with amino acids.

    PubMed

    Moriwaki, Hiroshi; Okabayashi, Masanori; Watanabe, Takehiro; Kawasaki, Hideya; Arakawa, Ryuichi

    2009-07-01

    Zinc pyrithione (ZnPT) is widely used as an antidandruff or antifouling reagent. However, this compound is considered toxic, such as the teratogenic effect, to aquatic lives, and it is important to clarify the mechanism of its toxicity. In this study, the interactions between ZnPT and amino acids were observed using electrospray ionization mass spectrometry (ESI-MS) in order to obtain information on the activity of ZnPT within the living body. The ZnPT complex ([ZnPT-ligand+Amino acid]+), in which the ligand of ZnPT was exchanged by the amino acid, was detected in ZnPT solutions mixed with one of 20 amino acids by ESI-MS. Histidine and cysteine, in particular, showed a high reactivity with ZnPT, while serine and glycine showed a low reactivity. The complexes of ZnPT and a peptide were also observed by the ESI-MS measurement of the solution containing ZnPT with the peptide. These results would be useful to understand the mechanism of ZnPT toxicities to living creatures.

  17. A rapid method for manual or automated purification of fluorescently labeled nucleic acids for sequencing, genotyping, and microarrays.

    PubMed

    Springer, Amy L; Booth, Lisa R; Braid, Michael D; Houde, Christiane M; Hughes, Karin A; Kaiser, Robert J; Pedrak, Casandra; Spicer, Douglas A; Stolyar, Sergey

    2003-03-01

    Fluorescent dyes provide specific, sensitive, and multiplexed detection of nucleic acids. To maximize sensitivity, fluorescently labeled reaction products (e.g., cycle sequencing or primer extension products) must be purified away from residual dye-labeled precursors. Successful high-throughput analyses require that this purification be reliable, rapid, and amenable to automation. Common methods for purifying reaction products involve several steps and require processes that are not easily automated. Prolinx, Inc. has devel oped RapXtract superparamagnetic separation technology affording rapid and easy-to-perform methods that yield high-quality product and are easily automated. The technology uses superparamagnetic particles that specifically remove unincorporated dye-labeled precursors. These particles are efficiently pelleted in the presence of a magnetic field, making them ideal for purification because of the rapid separations that they allow. RapXtract-purified sequencing reactions yield data with good signal and high Phred quality scores, and they work with various sequencing dye chemistries, including BigDye and near-infrared fluorescence IRDyes. RapXtract technology can also be used to purify dye primer sequencing reactions, primer extension reactions for genotyping analysis, and nucleic acid labeling reactions for microarray hybridization. The ease of use and versatility of RapXtract technology makes it a good choice for manual or automated purification of fluorescently labeled nucleic acids.

  18. Gene structure and amino acid sequence of Latimeria chalumnae (coelacanth) myelin DM20: phylogenetic relation of the fish.

    PubMed

    Tohyama, Y; Kasama-Yoshida, H; Sakuma, M; Kobayashi, Y; Cao, Y; Hasegawa, M; Kojima, H; Tamai, Y; Tanokura, M; Kurihara, T

    1999-07-01

    The structure of Latimeria chalumnae (coelacanth) proteolipid protein/DM20 gene excluding exon 1 was determined, and the amino acid sequence of Latimeria DM20 corresponding to exons 2-7 was deduced. The nucleotide sequence of exon 3 suggests that only DM20 isoform is expressed in Latimeria. The structure of proteolipid protein/DM20 gene is well preserved among human, dog, mouse, and Latimeria. Southern blot analysis indicates that Latimeria DM20 gene is a single-copy gene. When the amino acid sequences of DM20 were compared among various species, Latimeria was more similar to tetrapods than other fishes including lungfish, confirming the previous finding by immunoreactivity (Waehneldt and Malotka 1989 J. Neurochem. 52:1941-1943). However, when phylogenetic trees were constructed from the DM20 sequences, lungfish was clearly the closest to tetrapods. Latimeria was situated outside of lungfish by the maximum likelihood method. The apparent similarity of Latimeria DM20 to tetrapod proteolipid protein/DM20 is explained by the slow amino acid substitution rate of Latimeria DM20.

  19. Measuring the mass of a pre-main sequence binary star through the orbit of TWA5A

    SciTech Connect

    Konopacky, Q; Ghez, A; Duchene, G; McCabe, C; Macintosh, B

    2007-01-18

    We present the results of a five year monitoring campaign of the close binary TWA 5Aab in the TW Hydrae association, using speckle and adaptive optics on the W.M. Keck 10 m telescopes. These measurements were taken as part of our ongoing monitoring of pre-main sequence (PMS) binaries in an effort to increase the number of dynamically determined PMS masses and thereby calibrate the theoretical PMS evolutionary tracks. Our observations have allowed us to obtain the first determination of this system's astrometric orbit. We find an orbital period of 5.94 {+-} 0.09 years and a semi-major axis of 0.''066 {+-} 0.''005. Combining these results with a kinematic distance, we calculate a total mass of 0.71 {+-} 0.14 M{sub {circle_dot}} (D/44 pc){sup 3}. for this system. This mass measurement, as well as the estimated age of this system, are consistent to within 2{sigma} of all theoretical models considered. In this analysis, we properly account for correlated uncertainties, and show that while these correlations are generally ignored, they increase the formal uncertainties by up to a factor of five and therefore are important to incorporate. With only a few more years of observation, this type of measurement will allow the theoretical models to be distinguished.

  20. Peptides Labeled with Pyridinium Salts for Sensitive Detection and Sequencing by Electrospray Tandem Mass Spectrometry

    PubMed Central

    Waliczek, Mateusz; Kijewska, Monika; Rudowska, Magdalena; Setner, Bartosz; Stefanowicz, Piotr; Szewczuk, Zbigniew

    2016-01-01

    Mass spectrometric analysis of trace amounts of peptides may be problematic due to the insufficient ionization efficiency resulting in limited sensitivity. One of the possible ways to overcome this problem is the application of ionization enhancers. Herein we developed new ionization markers based on 2,4,6-triphenylpyridinium and 2,4,6-trimethylpyridinium salts. Using of inexpensive and commercially available pyrylium salt allows selective derivatization of primary amino groups, especially those sterically unhindered, such as ε-amino group of lysine. The 2,4,6-triphenylpyridinium modified peptides generate in MS/MS experiments an abundant protonated 2,4,6-triphenylpyridinium ion. This fragment is a promising reporter ion for the multiple reactions monitoring (MRM) analysis. In addition, the fixed positive charge of the pyridinium group enhances the ionization efficiency. Other advantages of the proposed ionization enhancers are the simplicity of derivatization of peptides and the possibility of convenient incorporation of isotopic labels into derivatized peptides. PMID:27892962

  1. E/S0 GALAXIES ON THE BLUE COLOR-STELLAR MASS SEQUENCE AT z = 0: FADING MERGERS OR FUTURE SPIRALS?

    SciTech Connect

    Kannappan, Sheila J.; Guie, Jocelly M.; Baker, Andrew J. E-mail: jocelly@mail.utexas.edu

    2009-08-15

    We identify a population of morphologically defined E/S0 galaxies lying on the locus of late-type galaxies in color-stellar mass space - the 'blue sequence' -at the present epoch. Using three samples (from the Nearby Field Galaxy Survey or NFGS, a merged HyperLeda/Sloan Digital Sky Survey/Two Micron All Sky Survey catalog, and the NYU Value-Added Galaxy Catalog), we analyze blue-sequence E/S0s with stellar masses {approx}>10{sup 8} M {sub sun}, arguing that individual objects may be evolving either up toward the red sequence or down into the blue sequence. Blue-sequence E/S0 galaxies become more common with decreasing stellar mass, comprising {approx}<2% of E/S0s near the 'shutdown mass' M{sub s} {approx} 1-2 x 10{sup 11} M {sub sun}, increasing to {approx}>5% near the 'bimodality mass' M{sub b} {approx} 3 x 10{sup 10} M {sub sun}, and sharply rising to {approx}> 20%-30% below the 'threshold mass' M{sub t} {approx} 4-6 x 10{sup 9} M {sub sun}, down to our completeness analysis limit at {approx}10{sup 9} M {sub sun}. The strong emergence of blue-sequence E/S0s below M{sub t} coincides with a previously reported global increase in mean atomic gas fractions below M{sub t} for galaxies of all types on both sequences, suggesting that the availability of cold gas may be basic to blue-sequence E/S0s' existence. Environmental analysis reveals that many sub-M{sub b} blue-sequence E/S0s reside in low-to-intermediate density environments. Thus, the bulk of the population we analyze appears distinct from the generally lower-mass cluster dE population; S0 morphologies with a range of bulge sizes are typical. In mass-radius and mass-{sigma} scaling relations, blue-sequence E/S0s are more similar to red-sequence E/S0s than to late-type galaxies, but they represent a transitional class. While some of them, especially in the high-mass range from M{sub b} to M{sub s} , resemble major-merger remnants that will likely fade onto the red sequence, most blue-sequence E/S0s below M{sub b

  2. Coronal Activity in Low-Mass Pre-Main Sequence Stars: NGC 2264

    NASA Technical Reports Server (NTRS)

    Tebbe, H. J.; Patten, B. M.

    2000-01-01

    We present the preliminary results of an analysis of ROSAT images in the region of the populous young (age approx. 3 Myr) star-forming region NGC 2264. The cluster was imaged with the ROSAT HRI in two sets of pointings -- one set near the central region of the cluster, centered on the star LW Mon, and the other set in the southern part of the cluster, centered near the star V428 Mon, just south of the Cone Nebula. In total 113 unique X-ray sources have been identified in the ROSAT images with signal-to-noise ratios greater than 3. The limiting luminosities (log Lx(ergs/sec)) for 3-sigma detections are estimated to be 30.18, 30.23, and 30.08 for the northern field, southern field, and overlap region between the two fields respectively. Extensive optical photometry, classification spectroscopy, and proper motions, obtained from recent ground-based surveys of this region, were used to identify the most likely optical counterpart to each X-ray source. Although most of our X-ray selected sample appears to be associated with NGC 2264 members, we find that the vast majority of the cluster membership was undetected in the ROSAT HRI survey. The X-ray cumulative luminosity function for solar-mass stars in NGC 2264 shows that most of the low-mass members probably have X-ray luminosities similar to those seen for the X-ray brightest members of older clusters such as IC 2391/IC 2602 (age approx. 50 Myr) and the Pleiades (age approx. 100 Myr). This research was funded in part by the SAO Summer Intern Program and NASA grant NAG5-8120.

  3. Purification of a marsupial insulin: amino-acid sequence of insulin from the eastern grey kangaroo Macropus giganteus.

    PubMed

    Treacy, G B; Shaw, D C; Griffiths, M E; Jeffrey, P D

    1989-03-24

    Insulin has been purified from kangaroo pancreas by acidic ethanol extraction, diethyl ether precipitation and gel filtration. The amino-acid sequence of this, the first marsupial insulin to be studied, is reported. It differs from human insulin by only four amino-acid substitutions, all in regions of the molecule previously known to be variable. However, it should be noted that one of these, asparagine for threonine at A8, has not been reported before. Computer comparisons of all 43 insulin sequences reported to date with kangaroo insulin show it to be most closely related to a group of mammalian insulins (dog, pig, cow, human) known to be of high biological potency. The measurement of blood glucose lowering in the rabbit by kangaroo insulin is consistent with this conclusion. Comparisons of amino-acid sequences of other proteins with their kangaroo counterparts show a greater difference, in line with the time of divergence of marsupials. The limited differences observed in insulin and cytochrome c suggest that their structures need to be closely conserved in order to maintain function.

  4. K-Pax2: Bayesian identification of cluster-defining amino acid positions in large sequence datasets

    PubMed Central

    Grad, Yonatan; Cobey, Sarah; Puranen, Juha Santeri; Corander, Jukka

    2015-01-01

    The recent growth in publicly available sequence data has introduced new opportunities for studying microbial evolution and spread. Because the pace of sequence accumulation tends to exceed the pace of experimental studies of protein function and the roles of individual amino acids, statistical tools to identify meaningful patterns in protein diversity are essential. Large sequence alignments from fast-evolving micro-organisms are particularly challenging to dissect using standard tools from phylogenetics and multivariate statistics because biologically relevant functional signals are easily masked by neutral variation and noise. To meet this need, a novel computational method is introduced that is easily executed in parallel using a cluster environment and can handle thousands of sequences with minimal subjective input from the user. The usefulness of this kind of machine learning is demonstrated by applying it to nearly 5000 haemagglutinin sequences of influenza A/H3N2.Antigenic and 3D structural mapping of the results show that the method can recover the major jumps in antigenic phenotype that occurred between 1968 and 2013 and identify specific amino acids associated with these changes. The method is expected to provide a useful tool to uncover patterns of protein evolution. PMID:28348810

  5. Complete genome sequence of Enterococcus mundtii QU 25, an efficient L-(+)-lactic acid-producing bacterium.

    PubMed

    Shiwa, Yuh; Yanase, Hiroaki; Hirose, Yuu; Satomi, Shohei; Araya-Kojima, Tomoko; Watanabe, Satoru; Zendo, Takeshi; Chibazakura, Taku; Shimizu-Kadota, Mariko; Yoshikawa, Hirofumi; Sonomoto, Kenji

    2014-08-01

    Enterococcus mundtii QU 25, a non-dairy bacterial strain of ovine faecal origin, can ferment both cellobiose and xylose to produce l-lactic acid. The use of this strain is highly desirable for economical l-lactate production from renewable biomass substrates. Genome sequence determination is necessary for the genetic improvement of this strain. We report the complete genome sequence of strain QU 25, primarily determined using Pacific Biosciences sequencing technology. The E. mundtii QU 25 genome comprises a 3 022 186-bp single circular chromosome (GC content, 38.6%) and five circular plasmids: pQY182, pQY082, pQY039, pQY024, and pQY003. In all, 2900 protein-coding sequences, 63 tRNA genes, and 6 rRNA operons were predicted in the QU 25 chromosome. Plasmid pQY024 harbours genes for mundticin production. We found that strain QU 25 produces a bacteriocin, suggesting that mundticin-encoded genes on plasmid pQY024 were functional. For lactic acid fermentation, two gene clusters were identified-one involved in the initial metabolism of xylose and uptake of pentose and the second containing genes for the pentose phosphate pathway and uptake of related sugars. This is the first complete genome sequence of an E. mundtii strain. The data provide insights into lactate production in this bacterium and its evolution among enterococci.

  6. Isolation, Amino Acid Sequence and Biological Activities of Novel Long-Chain Polyamine-Associated Peptide Toxins from the Sponge Axinyssa aculeata

    PubMed Central

    Matsunaga, Satoko; Jimbo, Mitsuru; Gill, Martin B.; Lash-Van Wyhe, L. Leanne; Murata, Michio; Nonomura, Ken’ichi; Swanson, Geoffrey T.

    2012-01-01

    A novel family of functionalized peptide toxins, aculeines (ACUs), was isolated from the marine sponge Axinyssa aculeate. ACUs are polypeptides with N-terminal residues that are modified by the addition of long-chain polyamines (LCPA). Aculeines were present in the sponge extract as a complex mixture with differing polyamine chain lengths and peptide structures. ACU-A and B, which were purified in this study, share a common polypeptide chain but differ in their N-terminal residue modifications. The amino acid sequence of the polypeptide portion of ACU-A and B was deduced from 3′ and 5′ RACE, and supported by Edman degradation and mass spectral analysis of peptide fragments. ACU induced convulsions upon intracerebroventricular (i.c.v.) injection in mice, and disrupted neuronal membrane integrity in electrophysiological assays. ACU also lysed erythrocytes with a potency that differed between animal species. Here we describe the isolation, amino acid sequence, and biological activity of this new group of cytotoxic sponge peptides. PMID:21830292

  7. The Human CCHC-type Zinc Finger Nucleic Acid-Binding Protein Binds G-Rich Elements in Target mRNA Coding Sequences and Promotes Translation.

    PubMed

    Benhalevy, Daniel; Gupta, Sanjay K; Danan, Charles H; Ghosal, Suman; Sun, Hong-Wei; Kazemier, Hinke G; Paeschke, Katrin; Hafner, Markus; Juranek, Stefan A

    2017-03-21

    The CCHC-type zinc finger nucleic acid-binding protein (CNBP/ZNF9) is conserved in eukaryotes and is essential for embryonic development in mammals. It has been implicated in transcriptional, as well as post-transcriptional, gene regulation; however, its nucleic acid ligands and molecular function remain elusive. Here, we use multiple systems-wide approaches to identify CNBP targets and function. We used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to identify 8,420 CNBP binding sites on 4,178 mRNAs. CNBP preferentially bound G-rich elements in the target mRNA coding sequences, most of which were previously found to form G-quadruplex and other stable structures in vitro. Functional analyses, including RNA sequencing, ribosome profiling, and quantitative mass spectrometry, revealed that CNBP binding did not influence target mRNA abundance but rather increased their translational efficiency. Considering that CNBP binding prevented G-quadruplex structure formation in vitro, we hypothesize that CNBP is supporting translation by resolving stable structures on mRNAs.

  8. Determinants of protein hyperthermostability: Purification and amino acid sequence of rubredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus and secondary structure of the zinc adduct by NMR

    SciTech Connect

    Blake, P.R.; Summers, M.F. ); Park, J.B.; Bryant, F.O.; Aono, Shigetoshi; Adams, M.W.W. ); Magnuson, J.K.; Eccleston, E.; Howard, J.B. )

    1991-11-12

    The purification, amino acid sequence, and two-dimensional {sup 1}H NMR results are reported for the rubredoxin (Rd) from the hyperthermophilic archaebacterium Pyrococcus furiosus, an organism that grows optimally at 100C. The molecular mass (5397 Da), iron content UV-vis spectrophotometric properties, and amino acid sequence are found to be typical of this class of redox protein. However, P. furiosus Rd is remarkably thermostable, being unaffected after incubation for 24 h at 95C. One- and two-dimensional {sup 1}H nuclear magnetic resonance spectra of the oxidized (Fe(III)Rd) and reduced (Fe(II)Rd) forms of P. furiosus Rd exhibited substantial paramagnetic line broadening, and this precluded detailed 3D structural studies. The apoprotein was not readily amenable to NMR studies due to apparent protein oxidation involving the free cysteine sulfhydryls. Secondary structural elements were determined from qualitative analysis of 2D Overhauser effect spectra. These structural elements are similar to those observed by X-ray crystallography for native Rd from the mesophile C. pasteurianum. From analysis of the secondary structure, potentially stabilizing electrostatic interactions involving the charged groups of residues Ala(1), Glu(14), and Glu(52) are proposed. These interactions, which are not present in rubredoxins from mesophilic organisms, may prevent the {beta}-sheet from unzipping' at elevated temperatures.

  9. Isolation and a partial amino acid sequence of insulin from the islet tissue of cod (Gadus callarias)

    PubMed Central

    Grant, P. T.; Reid, K. B. M.

    1968-01-01

    1. Insulin has been isolated by gel filtration and ion-exchange chromatography from extracts of the discrete islet tissue of cod. The final preparation yielded a single band on electrophoresis at two pH values. The biological potency was 11·5 international units/mg. in mouse-convulsion and other assay procedures. 2. Glycine and methionine were shown to be the N-terminal amino acids of the A and B chains respectively. An estimate of the molecular weight together with amino acid analyses indicated that cod insulin, like the bovine hormone, consists of 51 amino acid residues. In contrast, the amino acid composition differs markedly from bovine insulin. 3. Oxidation of insulin with performic acid yielded the A and B peptide chains, which were separated by ion-exchange chromatography. Sequence studies on smaller peptides isolated from enzymic digests or from dilute acetic acid hydrolysates of the two chains have established the sequential order of 14 of the 21 amino acid residues of the A chain and 25 of the 30 amino acid residues of the B chain. PMID:4866431

  10. Oxygen affinity and amino acid sequence of myoglobins from endothermic and ectothermic fish.

    PubMed

    Marcinek, D J; Bonaventura, J; Wittenberg, J B; Block, B A

    2001-04-01

    Myoglobin (Mb) buffers intracellular O2 and facilitates diffusion of O2 through the cell. These functions of Mb will be most effective when intracellular PO2 is near the partial pressure of oxygen at which Mb is half saturated (P50) of the molecule. We test the hypothesis that Mb oxygen affinity has evolved such that it is conserved when adjusted for body temperature among closely related animals. We measure oxygen P50s tonometrically and oxygen dissociation rate constants with stopped flow and generate amino acid sequence from cDNA of Mbs from fish with different body temperatures. P50s for the endothermic bluefin tuna, skipjack tuna, and blue marlin at 20 degrees C were 0.62 +/- 0.02, 0.59 +/- 0.01, 0.58 +/- 0.04 mmHg, respectively, and were significantly lower than those for ectothermic bonito (1.03 +/- 0.07 mmHg) and mackerel (1.39 +/- 0.03 mmHg). Because the oxygen affinity of Mb decreases with increasing temperature, the above differences in oxygen affinity between endothermic and ectothermic fish are reduced when adjusted for the in vivo muscle temperature of the animal. Oxygen dissociation rate constants at 20 degrees C for the endothermic species ranged from 34.1 to 49.3 s(-1), whereas those for mackerel and bonito were 102 and 62 s(-1), respectively. Correlated with the low oxygen affinity and fast dissociation kinetics of mackerel Mb is a substitution of alanine for proline that would likely result in a more flexible mackerel protein.

  11. Molecular cloning, nucleotide sequence, and abscisic acid induction of a suberization-associated highly anionic peroxidase.

    PubMed

    Roberts, E; Kolattukudy, P E

    1989-06-01

    A highly anionic peroxidase induced in suberizing cells was suggested to be the key enzyme involved in polymerization of phenolic monomers to generate the aromatic matrix of suberin. The enzyme encoded by a potato cDNA was found to be highly homologous to the anionic peroxidase induced in suberizing tomato fruit. A tomato genomic library was screened using the potato anionic peroxidase cDNA and one genomic clone was isolated that contained two tandemly oriented anionic peroxidase genes. These genes were sequenced and were 96% and 87% identical to the mRNA for potato anionic peroxidase. Both genes consist of three exons with the relative positions of their two introns being conserved between the two genes. Primer extension analysis showed that only one of the genes is expressed in the periderm of 3 day wound-healed tomato fruits. Southern blot analyses suggested that there are two copies each of the two highly homologous genes per haploid genome in both potato and tomato. Abscisic acid (ABA) induced the accumulation of the anionic peroxidase transcripts in potato and tomato callus tissues. Northern blots showed that peroxidase mRNA was detectable at 2 days and was maximal at 8 days after transfer of potato callus to solid agar media containing 10(-4) M ABA. The transcripts induced by ABA in both potato and tomato callus were identical in size to those induced in wound-healing potato tuber and tomato fruit. The anionic peroxidase peptide was detected in extracts of potato callus grown on the ABA-containing media by western blot analysis. The results support the suggestion that stimulation of suberization by ABA involves the induction of the highly anionic peroxidase.

  12. Complete genome sequence of the probiotic lactic acid bacterium Lactobacillus acidophilus NCFM

    PubMed Central

    Altermann, Eric; Russell, W. Michael; Azcarate-Peril, M. Andrea; Barrangou, Rodolphe; Buck, B. Logan; McAuliffe, Olivia; Souther, Nicole; Dobson, Alleson; Duong, Tri; Callanan, Michael; Lick, Sonja; Hamrick, Alice; Cano, Raul; Klaenhammer, Todd R.

    2005-01-01

    Lactobacillus acidophilus NCFM is a probiotic bacterium that has been produced commercially since 1972. The complete genome is 1,993,564 nt and devoid of plasmids. The average GC content is 34.71% with 1,864 predicted ORFs, of which 72.5% were functionally classified. Nine phage-related integrases were predicted, but no complete prophages were found. However, three unique regions designated as potential autonomous units (PAUs) were identified. These units resemble a unique structure and bear characteristics of both plasmids and phages. Analysis of the three PAUs revealed the presence of two R/M systems and a prophage maintenance system killer protein. A spacers interspersed direct repeat locus containing 32 nearly perfect 29-bp repeats was discovered and may provide a unique molecular signature for this organism. In silico analyses predicted 17 transposase genes and a chromosomal locus for lactacin B, a class II bacteriocin. Several mucus- and fibronectin-binding proteins, implicated in adhesion to human intestinal cells, were also identified. Gene clusters for transport of a diverse group of carbohydrates, including fructooligosaccharides and raffinose, were present and often accompanied by transcriptional regulators of the lacI family. For protein degradation and peptide utilization, the organism encoded 20 putative peptidases, homologs for PrtP and PrtM, and two complete oligopeptide transport systems. Nine two-component regulatory systems were predicted, some associated with determinants implicated in bacteriocin production and acid tolerance. Collectively, these features within the genome sequence of L. acidophilus are likely to contribute to the organisms' gastric survival and promote interactions with the intestinal mucosa and microbiota. PMID:15671160

  13. Quasispecies structure, cornerstone of hepatitis B virus infection: mass sequencing approach.

    PubMed

    Rodriguez-Frias, Francisco; Buti, Maria; Tabernero, David; Homs, Maria

    2013-11-07