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Sample records for acid sequence level

  1. 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.

  2. Composition for nucleic acid sequencing

    SciTech Connect

    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.

  3. 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.

  4. Chip-based sequencing nucleic acids

    SciTech Connect

    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.

  5. The phylogeny of acetic acid bacteria based on the partial sequences of 16S ribosomal RNA: the elevation of the subgenus Gluconoacetobacter to the generic level.

    PubMed

    Yamada, Y; Hoshino, K; Ishikawa, T

    1997-08-01

    Thirty-six strains of acetic acid bacteria classified in the genera Acetobacter, Gluconobacter, and Acidomonas were examined for their partial base sequences in positions 1220 through 1375, 156 bases, of 16S rRNA. The strains of the Q10-equipped Gluconobacter species examined were divided into two subgroups, which included the type strains of Gluconobacter oxydans, the type species of the genus Gluconobacter, and of a second species, Gluconobacter cerinus, respectively. The base differences numbered four between the two type strains. The strains of the Q9-equipped species examined classified in the type subgenus Acetobacter of the genus Acetobacter were not very distant phylogenetically from those of the genus Gluconobacter. The calculated number of base differences was 9-6 between the type strains of G. oxydans and G. cerinus and the type strains of Acetobacter aceti and Acetobacter pasteurianus. In contrast, the strains of the Q10-equipped species examined classified in the subgenus Gluconoacetobacter of the genus Acetobacter were very distant phylogenetically from those of the Acetobacter and Gluconobacter species mentioned above. The number of base differences was calculated to be 14-8. Furthermore, the strains of the methanol-assimilating, Q10-equipped species of the genus Acidomonas examined were located in phylogenetically isolated positions. The type strain of Acidomonas methanolica (identical to Acetobacter methanolicus), the type species of the genus Acidomonas, had 16-9 base differences. The data obtained here indicated that the members of the subgenus Gluconoacetobacter of the genus Acetobacter can be distinguished at the generic level. The new genus Gluconoacetobacter was proposed with the type species, Gluconoacetobacter liquefaciens, in recognition of the genus Acidomonas along with the genera Acetobacter and Gluconobacter in the classification of the acetic acid bacteria.

  6. Distinguishing proteins from arbitrary amino acid sequences.

    PubMed

    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

  7. 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.

  8. 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.

  9. Frontal Glutamate and γ-Aminobutyric Acid Levels and Their Associations With Mismatch Negativity and Digit Sequencing Task Performance in Schizophrenia

    PubMed Central

    Rowland, Laura M.; Summerfelt, Ann; Wijtenburg, S. Andrea; Du, Xiaoming; Chiappelli, Joshua J.; Krishna, Nithin; West, Jeffrey; Muellerklein, Florian; Kochunov, Peter; Hong, L. Elliot

    2016-01-01

    IMPORTANCE Auditory mismatch negativity (MMN) is a biomarker for schizophrenia thought to reflect glutamatergic N-methyl-d-aspartate receptor function and excitatory-inhibitory neurotransmission balance. However, the association of glutamate level with MMN has not been directly examined in patients with schizophrenia, to our knowledge. OBJECTIVE To investigate the contributions of glutamate and γ-aminobutyric acid (GABA) to MMN and digit sequencing task (DST) performance, an assessment of verbal working memory, in schizophrenia. DESIGN, SETTING, AND PARTICIPANTS Fifty-three control participants from the community and 45 persons with schizophrenia from outpatient clinics completed an electroencephalographic session for MMN, magnetic resonance spectroscopy for glutamate and GABA, and a DST. The study dates were July 2011 to May 2014, and the dates of our analysis were May 2014 to August 2015. MAIN OUTCOMES AND MEASURES Glutamate, GABA, the ratio of glutamine to glutamate, MMN amplitude, and DST. Structural equation modeling was used to test the effects of neurochemistry and MMN amplitude on DST performance. RESULTS The 45 persons with schizophrenia were a mean (SD) of 37.7 (12.8) years and the control participants were 37.1 (13.1) years. The schizophrenia group had a mean (SD) of 14.7 (12.1) years of illness. Mismatch negativity amplitude (F = 4.39, P = .04) and glutamate (F = 9.69, P = .002) were reduced in the schizophrenia group. Smaller MMN amplitude was significantly associated with lower GABA level (P = .008), lower glutamate level (P = .05), and higher ratio of glutamine to glutamate (P = .003). Reduced MMN amplitude was linked to poor verbal working memory in schizophrenia (P = .002). Modeling revealed that a proxy of glutamatergic function, indexed by the ratio of glutamine to glutamate, influenced a path from the ratio of glutamine to glutamate to MMN to verbal working memory (P = .38 [root-mean-square error of approximation, P < .001] by χ2 test

  10. Bovine Parathyroid Hormone: Amino Acid Sequence

    PubMed Central

    Brewer, H. Bryan; Ronan, Rosemary

    1970-01-01

    Bovine parathyroid hormone has been isolated in homogeneous form, and its complete amino acid sequence determined. The bovine hormone is a single chain, 84 amino acids long. It contains amino-terminal alanine, and carboxyl-terminal glutamine. The bovine parathyroid hormone is approximately three times the length of the newly discovered hormone, thyrocalcitonin, whose action is reciprocal to parathyroid hormone. Images PMID:5275384

  11. 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.

  12. 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.

  13. 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.

  14. Optimization of short amino acid sequences classifier

    NASA Astrophysics Data System (ADS)

    Barcz, Aleksy; Szymański, Zbigniew

    This article describes processing methods used for short amino acid sequences classification. The data processed are 9-symbols string representations of amino acid sequences, divided into 49 data sets - each one containing samples labeled as reacting or not with given enzyme. The goal of the classification is to determine for a single enzyme, whether an amino acid sequence would react with it or not. Each data set is processed separately. Feature selection is performed to reduce the number of dimensions for each data set. The method used for feature selection consists of two phases. During the first phase, significant positions are selected using Classification and Regression Trees. Afterwards, symbols appearing at the selected positions are substituted with numeric values of amino acid properties taken from the AAindex database. In the second phase the new set of features is reduced using a correlation-based ranking formula and Gram-Schmidt orthogonalization. Finally, the preprocessed data is used for training LS-SVM classifiers. SPDE, an evolutionary algorithm, is used to obtain optimal hyperparameters for the LS-SVM classifier, such as error penalty parameter C and kernel-specific hyperparameters. A simple score penalty is used to adapt the SPDE algorithm to the task of selecting classifiers with best performance measures values.

  15. 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.

  16. Molecular level understanding of resistance to nalidixic acid in Salmonella enteric serovar typhimurium associates with the S83F sequence type.

    PubMed

    Preethi, B; Ramanathan, K

    2016-01-01

    Nalidixic acid is an antibiotic drug used for treatment of Salmonellosis, a gastrointestinal infection. DNA gyrase subunit A (GyrA) of Salmonella typhimurium is the drug target for nalidixic acid. Resistance of GyrA to nalidixic acid, because of a point mutation in S. typhimurium, was recently reported. Substitution of Phe in place of Ser at locus 83 in GyrA of S. typhimurium has been experimentally associated with nalidixic acid resistance. Despite recent efforts, the mechanism of this resistance is not well understood. In this investigation we used computational techniques to address this shortcoming. Our results showed that contact with residue Arg 91 is certainly important for efficient binding of nalidixic acid to the target protein, and that mutation of this residue results in 180° rotation of the antibiotic in its binding pocket, around its own long axis. It is hoped these findings may enable development of new antibiotics against resistant forms of Salmonella. PMID:26329667

  17. 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...

  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. 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.

  20. On combining protein sequences and nucleic acid sequences in phylogenetic analysis: the homeobox protein case.

    PubMed

    Agosti, D; Jacobs, D; DeSalle, R

    1996-01-01

    Amino acid encoding genes contain character state information that may be useful for phylogenetic analysis on at least two levels. The nucleotide sequence and the translated amino acid sequences have both been employed separately as character states for cladistic studies of various taxa, including studies of the genealogy of genes in multigene families. In essence, amino acid sequences and nucleic acid sequences are two different ways of character coding the information in a gene. Silent positions in the nucleotide sequence (first or third positions in codons that can accrue change without changing the identity of the amino acid that the triplet codes for) may accrue change relatively rapidly and become saturated, losing the pattern of historical divergence. On the other hand, non-silent nucleotide alterations and their accompanying amino acid changes may evolve too slowly to reveal relationships among closely related taxa. In general, the dynamics of sequence change in silent and non-silent positions in protein coding genes result in homoplasy and lack of resolution, respectively. We suggest that the combination of nucleic acid and the translated amino acid coded character states into the same data matrix for phylogenetic analysis addresses some of the problems caused by the rapid change of silent nucleotide positions and overall slow rate of change of non-silent nucleotide positions and slowly changing amino acid positions. One major theoretical problem with this approach is the apparent non-independence of the two sources of characters. However, there are at least three possible outcomes when comparing protein coding nucleic acid sequences with their translated amino acids in a phylogenetic context on a codon by codon basis. First, the two character sets for a codon may be entirely congruent with respect to the information they convey about the relationships of a certain set of taxa. Second, one character set may display no information concerning a phylogenetic

  1. Predicting intrinsic disorder from amino acid sequence.

    PubMed

    Obradovic, Zoran; Peng, Kang; Vucetic, Slobodan; Radivojac, Predrag; Brown, Celeste J; Dunker, A Keith

    2003-01-01

    Blind predictions of intrinsic order and disorder were made on 42 proteins subsequently revealed to contain 9,044 ordered residues, 284 disordered residues in 26 segments of length 30 residues or less, and 281 disordered residues in 2 disordered segments of length greater than 30 residues. The accuracies of the six predictors used in this experiment ranged from 77% to 91% for the ordered regions and from 56% to 78% for the disordered segments. The average of the order and disorder predictions ranged from 73% to 77%. The prediction of disorder in the shorter segments was poor, from 25% to 66% correct, while the prediction of disorder in the longer segments was better, from 75% to 95% correct. Four of the predictors were composed of ensembles of neural networks. This enabled them to deal more efficiently with the large asymmetry in the training data through diversified sampling from the significantly larger ordered set and achieve better accuracy on ordered and long disordered regions. The exclusive use of long disordered regions for predictor training likely contributed to the disparity of the predictions on long versus short disordered regions, while averaging the output values over 61-residue windows to eliminate short predictions of order or disorder probably contributed to the even greater disparity for three of the predictors. This experiment supports the predictability of intrinsic disorder from amino acid sequence. PMID:14579347

  2. 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.

  3. 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.

  4. 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

  5. Nucleic acid sequence detection using multiplexed oligonucleotide PCR

    SciTech Connect

    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. 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.

  7. 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.

  8. From Artificial Amino Acids to Sequence-Defined Targeted Oligoaminoamides.

    PubMed

    Morys, Stephan; Wagner, Ernst; Lächelt, Ulrich

    2016-01-01

    Artificial oligoamino acids with appropriate protecting groups can be used for the sequential assembly of oligoaminoamides on solid-phase. With the help of these oligoamino acids multifunctional nucleic acid (NA) carriers can be designed and produced in highly defined topologies. Here we describe the synthesis of the artificial oligoamino acid Fmoc-Stp(Boc3)-OH, the subsequent assembly into sequence-defined oligomers and the formulation of tumor-targeted plasmid DNA (pDNA) polyplexes. PMID:27436323

  9. Segments of amino acid sequence similarity in beta-amylases.

    PubMed

    Friedberg, F; Rhodes, C

    1988-01-01

    In alpha-amylases from animals, plants and bacteria and in beta-amylases from plants and bacteria a number of segments exhibit amino acid sequence similarity specific to the alpha or to the beta type, respectively. In the case of the beta-amylases the similar sequence regions are extensive and they are disrupted only by short interspersed dissimilar regions. Close to the C terminus, however, no such sequence similarity exist. PMID:2464171

  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. The amino acid sequence of Staphylococcus aureus penicillinase.

    PubMed Central

    Ambler, R P

    1975-01-01

    The amino acid sequence of the penicillinase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) from Staphylococcus aureus strain PC1 was determined. The protein consists of a single polypeptide chain of 257 residues, and the sequence was determined by characterization of tryptic, chymotryptic, peptic and CNBr peptides, with some additional evidence from thermolysin and S. aureus proteinase peptides. A mistake in the preliminary report of the sequence is corrected; residues 113-116 are now thought to be -Lys-Lys-Val-Lys- rather than -Lys-Val-Lys-Lys-. Detailed evidence for the amino acid sequence has been deposited as Supplementary Publication SUP 50056 (91 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1975) 145, 5. PMID:1218078

  12. cDNA-derived amino acid sequences of myoglobins from nine species of whales and dolphins.

    PubMed

    Iwanami, Kentaro; Mita, Hajime; Yamamoto, Yasuhiko; Fujise, Yoshihiro; Yamada, Tadasu; Suzuki, Tomohiko

    2006-10-01

    We determined the myoglobin (Mb) cDNA sequences of nine cetaceans, of which six are the first reports of Mb sequences: sei whale (Balaenoptera borealis), Bryde's whale (Balaenoptera edeni), pygmy sperm whale (Kogia breviceps), Stejneger's beaked whale (Mesoplodon stejnegeri), Longman's beaked whale (Indopacetus pacificus), and melon-headed whale (Peponocephala electra), and three confirm the previously determined chemical amino acid sequences: sperm whale (Physeter macrocephalus), common minke whale (Balaenoptera acutorostrata) and pantropical spotted dolphin (Stenella attenuata). We found two types of Mb in the skeletal muscle of pantropical spotted dolphin: Mb I with the same amino acid sequence as that deposited in the protein database, and Mb II, which differs at two amino acid residues compared with Mb I. Using an alignment of the amino acid or cDNA sequences of cetacean Mb, we constructed a phylogenetic tree by the NJ method. Clustering of cetacean Mb amino acid and cDNA sequences essentially follows the classical taxonomy of cetaceans, suggesting that Mb sequence data is valid for classification of cetaceans at least to the family level. PMID:16962803

  13. The amino-acid sequence of kangaroo pancreatic ribonuclease.

    PubMed

    Gaastra, W; Welling, G W; Beintema, J J

    1978-05-01

    Red kangaroo (Macropus rufus) ribonuclease was isolated from pancreatic tissue by affinity chromatography. The amino acid sequence was determined by automatic sequencing of overlapping large fragments and by analysis of shorter peptides obtained by digestion with a number of proteolytic enzymes. The polypeptide chain consists of 122 amino acid residues. Compared to other ribonucleases, the N-terminal residue and residue 114 are deleted. In other pancreatic ribonucleases position 114 is occupied by a cis proline residue in an external loop at the surface of the molecule. Other remarkable substitutions are the presence of a tyrosine residue at position 123 instead of a serine which forms a hydrogen bond with the pyrimidine ring of a nucleotide substrate, and a number of hydrophobichydrophilic interchanges in the sequence 51-55, which forms part of an alpha-helix in bovine ribonuclease and exhibits few substitutions in the placental mammals. Kangaroo ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Val-Thr (62-64). The enzyme differs at about 35-40% of the positions from all other mammalian pancreatic ribonucleases sequenced to date, which is in agreement with the early divergence between the marsupials and the placental mammals. From fragmentary data a tentative sequence of red-necked wallaby (Macropus rufogriseus) pancreatic ribonuclease has been derived. Eight differences with the kangaroo sequence were found.

  14. Prebiotically plausible mechanisms increase compositional diversity of nucleic acid sequences

    PubMed Central

    Derr, Julien; Manapat, Michael L.; Rajamani, Sudha; Leu, Kevin; Xulvi-Brunet, Ramon; Joseph, Isaac; Nowak, Martin A.; Chen, Irene A.

    2012-01-01

    During the origin of life, the biological information of nucleic acid polymers must have increased to encode functional molecules (the RNA world). Ribozymes tend to be compositionally unbiased, as is the vast majority of possible sequence space. However, ribonucleotides vary greatly in synthetic yield, reactivity and degradation rate, and their non-enzymatic polymerization results in compositionally biased sequences. While natural selection could lead to complex sequences, molecules with some activity are required to begin this process. Was the emergence of compositionally diverse sequences a matter of chance, or could prebiotically plausible reactions counter chemical biases to increase the probability of finding a ribozyme? Our in silico simulations using a two-letter alphabet show that template-directed ligation and high concatenation rates counter compositional bias and shift the pool toward longer sequences, permitting greater exploration of sequence space and stable folding. We verified experimentally that unbiased DNA sequences are more efficient templates for ligation, thus increasing the compositional diversity of the pool. Our work suggests that prebiotically plausible chemical mechanisms of nucleic acid polymerization and ligation could predispose toward a diverse pool of longer, potentially structured molecules. Such mechanisms could have set the stage for the appearance of functional activity very early in the emergence of life. PMID:22319215

  15. The amino-acid sequence of kangaroo pancreatic ribonuclease.

    PubMed

    Gaastra, W; Welling, G W; Beintema, J J

    1978-05-01

    Red kangaroo (Macropus rufus) ribonuclease was isolated from pancreatic tissue by affinity chromatography. The amino acid sequence was determined by automatic sequencing of overlapping large fragments and by analysis of shorter peptides obtained by digestion with a number of proteolytic enzymes. The polypeptide chain consists of 122 amino acid residues. Compared to other ribonucleases, the N-terminal residue and residue 114 are deleted. In other pancreatic ribonucleases position 114 is occupied by a cis proline residue in an external loop at the surface of the molecule. Other remarkable substitutions are the presence of a tyrosine residue at position 123 instead of a serine which forms a hydrogen bond with the pyrimidine ring of a nucleotide substrate, and a number of hydrophobichydrophilic interchanges in the sequence 51-55, which forms part of an alpha-helix in bovine ribonuclease and exhibits few substitutions in the placental mammals. Kangaroo ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Val-Thr (62-64). The enzyme differs at about 35-40% of the positions from all other mammalian pancreatic ribonucleases sequenced to date, which is in agreement with the early divergence between the marsupials and the placental mammals. From fragmentary data a tentative sequence of red-necked wallaby (Macropus rufogriseus) pancreatic ribonuclease has been derived. Eight differences with the kangaroo sequence were found. PMID:658039

  16. Development of an expert system for amino acid sequence identification.

    PubMed

    Hu, L; Saulinskas, E F; Johnson, P; Harrington, P B

    1996-08-01

    An expert system for amino acid sequence identification has been developed. The algorithm uses heuristic rules developed by human experts in protein sequencing. The system is applied to the chromatographic data of phenylthiohydantoin-amino acids acquired from an automated sequencer. The peak intensities in the current cycle are compared with those in the previous cycle, while the calibration and succeeding cycles are used as ancillary identification criteria when necessary. The retention time for each chromatographic peak in each cycle is corrected by the corresponding peak in the calibration cycle at the same run. The main improvement of our system compared with the onboard software used by the Applied Biosystems 477A Protein/Peptide Sequencer is that each peak in each cycle is assigned an identification name according to the corrected retention time to be used for the comparison with different cycles. The system was developed from analyses of ribonuclease A and evaluated by runs of four other protein samples that were not used in rule development. This paper demonstrates that rules developed by human experts can be automatically applied to sequence assignment. The expert system performed more accurately than the onboard software of the protein sequencer, in that the misidentification rates for the expert system were around 7%, whereas those for the onboard software were between 13 and 21%.

  17. 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.

  18. The genome of RNA tumor viruses contains polyadenylic acid sequences.

    PubMed

    Green, M; Cartas, M

    1972-04-01

    The 70S genome of two RNA tumor viruses, murine sarcoma virus and avian myeloblastosis virus, binds to Millipore filters in buffer with high salt concentration and to glass fiber filters containing poly(U). These observations suggest that 70S RNA contains adenylic acid-rich sequences. When digested by pancreatic RNase, 70S RNA of murine sarcoma virus yielded poly(A) sequences that contain 91% adenylic acid. These poly(A) sequences sedimented as a relatively homogenous peak in sucrose gradients with a sedimentation coefficient of 4-5 S, but had a mobility during polyacrylamide gel electrophoresis that corresponds to molecules that sediment at 6-7 S. If we estimate a molecular weight for each sequence of 30,000-60,000 (100-200 nucleotides) and a molecular weight for viral 70S RNA of 3-12 million, each viral genome could contain 1-8 poly(A) sequences. Possible functions of poly(A) in the infecting viral RNA may include a role in the initiation of viral DNA or RNA synthesis, in protein maturation, or in the assembly of the viral genome.

  19. 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.

  20. Nucleic acid sequence design via efficient ensemble defect optimization.

    PubMed

    Zadeh, Joseph N; Wolfe, Brian R; Pierce, Niles A

    2011-02-01

    We describe an algorithm for designing the sequence of one or more interacting nucleic acid strands intended to adopt a target secondary structure at equilibrium. Sequence design is formulated as an optimization problem with the goal of reducing the ensemble defect below a user-specified stop condition. For a candidate sequence and a given target secondary structure, the ensemble defect is the average number of incorrectly paired nucleotides at equilibrium evaluated over the ensemble of unpseudoknotted secondary structures. To reduce the computational cost of accepting or rejecting mutations to a random initial sequence, candidate mutations are evaluated on the leaf nodes of a tree-decomposition of the target structure. During leaf optimization, defect-weighted mutation sampling is used to select each candidate mutation position with probability proportional to its contribution to the ensemble defect of the leaf. As subsequences are merged moving up the tree, emergent structural defects resulting from crosstalk between sibling sequences are eliminated via reoptimization within the defective subtree starting from new random subsequences. Using a Θ(N(3) ) dynamic program to evaluate the ensemble defect of a target structure with N nucleotides, this hierarchical approach implies an asymptotic optimality bound on design time: for sufficiently large N, the cost of sequence design is bounded below by 4/3 the cost of a single evaluation of the ensemble defect for the full sequence. Hence, the design algorithm has time complexity Ω(N(3) ). For target structures containing N ∈{100,200,400,800,1600,3200} nucleotides and duplex stems ranging from 1 to 30 base pairs, RNA sequence designs at 37°C typically succeed in satisfying a stop condition with ensemble defect less than N/100. Empirically, the sequence design algorithm exhibits asymptotic optimality and the exponent in the time complexity bound is sharp.

  1. Draft genome sequence of the docosahexaenoic acid producing thraustochytrid Aurantiochytrium sp. T66.

    PubMed

    Liu, Bin; Ertesvåg, Helga; Aasen, Inga Marie; Vadstein, Olav; Brautaset, Trygve; Heggeset, Tonje Marita Bjerkan

    2016-06-01

    Thraustochytrids are unicellular, marine protists, and there is a growing industrial interest in these organisms, particularly because some species, including strains belonging to the genus Aurantiochytrium, accumulate high levels of docosahexaenoic acid (DHA). Here, we report the draft genome sequence of Aurantiochytrium sp. T66 (ATCC PRA-276), with a size of 43 Mbp, and 11,683 predicted protein-coding sequences. The data has been deposited at DDBJ/EMBL/Genbank under the accession LNGJ00000000. The genome sequence will contribute new insight into DHA biosynthesis and regulation, providing a basis for metabolic engineering of thraustochytrids. PMID:27222814

  2. Draft genome sequence of the docosahexaenoic acid producing thraustochytrid Aurantiochytrium sp. T66.

    PubMed

    Liu, Bin; Ertesvåg, Helga; Aasen, Inga Marie; Vadstein, Olav; Brautaset, Trygve; Heggeset, Tonje Marita Bjerkan

    2016-06-01

    Thraustochytrids are unicellular, marine protists, and there is a growing industrial interest in these organisms, particularly because some species, including strains belonging to the genus Aurantiochytrium, accumulate high levels of docosahexaenoic acid (DHA). Here, we report the draft genome sequence of Aurantiochytrium sp. T66 (ATCC PRA-276), with a size of 43 Mbp, and 11,683 predicted protein-coding sequences. The data has been deposited at DDBJ/EMBL/Genbank under the accession LNGJ00000000. The genome sequence will contribute new insight into DHA biosynthesis and regulation, providing a basis for metabolic engineering of thraustochytrids.

  3. 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.

  4. The amino acid sequence of Escherichia coli cyanase.

    PubMed

    Chin, C C; Anderson, P M; Wold, F

    1983-01-10

    The amino acid sequence of the enzyme cyanase (cyanate hydrolase) from Escherichia coli has been determined by automatic Edman degradation of the intact protein and of its component peptides. The primary peptides used in the sequencing were produced by cyanogen bromide cleavage at the methionine residues, yielding 4 peptides plus free homoserine from the NH2-terminal methionine, and by trypsin cleavage at the 7 arginine residues after acetylation of the lysines. Secondary peptides required for overlaps and COOH-terminal sequences were produced by chymotrypsin or clostripain cleavage of some of the larger peptides. The complete sequence of the cyanase subunit consists of 156 amino acid residues (Mr 16,350). Based on the observation that the cysteine-containing peptide is obtained as a disulfide-linked dimer, it is proposed that the covalent structure of cyanase is made up of two subunits linked by a disulfide bond between the single cystine residue in each subunit. The native enzyme (Mr 150,000) then appears to be a complex of four or five such subunit dimers.

  5. Amino acid and cDNA sequences of lysozyme from Hyalophora cecropia

    PubMed Central

    Engström, Å.; Xanthopoulos, K. G.; Boman, H. G.; Bennich, H.

    1985-01-01

    The amino acid and cDNA sequences of lysozyme from the giant silk moth Hyalophora cecropia have been determined. This enzyme is one of several immune proteins produced by the diapausing pupae after injection of bacteria. Cecropia lysozyme is composed of 120 amino acids, has a mol. wt. of 13.8 kd and shows great similarity with vertebrate lysozymes of the chicken type. The amino acid residues responsible for the catalytic activity and for the binding of substrate are essentially conserved. Three allelic variants of the Cecropia enzyme are identified. A comparison of the chicken and the Cecropia lysozymes shows that there is a 40% identity at both the amino acid and the nucleotide level. Some evolutionary aspects of the sequence data are discussed. PMID:16453632

  6. Heavy metals influence on ascorbic acid level

    NASA Astrophysics Data System (ADS)

    Kamaldinov, E. V.; Patrashkov, S. A.; Batenyeva, E. V.; Korotkevich, O. S.

    2003-05-01

    It is well known that heavy metals (HM) are extremely dangerous pollutants influencing to metabolism in animals' organisms. The vitamin C is one of the most important metabolites taking part in many biochemical processes. We studied the influence of main essential HM-Zn and Cu as well as the based supertoxical elements - Cd and Pd on ascorbic acid level in serum. The studies were carried out in Tulinskoe farm of Novosibirsk region. The objects of investigations were piglets (2 month after weaning) and 6-month pigs of Early Ripe Meat breed. The levels of HM in bristle were found by stripping voltammetric analysis using the TA-2 analyzer. Vitamin C content was determined by I.P. Kondrakhin (1985) method using 2,2-dipyridyl. The significant negative correlations between Pb, Cd content and vitamin C (-0.46 ± 0.18, -0.47 ± 0.19) in 6-month pigs were determined. The tendencies of negative correlation between all HM levels in hair and ascorbic acid level in plasma of piglets were revealed. Thus, the obtained correlations let us to suppose that all studied HM influence on 1-gulono-gamma-lactone oxidase and other vitamin C metabolism enzymes activity.

  7. Sequence determinants of prokaryotic gene expression level under heat stress.

    PubMed

    Xiong, Heng; Yang, Yi; Hu, Xiao-Pan; He, Yi-Ming; Ma, Bin-Guang

    2014-11-01

    Prokaryotic gene expression is environment-dependent and temperature plays an important role in shaping the gene expression profile. Revealing the regulation mechanisms of gene expression pertaining to temperature has attracted tremendous efforts in recent years particularly owning to the yielding of transcriptome and proteome data by high-throughput techniques. However, most of the previous works concentrated on the characterization of the gene expression profile of individual organism and little effort has been made to disclose the commonality among organisms, especially for the gene sequence features. In this report, we collected the transcriptome and proteome data measured under heat stress condition from recently published literature and studied the sequence determinants for the expression level of heat-responsive genes on multiple layers. Our results showed that there indeed exist commonness and consistent patterns of the sequence features among organisms for the differentially expressed genes under heat stress condition. Some features are attributed to the requirement of thermostability while some are dominated by gene function. The revealed sequence determinants of bacterial gene expression level under heat stress complement the knowledge about the regulation factors of prokaryotic gene expression responding to the change of environmental conditions. Furthermore, comparisons to thermophilic adaption have been performed to reveal the similarity and dissimilarity of the sequence determinants for the response to heat stress and for the adaption to high habitat temperature, which elucidates the complex landscape of gene expression related to the same physical factor of temperature.

  8. 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.

  9. 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.

  10. Characterization and amino acid sequence of a fatty acid-binding protein from human heart.

    PubMed Central

    Offner, G D; Brecher, P; Sawlivich, W B; Costello, C E; Troxler, R F

    1988-01-01

    The complete amino acid sequence of a fatty acid-binding protein from human heart was determined by automated Edman degradation of CNBr, BNPS-skatole [3'-bromo-3-methyl-2-(2-nitrobenzenesulphenyl)indolenine], hydroxylamine, Staphylococcus aureus V8 proteinase, tryptic and chymotryptic peptides, and by digestion of the protein with carboxypeptidase A. The sequence of the blocked N-terminal tryptic peptide from citraconylated protein was determined by collisionally induced decomposition mass spectrometry. The protein contains 132 amino acid residues, is enriched with respect to threonine and lysine, lacks cysteine, has an acetylated valine residue at the N-terminus, and has an Mr of 14768 and an isoelectric point of 5.25. This protein contains two short internal repeated sequences from residues 48-54 and from residues 114-119 located within regions of predicted beta-structure and decreasing hydrophobicity. These short repeats are contained within two longer repeated regions from residues 48-60 and residues 114-125, which display 62% sequence similarity. These regions could accommodate the charged and uncharged moieties of long-chain fatty acids and may represent fatty acid-binding domains consistent with the finding that human heart fatty acid-binding protein binds 2 mol of oleate or palmitate/mol of protein. Detailed evidence for the amino acid sequences of the peptides has been deposited as Supplementary Publication SUP 50143 (23 pages) at the British Library Lending Division, Boston Spa, Yorkshire LS23 7BQ, U.K., from whom copies may be obtained as indicated in Biochem. J. (1988) 249, 5. PMID:3421901

  11. Accident sequence precursor analysis level 2/3 model development

    SciTech Connect

    Lui, C.H.; Galyean, W.J.; Brownson, D.A.

    1997-02-01

    The US Nuclear Regulatory Commission`s Accident Sequence Precursor (ASP) program currently uses simple Level 1 models to assess the conditional core damage probability for operational events occurring in commercial nuclear power plants (NPP). Since not all accident sequences leading to core damage will result in the same radiological consequences, it is necessary to develop simple Level 2/3 models that can be used to analyze the response of the NPP containment structure in the context of a core damage accident, estimate the magnitude of the resulting radioactive releases to the environment, and calculate the consequences associated with these releases. The simple Level 2/3 model development work was initiated in 1995, and several prototype models have been completed. Once developed, these simple Level 2/3 models are linked to the simple Level 1 models to provide risk perspectives for operational events. This paper describes the methods implemented for the development of these simple Level 2/3 ASP models, and the linkage process to the existing Level 1 models.

  12. New approaches for computer analysis of nucleic acid sequences.

    PubMed

    Karlin, S; Ghandour, G; Ost, F; Tavare, S; Korn, L J

    1983-09-01

    A new high-speed computer algorithm is outlined that ascertains within and between nucleic acid and protein sequences all direct repeats, dyad symmetries, and other structural relationships. Large repeats, repeats of high frequency, dyad symmetries of specified stem length and loop distance, and their distributions are determined. Significance of homologies is assessed by a hierarchy of permutation procedures. Applications are made to papovaviruses, the human papillomavirus HPV, lambda phage, the human and mouse mitochondrial genomes, and the human and mouse immunoglobulin kappa-chain genes. PMID:6577449

  13. Antibody-specific model of amino acid substitution for immunological inferences from alignments of antibody sequences.

    PubMed

    Mirsky, Alexander; Kazandjian, Linda; Anisimova, Maria

    2015-03-01

    Antibodies are glycoproteins produced by the immune system as a dynamically adaptive line of defense against invading pathogens. Very elegant and specific mutational mechanisms allow B lymphocytes to produce a large and diversified repertoire of antibodies, which is modified and enhanced throughout all adulthood. One of these mechanisms is somatic hypermutation, which stochastically mutates nucleotides in the antibody genes, forming new sequences with different properties and, eventually, higher affinity and selectivity to the pathogenic target. As somatic hypermutation involves fast mutation of antibody sequences, this process can be described using a Markov substitution model of molecular evolution. Here, using large sets of antibody sequences from mice and humans, we infer an empirical amino acid substitution model AB, which is specific to antibody sequences. Compared with existing general amino acid models, we show that the AB model provides significantly better description for the somatic evolution of mice and human antibody sequences, as demonstrated on large next generation sequencing (NGS) antibody data. General amino acid models are reflective of conservation at the protein level due to functional constraints, with most frequent amino acids exchanges taking place between residues with the same or similar physicochemical properties. In contrast, within the variable part of antibody sequences we observed an elevated frequency of exchanges between amino acids with distinct physicochemical properties. This is indicative of a sui generis mutational mechanism, specific to antibody somatic hypermutation. We illustrate this property of antibody sequences by a comparative analysis of the network modularity implied by the AB model and general amino acid substitution models. We recommend using the new model for computational studies of antibody sequence maturation, including inference of alignments and phylogenetic trees describing antibody somatic hypermutation in

  14. Diverse levels of sequence selectivity and catalytic efficiency of protein-tyrosine phosphatases.

    PubMed

    Selner, Nicholas G; Luechapanichkul, Rinrada; Chen, Xianwen; Neel, Benjamin G; Zhang, Zhong-Yin; Knapp, Stefan; Bell, Charles E; Pei, Dehua

    2014-01-21

    The sequence selectivity of 14 classical protein-tyrosine phosphatases (PTPs) (PTPRA, PTPRB, PTPRC, PTPRD, PTPRO, PTP1B, SHP-1, SHP-2, HePTP, PTP-PEST, TCPTP, PTPH1, PTPD1, and PTPD2) was systematically profiled by screening their catalytic domains against combinatorial peptide libraries. All of the PTPs exhibit similar preference for pY peptides rich in acidic amino acids and disfavor positively charged sequences but differ vastly in their degrees of preference/disfavor. Some PTPs (PTP-PEST, SHP-1, and SHP-2) are highly selective for acidic over basic (or neutral) peptides (by >10(5)-fold), whereas others (PTPRA and PTPRD) show no to little sequence selectivity. PTPs also have diverse intrinsic catalytic efficiencies (kcat/KM values against optimal substrates), which differ by >10(5)-fold due to different kcat and/or KM values. Moreover, PTPs show little positional preference for the acidic residues relative to the pY residue. Mutation of Arg47 of PTP1B, which is located near the pY-1 and pY-2 residues of a bound substrate, decreased the enzymatic activity by 3-18-fold toward all pY substrates containing acidic residues anywhere within the pY-6 to pY+5 region. Similarly, mutation of Arg24, which is situated near the C-terminus of a bound substrate, adversely affected the kinetic activity of all acidic substrates. A cocrystal structure of PTP1B bound with a nephrin pY(1193) peptide suggests that Arg24 engages in electrostatic interactions with acidic residues at the pY+1, pY+2, and likely other positions. These results suggest that long-range electrostatic interactions between positively charged residues near the PTP active site and acidic residues on pY substrates allow a PTP to bind acidic substrates with similar affinities, and the varying levels of preference for acidic sequences by different PTPs are likely caused by the different electrostatic potentials near their active sites. The implications of the varying sequence selectivity and intrinsic catalytic

  15. Abscisic Acid Levels and Seed Dormancy

    PubMed Central

    Sondheimer, E.; Tzou, D. S.; Galson, Eva C.

    1968-01-01

    Dormant seeds from Fraxinus species require cold-temperature after-ripening prior to germination. Earlier, we found that abscisic acid (ABA) will inhibit germination of excised nondormant embryos and that this can be reversed with a combination of gibberellic acid and kinetin. Using Milborrow's quantitative “racemate dilution” method the ABA concentration in 3 types of Fraxinus seed and pericarp were determined. While ABA was present in all tissues, the highest concentration was found in the seed and pericarp of dormant F. americana. During the chilling treatment of F. americana the ABA levels decreased 37% in the pericarp and 68% in the seed. The ABA concentration of the seed of the nondormant species, F. ornus, is as low as that found in F. americana seeds after cold treatment. Experiments with exogenously added ABA solutions indicate that it is unlikely that the ABA in the pericarp functions in the regulation of seed dormancy. However, the ABA in the seed does seem to have a regulatory role in germination. Images PMID:16656935

  16. Pyruvate decarboxylase from Pisum sativum. Properties, nucleotide and amino acid sequences.

    PubMed

    Mücke, U; Wohlfarth, T; Fiedler, U; Bäumlein, H; Rücknagel, K P; König, S

    1996-04-15

    To study the molecular structure and function of pyruvate decarboxylase (PDC) from plants the protein was isolated from pea seeds and partially characterised. The active enzyme which occurs in the form of higher oligomers consists of two different subunits appearing in SDS/PAGE and mass spectroscopy experiments. For further experiments, like X-ray crystallography, it was necessary to elucidate the protein sequence. Partial cDNA clones encoding pyruvate decarboxylase from seeds of Pisum sativum cv. Miko have been obtained by means of polymerase chain reaction techniques. The first sequences were found using degenerate oligonucleotide primers designated according to conserved amino acid sequences of known pyruvate decarboxylases. The missing parts of one cDNA were amplified applying the 3'- and 5'-rapid amplification of cDNA ends systems. The amino acid sequence deduced from the entire cDNA sequence displays strong similarity to pyruvate decarboxylases from other organisms, especially from plants. A molecular mass of 64 kDa was calculated for this protein correlating with estimations for the smaller subunit of the oligomeric enzyme. The PCR experiments led to at least three different clones representing the middle part of the PDC cDNA indicating the existence of three isozymes. Two of these isoforms could be confirmed on the protein level by sequencing tryptic peptides. Only anaerobically treated roots showed a positive signal for PDC mRNA in Northern analysis although the cDNA from imbibed seeds was successfully used for PCR.

  17. Progress towards DNA sequencing at the single molecule level

    SciTech Connect

    Goodwin, P.M.; Affleck, R.L.; Ambrose, W.P.

    1995-12-01

    We describe progress towards sequencing DNA at the single molecule level. Our technique involves incorporation of fluorescently tagged nucleotides into a targeted sequence, anchoring the labeled DNA strand in a flowing stream, sequential exonuclease digestion of the DNA strand, and efficient detection and identification of single tagged nucleotides. Experiments demonstrating strand specific exonuclease digestion of fluorescently labeled DNA anchored in flow as well as the detection of single cleaved fluorescently tagged nucleotides from a small number of anchored DNA fragments axe described. We find that the turnover rate of Esherichia coli exonuclease III on fluorescently labeled DNA in flow at 36{degree}C is {approximately}7 nucleotides per DNA strand per second, which is approximately the same as that measured for this enzyme on native DNA under static, saturated (excess enzyme) conditions. Experiments demonstrating the efficient detection of single fluorescent molecules delivered electrokinetically to a {approximately}3 pL probe volume are also described.

  18. Complete Genome Sequences of Escherichia coli O157:H7 Strains SRCC 1675 and 28RC, Which Vary in Acid Resistance

    PubMed Central

    Baranzoni, Gian Marco; Reichenberger, Erin R.; Kim, Gwang-Hee; Breidt, Frederick; Kay, Kathryn; Oh, Deog-Hwan

    2016-01-01

    The level of acid resistance among Escherichia coli O157:H7 strains varies, and strains with higher resistance to acid may have a lower infectious dose. The complete genome sequences belonging to two strains of Escherichia coli O157:H7 with different levels of acid resistance are presented here. PMID:27469964

  19. Complete genome sequences of Escherichia coli O157:H7 strains SRCC 1675 and 28RC that vary in acid resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The level of acid resistance among Escherichia coli O157:H7 strains varies, and strains with higher resistance to acid may have a lower infectious dose. The complete genome sequences belonging to two strains of Escherichia coli O157:H7 with different levels of acid resistance are presented....

  20. Complete Genome Sequences of Escherichia coli O157:H7 Strains SRCC 1675 and 28RC, Which Vary in Acid Resistance.

    PubMed

    Baranzoni, Gian Marco; Fratamico, Pina M; Reichenberger, Erin R; Kim, Gwang-Hee; Breidt, Frederick; Kay, Kathryn; Oh, Deog-Hwan

    2016-01-01

    The level of acid resistance among Escherichia coli O157:H7 strains varies, and strains with higher resistance to acid may have a lower infectious dose. The complete genome sequences belonging to two strains of Escherichia coli O157:H7 with different levels of acid resistance are presented here. PMID:27469964

  1. Predicting protein disorder by analyzing amino acid sequence

    PubMed Central

    Yang, Jack Y; Yang, Mary Qu

    2008-01-01

    Background Many protein regions and some entire proteins have no definite tertiary structure, presenting instead as dynamic, disorder ensembles under different physiochemical circumstances. These proteins and regions are known as Intrinsically Unstructured Proteins (IUP). IUP have been associated with a wide range of protein functions, along with roles in diseases characterized by protein misfolding and aggregation. Results Identifying IUP is important task in structural and functional genomics. We exact useful features from sequences and develop machine learning algorithms for the above task. We compare our IUP predictor with PONDRs (mainly neural-network-based predictors), disEMBL (also based on neural networks) and Globplot (based on disorder propensity). Conclusion We find that augmenting features derived from physiochemical properties of amino acids (such as hydrophobicity, complexity etc.) and using ensemble method proved beneficial. The IUP predictor is a viable alternative software tool for identifying IUP protein regions and proteins. PMID:18831799

  2. Heterogeneity of amino acid sequence in hippopotamus cytochrome c.

    PubMed

    Thompson, R B; Borden, D; Tarr, G E; Margoliash, E

    1978-12-25

    The amino acid sequences of chymotryptic and tryptic peptides of Hippopotamus amphibius cytochrome c were determined by a recent modification of the manual Edman sequential degradation procedure. They were ordered by comparison with the structure of the hog protein. The hippopotamus protein differs in three positions: serine, alanine, and glutamine replace alanine, glutamic acid, and lysine in positions 43, 92, and 100, respectively. Since the artiodactyl suborders diverged in the mid-Eocene some 50 million years ago, the fact that representatives of some of them show no differences in their cytochromes c (cow, sheep, and hog), while another exhibits as many as three such differences, verifies that even in relatively closely related lines of descent the rate at which cytochrome c changes in the course of evolution is not constant. Furthermore, 10.6% of the hippopotamus cytochrome c preparation was shown to contain isoleucine instead of valine at position 3, indicating that one of the four animals from which the protein was obtained was heterozygous in the cytochrome c gene. Such heterogeneity is a necessary condition of evolutionary variation and has not been previously observed in the cytochrome c of a wild mammalian population.

  3. Egg mercury levels decline with the laying sequence in charadriiformes

    SciTech Connect

    Becker, P.H. )

    1992-05-01

    Whereas pollutants do not differ in concentration among eggs of one clutch in some bird species, in gulls, terns and grebes several organochlorines show intraclutch variation: Concentrations increase with the laying sequence. Heavy metals, however, are not so intensively studied with respect to intraclutch variation. In contrast to lead and cadmium, mercury is accumulated in great quantities in eggs. Variation in mercury levels between the eggs of one clutch were low compared to interclutch variability in the White-tailed Sea Eagle (Haliaeetus albicilla) and the Peregrine (Falco peregrinus). In gulls, however, intraclutch variation was significant and characterized by higher mercury levels in the first than in subsequently laid eggs, which is the opposite to the trend in organochlorine levels. In this paper, the author reports on investigations of intraclutch variation in mercury levels in three Charadriiform-species, Herring Gull, Common Tern and Oystercatcher (Haematopus ostralegus). The results confirm those previously reported in gulls and point to the importance of the egg in reducing the females' mercury burden. 23 refs, 2 tabs.

  4. 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

  5. Exploring Sequence Characteristics Related to High-Level Production of Secreted Proteins in Aspergillus niger

    PubMed Central

    van den Berg, Bastiaan A.; Reinders, Marcel J. T.; Hulsman, Marc; Wu, Liang; Pel, Herman J.; Roubos, Johannes A.; de Ridder, Dick

    2012-01-01

    Protein sequence features are explored in relation to the production of over-expressed extracellular proteins by fungi. Knowledge on features influencing protein production and secretion could be employed to improve enzyme production levels in industrial bioprocesses via protein engineering. A large set, over 600 homologous and nearly 2,000 heterologous fungal genes, were overexpressed in Aspergillus niger using a standardized expression cassette and scored for high versus no production. Subsequently, sequence-based machine learning techniques were applied for identifying relevant DNA and protein sequence features. The amino-acid composition of the protein sequence was found to be most predictive and interpretation revealed that, for both homologous and heterologous gene expression, the same features are important: tyrosine and asparagine composition was found to have a positive correlation with high-level production, whereas for unsuccessful production, contributions were found for methionine and lysine composition. The predictor is available online at http://bioinformatics.tudelft.nl/hipsec. Subsequent work aims at validating these findings by protein engineering as a method for increasing expression levels per gene copy. PMID:23049690

  6. On human disease-causing amino acid variants: statistical study of sequence and structural patterns

    PubMed Central

    Alexov, Emil

    2015-01-01

    Statistical analysis was carried out on large set of naturally occurring human amino acid variations and it was demonstrated that there is a preference for some amino acid substitutions to be associated with diseases. At an amino acid sequence level, it was shown that the disease-causing variants frequently involve drastic changes of amino acid physico-chemical properties of proteins such as charge, hydrophobicity and geometry. Structural analysis of variants involved in diseases and being frequently observed in human population showed similar trends: disease-causing variants tend to cause more changes of hydrogen bond network and salt bridges as compared with harmless amino acid mutations. Analysis of thermodynamics data reported in literature, both experimental and computational, indicated that disease-causing variants tend to destabilize proteins and their interactions, which prompted us to investigate the effects of amino acid mutations on large databases of experimentally measured energy changes in unrelated proteins. Although the experimental datasets were linked neither to diseases nor exclusory to human proteins, the observed trends were the same: amino acid mutations tend to destabilize proteins and their interactions. Having in mind that structural and thermodynamics properties are interrelated, it is pointed out that any large change of any of them is anticipated to cause a disease. PMID:25689729

  7. Canine amino acid transport system Xc(-): cDNA sequence, distribution and cystine transport activity in lens epithelial cells.

    PubMed

    Maruo, Takuya; Kanemaki, Nobuyuki; Onda, Ken; Sato, Reiichiro; Ichihara, Nobuteru; Ochiai, Hideharu

    2014-04-01

    The cystine transport activity of a lens epithelial cell line originated from a canine mature cataract was investigated. The distinct cystine transport activity was observed, which was inhibited to 28% by extracellular 1 mM glutamate. The cDNA sequences of canine cysteine/glutamate exchanger (xCT) and 4F2hc were determined. The predicted amino acid sequences were 527 and 533 amino acid polypeptides, respectively. The amino acid sequences of canine xCT and 4F2hc showed high similarities (>80%) to those of humans. The expression of xCT in lens epithelial cell line was confirmed by western blot analysis. RT-PCR analysis revealed high level expression only in the brain, and it was below the detectable level in other tissues.

  8. 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.

  9. Advanced accident sequence precursor analysis level 2 models

    SciTech Connect

    Galyean, W.J.; Brownson, D.A.; Rempe, J.L.

    1996-03-01

    The U.S. Nuclear Regulatory Commission Accident Sequence Precursor program pursues the ultimate objective of performing risk significant evaluations on operational events (precursors) occurring in commercial nuclear power plants. To achieve this objective, the Office of Nuclear Regulatory Research is supporting the development of simple probabilistic risk assessment models for all commercial nuclear power plants (NPP) in the U.S. Presently, only simple Level 1 plant models have been developed which estimate core damage frequencies. In order to provide a true risk perspective, the consequences associated with postulated core damage accidents also need to be considered. With the objective of performing risk evaluations in an integrated and consistent manner, a linked event tree approach which propagates the front end results to back end was developed. This approach utilizes simple plant models that analyze the response of the NPP containment structure in the context of a core damage accident, estimate the magnitude and timing of a radioactive release to the environment, and calculate the consequences for a given release. Detailed models and results from previous studies, such as the NUREG-1150 study, are used to quantify these simple models. These simple models are then linked to the existing Level 1 models, and are evaluated using the SAPHIRE code. To demonstrate the approach, prototypic models have been developed for a boiling water reactor, Peach Bottom, and a pressurized water reactor, Zion.

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

    PubMed

    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

  11. 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

  12. Natural vs. random protein sequences: Discovering combinatorics properties on amino acid words.

    PubMed

    Santoni, Daniele; Felici, Giovanni; Vergni, Davide

    2016-02-21

    Casual mutations and natural selection have driven the evolution of protein amino acid sequences that we observe at present in nature. The question about which is the dominant force of proteins evolution is still lacking of an unambiguous answer. Casual mutations tend to randomize protein sequences while, in order to have the correct functionality, one expects that selection mechanisms impose rigid constraints on amino acid sequences. Moreover, one also has to consider that the space of all possible amino acid sequences is so astonishingly large that it could be reasonable to have a well tuned amino acid sequence indistinguishable from a random one. In order to study the possibility to discriminate between random and natural amino acid sequences, we introduce different measures of association between pairs of amino acids in a sequence, and apply them to a dataset of 1047 natural protein sequences and 10,470 random sequences, carefully generated in order to preserve the relative length and amino acid distribution of the natural proteins. We analyze the multidimensional measures with machine learning techniques and show that, to a reasonable extent, natural protein sequences can be differentiated from random ones.

  13. Natural vs. random protein sequences: Discovering combinatorics properties on amino acid words.

    PubMed

    Santoni, Daniele; Felici, Giovanni; Vergni, Davide

    2016-02-21

    Casual mutations and natural selection have driven the evolution of protein amino acid sequences that we observe at present in nature. The question about which is the dominant force of proteins evolution is still lacking of an unambiguous answer. Casual mutations tend to randomize protein sequences while, in order to have the correct functionality, one expects that selection mechanisms impose rigid constraints on amino acid sequences. Moreover, one also has to consider that the space of all possible amino acid sequences is so astonishingly large that it could be reasonable to have a well tuned amino acid sequence indistinguishable from a random one. In order to study the possibility to discriminate between random and natural amino acid sequences, we introduce different measures of association between pairs of amino acids in a sequence, and apply them to a dataset of 1047 natural protein sequences and 10,470 random sequences, carefully generated in order to preserve the relative length and amino acid distribution of the natural proteins. We analyze the multidimensional measures with machine learning techniques and show that, to a reasonable extent, natural protein sequences can be differentiated from random ones. PMID:26656109

  14. Physical activity as a determinant of fecal bile acid levels

    PubMed Central

    Wertheim, Betsy C.; Martínez, María Elena; Ashbeck, Erin L.; Roe, Denise J.; Jacobs, Elizabeth T.; Alberts, David S.; Thompson, Patricia A.

    2009-01-01

    Physical activity is protective against colon cancer, whereas colonic bile acid exposure is a suspected risk factor. While likely related, the association between physical activity and bile acid levels has not been well studied. Furthermore, the effect of triglycerides, which are known to modify bile acid levels, on this relationship has not been investigated. We conducted a cross-sectional analysis of baseline fecal bile acid levels for 735 colorectal adenoma formers obtained from participants in a phase III ursodeoxycholic acid chemoprevention trial. Compared to the lowest quartile of recreational physical activity duration, the highest quartile was associated with a 17% lower fecal bile acid concentration, adjusted for age, sex, dietary fiber intake, and body mass index (P = 0.042). Furthermore, consistent with a previously established relationship between serum triglyceride levels and bile acid metabolism, we stratified by triglyceride level and observed a 34% lower fecal bile acid concentration (highest versus lowest quartiles of physical activity) in individuals with low triglycerides (< 136 mg/dL; P = 0.002). In contrast, no association between physical activity and fecal bile acid concentration was observed for subjects with high triglycerides (≥ 136 mg/dL). Our results suggest that the biological mechanism responsible for the protective effect of physical activity on the incidence of colon cancer may be partially mediated by decreasing colonic bile acid exposure. However, this effect may be limited to individuals with lower triglyceride levels. PMID:19383885

  15. 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.

  16. Enzyme-free translation of DNA into sequence-defined synthetic polymers structurally unrelated to nucleic acids

    NASA Astrophysics Data System (ADS)

    Niu, Jia; Hili, Ryan; Liu, David R.

    2013-04-01

    The translation of DNA sequences into corresponding biopolymers enables the production, function and evolution of the macromolecules of life. In contrast, methods to generate sequence-defined synthetic polymers with similar levels of control have remained elusive. Here, we report the development of a DNA-templated translation system that enables the enzyme-free translation of DNA templates into sequence-defined synthetic polymers that have no necessary structural relationship with nucleic acids. We demonstrate the efficiency, sequence-specificity and generality of this translation system by oligomerizing building blocks including polyethylene glycol, α-(D)-peptides, and β-peptides in a DNA-programmed manner. Sequence-defined synthetic polymers with molecular weights of 26 kDa containing 16 consecutively coupled building blocks and 90 densely functionalized β-amino acid residues were translated from DNA templates using this strategy. We integrated the DNA-templated translation system developed here into a complete cycle of translation, coding sequence replication, template regeneration and re-translation suitable for the iterated in vitro selection of functional sequence-defined synthetic polymers unrelated in structure to nucleic acids.

  17. Advanced accident sequence precursor analysis level 1 models

    SciTech Connect

    Sattison, M.B.; Thatcher, T.A.; Knudsen, J.K.; Schroeder, J.A.; Siu, N.O.

    1996-03-01

    INEL has been involved in the development of plant-specific Accident Sequence Precursor (ASP) models for the past two years. These models were developed for use with the SAPHIRE suite of PRA computer codes. They contained event tree/linked fault tree Level 1 risk models for the following initiating events: general transient, loss-of-offsite-power, steam generator tube rupture, small loss-of-coolant-accident, and anticipated transient without scram. Early in 1995 the ASP models were revised based on review comments from the NRC and an independent peer review. These models were released as Revision 1. The Office of Nuclear Regulatory Research has sponsored several projects at the INEL this fiscal year to further enhance the capabilities of the ASP models. Revision 2 models incorporates more detailed plant information into the models concerning plant response to station blackout conditions, information on battery life, and other unique features gleaned from an Office of Nuclear Reactor Regulation quick review of the Individual Plant Examination submittals. These models are currently being delivered to the NRC as they are completed. A related project is a feasibility study and model development of low power/shutdown (LP/SD) and external event extensions to the ASP models. This project will establish criteria for selection of LP/SD and external initiator operational events for analysis within the ASP program. Prototype models for each pertinent initiating event (loss of shutdown cooling, loss of inventory control, fire, flood, seismic, etc.) will be developed. A third project concerns development of enhancements to SAPHIRE. In relation to the ASP program, a new SAPHIRE module, GEM, was developed as a specific user interface for performing ASP evaluations. This module greatly simplifies the analysis process for determining the conditional core damage probability for a given combination of initiating events and equipment failures or degradations.

  18. Amino acid sequence of horseshoe crab, Tachypleus tridentatus, striated muscle troponin C.

    PubMed

    Kobayashi, T; Kagami, O; Takagi, T; Konishi, K

    1989-05-01

    The amino acid sequence of troponin C obtained from horseshoe crab, Tachypleus tridentatus, striated muscle was determined by sequence analysis and alignments of chemically and enzymatically cleaved peptides. Troponin C is composed of 153 amino acid residues with a blocked N-terminus and contains no tryptophan or cysteine residue. The site I, one of the four Ca2+-binding sites, is considered to have lost its ability to bind Ca2+ owing to the replacements of certain amino acid residues.

  19. Serum Uric Acid Level among Acute Stroke Patients.

    PubMed

    Mohsin, M; Das, S N; Haque, M F; Shikha, S S; Bhuiyan, A S; Saha, P L; Das, B R; Chowdhury, M; Jahan, M K; Rahman, M M

    2016-04-01

    Stroke is one of the leading causes of death and disability in developed as well as developing countries like Bangladesh. Elevated serum uric acid levels may predict an increased risk for cerebro-vascular (CV) events including stroke. Aim of the study was to measure the serum uric acid level among stroke patients and determine the relationship between serum uric acid level and stroke. This descriptive, cross-sectional study was carried out in Department of Medicine, Mymensingh Medical College Hospital, Mymensingh, Bangladesh to measure serum uric acid level among 102 stroke patients in a period of one year by using non-probability sampling procedure. Finally, collected data were analyzed using SPSS software Version 17.0. It was observed that the mean age of patients was 60.87±8.05 years, of them 80(78.43%) patients were male and the rest 22(21.57%) were female. About 66(64.70%) of respondents were in age group 60 years and above, while 36(35.30%) were in age group 59 years and below. At least 23(22.55%) of stroke patients had elevated serum uric acid with a mean serum uric acid level of 5.18mg/dl and standard deviation 1.26mg/dl. About 23(27.38%) patients in ischemic stroke had elevated serum uric acid whereas 18(100%) patients in hemorrhagic stroke had normal uric acid level. Uric acid level was elevated in ischemic stroke than haemorrhagic stroke patients (p<0.001). High uric acid level may be considered as a risk factor in patients with acute ischemic stroke. PMID:27277350

  20. Ethanol Effects On Physiological Retinoic Acid Levels

    PubMed Central

    Napoli, Joseph L.

    2011-01-01

    Summary All-trans-retinoic acid (atRA) serves essential functions during embryogenesis and throughout post-natal vertebrate life. Insufficient or excess atRA causes teratogenic and/or toxic effects in the developing embryo: interference with atRA biosynthesis or signaling likely underlies some forms of cancer. Many symptoms of vitamin A (atRA precursor) deficiency and/or toxicity overlap with those of another pleiotropic agent—ethanol. These overlapping symptoms have prompted research to understand whether interference with atRA biosynthesis and/or action may explain (in part) pathology associated with excess ethanol consumption. Ethanol affects many aspects of retinoid metabolism and mechanisms of action site-specifically, but no robust data support inhibition of vitamin A metabolism, resulting in decreased atRA in vivo during normal vitamin A nutriture. Actually, ethanol either has no effect on or increases atRA at select sites. Despite this realization, insight into whether interactions between ethanol and retinoids represent cause vs. effect requires additional research. PMID:21766417

  1. [Serum uric acid levels and risk of developing preeclampsia].

    PubMed

    Corominas, Ana I; Balconi, Silvia M; Palermo, Mario; Maskin, Bernardo; Damiano, Alicia E

    2014-01-01

    It is well known that preeclampsia is associated to high uric acid levels, but the clinical assessment of this relationship is still under consideration. Our research was to evaluate if periodic doses of uric acid during pregnancy might help to identify a high risk group prior to the onset of preeclampsia. We conducted a retrospective investigation in 79 primary gestates with normal blood pressure and 79 women with preeclampsia who were assisted at Hospital Nacional Posadas during 2010. Serum uric acid levels, creatininemia, uremia, and proteinuria data from the clinical records of the pregnant women were considered. Uric acid levels were similar in both groups during the first half of gestation. However, as of the 20th week, uric acid increased 1.5-times in preeclamptic women with no changes in creatinine and urea, confirming that these patients had no renal complications. Furthermore, we noted that higher levels of uric acid correlated with low birth weight. We also observed that pregnant women with a family history of hypertension were more likely to develop this condition. Moreover, we did not find a direct relationship with the fetal sex or the appearance of clinical symptoms. The analytical evidence suggests that changes in uric acid concentrations may be due to metabolic alterations at the initial stages of preeclampsia. Therefore, we propose that monitoring levels of uric acid during pregnancy might contribute to the early control of this condition.

  2. A novel phytase with sequence similarity to purple acid phosphatases is expressed in cotyledons of germinating soybean seedlings.

    PubMed

    Hegeman, C E; Grabau, E A

    2001-08-01

    Phytic acid (myo-inositol hexakisphosphate) is the major storage form of phosphorus in plant seeds. During germination, stored reserves are used as a source of nutrients by the plant seedling. Phytic acid is degraded by the activity of phytases to yield inositol and free phosphate. Due to the lack of phytases in the non-ruminant digestive tract, monogastric animals cannot utilize dietary phytic acid and it is excreted into manure. High phytic acid content in manure results in elevated phosphorus levels in soil and water and accompanying environmental concerns. The use of phytases to degrade seed phytic acid has potential for reducing the negative environmental impact of livestock production. A phytase was purified to electrophoretic homogeneity from cotyledons of germinated soybeans (Glycine max L. Merr.). Peptide sequence data generated from the purified enzyme facilitated the cloning of the phytase sequence (GmPhy) employing a polymerase chain reaction strategy. The introduction of GmPhy into soybean tissue culture resulted in increased phytase activity in transformed cells, which confirmed the identity of the phytase gene. It is surprising that the soybean phytase was unrelated to previously characterized microbial or maize (Zea mays) phytases, which were classified as histidine acid phosphatases. The soybean phytase sequence exhibited a high degree of similarity to purple acid phosphatases, a class of metallophosphoesterases.

  3. Maternal folic acid supplementation to dams on marginal protein level alters brain fatty acid levels of their adult offspring.

    PubMed

    Rao, Shobha; Joshi, Sadhana; Kale, Anvita; Hegde, Mahabaleshwar; Mahadik, Sahebarao

    2006-05-01

    Studies on fetal programming of adult diseases have highlighted the importance of maternal nutrition during pregnancy. Folic acid and long-chain essential polyunsaturated fatty acids (LC-PUFAs) have independent effects on fetal growth. However, folic acid effects may also involve alteration of LC-PUFA metabolism. Because marginal deficiency of LC-PUFAs during critical periods of brain growth and development is associated with risks for adult diseases, it is highly relevant to investigate how maternal supplementation of such nutrients can alter brain fatty acid levels. We examined the impact of folic acid supplementation, conventionally used in maternal intervention, on brain essential fatty acid levels and plasma corticosterone concentrations in adult offspring at 11 months of age. Pregnant female rats from 4 groups (6 in each) were fed with casein diets either with 18 g protein/100 g diet (control diet) or treatment diets that were marginal in protein (MP), such as 12 g protein/100 g diet supplemented with 8 mg folic acid (FAS/MP), 12 g protein/100 g diet without folic acid (FAD/MP), or 12 g protein/100 g diet (MP) with 2 mg folic acid. Pups were weaned to a standard laboratory diet with 18 g protein/100 g diet. All male adult offspring in the FAS/MP group showed lower docosahexaenoic acid (P<.05) as compared with control adult offspring (6.04+/-2.28 vs 10.33+/-0.86 g/100 g fatty acids) and higher n-6/n-3 ratio (P<.05). Docosahexaenoic acid levels in FAS/MP adult offspring were also lower (P<.05) when compared with the MP group. Plasma corticosterone concentrations were higher (P<.05) in male adult offspring from the FAS/MP group compared with control as well as the MP adult offspring. Results suggest that maternal folic acid supplementation at MP intake decreased brain docosahexaenoic acid levels probably involving corticosterone increase. PMID:16631439

  4. Maternal folic acid supplementation to dams on marginal protein level alters brain fatty acid levels of their adult offspring.

    PubMed

    Rao, Shobha; Joshi, Sadhana; Kale, Anvita; Hegde, Mahabaleshwar; Mahadik, Sahebarao

    2006-05-01

    Studies on fetal programming of adult diseases have highlighted the importance of maternal nutrition during pregnancy. Folic acid and long-chain essential polyunsaturated fatty acids (LC-PUFAs) have independent effects on fetal growth. However, folic acid effects may also involve alteration of LC-PUFA metabolism. Because marginal deficiency of LC-PUFAs during critical periods of brain growth and development is associated with risks for adult diseases, it is highly relevant to investigate how maternal supplementation of such nutrients can alter brain fatty acid levels. We examined the impact of folic acid supplementation, conventionally used in maternal intervention, on brain essential fatty acid levels and plasma corticosterone concentrations in adult offspring at 11 months of age. Pregnant female rats from 4 groups (6 in each) were fed with casein diets either with 18 g protein/100 g diet (control diet) or treatment diets that were marginal in protein (MP), such as 12 g protein/100 g diet supplemented with 8 mg folic acid (FAS/MP), 12 g protein/100 g diet without folic acid (FAD/MP), or 12 g protein/100 g diet (MP) with 2 mg folic acid. Pups were weaned to a standard laboratory diet with 18 g protein/100 g diet. All male adult offspring in the FAS/MP group showed lower docosahexaenoic acid (P<.05) as compared with control adult offspring (6.04+/-2.28 vs 10.33+/-0.86 g/100 g fatty acids) and higher n-6/n-3 ratio (P<.05). Docosahexaenoic acid levels in FAS/MP adult offspring were also lower (P<.05) when compared with the MP group. Plasma corticosterone concentrations were higher (P<.05) in male adult offspring from the FAS/MP group compared with control as well as the MP adult offspring. Results suggest that maternal folic acid supplementation at MP intake decreased brain docosahexaenoic acid levels probably involving corticosterone increase.

  5. 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. PMID:24338313

  6. 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.

  7. tax and rex Sequences of bovine leukaemia virus from globally diverse isolates: rex amino acid sequence more variable than tax.

    PubMed

    McGirr, K M; Buehring, G C

    2005-02-01

    Bovine leukaemia virus (BLV) is an important agricultural problem with high costs to the dairy industry. Here, we examine the variation of the tax and rex genes of BLV. The tax and rex genes share 420 bases and have overlapping reading frames. The tax gene encodes a protein that functions as a transactivator of the BLV promoter, is required for viral replication, acts on cellular promoters, and is responsible for oncogenesis. The rex facilitates the export of viral mRNAs from the nucleus and regulates transcription. We have sequenced five new isolates of the tax/rex gene. We examined the five new and three previously published tax/rex DNA and predicted amino acid sequences of BLV isolates from cattle in representative regions worldwide. The highest variation among nucleic acid sequences for tax and rex was 7% and 5%, respectively; among predicted amino acid sequences for Tax and Rex, 9% and 11%, respectively. Significantly more nucleotide changes resulted in predicted amino acid changes in the rex gene than in the tax gene (P < or = 0.0006). This variability is higher than previously reported for any region of the viral genome. This research may also have implications for the development of Tax-based vaccines. PMID:15702995

  8. A nucleic acid sequence-based amplification system for detection of Listeria monocytogenes hlyA sequences.

    PubMed Central

    Blais, B W; Turner, G; Sooknanan, R; Malek, L T

    1997-01-01

    A nucleic acid sequence-based amplification system primarily targeting mRNA from the Listeria monocytogenes hlyA gene was developed. This system enabled the detection of low numbers (< 10 CFU/g) of L. monocytogenes cells inoculated into a variety of dairy and egg products after 48 h of enrichment in modified listeria enrichment broth. PMID:8979357

  9. Sequence Design for a Test Tube of Interacting Nucleic Acid Strands.

    PubMed

    Wolfe, Brian R; Pierce, Niles A

    2015-10-16

    We describe an algorithm for designing the equilibrium base-pairing properties of a test tube of interacting nucleic acid strands. A target test tube is specified as a set of desired "on-target" complexes, each with a target secondary structure and target concentration, and a set of undesired "off-target" complexes, each with vanishing target concentration. Sequence design is performed by optimizing the test tube ensemble defect, corresponding to the concentration of incorrectly paired nucleotides at equilibrium evaluated over the ensemble of the test tube. To reduce the computational cost of accepting or rejecting mutations to a random initial sequence, the structural ensemble of each on-target complex is hierarchically decomposed into a tree of conditional subensembles, yielding a forest of decomposition trees. Candidate sequences are evaluated efficiently at the leaf level of the decomposition forest by estimating the test tube ensemble defect from conditional physical properties calculated over the leaf subensembles. As optimized subsequences are merged toward the root level of the forest, any emergent defects are eliminated via ensemble redecomposition and sequence reoptimization. After successfully merging subsequences to the root level, the exact test tube ensemble defect is calculated for the first time, explicitly checking for the effect of the previously neglected off-target complexes. Any off-target complexes that form at appreciable concentration are hierarchically decomposed, added to the decomposition forest, and actively destabilized during subsequent forest reoptimization. For target test tubes representative of design challenges in the molecular programming and synthetic biology communities, our test tube design algorithm typically succeeds in achieving a normalized test tube ensemble defect ≤1% at a design cost within an order of magnitude of the cost of test tube analysis.

  10. 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.

  11. 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.

  12. High-level phylogeny of the Coleoptera inferred with mitochondrial genome sequences.

    PubMed

    Yuan, Ming-Long; Zhang, Qi-Lin; Zhang, Li; Guo, Zhong-Long; Liu, Yong-Jian; Shen, Yu-Ying; Shao, Renfu

    2016-11-01

    The Coleoptera (beetles) exhibits tremendous morphological, ecological, and behavioral diversity. To better understand the phylogenetics and evolution of beetles, we sequenced three complete mitogenomes from two families (Cleridae and Meloidae), which share conserved mitogenomic features with other completely sequenced beetles. We assessed the influence of six datasets and three inference methods on topology and nodal support within the Coleoptera. We found that both Bayesian inference and maximum likelihood with homogeneous-site models were greatly affected by nucleotide compositional heterogeneity, while the heterogeneous-site mixture model in PhyloBayes could provide better phylogenetic signals for the Coleoptera. The amino acid dataset generated more reliable tree topology at the higher taxonomic levels (i.e. suborders and series), where the inclusion of rRNA genes and the third positions of protein-coding genes improved phylogenetic inference at the superfamily level, especially under a heterogeneous-site model. We recovered the suborder relationships as (Archostemata+Adephaga)+(Myxophaga+Polyphaga). The series relationships within Polyphaga were recovered as (Scirtiformia+(Elateriformia+((Bostrichiformia+Scarabaeiformia+Staphyliniformia)+Cucujiformia))). All superfamilies within Cucujiformia were recovered as monophyletic. We obtained a cucujiform phylogeny of (Cleroidea+(Coccinelloidea+((Lymexyloidea+Tenebrionoidea)+(Cucujoidea+(Chrysomeloidea+Curculionoidea))))). This study showed that although tree topologies were sensitive to data types and inference methods, mitogenomic data could provide useful information for resolving the Coleoptera phylogeny at various taxonomic levels by using suitable datasets and heterogeneous-site models.

  13. High-level phylogeny of the Coleoptera inferred with mitochondrial genome sequences.

    PubMed

    Yuan, Ming-Long; Zhang, Qi-Lin; Zhang, Li; Guo, Zhong-Long; Liu, Yong-Jian; Shen, Yu-Ying; Shao, Renfu

    2016-11-01

    The Coleoptera (beetles) exhibits tremendous morphological, ecological, and behavioral diversity. To better understand the phylogenetics and evolution of beetles, we sequenced three complete mitogenomes from two families (Cleridae and Meloidae), which share conserved mitogenomic features with other completely sequenced beetles. We assessed the influence of six datasets and three inference methods on topology and nodal support within the Coleoptera. We found that both Bayesian inference and maximum likelihood with homogeneous-site models were greatly affected by nucleotide compositional heterogeneity, while the heterogeneous-site mixture model in PhyloBayes could provide better phylogenetic signals for the Coleoptera. The amino acid dataset generated more reliable tree topology at the higher taxonomic levels (i.e. suborders and series), where the inclusion of rRNA genes and the third positions of protein-coding genes improved phylogenetic inference at the superfamily level, especially under a heterogeneous-site model. We recovered the suborder relationships as (Archostemata+Adephaga)+(Myxophaga+Polyphaga). The series relationships within Polyphaga were recovered as (Scirtiformia+(Elateriformia+((Bostrichiformia+Scarabaeiformia+Staphyliniformia)+Cucujiformia))). All superfamilies within Cucujiformia were recovered as monophyletic. We obtained a cucujiform phylogeny of (Cleroidea+(Coccinelloidea+((Lymexyloidea+Tenebrionoidea)+(Cucujoidea+(Chrysomeloidea+Curculionoidea))))). This study showed that although tree topologies were sensitive to data types and inference methods, mitogenomic data could provide useful information for resolving the Coleoptera phylogeny at various taxonomic levels by using suitable datasets and heterogeneous-site models. PMID:27497607

  14. Update of PROFEAT: a web server for computing structural and physicochemical features of proteins and peptides from amino acid sequence.

    PubMed

    Rao, H B; Zhu, F; Yang, G B; Li, Z R; Chen, Y Z

    2011-07-01

    Sequence-derived structural and physicochemical features have been extensively used for analyzing and predicting structural, functional, expression and interaction profiles of proteins and peptides. PROFEAT has been developed as a web server for computing commonly used features of proteins and peptides from amino acid sequence. To facilitate more extensive studies of protein and peptides, numerous improvements and updates have been made to PROFEAT. We added new functions for computing descriptors of protein-protein and protein-small molecule interactions, segment descriptors for local properties of protein sequences, topological descriptors for peptide sequences and small molecule structures. We also added new feature groups for proteins and peptides (pseudo-amino acid composition, amphiphilic pseudo-amino acid composition, total amino acid properties and atomic-level topological descriptors) as well as for small molecules (atomic-level topological descriptors). Overall, PROFEAT computes 11 feature groups of descriptors for proteins and peptides, and a feature group of more than 400 descriptors for small molecules plus the derived features for protein-protein and protein-small molecule interactions. Our computational algorithms have been extensively tested and used in a number of published works for predicting proteins of specific structural or functional classes, protein-protein interactions, peptides of specific functions and quantitative structure activity relationships of small molecules. PROFEAT is accessible free of charge at http://bidd.cz3.nus.edu.sg/cgi-bin/prof/protein/profnew.cgi.

  15. Association of exome sequences with plasma C-reactive protein levels in >9000 participants

    PubMed Central

    Schick, Ursula M.; Auer, Paul L.; Bis, Joshua C.; Lin, Honghuang; Wei, Peng; Pankratz, Nathan; Lange, Leslie A.; Brody, Jennifer; Stitziel, Nathan O.; Kim, Daniel S.; Carlson, Christopher S.; Fornage, Myriam; Haessler, Jeffery; Hsu, Li; Jackson, Rebecca D.; Kooperberg, Charles; Leal, Suzanne M.; Psaty, Bruce M.; Boerwinkle, Eric; Tracy, Russell; Ardissino, Diego; Shah, Svati; Willer, Cristen; Loos, Ruth; Melander, Olle; Mcpherson, Ruth; Hovingh, Kees; Reilly, Muredach; Watkins, Hugh; Girelli, Domenico; Fontanillas, Pierre; Chasman, Daniel I.; Gabriel, Stacey B.; Gibbs, Richard; Nickerson, Deborah A.; Kathiresan, Sekar; Peters, Ulrike; Dupuis, Josée; Wilson, James G.; Rich, Stephen S.; Morrison, Alanna C.; Benjamin, Emelia J.; Gross, Myron D.; Reiner, Alex P.

    2015-01-01

    C-reactive protein (CRP) concentration is a heritable systemic marker of inflammation that is associated with cardiovascular disease risk. Genome-wide association studies have identified CRP-associated common variants associated in ∼25 genes. Our aims were to apply exome sequencing to (1) assess whether the candidate loci contain rare coding variants associated with CRP levels and (2) perform an exome-wide search for rare variants in novel genes associated with CRP levels. We exome-sequenced 6050 European-Americans (EAs) and 3109 African-Americans (AAs) from the NHLBI-ESP and the CHARGE consortia, and performed association tests of sequence data with measured CRP levels. In single-variant tests across candidate loci, a novel rare (minor allele frequency = 0.16%) CRP-coding variant (rs77832441-A; p.Thr59Met) was associated with 53% lower mean CRP levels (P = 2.9 × 10−6). We replicated the association of rs77832441 in an exome array analysis of 11 414 EAs (P = 3.0 × 10−15). Despite a strong effect on CRP levels, rs77832441 was not associated with inflammation-related phenotypes including coronary heart disease. We also found evidence for an AA-specific association of APOE-ε2 rs7214 with higher CRP levels. At the exome-wide significance level (P < 5.0 × 10−8), we confirmed associations for reported common variants of HNF1A, CRP, IL6R and TOMM40-APOE. In gene-based tests, a burden of rare/lower frequency variation in CRP in EAs (P ≤ 6.8 × 10−4) and in retinoic acid receptor-related orphan receptor α (RORA) in AAs (P = 1.7 × 10−3) were associated with CRP levels at the candidate gene level (P < 2.0 × 10−3). This inquiry did not elucidate novel genes, but instead demonstrated that variants distributed across the allele frequency spectrum within candidate genes contribute to CRP levels. PMID:25187575

  16. The amino acid sequence of elephant (Elephas maximus) myoglobin and the phylogeny of Proboscidea.

    PubMed

    Dene, H; Goodman, M; Romero-Herrera, A E

    1980-02-13

    The complete amino acid sequence of skeletal myoglobin from the Asian elephant (Elephas maximus) is reported. The functional significance of variations seen when this sequence is compared with that of sperm whale myoglobin is explored in the light of the crystallographic model available for the latter molecule. The phylogenetic implications of the elephant myoglobin amino acid sequence are evaluated by using the maximum parsimony technique. A similar analysis is also presented which incorporates all of the proteins sequenced from the elephant. These results are discussed with respect to current views on proboscidean phylogeny.

  17. An expanded sequence context model broadly explains variability in polymorphism levels across the human genome.

    PubMed

    Aggarwala, Varun; Voight, Benjamin F

    2016-04-01

    The rate of single-nucleotide polymorphism varies substantially across the human genome and fundamentally influences evolution and incidence of genetic disease. Previous studies have only considered the immediately flanking nucleotides around a polymorphic site--the site's trinucleotide sequence context--to study polymorphism levels across the genome. Moreover, the impact of larger sequence contexts has not been fully clarified, even though context substantially influences rates of polymorphism. Using a new statistical framework and data from the 1000 Genomes Project, we demonstrate that a heptanucleotide context explains >81% of variability in substitution probabilities, highlighting new mutation-promoting motifs at ApT dinucleotide, CAAT and TACG sequences. Our approach also identifies previously undocumented variability in C-to-T substitutions at CpG sites, which is not immediately explained by differential methylation intensity. Using our model, we present informative substitution intolerance scores for genes and a new intolerance score for amino acids, and we demonstrate clinical use of the model in neuropsychiatric diseases. PMID:26878723

  18. An expanded sequence context model broadly explains variability in polymorphism levels across the human genome

    PubMed Central

    Aggarwala, Varun; Voight, Benjamin F.

    2016-01-01

    The rate of single nucleotide polymorphism varies substantially across the human genome and fundamentally influences evolution and incidence of genetic disease. Previous studies have only considered the immediate flanking nucleotides around a polymorphic site –the site’s trinucleotide sequence context– to study polymorph levels across the genome. Moreover, the impact of larger sequence contexts has not been fully clarified, even though context substantially influences rates of polymorphism. Using a new statistical framework and data from the 1000 Genomes Project, we demonstrate that a heptanucleotide context explains >81% of variability in substitution probabilities, revealing new mutation-promoting motifs at ApT dinucleotide, CAAT, and TACG sequences. Our approach also identifies previously undocumented variability in C-to-T substitutions at CpG sites, which is not immediately explained by differential methylation intensity. Using our model, we present informative substitution intolerance scores for genes and a new intolerance score for amino acids, and we demonstrate clinical use of the model in neuropsychiatric diseases. PMID:26878723

  19. An expanded sequence context model broadly explains variability in polymorphism levels across the human genome.

    PubMed

    Aggarwala, Varun; Voight, Benjamin F

    2016-04-01

    The rate of single-nucleotide polymorphism varies substantially across the human genome and fundamentally influences evolution and incidence of genetic disease. Previous studies have only considered the immediately flanking nucleotides around a polymorphic site--the site's trinucleotide sequence context--to study polymorphism levels across the genome. Moreover, the impact of larger sequence contexts has not been fully clarified, even though context substantially influences rates of polymorphism. Using a new statistical framework and data from the 1000 Genomes Project, we demonstrate that a heptanucleotide context explains >81% of variability in substitution probabilities, highlighting new mutation-promoting motifs at ApT dinucleotide, CAAT and TACG sequences. Our approach also identifies previously undocumented variability in C-to-T substitutions at CpG sites, which is not immediately explained by differential methylation intensity. Using our model, we present informative substitution intolerance scores for genes and a new intolerance score for amino acids, and we demonstrate clinical use of the model in neuropsychiatric diseases.

  20. [Hydroxycinnamic acid levels of various batches from mugwort flowering tops].

    PubMed

    Fraisse, D; Carnat, A; Carnat, A-P; Guédon, D; Lamaison, J-L

    2003-07-01

    Dried flowering tops of 24 harvested batches (Artemisia vulgaris: 13; Artemisia verlotiorum: 11) and 12 batches of mugwort from commercial origin were examined. The levels of principal compounds averaged respectively: total hydroxycinnamic acids 6.09; 10.29 and 9.13%, chlorogenic acid 0.79; 2.05 and 1.35%, 1,5-dicaffeoylquinic acid 0.51; 4.01 and 1.25%, 3,5-dicaffeoylquinic acid 2.21; 1.25 and 2.60%. Specifications were discussed for an European Pharmacopoeial monography.

  1. [Hydroxycinnamic acid levels of various batches from mugwort flowering tops].

    PubMed

    Fraisse, D; Carnat, A; Carnat, A-P; Guédon, D; Lamaison, J-L

    2003-07-01

    Dried flowering tops of 24 harvested batches (Artemisia vulgaris: 13; Artemisia verlotiorum: 11) and 12 batches of mugwort from commercial origin were examined. The levels of principal compounds averaged respectively: total hydroxycinnamic acids 6.09; 10.29 and 9.13%, chlorogenic acid 0.79; 2.05 and 1.35%, 1,5-dicaffeoylquinic acid 0.51; 4.01 and 1.25%, 3,5-dicaffeoylquinic acid 2.21; 1.25 and 2.60%. Specifications were discussed for an European Pharmacopoeial monography. PMID:12843960

  2. Facile Analysis and Sequencing of Linear and Branched Peptide Boronic Acids by MALDI Mass Spectrometry

    PubMed Central

    Crumpton, Jason; Zhang, Wenyu; Santos, Webster

    2011-01-01

    Interest in peptides incorporating boronic acid moieties is increasing due to their potential as therapeutics/diagnostics for a variety of diseases such as cancer. The utility of peptide boronic acids may be expanded with access to vast libraries that can be deconvoluted rapidly and economically. Unfortunately, current detection protocols using mass spectrometry are laborious and confounded by boronic acid trimerization, which requires time consuming analysis of dehydration products. These issues are exacerbated when the peptide sequence is unknown, as with de novo sequencing, and especially when multiple boronic acid moieties are present. Thus, a rapid, reliable and simple method for peptide identification is of utmost importance. Herein, we report the identification and sequencing of linear and branched peptide boronic acids containing up to five boronic acid groups by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Protocols for preparation of pinacol boronic esters were adapted for efficient MALDI analysis of peptides. Additionally, a novel peptide boronic acid detection strategy was developed in which 2,5-dihydroxybenzoic acid (DHB) served as both matrix and derivatizing agent in a convenient, in situ, on-plate esterification. Finally, we demonstrate that DHB-modified peptide boronic acids from a single bead can be analyzed by MALDI-MSMS analysis, validating our approach for the identification and sequencing of branched peptide boronic acid libraries. PMID:21449540

  3. Evolution of an Enzyme from a Noncatalytic Nucleic Acid Sequence.

    PubMed

    Gysbers, Rachel; Tram, Kha; Gu, Jimmy; Li, Yingfu

    2015-01-01

    The mechanism by which enzymes arose from both abiotic and biological worlds remains an unsolved natural mystery. We postulate that an enzyme can emerge from any sequence of any functional polymer under permissive evolutionary conditions. To support this premise, we have arbitrarily chosen a 50-nucleotide DNA fragment encoding for the Bos taurus (cattle) albumin mRNA and subjected it to test-tube evolution to derive a catalytic DNA (DNAzyme) with RNA-cleavage activity. After only a few weeks, a DNAzyme with significant catalytic activity has surfaced. Sequence comparison reveals that seven nucleotides are responsible for the conversion of the noncatalytic sequence into the enzyme. Deep sequencing analysis of DNA pools along the evolution trajectory has identified individual mutations as the progressive drivers of the molecular evolution. Our findings demonstrate that an enzyme can indeed arise from a sequence of a functional polymer via permissive molecular evolution, a mechanism that may have been exploited by nature for the creation of the enormous repertoire of enzymes in the biological world today. PMID:26091540

  4. Multiple Amino Acid Sequence Alignment Nitrogenase Component 1: Insights into Phylogenetics and Structure-Function Relationships

    PubMed Central

    Howard, James B.; Kechris, Katerina J.; Rees, Douglas C.; Glazer, Alexander N.

    2013-01-01

    Amino acid residues critical for a protein's structure-function are retained by natural selection and these residues are identified by the level of variance in co-aligned homologous protein sequences. The relevant residues in the nitrogen fixation Component 1 α- and β-subunits were identified by the alignment of 95 protein sequences. Proteins were included from species encompassing multiple microbial phyla and diverse ecological niches as well as the nitrogen fixation genotypes, anf, nif, and vnf, which encode proteins associated with cofactors differing at one metal site. After adjusting for differences in sequence length, insertions, and deletions, the remaining >85% of the sequence co-aligned the subunits from the three genotypes. Six Groups, designated Anf, Vnf , and Nif I-IV, were assigned based upon genetic origin, sequence adjustments, and conserved residues. Both subunits subdivided into the same groups. Invariant and single variant residues were identified and were defined as “core” for nitrogenase function. Three species in Group Nif-III, Candidatus Desulforudis audaxviator, Desulfotomaculum kuznetsovii, and Thermodesulfatator indicus, were found to have a seleno-cysteine that replaces one cysteinyl ligand of the 8Fe:7S, P-cluster. Subsets of invariant residues, limited to individual groups, were identified; these unique residues help identify the gene of origin (anf, nif, or vnf) yet should not be considered diagnostic of the metal content of associated cofactors. Fourteen of the 19 residues that compose the cofactor pocket are invariant or single variant; the other five residues are highly variable but do not correlate with the putative metal content of the cofactor. The variable residues are clustered on one side of the cofactor, away from other functional centers in the three dimensional structure. Many of the invariant and single variant residues were not previously recognized as potentially critical and their identification provides the bases

  5. 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)

  6. Isolation and amino acid sequences of squirrel monkey (Saimiri sciurea) insulin and glucagon.

    PubMed Central

    Yu, J H; Eng, J; Yalow, R S

    1990-01-01

    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 pork 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 we describe the purification and amino acid sequences of squirrel monkey insulin and glucagon. We 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 our immunoassay system is only a few percent of that of human insulin. Squirrel monkey glucagon is identical with the usual glucagon found in Old World mammals, which predicts that the glucagons of other New World monkeys would not differ from the usual Old World mammalian glucagon. 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. PMID:2263627

  7. 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.

  8. Pancreatic ribonucleases of mammals with ruminant-like digestion. Amino-acid sequences of hippopotamus and sloth ribonucleases.

    PubMed

    Havinga, J; Beintema, J J

    1980-09-01

    High levels of pancreatic ribonucleases are found in ruminants, species that have a ruminant-like digestion and several species with coecal digestion. Pancreatic ribonucleases from several independently evolved species with ruminant-like digestion were investigated to test a hypothesis that glycosylation of ribonucleases may have some function in species with coecal digestion and that glycosylation of the enzyme may not be advantageous for ruminants. Ribonucleases from the hippopotamus, two-toed sloth and three-toed sloth were isolated by extraction with sulfuric acid and affinity chromatography. Complete amino acid sequences were determined for the ribonucleases from the hippopotamus and two-toed sloth and a partial sequence for the enzyme from the three-toed sloth. The amino acids 75-78 of hippopotamus ribonuclease were positioned by homology with other artiodactyl ribonucleases. In hippopotamus ribonuclease a heterogeneity was found at position 37, half of the molecules containing glutamine acid the other half lysine. Hippopotamus ribonuclease differs less from pig and bovine ribonuclease than these differ from each other, because more ancestral characteristics have been retained. Although hippopotamus ribonuclease contains all four Asn-X-Ser/Thr sequences previously found to be glycosylation sites in one or more pancreatic ribonucleases, only the sequence Ans-Met-Thr (34-36) is glycosylated in the variant with glutamine at position 37, while the variant with lysine at this position is carbohydrate-free. Both sloth ribonucleases are completely glycosylated at the sequence Ans-Met-Thr (34-36) with a simple type of carbohydrate chain. The amino acid sequence of two-toed sloth ribonuclease shows some interesting coupled replacements.

  9. Reaction sequences in simulated neutralized current acid waste slurry during processing with formic acid

    SciTech Connect

    Smith, H.D.; Wiemers, K.D.; Langowski, M.H.; Powell, M.R.; Larson, D.E.

    1993-11-01

    The Hanford Waste Vitrification Plant (HWVP) is being designed for the Department of Energy to immobilize high-level and transuranic wastes as glass for permanent disposal. Pacific Northwest Laboratory is supporting the HWVP design activities by conducting laboratory-scale studies using a HWVP simulated waste slurry. Conditions which affect the slurry processing chemistry were evaluated in terms of offgas composition and peak generation rate and changes in slurry composition. A standard offgas profile defined in terms of three reaction phases, decomposition of H{sub 2}CO{sub 3}, destruction of NO{sub 2}{sup {minus}}, and production of H{sub 2} and NH{sub 3} was used as a baseline against which changes were evaluated. The test variables include nitrite concentration, acid neutralization capacity, temperature, and formic acid addition rate. Results to date indicate that pH is an important parameter influencing the N{sub 2}O/NO{sub x} generation ratio; nitrite can both inhibit and activate rhodium as a catalyst for formic acid decomposition to CO{sub 2} and H{sub 2}; and a separate reduced metal phase forms in the reducing environment. These data are being compiled to provide a basis for predicting the HWVP feed processing chemistry as a function of feed composition and operation variables, recommending criteria for chemical adjustments, and providing guidelines with respect to important control parameters to consider during routine and upset plant operation.

  10. 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).

  11. 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). PMID:9836434

  12. The nucleotide sequence of HLA-B{sup *}2704 reveals a new amino acid substitution in exon 4 which is also present in HLA-B{sup *}2706

    SciTech Connect

    Rudwaleit, M.; Bowness, P.; Wordsworth, P.

    1996-12-31

    The HLA-B27 subtype HLA-B{sup *}2704 is virtually absent in Caucasians but common in Orientals, where it is associated with ankylosing spondylitis. The amino acid sequence of HLA-B{sup *}2704 has been established by peptide mapping and was shown to differ by two amino acids from HLA-B{sup *}2705, HLA-B{sup *}2704 is characterized by a serine for aspartic acid substitution at position 77 and glutamic acid for valine at position 152. To date, however, no nucleotide sequence confirming these changes at the DNA level has been published. 13 refs., 2 figs.

  13. Studies on monotreme proteins. VII. Amino acid sequence of myoglobin from the platypus, Ornithoryhynchus anatinus.

    PubMed

    Fisher, W K; Thompson, E O

    1976-03-01

    Myoglobin isolated from skeletal muscle of the platypus contains 153 amino acid residues. The complete amino acid sequence has been determined following cleavage with cyanogen bromide and further digestion of the four fragments with trypsin, chymotrypsin, pepsin and thermolysin. Sequences of the purified peptides were determined by the dansyl-Edman procedure. The amino acid sequence showed 25 differences from human myoglobin and 24 from kangaroo myoglobin. Amino acid sequences in myoglobins are more conserved than sequences in the alpha- and beta-globin chains, and platypus myoglobin shows a similar number of variations in sequence to kangaroo myoglobin when compared with myoglobin of other species. The date of divergence of the platypus from other mammals was estimated at 102 +/- 31 million years, based on the number of amino acid differences between species and allowing for mutations during the evolutionary period. This estimate differs widely from the estimate given by similar treatment of the alpha- and beta-chain sequences and a constant rate of mutation of globin chains is not supported. PMID:962722

  14. 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

  15. Stability of free amino acid levels in stressed Abarenicola pacifica

    SciTech Connect

    Augenfeld, J.M.; Anderson, J.W.

    1980-01-01

    A change in the composition of the free amino acid (FAA) pool of pelecypod molluscs under stress has been described. This response consists of a significant decrease in the level of glycine, while the taurine level remains constant, leading to a decrease in the total free amino acid level and a decrease in the glycine:taurine ratio. It has been suggested that this alteration in FAA composition may be useful as an indicator of physiological stress in a wide variety of invertebrates. This suggestion has been tested by exposing a burrowing polychaete, Abarenicola pacifica, to sediment artificially contaminated with Prudhoe Bay crude oil (PBC).

  16. 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. PMID:18521668

  17. Draft Genome Sequences of Two Novel Acidimicrobiaceae Members from an Acid Mine Drainage Biofilm Metagenome

    PubMed Central

    Pinto, Ameet J.; Sharp, Jonathan O.; Yoder, Michael J.

    2016-01-01

    Bacteria belonging to the family Acidimicrobiaceae are frequently encountered in heavy metal-contaminated acidic environments. However, their phylogenetic and metabolic diversity is poorly resolved. We present draft genome sequences of two novel and phylogenetically distinct Acidimicrobiaceae members assembled from an acid mine drainage biofilm metagenome. PMID:26769942

  18. Complete Genome Sequence of Streptomyces clavuligerus F613-1, an Industrial Producer of Clavulanic Acid.

    PubMed

    Cao, Guangxiang; Zhong, Chuanqing; Zong, Gongli; Fu, Jiafang; Liu, Zhong; Zhang, Guimin; Qin, Ronghuo

    2016-01-01

    Streptomyces clavuligerus strain F613-1 is an industrial strain with high-yield clavulanic acid production. In this study, the complete genome sequence of S. clavuligerus strain F613-1 was determined, including one linear chromosome and one linear plasmid, carrying numerous sets of genes involving in the biosynthesis of clavulanic acid.

  19. Complete Genome Sequence of Streptomyces clavuligerus F613-1, an Industrial Producer of Clavulanic Acid.

    PubMed

    Cao, Guangxiang; Zhong, Chuanqing; Zong, Gongli; Fu, Jiafang; Liu, Zhong; Zhang, Guimin; Qin, Ronghuo

    2016-01-01

    Streptomyces clavuligerus strain F613-1 is an industrial strain with high-yield clavulanic acid production. In this study, the complete genome sequence of S. clavuligerus strain F613-1 was determined, including one linear chromosome and one linear plasmid, carrying numerous sets of genes involving in the biosynthesis of clavulanic acid. PMID:27660792

  20. Complete Genome Sequence of Streptomyces clavuligerus F613-1, an Industrial Producer of Clavulanic Acid

    PubMed Central

    Zhong, Chuanqing; Zong, Gongli; Fu, Jiafang; Liu, Zhong; Zhang, Guimin; Qin, Ronghuo

    2016-01-01

    Streptomyces clavuligerus strain F613-1 is an industrial strain with high-yield clavulanic acid production. In this study, the complete genome sequence of S. clavuligerus strain F613-1 was determined, including one linear chromosome and one linear plasmid, carrying numerous sets of genes involving in the biosynthesis of clavulanic acid. PMID:27660792

  1. Predicted Molecular Effects of Sequence Variants Link to System Level of Disease.

    PubMed

    Reeb, Jonas; Hecht, Maximilian; Mahlich, Yannick; Bromberg, Yana; Rost, Burkhard

    2016-08-01

    Developments in experimental and computational biology are advancing our understanding of how protein sequence variation impacts molecular protein function. However, the leap from the micro level of molecular function to the macro level of the whole organism, e.g. disease, remains barred. Here, we present new results emphasizing earlier work that suggested some links from molecular function to disease. We focused on non-synonymous single nucleotide variants, also referred to as single amino acid variants (SAVs). Building upon OMIA (Online Mendelian Inheritance in Animals), we introduced a curated set of 117 disease-causing SAVs in animals. Methods optimized to capture effects upon molecular function often correctly predict human (OMIM) and animal (OMIA) Mendelian disease-causing variants. We also predicted effects of human disease-causing variants in the mouse model, i.e. we put OMIM SAVs into mouse orthologs. Overall, fewer variants were predicted with effect in the model organism than in the original organism. Our results, along with other recent studies, demonstrate that predictions of molecular effects capture some important aspects of disease. Thus, in silico methods focusing on the micro level of molecular function can help to understand the macro system level of disease. PMID:27536940

  2. Predicted Molecular Effects of Sequence Variants Link to System Level of Disease

    PubMed Central

    Bromberg, Yana; Rost, Burkhard

    2016-01-01

    Developments in experimental and computational biology are advancing our understanding of how protein sequence variation impacts molecular protein function. However, the leap from the micro level of molecular function to the macro level of the whole organism, e.g. disease, remains barred. Here, we present new results emphasizing earlier work that suggested some links from molecular function to disease. We focused on non-synonymous single nucleotide variants, also referred to as single amino acid variants (SAVs). Building upon OMIA (Online Mendelian Inheritance in Animals), we introduced a curated set of 117 disease-causing SAVs in animals. Methods optimized to capture effects upon molecular function often correctly predict human (OMIM) and animal (OMIA) Mendelian disease-causing variants. We also predicted effects of human disease-causing variants in the mouse model, i.e. we put OMIM SAVs into mouse orthologs. Overall, fewer variants were predicted with effect in the model organism than in the original organism. Our results, along with other recent studies, demonstrate that predictions of molecular effects capture some important aspects of disease. Thus, in silico methods focusing on the micro level of molecular function can help to understand the macro system level of disease. PMID:27536940

  3. 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.

  4. New Insights into Poly(Lactic-co-glycolic acid) Microstructure: Using Repeating Sequence Copolymers to Decipher Complex NMR and Thermal Behavior

    PubMed Central

    Stayshich, Ryan M.; Meyer, Tara Y.

    2012-01-01

    Sequence, which Nature uses to spectacular advantage, has not been fully exploited in synthetic copolymers. To investigate the effect of sequence and stereosequence on the physical properties of copolymers a family of complex isotactic, syndiotactic and atactic repeating sequence poly(lactic-co-glycolic acid) copolymers (RSC PLGAs) were prepared and their NMR and thermal behavior was studied. The unique suitability of polymers prepared from the bioassimilable lactic and glycolic acid monomers for biomedical applications makes them ideal candidates for this type of sequence engineering. Polymers with repeating units of LG, GLG and LLG (L = lactic, G = glycolic) with controlled and varied tacticities were synthesized by assembly of sequence specific, stereopure dimeric, trimeric and hexameric segmer units. Specifically labeled deuterated lactic and glycolic acid segmers were likewise prepared and polymerized. Molecular weights for the copolymers ranged from Mn = 12-40 kDa by size exclusion chromatography in THF. Although the effects of sequence-influenced solution conformation were visible in all resonances of the 1H and 13C NMR spectra, the diastereotopic methylene resonances in the 1H NMR (CDCl3) for the glycolic units of the copolymers proved most sensitive. An octad level of resolution, which corresponds to an astounding 31-atom distance between the most separated stereocenters, was observed in some mixed sequence polymers. Importantly, the level of sensitivity of a particular NMR resonance to small differences in sequence was found to depend on the sequence itself. Thermal properties were also correlated with sequence. PMID:20681726

  5. 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.

  6. 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.

  7. Amino acid sequence of a new mitochondrially synthesized proteolipid of the ATP synthase of Saccharomyces cerevisiae.

    PubMed Central

    Velours, J; Esparza, M; Hoppe, J; Sebald, W; Guerin, B

    1984-01-01

    The purification and the amino acid sequence of a proteolipid translated on ribosomes in yeast mitochondria is reported. This protein, which is a subunit of the ATP synthase, was purified by extraction with chloroform/methanol (2/1) and subsequent chromatography on phosphocellulose and reverse phase h.p.l.c. A mol. wt. of 5500 was estimated by chromatography on Bio-Gel P-30 in 80% formic acid. The complete amino acid sequence of this protein was determined by automated solid phase Edman degradation of the whole protein and of fragments obtained after cleavage with cyanogen bromide. The sequence analysis indicates a length of 48 amino acid residues. The calculated mol. wt. of 5870 corresponds to the value found by gel chromatography. This polypeptide contains three basic residues and no negatively charged side chain. The three basic residues are clustered at the C terminus. The primary structure of this protein is in full agreement with the predicted amino acid sequence of the putative polypeptide encoded by the mitochondrial aap1 gene recently discovered in Saccharomyces cerevisiae. Moreover, this protein shows 50% homology with the amino acid sequence of a putative polypeptide encoded by an unidentified reading frame also discovered near the mitochondrial ATPase subunit 6 gene in Aspergillus nidulans. Images Fig. 2. PMID:6323165

  8. Early postnatal docosahexaenoic acid levels and improved preterm brain development

    PubMed Central

    Tam, Emily W.Y.; Chau, Vann; Barkovich, A. James; Ferriero, Donna M.; Miller, Steven P.; Rogers, Elizabeth E.; Grunau, Ruth E.; Synnes, Anne R.; Xu, Duan; Foong, Justin; Brant, Rollin; Innis, Sheila M.

    2015-01-01

    Background Preterm birth has a dramatic impact on polyunsaturated fatty acid exposures for the developing brain. This study examined the association between postnatal fatty acid levels and measures of brain injury and development, as well as outcomes. Methods A cohort of 60 preterm newborns (24–32 weeks GA) was assessed using early and near-term MRI studies. Red blood cell fatty acid composition was analyzed coordinated with each scan. Outcome at a mean of 33 months corrected age was assessed using the Bayley Scales of Infant Development, 3rd edition. Results Adjusting for confounders, a 1% increase in postnatal docosahexaenoic acid (DHA) levels at early MRI was associated with 4.3-fold decreased odds of intraventricular hemorrhage, but was not associated with white matter injury or cerebellar haemorrhage. Higher DHA and lower linoleic acid (LA) levels at early MRI were associated with lower diffusivity in white matter tracts, and corresponding improved developmental scores in follow-up. Conclusion Higher DHA and lower LA levels in the first few weeks of life are associated with decreased IVH, improved microstructural brain development, and improved outcomes in preterm born children. Early, and possibly antenatal, intervention in high-risk pregnancies needs to be studied for potential benefits in preterm developmental outcomes. PMID:26761122

  9. 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. PMID:2708331

  10. Homology of amino acid sequences of rat liver cathepsins B and H with that of papain.

    PubMed Central

    Takio, K; Towatari, T; Katunuma, N; Teller, D C; Titani, K

    1983-01-01

    The amino acid sequences of rat liver lysosomal thiol endopeptidases, cathepsins B and H, are presented and compared with that of the plant thiol protease papain. The 252-residue sequence of cathepsin B and the 220-residue sequence of cathepsin H were determined largely by automated Edman degradation of their intact polypeptide chains and of the two chains of each enzyme generated by limited proteolysis. Subfragments of the chains were produced by enzymatic digestion and by chemical cleavage of methionyl and tryptophanyl bonds. Comparison of the amino acid sequences of cathepsins B and H with each other and with that of papain demonstrates a striking homology among their primary structures. Sequence identity is extremely high in regions which, according to the three-dimensional structure of papain, constitute the catalytic site. The results not only reveal the first structural features of mammalian thiol endopeptidases but also provide insight into the evolutionary relationships among plant and mammalian thiol proteases. PMID:6574504

  11. Specialized Mathematics Courses, Secondary Level: Learning Objectives, Scope and Sequence.

    ERIC Educational Resources Information Center

    Lehigh County Community Coll., Schnecksville, PA.

    In this document, detailed objectives for the following courses are listed: Algebra I, Algebra II, Geometry, Trigonometry, Analytic Geometry, Advanced Mathematics, Personal Business Mathematics, and Business Mathematics. Each objective is keyed to an expected level of achievement: awareness, knowledge, application, mastery, reinforcement, and…

  12. Association of Renal Manifestations with Serum Uric Acid in Korean Adults with Normal Uric Acid Levels

    PubMed Central

    Jung, Dong-Hyuk; Lee, Yong-Jae; Lee, Hye-Ree; Lee, Jung-Hyun

    2010-01-01

    Several studies have reported that hyperuricemia is associated with the development of hypertension and cardiovascular disease. Increasing evidences also suggest that hyperuricemia may have a pathogenic role in the progression of renal disease. Paradoxically, uric acid is also widely accepted to have antioxidant activity in experimental studies. We aimed to investigate the association between glomerular filtration rate (GFR) and uric acid in healthy individuals with a normal serum level of uric acid. We examined renal function determined by GFR and uric acid in 3,376 subjects (1,896 men; 1,480 women; aged 20-80 yr) who underwent medical examinations at Gangnam Severance Hospital from November 2006 to June 2007. Determinants for renal function and uric acid levels were also investigated. In both men and women, GFR was negatively correlated with systolic and diastolic blood pressures, fasting plasma glucose, total cholesterol, uric acid, log transformed C reactive protein, and log transformed triglycerides. In multivariate regression analysis, total uric acid was found to be an independent factor associated with estimated GFR in both men and women. This result suggests that uric acid appears to contribute to renal impairment in subjects with normal serum level of uric acid. PMID:21165292

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

    PubMed Central

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

    1987-01-01

    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 approximately equal to 40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share approximately 40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and approximately 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. Images PMID:3110773

  14. 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.

  15. DNA Sequence and Expression Variation of Hop (Humulus lupulus) Valerophenone Synthase (VPS), a Key Gene in Bitter Acid Biosynthesis

    PubMed Central

    Castro, Consuelo B.; Whittock, Lucy D.; Whittock, Simon P.; Leggett, Grey; Koutoulis, Anthony

    2008-01-01

    Background The hop plant (Humulus lupulus) is a source of many secondary metabolites, with bitter acids essential in the beer brewing industry and others having potential applications for human health. This study investigated variation in DNA sequence and gene expression of valerophenone synthase (VPS), a key gene in the bitter acid biosynthesis pathway of hop. Methods Sequence variation was studied in 12 varieties, and expression was analysed in four of the 12 varieties in a series across the development of the hop cone. Results Nine single nucleotide polymorphisms (SNPs) were detected in VPS, seven of which were synonymous. The two non-synonymous polymorphisms did not appear to be related to typical bitter acid profiles of the varieties studied. However, real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of VPS expression during hop cone development showed a clear link with the bitter acid content. The highest levels of VPS expression were observed in two triploid varieties, ‘Symphony’ and ‘Ember’, which typically have high bitter acid levels. Conclusions In all hop varieties studied, VPS expression was lowest in the leaves and an increase in expression was consistently observed during the early stages of cone development. PMID:18519445

  16. Influence of educational level on determinants of folic acid use.

    PubMed

    van der Pal-de Bruin, Karin M; de Walle, Hermien E K; de Rover, Carolien M; Jeeninga, Wendy; Cornel, Martina C; de Jong-van den Berg, Lolkje T W; Buitendijk, Simone E; Paulussen, Theo G W M

    2003-07-01

    In The Netherlands, periconceptional folic acid use to prevent neural tube defects was promoted through a national 'Folic Acid Campaign'. In two regions, a local campaign supplemented the national campaign to increase the chances of reaching women with low socio-economic status (SES). A framework of outcome criteria, defined as awareness knowledge, perceived safety, attitudes and subjective norms, was developed to evaluate the effectiveness of the two local campaigns. Data were gathered by means of two cross-sectional studies conducted just before and 1 year after the campaigns took place. Before the campaigns were conducted, there were already differences in all effect criteria and folic acid use between women of different educational levels, mostly in favour of women with a high level of education. Although both educational campaigns appeared to have a positive impact on all outcome criteria, they failed to reduce the existing differences in these outcome criteria between women of different educational levels. Folic acid use can be promoted effectively by mass media campaigns, certainly in a large group of women with no prior knowledge of the health benefits associated with periconceptional folic acid use. However, in order to achieve more equal health outcomes among women of low and high SES, it seems that more tailored interventions for women of low SES are needed.

  17. Amino acid sequence heterogeneity of the chromosomal encoded Borrelia burgdorferi sensu lato major antigen P100.

    PubMed

    Fellinger, W; Farencena, A; Redl, B; Sambri, V; Cevenini, R; Stöffler, G

    1995-04-01

    The entire nucleotide sequence of the chromosomal encoded major antigen p100 of the European Borrelia garinii isolate B29 was determined and the deduced amino acid sequence was compared to the homologous antigen p83 of the North American Borrelia burgdorferi sensu stricto strain B31 and the p100 of the European Borrelia afzelii (group VS461) strain PKo. p100 of strain B29 shows 87% amino acid sequence identity to strain B31 and 79.2% to strain PKo, p100 of strain B31 and PKo shows 62.5% identity to each other. In addition, partial nucleotide sequences of the most heterogeneous region of the p100 gene of two other Borrelia garinii isolates (PBi and VS286) have been determined and the deduced amino acid sequences were compared with all p100 of Borrelia garinii published so far. We found an amino acid sequence identity between 88.6 and 100% within the same genospecies. The N-terminal part of the p100 proteins is highly conserved whereas a striking heterogeneous region within the C-terminal part of the proteins was observed.

  18. Increased isoprostane levels in oleic acid-induced lung injury

    SciTech Connect

    Ono, Koichi; Koizumi, Tomonobu; Tsushima, Kenji; Yoshikawa, Sumiko; Yokoyama, Toshiki; Nakagawa, Rikimaru; Obata, Toru

    2009-10-16

    The present study was performed to examine a role of oxidative stress in oleic acid-induced lung injury model. Fifteen anesthetized sheep were ventilated and instrumented with a lung lymph fistula and vascular catheters for blood gas analysis and measurement of isoprostanes (8-epi prostaglandin F2{alpha}). Following stable baseline measurements, oleic acid (0.08 ml/kg) was administered and observed 4 h. Isoprostane was measured by gas chromatography mass spectrometry with the isotope dilution method. Isoprostane levels in plasma and lung lymph were significantly increased 2 h after oleic acid administration and then decreased at 4 h. The percent increases in isoprostane levels in plasma and lung lymph at 2 h were significantly correlated with deteriorated oxygenation at the same time point, respectively. These findings suggest that oxidative stress is involved in the pathogenesis of the pulmonary fat embolism-induced acute lung injury model in sheep and that the increase relates with the deteriorated oxygenation.

  19. Relationship between Uric Acid Level and Achievement Motivation. Final Report.

    ERIC Educational Resources Information Center

    Mueller, Ernst F.; French, John R. P., Jr.

    In an investigation of the relationship of uric acid (a metabolic end product) to achievement, this study hypothesized that a person's serum urate level (a factor often associated with gout) is positively related to achievement need as well as indicators of actual achievement. (Speed of promotion and number of yearly publications were chosen as…

  20. Rational design of translational pausing without altering the amino acid sequence dramatically promotes soluble protein expression: a strategic demonstration.

    PubMed

    Chen, Wei; Jin, Jingjie; Gu, Wei; Wei, Bo; Lei, Yun; Xiong, Sheng; Zhang, Gong

    2014-11-10

    The production of many pharmaceutical and industrial proteins in prokaryotic hosts is hindered by the insolubility of industrial expression products resulting from misfolding. Even with a correct primary sequence, an improper translation elongation rate in a heterologous expression system is an important cause of misfolding. In silico analysis revealed that most of the endogenous Escherichia coli genes display translational pausing sites that promote correct folding, and almost 1/5 genes have pausing sites at the 3'-termini of their coding sequence. Therefore, we established a novel strategy to efficiently promote the expression of soluble and active proteins without altering the amino acid sequence or expression conditions. This strategy uses the rational design of translational pausing based on structural information solely through synonymous substitutions, i.e. no change on the amino acids sequence. We demonstrated this strategy on a promising antiviral candidate, Cyanovirin-N (CVN), which could not be efficiently expressed in any previously reported system. By introducing silent mutations, we increased the soluble expression level in E. coli by 2000-fold without altering the CVN protein sequence, and the specific activity was slightly higher for the optimized CVN than for the wild-type variant. This strategy introduces new possibilities for the production of bioactive recombinant proteins.

  1. 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.

  2. 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.

  3. Evolution of vertebrate IgM: complete amino acid sequence of the constant region of Ambystoma mexicanum mu chain deduced from cDNA sequence.

    PubMed

    Fellah, J S; Wiles, M V; Charlemagne, J; Schwager, J

    1992-10-01

    cDNA clones coding for the constant region of the Mexican axolotl (Ambystoma mexicanum) mu heavy immunoglobulin chain were selected from total spleen RNA, using a cDNA polymerase chain reaction technique. The specific 5'-end primer was an oligonucleotide homologous to the JH segment of Xenopus laevis mu chain. One of the clones, JHA/3, corresponded to the complete constant region of the axolotl mu chain, consisting of a 1362-nucleotide sequence coding for a polypeptide of 454 amino acids followed in 3' direction by a 179-nucleotide untranslated region and a polyA+ tail. The axolotl C mu is divided into four typical domains (C mu 1-C mu 4) and can be aligned with the Xenopus C mu with an overall identity of 56% at the nucleotide level. Percent identities were particularly high between C mu 1 (59%) and C mu 4 (71%). The C-terminal 20-amino acid segment which constitutes the secretory part of the mu chain is strongly homologous to the equivalent sequences of chondrichthyans and of other tetrapods, including a conserved N-linked oligosaccharide, the penultimate cysteine and the C-terminal lysine. The four C mu domains of 13 vertebrate species ranging from chondrichthyans to mammals were aligned and compared at the amino acid level. The significant number of mu-specific residues which are conserved into each of the four C mu domains argues for a continuous line of evolution of the vertebrate mu chain. This notion was confirmed by the ability to reconstitute a consistent vertebrate evolution tree based on the phylogenic parsimony analysis of the C mu 4 sequences. PMID:1382992

  4. 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. PMID:22449695

  5. The amino acid sequence of mitogenic lectin-B from the roots of pokeweed (Phytolacca americana).

    PubMed

    Yamaguchi, K; Yurino, N; Kino, M; Ishiguro, M; Funatsu, G

    1997-04-01

    The complete amino acid sequence of pokeweed lectin-B (PL-B) has been analyzed by first sequencing seven lysylendopeptidase peptides derived from the reduced and S-pyridylethylated PL-B and then connecting them by analyzing the arginylendopeptidase peptides from the reduced and S-carboxymethylated PL-B. PL-B consists of 295 amino acid residues and two oligosaccharides linked to Asn96 and Asn139, and has a molecular mass of 34,493 Da. PL-B is composed of seven repetitive chitin-binding domains having 48-79% sequence homology with each other. Twelve amino acid residues including eight cysteine residues in these domains are absolutely conserved in all other chitin-binding domains of plant lectins and class I chitinases. Also, it was strongly suggested that the extremely high hemagglutinating and mitogenic activities of PL-B may be ascribed to its seven-domain structure.

  6. ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids.

    PubMed

    Ashkenazy, Haim; Erez, Elana; Martz, Eric; Pupko, Tal; Ben-Tal, Nir

    2010-07-01

    It is informative to detect highly conserved positions in proteins and nucleic acid sequence/structure since they are often indicative of structural and/or functional importance. ConSurf (http://consurf.tau.ac.il) and ConSeq (http://conseq.tau.ac.il) are two well-established web servers for calculating the evolutionary conservation of amino acid positions in proteins using an empirical Bayesian inference, starting from protein structure and sequence, respectively. Here, we present the new version of the ConSurf web server that combines the two independent servers, providing an easier and more intuitive step-by-step interface, while offering the user more flexibility during the process. In addition, the new version of ConSurf calculates the evolutionary rates for nucleic acid sequences. The new version is freely available at: http://consurf.tau.ac.il/.

  7. Amino acid repeats cause extraordinary coding sequence variation in the social amoeba Dictyostelium discoideum.

    PubMed

    Scala, Clea; Tian, Xiangjun; Mehdiabadi, Natasha J; Smith, Margaret H; Saxer, Gerda; Stephens, Katie; Buzombo, Prince; Strassmann, Joan E; Queller, David C

    2012-01-01

    Protein sequences are normally the most conserved elements of genomes owing to purifying selection to maintain their functions. We document an extraordinary amount of within-species protein sequence variation in the model eukaryote Dictyostelium discoideum stemming from triplet DNA repeats coding for long strings of single amino acids. D. discoideum has a very large number of such strings, many of which are polyglutamine repeats, the same sequence that causes various human neurological disorders in humans, like Huntington's disease. We show here that D. discoideum coding repeat loci are highly variable among individuals, making D. discoideum a candidate for the most variable proteome. The coding repeat loci are not significantly less variable than similar non-coding triplet repeats. This pattern is consistent with these amino-acid repeats being largely non-functional sequences evolving primarily by mutation and drift. PMID:23029418

  8. 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.

  9. Shark myoglobins. II. Isolation, characterization and amino acid sequence of myoglobin from Galeorhinus japonicus.

    PubMed

    Suzuki, T; Suzuki, T; Yata, T

    1985-01-01

    Native oxymyoglobin (MbO2) was isolated from red muscle of G. japonicus by chromatographic separation from metmyoglobin (metMb) on DEAE-cellulose and the amino acid sequence of the major chain was determined with the aid of sequence homology with that of G. australis. It was shown to differ in amino acid sequence from that of G. australis by 10 replacements, to be acetylated at the amino terminus and to contain glutamine at the distal (E7) residue. It was also shown to have a spectrum very similar to that of mammalian MbO2. However, the pH-dependence for the autoxidation of MbO2 was seen to be quite different from that of sperm whale (Physeter catodon) MbO2. Although the sequence homology between sperm whale and G. japonicus myoglobins is about 40%, their hydropathy profiles were very similar, indicating that they have a similar geometry in their globin folding.

  10. Puget Sound acidity levels drop after ASARCO shutdown

    SciTech Connect

    Not Available

    1987-07-01

    The levels of acidity in Puget Sound region rainfall have decreased significantly since the shutdown of the ASARCO copper smelter in Tacoma, Washington, according to a study funded by the US Environmental Protection Agency. Results indicate that sulfate and hydrogen ion concentrations obtained from samples taken before the closure were significantly different than those collected after the shutdown. Rainwater samples collected downwind during smelter operation were also significantly different from those collected upwind. Sulfur dioxide is considered to be one of the principal contributors to acid rain. The smelter was a major source of sulfur dioxide emissions in the Puget Sound region before it shut down in March 1985.

  11. Conversion of amino-acid sequence in proteins to classical music: search for auditory patterns

    PubMed Central

    2007-01-01

    We have converted genome-encoded protein sequences into musical notes to reveal auditory patterns without compromising musicality. We derived a reduced range of 13 base notes by pairing similar amino acids and distinguishing them using variations of three-note chords and codon distribution to dictate rhythm. The conversion will help make genomic coding sequences more approachable for the general public, young children, and vision-impaired scientists. PMID:17477882

  12. Visible sensing of nucleic acid sequences using a genetically encodable unmodified mRNA probe.

    PubMed

    Narita, Atsushi; Ogawa, Kazumasa; Sando, Shinsuke; Aoyama, Yasuhiro

    2006-01-01

    We previously reported a molecular beacon-mRNA (MB-mRNA) strategy for nucleic acid detection/sensing in a cell-free translation system using unmodified RNA as a probe. Here in this presentation, we report that a combination with RNase H activity, which induces an additional process of irreversible cleavage of MB-domain, achieves an improved sequence selectivity (one nucleotide selectivity) and an enhanced sensitivity. This improved system finally enabled visible sensing of target nucleic acid sequence at a single nucleotide resolution under isothermal conditions.

  13. High Level Waste System Impacts from Acid Dissolution of Sludge

    SciTech Connect

    KETUSKY, EDWARD

    2006-04-20

    This research evaluates the ability of OLI{copyright} equilibrium based software to forecast Savannah River Site High Level Waste system impacts from oxalic acid dissolution of Tank 1-15 sludge heels. Without further laboratory and field testing, only the use of oxalic acid can be considered plausible to support sludge heel dissolution on multiple tanks. Using OLI{copyright} and available test results, a dissolution model is constructed and validated. Material and energy balances, coupled with the model, identify potential safety concerns. Overpressurization and overheating are shown to be unlikely. Corrosion induced hydrogen could, however, overwhelm the tank ventilation. While pH adjustment can restore the minimal hydrogen generation, resultant precipitates will notably increase the sludge volume. OLI{copyright} is used to develop a flowsheet such that additional sludge vitrification canisters and other negative system impacts are minimized. Sensitivity analyses are used to assess the processability impacts from variations in the sludge/quantities of acids.

  14. Altered Serum Uric Acid Level in Lichen Planus Patients

    PubMed Central

    Chakraborti, Goutam; Biswas, Rabindranath; Chakraborti, Sandip; Sen, Pradyot Kumar

    2014-01-01

    Background: Lichen planus (LP) is a common disorder whose etiopathogenesis is not clear. Recently, it has been suggested that increased reactive oxygen species (ROS) play important roles in the underlying mechanism of LP. Objectives: The principal aim of this study was to evaluate serum uric acid (UA) levels as a measure of the antioxidant defense status in LP patients. Methods: Serum UA levels were determined in 58 LP patients and 61 controls. Results: Serum UA levels were significantly decreased in patients with respect to controls. Moreover, serum UA level was decreased according to increasing duration of disease. Conclusions: The results of our study suggest that LP is associated with decrease of UA levels in serum. UA may be a potential, useful biomarker of antioxidant status in LP for elaboration of treatment strategy and monitoring. PMID:25484383

  15. 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.

  16. AcalPred: a sequence-based tool for discriminating between acidic and alkaline enzymes.

    PubMed

    Lin, Hao; Chen, Wei; Ding, Hui

    2013-01-01

    The structure and activity of enzymes are influenced by pH value of their surroundings. Although many enzymes work well in the pH range from 6 to 8, some specific enzymes have good efficiencies only in acidic (pH<5) or alkaline (pH>9) solution. Studies have demonstrated that the activities of enzymes correlate with their primary sequences. It is crucial to judge enzyme adaptation to acidic or alkaline environment from its amino acid sequence in molecular mechanism clarification and the design of high efficient enzymes. In this study, we developed a sequence-based method to discriminate acidic enzymes from alkaline enzymes. The analysis of variance was used to choose the optimized discriminating features derived from g-gap dipeptide compositions. And support vector machine was utilized to establish the prediction model. In the rigorous jackknife cross-validation, the overall accuracy of 96.7% was achieved. The method can correctly predict 96.3% acidic and 97.1% alkaline enzymes. Through the comparison between the proposed method and previous methods, it is demonstrated that the proposed method is more accurate. On the basis of this proposed method, we have built an online web-server called AcalPred which can be freely accessed from the website (http://lin.uestc.edu.cn/server/AcalPred). We believe that the AcalPred will become a powerful tool to study enzyme adaptation to acidic or alkaline environment.

  17. AcalPred: A Sequence-Based Tool for Discriminating between Acidic and Alkaline Enzymes

    PubMed Central

    Lin, Hao; Chen, Wei; Ding, Hui

    2013-01-01

    The structure and activity of enzymes are influenced by pH value of their surroundings. Although many enzymes work well in the pH range from 6 to 8, some specific enzymes have good efficiencies only in acidic (pH<5) or alkaline (pH>9) solution. Studies have demonstrated that the activities of enzymes correlate with their primary sequences. It is crucial to judge enzyme adaptation to acidic or alkaline environment from its amino acid sequence in molecular mechanism clarification and the design of high efficient enzymes. In this study, we developed a sequence-based method to discriminate acidic enzymes from alkaline enzymes. The analysis of variance was used to choose the optimized discriminating features derived from g-gap dipeptide compositions. And support vector machine was utilized to establish the prediction model. In the rigorous jackknife cross-validation, the overall accuracy of 96.7% was achieved. The method can correctly predict 96.3% acidic and 97.1% alkaline enzymes. Through the comparison between the proposed method and previous methods, it is demonstrated that the proposed method is more accurate. On the basis of this proposed method, we have built an online web-server called AcalPred which can be freely accessed from the website (http://lin.uestc.edu.cn/server/AcalPred). We believe that the AcalPred will become a powerful tool to study enzyme adaptation to acidic or alkaline environment. PMID:24130738

  18. B-MIC: An Ultrafast Three-Level Parallel Sequence Aligner Using MIC.

    PubMed

    Cui, Yingbo; Liao, Xiangke; Zhu, Xiaoqian; Wang, Bingqiang; Peng, Shaoliang

    2016-03-01

    Sequence alignment is the central process for sequence analysis, where mapping raw sequencing data to reference genome. The large amount of data generated by NGS is far beyond the process capabilities of existing alignment tools. Consequently, sequence alignment becomes the bottleneck of sequence analysis. Intensive computing power is required to address this challenge. Intel recently announced the MIC coprocessor, which can provide massive computing power. The Tianhe-2 is the world's fastest supercomputer now equipped with three MIC coprocessors each compute node. A key feature of sequence alignment is that different reads are independent. Considering this property, we proposed a MIC-oriented three-level parallelization strategy to speed up BWA, a widely used sequence alignment tool, and developed our ultrafast parallel sequence aligner: B-MIC. B-MIC contains three levels of parallelization: firstly, parallelization of data IO and reads alignment by a three-stage parallel pipeline; secondly, parallelization enabled by MIC coprocessor technology; thirdly, inter-node parallelization implemented by MPI. In this paper, we demonstrate that B-MIC outperforms BWA by a combination of those techniques using Inspur NF5280M server and the Tianhe-2 supercomputer. To the best of our knowledge, B-MIC is the first sequence alignment tool to run on Intel MIC and it can achieve more than fivefold speedup over the original BWA while maintaining the alignment precision.

  19. The value of short amino acid sequence matches for prediction of protein allergenicity.

    PubMed

    Silvanovich, Andre; Nemeth, Margaret A; Song, Ping; Herman, Rod; Tagliani, Laura; Bannon, Gary A

    2006-03-01

    Typically, genetically engineered crops contain traits encoded by one or a few newly expressed proteins. The allergenicity assessment of newly expressed proteins is an important component in the safety evaluation of genetically engineered plants. One aspect of this assessment involves sequence searches that compare the amino acid sequence of the protein to all known allergens. Analyses are performed to determine the potential for immunologically based cross-reactivity where IgE directed against a known allergen could bind to the protein and elicit a clinical reaction in sensitized individuals. Bioinformatic searches are designed to detect global sequence similarity and short contiguous amino acid sequence identity. It has been suggested that potential allergen cross-reactivity may be predicted by identifying matches as short as six to eight contiguous amino acids between the protein of interest and a known allergen. A series of analyses were performed, and match probabilities were calculated for different size peptides to determine if there was a scientifically justified search window size that identified allergen sequence characteristics. Four probability modeling methods were tested: (1) a mock protein and a mock allergen database, (2) a mock protein and genuine allergen database, (3) a genuine allergen and genuine protein database, and (4) a genuine allergen and genuine protein database combined with a correction for repeating peptides. These analyses indicated that searches for short amino acid sequence matches of eight amino acids or fewer to identify proteins as potential cross-reactive allergens is a product of chance and adds little value to allergy assessments for newly expressed proteins.

  20. Comparison of the amino acid sequence of the major immunogen from three serotypes of foot and mouth disease virus.

    PubMed Central

    Makoff, A J; Paynter, C A; Rowlands, D J; Boothroyd, J C

    1982-01-01

    Cloned cDNA molecules from three serotypes of FMDV have been sequenced around the VP1-coding region. The predicted amino acid sequences for VP1 were compared with the published sequences and variable regions identified. The amino acid sequences were also analysed for hydrophilic regions. Two of the variable regions, numbered 129-160 and 193-204 overlapped hydrophilic regions, and were therefore identified as potentially immunogenic. These regions overlap regions shown by others to be immunogenic. PMID:6298715

  1. Non-redundant patent sequence databases with value-added annotations at two levels.

    PubMed

    Li, Weizhong; McWilliam, Hamish; de la Torre, Ana Richart; Grodowski, Adam; Benediktovich, Irina; Goujon, Mickael; Nauche, Stephane; Lopez, Rodrigo

    2010-01-01

    The European Bioinformatics Institute (EMBL-EBI) provides public access to patent data, including abstracts, chemical compounds and sequences. Sequences can appear multiple times due to the filing of the same invention with multiple patent offices, or the use of the same sequence by different inventors in different contexts. Information relating to the source invention may be incomplete, and biological information available in patent documents elsewhere may not be reflected in the annotation of the sequence. Search and analysis of these data have become increasingly challenging for both the scientific and intellectual-property communities. Here, we report a collection of non-redundant patent sequence databases, which cover the EMBL-Bank nucleotides patent class and the patent protein databases and contain value-added annotations from patent documents. The databases were created at two levels by the use of sequence MD5 checksums. Sequences within a level-1 cluster are 100% identical over their whole length. Level-2 clusters were defined by sub-grouping level-1 clusters based on patent family information. Value-added annotations, such as publication number corrections, earliest publication dates and feature collations, significantly enhance the quality of the data, allowing for better tracking and cross-referencing. The databases are available format: http://www.ebi.ac.uk/patentdata/nr/.

  2. Quantitative detection of Aspergillus spp. by real-time nucleic acid sequence-based amplification.

    PubMed

    Zhao, Yanan; Perlin, David S

    2013-01-01

    Rapid and quantitative detection of Aspergillus from clinical samples may facilitate an early diagnosis of invasive pulmonary aspergillosis (IPA). As nucleic acid-based detection is a viable option, we demonstrate that Aspergillus burdens can be rapidly and accurately detected by a novel real-time nucleic acid assay other than qPCR by using the combination of nucleic acid sequence-based amplification (NASBA) and the molecular beacon (MB) technology. Here, we detail a real-time NASBA assay to determine quantitative Aspergillus burdens in lungs and bronchoalveolar lavage (BAL) fluids of rats with experimental IPA.

  3. Draft Genome Sequence of the Butyric Acid Producer Clostridium tyrobutyricum Strain CIP I-776 (IFP923)

    PubMed Central

    Clément, Benjamin; Lopes Ferreira, Nicolas

    2016-01-01

    Here, we report the draft genome sequence of Clostridium tyrobutyricum CIP I-776 (IFP923), an efficient producer of butyric acid. The genome consists of a single chromosome of 3.19 Mb and provides useful data concerning the metabolic capacities of the strain. PMID:26941139

  4. Amino acid sequence of the encephalitogenic basic protein from human myelin

    PubMed Central

    Carnegie, P. R.

    1971-01-01

    Myelin from the central nervous system contains an unusual basic protein, which can induce experimental autoimmune encephalomyelitis. The basic protein from human brain was digested with trypsin and other enzymes and the sequence of the 170 amino acids was determined. The localization of the encephalitogenic determinants was described. Possible roles for the protein in the structure and function of myelin are discussed. PMID:4108501

  5. Sequence-specific formation of d-amino acids in a monoclonal antibody during light exposure.

    PubMed

    Mozziconacci, Olivier; Schöneich, Christian

    2014-11-01

    The photoirradiation of a monoclonal antibody 1 (mAb1) at λ = 254 nm and λmax = 305 nm resulted in the sequence-specific generation of d-Val, d-Tyr, and potentially d-Ala and d-Arg, in the heavy chain sequence [95-101] YCARVVY. d-Amino acid formation is most likely the product of reversible intermediary carbon-centered radical formation at the (α)C-positions of the respective amino acids ((α)C(•) radicals) through the action of Cys thiyl radicals (CysS(•)). The latter can be generated photochemically either through direct homolysis of cystine or through photoinduced electron transfer from Trp and/or Tyr residues. The potential of mAb1 sequences to undergo epimerization was first evaluated through covalent H/D exchange during photoirradiation in D2O, and proteolytic peptides exhibiting deuterium incorporation were monitored by HPLC-MS/MS analysis. Subsequently, mAb1 was photoirradiated in H2O, and peptides, for which deuterium incorporation in D2O had been documented, were purified by HPLC and subjected to hydrolysis and amino acid analysis. Importantly, not all peptide sequences which incorporated deuterium during photoirradiation in D2O also exhibited photoinduced d-amino acid formation. For example, the heavy chain sequence [12-18] VQPGGSL showed significant deuterium incorporation during photoirradiation in D2O, but no photoinduced formation of d-amino acids was detected. Instead this sequence contained ca. 22% d-Val in both a photoirradiated and a control sample. This observation could indicate that d-Val may have been generated either during production and/or storage or during sample preparation. While sample preparation did not lead to the formation of d-Val or other d-amino acids in the control sample for the heavy chain sequence [95-101] YCARVVY, we may have to consider that during hydrolysis N-terminal residues (such as in VQPGGSL) may be more prone to epimerization. We conclude that the photoinduced, radical-dependent formation of d-amino acids

  6. The complete amino acid sequence of chitinase-B from the leaves of pokeweed (Phytolacca americana).

    PubMed

    Tanigawa, M; Yamagami, T; Funatsu, G

    1995-05-01

    The complete amino acid sequence of pokeweed leaf chitinase-B (PLC-B) has been determined by first sequencing all 19 tryptic peptides derived from the reduced and S-carboxymethylated (RCm-) PLC-B and then connecting them by analyzing the chymotryptic peptides from three fragments produced by cyanogen bromide cleavage of RCm-PLC-B. PLC-B consists of 274 amino acid residues and has a molecular mass of 29,473 Da. Six cysteine residues are linked by disulfide bonds between Cys20 and Cys67, Cys50 and Cys57, and Cys159 and Cys188. From 58-68% sequence homology of PLC-B with five class III chitinases, it was concluded that PLC-B is a basic class III chitinase.

  7. Does high serum uric acid level cause aspirin resistance?

    PubMed

    Yildiz, Bekir S; Ozkan, Emel; Esin, Fatma; Alihanoglu, Yusuf I; Ozkan, Hayrettin; Bilgin, Murat; Kilic, Ismail D; Ergin, Ahmet; Kaftan, Havane A; Evrengul, Harun

    2016-06-01

    In patients with coronary artery disease (CAD), though aspirin inhibits platelet activation and reduces atherothrombotic complications, it does not always sufficiently inhibit platelet function, thereby causing a clinical situation known as aspirin resistance. As hyperuricemia activates platelet turnover, aspirin resistance may be specifically induced by increased serum uric acid (SUA) levels. In this study, we thus investigated the association between SUA level and aspirin resistance in patients with CAD. We analyzed 245 consecutive patients with stable angina pectoris (SAP) who in coronary angiography showed more than 50% occlusion in a major coronary artery. According to aspirin resistance, two groups were formed: the aspirin resistance group (Group 1) and the aspirin-sensitive group (Group 2). Compared with those of Group 2, patients with aspirin resistance exhibited significantly higher white blood cell counts, neutrophil counts, neutrophil-to-lymphocyte ratios, SUA levels, high-sensitivity C-reactive protein levels, and fasting blood glucose levels. After multivariate analysis, a high level of SUA emerged as an independent predictor of aspirin resistance. The receiver-operating characteristic analysis provided a cutoff value of 6.45 mg/dl for SUA to predict aspirin resistance with 79% sensitivity and 65% specificity. Hyperuricemia may cause aspirin resistance in patients with CAD and high SUA levels may indicate aspirin-resistant patients. Such levels should thus recommend avoiding heart attack and stroke by adjusting aspirin dosage. PMID:26656902

  8. Life-history evolution at the molecular level: adaptive amino acid composition of avian vitellogenins

    PubMed Central

    Hughes, Austin L.

    2015-01-01

    Avian genomes typically encode three distinct vitellogenin (VTG) egg yolk proteins (VTG1, VTG2 and VTG3), which arose by gene duplication prior to the most recent common ancestor of birds. Analysis of VTG sequences from 34 avian species in a phylogenetic framework supported the hypothesis that VTG amino acid composition has co-evolved with embryo incubation time. Embryo incubation time was positively correlated with the proportions of dietary essential amino acids (EAAs) in VTG1 and VTG2, and with the proportion of sulfur-containing amino acids in VTG3. These patterns were seen even when only semi-altricial and/or altricial species were considered, suggesting that the duration of embryo incubation is a major selective factor on the amino acid composition of VTGs, rather than developmental mode alone. The results are consistent with the hypothesis that the level of EAAs provided to the egg represents an adaptation to the loss of amino acids through breakdown over the course of incubation and imply that life-history phenotypes and VTG amino acid composition have co-evolved throughout the evolutionary history of birds. PMID:26224713

  9. Fad7 gene identification and fatty acids phenotypic variation in an olive collection by EcoTILLING and sequencing approaches.

    PubMed

    Sabetta, Wilma; Blanco, Antonio; Zelasco, Samanta; Lombardo, Luca; Perri, Enzo; Mangini, Giacomo; Montemurro, Cinzia

    2013-08-01

    The ω-3 fatty acid desaturases (FADs) are enzymes responsible for catalyzing the conversion of linoleic acid to α-linolenic acid localized in the plastid or in the endoplasmic reticulum. In this research we report the genotypic and phenotypic variation of Italian Olea europaea L. germoplasm for the fatty acid composition. The phenotypic oil characterization was followed by the molecular analysis of the plastidial-type ω-3 FAD gene (fad7) (EC 1.14.19), whose full-length sequence has been here identified in cultivar Leccino. The gene consisted of 2635 bp with 8 exons and 5'- and 3'-UTRs of 336 and 282 bp respectively, and showed a high level of heterozygousity (1/110 bp). The natural allelic variation was investigated both by a LiCOR EcoTILLING assay and the PCR product direct sequencing. Only three haplotypes were identified among the 96 analysed cultivars, highlighting the strong degree of conservation of this gene. PMID:23685785

  10. Concerted evolution at the population level: pupfish HindIII satellite DNA sequences.

    PubMed Central

    Elder, J F; Turner, B J

    1994-01-01

    The canonical monomers (approximately 170 bp) of an abundant (1.9 x 10(6) copies per diploid genome) satellite DNA sequence family in the genome of Cyprinodon variegatus, a "pupfish" that ranges along the Atlantic coast from Cape Cod to central Mexico, are divergent in base sequence in 10 of 12 samples collected from natural populations. The divergence involves substitutions, deletions, and insertions, is marked in scope (mean pairwise sequence similarity = 61.6%; range = 35-95.9%), is largely confined to the 3' half of the monomer, and is not correlated with the distance among collecting sites. Repetitive cloning and direct genomic sequencing experiments failed to detect intrapopulation and intraindividual variation, suggesting high levels of sequence homogeneity within populations. The satellite sequence has therefore undergone "concerted evolution," at the level of the local population. Concerted evolution has previously almost always been discussed in terms of the divergence of species or higher taxa; its intraspecific occurrence apparently has not been reported previously. The generality of the observation is difficult to evaluate, for although satellite DNAs from a large number of organisms have been studied in detail, there appear to be little or no other data on their sequence variation in natural populations. The relationship (if any) between concerted, population level, satellite DNA divergence and the extent of gene flow/genetic isolation among conspecific natural populations remains to be established. Images PMID:8302879

  11. Allelic polymorphism in arabian camel ribonuclease and the amino acid sequence of bactrian camel ribonuclease.

    PubMed

    Welling, G W; Mulder, H; Beintema, J J

    1976-04-01

    Pancreatic ribonucleases from several species (whitetail deer, roe deer, guinea pig, and arabian camel) exhibit more than one amino acid at particular positions in their amino acid sequences. Since these enzymes were isolated from pooled pancreas, the origin of this heterogeneity is not clear. The pancreatic ribonucleases from 11 individual arabian camels (Camelus dromedarius) have been investigated with respect to the lysine-glutamine heterogeneity at position 103 (Welling et al., 1975). Six ribonucleases showed only one basic band and five showed two bands after polyacrylamide gel electrophoresis, suggesting a gene frequency of about 0.75 for the Lys gene and about 0.25 for the Gln gene. The amino acid sequence of bactrian camel (Camelus bactrianus) ribonuclease isolated from individual pancreatic tissue was determined and compared with that of arabian camel ribonuclease. The only difference was observed at position 103. In the ribonucleases from two unrelated bactrian camels, only glutamine was observed at that position. PMID:962846

  12. Serum fluoride and sialic acid levels in osteosarcoma.

    PubMed

    Sandhu, R; Lal, H; Kundu, Z S; Kharb, S

    2011-12-01

    Osteosarcoma is a rare malignant bone tumor most commonly occurring in children and young adults presenting with painful swelling. Various etiological factors for osteosarcoma are ionizing radiation, family history of bone disorders and cancer, chemicals (fluoride, beryllium, and vinyl chloride), and viruses. Status of fluoride levels in serum of osteosarcoma is still not clear. Recent reports have indicated that there is a link between fluoride exposure and osteosarcoma. Glycoproteins and glycosaminoglycans are an integral part of bone and prolonged exposure to fluoride for long duration has been shown to cause degradation of collagen and ground substance in bones. The present study was planned to analyze serum fluoride, sialic acid, calcium, phosphorus, and alkaline phosphatase levels in 25 patients of osteosarcoma and age- and sex-matched subjects with bone-forming tumours other than osteosarcoma and musculo-skeletal pain (controls, 25 each). Fluoride levels were analyzed by ISE and sialic acid was analyzed by Warren's method. Mean serum fluoride concentration was found to be significantly higher in patients with osteosarcoma as compared to the other two groups. The mean value of flouride in patients with other bone-forming tumors was approximately 50% of the group of osteosarcoma; however, it was significantly higher when compared with patients of group I. Serum sialic acid concentration was found to be significantly raised in patients with osteosarcoma as well as in the group with other bone-forming tumors as compared to the group of controls. There was, however, no significant difference in the group of patients of osteosarcoma when compared with group of patients with other bone-forming tumors. These results showing higher level of fluoride with osteosarcoma compared to others suggesting a role of fluoride in the disease.

  13. Prognostic Significance of Uric Acid Levels in Ischemic Stroke Patients.

    PubMed

    Zhang, Xia; Huang, Zhi-Chao; Lu, Tao-Sheng; You, Shou-Jiang; Cao, Yong-Jun; Liu, Chun-Feng

    2016-01-01

    The importance and function of serum uric acid (UA) levels in patients with cardiovascular disease or stroke are unclear. We sought to evaluate the appropriate UA levels for stroke patients and the association between endogenous UA levels and clinical outcomes in acute ischemic stroke (AIS) patients, particularly regarding the possible interaction between gender and UA levels with respect to AIS prognosis. We examined 303 patients who had an onset of ischemic stroke within 48 h. Of those, 101 patients received thrombolytic treatment. Serum UA (μmol/L) levels were measured the second morning after admission. Patient prognosis was evaluated 90 days after clinical onset by modified Rankin Scale. Patients were divided into four groups according to serum UA quartiles. A binary multivariate logistic regression model was used to assess clinical relevance in regard to functional outcome and endogenous UA levels. Analysis of subgroups by gender and normal glomerular filtration rate were also been done. Poor functional outcome was associated with older age, history of atrial fibrillation, or higher baseline National Institutes of Health Stroke Scale scores. After adjustment for potential confounders, patients with higher UA levels (>380 μmol/L) or lower UA levels (≤250 μmol/L) were 2-3 times more likely to have a poor outcome (OR 2.95, 95% CI 1.14-7.61; OR 2.78, 95% CI 1.02-7.58, respectively) compared to the baseline group (UA level 316-380 μmol/L). The same results were observed in thrombolyzed patients. Patients with high and low UA levels were 9-18 times more likely to having poor outcomes compared to the baseline group (UA level: 316-380 μmol/L; OR 18.50, 95% CI: 2.041-167.67; OR 9.66, 95% CI 1.42-65.88, respectively). In men, patients with high UA levels were 6 times more likely to have poor outcomes compared to the baseline group (UA level: 279-334 μmol/L; OR 6.10, 95% CI 1.62-22.93). However, female patients with UA level 271-337 μmol/L were seven times more

  14. Nucleotide sequence of Crithidia fasciculata cytosol 5S ribosomal ribonucleic acid.

    PubMed

    MacKay, R M; Gray, M W; Doolittle, W F

    1980-11-11

    The complete nucleotide sequence of the cytosol 5S ribosomal ribonucleic acid of the trypanosomatid protozoan Crithidia fasciculata has been determined by a combination of T1-oligonucleotide catalog and gel sequencing techniques. The sequence is: GAGUACGACCAUACUUGAGUGAAAACACCAUAUCCCGUCCGAUUUGUGAAGUUAAGCACC CACAGGCUUAGUUAGUACUGAGGUCAGUGAUGACUCGGGAACCCUGAGUGCCGUACUCCCOH. This 5S ribosomal RNA is unique in having GAUU in place of the GAAC or GAUC found in all other prokaryotic and eukaryotic 5S RNAs, and thought to be involved in interactions with tRNAs. Comparisons to other eukaryotic cytosol 5S ribosomal RNA sequences indicate that the four major eukaryotic kingdoms (animals, plants, fungi, and protists) are about equally remote from each other, and that the latter kingdom may be the most internally diverse.

  15. Pattern recognition in nucleic acid sequences. II. An efficient method for finding locally stable secondary structures.

    PubMed Central

    Kanehisa, M I; Goad, W B

    1982-01-01

    We present a method for calculating all possible single hairpin loop secondary structures in a nucleic acid sequence by the order of N2 operations where N is the total number of bases. Each structure may contain any number of bulges and internal loops. Most natural sequences are found to be indistinguishable from random sequences in the potential of forming secondary structures, which is defined by the frequency of possible secondary structures calculated by the method. There is a strong correlation between the higher G+C content and the higher structure forming potential. Interestingly, the removal of intervening sequences in mRNAs is almost always accompanied by an increase in the G+C content, which may suggest an involvement of structural stabilization in the mRNA maturation. PMID:6174936

  16. Light-Induced Alterations in Basil Ganglia Kynurenic Acid Levels

    NASA Technical Reports Server (NTRS)

    Sroufe, Angela E.; Whittaker, J. A.; Patrickson, J. W.; Orr, M. C.

    1997-01-01

    The metabolic synthesis, release and breakdown of several known CNS neurotransmitters have been shown to follow a circadian pattern entrained to the environmental light/dark cycle. The levels of excitatory amino acid (EAA) transmitters such as glutamate, have been shown to vary with environmental lighting conditions. Kynurenic Acid (KA), an endogenous tryptophan metabolite and glutamate receptor antagonist, has been reported to have neuroprotective effects against EAA-induced excitotoxic cell damage. Changes in KA's activity within the mammalian basal ganglia has been proposed as being contributory to neurotoxicity in Huntington's Disease. It is not known whether CNS KA levels follow a circadian pattern or exhibit light-induced fluctuations. However, because the symptoms of certain degenerative motor disorders seem to fluctuate with daily 24 hour rhythm, we initiated studies to determine if basal ganglia KA were influenced by the daily light/dark cycle and could influence motor function. Therefore in this study, HPLC-EC was utilized to determine if basal ganglia KA levels in tissue extracts from adult male Long-Evans rats (200-250g) entrained to 24 and 48 hours constant light and dark conditions, respectively. Samples were taken one hour before the onset of the subjective day and one hour prior to the onset of the subjective night in order to detect possible phase differences in KA levels and to allow for accumulation of factors expressed in association with the light or dark phase. Data analysis revealed that KA levels in the basal ganglia vary with environmental lighting conditions; being elevated generally during the dark. Circadian phase differences in KA levels were also evident during the subjective night and subjective day, respectively. Results from these studies are discussed with respect to potential cyclic changes in neuronal susceptibility to excitotoxic damage during the daily 24 hour cycle and its possible relevance to future therapeutic approaches in

  17. Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization.

    PubMed

    Anahtar, Melis N; Bowman, Brittany A; Kwon, Douglas S

    2016-01-01

    There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information. PMID:27168460

  18. Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

    PubMed Central

    Anahtar, Melis N.; Bowman, Brittany A.; Kwon, Douglas S.

    2016-01-01

    There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information. PMID:27168460

  19. Design of nucleic acid sequences for DNA computing based on a thermodynamic approach.

    PubMed

    Tanaka, Fumiaki; Kameda, Atsushi; Yamamoto, Masahito; Ohuchi, Azuma

    2005-01-01

    We have developed an algorithm for designing multiple sequences of nucleic acids that have a uniform melting temperature between the sequence and its complement and that do not hybridize non-specifically with each other based on the minimum free energy (DeltaG (min)). Sequences that satisfy these constraints can be utilized in computations, various engineering applications such as microarrays, and nano-fabrications. Our algorithm is a random generate-and-test algorithm: it generates a candidate sequence randomly and tests whether the sequence satisfies the constraints. The novelty of our algorithm is that the filtering method uses a greedy search to calculate DeltaG (min). This effectively excludes inappropriate sequences before DeltaG (min) is calculated, thereby reducing computation time drastically when compared with an algorithm without the filtering. Experimental results in silico showed the superiority of the greedy search over the traditional approach based on the hamming distance. In addition, experimental results in vitro demonstrated that the experimental free energy (DeltaG (exp)) of 126 sequences correlated well with DeltaG (min) (|R| = 0.90) than with the hamming distance (|R| = 0.80). These results validate the rationality of a thermodynamic approach. We implemented our algorithm in a graphic user interface-based program written in Java.

  20. 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.

  1. 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)

    SciTech Connect

    Patel, Kamlesh D; SNL,

    2012-06-01

    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.

  2. 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)

  3. 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.

  4. Complete amino acid sequence of a human monocyte chemoattractant, a putative mediator of cellular immune reactions.

    PubMed Central

    Robinson, E A; Yoshimura, T; Leonard, E J; Tanaka, S; Griffin, P R; Shabanowitz, J; Hunt, D F; Appella, E

    1989-01-01

    In a study of the structural basis for leukocyte specificity of chemoattractants, we determined the complete amino acid sequence of human glioma-derived monocyte chemotactic factor (GDCF-2), a peptide that attracts human monocytes but not neutrophils. The choice of a tumor cell product for analysis was dictated by its relative abundance and an amino acid composition indistinguishable from that of lymphocyte-derived chemotactic factor (LDCF), the agonist thought to account for monocyte accumulation in cellular immune reactions. By a combination of Edman degradation and mass spectrometry, it was established that GDCF-2 comprises 76 amino acid residues, commencing at the N terminus with pyroglutamic acid. The peptide contains four half-cystines, at positions 11, 12, 36, and 52, which create a pair of loops, clustered at the disulfide bridges. The relative positions of the half-cystines are almost identical to those of monocyte-derived neutrophil chemotactic factor (MDNCF), a peptide of similar mass but with only 24% sequence identity to GDCF. Thus, GDCF and MDNCF have a similar gross secondary structure because of the loops formed by the clustered disulfides, and their different leukocyte specificities are most likely determined by the large differences in primary sequence. PMID:2648385

  5. Amino acid sequences of lower vertebrate parvalbumins and their evolution: parvalbumins of boa, turtle, and salamander.

    PubMed

    Maeda, N; Zhu, D X; Fitch, W M

    1984-11-01

    One major parvalbumin each was isolated from the skeletal muscle of two reptiles, a boa snake, Boa constrictor, and a map turtle, Graptemys geographica, while two parvalbumins were isolated from an amphibian, the salamander Amphiuma means. The amino acid sequences of all four parvalbumins were determined from the sequences of their tryptic peptides, which were ordered partially by homology to other parvalbumins. Phylogenetic study of these and 16 other parvalbumin sequences revealed that the turtle parvalbumin belongs to beta lineage, while the salamander sequences belong, one each, to the alpha and beta lineages defined by Goodman and Pechère (1977). Boa parvalbumin, however, while belonging to the beta lineage, clusters within the fish in all reasonably parsimonious trees. The most parsimonious trees show many parallel or back mutations in the evolution of many parvalbumin residues, although the residues responsible for Ca2+ binding are very well conserved. These most parsimonious trees show an actinopterygian rather than a crossoptyrigian origin of the tetrapods in both the alpha and beta groups. One of two electric eel parvalbumins is evolving more than 10 times faster than its paralogous partner, suggesting it may be on its way to becoming a pseudogene. It is concluded that varying rates of amino acid replacement, much homoplasy, considerable gene duplication, plus complicated lineages make the set of parvalbumin sequences unsuitable for systematic study of the origin of the tetrapods and other higher-taxa divergence, although it may be suitable within a genus or family.

  6. Sequence analysis of four acidic beta-crystallin subunits of amphibian lenses: phylogenetic comparison between beta- and gamma-crystallins.

    PubMed

    Lu, S F; Pan, F M; Chiou, S H

    1996-04-16

    beta-Crystallins composed of the most heterogeneous group of subunit chains among the three major crystallin families of vertebrates, i.e. alpha-, beta- and gamma-crystallins, are less well understood at the structural and functional levels than the other two. They comprise a multigene family with at least three basic (betaB1-3) and four acidic (betaA1-4) subunit polypeptides. In order to facilitate the determination of the primary sequences of all these ubiquitous crystallin subunits present in all vertebrate species, cDNA mixture was synthesized from the poly(A)+ mRNA isolated from bullfrog eye lenses. We report here a protocol of Rapid Amplification of cDNA Ends (RACE) was used to amplify cDNAs encoding beta-crystallin acidic subunit polypeptides by polymerase chain reaction (PCR). Four complete full-length reading frames with two each of 597 and 648 base pairs, which cover four deduced protein sequences of 198 (betaA1-1 and betaA1-2) and 215 (betaA3-1 and betaA3-2) amino acids including the universal initiating methionine, were revealed by nucleotide sequencing. They show about 96-98% sequence similarity among themselves and 76-80%, 80-83% to the homologous betaA1/A3 crystallins of bovine and human species respectively, revealing the close structural relationship among acidic subunits of all beta-crystallins even from remotely related species. In this study a phylogenetic comparison based on amino-acid sequences of various betaA1/A3 crystallins plus the major basic beta-crystallin (betaBp) and gamma-crystallin from different vertebrate species is made using a combination of distance matrix and approximate parsimony methods, which correctly groups these betaA crystallin chains together as one family distinct from basic beta-crystallins and gamma-crystallin and further corroborates the supposition that beta- and gamma-crystallins form a superfamily with a common ancestry.

  7. 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.

  8. Nucleotide and amino acid sequences of human intestinal alkaline phosphatase: close homology to placental alkaline phosphatase

    SciTech Connect

    Henthorn, P.S.; Raducha, M.; Edwards, Y.H.; Weiss, M.J.; Slaughter, C.; Lafferty, M.A.; Harris, H.

    1987-03-01

    A cDNA clone for human adult intestinal alkaline phosphatase (ALP) (orthophosphoric-monoester phosphohydrolase (alkaline optimum); EC 3.1.3.1) was isolated from a lambdagt11 expression library. The cDNA insert of this clone is 2513 base pairs in length and contains an open reading frame that encodes a 528-amino acid polypeptide. This deduced polypeptide contains the first 40 amino acids of human intestinal ALP, as determined by direct protein sequencing. Intestinal ALP shows 86.5% amino acid identity to placental (type 1) ALP and 56.6% amino acid identity to liver/bone/kidney ALP. In the 3'-untranslated regions, intestinal and placental ALP cDNAs are 73.5% identical (excluding gaps). The evolution of this multigene enzyme family is discussed.

  9. Association between serum folic acid level and erectile dysfunction.

    PubMed

    Karabakan, M; Erkmen, A E; Guzel, O; Aktas, B K; Bozkurt, A; Akdemir, S

    2016-06-01

    This study measured the serum folic acid (FA) level in patients with erectile dysfunction (ED) and evaluated the possible association between the serum FA level and erectile function. The study divided 120 patients with ED into 3 groups of 40 patients each: those with severe, moderate and mild ED. Forty healthy men served as controls. Fasting serum samples were obtained, and the total testosterone, cholesterol and FA levels were measured using chemiluminescent immunoassays. There were no significant differences in the mean age, mean body mass index or mean serum total testosterone and cholesterol levels among the three ED groups and controls (P > 0.05). The mean serum FA concentrations were 7.2 ± 3.7, 7.1 ± 3.2, 10.2 ± 4.6 and 10.7 ± 4.6 ng ml(-1) in the severe, moderate and mild ED and control groups respectively. The mean serum FA concentration was significantly higher in the control group than in the severe and moderate ED groups (both P < 0.001), but not the mild ED group (P = 0.95). Considering the significant differences in the serum FA levels between the control and ED groups, serum FA deficiency might reflect the severity of ED.

  10. Ocean acidification increases fatty acids levels of larval fish.

    PubMed

    Díaz-Gil, Carlos; Catalán, Ignacio A; Palmer, Miquel; Faulk, Cynthia K; Fuiman, Lee A

    2015-07-01

    Rising levels of anthropogenic carbon dioxide in the atmosphere are acidifying the oceans and producing diverse and important effects on marine ecosystems, including the production of fatty acids (FAs) by primary producers and their transfer through food webs. FAs, particularly essential FAs, are necessary for normal structure and function in animals and influence composition and trophic structure of marine food webs. To test the effect of ocean acidification (OA) on the FA composition of fish, we conducted a replicated experiment in which larvae of the marine fish red drum (Sciaenops ocellatus) were reared under a climate change scenario of elevated CO2 levels (2100 µatm) and under current control levels (400 µatm). We found significantly higher whole-body levels of FAs, including nine of the 11 essential FAs, and altered relative proportions of FAs in the larvae reared under higher levels of CO2. Consequences of this effect of OA could include alterations in performance and survival of fish larvae and transfer of FAs through food webs. PMID:26179801

  11. Ocean acidification increases fatty acids levels of larval fish

    PubMed Central

    Díaz-Gil, Carlos; Catalán, Ignacio A.; Palmer, Miquel; Faulk, Cynthia K.; Fuiman, Lee A.

    2015-01-01

    Rising levels of anthropogenic carbon dioxide in the atmosphere are acidifying the oceans and producing diverse and important effects on marine ecosystems, including the production of fatty acids (FAs) by primary producers and their transfer through food webs. FAs, particularly essential FAs, are necessary for normal structure and function in animals and influence composition and trophic structure of marine food webs. To test the effect of ocean acidification (OA) on the FA composition of fish, we conducted a replicated experiment in which larvae of the marine fish red drum (Sciaenops ocellatus) were reared under a climate change scenario of elevated CO2 levels (2100 µatm) and under current control levels (400 µatm). We found significantly higher whole-body levels of FAs, including nine of the 11 essential FAs, and altered relative proportions of FAs in the larvae reared under higher levels of CO2. Consequences of this effect of OA could include alterations in performance and survival of fish larvae and transfer of FAs through food webs. PMID:26179801

  12. Ocean acidification increases fatty acids levels of larval fish.

    PubMed

    Díaz-Gil, Carlos; Catalán, Ignacio A; Palmer, Miquel; Faulk, Cynthia K; Fuiman, Lee A

    2015-07-01

    Rising levels of anthropogenic carbon dioxide in the atmosphere are acidifying the oceans and producing diverse and important effects on marine ecosystems, including the production of fatty acids (FAs) by primary producers and their transfer through food webs. FAs, particularly essential FAs, are necessary for normal structure and function in animals and influence composition and trophic structure of marine food webs. To test the effect of ocean acidification (OA) on the FA composition of fish, we conducted a replicated experiment in which larvae of the marine fish red drum (Sciaenops ocellatus) were reared under a climate change scenario of elevated CO2 levels (2100 µatm) and under current control levels (400 µatm). We found significantly higher whole-body levels of FAs, including nine of the 11 essential FAs, and altered relative proportions of FAs in the larvae reared under higher levels of CO2. Consequences of this effect of OA could include alterations in performance and survival of fish larvae and transfer of FAs through food webs.

  13. Bovine thrombospondin-2: complete complementary deoxyribonucleic acid sequence and immunolocalization in the external zones of the adrenal cortex.

    PubMed

    Danik, M; Chinn, A M; Lafeuillade, B; Keramidas, M; Aguesse-Germon, S; Penhoat, A; Chen, H; Mosher, D F; Chambaz, E M; Feige, J J

    1999-06-01

    Given the variety of biological functions in the adrenal cortex that are controlled by ACTH, we hypothesized that some extracellular proteins act as biological relays for this systemic hormone. One candidate protein [corticotropin-induced secreted protein (CISP)] was purified from the conditioned medium of bovine adrenocortical cells on the basis of a 5- to 14-fold increase in its synthesis after the addition of ACTH. We report here the cloning of overlapping complementary DNAs that span the sequence encoding the full-length protein (1170 amino acids). The deduced CISP protein sequence is 89% identical to that of human thrombospondin-2 (TSP2), but only 61% identical to that of bovine TSP1, confirming that CISP is the bovine ortholog of TSP2. The bovine TSP2 sequence aligned perfectly with human, mouse, and chicken TSP2 sequences, except for a gap of 2 amino acids located in a linker region. All 58 cysteine residues that are conserved in other species were present in the bovine sequence as well as most of the functional domains. Most endocrine tissues (adrenal cortex, testis, ovary, and placenta) appeared to express TSP2, as determined by Western blot analysis. The highest levels of TSP2 protein were found in the adrenal cortex, followed by the heart, spleen, brain, and kidney. A differential extent of N-glycosylation or tissular proteolytic maturation may be responsible for the mol wt differences observed between bovine TSP2 detected in the medium from primary cultures and that in fresh tissue extracts. The immunohistochemical analysis of the distribution of TSP2 in the bovine adrenal gland revealed that the protein is much more abundant in the external zones (zona glomerulosa and zona fasciculata) than in the internal reticularis zone, a pattern similar to that reported for ACTH receptors. This distribution clearly suggests that TSP2 is a candidate relay protein for a subset of ACTH actions in the adrenal cortex. PMID:10342868

  14. 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.

  15. 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.

  16. The complete amino acid sequence of lectin-C from the roots of pokeweed (Phytolacca americana).

    PubMed

    Yamaguchi, K; Mori, A; Funatsu, G

    1995-07-01

    The complete amino acid sequence of pokeweed lectin-C (PL-C) consisting of 126 residues has been determined. PL-C is an acidic simple protein with molecular mass of 13,747 Da and consists of three cysteine-rich domains with 51-63% homology. PL-C shows homology to chitin-binding proteins such as wheat germ agglutinin, and all eight cysteine residues in the three domains of PL-C are completely conserved in all other chitin-binding domains.

  17. Amino-acid sequence of a cooperative, dimeric myoglobin from the gastropod mollusc, Buccinum undatum L.

    PubMed

    Wen, D; Laursen, R A

    1994-10-19

    The complete amino-acid sequence of a dimeric myoglobin from the radular mussel of the gastropod mollusc, Buccinum undatum L. has been determined. The globin, which shows cooperative binding of oxygen, contains 146 amino acids, is N-terminal aminoacetylated, and has histidine residues at position 65 and 97, corresponding to the heme-binding histidines seen in mammalian myoglobins. It shows about 75% and 50% homology, respectively, with the dimeric molluscan myoglobins from Busycon canaliculatum and Cerithidea rhizophorarum, the former of which also shows weak cooperatively, but much less similarity to other species of myoglobin and hemoglobin.

  18. What is the relationship between gestational age and docosahexaenoic acid (DHA) and arachidonic acid (ARA) levels?

    PubMed

    Baack, Michelle L; Puumala, Susan E; Messier, Stephen E; Pritchett, Deborah K; Harris, William S

    2015-09-01

    Long chain polyunsaturated fatty acids (LCPUFA) including docosahexaenoic acid (DHA) and arachidonic acid (ARA) are increasingly transferred from mother to fetus late in pregnancy. Infants born before this transfer is complete are at risk for deficiency. This study determines the relationship between gestational age (GA) and circulating LCPUFA levels to better understand the unique needs of premature infants born at various GAs. Whole blood was collected within the first 7 days of life from 60 preterm (≤34 weeks GA) and 30 term infants (≥38 weeks GA) and FA levels were analyzed. Since concurrent intravenous lipid emulsion can skew composition data, blood LCPUFA concentrations were also measured. Levels were compared among groups, and linear regression models were used to examine the association between FA composition and GA. Preterm infants had significantly lower DHA and ARA levels than term peers, and whether assessed as concentrations or compositions, both directly correlated with GA (p<0.0001). Moreover, FA comparisons suggest that premature infants have impaired synthesis of LCPUFAs from precursors and may require preformed DHA and ARA. This study confirms that essential FA status is strongly related to GA, and that those babies born the earliest are at the greatest risk of LCPUFA deficiency.

  19. What is the Relationship between Gestational Age and Docosahexaenoic Acid (DHA) and Arachidonic Acid (ARA) Levels?

    PubMed Central

    Baack, Michelle L; Puumala, Susan E; Messier, Stephen E; Pritchett, Deborah K; Harris, William S

    2015-01-01

    Long chain polyunsaturated fatty acids (LCPUFA) including docosahexaenoic acid (DHA) and arachidonic acid (ARA) are increasingly transferred from mother to fetus late in pregnancy. Infants born before this transfer is complete are at risk for deficiency. This study determines the relationship between gestational age (GA) and circulating LCPUFA levels to better understand the unique needs of premature infants born at various GAs. Whole blood was collected within the first 7 days of life from 60 preterm (≤34 weeks GA) and 30 term infants (≥38 weeks GA) and FA levels were analyzed. Since concurrent intravenous lipid emulsion can skew composition data, blood LCPUFA concentrations were also measured. Levels were compared among groups, and linear regression models were used to examine the association between FA composition and GA. Preterm infants had significantly lower DHA and ARA levels than term peers, and whether assessed as concentrations or compositions, both directly correlated with GA (p<0.0001). Moreover, FA comparisons suggest that premature infants have impaired synthesis of LCPUFAs from precursors and may require preformed DHA and ARA. This study confirms that essential FA status is strongly related to GA, and that those babies born the earliest are at the greatest risk of LCPUFA deficiency. PMID:26205427

  20. Population sequencing of two endocannabinoid metabolic genes identifies rare and common regulatory variants associated with extreme obesity and metabolite level

    PubMed Central

    2010-01-01

    Background Targeted re-sequencing of candidate genes in individuals at the extremes of a quantitative phenotype distribution is a method of choice to gain information on the contribution of rare variants to disease susceptibility. The endocannabinoid system mediates signaling in the brain and peripheral tissues involved in the regulation of energy balance, is highly active in obese patients, and represents a strong candidate pathway to examine for genetic association with body mass index (BMI). Results We sequenced two intervals (covering 188 kb) encoding the endocannabinoid metabolic enzymes fatty-acid amide hydrolase (FAAH) and monoglyceride lipase (MGLL) in 147 normal controls and 142 extremely obese cases. After applying quality filters, we called 1,393 high quality single nucleotide variants, 55% of which are rare, and 143 indels. Using single marker tests and collapsed marker tests, we identified four intervals associated with BMI: the FAAH promoter, the MGLL promoter, MGLL intron 2, and MGLL intron 3. Two of these intervals are composed of rare variants and the majority of the associated variants are located in promoter sequences or in predicted transcriptional enhancers, suggesting a regulatory role. The set of rare variants in the FAAH promoter associated with BMI is also associated with increased level of FAAH substrate anandamide, further implicating a functional role in obesity. Conclusions Our study, which is one of the first reports of a sequence-based association study using next-generation sequencing of candidate genes, provides insights into study design and analysis approaches and demonstrates the importance of examining regulatory elements rather than exclusively focusing on exon sequences. PMID:21118518

  1. 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

  2. Amino acid sequence of human cholinesterase. Annual report, 30 September 1984-30 September 1985

    SciTech Connect

    Lockridge, O.

    1985-10-01

    The active-site serine residue is located 198 amino acids from the N-terminal. The active-site peptide was isolated from three different genetic types of human serum cholinesterase: from usual, atypical, and atypical-silent genotypes. It was found that the amino acid sequence of the active-site peptide was identical in all three genotypes. Comparison of the complete sequences of cholinesterase from human serum and acetylcholinesterase from the electric organ of Torpedo californica shows an identity of 53%. Cholinesterase is of interest to the Department of Defense because cholinesterase protects against organophosphate poisons of the type used in chemical warfare. The structural results presented here will serve as the basis for cloning the gene for cholinesterase. The potential uses of large amounts of cholinesterase would be for cleaning up spills of organophosphates and possibly for detoxifying exposed personnel.

  3. Amino acid sequence differences in pancreatic ribonucleases from water buffalo breeds from Indonesia and Italy.

    PubMed

    Sidik, A; Martena, B; Beintema, J J

    1979-12-01

    The amino acid sequences of the pancreatic ribonucleases from river-breed water buffaloes from Italy and swamp-breed water buffaloes from Indonesia differ at three positions. One of the differences involves a replacement of asparagine-34, with covalently attached carbohydrate on all molecules, in the river-breed enzyme by serine in the swamp-breed enzyme. The ribonuclease content of the pancreas differs considerably between breeds and is lower in river buffaloes. A ribonuclease preparation from two swamp buffaloes contained a minor glycosylated component. Preliminary evidence was obtained that the amino acid sequence of this component has factors in common with the main component of the swamp-breed ribonuclease and with the river-breed enzyme.

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

    NASA Astrophysics Data System (ADS)

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

    2016-10-01

    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.

  5. 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.

  6. Comparisons of the Distribution of Nucleotides and Common Sequences in Deoxyribonucleic Acid from Selected Bacteriophages

    PubMed Central

    Skalka, A.; Hanson, P.

    1972-01-01

    Results from comparisons of deoxyribonucleic acid (DNA) from several classes of bacteriophages suggest that most phage chromosomes contain either a homogeneous distribution of nucleotides or are made up of a few, rather large segments of different quanine plus cytosine (G + C) contents which are internally homogeneous. Among those temperate phages tested, most contained segmented DNA. Comparisons of sequence similarities among segments from lambdoid phage DNA species revealed the following order in relatedness to λ: 82 (and 434) > 21 > 424 > φ80. Most common sequences are found in the highest G + C segments, which in λ contain head and tail genes. Hybridization tests with λ and 186 or P2 DNA species verified that the lambdoids and 186 and P2 belong to two distinct groups. There are fewer homologous sequences between the DNA species of coliphages λ and P2 or 186 than there are between the DNA species of coliphage λ and salmonella phage P22. PMID:4553679

  7. Structure of the fully modified left-handed cyclohexene nucleic acid sequence GTGTACAC.

    PubMed

    Robeyns, Koen; Herdewijn, Piet; Van Meervelt, Luc

    2008-02-13

    CeNA oligonucleotides consist of a phosphorylated backbone where the deoxyribose sugars are replaced by cyclohexene moieties. The X-ray structure determination and analysis of a fully modified octamer sequence GTGTACAC, which is the first crystal structure of a carbocyclic-based nucleic acid, is presented. This particular sequence was built with left-handed building blocks and crystallizes as a left-handed double helix. The helix can be characterized as belonging to the (mirrored) A-type family. Crystallographic data were processed up to 1.53 A, and the octamer sequence crystallizes in the space group R32. The sugar puckering is found to adopt the 3H2 half-chair conformation which mimics the C3'-endo conformation of the ribose sugar. The double helices stack on top of each other to form continuous helices, and static disorder is observed due to this end-to-end stacking.

  8. Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

    PubMed

    Papayannopoulos, I A; Biemann, K

    1992-02-01

    The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.

  9. Level sequence and splitting identification of closely spaced energy levels by angle-resolved analysis of fluorescence light

    NASA Astrophysics Data System (ADS)

    Wu, Z. W.; Volotka, A. V.; Surzhykov, A.; Dong, C. Z.; Fritzsche, S.

    2016-06-01

    The angular distribution and linear polarization of the fluorescence light following the resonant photoexcitation is investigated within the framework of density matrix and second-order perturbation theory. Emphasis has been placed on "signatures" for determining the level sequence and splitting of intermediate (partially) overlapping resonances, if analyzed as a function of photon energy of incident light. Detailed computations within the multiconfiguration Dirac-Fock method have been performed, especially for the 1 s22 s22 p63 s ,Ji=1 /2 +γ1→(1s22 s 2 p63 s ) 13 p3 /2,J =1 /2 ,3 /2 →1 s22 s22 p63 s ,Jf=1 /2 +γ2 photoexcitation and subsequent fluorescence emission of atomic sodium. A remarkably strong dependence of the angular distribution and linear polarization of the γ2 fluorescence emission is found upon the level sequence and splitting of the intermediate (1s22 s 2 p63 s ) 13 p3 /2,J =1 /2 ,3 /2 overlapping resonances owing to their finite lifetime (linewidth). We therefore suggest that accurate measurements of the angular distribution and linear polarization might help identify the sequence and small splittings of closely spaced energy levels, even if they cannot be spectroscopically resolved.

  10. Fatty Acid Profile and Unigene-Derived Simple Sequence Repeat Markers in Tung Tree (Vernicia fordii)

    PubMed Central

    Zhang, Lin; Jia, Baoguang; Tan, Xiaofeng; Thammina, Chandra S.; Long, Hongxu; Liu, Min; Wen, Shanna; Song, Xianliang; Cao, Heping

    2014-01-01

    Tung tree (Vernicia fordii) provides the sole source of tung oil widely used in industry. Lack of fatty acid composition and molecular markers hinders biochemical, genetic and breeding research. The objectives of this study were to determine fatty acid profiles and develop unigene-derived simple sequence repeat (SSR) markers in tung tree. Fatty acid profiles of 41 accessions showed that the ratio of α-eleostearic acid was increasing continuously with a parallel trend to the amount of tung oil accumulation while the ratios of other fatty acids were decreasing in different stages of the seeds and that α-eleostearic acid (18∶3) consisted of 77% of the total fatty acids in tung oil. Transcriptome sequencing identified 81,805 unigenes from tung cDNA library constructed using seed mRNA and discovered 6,366 SSRs in 5,404 unigenes. The di- and tri-nucleotide microsatellites accounted for 92% of the SSRs with AG/CT and AAG/CTT being the most abundant SSR motifs. Fifteen polymorphic genic-SSR markers were developed from 98 unigene loci tested in 41 cultivated tung accessions by agarose gel and capillary electrophoresis. Genbank database search identified 10 of them putatively coding for functional proteins. Quantitative PCR demonstrated that all 15 polymorphic SSR-associated unigenes were expressed in tung seeds and some of them were highly correlated with oil composition in the seeds. Dendrogram revealed that most of the 41 accessions were clustered according to the geographic region. These new polymorphic genic-SSR markers will facilitate future studies on genetic diversity, molecular fingerprinting, comparative genomics and genetic mapping in tung tree. The lipid profiles in the seeds of 41 tung accessions will be valuable for biochemical and breeding studies. PMID:25167054

  11. Characterization of DNA-binding sequences for CcaR in the cephamycin-clavulanic acid supercluster of Streptomyces clavuligerus.

    PubMed

    Santamarta, I; López-García, M T; Kurt, A; Nárdiz, N; Alvarez-Álvarez, R; Pérez-Redondo, R; Martín, J F; Liras, P

    2011-08-01

    RT-PCR analysis of the genes in the clavulanic acid cluster revealed three transcriptional polycistronic units that comprised the ceaS2-bls2-pah2-cas2, cyp-fd-orf12-orf13 and oppA2-orf16 genes, whereas oat2, car, oppA1, claR, orf14, gcaS and pbpA were expressed as monocistronic transcripts. Quantitative RT-PCR of Streptomyces clavuligerus ATCC 27064 and the mutant S. clavuligerus ccaR::aph showed that, in the mutant, there was a 1000- to 10,000-fold lower transcript level for the ceaS2 to cas2 polycistronic transcript that encoded CeaS2, the first enzyme of the clavulanic acid pathway that commits arginine to clavulanic acid biosynthesis. Smaller decreases in expression were observed in the ccaR mutant for other genes in the cluster. Two-dimensional electrophoresis and MALDI-TOF analysis confirmed the absence in the mutant strain of proteins CeaS2, Bls2, Pah2 and Car that are required for clavulanic acid biosynthesis, and CefF and IPNS that are required for cephamycin biosynthesis. Gel shift electrophoresis using recombinant r-CcaR protein showed that it bound to the ceaS2 and claR promoter regions in the clavulanic acid cluster, and to the lat, cefF, cefD-cmcI and ccaR promoter regions in the cephamycin C gene cluster. Footprinting experiments indicated that triple heptameric conserved sequences were protected by r-CcaR, and allowed identification of heptameric sequences as CcaR binding sites.

  12. Some properties and amino acid sequence of plastocyanin from a green alga, Ulva arasakii.

    PubMed

    Yoshizaki, F; Fukazawa, T; Mishina, Y; Sugimura, Y

    1989-08-01

    Plastocyanin was purified from a multicellular, marine green alga, Ulva arasakii, by conventional methods to homogeneity. The oxidized plastocyanin showed absorption maxima at 252, 276.8, 460, 595.3, and 775 nm, and shoulders at 259, 265, 269, and 282.5 nm; the ratio A276.8/A595.3 was 1.5. The midpoint redox potential was determined to be 0.356 V at pH 7.0 with a ferri- and ferrocyanide system. The molecular weight was estimated to be 10,200 and 11,000 by SDS-PAGE and by gel filtration, respectively. U. arasakii also has a small amount of cytochrome c6, like Enteromorpha prolifera. The amino acid sequence of U. arasakii plastocyanin was determined by Edman degradation and by carboxypeptidase digestion of the plastocyanin, six tryptic peptides, and five staphylococcal protease peptides. The plastocyanin contained 98 amino acid residues, giving a molecular weight of 10,236 including one copper atom. The complete sequence is as follows: AQIVKLGGDDGALAFVPSKISVAAGEAIEFVNNAGFPHNIVFDEDAVPAGVDADAISYDDYLNSKGETV VRKLSTPGVY G VYCEPHAGAGMKMTITVQ. The sequence of U. arasakii plastocyanin is closet to that of the E. prolifera protein (85% homology). A phylogenetic tree of five algal and two higher plant plastocyanins was constructed by comparing the amino acid differences. The branching order is considered to be as follows: a blue-green alga, unicellular green algae, multicellular green algae, and higher plants. PMID:2509442

  13. 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. PMID:23485423

  14. Complete amino acid sequence of chitinase-A from leaves of pokeweed (Phytolacca americana).

    PubMed

    Yamagami, T; Tanigawa, M; Ishiguro, M; Funatsu, G

    1998-04-01

    The complete amino acid sequence of pokeweed leaf chitinase-A was determined. First all 11 tryptic peptides from the reduced and S-carboxymethylated form of the enzyme were sequenced. Then the same form of the enzyme was cleaved with cyanogen bromide, giving three fragments. The fragments were digested with chymotrypsin or Staphylococcus aureus V8 protease. Last, the 11 tryptic peptides were put in order. Of seven cysteine residues, six were linked by disulfide bonds (between Cys25 and Cys74, Cys89 and Cys98, and Cys195 and Cys208); Cys176 was free. The enzyme consisted of 208 amino acid residues and had a molecular weight of 22,391. It consisted of only one polypeptide chain without a chitin-binding domain. The length of the chain was almost the same as that of the catalytic domains of class IL chitinases. These findings suggested that this enzyme is a new kind of class IIL chitinase, although its sequence resembles that of catalytic domains of class IL chitinases more than that of the class IIL chitinases reported so far. Discussion on the involvement of specific tryptophan residue in the active site of PLC-A is also given based on the sequence similarity with rye seed chitinase-c.

  15. Metazoan remaining genes for essential amino acid biosynthesis: sequence conservation and evolutionary analyses.

    PubMed

    Costa, Igor R; Thompson, Julie D; Ortega, José Miguel; Prosdocimi, Francisco

    2014-12-24

    Essential amino acids (EAA) consist of a group of nine amino acids that animals are unable to synthesize via de novo pathways. Recently, it has been found that most metazoans lack the same set of enzymes responsible for the de novo EAA biosynthesis. Here we investigate the sequence conservation and evolution of all the metazoan remaining genes for EAA pathways. Initially, the set of all 49 enzymes responsible for the EAA de novo biosynthesis in yeast was retrieved. These enzymes were used as BLAST queries to search for similar sequences in a database containing 10 complete metazoan genomes. Eight enzymes typically attributed to EAA pathways were found to be ubiquitous in metazoan genomes, suggesting a conserved functional role. In this study, we address the question of how these genes evolved after losing their pathway partners. To do this, we compared metazoan genes with their fungal and plant orthologs. Using phylogenetic analysis with maximum likelihood, we found that acetolactate synthase (ALS) and betaine-homocysteine S-methyltransferase (BHMT) diverged from the expected Tree of Life (ToL) relationships. High sequence conservation in the paraphyletic group Plant-Fungi was identified for these two genes using a newly developed Python algorithm. Selective pressure analysis of ALS and BHMT protein sequences showed higher non-synonymous mutation ratios in comparisons between metazoans/fungi and metazoans/plants, supporting the hypothesis that these two genes have undergone non-ToL evolution in animals.

  16. 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

  17. [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.

  18. Genetic architecture of vitamin B12 and folate levels uncovered applying deeply sequenced large datasets.

    PubMed

    Grarup, Niels; Sulem, Patrick; Sandholt, Camilla H; Thorleifsson, Gudmar; Ahluwalia, Tarunveer S; Steinthorsdottir, Valgerdur; Bjarnason, Helgi; Gudbjartsson, Daniel F; Magnusson, Olafur T; Sparsø, Thomas; Albrechtsen, Anders; Kong, Augustine; Masson, Gisli; Tian, Geng; Cao, Hongzhi; Nie, Chao; Kristiansen, Karsten; Husemoen, Lise Lotte; Thuesen, Betina; Li, Yingrui; Nielsen, Rasmus; Linneberg, Allan; Olafsson, Isleifur; Eyjolfsson, Gudmundur I; Jørgensen, Torben; Wang, Jun; Hansen, Torben; Thorsteinsdottir, Unnur; Stefánsson, Kari; Pedersen, Oluf

    2013-06-01

    Genome-wide association studies have mainly relied on common HapMap sequence variations. Recently, sequencing approaches have allowed analysis of low frequency and rare variants in conjunction with common variants, thereby improving the search for functional variants and thus the understanding of the underlying biology of human traits and diseases. Here, we used a large Icelandic whole genome sequence dataset combined with Danish exome sequence data to gain insight into the genetic architecture of serum levels of vitamin B(12) (B12) and folate. Up to 22.9 million sequence variants were analyzed in combined samples of 45,576 and 37,341 individuals with serum B(12) and folate measurements, respectively. We found six novel loci associating with serum B(12) (CD320, TCN2, ABCD4, MMAA, MMACHC) or folate levels (FOLR3) and confirmed seven loci for these traits (TCN1, FUT6, FUT2, CUBN, CLYBL, MUT, MTHFR). Conditional analyses established that four loci contain additional independent signals. Interestingly, 13 of the 18 identified variants were coding and 11 of the 13 target genes have known functions related to B(12) and folate pathways. Contrary to epidemiological studies we did not find consistent association of the variants with cardiovascular diseases, cancers or Alzheimer's disease although some variants demonstrated pleiotropic effects. Although to some degree impeded by low statistical power for some of these conditions, these data suggest that sequence variants that contribute to the population diversity in serum B(12) or folate levels do not modify the risk of developing these conditions. Yet, the study demonstrates the value of combining whole genome and exome sequencing approaches to ascertain the genetic and molecular architectures underlying quantitative trait associations.

  19. Genetic Architecture of Vitamin B12 and Folate Levels Uncovered Applying Deeply Sequenced Large Datasets

    PubMed Central

    Thorleifsson, Gudmar; Ahluwalia, Tarunveer S.; Steinthorsdottir, Valgerdur; Bjarnason, Helgi; Gudbjartsson, Daniel F.; Magnusson, Olafur T.; Sparsø, Thomas; Albrechtsen, Anders; Kong, Augustine; Masson, Gisli; Tian, Geng; Cao, Hongzhi; Nie, Chao; Kristiansen, Karsten; Husemoen, Lise Lotte; Thuesen, Betina; Li, Yingrui; Nielsen, Rasmus; Linneberg, Allan; Olafsson, Isleifur; Eyjolfsson, Gudmundur I.; Jørgensen, Torben; Wang, Jun; Hansen, Torben; Thorsteinsdottir, Unnur; Stefánsson, Kari; Pedersen, Oluf

    2013-01-01

    Genome-wide association studies have mainly relied on common HapMap sequence variations. Recently, sequencing approaches have allowed analysis of low frequency and rare variants in conjunction with common variants, thereby improving the search for functional variants and thus the understanding of the underlying biology of human traits and diseases. Here, we used a large Icelandic whole genome sequence dataset combined with Danish exome sequence data to gain insight into the genetic architecture of serum levels of vitamin B12 (B12) and folate. Up to 22.9 million sequence variants were analyzed in combined samples of 45,576 and 37,341 individuals with serum B12 and folate measurements, respectively. We found six novel loci associating with serum B12 (CD320, TCN2, ABCD4, MMAA, MMACHC) or folate levels (FOLR3) and confirmed seven loci for these traits (TCN1, FUT6, FUT2, CUBN, CLYBL, MUT, MTHFR). Conditional analyses established that four loci contain additional independent signals. Interestingly, 13 of the 18 identified variants were coding and 11 of the 13 target genes have known functions related to B12 and folate pathways. Contrary to epidemiological studies we did not find consistent association of the variants with cardiovascular diseases, cancers or Alzheimer's disease although some variants demonstrated pleiotropic effects. Although to some degree impeded by low statistical power for some of these conditions, these data suggest that sequence variants that contribute to the population diversity in serum B12 or folate levels do not modify the risk of developing these conditions. Yet, the study demonstrates the value of combining whole genome and exome sequencing approaches to ascertain the genetic and molecular architectures underlying quantitative trait associations. PMID:23754956

  20. Characterization of high-level expression and sequencing of the Escherichia coli K-12 cynS gene encoding cyanase.

    PubMed

    Sung, Y C; Anderson, P M; Fuchs, J A

    1987-11-01

    Restriction fragments containing the gene encoding cyanase, cynS, without its transcriptional regulatory sequences were placed downstream of lac and tac promoters in various pUC derivatives to maximize production of cyanase. Plasmid pSJ105, which contains the cynS gene and an upstream open reading frame, gave the highest expression of cyanase. Approximately 50% of the total soluble protein in stationary-phase cultures of a lac-deleted strain containing plasmid pSJ105 was cyanase. The inserted DNA fragment of pSJ105 was transferred into pUC18 derivatives that contain a hybrid tac promoter, instead of the lac promoter, and a strong terminator to generate pSJ124. Stationary-phase cultures of JM101 containing plasmid pSJ124 overexpressed a similar level of cyanase. In JM101(pSJ124), maximum production of cyanase could be obtained either by induction with isopropyl-beta-D-thiogalactopyranoside (IPTG) for 3 h or by growth without IPTG into late stationary phase. The latter conditions resulted in a 10- to 20-fold increase in plasmid content and presumably titration of the lac repressor. The nucleotide sequence of the cloned cynS gene from Escherichia coli K-12 was determined. The predicted amino acid sequence differed from the known amino acid sequence of cyanase isolated from a B strain by four residues. However, overexpressed cyanase was purified to homogeneity, and a comparison of the enzymes from the two sources indicated that they did not differ with respect to physical and kinetic properties. The cynS gene was located next to the lac operon, and the direction of cynS transcription was opposite that of lac.

  1. Draft Genome Sequences of 11 Salmonella enterica Strains with Variable Levels of Barotolerance.

    PubMed

    Ronholm, Jennifer; Petronella, Nicholas; Tamber, Sandeep

    2016-01-01

    The diversity of the genus Salmonella is reflected in the physiological adaptations used by its members in response to stressors such as high pressure. Here we report the draft whole genome sequences of 11 Salmonella enterica strains, five sensitive strains and six demonstrating high levels of pressure resistance. PMID:27660773

  2. Draft Genome Sequences of 11 Salmonella enterica Strains with Variable Levels of Barotolerance

    PubMed Central

    Ronholm, Jennifer; Petronella, Nicholas

    2016-01-01

    The diversity of the genus Salmonella is reflected in the physiological adaptations used by its members in response to stressors such as high pressure. Here we report the draft whole genome sequences of 11 Salmonella enterica strains, five sensitive strains and six demonstrating high levels of pressure resistance. PMID:27660773

  3. Unsaturated fatty acids supplementation reduces blood lead level in rats.

    PubMed

    Skoczyńska, Anna; Wojakowska, Anna; Nowacki, Dorian; Bobak, Łukasz; Turczyn, Barbara; Smyk, Beata; Szuba, Andrzej; Trziszka, Tadeusz

    2015-01-01

    Some dietary factors could inhibit lead toxicity. The aim of this study was to evaluate the effect of dietary compounds rich in unsaturated fatty acids (FA) on blood lead level, lipid metabolism, and vascular reactivity in rats. Serum metallothionein and organs' lead level were evaluated with the aim of assessing the possible mechanism of unsaturated FA impact on blood lead level. For three months, male Wistar rats that were receiving drinking water with (100 ppm Pb) or without lead acetate were supplemented per os daily with virgin olive oil or linseed oil (0.2 mL/kg b.w.) or egg derived lecithin fraction: "super lecithin" (50 g/kg b.w.). Mesenteric artery was stimulated ex vivo by norepinephrine (NE) administered at six different doses. Lecithin supplementation slightly reduced pressor responses of artery to NE. Lead administered to rats attenuated the beneficial effect of unsaturated FA on lipid metabolism and vascular reactivity to adrenergic stimulation. On the other hand, the super lecithin and linseed oil that were characterized by low omega-6 to omega-3 ratio (about 1) reduced the blood lead concentration. This effect was observed in lead poisoned rats (p < 0.0001) and also in rats nonpoisoned with lead (p < 0.05). PMID:26075218

  4. Unsaturated Fatty Acids Supplementation Reduces Blood Lead Level in Rats

    PubMed Central

    Skoczyńska, Anna; Wojakowska, Anna; Nowacki, Dorian; Bobak, Łukasz; Turczyn, Barbara; Smyk, Beata; Szuba, Andrzej; Trziszka, Tadeusz

    2015-01-01

    Some dietary factors could inhibit lead toxicity. The aim of this study was to evaluate the effect of dietary compounds rich in unsaturated fatty acids (FA) on blood lead level, lipid metabolism, and vascular reactivity in rats. Serum metallothionein and organs' lead level were evaluated with the aim of assessing the possible mechanism of unsaturated FA impact on blood lead level. For three months, male Wistar rats that were receiving drinking water with (100 ppm Pb) or without lead acetate were supplemented per os daily with virgin olive oil or linseed oil (0.2 mL/kg b.w.) or egg derived lecithin fraction: “super lecithin” (50 g/kg b.w.). Mesenteric artery was stimulated ex vivo by norepinephrine (NE) administered at six different doses. Lecithin supplementation slightly reduced pressor responses of artery to NE. Lead administered to rats attenuated the beneficial effect of unsaturated FA on lipid metabolism and vascular reactivity to adrenergic stimulation. On the other hand, the super lecithin and linseed oil that were characterized by low omega-6 to omega-3 ratio (about 1) reduced the blood lead concentration. This effect was observed in lead poisoned rats (p < 0.0001) and also in rats nonpoisoned with lead (p < 0.05). PMID:26075218

  5. 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

  6. 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.

  7. 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

  8. [Partial sequence homology of FtsZ in phylogenetics analysis of lactic acid bacteria].

    PubMed

    Zhang, Bin; Dong, Xiu-zhu

    2005-10-01

    FtsZ is a structurally conserved protein, which is universal among the prokaryotes. It plays a key role in prokaryote cell division. A partial fragment of the ftsZ gene about 800bp in length was amplified and sequenced and a partial FtsZ protein phylogenetic tree for the lactic acid bacteria was constructed. By comparing the FtsZ phylogenetic tree with the 16S rDNA tree, it was shown that the two trees were similar in topology. Both trees revealed that Pediococcus spp. were closely related with L. casei group of Lactobacillus spp. , but less related with other lactic acid cocci such as Enterococcus and Streptococcus. The results also showed that the discriminative power of FtsZ was higher than that of 16S rDNA for either inter-species or inter-genus and could be a very useful tool in species identification of lactic acid bacteria. PMID:16342751

  9. 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.

  10. 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. PMID:1368578

  11. Low irradiances affect abscisic acid, indole-3-acidic acid, and cytokinin levels of wheat (Triticum aestivum L.) tissues

    NASA Technical Reports Server (NTRS)

    Nan, R.; Carman, J. G.; Salisbury, F. B.

    1999-01-01

    Wheat (Triticum aestivum L.) plants were grown under four irradiance levels: 1,400, 400, 200, and 100 micromol m-2 s-1. Leaves and roots were sampled before, during, and after the boot stage, and levels of abscisic acid (ABA), indole-3-acetic acid (IAA), zeatin, zeatin riboside, dihydrozeatin, dihydrozeatin riboside, isopentenyl adenine, and isopentenyl adenosine were quantified using noncompetitive indirect ELISA systems. Levels of IAA in leaves and roots of plants exposed to 100 micromol m-2 s-1 of irradiance were 0.7 and 2.9 micromol kg-1 dry mass (DM), respectively. These levels were 0.2 and 1.0 micromol kg-1 DM, respectively, when plants were exposed to 1,400 micromol m-2 s-1. Levels of ABA in leaves and roots of plants exposed to 100 micromol m-2 s-1 were 0.65 and 0.55 micromol kg-1 DM, respectively. They were 0.24 micromol kg-1 DM (both leaves and roots) when plants were exposed to 1,400 micromol m-2 s-1. Levels of isopentenyl adenosine in leaves (24.3 nmol kg-1 DM) and roots (29.9 nmol kg-1 DM) were not affected by differences in the irradiance regime. Similar values were obtained in a second experiment. Other cytokinins could not be detected (<10 nmol kg 1 DM) in either experiment with the sample sizes used (150-600 mg DM for roots and shoots, respectively).

  12. Sequence, internal homology and high-level expression of the gene for a DNA-(cytosine N4)-methyltransferase, M.Pvu II.

    PubMed Central

    Tao, T; Walter, J; Brennan, K J; Cotterman, M M; Blumenthal, R M

    1989-01-01

    The base sequence of the pvuIIM gene has been determined. This gene codes for a DNA-(cytosine N4)-methyltransferase, M.Pvu II. The base sequence contains a single large open reading frame that predicts a 38.3kDa polypeptide, consistent with experimental data. The pvuIIM gene contains some sequences common to DNA methyltransferases in general, but includes none of the sequences specifically conserved among DNA-(cytosine 5)-methyltransferases. The pvuIIM sequence also reveals an internal homology at the amino acid level, each half of which spans over 100 amino acids and is itself homologous to the sequences of some DNA-(adenine N6)-methyltransferases. A derivative of the pvuIIM plasmid was constructed to allow high-level production of M.Pvu II. Specifically, the composite Ptac promoter was inserted 5' to pvuIIM, intervening DNA was deleted, and the resulting construct was used to transform an mcrB laclq strain of Escherichia coli. When this transformant was induced with isopropyl-B-D-galactopyranoside (IPTG), growth rapidly ceased and M.Pvu II accumulated to the point of comprising over 10% of the total soluble protein. Images PMID:2662138

  13. N-terminal amino acid sequences and some characteristics of fibrinolytic/hemorrhagic metalloproteinases purified from Bothrops jararaca venom.

    PubMed

    Maruyama, Masugi; Sugiki, Masahiko; Anai, Keita; Yoshida, Etsuo

    2002-08-01

    We determined the N-terminal amino acid sequences of the fibrinolytic/hemorrhagic metalloproteinases (jararafibrases I, III and IV) purified from Bothrops jararaca venom. The N-terminal amino acid sequences of jararafibrase I and its degradation products were identical to those of jararhagin, another hemorrhagic metalloproteinase purified from the same snake venom. Together with enzymatic and immunological properties, we concluded that those two enzymes are identical. The N-terminal amino acid sequence of jararafibrase III was quite similar to C-type lectin isolated from Crotalus atrox, and the protein had a hemagglutinating activity on intact rat red blood cells. PMID:12165326

  14. Alterations of Amino Acid Level in Depressed Rat Brain

    PubMed Central

    Yang, Pei; Li, Xuechun; Tian, Jingchen; Jing, Fu; Qu, Changhai; Lin, Longfei; Zhang, Hui

    2014-01-01

    Amino-acid neurotransmitter system dysfunction plays a major role in the pathophysiology of depression. Several studies have demonstrated the potential of amino acids as a source of neuro-specific biomarkers could be used in future diagnosis of depression. Only partial amino acids such as glycine and asparagine were determined from certain parts of rats' brain included hippocampi and cerebral cortex in previous studies. However, according to systematic biology, amino acids in different area of brain are interacted and interrelated. Hence, the determination of 34 amino acids through entire rats' brain was conducted in this study in order to demonstrate more possibilities for biomarkers of depression by discovering other potential amino acids in more areas of rats' brain. As a result, 4 amino acids (L-aspartic acid, L-glutamine, taurine and γ-amino-n-butyric acid) among 34 were typically identified as potentially primary biomarkers of depression by data statistics. Meanwhile, an antidepressant called Fluoxetine was employed to verify other potential amino acids which were not identified by data statistics. Eventually, we found L-α-amino-adipic acid could also become a new potentially secondary biomarker of depression after drug validation. In conclusion, we suggested that L-aspartic acid, L-glutamine, taurine, γ-amino-n-butyric acid and L-α-amino-adipic acid might become potential biomarkers for future diagnosis of depression and development of antidepressant. PMID:25352755

  15. Protein sequence analysis by incorporating modified chaos game and physicochemical properties into Chou's general pseudo amino acid composition.

    PubMed

    Xu, Chunrui; Sun, Dandan; Liu, Shenghui; Zhang, Yusen

    2016-10-01

    In this contribution we introduced a novel graphical method to compare protein sequences. By mapping a protein sequence into 3D space based on codons and physicochemical properties of 20 amino acids, we are able to get a unique P-vector from the 3D curve. This approach is consistent with wobble theory of amino acids. We compute the distance between sequences by their P-vectors to measure similarities/dissimilarities among protein sequences. Finally, we use our method to analyze four datasets and get better results compared with previous approaches. PMID:27375218

  16. Late Cretaceous base level lowering in Campanian and Maastrichtian depositional sequences, Kure Beach, North Carolina

    USGS Publications Warehouse

    Harris, W.B.; ,

    2006-01-01

    Campanian through Maastrichtian mixed carbonate and siliciclastic sediments in a 422 m continuous core drilled at Kure Beach, NC provide a record of sea-level change. Based on lithology and stratigraphy, depositional sequences are defined, and calcareous nannofossil zones and 87Sr/86Sr ratios and corresponding ages using the LOWESS Table determined. Campanian and Maastrichtian sediments comprise six depositional sequences. The oldest is Tar Heel 1 and contains calcareous nannofossils that indicate assignment to the upper part of Zones CC18a, CC18c and the lower part of CC19. 87Sr/86 Sr ratios indicate ages from 83.2 to 80.0 Ma or lower Campanian. Tar Heel II contains calcareous nannofossils that indicate assignment to the upper part of Zone CC 19 and CC20. 87Sr/86Sr ratios indicate ages from 78.0 to 76.3 Ma or middle Campanian. Donoho Creek I and II are thin and contain calcareous nannofossils referable to upper Zone CC21 and Zone CC22, and to CC23, respectively. The top of Donoho Creek II marks the Campanian-Maastrichtian boundary. Donoho Creek I 87Sr/86Sr ratios cluster into two groups, and provide ages from 78.0 to 76.2 Ma and 73.7 to 72.3 Ma, respectively. 87Sr/86Sr ratios in Donoho Creek II indicate ages from 71.4 to 69.6 Ma. Two Maastrichtian sequences are present; the lowermost Peedee I contains calcareous nannofossils that place it in Zones CC25a and CC25b. 87Sr/86Sr r ratios indicate an age from 69.3 to 66.9 Ma or late Maastrichtian. Peedee II is assigned to calcareous nannofossil Zone CC26a. 87Sr/86Sr ratios indicate ages from 66.4 to 65.2 Ma or late Maastrichtian. The four Campanian sequences correlate to three depositional sequences in New Jersey; the sequence boundary between upper Campanian Donoho Creek I and Donoho Creek II is not recognized in New Jersey. This boundary is interpreted to result from Gulf Stream impingement and subsequent erosion on the outer shelf. The two Maastrichtian sequences recognized in the Kure Beach core correlate to the two

  17. Momentum sequence and environmental climate influence levels of perceived psychological momentum within a sport competition.

    PubMed

    Briki, Walid; Markman, Keith D; Coudevylle, Guillaume; Sinnapah, Stéphane; Hue, Olivier

    2016-01-01

    The present study examined the influence of momentum sequence (positive vs. negative) and environmental climate (hot-wet vs. neutral) on supporters' (i.e. virtual observers') reported levels of perceived psychological momentum (PM) during a simulated cycling competition. Participants supported one of two competing cyclists involved in a race that was displayed on a screen in a lecture hall. The race scenario was manipulated so that the supported cyclist appeared to undergo either a positive or negative momentum sequence. In addition, participants were either exposed to a hot-wet environmental climate or to a neutral environmental climate while observing the race scenario. According to the results, reported levels of PM were higher in the positive momentum sequence condition than in the negative momentum sequence condition, consistent with the notion that supporters' PM is influenced by a positivity bias, and reported levels of PM were also found to be higher in the hot-wet climate condition than in the neutral climate condition, consistent with the notion that environmental climate is a contextual factor that influences PM through the operation of a causal augmenting mechanism.

  18. Serum uric acid levels during leprosy reaction episodes

    PubMed Central

    Alves-Junior, Eduardo R.; Arruda, Talita A.; Lopes, Jose C.; Fontes, Cor J.F.

    2016-01-01

    Background. Leprosy reactions are acute inflammatory episodes that occur mainly in the multibacillary forms of the disease. The reactions are classified as type 1 (reverse reaction) or type 2 (erythema nodosum leprosum). Leprosy-associated oxidative stress has been widely demonstrated. Several recent studies have shown uric acid (UA) to have antioxidative effects under pathologic conditions. The objective of this study was to assess serum levels of UA in patients with leprosy reactions, with the aim of monitoring their levels before and after treatment, compared with levels in leprosy patients without reactions. Methods. The study included patients aged 18–69 years assisted at a leprosy treatment reference center in the Central Region of Brazil. Patients who were pregnant; were using immunosuppressant drugs or immunobiologicals; or had an autoimmune disease, human immunodeficiency virus infection, acquired immune deficiency syndrome, or tuberculosis were excluded. Upon recruitment, all individuals were clinically assessed for skin lesions and neural or systemic impairment. Some patients had already completed treatment for leprosy, while others were still undergoing treatment or had initiated treatment after being admitted. The treatment of the reactional episode was started only after the initial evaluation. Laboratory assessments were performed upon admission (baseline) and at approximately 30 and 60 days (time points 1 and 2, respectively). Results. A total of 123 leprosy patients were recruited between June 2012 and June 2015; among them, 56, 42, and 25 presented with type 1, type 2, and no reactions, respectively. Serum UA levels were significantly reduced in patients with type 2 leprosy reactions compared with patients in the control group and remained lower in the two subsequent assessments, after initiation of anti-reaction treatments, with similar values to those recorded before the treatment. Discussion. The decreased serum UA levels in patients with

  19. SigniSite: Identification of residue-level genotype-phenotype correlations in protein multiple sequence alignments.

    PubMed

    Jessen, Leon Eyrich; Hoof, Ilka; Lund, Ole; Nielsen, Morten

    2013-07-01

    Identifying which mutation(s) within a given genotype is responsible for an observable phenotype is important in many aspects of molecular biology. Here, we present SigniSite, an online application for subgroup-free residue-level genotype-phenotype correlation. In contrast to similar methods, SigniSite does not require any pre-definition of subgroups or binary classification. Input is a set of protein sequences where each sequence has an associated real number, quantifying a given phenotype. SigniSite will then identify which amino acid residues are significantly associated with the data set phenotype. As output, SigniSite displays a sequence logo, depicting the strength of the phenotype association of each residue and a heat-map identifying 'hot' or 'cold' regions. SigniSite was benchmarked against SPEER, a state-of-the-art method for the prediction of specificity determining positions (SDP) using a set of human immunodeficiency virus protease-inhibitor genotype-phenotype data and corresponding resistance mutation scores from the Stanford University HIV Drug Resistance Database, and a data set of protein families with experimentally annotated SDPs. For both data sets, SigniSite was found to outperform SPEER. SigniSite is available at: http://www.cbs.dtu.dk/services/SigniSite/. PMID:23761454

  20. Purification to homogeneity and amino acid sequence analysis of two anionic species of human interleukin 1

    PubMed Central

    1986-01-01

    Two anionic species of human IL-1 have been purified to homogeneity. These molecules were characterized as having pI of 5.4 and 5.2 and molecular weights identical to IL-1/6.8 (17,500). The specific activities of IL-1/5.4 and IL-1/5.2, as measured in the mouse thymocyte co-mitogenic assay, were identical to that of IL-1/6.8, namely 1.2 X 10(7) U/mg, with half-maximal stimulation observed at 2 X 10(-11) M. IL- 1/5.4 and IL-1/5.2 were found to be antigenically distinct from IL- 1/6.8 in an ELISA. IL-1/5.4 was structurally distinct from IL-1/6.8 based on reverse-phase HPLC or CNBr peptides. Intact IL-1/5.2 and three intact CNBr peptides of IL-1/5.4 were sequenced, with the identification of 74 amino acid residues. These sequences were found to correspond exactly with the amino acid sequence deduced from the IL-1- alpha cDNA reported by March et al. PMID:3487613

  1. Protein meta-functional signatures from combining sequence, structure, evolution, and amino acid property information.

    PubMed

    Wang, Kai; Horst, Jeremy A; Cheng, Gong; Nickle, David C; Samudrala, Ram

    2008-09-26

    Protein function is mediated by different amino acid residues, both their positions and types, in a protein sequence. Some amino acids are responsible for the stability or overall shape of the protein, playing an indirect role in protein function. Others play a functionally important role as part of active or binding sites of the protein. For a given protein sequence, the residues and their degree of functional importance can be thought of as a signature representing the function of the protein. We have developed a combination of knowledge- and biophysics-based function prediction approaches to elucidate the relationships between the structural and the functional roles of individual residues and positions. Such a meta-functional signature (MFS), which is a collection of continuous values representing the functional significance of each residue in a protein, may be used to study proteins of known function in greater detail and to aid in experimental characterization of proteins of unknown function. We demonstrate the superior performance of MFS in predicting protein functional sites and also present four real-world examples to apply MFS in a wide range of settings to elucidate protein sequence-structure-function relationships. Our results indicate that the MFS approach, which can combine multiple sources of information and also give biological interpretation to each component, greatly facilitates the understanding and characterization of protein function.

  2. Em polypeptide and its messenger RNA levels are modulated by abscisic acid during embryogenesis in wheat.

    PubMed

    Williamson, J D; Quatrano, R S; Cuming, A C

    1985-10-15

    The effect of abscisic acid (ABA) on the expression of the 'early-methionine-labeled' (Em) polypeptide was examined in cultured, immature wheat (Triticum aestivum, L.) embryos and in developing embryos in planta. A complementary DNA (cDNA) library was constructed from poly(A)-rich RNA from immature embryos cultured in the presence of ABA. ABA-enhanced sequences were first identified by differential colony-blot hybridization, and then verified using RNA slot-blot analysis. Dot-blot hybridization showed that one clone, p1015, was homologous to the previously isolated Em cDNA, pWG432. Electrophoretic analysis of the hybrid-select translation product of p1015 confirmed its identity as an Em sequence. Comparison of the p1015 cDNA insert size and the Em message size, from northern blot analysis, showed that p1015 contained about 87% of the Em sequence. RNA slot-blot analysis and protein electrophoresis showed that Em message, but not Em protein, accumulated at a low, basal level in immature embryos in the absence of ABA. Neither Em message nor Em protein was seen in three-day germinated seedlings. Steady-state levels of Em message and protein increased in immature embryos in the presence of ABA, both in culture and in planta. Regulation appeared to be primarily at the level of transcription or specific message stability. Regulation may also involve specific protein stability, since synthesis of Em protein continued in immature embryos in the absence of ABA, but Em protein did not accumulate in detectable amounts. We conclude that ABA specifically modulates Em message and protein levels in immature embryos, but is probably not responsible for the embryogenic specificity of Em expression.

  3. Bacteria obtained from a sequencing batch reactor that are capable of growth on dehydroabietic acid.

    PubMed

    Mohn, W W

    1995-06-01

    Eleven isolates capable of growth on the resin acid dehydroabietic acid (DhA) were obtained from a sequencing batch reactor designed to treat a high-strength process stream from a paper mill. The isolates belonged to two groups, represented by strains DhA-33 and DhA-35, which were characterized. In the bioreactor, bacteria like DhA-35 were more abundant than those like DhA-33. The population in the bioreactor of organisms capable of growth on DhA was estimated to be 1.1 x 10(6) propagules per ml, based on a most-probable-number determination. Analysis of small-subunit rRNA partial sequences indicated that DhA-33 was most closely related to Sphingomonas yanoikuyae (Sab = 0.875) and that DhA-35 was most closely related to Zoogloea ramigera (Sab = 0.849). Both isolates additionally grew on other abietanes, i.e., abietic and palustric acids, but not on the pimaranes, pimaric and isopimaric acids. For DhA-33 and DhA-35 with DhA as the sole organic substrate, doubling times were 2.7 and 2.2 h, respectively, and growth yields were 0.30 and 0.25 g of protein per g of DhA, respectively. Glucose as a cosubstrate stimulated growth of DhA-33 on DhA and stimulated DhA degradation by the culture. Pyruvate as a cosubstrate did not stimulate growth of DhA-35 on DhA and reduced the specific rate of DhA degradation of the culture. DhA induced DhA and abietic acid degradation activities in both strains, and these activities were heat labile. Cell suspensions of both strains consumed DhA at a rate of 6 mumol mg of protein-1 h-1.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Mutation-selection models of coding sequence evolution with site-heterogeneous amino acid fitness profiles.

    PubMed

    Rodrigue, Nicolas; Philippe, Hervé; Lartillot, Nicolas

    2010-03-01

    Modeling the interplay between mutation and selection at the molecular level is key to evolutionary studies. To this end, codon-based evolutionary models have been proposed as pertinent means of studying long-range evolutionary patterns and are widely used. However, these approaches have not yet consolidated results from amino acid level phylogenetic studies showing that selection acting on proteins displays strong site-specific effects, which translate into heterogeneous amino acid propensities across the columns of alignments; related codon-level studies have instead focused on either modeling a single selective context for all codon columns, or a separate selective context for each codon column, with the former strategy deemed too simplistic and the latter deemed overparameterized. Here, we integrate recent developments in nonparametric statistical approaches to propose a probabilistic model that accounts for the heterogeneity of amino acid fitness profiles across the coding positions of a gene. We apply the model to a dozen real protein-coding gene alignments and find it to produce biologically plausible inferences, for instance, as pertaining to site-specific amino acid constraints, as well as distributions of scaled selection coefficients. In their account of mutational features as well as the heterogeneous regimes of selection at the amino acid level, the modeling approaches studied here can form a backdrop for several extensions, accounting for other selective features, for variable population size, or for subtleties of mutational features, all with parameterizations couched within population-genetic theory. PMID:20176949

  5. Development of a SCAR (sequence-characterised amplified region) marker for acid resistance-related gene in Lactobacillus plantarum.

    PubMed

    Liu, Shu-Wen; Li, Kai; Yang, Shi-Ling; Tian, Shu-Fen; He, Ling

    2015-03-01

    A sequence characterised amplified region marker was developed to determine an acid resistance-related gene in Lactobacillus plantarum. A random amplified polymorphic DNA marker named S116-680 was reported to be closely related to the acid resistance of the strains. The DNA band corresponding to this marker was cloned and sequenced with the induction of specific designed PCR primers. The results of PCR test helped to amplify a clear specific band of 680 bp in the tested acid-resistant strains. S116-680 marker would be useful to explore the acid-resistant mechanism of L. plantarum and to screen desirable malolactic fermentation strains.

  6. 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.

  7. Lower serum uric acid level predicts mortality in dialysis patients

    PubMed Central

    Bae, Eunjin; Cho, Hyun-Jeong; Shin, Nara; Kim, Sun Moon; Yang, Seung Hee; Kim, Dong Ki; Kim, Yong-Lim; Kang, Shin-Wook; Yang, Chul Woo; Kim, Nam Ho; Kim, Yon Su; Lee, Hajeong

    2016-01-01

    Abstract We evaluated the impact of serum uric acid (SUA) on mortality in patients with chronic dialysis. A total of 4132 adult patients on dialysis were enrolled prospectively between August 2008 and September 2014. Among them, we included 1738 patients who maintained dialysis for at least 3 months and had available SUA in the database. We categorized the time averaged-SUA (TA-SUA) into 5 groups: <5.5, 5.5–6.4, 6.5–7.4, 7.5–8.4, and ≥8.5 mg/dL. Cox regression analysis was used to calculate the hazard ratio (HR) of all-cause mortality according to SUA group. The mean TA-SUA level was slightly higher in men than in women. Patients with lower TA-SUA level tended to have lower body mass index (BMI), phosphorus, serum albumin level, higher proportion of diabetes mellitus (DM), and higher proportion of malnourishment on the subjective global assessment (SGA). During a median follow-up of 43.9 months, 206 patients died. Patients with the highest SUA had a similar risk to the middle 3 TA-SUA groups, but the lowest TA-SUA group had a significantly elevated HR for mortality. The lowest TA-SUA group was significantly associated with increased all-cause mortality (adjusted HR, 1.720; 95% confidence interval, 1.007–2.937; P = 0.047) even after adjusting for demographic, comorbid, nutritional covariables, and medication use that could affect SUA levels. This association was prominent in patients with well nourishment on the SGA, a preserved serum albumin level, a higher BMI, and concomitant DM although these parameters had no significant interaction in the TA-SUA-mortality relationship except DM. In conclusion, a lower TA-SUA level <5.5 mg/dL predicted all-cause mortality in patients with chronic dialysis. PMID:27310949

  8. Phylum-Level Conservation of Regulatory Information in Nematodes despite Extensive Non-coding Sequence Divergence

    PubMed Central

    Gordon, Kacy L.; Arthur, Robert K.; Ruvinsky, Ilya

    2015-01-01

    Gene regulatory information guides development and shapes the course of evolution. To test conservation of gene regulation within the phylum Nematoda, we compared the functions of putative cis-regulatory sequences of four sets of orthologs (unc-47, unc-25, mec-3 and elt-2) from distantly-related nematode species. These species, Caenorhabditis elegans, its congeneric C. briggsae, and three parasitic species Meloidogyne hapla, Brugia malayi, and Trichinella spiralis, represent four of the five major clades in the phylum Nematoda. Despite the great phylogenetic distances sampled and the extensive sequence divergence of nematode genomes, all but one of the regulatory elements we tested are able to drive at least a subset of the expected gene expression patterns. We show that functionally conserved cis-regulatory elements have no more extended sequence similarity to their C. elegans orthologs than would be expected by chance, but they do harbor motifs that are important for proper expression of the C. elegans genes. These motifs are too short to be distinguished from the background level of sequence similarity, and while identical in sequence they are not conserved in orientation or position. Functional tests reveal that some of these motifs contribute to proper expression. Our results suggest that conserved regulatory circuitry can persist despite considerable turnover within cis elements. PMID:26020930

  9. Epigenetics and cortical spreading depression: changes of DNA methylation level at retrotransposon sequences.

    PubMed

    Drongitis, Denise; Rainone, Sara; Piscopo, Marina; Viggiano, Emanuela; Viggiano, Alessandro; De Luca, Bruno; Fucci, Laura; Donizetti, Aldo

    2016-08-01

    Cortical spreading depression (CSD) is an evolutionarily conserved phenomenon that involves a slow and self-propagating depolarization wave associated with spontaneous depression of electrical neuronal activity. CSD plays a central role in the pathophysiology of several brain diseases and is considered to be able to promote "Preconditioning". This phenomenon consists of the brain protecting itself against future injury by adaptation. Understanding of the molecular mechanisms underlying Preconditioning has significant clinical implications. We have already proposed that the long-lasting effects of CSD could be related to silencing of retrotransposon sequences by histone methylation. We analyzed DNA methylation of two retrotransposon sequences, LINE1 and L1, and their corresponding expression pattern after CSD induction. Based on immunoprecipitation assay of the methylated DNA (meDIP), we demonstrated hypermethylation of both sequences in preconditioned rat brain cortex compared with a control 24 h after CSD induction. Using quantitative PCR, we also showed that CSD induction caused a decrease of the transcript level of both retrotransposon sequences. Our data are consistent with the hypothesis of epigenetic modifications in Preconditioning-dependent neuroprotection by increasing genome stability via the silencing of retrotransposon sequences. PMID:27169424

  10. Phylum-Level Conservation of Regulatory Information in Nematodes despite Extensive Non-coding Sequence Divergence.

    PubMed

    Gordon, Kacy L; Arthur, Robert K; Ruvinsky, Ilya

    2015-05-01

    Gene regulatory information guides development and shapes the course of evolution. To test conservation of gene regulation within the phylum Nematoda, we compared the functions of putative cis-regulatory sequences of four sets of orthologs (unc-47, unc-25, mec-3 and elt-2) from distantly-related nematode species. These species, Caenorhabditis elegans, its congeneric C. briggsae, and three parasitic species Meloidogyne hapla, Brugia malayi, and Trichinella spiralis, represent four of the five major clades in the phylum Nematoda. Despite the great phylogenetic distances sampled and the extensive sequence divergence of nematode genomes, all but one of the regulatory elements we tested are able to drive at least a subset of the expected gene expression patterns. We show that functionally conserved cis-regulatory elements have no more extended sequence similarity to their C. elegans orthologs than would be expected by chance, but they do harbor motifs that are important for proper expression of the C. elegans genes. These motifs are too short to be distinguished from the background level of sequence similarity, and while identical in sequence they are not conserved in orientation or position. Functional tests reveal that some of these motifs contribute to proper expression. Our results suggest that conserved regulatory circuitry can persist despite considerable turnover within cis elements.

  11. 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

  12. 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. PMID:26301592

  13. Higher transcription levels in ascorbic acid biosynthetic and recycling genes were associated with higher ascorbic acid accumulation in blueberry.

    PubMed

    Liu, Fenghong; Wang, Lei; Gu, Liang; Zhao, Wei; Su, Hongyan; Cheng, Xianhao

    2015-12-01

    In our preliminary study, the ripe fruits of two highbush blueberry (Vaccinium corymbosum L.) cultivars, cv 'Berkeley' and cv 'Bluecrop', were found to contain different levels of ascorbic acid. However, factors responsible for these differences are still unknown. In the present study, ascorbic acid content in fruits was compared with expression profiles of ascorbic acid biosynthetic and recycling genes between 'Bluecrop' and 'Berkeley' cultivars. The results indicated that the l-galactose pathway was the predominant route of ascorbic acid biosynthesis in blueberry fruits. Moreover, higher expression levels of the ascorbic acid biosynthetic genes GME, GGP, and GLDH, as well as the recycling genes MDHAR and DHAR, were associated with higher ascorbic acid content in 'Bluecrop' compared with 'Berkeley', which indicated that a higher efficiency ascorbic acid biosynthesis and regeneration was likely to be responsible for the higher ascorbic acid accumulation in 'Bluecrop'.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. A new antifungal peptide from the seeds of Phytolacca americana: characterization, amino acid sequence and cDNA cloning.

    PubMed

    Shao, F; Hu, Z; Xiong, Y M; Huang, Q Z; WangCG; Zhu, R H; Wang, D C

    1999-03-19

    An antifungal peptide from seeds of Phytolacca americana, designated PAFP-s, has been isolated. The peptide is highly basic and consists of 38 residues with three disulfide bridges. Its molecular mass of 3929.0 was determined by mass spectrometry. The complete amino acid sequence was obtained from automated Edman degradation, and cDNA cloning was successfully performed by 3'-RACE. The deduced amino acid sequence of a partial cDNA corresponded to the amino acid sequence from chemical sequencing. PAFP-s exhibited a broad spectrum of antifungal activity, and its activities differed among various fungi. PAFP-s displayed no inhibitory activity towards Escherichia coli. PAFP-s shows significant sequence similarities and the same cysteine motif with Mj-AMPs, antimicrobial peptides from seeds of Mirabilis jalapa belonging to the knottin-type antimicrobial peptide.

  19. Amino acid sequence and variant forms of favin, a lectin from Vicia faba.

    PubMed

    Hopp, T P; Hemperly, J J; Cunningham, B A

    1982-04-25

    We have determined the complete amino acid sequence (182 residues) of the beta chain of favin, the glucose-binding lectin from fava beans (Vicia faba), and have established that the carbohydrate moiety is attached to Asn 168. Together with the sequence of the alpha chain previously reported (Hemperly, J. J., Hopp, T. P., Becker, J. W., and Cunningham, B. A. (1979) J. Biol. Chem. 254, 6803-6810), these data complete the analysis of the primary structure of the lectin. We have also examined minor polypeptides that appear in all preparations of favin. Two lower molecular weight species (Mr = 9,500-11,600) appear to be fragments of the beta chain resulting from cleavage following Asn 76, whereas six high molecular weight forms (Mr = 25,000 or greater) appear to include aggregates of the beta chain and possibly some alternative products of chain processing. PMID:7068646

  20. Pyrosequencing on templates generated by asymmetric nucleic acid sequence-based amplification (asymmetric-NASBA).

    PubMed

    Jia, Huning; Chen, Zhiyao; Wu, Haiping; Ye, Hui; Yan, Zhengyu; Zhou, Guohua

    2011-12-21

    Pyrosequencing is an ideal tool for verifying the sequence of amplicons. To enable pyrosequencing on amplicons from nucleic acid sequence-based amplification (NASBA), asymmetric NASBA with unequal concentrations of T7 promoter primer and reverse transcription primer was proposed. By optimizing the ratio of two primers and the concentration of dNTPs and NTPs, the amount of single-stranded cDNA in the amplicons from asymmetric NASBA was found increased 12 times more than the conventional NASBA through the real-time detection of a molecular beacon specific to cDNA of interest. More than 20 bases have been successfully detected by pyrosequencing on amplicons from asymmetric NASBA using Human parainfluenza virus (HPIV) as an amplification template. The primary results indicate that the combination of NASBA with a pyrosequencing system is practical, and should open a new field in clinical diagnosis.

  1. 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.

  2. Upper Cretaceous sequences and sea-level history, New Jersey Coastal Plain

    USGS Publications Warehouse

    Miller, K.G.; Sugarman, P.J.; Browning, J.V.; Kominz, M.A.; Olsson, R.K.; Feigenson, M.D.; Hernandez, J.C.

    2004-01-01

    We developed a Late Cretaceous sealevel estimate from Upper Cretaceous sequences at Bass River and Ancora, New Jersey (ODP [Ocean Drilling Program] Leg 174AX). We dated 11-14 sequences by integrating Sr isotope and biostratigraphy (age resolution ??0.5 m.y.) and then estimated paleoenvironmental changes within the sequences from lithofacies and biofacies analyses. Sequences generally shallow upsection from middle-neritic to inner-neritic paleodepths, as shown by the transition from thin basal glauconite shelf sands (transgressive systems tracts [TST]), to medial-prodelta silty clays (highstand systems tracts [HST]), and finally to upper-delta-front quartz sands (HST). Sea-level estimates obtained by backstripping (accounting for paleodepth variations, sediment loading, compaction, and basin subsidence) indicate that large (>25 m) and rapid (???1 m.y.) sea-level variations occurred during the Late Cretaceous greenhouse world. The fact that the timing of Upper Cretaceous sequence boundaries in New Jersey is similar to the sea-level lowering records of Exxon Production Research Company (EPR), northwest European sections, and Russian platform outcrops points to a global cause. Because backstripping, seismicity, seismic stratigraphic data, and sediment-distribution patterns all indicate minimal tectonic effects on the New Jersey Coastal Plain, we interpret that we have isolated a eustatic signature. The only known mechanism that can explain such global changes-glacio-eustasy-is consistent with foraminiferal ??18O data. Either continental ice sheets paced sea-level changes during the Late Cretaceous, or our understanding of causal mechanisms for global sea-level change is fundamentally flawed. Comparison of our eustatic history with published ice-sheet models and Milankovitch predictions suggests that small (5-10 ?? 106 km3), ephemeral, and areally restricted Antarctic ice sheets paced the Late Cretaceous global sea-level change. New Jersey and Russian eustatic estimates

  3. ADS genes for reducing saturated fatty acid levels in seed oils

    DOEpatents

    Heilmann, Ingo H.; Shanklin, John

    2010-02-02

    The present invention relates to enzymes involved in lipid metabolism. In particular, the present invention provides coding sequences for Arabidopsis Desaturases (ADS), the encoded ADS polypeptides, and methods for using the sequences and encoded polypeptides, where such methods include decreasing and increasing saturated fatty acid content in plant seed oils.

  4. ADS genes for reducing saturated fatty acid levels in seed oils

    DOEpatents

    Heilmann, Ingo H; Shanklin, John

    2014-03-18

    The present invention relates to enzymes involved in lipid metabolism. In particular, the present invention provides coding sequences for Arabidopsis Desaturases (ADS), the encoded ADS polypeptides, and methods for using the sequences and encoded polypeptides, where such methods include decreasing and increasing saturated fatty acid content in plant seed oils.

  5. The amino-acid sequences of sculpin islet somatostatin-28 and peptide YY.

    PubMed

    Cutfield, S M; Carne, A; Cutfield, J F

    1987-04-01

    Two pancreatic peptides, somatostatin-28 and peptide YY, have been isolated from the Brockmann bodies of the teleost fish Cottus scorpius (daddy sculpin). Following purification by reverse-phase HPLC, each peptide was sequenced completely through to the carboxyl-terminus by gas-phase Edman degradation. Somatostatin-28 was the major form of somatostatin detected and is similar to the gene II product from anglerfish. Peptide YY (36 amino acids) more closely resembles porcine neuropeptide YY and intestinal peptide YY than it does the pancreatic polypeptides. PMID:2883025

  6. 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.

  7. Sequence selective double strand DNA cleavage by peptide nucleic acid (PNA) targeting using nuclease S1.

    PubMed Central

    Demidov, V; Frank-Kamenetskii, M D; Egholm, M; Buchardt, O; Nielsen, P E

    1993-01-01

    A novel method for sequence specific double strand DNA cleavage using PNA (peptide nucleic acid) targeting is described. Nuclease S1 digestion of double stranded DNA gives rise to double strand cleavage at an occupied PNA strand displacement binding site, and under optimized conditions complete cleavage can be obtained. The efficiency of this cleavage is more than 10 fold enhanced when a tandem PNA site is targeted, and additionally enhanced if this site is in trans rather than in cis orientation. Thus in effect, the PNA targeting makes the single strand specific nuclease S1 behave like a pseudo restriction endonuclease. Images PMID:8502550

  8. 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.

  9. WinGene/WinPep: user-friendly software for the analysis of amino acid sequences.

    PubMed

    Hennig, L

    1999-06-01

    WinGene1.0/WinPep1.2 is a pair of Microsoft Windows programs designed to read nucleotide or amino acid sequence data. These versatile programs have the following capabilities: (i) searches for open reading frames and their translation, (ii) assisting the design of primers for PCR and (iii) calculation of molecular weight, isoelectric point and molar absorbtion coefficients of polypeptides. Furthermore, hydropathic plots and helical wheel displays are easily produced. The programs run with an intuitive Windows interface, contain a comprehensive help file and enable data exchange with other applications by means of the Copy&Paste command. The software is free for academic and noncommercial users.

  10. Amino acid sequence coevolution in the insect bursicon ligand-receptor system.

    PubMed

    Hughes, Austin L

    2012-06-01

    The pattern of amino acid residue replacement in the components of the bursicon signaling system (involving the BURSα/BURSβ heterodimer and its receptor BURSrec) was reconstructed across a phylogeny of 17 insect species, in order to test for the co-occurrence of replacements at sets of individual sites. Sets of three or more branches with perfectly concordant changes occurred to a greater extent than expected by chance, given the observed level of amino acid change. The latter sites (SPC sites) were found to have distinctive characteristics: (1) the mean number of changes was significantly lower at SPC sites than that at other sites with multiple changes; (2) SPC sites had a significantly greater tendency toward parallel amino acid changes than other sites with multiple changes, but no greater tendency toward convergent changes; and (3) parallel changes tended to involve relatively similar amino acids, as indicated by relatively low mean chemical distances. The results implicated functional constraint, permitting only a limited subset of amino acids in a given site, as a major factor in causing both parallel amino acid replacement and coordinated amino acid changes in different sites of the same protein and of interacting proteins in this system.

  11. Purification and amino acid sequence of aminopeptidase P from pig kidney.

    PubMed

    Vergas Romero, C; Neudorfer, I; Mann, K; Schäfer, W

    1995-04-01

    Aminopeptidase P from kidney cortex was purified in high yield (recovery greater than or equal to 20%) by a series of column chromatographic steps after solubilization of the membrane-bound glycoprotein with n-butanol. A coupled enzymic assay, using Gly-Pro-Pro-NH-Nap as substrate and dipeptidyl-peptidase IV as auxilliary enzyme, was used to monitor the purification. The purification procedure yielded two forms of aminopeptidase P differing in their carbohydrate composition (glycoforms). Both enzyme preparations were homogeneous as assessed by SDS/PAGE silver staining, and isoelectric focusing. Both forms possessed the same substrate specificity, catalysed the same reaction, and consisted of identical protein chains. The amino acid sequence determined by Edman degradation and mass spectrometry consisted of 623 amino acids. Six N-glycosylation sites, all contained in the N-terminal half of the protein, were characterized. PMID:7744038

  12. Mass spectrometric detection of the amino acid sequence polymorphism of the hepatitis C virus antigen.

    PubMed

    Kaysheva, A L; Ivanov, Yu D; Frantsuzov, P A; Krohin, N V; Pavlova, T I; Uchaikin, V F; Konev, V А; Kovalev, O B; Ziborov, V S; Archakov, A I

    2016-03-01

    A method for detection and identification of the hepatitis C virus antigen (HCVcoreAg) in human serum with consideration for possible amino acid substitutions is proposed. The method is based on a combination of biospecific capturing and concentrating of the target protein on the surface of the chip for atomic force microscope (AFM chip) with subsequent protein identification by tandem mass spectrometric (MS/MS) analysis. Biospecific AFM-capturing of viral particles containing HCVcoreAg from serum samples was performed by use of AFM chips with monoclonal antibodies (anti-HCVcore) covalently immobilized on the surface. Biospecific complexes were registered and counted by AFM. Further MS/MS analysis allowed to reliably identify the HCVcoreAg in the complexes formed on the AFM chip surface. Analysis of MS/MS spectra, with the account taken of the possible polymorphisms in the amino acid sequence of the HCVcoreAg, enabled us to increase the number of identified peptides.

  13. Heavy metal levels and delta-amino-levulinic acid dehydrase levels in peripheral polyneuropathy.

    PubMed

    Melgaard, B; Clausen, J; Rastogi, S C

    1976-04-01

    On the basis of assay of heavy metals in whole blood (lead, cadimum, chromium, copper, nickel and manganese) and delta-amino-levulinic-acid dehydrase (ALA-D) and ALA in urine in a normal Danish population, the levels of these clinco-chemical factors were assayed in 23 patients with peripheral neuropathy of unknown etiology. All patients studied showed electro-physiological sign of denervation and/or reduced motor or sensoric nerve conduction velocity. Cadmium and manganese were never found to be increased. In all but four patients, an increase of one or more heavy metals was found. Ten patients showed raised levels of two or more metals, the dominant metal being lead (10 cases), nine patients showed increased in chromium. A significant corrleation was found between increasing lead levels and decreasing ALA-D activity. Although normal concentrations of manganese were found, correlation analysis revealed a significant correlation between increased manganese and decreased ALA-D. The raised values of heavy metals could not be traced to occupational or other exposure to heavy metals and the increased values were not related to tobacco consumption. The findings are discussed in relation to known data on neuropathy and the results seem to indicate a multifactorial patholgenesis of the disease. Among factors contributing to the precipitation of the syndrome may be raised levels of heavy metals.

  14. Draft Genome Sequence of Bacillus subtilis subsp. natto Strain CGMCC 2108, a High Producer of Poly-γ-Glutamic Acid

    PubMed Central

    Tan, Siyuan; Su, Anping; Zhang, Chen; Ren, Yuanyuan

    2016-01-01

    Here, we report the 4.1-Mb draft genome sequence of Bacillus subtilis subsp. natto strain CGMCC 2108, a high producer of poly-γ-glutamic acid (γ-PGA). This sequence will provide further help for the biosynthesis of γ-PGA and will greatly facilitate research efforts in metabolic engineering of B. subtilis subsp. natto strain CGMCC 2108. PMID:27231363

  15. WAViS server for handling, visualization and presentation of multiple alignments of nucleotide or amino acids sequences.

    PubMed

    Zika, Radek; Paces, Jan; Pavlícek, Adam; Paces, Václav

    2004-07-01

    Web Alignment Visualization Server contains a set of web-tools designed for quick generation of publication-quality color figures of multiple alignments of nucleotide or amino acids sequences. It can be used for identification of conserved regions and gaps within many sequences using only common web browsers. The server is accessible at http://wavis.img.cas.cz.

  16. ANTICALIgN: visualizing, editing and analyzing combined nucleotide and amino acid sequence alignments for combinatorial protein engineering.

    PubMed

    Jarasch, Alexander; Kopp, Melanie; Eggenstein, Evelyn; Richter, Antonia; Gebauer, Michaela; Skerra, Arne

    2016-07-01

    ANTIC ALIGN: is an interactive software developed to simultaneously visualize, analyze and modify alignments of DNA and/or protein sequences that arise during combinatorial protein engineering, design and selection. ANTIC ALIGN: combines powerful functions known from currently available sequence analysis tools with unique features for protein engineering, in particular the possibility to display and manipulate nucleotide sequences and their translated amino acid sequences at the same time. ANTIC ALIGN: offers both template-based multiple sequence alignment (MSA), using the unmutated protein as reference, and conventional global alignment, to compare sequences that share an evolutionary relationship. The application of similarity-based clustering algorithms facilitates the identification of duplicates or of conserved sequence features among a set of selected clones. Imported nucleotide sequences from DNA sequence analysis are automatically translated into the corresponding amino acid sequences and displayed, offering numerous options for selecting reading frames, highlighting of sequence features and graphical layout of the MSA. The MSA complexity can be reduced by hiding the conserved nucleotide and/or amino acid residues, thus putting emphasis on the relevant mutated positions. ANTIC ALIGN: is also able to handle suppressed stop codons or even to incorporate non-natural amino acids into a coding sequence. We demonstrate crucial functions of ANTIC ALIGN: in an example of Anticalins selected from a lipocalin random library against the fibronectin extradomain B (ED-B), an established marker of tumor vasculature. Apart from engineered protein scaffolds, ANTIC ALIGN: provides a powerful tool in the area of antibody engineering and for directed enzyme evolution.

  17. 3-d structure-based amino acid sequence alignment of esterases, lipases and related proteins

    SciTech Connect

    Gentry, M.K.; Doctor, B.P.; Cygler, M.; Schrag, J.D.; Sussman, J.L.

    1993-05-13

    Acetylcholinesterase and butyrylcholinesterase, enzymes with potential as pretreatment drugs for organophosphate toxicity, are members of a larger family of homologous proteins that includes carboxylesterases, cholesterol esterases, lipases, and several nonhydrolytic proteins. A computer-generated alignment of 18 of the proteins, the acetylcholinesases, butyrylcholinesterases, carboxylesterases, some esterases, and the nonenzymatic proteins has been previously presented. More recently, the three-dimensional structures of two enzymes enzymes in this group, acetylcholinesterase from Torpedo californica and lipase from Geotrichum candidum, have been determined. Based on the x-ray structures and the superposition of these two enzymes, it was possible to obtain an improved amino acid sequence alignment of 32 members of this family of proteins. Examination of this alignment reveals that 24 amino acids are invariant in all of the hydrolytic proteins, and an additional 49 are well conserved. Conserved amino acids include those of the active site, the disulfide bridges, the salt bridges, in the core of the proteins, and at the edges of secondary structural elements. Comparison of the three-dimensional structures makes it possible to find a well-defined structural basis for the conservation of many of these amino acids.

  18. High nucleotide and amino acid sequence similarities in tumour necrosis factor-alpha amongst Indian buffalo (Bubalus bubalis), Indian cattle (Bos indicus) and other ruminants.

    PubMed

    Gupta, P K; Bind, R B; Walunj, S S; Saini, M

    2004-08-01

    Tumour necrosis factor-alpha (TNF-alpha) mRNA from Indian water buffalo (Bubalus bubalis) and Indian cattle (Bos indicus) was reverse transcribed and amplified using reverse transcriptase-polymerase chain reaction (RT-PCR). The nucleotide sequences of cDNAs were determined after cloning into pGEM-T-Easy vector (Promega, Madison, WI) and compared with reported nucleotide sequences of TNF-alpha cDNA from other species. The nucleotide sequences of TNF-alpha from Indian cattle revealed significantly high similarities at nucleotide (99.2%) and amino acid (100%) levels with those of cattle (Bos taurus; Zebu). The sequences from buffalo had 98.4% nucleotide and 99.1% amino acid similarities with Indian cattle, indicating functional cross-reactivity. One amino acid deletion at position 63 and one substitution (A-->P) at position 64 were observed in buffalo compared with Indian cattle. The amino acid deletion at position 63 was predicted due to differences in pre-mRNA splicing.

  19. Correlations Between Amino Acids at Different Sites in Local Sequences of Protein Fragments with Given Structural Patterns

    NASA Astrophysics Data System (ADS)

    Lu, Wen; Liu, Hai-yan

    2007-02-01

    Ample evidence suggests that the local structures of peptide fragments in native proteins are to some extent encoded by their local sequences. Detecting such local correlations is important but it is still an open question what would be the most appropriate method. This is partly because conventional sequence analyses treat amino acid preferences at each site of a protein sequence independently, while it is often the inter-site interactions that bring about local sequence-structure correlations. Here a new scheme is introduced to capture the correlation between amino acid preferences at different sites for different local structure types. A library of nine-residue fragments is constructed, and the fragments are divided into clusters based on their local structures. For each local structure cluster or type, chi-square tests are used to identify correlated preferences of amino acid combinations at pairs of sites. A score function is constructed including both the single site amino acid preferences and the dual-site amino acid combination preferences, which can be used to identify whether a sequence fragment would have a strong tendency to form a particular local structure in native proteins. The results show that, given a local structure pattern, dual-site amino acid combinations contain different information from single site amino acid preferences. Representative examples show that many of the statistically identified correlations agree with previously-proposed heuristic rules about local sequence-structure correlations, or are consistent with physical-chemical interactions required to stabilize particular local structures. Results also show that such dual-site correlations in the score function significantly improves the Z-score matching a sequence fragment to its native local structure relative to non-native local structures, and certain local structure types are highly predictable from the local sequence alone if inter-site correlations are considered.

  20. Pleiotropic constraints, expression level, and the evolution of miRNA sequences.

    PubMed

    Jovelin, Richard

    2013-12-01

    Post-transcriptional gene regulation mediated by microRNAs (miRNAs) plays critical roles during development by modulating gene expression and conferring robustness to stochastic errors. Phylogenetic analyses suggest that miRNA acquisition could play a role in phenotypic innovation. Moreover, miRNA-induced regulation strongly impacts genome evolution, increasing selective constraints on 3'UTRs, protein sequences, and expression level divergence. Thus, it is essential to understand the factors governing sequence evolution for this important class of regulatory molecules. Investigation of the patterns of molecular evolution at miRNA loci have been limited in Caenorhabditis elegans because of the lack of a close outgroup. Instead, I used Caenorhabditis briggsae as the focus point of this study because of its close relationship to Caenorhabditis sp. 9. I also corroborated the patterns of sequence evolution in Caenorhabditis using published orthologous relationships among miRNAs in Drosophila. In nematodes and in flies, miRNA sequence divergence is not influenced by the genomic neighborhood (i.e., intronic or intergenic) but is nevertheless affected by the genomic context because X-linked miRNAs evolve faster than autosomal miRNAs. However, this effect of chromosomal linkage can be explained by differential expression levels rather than a fast-X effect. The results presented here support a universal negative relationship between rates of molecular evolution and expression level, and suggest that mutations in highly expressed miRNAs are more likely to be deleterious because they potentially affect a larger number of target genes. Finally, I show that many single family member miRNAs evolve faster than miRNAs from multigene families and have limited functional scope, suggesting that they are not strongly integrated in gene regulatory networks.

  1. Draft Genome Sequences of Gluconobacter cerinus CECT 9110 and Gluconobacter japonicus CECT 8443, Acetic Acid Bacteria Isolated from Grape Must

    PubMed Central

    Sainz, Florencia

    2016-01-01

    We report here the draft genome sequences of Gluconobacter cerinus strain CECT9110 and Gluconobacter japonicus CECT8443, acetic acid bacteria isolated from grape must. Gluconobacter species are well known for their ability to oxidize sugar alcohols into the corresponding acids. Our objective was to select strains to oxidize effectively d-glucose. PMID:27365351

  2. Chicoric Acid Levels in Basil (Ocimum basilicum L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recently, we reported the presence of chicoric acid in basil leaves (confirmed by co-chromatography with purchased standard). Chicoric acid being the chief phenolic of the Echinacea purpurea plant which is popularly consumed as a dietary supplement. For this study, basil products commonly purchased ...

  3. 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

  4. 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.

  5. Detection of piscine nodaviruses by real-time nucleic acid sequence based amplification (NASBA).

    PubMed

    Starkey, William G; Millar, Rose Mary; Jenkins, Mary E; Ireland, Jacqueline H; Muir, K Fiona; Richards, Randolph H

    2004-05-01

    Nucleic acid sequence based amplification (NASBA) is an isothermal nucleic acid amplification procedure based on target-specific primers and probes, and the co-ordinated activity of 3 enzymes: AMV reverse transcriptase, RNase H, and T7 RNA polymerase. We have developed a real-time NASBA procedure for detection of piscine nodaviruses, which have emerged as major pathogens of marine fish. Viral RNA was isolated by guanidine thiocyanate lysis followed by purification on silica particles. Primers were designed to target sequences in the nodavirus capsid protein gene, yielding an amplification product of 120 nucleotides. Amplification products were detected in real-time with a molecular beacon (FAM labelled/methyl-red quenched) that recognised an internal region of the target amplicon. Amplification and detection were performed at 41 degrees C for 90 min in a Corbett Research Rotorgene. Based on the detection of cell culture-derived nodavirus, and a synthetic RNA target, the real-time NASBA procedure was approximately 100-fold more sensitive than single-tube RT-PCR. When used to test a panel of 37 clinical samples (negative, n = 18; positive, n = 19), the real-time NASBA assay correctly identified all 18 negative and 19 positive samples. In comparison, the RT-PCR procedure identified all 18 negative samples, but only 16 of the positive samples. These results suggest that real-time NASBA may represent a sensitive and specific diagnostic procedure for piscine nodaviruses.

  6. From amino acid sequence to bioactivity: The biomedical potential of antitumor peptides.

    PubMed

    Blanco-Míguez, Aitor; Gutiérrez-Jácome, Alberto; Pérez-Pérez, Martín; Pérez-Rodríguez, Gael; Catalán-García, Sandra; Fdez-Riverola, Florentino; Lourenço, Anália; Sánchez, Borja

    2016-06-01

    Chemoprevention is the use of natural and/or synthetic substances to block, reverse, or retard the process of carcinogenesis. In this field, the use of antitumor peptides is of interest as, (i) these molecules are small in size, (ii) they show good cell diffusion and permeability, (iii) they affect one or more specific molecular pathways involved in carcinogenesis, and (iv) they are not usually genotoxic. We have checked the Web of Science Database (23/11/2015) in order to collect papers reporting on bioactive peptide (1691 registers), which was further filtered searching terms such as "antiproliferative," "antitumoral," or "apoptosis" among others. Works reporting the amino acid sequence of an antiproliferative peptide were kept (60 registers), and this was complemented with the peptides included in CancerPPD, an extensive resource for antiproliferative peptides and proteins. Peptides were grouped according to one of the following mechanism of action: inhibition of cell migration, inhibition of tumor angiogenesis, antioxidative mechanisms, inhibition of gene transcription/cell proliferation, induction of apoptosis, disorganization of tubulin structure, cytotoxicity, or unknown mechanisms. The main mechanisms of action of those antiproliferative peptides with known amino acid sequences are presented and finally, their potential clinical usefulness and future challenges on their application is discussed.

  7. 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

    1989-07-01

    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.

  8. Molecular cloning, encoding sequence, and expression of vaccinia virus nucleic acid-dependent nucleoside triphosphatase gene.

    PubMed

    Rodriguez, J F; Kahn, J S; Esteban, M

    1986-12-01

    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.

  9. NR-2L: a two-level predictor for identifying nuclear receptor subfamilies based on sequence-derived features.

    PubMed

    Wang, Pu; Xiao, Xuan; Chou, Kuo-Chen

    2011-01-01

    Nuclear receptors (NRs) are one of the most abundant classes of transcriptional regulators in animals. They regulate diverse functions, such as homeostasis, reproduction, development and metabolism. Therefore, NRs are a very important target for drug development. Nuclear receptors form a superfamily of phylogenetically related proteins and have been subdivided into different subfamilies due to their domain diversity. In this study, a two-level predictor, called NR-2L, was developed that can be used to identify a query protein as a nuclear receptor or not based on its sequence information alone; if it is, the prediction will be automatically continued to further identify it among the following seven subfamilies: (1) thyroid hormone like (NR1), (2) HNF4-like (NR2), (3) estrogen like, (4) nerve growth factor IB-like (NR4), (5) fushi tarazu-F1 like (NR5), (6) germ cell nuclear factor like (NR6), and (7) knirps like (NR0). The identification was made by the Fuzzy K nearest neighbor (FK-NN) classifier based on the pseudo amino acid composition formed by incorporating various physicochemical and statistical features derived from the protein sequences, such as amino acid composition, dipeptide composition, complexity factor, and low-frequency Fourier spectrum components. As a demonstration, it was shown through some benchmark datasets derived from the NucleaRDB and UniProt with low redundancy that the overall success rates achieved by the jackknife test were about 93% and 89% in the first and second level, respectively. The high success rates indicate that the novel two-level predictor can be a useful vehicle for identifying NRs and their subfamilies. As a user-friendly web server, NR-2L is freely accessible at either http://icpr.jci.edu.cn/bioinfo/NR2L or http://www.jci-bioinfo.cn/NR2L. Each job submitted to NR-2L can contain up to 500 query protein sequences and be finished in less than 2 minutes. The less the number of query proteins is, the shorter the time will

  10. Gut Microbial Fatty Acid Metabolites Reduce Triacylglycerol Levels in Hepatocytes.

    PubMed

    Nanthirudjanar, Tharnath; Furumoto, Hidehiro; Zheng, Jiawen; Kim, Young-Il; Goto, Tsuyoshi; Takahashi, Nobuyuki; Kawada, Teruo; Park, Si-Bum; Hirata, Akiko; Kitamura, Nahoko; Kishino, Shigenobu; Ogawa, Jun; Hirata, Takashi; Sugawara, Tatsuya

    2015-11-01

    Hydroxy and oxo fatty acids were recently found to be produced as intermediates during gut microbial fatty acid metabolism. Lactobacillus plantarum produces these fatty acids from unsaturated fatty acids such as linoleic acid. In this study, we investigated the effects of these gut microbial fatty acid metabolites on the lipogenesis in liver cells. We screened their effect on sterol regulatory element binding protein-1c (SREBP-1c) expression in HepG2 cells treated with a synthetic liver X receptor α (LXRα) agonist (T0901317). The results showed that 10-hydroxy-12(Z)-octadecenoic acid (18:1) (HYA), 10-hydroxy-6(Z),12(Z)-octadecadienoic acid (18:2) (γHYA), 10-oxo-12(Z)-18:1 (KetoA), and 10-oxo-6(Z),12(Z)-18:2 (γKetoA) significantly decreased SREBP-1c mRNA expression induced by T0901317. These fatty acids also downregulated the mRNA expression of lipogenic genes by suppressing LXRα activity and inhibiting SREBP-1 maturation. Oral administration of KetoA, which effectively reduced triacylglycerol accumulation and acetyl-CoA carboxylase 2 (ACC2) expression in HepG2 cells, for 2 weeks significantly decreased Srebp-1c, Scd-1, and Acc2 expression in the liver of mice fed a high-sucrose diet. Our findings suggest that the hypolipidemic effect of the fatty acid metabolites produced by L. plantarum can be exploited in the treatment of cardiovascular diseases or dyslipidemia. PMID:26399511

  11. Complete amino acid sequence of the myoglobin from the Pacific spotted dolphin, Stenella attenuata graffmani.

    PubMed

    Jones, B N; Wang, C C; Dwulet, F E; Lehman, L D; Meuth, J L; Bogardt, R A; Gurd, F R

    1979-04-25

    The complete amino acid sequence of the major component myoglobin from the Pacific spotted dolphin, Stenella attenuata graffmani, was determined by the automated Edman degradation of several large peptides obtained by specific cleavage of the protein. The acetimidated apomyoglobin was selectively cleaved at its two methionyl residues with cyanogen bromide and at its three arginyl residues by trypsin. By subjecting four of these peptides and the apomyoglobin to automated Edman degradation, over 80% of the primary structure of the protein was obtained. The remainder of the covalent structure was determined by the sequence analysis of peptides that resulted from further digestion of the central cyanogen bromide fragment. This fragment was cleaved at its glutamyl residues with staphylococcal protease and its lysyl residues with trypsin. The action of trypsin was restricted to the lysyl residues by chemical modification of the single arginyl residue of the fragment with 1,2-cyclohexanedione. The primary structure of this myoglobin proved to be identical with that from the Atlantic bottlenosed dolphin and Pacific common dolphin but differs from the myoglobins of the killer whale and pilot whale at two positions. The above sequence identities and differences reflect the close taxonomic relationship of these five species of Cetacea. PMID:454657

  12. 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.

  13. Deleted copy number variation of Hanwoo and Holstein using next generation sequencing at the population level

    PubMed Central

    2014-01-01

    Background Copy number variation (CNV), a source of genetic diversity in mammals, has been shown to underlie biological functions related to production traits. Notwithstanding, there have been few studies conducted on CNVs using next generation sequencing at the population level. Results Illumina NGS data was obtained for ten Holsteins, a dairy cattle, and 22 Hanwoo, a beef cattle. The sequence data for each of the 32 animals varied from 13.58-fold to almost 20-fold coverage. We detected a total of 6,811 deleted CNVs across the analyzed individuals (average length = 2732.2 bp) corresponding to 0.74% of the cattle genome (18.6 Mbp of variable sequence). By examining the overlap between CNV deletion regions and genes, we selected 30 genes with the highest deletion scores. These genes were found to be related to the nervous system, more specifically with nervous transmission, neuron motion, and neurogenesis. We regarded these genes as having been effected by the domestication process. Further analysis of the CNV genotyping information revealed 94 putative selected CNVs and 954 breed-specific CNVs. Conclusions This study provides useful information for assessing the impact of CNVs on cattle traits using NGS at the population level. PMID:24673797

  14. Sequence-Level Analysis of the Major European Huntington Disease Haplotype.

    PubMed

    Lee, Jong-Min; Kim, Kyung-Hee; Shin, Aram; Chao, Michael J; Abu Elneel, Kawther; Gillis, Tammy; Mysore, Jayalakshmi Srinidhi; Kaye, Julia A; Zahed, Hengameh; Kratter, Ian H; Daub, Aaron C; Finkbeiner, Steven; Li, Hong; Roach, Jared C; Goodman, Nathan; Hood, Leroy; Myers, Richard H; MacDonald, Marcy E; Gusella, James F

    2015-09-01

    Huntington disease (HD) reflects the dominant consequences of a CAG-repeat expansion in HTT. Analysis of common SNP-based haplotypes has revealed that most European HD subjects have distinguishable HTT haplotypes on their normal and disease chromosomes and that ∼50% of the latter share the same major HD haplotype. We reasoned that sequence-level investigation of this founder haplotype could provide significant insights into the history of HD and valuable information for gene-targeting approaches. Consequently, we performed whole-genome sequencing of HD and control subjects from four independent families in whom the major European HD haplotype segregates with the disease. Analysis of the full-sequence-based HTT haplotype indicated that these four families share a common ancestor sufficiently distant to have permitted the accumulation of family-specific variants. Confirmation of new CAG-expansion mutations on this haplotype suggests that unlike most founders of human disease, the common ancestor of HD-affected families with the major haplotype most likely did not have HD. Further, availability of the full sequence data validated the use of SNP imputation to predict the optimal variants for capturing heterozygosity in personalized allele-specific gene-silencing approaches. As few as ten SNPs are capable of revealing heterozygosity in more than 97% of European HD subjects. Extension of allele-specific silencing strategies to the few remaining homozygous individuals is likely to be achievable through additional known SNPs and discovery of private variants by complete sequencing of HTT. These data suggest that the current development of gene-based targeting for HD could be extended to personalized allele-specific approaches in essentially all HD individuals of European ancestry. PMID:26320893

  15. Use of high throughput sequencing to observe genome dynamics at a single cell level

    PubMed Central

    Parkhomchuk, D.; Amstislavskiy, V.; Soldatov, A.; Ogryzko, V.

    2009-01-01

    With the development of high throughput sequencing technology, it becomes possible to directly analyze mutation distribution in a genome-wide fashion, dissociating mutation rate measurements from the traditional underlying assumptions. Here, we sequenced several genomes of Escherichia coli from colonies obtained after chemical mutagenesis and observed a strikingly nonrandom distribution of the induced mutations. These include long stretches of exclusively G to A or C to T transitions along the genome and orders of magnitude intra- and intergenomic differences in mutation density. Whereas most of these observations can be explained by the known features of enzymatic processes, the others could reflect stochasticity in the molecular processes at the single-cell level. Our results demonstrate how analysis of the molecular records left in the genomes of the descendants of an individual mutagenized cell allows for genome-scale observations of fixation and segregation of mutations, as well as recombination events, in the single genome of their progenitor. PMID:19934054

  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. Convergent Evolution of Hemoglobin Function in High-Altitude Andean Waterfowl Involves Limited Parallelism at the Molecular Sequence Level.

    PubMed

    Natarajan, Chandrasekhar; Projecto-Garcia, Joana; Moriyama, Hideaki; Weber, Roy E; Muñoz-Fuentes, Violeta; Green, Andy J; Kopuchian, Cecilia; Tubaro, Pablo L; Alza, Luis; Bulgarella, Mariana; Smith, Matthew M; Wilson, Robert E; Fago, Angela; McCracken, Kevin G; Storz, Jay F

    2015-12-01

    A fundamental question in evolutionary genetics concerns the extent to which adaptive phenotypic convergence is attributable to convergent or parallel changes at the molecular sequence level. Here we report a comparative analysis of hemoglobin (Hb) function in eight phylogenetically replicated pairs of high- and low-altitude waterfowl taxa to test for convergence in the oxygenation properties of Hb, and to assess the extent to which convergence in biochemical phenotype is attributable to repeated amino acid replacements. Functional experiments on native Hb variants and protein engineering experiments based on site-directed mutagenesis revealed the phenotypic effects of specific amino acid replacements that were responsible for convergent increases in Hb-O2 affinity in multiple high-altitude taxa. In six of the eight taxon pairs, high-altitude taxa evolved derived increases in Hb-O2 affinity that were caused by a combination of unique replacements, parallel replacements (involving identical-by-state variants with independent mutational origins in different lineages), and collateral replacements (involving shared, identical-by-descent variants derived via introgressive hybridization). In genome scans of nucleotide differentiation involving high- and low-altitude populations of three separate species, function-altering amino acid polymorphisms in the globin genes emerged as highly significant outliers, providing independent evidence for adaptive divergence in Hb function. The experimental results demonstrate that convergent changes in protein function can occur through multiple historical paths, and can involve multiple possible mutations. Most cases of convergence in Hb function did not involve parallel substitutions and most parallel substitutions did not affect Hb-O2 affinity, indicating that the repeatability of phenotypic evolution does not require parallelism at the molecular level.

  18. Convergent Evolution of Hemoglobin Function in High-Altitude Andean Waterfowl Involves Limited Parallelism at the Molecular Sequence Level

    PubMed Central

    Natarajan, Chandrasekhar; Projecto-Garcia, Joana; Moriyama, Hideaki; Weber, Roy E.; Muñoz-Fuentes, Violeta; Green, Andy J.; Kopuchian, Cecilia; Tubaro, Pablo L.; Alza, Luis; Bulgarella, Mariana; Smith, Matthew M.; Wilson, Robert E.; Fago, Angela; McCracken, Kevin G.; Storz, Jay F.

    2015-01-01

    A fundamental question in evolutionary genetics concerns the extent to which adaptive phenotypic convergence is attributable to convergent or parallel changes at the molecular sequence level. Here we report a comparative analysis of hemoglobin (Hb) function in eight phylogenetically replicated pairs of high- and low-altitude waterfowl taxa to test for convergence in the oxygenation properties of Hb, and to assess the extent to which convergence in biochemical phenotype is attributable to repeated amino acid replacements. Functional experiments on native Hb variants and protein engineering experiments based on site-directed mutagenesis revealed the phenotypic effects of specific amino acid replacements that were responsible for convergent increases in Hb-O2 affinity in multiple high-altitude taxa. In six of the eight taxon pairs, high-altitude taxa evolved derived increases in Hb-O2 affinity that were caused by a combination of unique replacements, parallel replacements (involving identical-by-state variants with independent mutational origins in different lineages), and collateral replacements (involving shared, identical-by-descent variants derived via introgressive hybridization). In genome scans of nucleotide differentiation involving high- and low-altitude populations of three separate species, function-altering amino acid polymorphisms in the globin genes emerged as highly significant outliers, providing independent evidence for adaptive divergence in Hb function. The experimental results demonstrate that convergent changes in protein function can occur through multiple historical paths, and can involve multiple possible mutations. Most cases of convergence in Hb function did not involve parallel substitutions and most parallel substitutions did not affect Hb-O2 affinity, indicating that the repeatability of phenotypic evolution does not require parallelism at the molecular level. PMID:26637114

  19. Sea-level changes and the Middle-Upper Devonian sequence in the Baltic basin

    NASA Astrophysics Data System (ADS)

    Lukševičs, E.; Stinkulis, Ä.¢.

    2012-04-01

    Lithostratigraphic succession of the Middle-Upper Devonian of Baltic, represented by siliciclastic and carbonate deposits of the wide shallow epeiric sea, is well-established. The sequence of biotic and sea-level changes has been studied in detail for about half a century. However, it was rarely tried to compare the sea-level curve for the Baltic with the curve of the global sea-level changes and correlate the succession with the sequence of the Devonian global events well established mainly in the marine record. New facies analysis and biostratigraphical and taphonomical studies together with the re-evaluation of signatures of the world-wide events using sedimentological and palaeontological data enables better understanding of the development of the Middle-Late Devonian basins of the Baltic area. Indications of such events as eustatic fall of the sea-level close to the Givetian-Frasnian and Frasnian-Famennian boundaries, several small-scale transgressions during Famennian interglacials are rather clearly traceable within the sequence. Distribution of deposits, biotas and facies suggests diminishing of the depositional area since the maximum transgression in the earliest Frasnian thus demonstrating good coincidence with the global sea level curve (Haq & Schutter 2008). The retreat of depocentre of the palaeobasin in westerly direction during the late Frasnian and the Famennian likely was caused by tectonic subsidence at the western part of the east Baltics. The most significant event levels identified within the section are the extended Taghanic onlap (middle Givetian) evidenced by dolocretes in the upper part of the Burtnieki Fm; significant drop of the sea level during the earliest Frasnian indicated by widely distributed dolocretes in the top of the Amata Fm that points to the possible position of the Givetian/Frasnian boundary below the Amata; level of the Dubnik RS with extensive gypsum deposits and non-oxidised organic matter of the Salaspils Fm evidencing

  20. Effect of supplemental folic acid on valproic acid-induced embryotoxicity and tissue zinc levels in vivo.

    PubMed

    Hansen, D K; Grafton, T F; Dial, S L; Gehring, T A; Siitonen, P H

    1995-11-01

    Valproic acid (VPA) is an anti-convulsant drug known to cause spina bifida in humans. Administration of the vitamin, folic acid, has been shown to decrease the recurrence and possibly also the occurrence of neural tube defects, primarily spina bifida, in humans. Additionally, treatment with a derivative (folinic acid) of folic acid has been reported to decrease the frequency of VPA-induced exencephaly in mice treated with the drug in vivo. A protective effect by folinic acid has not been observed in vitro. The purpose of this investigation was to reexamine the ability of folinic acid to decrease the incidence of VPA-induced neural tube defects in vivo. We also examined the effect of increased intake of folic acid on zinc levels in various maternal and embryonic tissues. Folinic acid, whether administered by intraperitoneal injection or in osmotic mini-pumps, did not decrease the number of mouse fetuses with VPA-induced exencephaly. Dietary supplementation with 10-20 times the daily required intake of folic acid in rodents also failed to decrease the embryotoxicity of VPA. Such dietary supplementation had no effect on zinc levels in maternal liver, brain, or kidney, nor in embryonic tissues. These results indicate that folic acid is not able to reverse the embryotoxicity induced by the anticonvulsant, that there is no apparent effect of high dietary folate intake on maternal or embryonic zinc levels and suggest that folate is probably not involved in the mechanism of VPA-induced embryotoxicity. PMID:8838251

  1. Sequence-Specific Electrical Purification of Nucleic Acids with Nanoporous Gold Electrodes.

    PubMed

    Daggumati, Pallavi; Appelt, Sandra; Matharu, Zimple; Marco, Maria L; Seker, Erkin

    2016-06-22

    Nucleic-acid-based biosensors have enabled rapid and sensitive detection of pathogenic targets; however, these devices often require purified nucleic acids for analysis since the constituents of complex biological fluids adversely affect sensor performance. This purification step is typically performed outside the device, thereby increasing sample-to-answer time and introducing contaminants. We report a novel approach using a multifunctional matrix, nanoporous gold (np-Au), which enables both detection of specific target sequences in a complex biological sample and their subsequent purification. The np-Au electrodes modified with 26-mer DNA probes (via thiol-gold chemistry) enabled sensitive detection and capture of complementary DNA targets in the presence of complex media (fetal bovine serum) and other interfering DNA fragments in the range of 50-1500 base pairs. Upon capture, the noncomplementary DNA fragments and serum constituents of varying sizes were washed away. Finally, the surface-bound DNA-DNA hybrids were released by electrochemically cleaving the thiol-gold linkage, and the hybrids were iontophoretically eluted from the nanoporous matrix. The optical and electrophoretic characterization of the analytes before and after the detection-purification process revealed that low target DNA concentrations (80 pg/μL) can be successfully detected in complex biological fluids and subsequently released to yield pure hybrids free of polydisperse digested DNA fragments and serum biomolecules. Taken together, this multifunctional platform is expected to enable seamless integration of detection and purification of nucleic acid biomarkers of pathogens and diseases in miniaturized diagnostic devices.

  2. Amino acid sequence analysis and characterization of a ribonuclease from starfish Asterias amurensis.

    PubMed

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

    2016-09-01

    The aim of this study was to phylogenetically characterize the location of the RNase T2 enzyme in the starfish (Asterias amurensis). We isolated an RNase T2 ribonuclease (RNase Aa) from the ovaries of starfish and determined its amino acid sequence by protein chemistry and cloning cDNA encoding RNase Aa. The isolated protein had 231 amino acid residues, a predicted molecular mass of 25,906 Da, and an optimal pH of 5.0. RNase Aa preferentially released guanylic acid from the RNA. The catalytic sites of the RNase T2 family are conserved in RNase Aa; furthermore, the distribution of the cysteine residues in RNase Aa is similar to that in other animal and plant T2 RNases. RNase Aa is cleaved at two points: 21 residues from the N-terminus and 29 residues from the C-terminus; however, both fragments may remain attached to the protein via disulfide bridges, leading to the maintenance of its conformation, as suggested by circular dichroism spectrum analysis. The phylogenetic analysis revealed that starfish RNase Aa is evolutionarily an intermediate between protozoan and oyster RNases. PMID:26920046

  3. Isolation and amino acid sequence of crustacean hyperglycemic hormone precursor-related peptides.

    PubMed

    Tensen, C P; Verhoeven, A H; Gaus, G; Janssen, K P; Keller, R; Van Herp, F

    1991-01-01

    The crustacean hyperglycemic hormone (CHH) is synthesized as part of a larger preprohormone in which the sequence of CHH is N-terminally flanked by a peptide for which the name CPRP (CHH precursor-related peptide) is proposed. Both CHH and CPRP are present in the sinus gland, the neurohemal organ of neurosecretory cells located in the eyestalk of decapod crustaceans. This paper describes the isolation and sequence analysis of CPRPs isolated from sinus glands of the crab Carcinus maenas, the crayfish Orconectes limosus and the lobster Homarus americanus. The published sequence of "peptide H" isolated from the land crab, Cardisoma carnifex, has now been recognized as a CPRP in this species. Sequence comparison reveals a high level of identity for the N-terminal region (residues 1-13) between all four peptides, while identity in the C-terminal domain is high between lobster and crayfish CPRP on the one hand, and between both crab species on the other. Conserved N-terminal residues include a putative monobasic processing site at position 11, which suggests that CPRP may be a biosynthetic intermediate from which a potentially bioactive decapeptide can be derived.

  4. Fatty acid profiles in relation to triglyceride level in the liver of dairy cows.

    PubMed

    Sato, Hiroshi; Mohamed, Tharwat; Goto, Akiko; Oikawa, Shin; Kurosawa, Takashi

    2004-01-01

    To elucidate possible relationships between triglyceride (TG) levels and fatty acid composition in bovine liver, hepatic TG and seven individual fatty acids were measured in 23 Holstein dairy cows, of them 6 are healthy. Liver TG level was greater than 3 % in 12 cows which were ruled fatty liver. Palmitic and oleic acid proportions were significantly higher in fatty liver cows than in the healthy cows, while stearic acid was lower in fatty liver cows. With increased liver TG, stearic acid proportions decreased dramatically. Results indicate that hepatic lipidosis markedly alters the proportions of the various fatty acids in the liver of dairy cows.

  5. Arthropod Phylogenetics in Light of Three Novel Millipede (Myriapoda: Diplopoda) Mitochondrial Genomes with Comments on the Appropriateness of Mitochondrial Genome Sequence Data for Inferring Deep Level Relationships

    PubMed Central

    Brewer, Michael S.; Swafford, Lynn; Spruill, Chad L.; Bond, Jason E.

    2013-01-01

    Background Arthropods are the most diverse group of eukaryotic organisms, but their phylogenetic relationships are poorly understood. Herein, we describe three mitochondrial genomes representing orders of millipedes for which complete genomes had not been characterized. Newly sequenced genomes are combined with existing data to characterize the protein coding regions of myriapods and to attempt to reconstruct the evolutionary relationships within the Myriapoda and Arthropoda. Results The newly sequenced genomes are similar to previously characterized millipede sequences in terms of synteny and length. Unique translocations occurred within the newly sequenced taxa, including one half of the Appalachioria falcifera genome, which is inverted with respect to other millipede genomes. Across myriapods, amino acid conservation levels are highly dependent on the gene region. Additionally, individual loci varied in the level of amino acid conservation. Overall, most gene regions showed low levels of conservation at many sites. Attempts to reconstruct the evolutionary relationships suffered from questionable relationships and low support values. Analyses of phylogenetic informativeness show the lack of signal deep in the trees (i.e., genes evolve too quickly). As a result, the myriapod tree resembles previously published results but lacks convincing support, and, within the arthropod tree, well established groups were recovered as polyphyletic. Conclusions The novel genome sequences described herein provide useful genomic information concerning millipede groups that had not been investigated. Taken together with existing sequences, the variety of compositions and evolution of myriapod mitochondrial genomes are shown to be more complex than previously thought. Unfortunately, the use of mitochondrial protein-coding regions in deep arthropod phylogenetics appears problematic, a result consistent with previously published studies. Lack of phylogenetic signal renders the

  6. Variation in seed fatty acid composition and sequence divergence in the FAD2 gene coding region between wild and cultivated sesame.

    PubMed

    Chen, Zhenbang; Tonnis, Brandon; Morris, Brad; Wang, Richard B; Zhang, Amy L; Pinnow, David; Wang, Ming Li

    2014-12-01

    Sesame germplasm harbors genetic diversity which can be useful for sesame improvement in breeding programs. Seven accessions with different levels of oleic acid were selected from the entire USDA sesame germplasm collection (1232 accessions) and planted for morphological observation and re-examination of fatty acid composition. The coding region of the FAD2 gene for fatty acid desaturase (FAD) in these accessions was also sequenced. Cultivated sesame accessions flowered and matured earlier than the wild species. The cultivated sesame seeds contained a significantly higher percentage of oleic acid (40.4%) than the seeds of the wild species (26.1%). Nucleotide polymorphisms were identified in the FAD2 gene coding region between wild and cultivated species. Some nucleotide polymorphisms led to amino acid changes, one of which was located in the enzyme active site and may contribute to the altered fatty acid composition. Based on the morphology observation, chemical analysis, and sequence analysis, it was determined that two accessions were misnamed and need to be reclassified. The results obtained from this study are useful for sesame improvement in molecular breeding programs.

  7. Impacts on microbial communities and cultivable isolates from groundwater contaminated with high levels of nitric acid-uranium waste.

    PubMed

    Fields, Matthew W; Yan, Tingfen; Rhee, Sung-Keun; Carroll, Susan L; Jardine, Phil M; Watson, David B; Criddle, Craig S; Zhou, Jizhong

    2005-08-01

    Microbial communities were characterized at contaminated sites that had elevated levels of nitrate, nickel, aluminum, and uranium (up to 690 mM, 310 microM, 42 mM, and 30 microM, respectively). The bacterial community structure based upon clonal libraries of the SSU rRNA genes (screened clones = 876) was diverse at the background site, but the three acidic samples had decreased diversity and the majority of clones were closely related to Azoarcus and Pseudomonas species. Arthrobacter and Novosphingobium sequences were recovered from the background samples but not the acidic sites, and similar pseudomonad populations were present at the background and acidic sites albeit at different relative abundances. Heterologous sequence coverage analyses indicated the microbial communities at the contaminated sites were very similar (p = 0.001) but different from the background site. Bacterial isolates (n = 67) classified as beta-or gamma-Proteobacteria, high G+C Gram-positive or low G+C Gram-positive were obtained from the background and one contaminated sample, and some of the isolates had less than 95% sequence identity with previously observed microorganisms. Despite variations in nitrate and heavy metal levels and different proximities to the source ponds, the three acidic samples had similar microbial populations. However, the least contaminated site (lowest nitrate and aluminum) had increased diversity compared to the other acidic samples. The results suggested that the combined contamination has decreased the microbial diversity, and Azoarcus populations were observed at a drastically increased frequency compared to the background site that had a more even distribution of multiple taxa.

  8. Salivary Sialic Acid Levels in Smokeless Tobacco Users

    PubMed Central

    Farhad Mollashahi, Leila; Honarmand, Marieh; Nakhaee, Alireza; Mollashahi, Ghasem

    2016-01-01

    Background Smokeless tobacco chewing is one of the known risk factors for oral cancer. It is consumed widely by residents of southeastern Iran. Objectives In this study, salivary free and total sialic acid, and total protein were compared in paan consumers and non-consumers. Patients and Methods In this cross-sectional study, unstimulated saliva of 94 subjects (44 paan consumers and 50 non-consumers) who were referred to the oral medicine department of the dentistry school of Zahedan were collected. Salivary free and total sialic acid, and total protein concentration were measured by standard biochemical methods, and the obtained data were analyzed using SPSS 20 through the non-parametric Mann-Whitney test. Results The concentration of salivary free sialic acid (23.21 ± 18.98 mg/L) was significantly increased in paan consumers. The concentration of salivary Total sialic acid (TSA) (39.57 ± 26.58 mg/L) and total protein (0.77 ± 0.81 mg/mL) showed increases in paan consumers, however, the results were not statistically significant. Conclusions Salivary free and total sialic acid, and total protein were higher in the paan consumers compared to non-consumers. Due to the carcinogenic effect of smokeless tobacco, measurement of these parameters in saliva may be useful in early detection of oral cancer. PMID:27622172

  9. Salivary Sialic Acid Levels in Smokeless Tobacco Users

    PubMed Central

    Farhad Mollashahi, Leila; Honarmand, Marieh; Nakhaee, Alireza; Mollashahi, Ghasem

    2016-01-01

    Background Smokeless tobacco chewing is one of the known risk factors for oral cancer. It is consumed widely by residents of southeastern Iran. Objectives In this study, salivary free and total sialic acid, and total protein were compared in paan consumers and non-consumers. Patients and Methods In this cross-sectional study, unstimulated saliva of 94 subjects (44 paan consumers and 50 non-consumers) who were referred to the oral medicine department of the dentistry school of Zahedan were collected. Salivary free and total sialic acid, and total protein concentration were measured by standard biochemical methods, and the obtained data were analyzed using SPSS 20 through the non-parametric Mann-Whitney test. Results The concentration of salivary free sialic acid (23.21 ± 18.98 mg/L) was significantly increased in paan consumers. The concentration of salivary Total sialic acid (TSA) (39.57 ± 26.58 mg/L) and total protein (0.77 ± 0.81 mg/mL) showed increases in paan consumers, however, the results were not statistically significant. Conclusions Salivary free and total sialic acid, and total protein were higher in the paan consumers compared to non-consumers. Due to the carcinogenic effect of smokeless tobacco, measurement of these parameters in saliva may be useful in early detection of oral cancer.

  10. Assessment of Folic Acid Supplementation in Pregnant Women by Estimation of Serum Levels of Tetrahydrofolic Acid, Dihydrofolate Reductase, and Homocysteine.

    PubMed

    Naithani, Manisha; Saxena, Vartika; Mirza, Anissa Atif; Kumari, Ranjeeta; Sharma, Kapil; Bharadwaj, Jyoti

    2016-01-01

    Background. Status of folic acid use in pregnant women of the hilly regions in North India was little known. This study was carried out to assess the folic acid use and estimate folate metabolites in pregnant women of this region. Materials and Methods. This cross-sectional study is comprised of 76 pregnant women, whose folic acid supplementation was assessed by a questionnaire and serum levels of homocysteine, tetrahydrofolic acid (THFA), and dihydrofolate reductase (DHFR) were estimated using Enzyme Linked Immunoassays. Results. The study data revealed awareness of folic acid use during pregnancy was present in 46.1% and 23.7% were taking folic acid supplements. The study depicted that there was no statistically significant difference between serum levels of THFA and DHFR in pregnant women with and without folic acid supplements (p = 0.790). Hyperhomocysteinemia was present in 15.78% of the participants. Conclusion. Less awareness about folic acid supplementation and low use of folic acid by pregnant women were observed in this region. Sufficient dietary ingestion may suffice for the escalated requirements in pregnancy, but since this cannot be ensured, hence folic acid supplementation should be made as an integral part of education and reproductive health programs for its better metabolic use, growth, and development of fetus.

  11. Assessment of Folic Acid Supplementation in Pregnant Women by Estimation of Serum Levels of Tetrahydrofolic Acid, Dihydrofolate Reductase, and Homocysteine

    PubMed Central

    Saxena, Vartika; Mirza, Anissa Atif; Kumari, Ranjeeta; Sharma, Kapil; Bharadwaj, Jyoti

    2016-01-01

    Background. Status of folic acid use in pregnant women of the hilly regions in North India was little known. This study was carried out to assess the folic acid use and estimate folate metabolites in pregnant women of this region. Materials and Methods. This cross-sectional study is comprised of 76 pregnant women, whose folic acid supplementation was assessed by a questionnaire and serum levels of homocysteine, tetrahydrofolic acid (THFA), and dihydrofolate reductase (DHFR) were estimated using Enzyme Linked Immunoassays. Results. The study data revealed awareness of folic acid use during pregnancy was present in 46.1% and 23.7% were taking folic acid supplements. The study depicted that there was no statistically significant difference between serum levels of THFA and DHFR in pregnant women with and without folic acid supplements (p = 0.790). Hyperhomocysteinemia was present in 15.78% of the participants. Conclusion. Less awareness about folic acid supplementation and low use of folic acid by pregnant women were observed in this region. Sufficient dietary ingestion may suffice for the escalated requirements in pregnancy, but since this cannot be ensured, hence folic acid supplementation should be made as an integral part of education and reproductive health programs for its better metabolic use, growth, and development of fetus. PMID:27064332

  12. 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.

  13. Amino acid substitutions in genetic variants of human serum albumin and in sequences inferred from molecular cloning.

    PubMed

    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 NaDodSO4/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.

  14. Amino acid sequences of neuropeptides in the sinus gland of the land crab Cardisoma carnifex: a novel neuropeptide proteolysis site.

    PubMed

    Newcomb, R W

    1987-08-01

    The sinus gland is a major neurosecretory structure in Crustacea. Five peptides, labeled C, D, E, F, and I, isolated from the sinus gland of the land crab have been hypothesized to arise from the incomplete proteolysis at two internal sites on a single biosynthetic intermediate peptide "H", based on amino acid composition additivities and pulse-chase radiolabeling studies. The presence of only a single major precursor for the sinus gland peptides implies that peptide H may be synthesized on a common precursor with crustacean hyperglycemic hormone forms, "J" and "L," and a peptide, "K," similar to peptides with molt inhibiting activity. Here I report amino acid sequences of these peptides. The amino terminal sequence of the parent peptide, H, (and the homologous fragments) proved refractory to Edman degradation. Data from amino acid analysis and carboxypeptidase digestion of the naturally occurring fragments and of fragments produced by endopeptidase digestion were used together with Edman degradation to obtain the sequences. Amino acid analysis of fragments of the naturally occurring "overlap" peptides (those produced by internal cleavage at one site on H) was used to obtain the sequences across the cleavage sites. The amino acid sequence of the land crab peptide H is Arg-Ser-Ala-Asp-Gly-Phe-Gly-Arg-Met-Glu-Ser-Leu-Leu-Thr-Ser-Leu-Arg-Gly- Ser-Ala-Glu- Ser-Pro-Ala-Ala-Leu-Gly-Glu-Ala-Ser-Ala-Ala-His-Pro-Leu-Glu. In vivo cleavage at one site involves excision of arginine from the sequence Leu-Arg-Gly, whereas cleavage at the other site involves excision of serine from the sequence Glu-Ser-Leu. Proteolysis at the latter sequence has not been previously reported in intact secretory granules. The aspartate at position 4 is possibly covalently modified.

  15. Boronic acid functionalized peptidyl synthetic lectins: Combinatorial library design, peptide sequencing, and selective glycoprotein recognition

    PubMed Central

    Bicker, Kevin L.; Sun, Jing; Lavigne, John J.; Thompson, Paul R.

    2011-01-01

    Aberrant glycosylation of cell membrane and secreted glycoproteins is a hallmark of various disease states, including cancer. The natural lectins currently used in the recognition of these glycoproteins are costly, difficult to produce, and unstable towards rigorous use. Herein we describe the design and synthesis of several boronic acid functionalized peptide-based synthetic lectin (SL) libraries, as well as the optimized methodology for obtaining peptide sequences of these SLs. SL libraries were subsequently used to identify SLs with as high as 5-fold selectivity for various glycoproteins. SLs will inevitably find a role in cancer diagnositics, given that they do not suffer from the drawbacks of natural lectins and that the combinatorial nature of these libraries allows for the identification of an SL for nearly any glycosylated biomolecule. PMID:21405093

  16. Kinetics of amyloid aggregation of mammal apomyoglobins and correlation with their amino acid sequences.

    PubMed

    Vilasi, Silvia; Dosi, Roberta; Iannuzzi, Clara; Malmo, Clorinda; Parente, Augusto; Irace, Gaetano; Sirangelo, Ivana

    2006-03-01

    In protein deposition disorders, a normally soluble protein is deposited as insoluble aggregates, referred to as amyloid. The intrinsic effects of specific mutations on the rates of protein aggregation and amyloid formation of unfolded polypeptide chains can be correlated with changes in hydrophobicity, propensity to convert alpha-helical to beta sheet conformation and charge. In this paper, we report the aggregation rates of buffalo, horse and bovine apomyoglobins. The experimental values were compared with the theoretical ones evaluated considering the amino acid differences among the sequences. Our results show that the mutations which play critical roles in the rate-determining step of apomyoglobin aggregation are those located within the N-terminal region of the molecule.

  17. GAWK, a novel human pituitary polypeptide: isolation, immunocytochemical localization and complete amino acid sequence.

    PubMed

    Benjannet, S; Leduc, R; Lazure, C; Seidah, N G; Marcinkiewicz, M; Chrétien, M

    1985-01-16

    During the course of reverse-phase high pressure liquid chromatography (RP-HPLC) purification of a postulated big ACTH (1) from human pituitary gland extracts, a highly purified peptide bearing no resemblance to any known polypeptide was isolated. The complete sequence of this 74 amino acid polypeptide, called GAWK, has been determined. Search on a computer data bank on the possible homology to any known protein or fragment, using a mutation data matrix, failed to reveal any homology greater than 30%. An antibody produced against a synthetic fragment allowed us to detect several immunoreactive forms. The antisera also enabled us to localize the polypeptide, by immunocytochemistry, in the anterior lobe of the pituitary gland.

  18. 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.

  19. 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.

  20. Purification, properties and complete amino acid sequence of the ferredoxin from a green alga, Chlamydomonas reinhardtii.

    PubMed

    Schmitter, J M; Jacquot, J P; de Lamotte-Guéry, F; Beauvallet, C; Dutka, S; Gadal, P; Decottignies, P

    1988-03-01

    The ferredoxin was purified from the green alga, Chlamydomonas reinhardtii. The protein showed typical absorption and circular dichroism spectra of a [2Fe-2S] ferredoxin. When compared with spinach ferredoxin, the C. reinhardtii protein was less effective in the catalysis of NADP+ photoreduction, but its activity was higher in the light activation of C. reinhardtii malate dehydrogenase (NADP). The complete amino acid sequence was determined by automated Edman degradation of the whole protein and of peptides obtained by trypsin and chymotrypsin digestions and by CNBr cleavage. The protein consists of 94 residues, with Tyr at both NH2 and COOH termini. The positions of the four cysteines binding the two iron atoms are similar to those found in other [2Fe-2S] ferredoxins. The primary structure of C. reinhardtii ferredoxin showed a great homology (about 80%) with ferredoxins from two other green algae.

  1. Real-time nucleic acid sequence-based amplification in nanoliter volumes.

    PubMed

    Gulliksen, Anja; Solli, Lars; Karlsen, Frank; Rogne, Henrik; Hovig, Eivind; Nordstrøm, Trine; Sirevåg, Reidun

    2004-01-01

    Real-time nucleic acid sequence-based amplification (NASBA) is an isothermal method specifically designed for amplification of RNA. Fluorescent molecular beacon probes enable real-time monitoring of the amplification process. Successful identification, utilizing the real-time NASBA technology, was performed on a microchip with oligonucleotides at a concentration of 1.0 and 0.1 microM, in 10- and 50-nL reaction chambers, respectively. The microchip was developed in a silicon-glass structure. An instrument providing thermal control and an optical detection system was built for amplification readout. Experimental results demonstrate distinct amplification processes. Miniaturized real-time NASBA in microchips makes high-throughput diagnostics of bacteria, viruses, and cancer markers possible, at reduced cost and without contamination.

  2. Real-time nucleic acid sequence-based amplification assay for detection of hepatitis A virus.

    PubMed

    Abd el-Galil, Khaled H; el-Sokkary, M A; Kheira, S M; Salazar, Andre M; Yates, Marylynn V; Chen, Wilfred; Mulchandani, Ashok

    2005-11-01

    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.

  3. Detection of infectious salmon anaemia virus by real-time nucleic acid sequence based amplification.

    PubMed

    Starkey, William G; Smail, David A; Bleie, Hogne; Muir, K Fiona; Ireland, Jacqueline H; Richards, Randolph H

    2006-10-17

    We have developed a real-time nucleic acid sequence based amplification (NASBA) procedure for detection of infectious salmon anaemia virus (ISAV). Primers were designed to target a 124 nucleotide region of ISAV genome segment 8. Amplification products were detected in real-time with a molecular beacon (carboxyfluorescin [FAM]-labelled and methyl-red quenched) that recognised an internal region of the target amplicon. Amplification and detection were performed at 41 degrees C for 90 min in a Corbett Research Rotorgene. The real-time NASBA assay was compared to a conventional RT-PCR for ISAV detection. From a panel of 45 clinical samples, both assays detected ISAV in the same 19 samples. Based on the detection of a synthetic RNA target, the real-time NASBA procedure was approximately 100x more sensitive than conventional RT-PCR. These results suggest that real-time NASBA may represent a useful diagnostic procedure for ISAV.

  4. 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.

  5. Identification of multiple lipid genes with modifications in expression and sequence associated with the evolution of hydroxy fatty acid accumulation in Physaria fendleri.

    PubMed

    Horn, Patrick J; Liu, Jinjie; Cocuron, Jean-Christophe; McGlew, Kathleen; Thrower, Nicholas A; Larson, Matt; Lu, Chaofu; Alonso, Ana P; Ohlrogge, John

    2016-05-01

    Two Brassicaceae species, Physaria fendleri and Camelina sativa, are genetically very closely related to each other and to Arabidopsis thaliana. Physaria fendleri seeds contain over 50% hydroxy fatty acids (HFAs), while Camelina sativa and Arabidopsis do not accumulate HFAs. To better understand how plants evolved new biochemical pathways with the capacity to accumulate high levels of unusual fatty acids, transcript expression and protein sequences of developing seeds of Physaria fendleri, wild-type Camelina sativa, and Camelina sativa expressing a castor bean (Ricinus communis) hydroxylase were analyzed. A number of potential evolutionary adaptations within lipid metabolism that probably enhance HFA production and accumulation in Physaria fendleri, and, in their absence, limit accumulation in transgenic tissues were revealed. These adaptations occurred in at least 20 genes within several lipid pathways from the onset of fatty acid synthesis and its regulation to the assembly of triacylglycerols. Lipid genes of Physaria fendleri appear to have co-evolved through modulation of transcriptional abundances and alterations within protein sequences. Only a handful of genes showed evidence for sequence adaptation through gene duplication. Collectively, these evolutionary changes probably occurred to minimize deleterious effects of high HFA amounts and/or to enhance accumulation for physiological advantage. These results shed light on the evolution of pathways for novel fatty acid production in seeds, help explain some of the current limitations to accumulation of HFAs in transgenic plants, and may provide improved strategies for future engineering of their production.

  6. Parameters of proteome evolution from histograms of amino-acid sequence identities of paralogous proteins

    PubMed Central

    Axelsen, Jacob Bock; Yan, Koon-Kiu; Maslov, Sergei

    2007-01-01

    Background The evolution of the full repertoire of proteins encoded in a given genome is mostly driven by gene duplications, deletions, and sequence modifications of existing proteins. Indirect information about relative rates and other intrinsic parameters of these three basic processes is contained in the proteome-wide distribution of sequence identities of pairs of paralogous proteins. Results We introduce a simple mathematical framework based on a stochastic birth-and-death model that allows one to extract some of this information and apply it to the set of all pairs of paralogous proteins in H. pylori, E. coli, S. cerevisiae, C. elegans, D. melanogaster, and H. sapiens. It was found that the histogram of sequence identities p generated by an all-to-all alignment of all protein sequences encoded in a genome is well fitted with a power-law form ~ p-γ with the value of the exponent γ around 4 for the majority of organisms used in this study. This implies that the intra-protein variability of substitution rates is best described by the Gamma-distribution with the exponent α ≈ 0.33. Different features of the shape of such histograms allow us to quantify the ratio between the genome-wide average deletion/duplication rates and the amino-acid substitution rate. Conclusion We separately measure the short-term ("raw") duplication and deletion rates rdup∗, rdel∗ which include gene copies that will be removed soon after the duplication event and their dramatically reduced long-term counterparts rdup, rdel. High deletion rate among recently duplicated proteins is consistent with a scenario in which they didn't have enough time to significantly change their functional roles and thus are to a large degree disposable. Systematic trends of each of the four duplication/deletion rates with the total number of genes in the genome were analyzed. All but the deletion rate of recent duplicates rdel∗ were shown to systematically increase with Ngenes. Abnormally flat shapes

  7. Trypsin inhibitors from ridged gourd (Luffa acutangula Linn.) seeds: purification, properties, and amino acid sequences.

    PubMed

    Haldar, U C; Saha, S K; Beavis, R C; Sinha, N K

    1996-02-01

    Two trypsin inhibitors, LA-1 and LA-2, have been isolated from ridged gourd (Luffa acutangula Linn.) seeds and purified to homogeneity by gel filtration followed by ion-exchange chromatography. The isoelectric point is at pH 4.55 for LA-1 and at pH 5.85 for LA-2. The Stokes radius of each inhibitor is 11.4 A. The fluorescence emission spectrum of each inhibitor is similar to that of the free tyrosine. The biomolecular rate constant of acrylamide quenching is 1.0 x 10(9) M-1 sec-1 for LA-1 and 0.8 x 10(9) M-1 sec-1 for LA-2 and that of K2HPO4 quenching is 1.6 x 10(11) M-1 sec-1 for LA-1 and 1.2 x 10(11) M-1 sec-1 for LA-2. Analysis of the circular dichroic spectra yields 40% alpha-helix and 60% beta-turn for La-1 and 45% alpha-helix and 55% beta-turn for LA-2. Inhibitors LA-1 and LA-2 consist of 28 and 29 amino acid residues, respectively. They lack threonine, alanine, valine, and tryptophan. Both inhibitors strongly inhibit trypsin by forming enzyme-inhibitor complexes at a molar ratio of unity. A chemical modification study suggests the involvement of arginine of LA-1 and lysine of LA-2 in their reactive sites. The inhibitors are very similar in their amino acid sequences, and show sequence homology with other squash family inhibitors. PMID:8924202

  8. Microfluidic platform for isolating nucleic acid targets using sequence specific hybridization

    PubMed Central

    Wang, Jingjing; Morabito, Kenneth; Tang, Jay X.; Tripathi, Anubhav

    2013-01-01

    The separation of target nucleic acid sequences from biological samples has emerged as a significant process in today's diagnostics and detection strategies. In addition to the possible clinical applications, the fundamental understanding of target and sequence specific hybridization on surface modified magnetic beads is of high value. In this paper, we describe a novel microfluidic platform that utilizes a mobile magnetic field in static microfluidic channels, where single stranded DNA (ssDNA) molecules are isolated via nucleic acid hybridization. We first established efficient isolation of biotinylated capture probe (BP) using streptavidin-coated magnetic beads. Subsequently, we investigated the hybridization of target ssDNA with BP bound to beads and explained these hybridization kinetics using a dual-species kinetic model. The number of hybridized target ssDNA molecules was determined to be about 6.5 times less than that of BP on the bead surface, due to steric hindrance effects. The hybridization of target ssDNA with non-complementary BP bound to bead was also examined, and non-specific hybridization was found to be insignificant. Finally, we demonstrated highly efficient capture and isolation of target ssDNA in the presence of non-target ssDNA, where as low as 1% target ssDNA can be detected from mixture. The microfluidic method described in this paper is significantly relevant and is broadly applicable, especially towards point-of-care biological diagnostic platforms that require binding and separation of known target biomolecules, such as RNA, ssDNA, or protein. PMID:24404041

  9. Detection of Vibrio cholerae by real-time nucleic acid sequence-based amplification.

    PubMed

    Fykse, Else M; Skogan, Gunnar; Davies, William; Olsen, Jaran Strand; Blatny, Janet M

    2007-03-01

    A multitarget molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assay for the specific detection of Vibrio cholerae has been developed. The genes encoding the cholera toxin (ctxA), the toxin-coregulated pilus (tcpA; colonization factor), the ctxA toxin regulator (toxR), hemolysin (hlyA), and the 60-kDa chaperonin product (groEL) were selected as target sequences for detection. The beacons for the five different genetic targets were evaluated by serial dilution of RNA from V. cholerae cells. RNase treatment of the nucleic acids eliminated all NASBA, whereas DNase treatment had no effect, showing that RNA and not DNA was amplified. The specificity of the assay was investigated by testing several isolates of V. cholerae, other Vibrio species, and Bacillus cereus, Salmonella enterica, and Escherichia coli strains. The toxR, groEL, and hlyA beacons identified all V. cholerae isolates, whereas the ctxA and tcpA beacons identified the O1 toxigenic clinical isolates. The NASBA assay detected V. cholerae at 50 CFU/ml by using the general marker groEL and tcpA that specifically indicates toxigenic strains. A correlation between cell viability and NASBA was demonstrated for the ctxA, toxR, and hlyA targets. RNA isolated from different environmental water samples spiked with V. cholerae was specifically detected by NASBA. These results indicate that NASBA can be used in the rapid detection of V. cholerae from various environmental water samples. This method has a strong potential for detecting toxigenic strains by using the tcpA and ctxA markers. The entire assay including RNA extraction and NASBA was completed within 3 h.

  10. 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.

  11. A single molecular beacon probe is sufficient for the analysis of multiple nucleic acid sequences.

    PubMed

    Gerasimova, Yulia V; Hayson, Aaron; Ballantyne, Jack; Kolpashchikov, Dmitry M

    2010-08-16

    Molecular beacon (MB) probes are dual-labeled hairpin-shaped oligodeoxyribonucleotides that are extensively used for real-time detection of specific RNA/DNA analytes. In the MB probe, the loop fragment is complementary to the analyte: therefore, a unique probe is required for the analysis of each new analyte sequence. The conjugation of an oligonucleotide with two dyes and subsequent purification procedures add to the cost of MB probes, thus reducing their application in multiplex formats. Here we demonstrate how one MB probe can be used for the analysis of an arbitrary nucleic acid. The approach takes advantage of two oligonucleotide adaptor strands, each of which contains a fragment complementary to the analyte and a fragment complementary to an MB probe. The presence of the analyte leads to association of MB probe and the two DNA strands in quadripartite complex. The MB probe fluorescently reports the formation of this complex. In this design, the MB does not bind the analyte directly; therefore, the MB sequence is independent of the analyte. In this study one universal MB probe was used to genotype three human polymorphic sites. This approach promises to reduce the cost of multiplex real-time assays and improve the accuracy of single-nucleotide polymorphism genotyping.

  12. Modulation of sialic acid levels among some organs during insulin resistance or hyperglycemic states.

    PubMed

    Ibrahim, Mohammed Auwal; Abdulkadir, Aisha; Onojah, Alice; Sani, Lawal; Adamu, Auwal; Abdullahi, Hadiza

    2016-01-01

    Previous studies have suggested a possible connection between insulin resistance and chronic hyperglycemia with membrane sialic acid content. In this study, the effects of high (20% ad libitum) fructose and glucose feeding on the sialic acid levels of some organs were investigated in rats. The blood glucose levels of the high fructose- and glucose-fed rats were consistently and significantly (P < 0.05) higher than the normal control throughout the experiment. Free serum sialic acid and total hepatic sialic acid levels were elevated in the high fructose- and glucose-fed rats compared to normal control, but only the data for the high glucose-fed group were significantly (P < 0.05) different from the normal control. Conversely, a significant (P < 0.05) decrease in the pancreatic sialic acid level was observed in high glucose-fed group compared to normal control. Also, the high fructose-fed rats had lower, but insignificant (P > 0.05), pancreatic sialic acid level than the normal control. On the other hand, high fructose and glucose feeding did not significantly (P > 0.05) affect the sialic acid levels of the skeletal muscle and heart, though a tendency to increase the sialic acid level was evident in the heart. In the kidney, the sialic acid level was significantly (P < 0.05) increased in both high fructose- and glucose-fed groups. It was concluded that the liver and kidney tend to stimulate sialic acid synthesis, while the pancreas downregulate sialic acids synthesis and/or promote sialic acid release from glycoconjugates. Also, these organs may contribute to high-serum sialic acid level observed during diabetes. PMID:26468092

  13. Extracellular DNA amplicon sequencing reveals high levels of benthic eukaryotic diversity in the central Red Sea.

    PubMed

    Pearman, John K; Irigoien, Xabier; Carvalho, Susana

    2016-04-01

    The present study aims to characterize the benthic eukaryotic biodiversity patterns at a coarse taxonomic level in three areas of the central Red Sea (a lagoon, an offshore area in Thuwal and a shallow coastal area near Jeddah) based on extracellular DNA. High-throughput amplicon sequencing targeting the V9 region of the 18S rRNA gene was undertaken for 32 sediment samples. High levels of alpha-diversity were detected with 16,089 operational taxonomic units (OTUs) being identified. The majority of the OTUs were assigned to Metazoa (29.2%), Alveolata (22.4%) and Stramenopiles (17.8%). Stramenopiles (Diatomea) and Alveolata (Ciliophora) were frequent in a lagoon and in shallower coastal stations, whereas metazoans (Arthropoda: Maxillopoda) were dominant in deeper offshore stations. Only 24.6% of total OTUs were shared among all areas. Beta-diversity was generally lower between the lagoon and Jeddah (nearshore) than between either of those and the offshore area, suggesting a nearshore-offshore biodiversity gradient. The current approach allowed for a broad-range of benthic eukaryotic biodiversity to be analysed with significantly less labour than would be required by other traditional taxonomic approaches. Our findings suggest that next generation sequencing techniques have the potential to provide a fast and standardised screening of benthic biodiversity at large spatial and temporal scales.

  14. Higher-level phylogeny of paraneopteran insects inferred from mitochondrial genome sequences

    PubMed Central

    Li, Hu; Shao, Renfu; Song, Nan; Song, Fan; Jiang, Pei; Li, Zhihong; Cai, Wanzhi

    2015-01-01

    Mitochondrial (mt) genome data have been proven to be informative for animal phylogenetic studies but may also suffer from systematic errors, due to the effects of accelerated substitution rate and compositional heterogeneity. We analyzed the mt genomes of 25 insect species from the four paraneopteran orders, aiming to better understand how accelerated substitution rate and compositional heterogeneity affect the inferences of the higher-level phylogeny of this diverse group of hemimetabolous insects. We found substantial heterogeneity in base composition and contrasting rates in nucleotide substitution among these paraneopteran insects, which complicate the inference of higher-level phylogeny. The phylogenies inferred with concatenated sequences of mt genes using maximum likelihood and Bayesian methods and homogeneous models failed to recover Psocodea and Hemiptera as monophyletic groups but grouped, instead, the taxa that had accelerated substitution rates together, including Sternorrhyncha (a suborder of Hemiptera), Thysanoptera, Phthiraptera and Liposcelididae (a family of Psocoptera). Bayesian inference with nucleotide sequences and heterogeneous models (CAT and CAT + GTR), however, recovered Psocodea, Thysanoptera and Hemiptera each as a monophyletic group. Within Psocodea, Liposcelididae is more closely related to Phthiraptera than to other species of Psocoptera. Furthermore, Thysanoptera was recovered as the sister group to Hemiptera. PMID:25704094

  15. Higher-level phylogeny of paraneopteran insects inferred from mitochondrial genome sequences.

    PubMed

    Li, Hu; Shao, Renfu; Song, Nan; Song, Fan; Jiang, Pei; Li, Zhihong; Cai, Wanzhi

    2015-02-23

    Mitochondrial (mt) genome data have been proven to be informative for animal phylogenetic studies but may also suffer from systematic errors, due to the effects of accelerated substitution rate and compositional heterogeneity. We analyzed the mt genomes of 25 insect species from the four paraneopteran orders, aiming to better understand how accelerated substitution rate and compositional heterogeneity affect the inferences of the higher-level phylogeny of this diverse group of hemimetabolous insects. We found substantial heterogeneity in base composition and contrasting rates in nucleotide substitution among these paraneopteran insects, which complicate the inference of higher-level phylogeny. The phylogenies inferred with concatenated sequences of mt genes using maximum likelihood and Bayesian methods and homogeneous models failed to recover Psocodea and Hemiptera as monophyletic groups but grouped, instead, the taxa that had accelerated substitution rates together, including Sternorrhyncha (a suborder of Hemiptera), Thysanoptera, Phthiraptera and Liposcelididae (a family of Psocoptera). Bayesian inference with nucleotide sequences and heterogeneous models (CAT and CAT + GTR), however, recovered Psocodea, Thysanoptera and Hemiptera each as a monophyletic group. Within Psocodea, Liposcelididae is more closely related to Phthiraptera than to other species of Psocoptera. Furthermore, Thysanoptera was recovered as the sister group to Hemiptera.

  16. EXOZODIACAL DUST LEVELS FOR NEARBY MAIN-SEQUENCE STARS: A SURVEY WITH THE KECK INTERFEROMETER NULLER

    SciTech Connect

    Millan-Gabet, R.; Serabyn, E.; Mennesson, B.; Traub, W. A.; Stapelfeldt, K.; Bryden, G.; Colavita, M. M.; Booth, A. J.; Barry, R. K.; Danchi, W. C.; Kuchner, M.; Stark, C. C.; Ragland, S.; Hrynevych, M.; Woillez, J.

    2011-06-10

    The Keck Interferometer Nuller (KIN) was used to survey 25 nearby main-sequence stars in the mid-infrared, in order to assess the prevalence of warm circumstellar (exozodiacal) dust around nearby solar-type stars. The KIN measures circumstellar emission by spatially blocking the star but transmitting the circumstellar flux in a region typically 0.1-4 AU from the star. We find one significant detection ({eta} Crv), two marginal detections ({gamma} Oph and {alpha} Aql), and 22 clear non-detections. Using a model of our own solar system's zodiacal cloud, scaled to the luminosity of each target star, we estimate the equivalent number of target zodis needed to match our observations. Our three zodi detections are {eta} Crv (1250 {+-} 260), {gamma} Oph (200 {+-} 80), and {alpha} Aql (600 {+-} 200), where the uncertainties are 1{sigma}. The 22 non-detected targets have an ensemble weighted average consistent with zero, with an average individual uncertainty of 160 zodis (1{sigma}). These measurements represent the best limits to date on exozodi levels for a sample of nearby main-sequence stars. A statistical analysis of the population of 23 stars not previously known to contain circumstellar dust (excluding {eta} Crv and {gamma} Oph) suggests that, if the measurement errors are uncorrelated (for which we provide evidence) and if these 23 stars are representative of a single class with respect to the level of exozodi brightness, the mean exozodi level for the class is <150 zodis (3{sigma} upper limit, corresponding to 99% confidence under the additional assumption that the measurement errors are Gaussian). We also demonstrate that this conclusion is largely independent of the shape and mean level of the (unknown) true underlying exozodi distribution.

  17. High levels of stearic acid, palmitoleic acid, and dihomo-γ-linolenic acid and low levels of linoleic acid in serum cholesterol ester are associated with high insulin resistance.

    PubMed

    Kurotani, Kayo; Sato, Masao; Ejima, Yuko; Nanri, Akiko; Yi, Siyan; Pham, Ngoc Minh; Akter, Shamima; Poudel-Tandukar, Kalpana; Kimura, Yasumi; Imaizumi, Katsumi; Mizoue, Tetsuya

    2012-09-01

    The association of fatty acid composition with insulin resistance and type 2 diabetes has been reported in Western populations, but there is limited evidence of this association among the Japanese, whose populace consume large amounts of fish. To test the hypothesis that high palmitic, palmitoleic, and dihomo-γ-linolenic acids and low levels of linoleic and n-3 fatty acids are associated with higher insulin resistance among the Japanese, the authors investigated the relationship between serum fatty acid composition and serum C-peptide concentrations in 437 Japanese employees aged 21 to 67 years who participated in a workplace health examination. Serum cholesterol ester and phospholipid fatty acid compositions were measured by gas-liquid chromatography. Desaturase activity was estimated by fatty acid product-to-precursor ratios. A multiple regression was used to assess the association between fatty acid and C-peptide concentrations. C-peptide concentrations were associated inversely with linoleic acid levels in cholesterol ester and phospholipid (P for trend = .01 and .02, respectively) and positively with stearic and palmitoleic acids in cholesterol ester (P for trend =.02 and .006, respectively) and dihomo-γ-linolenic acid in cholesterol ester and phospholipid (P for trend < .0001 for both). C-peptide concentrations were not associated with n-3 polyunsaturated fatty acids. C-peptide concentrations significantly increased as δ-9-desaturase (16:1 n-7/16:0) and δ-6-desaturase (18:3 n-6/18:2 n-6) increased (P for trend = .01 and .03, respectively) and δ-5-desaturase (20:4 n-6/20:3 n-6) decreased (P for trend = .004). In conclusion, a fatty acid pattern with high levels of serum stearic, palmitoleic, or dihomo-γ-linolenic acids; δ-9-desaturase (16:1 n-7/16:0) or δ-6-desaturase (18:3 n-6/18:2 n-6) activities; and low levels of serum linoleic acid or δ-5-desaturase (20:4 n-6/20:3 n-6) activity might be associated with higher insulin resistance in Japanese adults.

  18. 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.

  19. Lactic acid production from potato peel waste by anaerobic sequencing batch fermentation using undefined mixed culture.

    PubMed

    Liang, Shaobo; McDonald, Armando G; Coats, Erik R

    2015-11-01

    Lactic acid (LA) is a necessary industrial feedstock for producing the bioplastic, polylactic acid (PLA), which is currently produced by pure culture fermentation of food carbohydrates. This work presents an alternative to produce LA from potato peel waste (PPW) by anaerobic fermentation in a sequencing batch reactor (SBR) inoculated with undefined mixed culture from a municipal wastewater treatment plant. A statistical design of experiments approach was employed using set of 0.8L SBRs using gelatinized PPW at a solids content range from 30 to 50 g L(-1), solids retention time of 2-4 days for yield and productivity optimization. The maximum LA production yield of 0.25 g g(-1) PPW and highest productivity of 125 mg g(-1) d(-1) were achieved. A scale-up SBR trial using neat gelatinized PPW (at 80 g L(-1) solids content) at the 3 L scale was employed and the highest LA yield of 0.14 g g(-1) PPW and a productivity of 138 mg g(-1) d(-1) were achieved with a 1 d SRT.

  20. Amino acid sequence surrounding the chondroitin sulfate attachment site of thrombomodulin regulates chondroitin polymerization.

    PubMed

    Izumikawa, Tomomi; Kitagawa, Hiroshi

    2015-05-01

    Thrombomodulin (TM) is a cell-surface glycoprotein and a critical mediator of endothelial anticoagulant function. TM exists as both a chondroitin sulfate (CS) proteoglycan (PG) form and a non-PG form lacking a CS chain (α-TM); therefore, TM can be described as a part-time PG. Previously, we reported that α-TM bears an immature, truncated linkage tetrasaccharide structure (GlcAβ1-3Galβ1-3Galβ1-4Xyl). However, the biosynthetic mechanism to generate part-time PGs remains unclear. In this study, we used several mutants to demonstrate that the amino acid sequence surrounding the CS attachment site influences the efficiency of chondroitin polymerization. In particular, the presence of acidic residues surrounding the CS attachment site was indispensable for the elongation of CS. In addition, mutants defective in CS elongation did not exhibit anti-coagulant activity, as in the case with α-TM. Together, these data support a model for CS chain assembly in which specific core protein determinants are recognized by a key biosynthetic enzyme involved in chondroitin polymerization.

  1. Lactic acid production from potato peel waste by anaerobic sequencing batch fermentation using undefined mixed culture.

    PubMed

    Liang, Shaobo; McDonald, Armando G; Coats, Erik R

    2015-11-01

    Lactic acid (LA) is a necessary industrial feedstock for producing the bioplastic, polylactic acid (PLA), which is currently produced by pure culture fermentation of food carbohydrates. This work presents an alternative to produce LA from potato peel waste (PPW) by anaerobic fermentation in a sequencing batch reactor (SBR) inoculated with undefined mixed culture from a municipal wastewater treatment plant. A statistical design of experiments approach was employed using set of 0.8L SBRs using gelatinized PPW at a solids content range from 30 to 50 g L(-1), solids retention time of 2-4 days for yield and productivity optimization. The maximum LA production yield of 0.25 g g(-1) PPW and highest productivity of 125 mg g(-1) d(-1) were achieved. A scale-up SBR trial using neat gelatinized PPW (at 80 g L(-1) solids content) at the 3 L scale was employed and the highest LA yield of 0.14 g g(-1) PPW and a productivity of 138 mg g(-1) d(-1) were achieved with a 1 d SRT. PMID:25708409

  2. 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.

  3. 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.

  4. The amino-acid sequence of the alpha-crystallin A chains of red kangaroo and Virginia opossum.

    PubMed

    De Jong, W W; Terwindt, E C

    1976-08-16

    The amino acid sequence of the A chain of the eye lens protein alpha-crystallin from the red kangaroo (Macropus rufus) was completely determined by manual Edman degradation of tryptic, thermolytic and cyanogen bromide peptides. The sequence of the alpha-crystallin A chain from the Virginia opossum (Didelphis marsupialis) was deduced from amino acid analyses and partial Edman degradation of peptides. The 173-residue A chains of kangaroo and opossum differ in six positions, whereas comparison with the bovine alpha-crystallin A chain reveals 17 and 22 substitutions, respectively. Most substitutions occur in the COOH-terminal part of the chain.

  5. Homology analyses of the protein sequences of fatty acid synthases from chicken liver, rat mammary gland, and yeast

    SciTech Connect

    Chang, Soo-Ik ); Hammes, G.G. )

    1989-11-01

    Homology analyses of the protein sequences of chicken liver and rat mammary gland fatty acid synthases were carried out. The amino acid sequences of the chicken and rat enzymes are 67% identical. If conservative substitutions are allowed, 78% of the amino acids are matched. A region of low homologies exists between the functional domains, in particular around amino acid residues 1059-1264 of the chicken enzyme. Homologies between the active sites of chicken and rat and of chicken and yeast enzymes have been analyzed by an alignment method. A high degree of homology exists between the active sites of the chicken and rat enzymes. However, the chicken and yeast enzymes show a lower degree of homology. The DADPH-binding dinucleotide folds of the {beta}-ketoacyl reductase and the enoyl reductase sites were identified by comparison with a known consensus sequence for the DADP- and FAD-binding dinucleotide folds. The active sites of all of the enzymes are primarily in hydrophobic regions of the protein. This study suggests that the genes for the functional domains of fatty acid synthase were originally separated, and these genes were connected to each other by using different connecting nucleotide sequences in different species. An alternative explanation for the differences in rat and chicken is a common ancestry and mutations in the joining regions during evolution.

  6. New alleles of FATB-1A to reduce palmitic acid levels in soybean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In wild-type soybeans, palmitic acid typically constitutes 10% of the total seed oil. Palmitic acid is a saturated fat linked to increased cholesterol levels, and reducing levels of saturated fats in soybean oil has been a breeding target. To identify novel and useful variation that could help in re...

  7. Plasma Amino Acid Levels in Children with Autism and Their Families.

    ERIC Educational Resources Information Center

    Aldred, Sarah; Moore, Kieran M.; Fitzgerald, Michael; Waring, Rosemary H.

    2003-01-01

    Plasma amino acid levels were measured in autistic (n=12), Asperger syndrome (n=11) patients, their parents and siblings. Patients with autism or Asperger syndrome and their siblings and parents all had raised glutamic acid, phenyalanine, asparagine, tyrosine, alanine, and lysine levels than age-matched controls. Results suggest dysregulated amino…

  8. Polyvinyl-alcohol-based magnetic beads for rapid and efficient separation of specific or unspecific nucleic acid sequences

    NASA Astrophysics Data System (ADS)

    Oster, Jürgen; Parker, Jeffrey; à Brassard, Lothar

    2001-01-01

    The versatile application of polyvinyl-alcohol-based magnetic M-PVA beads is demonstrated in the separation of genomic DNA, sequence specific nucleic acid purification, and binding of bacteria for subsequent DNA extraction and detection. It is shown that nucleic acids can be obtained in high yield and purity using M-PVA beads, making sample preparation efficient, fast and highly adaptable for automation processes.

  9. What is the intrapatient variability of mycophenolic acid trough levels?

    PubMed

    Todorova, Ekaterina K; Huang, Shih-Han S; Kobrzynski, Marta C; Filler, Guido

    2015-11-01

    TDM of MPA, the active compound of MMF, is rarely used despite its substantial intra- and interpatient variability. Little is known about the utility of long-term MPA TDM. Data are expressed as mean (one standard deviation). All available data from 27 renal transplant recipients (mean age at transplantation: 7.7 [5.0] yr) with an average follow-up of 9.3 (4.6) yr were analyzed. MPA levels were measured using the EMIT. GFR was measured using cystatin C and eGFR was calculated using the Filler formula. Intrapatient CV of the trough level was calculated as the ratio of the mean divided by one standard deviation. Mean cystatin C eGFR was 56.9 (24.4) mL/min/1.73 m(2) . There was a weak but significant correlation between the MPA trough level and the AUC (Spearman r = 0.6592, p < 0.0001). A total of 1964 MPA trough levels (73 [45]/patient) were measured, as compared to 3462 Tac trough levels (144 [71]/patient). The average MPA trough level was 3.01 (1.26) mg/L and the average trough Tac level was 7.3 (1.8) ng/mL. Intrapatient CV was statistically higher (p = 0.00093) for MPA at 0.68 (0.29) when compared to Tac with a CV of 0.46 (0.12). CV did not correlate with eGFR. Intrapatient MPA trough level CV is significantly higher than for Tac, while CV for both MPA and Tac was high. MPA trough level monitoring may be a feasible monitoring option to improve patient exposure and possibly outcomes. PMID:26201386

  10. What is the intrapatient variability of mycophenolic acid trough levels?

    PubMed

    Todorova, Ekaterina K; Huang, Shih-Han S; Kobrzynski, Marta C; Filler, Guido

    2015-11-01

    TDM of MPA, the active compound of MMF, is rarely used despite its substantial intra- and interpatient variability. Little is known about the utility of long-term MPA TDM. Data are expressed as mean (one standard deviation). All available data from 27 renal transplant recipients (mean age at transplantation: 7.7 [5.0] yr) with an average follow-up of 9.3 (4.6) yr were analyzed. MPA levels were measured using the EMIT. GFR was measured using cystatin C and eGFR was calculated using the Filler formula. Intrapatient CV of the trough level was calculated as the ratio of the mean divided by one standard deviation. Mean cystatin C eGFR was 56.9 (24.4) mL/min/1.73 m(2) . There was a weak but significant correlation between the MPA trough level and the AUC (Spearman r = 0.6592, p < 0.0001). A total of 1964 MPA trough levels (73 [45]/patient) were measured, as compared to 3462 Tac trough levels (144 [71]/patient). The average MPA trough level was 3.01 (1.26) mg/L and the average trough Tac level was 7.3 (1.8) ng/mL. Intrapatient CV was statistically higher (p = 0.00093) for MPA at 0.68 (0.29) when compared to Tac with a CV of 0.46 (0.12). CV did not correlate with eGFR. Intrapatient MPA trough level CV is significantly higher than for Tac, while CV for both MPA and Tac was high. MPA trough level monitoring may be a feasible monitoring option to improve patient exposure and possibly outcomes.

  11. The Association between Marine n-3 Polyunsaturated Fatty Acid Levels and Survival after Renal Transplantation

    PubMed Central

    Jenssen, Trond; Hartmann, Anders; Diep, Lien M.; Dahle, Dag O.; Reisæter, Anna V.; Bjerve, Kristian S.; Christensen, Jeppe H.; Schmidt, Erik B.; Svensson, My

    2015-01-01

    Background and objectives Several studies have reported beneficial cardiovascular effects of marine n-3 polyunsaturated fatty acids. To date, no large studies have investigated the potential benefits of marine n-3 polyunsaturated fatty acids in recipients of renal transplants. Design, setting, participants, & measurements In this observational cohort study of 1990 Norwegian recipients of renal transplants transplanted between 1999 and 2011, associations between marine n-3 polyunsaturated fatty acid levels and mortality were investigated by stratified analysis and multivariable Cox proportional hazard regression analysis adjusting for traditional and transplant-specific mortality risk factors. Marine n-3 polyunsaturated fatty acid levels in plasma phospholipids were measured by gas chromatography in a stable phase 10 weeks after transplantation. Results There were 406 deaths (20.4%) during a median follow-up period of 6.8 years. Mortality rates were lower in patients with high marine n-3 polyunsaturated fatty acid levels (≥7.95 weight percentage) compared with low levels (<7.95 weight percentage) for all age categories (pooled mortality rate ratio estimate, 0.69; 95% confidence interval, 0.57 to 0.85). When divided into quartiles according to marine n-3 polyunsaturated fatty acid levels, patients in the upper quartile compared with the lower quartile had a 56% lower risk of death (adjusted hazard ratio, 0.44; 95% confidence interval, 0.26 to 0.75) using multivariable Cox proportional hazard regression analysis. There was a lower hazard ratio for death from cardiovascular disease with high levels of marine n-3 polyunsaturated fatty acid and a lower hazard ratio for death from infectious disease with high levels of the marine n-3 polyunsaturated fatty acid eicosapentaenoic acid, whereas there was no association between total or individual marine n-3 polyunsaturated fatty acid levels and cancer mortality. Conclusions Higher plasma phospholipid marine n-3

  12. Serum levels of folic acid and vitamin B12 in Korean patients with vitiligo.

    PubMed

    Kim, S M; Kim, Y K; Hann, S K

    1999-06-01

    The association of vitiligo and pernicious anemia has been previously documented. The low levels of folic acid and vitamin B12 were thought to be related to vitiligo. To date, there have been very few reports about the serum levels of folic acid and vitamin B12 in patients with vitiligo. Using radioimmunoassay, we measured the serum levels of folic acid and vitamin B12 in 100 Korean patients with vitiligo. The mean serum levels of folic acid and vitamin B12 were 6.31 +/- 2.82 ng/ml and 630.25 +/- 230.94 pg/ml, respectively, in patients with vitiligo. These levels showed no significant difference compared to the normal control group, suggesting that folic acid and vitamin B12 do not appear to play a role in the pathogenesis of vitiligo.

  13. S-matrix Calculations of Energy Levels of the Lithium Isoelectronic Sequence

    SciTech Connect

    sapirstein, J; Cheng, K T

    2010-11-02

    A QED approach to the calculation of the spectra of the lithium isoelectronic sequence is implemented. A modified Furry representation based on the Kohn-Sham potential is used to evaluate all one- and two-photon diagrams with the exception of the two-loop Lamb shift. Three-photon diagrams are estimated with Hamiltonian methods. After incorporating recent calculations of the two-loop Lamb shift and recoil corrections a comprehensive tabulation of the 2s, 2p{sub 1/2} and 2p{sub 3/2} energy levels as well as the 2s - 2p{sub 1/2} and 2s - 2p{sub 3/2} transition energies for Z = 10 - 100 is presented.

  14. S-matrix calculations of energy levels of the lithium isoelectronic sequence

    NASA Astrophysics Data System (ADS)

    Sapirstein, J.; Cheng, K. T.

    2012-06-01

    A QED approach to the calculation of the spectra of the lithium isoelectronic sequence is implemented. A modified Furry representation based on the Kohn-Sham potential is used to evaluate all one- and two-photon diagrams with the exception of the two-loop Lamb shift. Three-photon diagrams are estimated with Hamiltonian methods. After incorporating recent calculations of the two-loop Lamb shift and recoil corrections a comprehensive tabulation of the 2s, 2p1/2 and 2p3/2 energy levels as well as the 2s-2p1/2 and 2s-2p3/2 transition energies for Z=10-100 is presented.

  15. S-matrix calculations of energy levels of the lithium isoelectronic sequence

    NASA Astrophysics Data System (ADS)

    Sapirstein, J.; Cheng, K. T.

    2011-01-01

    A QED approach to the calculation of the spectra of the lithium isoelectronic sequence is implemented. A modified Furry representation based on the Kohn-Sham potential is used to evaluate all one- and two-photon diagrams with the exception of the two-loop Lamb shift. Three-photon diagrams are estimated with Hamiltonian methods. After incorporating recent calculations of the two-loop Lamb shift and recoil corrections, a comprehensive tabulation of the 2s, 2p1/2, and 2p3/2 energy levels as well as the 2s-2p1/2 and 2s-2p3/2 transition energies for Z=10-100 is presented.

  16. Rare Variants Association Analysis in Large-Scale Sequencing Studies at the Single Locus Level

    PubMed Central

    Lu, Wenbin; Tzeng, Jung-Ying

    2016-01-01

    Genetic association analyses of rare variants in next-generation sequencing (NGS) studies are fundamentally challenging due to the presence of a very large number of candidate variants at extremely low minor allele frequencies. Recent developments often focus on pooling multiple variants to provide association analysis at the gene instead of the locus level. Nonetheless, pinpointing individual variants is a critical goal for genomic researches as such information can facilitate the precise delineation of molecular mechanisms and functions of genetic factors on diseases. Due to the extreme rarity of mutations and high-dimensionality, significances of causal variants cannot easily stand out from those of noncausal ones. Consequently, standard false-positive control procedures, such as the Bonferroni and false discovery rate (FDR), are often impractical to apply, as a majority of the causal variants can only be identified along with a few but unknown number of noncausal variants. To provide informative analysis of individual variants in large-scale sequencing studies, we propose the Adaptive False-Negative Control (AFNC) procedure that can include a large proportion of causal variants with high confidence by introducing a novel statistical inquiry to determine those variants that can be confidently dispatched as noncausal. The AFNC provides a general framework that can accommodate for a variety of models and significance tests. The procedure is computationally efficient and can adapt to the underlying proportion of causal variants and quality of significance rankings. Extensive simulation studies across a plethora of scenarios demonstrate that the AFNC is advantageous for identifying individual rare variants, whereas the Bonferroni and FDR are exceedingly over-conservative for rare variants association studies. In the analyses of the CoLaus dataset, AFNC has identified individual variants most responsible for gene-level significances. Moreover, single-variant results

  17. Hydroxamic acid derivatives of mycophenolic acid inhibit histone deacetylase at the cellular level.

    PubMed

    Batovska, Daniela I; Kim, Dong Hoon; Mitsuhashi, Shinya; Cho, Yoon Sun; Kwon, Ho Jeong; Ubukata, Makoto

    2008-10-01

    Mycophenolic acid (MPA, 1), an inhibitor of IMP-dehydrogenase (IMPDH) and a latent PPARgamma agonist, is used as an effective immunosuppressant for clinical transplantation and recently entered clinical trials in advanced multiple myeloma patients. On the other hand, suberoylanilide hydroxamic acid (SAHA), a non-specific histone deacetylase (HDAC) inhibitor, has been approved for treating cutaneous T-cell lymphoma. MPA seemed to bear a cap, a linker, and a weak metal-binding site as a latent inhibitor of HDAC. Therefore, the hydroxamic acid derivatives of mycophenolic acid having an effective metal-binding site, mycophenolic hydroxamic acid (MPHA, 2), 7-O-acetyl mycophenolic acid (7-O-Ac MPHA, 3), and 7-O-lauroyl mycophenolic hydroxamic acid (7-O-L MPHA, 4) were designed and synthesized. All these compounds inhibited histone deacetylase with IC50 values of 1, 0.9 and 0.5 microM, and cell proliferation at concentrations of 2, 1.5 and 1 microM, respectively. PMID:18838793

  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. Detection of Dengue Viral RNA Using a Nucleic Acid Sequence-Based Amplification Assay

    PubMed Central

    Wu, Shuenn-Jue L.; Lee, Eun Mi; Putvatana, Ravithat; Shurtliff, Roxanne N.; Porter, Kevin R.; Suharyono, Wuryadi; Watts, Douglas M.; King, Chwan-Chuen; Murphy, Gerald S.; Hayes, Curtis G.; Romano, Joseph W.

    2001-01-01

    Faster techniques are needed for the early diagnosis of dengue fever and dengue hemorrhagic fever during the acute viremic phase of infection. An isothermal nucleic acid sequence-based amplification (NASBA) assay was optimized to amplify viral RNA of all four dengue virus serotypes by a set of universal primers and to type the amplified products by serotype-specific capture probes. The NASBA assay involved the use of silica to extract viral nucleic acid, which was amplified without thermocycling. The amplified product was detected by a probe-hybridization method that utilized electrochemiluminescence. Using normal human plasma spiked with dengue viruses, the NASBA assay had a detection threshold of 1 to 10 PFU/ml. The sensitivity and specificity of the assay were determined by testing 67 dengue virus-positive and 21 dengue virus-negative human serum or plasma samples. The “gold standard” used for comparison and evaluation was the mosquito C6/36 cell culture assay followed by an immunofluorescent assay. Viral infectivity titers in test samples were also determined by a direct plaque assay in Vero cells. The NASBA assay was able to detect dengue viral RNA in the clinical samples at plaque titers below 25 PFU/ml (the detection limit of the plaque assay). Of the 67 samples found positive by the C6/36 assay, 66 were found positive by the NASBA assay, for a sensitivity of 98.5%. The NASBA assay had a specificity of 100% based on the negative test results for the 21 normal human serum or plasma samples. These results indicate that the NASBA assay is a promising assay for the early diagnosis of dengue infections. PMID:11473994

  20. Hippuric Acid Levels in Paint Workers at Steel Furniture Manufacturers in Thailand

    PubMed Central

    Decharat, Somsiri

    2014-01-01

    Background The aims of this study were to determine hippuric acid levels in urine samples, airborne toluene levels, acute and chronic neurological symptoms, and to describe any correlation between urinary hippuric acid and airborne toluene. Methods The hippuric acid concentration in the urine of 87 paint workers exposed to toluene at work (exposed group), and 87 nonexposed people (control group) was studied. Study participants were selected from similar factories in the same region. Urine samples were collected at the end of a shift and analyzed for hippuric acid by high performance liquid chromatography. Air samples for the estimation of toluene exposure were collected with diffusive personal samplers and the toluene quantified using gas–liquid chromatography. The two groups were also interviewed and observed about their work practices and health. Results The median of the 87 airborne toluene levels was 55 ppm (range, 12–198 ppm). The median urinary hippuric acid level was 800 mg/g creatinine (range, 90–2547 mg/g creatinine). A statistically significant positive correlation was found between airborne toluene exposure and urine hippuric acid levels (r = 0.548, p < 0.01). Workers with acute symptoms had significantly higher hippuric acid levels than those who did not (p < 0.05). It was concluded that there was a significant correlation between toluene exposure, hippuric acid levels, and health (p < 0.001). Conclusion There appears to be a significant correlation between workers exposure to toluene at work, their urine hippuric acid levels, and resulting symptoms of poor health. Improvements in working conditions and occupational health education are required at these workplaces. There was good correlation between urinary hippuric acid and airborne toluene levels. PMID:25516817

  1. Draft Genome Sequence of Lactobacillus delbrueckii subsp. bulgaricus CFL1, a Lactic Acid Bacterium Isolated from French Handcrafted Fermented Milk

    PubMed Central

    Meneghel, Julie; Irlinger, Françoise; Loux, Valentin; Vidal, Marie; Passot, Stéphanie; Béal, Catherine; Layec, Séverine

    2016-01-01

    Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) is a lactic acid bacterium widely used for the production of yogurt and cheeses. Here, we report the genome sequence of L. bulgaricus CFL1 to improve our knowledge on its stress-induced damages following production and end-use processes. PMID:26941141

  2. Genome Sequence of a Candidate World Health Organization Reference Strain of Zika Virus for Nucleic Acid Testing

    PubMed Central

    Trösemeier, Jan-Hendrik; Musso, Didier; Blümel, Johannes; Thézé, Julien; Pybus, Oliver G.

    2016-01-01

    We report here the sequence of a candidate reference strain of Zika virus (ZIKV) developed on behalf of the World Health Organization (WHO). The ZIKV reference strain is intended for use in nucleic acid amplification (NAT)-based assays for the detection and quantification of ZIKV RNA. PMID:27587826

  3. Draft Genome Sequence of Burkholderia stabilis LA20W, a Trehalose Producer That Uses Levulinic Acid as a Substrate

    PubMed Central

    Sato, Yuya; Koike, Hideaki; Kondo, Susumu; Hori, Tomoyuki; Kanno, Manabu; Kimura, Nobutada; Morita, Tomotake; Kirimura, Kohtaro

    2016-01-01

    Burkholderia stabilis LA20W produces trehalose using levulinic acid (LA) as a substrate. Here, we report the 7.97-Mb draft genome sequence of B. stabilis LA20W, which will be useful in investigations of the enzymes involved in LA metabolism and the mechanism of LA-induced trehalose production. PMID:27491978

  4. Draft Genome Sequence of Lactobacillus delbrueckii subsp. bulgaricus CFL1, a Lactic Acid Bacterium Isolated from French Handcrafted Fermented Milk.

    PubMed

    Meneghel, Julie; Dugat-Bony, Eric; Irlinger, Françoise; Loux, Valentin; Vidal, Marie; Passot, Stéphanie; Béal, Catherine; Layec, Séverine; Fonseca, Fernanda

    2016-01-01

    Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) is a lactic acid bacterium widely used for the production of yogurt and cheeses. Here, we report the genome sequence of L. bulgaricus CFL1 to improve our knowledge on its stress-induced damages following production and end-use processes. PMID:26941141

  5. Draft Genome Sequence of Acetobacter tropicalis Type Strain NBRC16470, a Producer of Optically Pure d-Glyceric Acid

    PubMed Central

    Koike, Hideaki; Sato, Shun; Morita, Tomotake; Fukuoka, Tokuma

    2014-01-01

    Here we report the 3.7-Mb draft genome sequence of Acetobacter tropicalis NBRC16470T, which can produce optically pure d-glyceric acid (d-GA; 99% enantiomeric excess) from raw glycerol feedstock derived from biodiesel fuel production processes. PMID:25523780

  6. Complete genome sequence of Lactobacillus plantarum ZS2058, a probiotic strain with high conjugated linoleic acid production ability.

    PubMed

    Yang, Bo; Chen, Haiqin; Tian, Fengwei; Zhao, Jianxin; Gu, Zhennan; Zhang, Hao; Chen, Yong Q; Chen, Wei

    2015-11-20

    Lactobacillus plantarum ZS2058 was isolated from sauerkraut and identified to synthesize the beneficial metabolite conjugated linoleic acid. The genome contains a 319,7363-bp chromosome and three plasmids. The sequence will facilitate identification and characterization of the genetic determinants for its putative biological benefits.

  7. Draft Genome Sequence of Cutaneotrichosporon curvatus DSM 101032 (Formerly Cryptococcus curvatus), an Oleaginous Yeast Producing Polyunsaturated Fatty Acids

    PubMed Central

    Hofmeyer, Thomas; Hackenschmidt, Silke; Nadler, Florian; Thürmer, Andrea; Daniel, Rolf

    2016-01-01

    Cutaneotrichosporon curvatus DSM 101032 is an oleaginous yeast that can be isolated from various habitats and is capable of producing substantial amounts of polyunsaturated fatty acids. Here, we present the first draft genome sequence of any C. curvatus species. PMID:27174275

  8. Draft Genome Sequence of Lactobacillus delbrueckii subsp. bulgaricus CFL1, a Lactic Acid Bacterium Isolated from French Handcrafted Fermented Milk.

    PubMed

    Meneghel, Julie; Dugat-Bony, Eric; Irlinger, Françoise; Loux, Valentin; Vidal, Marie; Passot, Stéphanie; Béal, Catherine; Layec, Séverine; Fonseca, Fernanda

    2016-03-03

    Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) is a lactic acid bacterium widely used for the production of yogurt and cheeses. Here, we report the genome sequence of L. bulgaricus CFL1 to improve our knowledge on its stress-induced damages following production and end-use processes.

  9. Genome Sequence of a Candidate World Health Organization Reference Strain of Zika Virus for Nucleic Acid Testing.

    PubMed

    Trösemeier, Jan-Hendrik; Musso, Didier; Blümel, Johannes; Thézé, Julien; Pybus, Oliver G; Baylis, Sally A

    2016-01-01

    We report here the sequence of a candidate reference strain of Zika virus (ZIKV) developed on behalf of the World Health Organization (WHO). The ZIKV reference strain is intended for use in nucleic acid amplification (NAT)-based assays for the detection and quantification of ZIKV RNA. PMID:27587826

  10. Ultra high-throughput nucleic acid sequencing as a tool for virus discovery in the turkey gut.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recently, the use of the next generation of nucleic acid sequencing technology (i.e., 454 pyrosequencing, as developed by Roche/454 Life Sciences) has allowed an in-depth look at the uncultivated microorganisms present in complex environmental samples, including samples with agricultural importance....

  11. Genome Sequence of a Candidate World Health Organization Reference Strain of Zika Virus for Nucleic Acid Testing.

    PubMed

    Trösemeier, Jan-Hendrik; Musso, Didier; Blümel, Johannes; Thézé, Julien; Pybus, Oliver G; Baylis, Sally A

    2016-01-01

    We report here the sequence of a candidate reference strain of Zika virus (ZIKV) developed on behalf of the World Health Organization (WHO). The ZIKV reference strain is intended for use in nucleic acid amplification (NAT)-based assays for the detection and quantification of ZIKV RNA.

  12. Purification, properties, and partial amino acid sequences of thermostable xylanases from Streptomyces thermoviolaceus OPC-520

    SciTech Connect

    Tsujibo, Hiroshi; Miyamoto, Katsushiro; Kuda, Takashi; Minami, Kazushi; Sakamoto, Takashi; Inamori, Yoshihiko ); Hasegawa, Toru )

    1992-01-01

    Two types of xylanases (1,4-{beta}-D-xylan xylanohydrolase, EC 3.2.1.8) were isolated from the culture filtrate of a thermophilic actinomycete, Streptomyces thermoviolaceus OPC-520. The enzymes (STX-I and STX-II) were purified by chromatography with DEAE-Toyopearl 650 M, CM-Toyopearl 650 M, Sephadex G-75, Phenyl-Toyopearl 650 M, and Mono Q HR. The purified enzymes showed single bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of STX-I and STX-II were 54,000 and 33,000, respectively. The pIs were 4.2 (STX-I) and 8.0 (STX-II). The optimum pH levels for the activity of STX-I and STX-II were pH 7.0. The optimum temperature for the activity of STX-I was 70C, and that for the activity of STX-II was 60C. The enzymes were completely inhibited by N-bromosuccinimide. The enzymes degraded xylan, producing xylose and xylobiose as the predominant products, indicating that they were endoxylanases. STX-I showed high sequence homology with the exoglucanase from Cellulomonas fimi (47% homology), and STX-II showed high sequence homology with the xylanase from Bacillus pumilus (46% homology).

  13. Amino acid sequence of Coprinus macrorhizus peroxidase and cDNA sequence encoding Coprinus cinereus peroxidase. A new family of fungal peroxidases.

    PubMed

    Baunsgaard, L; Dalbøge, H; Houen, G; Rasmussen, E M; Welinder, K G

    1993-04-01

    Sequence analysis and cDNA cloning of Coprinus peroxidase (CIP) were undertaken to expand the understanding of the relationships of structure, function and molecular genetics of the secretory heme peroxidases from fungi and plants. Amino acid sequencing of Coprinus macrorhizus peroxidase, and cDNA sequencing of Coprinus cinereus peroxidase showed that the mature proteins are identical in amino acid sequence, 343 residues in size and preceded by a 20-residue signal peptide. Their likely identity to peroxidase from Arthromyces ramosus is discussed. CIP has an 8-residue, glycine-rich N-terminal extension blocked with a pyroglutamate residue which is absent in other fungal peroxidases. The presence of pyroglutamate, formed by cyclization of glutamine, and the finding of a minor fraction of a variant form lacking the N-terminal residue, indicate that signal peptidase cleavage is followed by further enzymic processing. CIP is 40-45% identical in amino-acid sequence to 11 lignin peroxidases from four fungal species, and 42-43% identical to the two known Mn-peroxidases. Like these white-rot fungal peroxidases, CIP has an additional segment of approximately 40 residues at the C-terminus which is absent in plant peroxidases. Although CIP is much more similar to horseradish peroxidase (HRP C) in substrate specificity, specific activity and pH optimum than to white-rot fungal peroxidases, the sequences of CIP and HRP C showed only 18% identity. Hence, CIP qualifies as the first member of a new family of fungal peroxidases. The nine invariant residues present in all plant, fungal and bacterial heme peroxidases are also found in CIP. The present data support the hypothesis that only one chromosomal CIP gene exists. In contrast, a large number of secretory plant and fungal peroxidases are expressed from several peroxidase gene clusters. Analyses of three batches of CIP protein and of 49 CIP clones revealed the existence of only two highly similar alleles indicating less

  14. Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

    PubMed Central

    Fujii, Yuki; Tanaka, Shiho; Otsuki, Manami; Hoshino, Yasushi; Morimoto, Chinatsu; Kotani, Takuya; Harashima, Yuko; Endo, Haruka; Yoshizawa, Yasutaka; Sato, Ryoichi

    2012-01-01

    Characterizing the binding mechanism of Bt (Bacillus thuringiensis) Cry toxin to the cadherin receptor is indispensable to understanding the specific insecticidal activity of this toxin. To this end, we constructed 30 loop mutants by randomly inserting four serial amino acids covering all four receptor binding loops (loops α8, 1, 2 and 3) and analysed their binding affinities for Bombyx mori cadherin receptors via Biacore. High binding affinities were confirmed for all 30 mutants containing loop sequences that differed from those of wild-type. Insecticidal activities were confirmed in at least one mutant from loops 1, 2 and 3, suggesting that there is no critical amino acid sequence for the binding of the four loops to BtR175. When two mutations at different loops were integrated into one molecule, no reduction in binding affinity was observed compared with wild-type sequences. Based on these results, we discussed the binding mechanism of Cry toxin to cadherin protein. PMID:23145814

  15. The amino acid sequence of protein SCMK-B2C from the high-sulphur fraction of wool keratin.

    PubMed

    Elleman, T C

    1972-08-01

    1. The amino acid sequence of a protein from the reduced and carboxymethylated high-sulphur fraction of wool has been determined. 2. The sequence of this S-carboxymethylkerateine (SCMK-B2C) of 151 amino acid residues displays much internal homology and an unusual residue distribution. Thus a ten-residue sequence occurs four times near the N-terminus and five times near the C-terminus with few changes. These regions contain much of the molecule's half-cystine, whereas between them there is a region of 19 residues that are mainly small and devoid of cystine and proline. 3. Certain models of the wool fibre based on its mechanical and physical properties propose a matrix of small compact globular units linked together to form beaded chains. The unusual distribution of the component residues of protein SCMK-B2C suggests structures in the wool-fibre matrix compatible with certain features of the proposed models.

  16. Sequence-Specific Recognition of MicroRNAs and Other Short Nucleic Acids with Solid-State Nanopores.

    PubMed

    Zahid, Osama K; Wang, Fanny; Ruzicka, Jan A; Taylor, Ethan W; Hall, Adam R

    2016-03-01

    The detection and quantification of short nucleic acid sequences has many potential applications in studying biological processes, monitoring disease initiation and progression, and evaluating environmental systems, but is challenging by nature. We present here an assay based on the solid-state nanopore platform for the identification of specific sequences in solution. We demonstrate that hybridization of a target nucleic acid with a synthetic probe molecule enables discrimination between duplex and single-stranded molecules with high efficacy. Our approach requires limited preparation of samples and yields an unambiguous translocation event rate enhancement that can be used to determine the presence and abundance of a single sequence within a background of nontarget oligonucleotides. PMID:26824296

  17. Sequence of cDNA for rat cystathionine gamma-lyase and comparison of deduced amino acid sequence with related Escherichia coli enzymes.

    PubMed Central

    Erickson, P F; Maxwell, I H; Su, L J; Baumann, M; Glode, L M

    1990-01-01

    A cDNA clone for cystathionine gamma-lyase was isolated from a rat cDNA library in lambda gt11 by screening with a monospecific antiserum. The identity of this clone, containing 600 bp proximal to the 3'-end of the gene, was confirmed by positive hybridization selection. Northern-blot hybridization showed the expected higher abundance of the corresponding mRNA in liver than in brain. Two further cDNA clones from a plasmid pcD library were isolated by colony hybridization with the first clone and were found to contain inserts of 1600 and 1850 bp. One of these was confirmed as encoding cystathionine gamma-lyase by hybridization with two independent pools of oligodeoxynucleotides corresponding to partial amino acid sequence information for cystathionine gamma-lyase. The other clone (estimated to represent all but 8% of the 5'-end of the mRNA) was sequenced and its deduced amino acid sequence showed similarity to those of the Escherichia coli enzymes cystathionine beta-lyase and cystathionine gamma-synthase throughout its length, especially to that of the latter. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. PMID:2201285

  18. 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.

  19. The cDNA-derived amino acid sequence of hemoglobin II from Lucina pectinata.

    PubMed

    Torres-Mercado, Elineth; Renta, Jessicca Y; Rodríguez, Yolanda; López-Garriga, Juan; Cadilla, Carmen L

    2003-11-01

    Hemoglobin II from the clam Lucina pectinata is an oxygen-reactive protein with a unique structural organization in the heme pocket involving residues Gln65 (E7), Tyr30 (B10), Phe44 (CD1), and Phe69 (E11). We employed the reverse transcriptase-polymerase chain reaction (RT-PCR) and methods to synthesize various cDNA(HbII). An initial 300-bp cDNA clone was amplified from total RNA by RT-PCR using degenerate oligonucleotides. Gene-specific primers derived from the HbII-partial cDNA sequence were used to obtain the 5' and 3' ends of the cDNA by RACE. The length of the HbII cDNA, estimated from overlapping clones, was approximately 2114 bases. Northern blot analysis revealed that the mRNA size of HbII agrees with the estimated size using cDNA data. The coding region of the full-length HbII cDNA codes for 151 amino acids. The calculated molecular weight of HbII, including the heme group and acetylated N-terminal residue, is 17,654.07 Da.

  20. Compared with stearic acid, palmitic acid increased the yield of milk fat and improved feed efficiency across production level of cows.

    PubMed

    Rico, J E; Allen, M S; Lock, A L

    2014-02-01

    The effects of dietary palmitic and stearic acids on feed intake, yields of milk and milk components, and feed efficiency of dairy cows were evaluated in an experiment with a crossover arrangement of treatments with a covariate period. Cows with a wide range of milk production (38 to 65 kg/d) were used to determine if response to fat supplementation varied according to production level. Thirty-two Holstein cows (143 ± 61 d in milk) were assigned randomly to a treatment sequence within level of milk production. Treatments were diets supplemented (2% of diet dry matter) with palmitic acid (PA; 97.9% C16:0) or stearic acid (SA; 97.4% C18:0). Treatment periods were 21 d and cows were fed a nonfat supplemented diet for 14 d immediately before the first treatment period. The final 4d of each period were used for sample and data collection. Milk production measured during the covariate period (preliminary milk yield) was used as the covariate. No interactions were detected between treatment and preliminary milk yield for the production response variables measured. Compared with SA, the PA treatment increased milk fat concentration (3.66 vs. 3.55%) and yield (1.68 vs. 1.59 kg/d), and 3.5% fat-corrected milk yield (47.5 vs. 45.6 kg/d). Treatment did not affect dry matter intake, milk yield, milk protein yield, body weight, or body condition score. Milk protein concentration was lower for PA compared with SA treatment (3.24 vs. 3.29%). The PA treatment increased feed efficiency (3.5% fat-corrected milk yield/dry matter intake) compared with SA (1.48 vs. 1.40). The increase in milk fat yield by PA was entirely accounted for by a 24% increase in 16-carbon fatty acid output into milk. Yields of de novo (3.2%) and preformed fatty acids (2.9%) were only slightly decreased by PA relative to SA. The PA treatment increased plasma concentration of nonesterified fatty acids (96.3 vs. 88.2 μEq/L) and glucose (56.6 vs. 55.7 mg/dL) compared with SA, but insulin and

  1. 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

  2. Next-generation re-sequencing of genes involved in increased platelet reactivity in diabetic patients on acetylsalicylic acid.

    PubMed

    Postula, Marek; Janicki, Piotr K; Eyileten, Ceren; Rosiak, Marek; Kaplon-Cieslicka, Agnieszka; Sugino, Shigekazu; Wilimski, Radosław; Kosior, Dariusz A; Opolski, Grzegorz; Filipiak, Krzysztof J; Mirowska-Guzel, Dagmara

    2016-06-01

    The objective of this study was to investigate whether rare missense genetic variants in several genes related to platelet functions and acetylsalicylic acid (ASA) response are associated with the platelet reactivity in patients with diabetes type 2 (T2D) on ASA therapy. Fifty eight exons and corresponding introns of eight selected genes, including PTGS1, PTGS2, TXBAS1, PTGIS, ADRA2A, ADRA2B, TXBA2R, and P2RY1 were re-sequenced in 230 DNA samples from T2D patients by using a pooled PCR amplification and next-generation sequencing by Illumina HiSeq2000. The observed non-synonymous variants were confirmed by individual genotyping of 384 DNA samples comprising of the individuals from the original discovery pools and additional verification cohort of 154 ASA-treated T2DM patients. The association between investigated phenotypes (ASA induced changes in platelets reactivity by PFA-100, VerifyNow and serum thromboxane B2 level [sTxB2]), and accumulation of rare missense variants (genetic burden) in investigated genes was tested using statistical collapsing tests. We identified a total of 35 exonic variants, including 3 common missense variants, 15 rare missense variants, and 17 synonymous variants in 8 investigated genes. The rare missense variants exhibited statistically significant difference in the accumulation pattern between a group of patients with increased and normal platelet reactivity based on PFA-100 assay. Our study suggests that genetic burden of the rare functional variants in eight genes may contribute to differences in the platelet reactivity measured with the PFA-100 assay in the T2DM patients treated with ASA. PMID:26599574

  3. Chemical interactions between amino acid and RNA: multiplicity of the levels of specificity explains origin of the genetic code

    NASA Astrophysics Data System (ADS)

    Seligmann, Hervé; Amzallag, Nissim

    2002-11-01

    The emergence of the genetic code remains an enigma. Proposed mechanisms are based on random, historical, thermodynamic and natural selection. However, they introduce chance as a key factor for overcoming the difficulties encountered by the model. We propose here a model in which three successive levels of chemical specificity generated the nucleotide assignments of amino acids in the genetic code. The first level results from hydrophobic and stereospecific interactions between amino acids and short oligonucleotides (termed oligons). The second and third levels of specificity are determined by conditions of energy transfer from loaded oligons (amino acid-oligomer covalently linked) to formation of phosphodiester bond (second level of specificity) and peptidic bond (third level of specificity), while these reactions are catalyzed by RNA templates. This model is sustained by the relationships observed between dipole moments of the nucleotides (forming the anticodon) and reactivity of the amino acyl linkage of the loaded oligon. Moreover, analysis of modern tRNAs reveals that they were probably generated by loose duplication of the nucleotide sequence forming the oligons, after emergence of the 'genetic code.' Indeed, the similarity of nucleotide composition with that of the anticodon decreases with the tRNA domain's distance from the anticodon, but the acceptor stem is relatively more similar to the anticodon than other stems closer to it. This would be because energy transfer constraints that existed between anticodon and amino acid in prebiotic loaded oligonucleotides still affect the structures of modern tRNA acceptor stems. In the model presented, the genetic code is inherent to the most archaic 'molecular physiology' in protolife, even before emergence of a functional 'protein world.' Simple physical processes, in which a level of specificity is integrated in an emerging meta-structure expressing new properties, generate a parsimonious and realistic explanation

  4. Milk production responses to dietary stearic acid vary by production level in dairy cattle.

    PubMed

    Piantoni, P; Lock, A L; Allen, M S

    2015-03-01

    Effects of stearic acid supplementation on feed intake and metabolic and production responses of dairy cows with a wide range of milk production (32.2 to 64.4 kg/d) were evaluated in a crossover design experiment with a covariate period. Thirty-two multiparous Holstein cows (142±55 d in milk) were assigned randomly within level of milk yield to treatment sequence. Treatments were diets supplemented (2% of diet dry matter) with stearic acid (SA; 98% C18:0) or control (soyhulls). The diets were based on corn silage and alfalfa and contained 24.5% forage neutral detergent fiber, 25.1% starch, and 17.3% crude protein. Treatment periods were 21 d with the final 4 d used for data and sample collection. Compared with the control, SA increased dry matter intake (DMI; 26.1 vs. 25.2 kg/d) and milk yield (40.2 vs. 38.5 kg/d). Stearic acid had no effect on the concentration of milk components but increased yields of fat (1.42 vs. 1.35 kg/d), protein (1.19 vs. 1.14 kg/d), and lactose (1.96 vs. 1.87 kg/d). The SA treatment increased 3.5% fat-corrected milk (3.5% FCM; 40.5 vs. 38.6 kg/d) but did not affect feed efficiency (3.5% FCM/DMI, 1.55 vs. 1.53), body weight, or body condition score compared with the control. Linear interactions between treatment and level of milk yield during the covariate period were detected for DMI and yields of milk, fat, protein, lactose, and 3.5% FCM; responses to SA were positively related to milk yield of cows. The SA treatment increased crude protein digestibility (67.4 vs. 65.5%), tended to increase neutral detergent fiber digestibility (43.6 vs. 42.3%), decreased fatty acid (FA) digestibility (56.6 vs. 76.1%), and did not affect organic matter digestibility. Fatty acid yield response, calculated as the additional FA yield secreted in milk per unit of additional FA intake, was only 13.3% for total FA and 8.2% for C18:0 plus cis-9 C18:1. Low estimated digestibility of the SA supplement was at least partly responsible for the low FA yield response

  5. Molecular characterization of the level of sialic acids N-acetylneuraminic acid, N-glycolylneuraminic acid, and ketodeoxynonulosonic acid in porcine milk during lactation.

    PubMed

    Jahan, M; Wynn, P C; Wang, B

    2016-10-01

    Sialic acids (Sia) are key monosaccharide constituents of sialylated glycoproteins (Sia-GP), human sialylated milk oligosaccharide (Sia-MOS), and gangliosides. Human milk sialylated glycoconjugates (Sia-GC) are bioactive compounds known to act as prebiotics and promote neurodevelopment, immune function, and gut maturation in newborns. Only limited data are available on the Sia content of porcine milk. The objective of this study was to quantitatively determine the total level of Sia N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), and ketodeoxynonulosonic acid (KDN) in porcine milk and to compare these levels in gilt and sow milk during lactation. Milk from 8 gilts and 22 sows was collected at 3 stages of lactation (colostrum, transition, and mature milk). Standard and experimental samples were derivatized using 1,2-diamino-4,5-methylenedioxy-benzene and analyzed by ultra-high-performance liquid chromatography using a fluorescence detector. The following new findings are reported: (1) Gilt and sow milk contained significant levels of total Sia, with the highest concentration in colostrum (1,238.5 mg/L), followed by transition milk (778.3 mg/L) and mature milk (347.2 mg/L); (2) during lactation, the majority of Sia was conjugated to Sia-GP (41-46%), followed by Sia-MOS (31-42%) and a smaller proportion in gangliosides (12-28%); (3) Neu5Ac was the major form of Sia (93-96%), followed by Neu5Gc (3-6%) and then KDN (1-2%), irrespective of milk fraction or stage of lactation; (4) the concentration of Sia in Sia-GP and Sia-MOS showed a significant decline during lactation, but the level of ganglioside Sia remained relatively constant; (5) mature gilt milk contained a significantly higher concentration of Sia-GP than sow milk. The high concentration of total Sia in porcine milk suggests that Sia-GC are important nutrients that contribute to the optimization of neurodevelopment, immune function, and growth and development in piglets. These findings

  6. 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.

  7. Molecular cloning, coding nucleotides and the deduced amino acid sequence of P-450BM-1 from Bacillus megaterium.

    PubMed

    He, J S; Ruettinger, R T; Liu, H M; Fulco, A J

    1989-12-22

    The gene encoding barbiturate-inducible cytochrome P-450BM-1 from Bacillus megaterium ATCC 14581 has been cloned and sequenced. An open reading frame in the 1.9 kb of cloned DNA correctly predicted the NH2-terminal sequence of P-450BM-1 previously determined by protein sequencing, and, in toto, predicted a polypeptide of 410 amino acid residues with an Mr of 47,439. The sequence is most, but less than 27%, similar to that of P-450CAM from Pseudomonas putida, so that P-450BM-1 clearly belongs to a new P-450-gene family, distinct especially from that of the P-450 domain of P-450BM-3, a barbiturate-inducible single polypeptide cytochrome P-450:NADPH-P-450 reductase from the same strain of B. megaterium (Ruettinger, R.T., Wen, L.-P. and Fulco, A.J. (1989) J. Biol. Chem. 264, 10987-10995). PMID:2597681

  8. Inter-level Scaffolding and Sequences of Representational Activities in Teaching a Chemical System with Graphical Simulations

    NASA Astrophysics Data System (ADS)

    Li, Na; Black, John B.

    2016-05-01

    Chemistry knowledge can be represented at macro-, micro- and symbolic levels, and learning a chemistry topic requires students to engage in multiple representational activities. This study focused on scaffolding for inter-level connection-making in learning chemistry knowledge with graphical simulations. We also tested whether different sequences of representational activities produced different student learning outcomes in learning a chemistry topic. A sample of 129 seventh graders participated in this study. In a simulation-based environment, participants completed three representational activities to learn several ideal gas law concepts. We conducted a 2 × 3 factorial design experiment. We compared two scaffolding conditions: (1) the inter-level scaffolding condition in which participants received inter-level questions and experienced the dynamic link function in the simulation-based environment and (2) the intra-level scaffolding condition in which participants received intra-level questions and did not experience the dynamic link function. We also compared three different sequences of representational activities: macro-symbolic-micro, micro-symbolic-macro and symbolic-micro-macro. For the scaffolding variable, we found that the inter-level scaffolding condition produced significantly better performance in both knowledge comprehension and application, compared to the intra-level scaffolding condition. For the sequence variable, we found that the macro-symbolic-micro sequence produced significantly better knowledge comprehension performance than the other two sequences; however, it did not benefit knowledge application performance. There was a trend that the treatment group who experienced inter-level scaffolding and the micro-symbolic-macro sequence achieved the best knowledge application performance.

  9. Inter-level Scaffolding and Sequences of Representational Activities in Teaching a Chemical System with Graphical Simulations

    NASA Astrophysics Data System (ADS)

    Li, Na; Black, John B.

    2016-10-01

    Chemistry knowledge can be represented at macro-, micro- and symbolic levels, and learning a chemistry topic requires students to engage in multiple representational activities. This study focused on scaffolding for inter-level connection-making in learning chemistry knowledge with graphical simulations. We also tested whether different sequences of representational activities produced different student learning outcomes in learning a chemistry topic. A sample of 129 seventh graders participated in this study. In a simulation-based environment, participants completed three representational activities to learn several ideal gas law concepts. We conducted a 2 × 3 factorial design experiment. We compared two scaffolding conditions: (1) the inter- level scaffolding condition in which participants received inter-level questions and experienced the dynamic link function in the simulation-based environment and (2) the intra- level scaffolding condition in which participants received intra-level questions and did not experience the dynamic link function. We also compared three different sequences of representational activities: macro-symbolic-micro, micro-symbolic-macro and symbolic-micro-macro. For the scaffolding variable, we found that the inter- level scaffolding condition produced significantly better performance in both knowledge comprehension and application, compared to the intra- level scaffolding condition. For the sequence variable, we found that the macro-symbolic-micro sequence produced significantly better knowledge comprehension performance than the other two sequences; however, it did not benefit knowledge application performance. There was a trend that the treatment group who experienced inter- level scaffolding and the micro-symbolic-macro sequence achieved the best knowledge application performance.

  10. Use of whole-genus genome sequence data to develop a multilocus sequence typing tool that accurately identifies Yersinia isolates to the species and subspecies levels.

    PubMed

    Hall, Miquette; Chattaway, Marie A; Reuter, Sandra; Savin, Cyril; Strauch, Eckhard; Carniel, Elisabeth; Connor, Thomas; Van Damme, Inge; Rajakaruna, Lakshani; Rajendram, Dunstan; Jenkins, Claire; Thomson, Nicholas R; McNally, Alan

    2015-01-01

    The genus Yersinia is a large and diverse bacterial genus consisting of human-pathogenic species, a fish-pathogenic species, and a large number of environmental species. Recently, the phylogenetic and population structure of the entire genus was elucidated through the genome sequence data of 241 strains encompassing every known species in the genus. Here we report the mining of this enormous data set to create a multilocus sequence typing-based scheme that can identify Yersinia strains to the species level to a level of resolution equal to that for whole-genome sequencing. Our assay is designed to be able to accurately subtype the important human-pathogenic species Yersinia enterocolitica to whole-genome resolution levels. We also report the validation of the scheme on 386 strains from reference laboratory collections across Europe. We propose that the scheme is an important molecular typing system to allow accurate and reproducible identification of Yersinia isolates to the species level, a process often inconsistent in nonspecialist laboratories. Additionally, our assay is the most phylogenetically informative typing scheme available for Y. enterocolitica.

  11. Use of Whole-Genus Genome Sequence Data To Develop a Multilocus Sequence Typing Tool That Accurately Identifies Yersinia Isolates to the Species and Subspecies Levels

    PubMed Central

    Hall, Miquette; Chattaway, Marie A.; Reuter, Sandra; Savin, Cyril; Strauch, Eckhard; Carniel, Elisabeth; Connor, Thomas; Van Damme, Inge; Rajakaruna, Lakshani; Rajendram, Dunstan; Jenkins, Claire; Thomson, Nicholas R.

    2014-01-01

    The genus Yersinia is a large and diverse bacterial genus consisting of human-pathogenic species, a fish-pathogenic species, and a large number of environmental species. Recently, the phylogenetic and population structure of the entire genus was elucidated through the genome sequence data of 241 strains encompassing every known species in the genus. Here we report the mining of this enormous data set to create a multilocus sequence typing-based scheme that can identify Yersinia strains to the species level to a level of resolution equal to that for whole-genome sequencing. Our assay is designed to be able to accurately subtype the important human-pathogenic species Yersinia enterocolitica to whole-genome resolution levels. We also report the validation of the scheme on 386 strains from reference laboratory collections across Europe. We propose that the scheme is an important molecular typing system to allow accurate and reproducible identification of Yersinia isolates to the species level, a process often inconsistent in nonspecialist laboratories. Additionally, our assay is the most phylogenetically informative typing scheme available for Y. enterocolitica. PMID:25339391

  12. Sample Prep, Workflow Automation and Nucleic Acid Fractionation for Next Generation Sequencing

    SciTech Connect

    Roskey, Mark

    2010-06-03

    Mark Roskey of Caliper LifeSciences discusses how the company's technologies fit into the next generation sequencing workflow on June 3, 2010 at the "Sequencing, Finishing, Analysis in the Future" meeting in Santa Fe, NM

  13. Unusually high levels of n-6 polyunsaturated fatty acids in whale sharks and reef manta rays.

    PubMed

    Couturier, L I E; Rohner, C A; Richardson, A J; Pierce, S J; Marshall, A D; Jaine, F R A; Townsend, K A; Bennett, M B; Weeks, S J; Nichols, P D

    2013-10-01

    Fatty acid (FA) signature analysis has been increasingly used to assess dietary preferences and trophodynamics in marine animals. We investigated FA signatures of connective tissue of the whale shark Rhincodon typus and muscle tissue of the reef manta ray Manta alfredi. We found high levels of n-6 polyunsaturated fatty acids (PUFA), dominated by arachidonic acid (20:4n-6; 12-17 % of total FA), and comparatively lower levels of the essential n-3 PUFA-eicosapentaenoic acid (20:5n-3; ~1 %) and docosahexaenoic acid (22:6n-3; 3-10 %). Whale sharks and reef manta rays are regularly observed feeding on surface aggregations of coastal crustacean zooplankton during the day, which generally have FA profiles dominated by n-3 PUFA. The high levels of n-6 PUFA in both giant elasmobranchs raise new questions about the origin of their main food source.

  14. Effects of toxic work environments on sperm quality and ascorbic acid levels

    SciTech Connect

    Dawson, E.B.; Harris, W.A.; Powell, L.C. )

    1990-02-26

    Surveys have shown that toxic work environments lower sperm quality, and controlled studies indicate that ascorbic acid supplementation improves sperm viability and agglutination. The sperm quality of 50 subjects each from: (1) office workers, (2) a lead smelter, (3) petroleum refineries, and (4) a herbicide plant were compared with serum and semen ascorbic acid levels. The sperm characteristics studied were: count as million/ml and as percent; viability, motility, clumping, and abnormal morphology. The serum ascorbic acid levels were directly proportional to sperm viability and inversely correlated to clumping of all groups. Moreover, serum ascorbic acid levels were also inversely correlated to twin tail and amorphous forms of abnormal sperm morphology. The results of the study indicate that toxic environments depress sperm quality and suggest that ascorbic acid supplementation will improve sperm quality and fertility.

  15. Detection and quantification of the red tide dinoflagellate Karenia brevis by real-time nucleic acid sequence-based amplification.

    PubMed

    Casper, Erica T; Paul, John H; Smith, Matthew C; Gray, Michael

    2004-08-01

    Nucleic acid sequence-based amplification (NASBA) is an isothermal method of RNA amplification that has been previously used in clinical diagnostic testing. A real-time NASBA assay has been developed for the detection of rbcL mRNA from the red tide dinoflagellate Karenia brevis. This assay is sensitive to one K. brevis cell and 1.0 fg of in vitro transcript, with occasional detection of lower concentrations of transcript. The assay did not detect rbcL mRNA from a wide range of nontarget organisms and environmental clones, while 10 strains (all tested) of K. brevis were detected. By the use of standard curves based on time to positivity, concentrations of K. brevis in environmental samples were predicted by NASBA and classified into different levels of blooms per the Florida Fish and Wildlife Conservation Commission (FWC) system. NASBA classification matched FWC classification (based on cell counts) 72% of the time. Those samples that did not match were off by only one class. NASBA is sensitive, rapid, and effective and may be used as an additional or alternative method to detect and quantify K. brevis in the marine environment. PMID:15294808

  16. The predicted amino acid sequence of alpha-internexin is that of a novel neuronal intermediate filament protein.

    PubMed Central

    Fliegner, K H; Ching, G Y; Liem, R K

    1990-01-01

    Our laboratory recently isolated and began to characterize a 66 kd rat brain cytoskeletal protein, dubbed alpha-internexin for its interactions in vitro with several other cytoskeletal proteins. Although alpha-internexin bore several of the characteristics of intermediate filament (IF) proteins, including the recognition by an antibody reactive with all IF proteins, it did not polymerize into 10 nm filaments under the conditions tested. Here we show that the predicted amino acid sequence of a cDNA encoding alpha-internexin shows the latter to be an IF protein, probably most closely related to the neurofilament proteins. Northern blotting shows that alpha-internexin expression is brain specific, and that rat brain alpha-internexin mRNA levels are maximal prior to birth and decline into adulthood, while the converse is seen for NF-L, the low molecular weight neurofilament subunit, suggesting that these two proteins play different roles in the developing brain. Images Fig. 1. Fig. 3. Fig. 5. PMID:2311576

  17. Plasma levels of ursodeoxycholic acid in black bears, Ursus americanus: seasonal changes.

    PubMed

    Solá, Susana; Garshelis, David L; Amaral, Joana D; Noyce, Karen V; Coy, Pam L; Steer, Clifford J; Iaizzo, Paul A; Rodrigues, Cecília M P

    2006-06-01

    To date, no other studies have examined the seasonal changes in circulating levels of various bile acids in the plasma of wild North American black bears, Ursus americanus. Using gas chromatography, bile acid concentrations were measured in plasma samples obtained during either early or late hibernation, and during summer active periods. Thus, specific compositional changes from individual animals were examined through a given year. Total bile acid concentrations in the plasma of these normal animals were found to range between 0.2 and 3.1 micromol/L (0.9 +/- 0.2 micromol/L, mean +/- SEM). Cholic, ursodeoxycholic and chenodeoxycholic acids were the major bile acid species identified. Ursodeoxycholic acid represented 28.0 +/- 2.6% of the total bile acid pool. Deoxycholic and lithocholic acids were found only in small amounts. In addition, total bile acid concentrations were lower in plasma samples obtained during hibernation compared with those obtained during summer active periods (0.6 +/- 0.1 and 1.2 +/- 0.4 micromol/L, respectively; p < 0.05). However, the relative proportion of ursodeoxycholic acid, was significantly greater in winter than in summer (31.5 +/- 3.2% and 22.2 +/- 4.5%, p < 0.05). Finally, taurine-conjugated bile acids were the predominant species in bear plasma, accounting for >67% of the total bile acids. These data demonstrate that ursodeoxycholic acid is a major bile acid in black bear plasma, mostly conjugated with taurine. Further, the finding of seasonal variation in plasma bile acid composition provides evidence to support the possible role that ursodeoxycholic acid may play in cellular protection in hibernating black bears. PMID:16571381

  18. 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.

  19. 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. PMID:23637504

  20. 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.

  1. Amino acid sequence and structural properties of protein p12, an African swine fever virus attachment protein.

    PubMed Central

    Alcamí, A; Angulo, A; López-Otín, C; Muñoz, M; Freije, J M; Carrascosa, A L; Viñuela, E

    1992-01-01

    The gene encoding the African swine fever virus protein p12, which is involved in virus attachment to the host cell, has been mapped and sequenced in the genome of the Vero-adapted virus strain BA71V. The determination of the N-terminal amino acid sequence and the hybridization of oligonucleotide probes derived from this sequence to cloned restriction fragments allowed the mapping of the gene in fragment EcoRI-O, located in the central region of the viral genome. The DNA sequence of an EcoRI-XbaI fragment showed an open reading frame which is predicted to encode a polypeptide of 61 amino acids. The expression of this open reading frame in rabbit reticulocyte lysates and in Escherichia coli gave rise to a 12-kDa polypeptide that was immunoprecipitated with a monoclonal antibody specific for protein p12. The hydrophilicity profile indicated the existence of a stretch of 22 hydrophobic residues in the central part that may anchor the protein in the virus envelope. Three forms of the protein with apparent molecular masses of 17, 12, and 10 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis have been observed, depending on the presence of 2-mercaptoethanol and alkylation with 4-vinylpyridine, indicating that disulfide bonds are responsible for the multimerization of the protein. This result was in agreement with the existence of a cysteine-rich domain in the C-terminal region of the predicted amino acid sequence. The protein was synthesized at late times of infection, and no posttranslational modifications such as glycosylation, phosphorylation, or fatty acid acylation were detected. Images PMID:1583732

  2. Dissolution of Simulated and Radioactive Savannah River Site High-Level Waste Sludges with Oxalic Acid & Citric Acid Solutions

    SciTech Connect

    STALLINGS, MARY

    2004-07-08

    sludge solids. We recommend that these results be evaluated further to determine if these solutions contain sufficient neutron poisons. We observed low general corrosion rates in tests in which carbon steel coupons were contacted with solutions of oxalic acid, citric acid and mixtures of oxalic and citric acids. Wall thinning can be minimized by maintaining short contact times with these acid solutions. We recommend additional testing with oxalic and oxalic/citric acid mixtures to measure dissolution performance of sludges that have not been previously dried. This testing should include tests to clearly ascertain the effects of total acid strength and metal complexation on dissolution performance. Further work should also evaluate the downstream impacts of citric acid on the SRS High-Level Waste System (e.g., radiochemical separations in the Salt Waste Processing Facility and addition of organic carbon in the Saltstone and Defense Waste Processing facilities).

  3. 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.

  4. Two-Level Adaptive Algebraic Multigrid for a Sequence of Problems with Slowly Varying Random Coefficients [Adaptive Algebraic Multigrid for Sequence of Problems with Slowly Varying Random Coefficients

    SciTech Connect

    Kalchev, D.; Ketelsen, C.; Vassilevski, P. S.

    2013-11-07

    Our paper proposes an adaptive strategy for reusing a previously constructed coarse space by algebraic multigrid to construct a two-level solver for a problem with nearby characteristics. Furthermore, a main target application is the solution of the linear problems that appear throughout a sequence of Markov chain Monte Carlo simulations of subsurface flow with uncertain permeability field. We demonstrate the efficacy of the method with extensive set of numerical experiments.

  5. Amino acid sequence of an intracellular, phosphate-starvation-induced ribonuclease from cultured tomato (Lycopersicon esculentum) cells.

    PubMed

    Löffler, A; Glund, K; Irie, M

    1993-06-15

    The primary structure of an intracellular ribonuclease (RNase LX) from cultured tomato (Lycopersicon esculentum) cells has been determined. Previous studies have shown that the protein is located inside the tomato cells but outside the vacuoles and that its synthesis is induced after depleting the cells for phosphate [Löffler, A., Abel, S., Jost, W., Beintema, J. J., Glund, K. (1992) Plant Physiol. 98, 1472-1478]. Sequence analysis was carried out by analysis of peptides isolated after enzymatic and chemical cleavage of the protein. RNase LX consists of 213 amino acids and has a molecular mass of 24300 Da and an isoelectric point of 5.33. The enzyme contains 10 half-cystines and there are no potential N-glycosylation sites detectable in the sequence. RNase LX, as compared to an extracellular tomato RNase (RNase LE), which is also phosphate regulated and the amino acid sequence of which was recently established [Jost, W., Bak, H., Glund, K., Terpstra, P. & Beintema, J. J. (1991) Eur. J. Biochem. 198, 1-6] has 60% of all amino acids identical and in identical positions, revealing a high degree of similarity between both proteins. In contrast to RNase LE, RNase LX has a C-terminal extension of nine amino acids. The C-terminal tetrapeptide HDEF may be a retention signal of the protein in the endoplasmic reticulum. PMID:8319673

  6. 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.

  7. 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.

  8. The CYP51F1 Gene of Leptographium qinlingensis: Sequence Characteristic, Phylogeny and Transcript Levels.

    PubMed

    Dai, Lulu; Li, Zhumei; Yu, Jiamin; Ma, Mingyuan; Zhang, Ranran; Chen, Hui; Pham, Thanh

    2015-05-26

    Leptographium qinlingensis is a fungal associate of the Chinese white pine beetle (Dendroctonus armandi) and a pathogen of the Chinese white pine (Pinus armandi) that must overcome the terpenoid oleoresin defenses of host trees. L. qinlingensis responds to monoterpene flow with abundant mechanisms that include export and the use of these compounds as a carbon source. As one of the fungal cytochrome P450 proteins (CYPs), which play important roles in general metabolism, CYP51 (lanosterol 14-α demethylase) can catalyze the biosynthesis of ergosterol and is a target for antifungal drug. We have identified an L. qinlingensis CYP51F1 gene, and the phylogenetic analysis shows the highest homology with the 14-α-demethylase sequence from Grosmannia clavigera (a fungal associate of Dendroctonus ponderosae). The transcription level of CYP51F1 following treatment with terpenes and pine phloem extracts was upregulated, while using monoterpenes as the only carbon source led to the downregulation of CYP5F1 expression. The homology modeling structure of CYP51F1 is similar to the structure of the lanosterol 14-α demethylase protein of Saccharomyces cerevisiae YJM789, which has an N-terminal membrane helix 1 (MH1) and transmembrane helix 1 (TMH1). The minimal inhibitory concentrations (MIC) of terpenoid and azole fungicides (itraconazole (ITC)) and the docking of terpenoid molecules, lanosterol and ITC in the protein structure suggested that CYP51F1 may be inhibited by terpenoid molecules by competitive binding with azole fungicides.

  9. 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.

  10. 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

  11. Classification of protein motifs based on subcellular localization uncovers evolutionary relationships at both sequence and functional levels

    PubMed Central

    2013-01-01

    Background Most proteins have evolved in specific cellular compartments that limit their functions and potential interactions. On the other hand, motifs define amino acid arrangements conserved between protein family members and represent powerful tools for assigning function to protein sequences. The ideal motif would identify all members of a protein family but in practice many motifs identify both family members and unrelated proteins, referred to as True Positive (TP) and False Positive (FP) sequences, respectively. Results To address the relationship between protein motifs, protein function and cellular localization, we systematically assigned subcellular localization data to motif sequences from the comprehensive PROSITE sequence motif database. Using this data we analyzed relationships between localization and function. We find that TPs and FPs have a strong tendency to localize in different compartments. When multiple localizations are considered, TPs are usually distributed between related cellular compartments. We also identified cases where FPs are concentrated in particular subcellular regions, indicating possible functional or evolutionary relationships with TP sequences of the same motif. Conclusions Our findings suggest that the systematic examination of subcellular localization has the potential to uncover evolutionary and functional relationships between motif-containing sequences. We believe that this type of analysis complements existing motif annotations and could aid in their interpretation. Our results shed light on the evolution of cellular organelles and potentially establish the basis for new subcellular localization and function prediction algorithms. PMID:23865897

  12. Identification, characterization, and complete amino acid sequence of the conjugation-inducing glycoprotein (blepharmone) in the ciliate Blepharisma japonicum

    PubMed Central

    Sugiura, Mayumi; Harumoto, Terue

    2001-01-01

    Conjugation in Blepharisma japonicum is induced by interaction between complementary mating-types I and II, which excrete blepharmone (gamone 1) and blepharismone (gamone 2), respectively. Gamone 1 transforms type II cells such that they can unite, and gamone 2 similarly transforms type I cells. Moreover, each gamone promotes the production of the other gamone. Gamone 2 has been identified as calcium-3-(2′-formylamino-5′-hydroxy-benzoyl) lactate and has been synthesized chemically. Gamone 1 was isolated and characterized as a glycoprotein of 20–30 kDa containing 175 amino acids and 6 sugars. However, the amino acid sequence and arrangement of sugars in this gamone are still unknown. To determine partial amino acid sequences of gamone 1, we established a method of isolation based on the finding that this glycoprotein can be concentrated by a Con A affinity column. Gamone 1 is extremely unstable and loses its biological activity once adsorbed to any of the columns that we tested. By using a Con A affinity column and native PAGE, we detected a 30-kDa protein corresponding to gamone 1 activity and determined the partial amino acid sequences of the four peptides. To isolate gamone 1 cDNA, we isolated mRNA from mating-type I cells stimulated by synthetic gamone 2 and then performed rapid amplification of cDNA ends procedures by using gene-specific primers and cloned cDNA of gamone 1. The cDNA sequence contains an ORF of 305 amino acids and codes a possibly novel protein. We also estimated the arrangement of sugars by comparing the affinity to various lectin columns. PMID:11724922

  13. Identification, characterization, and complete amino acid sequence of the conjugation-inducing glycoprotein (blepharmone) in the ciliate Blepharisma japonicum.

    PubMed

    Sugiura, M; Harumoto, T

    2001-12-01

    Conjugation in Blepharisma japonicum is induced by interaction between complementary mating-types I and II, which excrete blepharmone (gamone 1) and blepharismone (gamone 2), respectively. Gamone 1 transforms type II cells such that they can unite, and gamone 2 similarly transforms type I cells. Moreover, each gamone promotes the production of the other gamone. Gamone 2 has been identified as calcium-3-(2'-formylamino-5'-hydroxy-benzoyl) lactate and has been synthesized chemically. Gamone 1 was isolated and characterized as a glycoprotein of 20-30 kDa containing 175 amino acids and 6 sugars. However, the amino acid sequence and arrangement of sugars in this gamone are still unknown. To determine partial amino acid sequences of gamone 1, we established a method of isolation based on the finding that this glycoprotein can be concentrated by a Con A affinity column. Gamone 1 is extremely unstable and loses its biological activity once adsorbed to any of the columns that we tested. By using a Con A affinity column and native PAGE, we detected a 30-kDa protein corresponding to gamone 1 activity and determined the partial amino acid sequences of the four peptides. To isolate gamone 1 cDNA, we isolated mRNA from mating-type I cells stimulated by synthetic gamone 2 and then performed rapid amplification of cDNA ends procedures by using gene-specific primers and cloned cDNA of gamone 1. The cDNA sequence contains an ORF of 305 amino acids and codes a possibly novel protein. We also estimated the arrangement of sugars by comparing the affinity to various lectin columns.

  14. Effect of dietary ascorbic acid supplementation level on productivity, mortality, and carcass characteristics of Venda chickens.

    PubMed

    Malebane, Ingrid M; Ng'ambi, Jones Wilfred; Norris, David; Mbajiorgu, Christian

    2010-12-01

    Two experiments were carried out to determine the effect of dietary ascorbic acid supplementation levels on productivity, carcass characteristics, and mortality of indigenous Venda chickens. The first experiment determined the effect of dietary ascorbic acid supplementation levels on productivity and mortality rate of 175 unsexed Venda chickens between 1 and 6 weeks old. The second experiment determined the effect of dietary ascorbic acid supplementation levels on productivity, carcass characteristics, and mortality rate of 140 female Venda chickens between 8 and 13 weeks old. A completely randomized design was used in both experiments. Supplementation of grower diets with ascorbic acid ranged from 0 to 2,000 mg per kg DM feed in both experiments. Levels of ascorbic acid supplementation for optimum feed intake, feed conversion ratio, growth rate, live weight, and breast meat yield were determined using a quadratic equation. The optimal dietary ascorbic acid supplementation levels for feed conversion ratio, growth rate, and live weight of Venda chickens during the starter phase were 1,050, 1,301, and 1,500 mg/kg DM feed, while, at the grower phase, the optimal supplementation levels for feed conversion ratio, growth rate, live weight, and breast meat yield were 1,000, 1,250, 1,482, and 769 mg/kg DM feed, respectively. Results indicate that different levels of ascorbic acid supplementation optimized feed conversion ratio, growth rate, and live weight of Venda chickens at each growth phase. However, levels of ascorbic acid supplementation for optimum feed conversion ratio, growth rate, and live weight were higher than that for breast meat yield. These findings have implications on ration formulation for Venda chickens.

  15. Complete nucleotide sequence analysis of plasmids in strains of Staphylococcus aureus clone USA300 reveals a high level of identity among isolates with closely related core genome sequences.

    PubMed

    Kennedy, Adam D; Porcella, Stephen F; Martens, Craig; Whitney, Adeline R; Braughton, Kevin R; Chen, Liang; Craig, Carly T; Tenover, Fred C; Kreiswirth, Barry N; Musser, James M; DeLeo, Frank R

    2010-12-01

    A community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strain known as pulsed-field type USA300 (USA300) is epidemic in the United States. Previous comparative whole-genome sequencing studies demonstrated that there has been recent clonal emergence of a subset of USA300 isolates, which comprise the epidemic clone. Although the core genomes of these isolates are closely related, the level of diversity among USA300 plasmids was not resolved. Inasmuch as these plasmids might contribute to significant gene diversity among otherwise closely related USA300 isolates, we performed de novo sequencing of endogenous plasmids from 10 previously characterized USA300 clinical isolates obtained from different geographic locations in the United States. All isolates tested contained small (2- to 3-kb) and/or large (27- to 30-kb) plasmids. The large plasmids encoded heavy metal and/or antimicrobial resistance elements, including those that confer resistance to cadmium, bacitracin, macrolides, penicillin, kanamycin, and streptothricin, although all isolates were sensitive to minocycline, doxycycline, trimethoprim-sulfamethoxazole, vancomycin, teicoplanin, and linezolid. One of the USA300 isolates contained an archaic plasmid that encoded staphylococcal enterotoxins R, J, and P. Notably, the large plasmids (27 to 28 kb) from 8 USA300 isolates--those that comprise the epidemic USA300 clone--were virtually identical (99% identity) and similar to a large plasmid from strain USA300_TCH1516 (a previously sequenced USA300 strain from Houston, TX). These plasmids are largely divergent from the 37-kb plasmid of FPR3757, the first sequenced USA300 strain. The high level of plasmid sequence identity among the majority of closely related USA300 isolates is consistent with the recent clonal emergence hypothesis for USA300.

  16. Relationship of sea level changes and intrabasinal tectonics on upper Cretaceous depositional sequences, Tremp basin, Pyrenees, Spain

    SciTech Connect

    Simo, A.

    1988-02-01

    Well-exposed Upper Cretaceous rocks in the Tremp basin display the effects of subsidence and sea level changes. Subsidence and a net sea level rise control the sequence geometry and the cyclicity of transgressive-regressive units. In addition, subsidence, sea level, and sediment supply control the facies framework. The Tremp basin, where specific depositional processes can be related to basin evolution, is proposed as a reference section to compare tectonism vs. sea level changes. Five depositional sequences recognized are seismic-stratigraphic in scale and are correlatives of global rhythms. Sequences are separated by unconformable or correlative conformable surfaces. Most of the surfaces in the area are concordant, characterized by a sharp transition upward from shallower to deeper facies. Sea level drops exposed the inner shelf but not the shelf margin; they did, however, result in submarine erosion and basin-restricted wedge deposition. The three oldest sequences are carbonate-rich. Each sequence exhibits very little coastal onlap; however, each successively younger carbonate platform backsteps the former. Thermal subsidence (6.3 cm/1000 yr) created depositional space in the basin, and uplift reduced shelf onlap. The fourth sequence is carbonate-rich on the shelf and siliciclastic-rich in the basin. Subsidence first increased (shelf = 5.13 cm/1000 yr; basin = 15.4 cm/1000 yr) because of thrusting west of the area, resulting in coastal onlap over 60 km and accompanying backstepping of the carbonate platform. Subsidence later decreased, resulting in platform progradation. The fifth depositional sequence shows an overall siliciclastic regression, in response to regional thrusting and uplift.

  17. Relationship of sea level changes and intrabasinal tectonics on Upper Cretaceous depositional sequences, Tremp Basin, Pyrenees, Spain

    SciTech Connect

    Simo, A.

    1988-01-01

    Well-exposed Upper Cretaceous rocks in the Tremp basin display the effects of subsidence and sea level changes. Subsidence and a net sea level rise control the sequence geometry and the cyclicity of transgressive-regressive units. In addition, subsidence, sea level, and sediment supply control the facies framework. The Tremp basin, where specific depositional processes can be related to basin evolution, is proposed as a reference section to compare tectonism vs. sea level changes. Five depositional sequences recognized are seismic-stratigraphic in scale and are correlatives of global rhythms. Sequences are separated by uncomfortable or correlative conformable surfaces. Most of the surfaces in the area are concordant, characterized by a sharp transition upward from shallower to deeper facies. Sea level drops exposed the inner shelf but not the shelf margin; they did, however, result in submarine erosion and basin-restricted wedge deposition. The three oldest sequences are carbonate-rich. Each sequence exhibits very little coastal onlap; however, each successively younger carbonate platform backsteps the former. Thermal subsidence (6.3cm/1,000 yr) created depositional space space in the basin, and uplift reduced shelf onlap. The fourth sequence is carbonate-rich on the shelf and siliciclastic-rich in the basin. Subsidence first increased (shelf = 5.13 cm/1,000 yr; basin = 15/4 cm/1,000 yr) because of thrusting west of the area, resulting in coastal onlap over 60 km and accompanying backstepping of the carbonate platform. Subsidence later decreased, resulting in platform progradation. The fifth depositional sequence shows an overall siliciclastic regression, in response to regional thrusting and uplift.

  18. Analysis of methylphosphonic acid, ethyl methylphosphonic acid and isopropyl methylphosphonic acid at low microgram per liter levels in groundwater.

    PubMed

    Sega, G A; Tomkins, B A; Griest, W H

    1997-11-28

    A method is described for determining methylphosphonic acid, ethyl methylphosphonic acid and isopropyl methylphosphonic acid, which are hydrolysis products of the nerve agents VX (S-2-diisopropylaminoethyl O-ethyl methylphosphonothiolate) and GB (sarin, isopropylmethyl phosphonofluoridate). The analytes are extracted from 50 ml groundwater using a solid-phase extraction column packed with 500 mg of silica with a bonded quaternary amine phase, and are eluted and derivatized with methanolic trimethylphenylammonium hydroxide. Separation and quantitation are achieved using a capillary column gas chromatograph equipped with a flame photometric detector operated in its phosphorus-selective mode. Two independent statistically-unbiased procedures were employed to determine the detection limits, which ranged between 3 and 9 micrograms/l, for the three analytes. PMID:9435117

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

    PubMed

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

    1990-03-01

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

  20. Effects of cochlear ablation on amino acid levels in the rat cochlear nucleus and superior olive.

    PubMed

    Godfrey, Donald A; Jin, Yong-Ming; Liu, Xiaochen; Godfrey, Matthew A

    2014-03-01

    Amino acids have important roles in the chemistry of the auditory system, including communication among neurons. There is much evidence for glutamate as a neurotransmitter from auditory nerve fibers to cochlear nucleus neurons. Previous studies in rodents have examined effects of removal of auditory nerve input by cochlear ablation on levels, uptake and release of glutamate in cochlear nucleus subdivisions, as well as on glutamate receptors. Effects have also been reported on uptake and release of γ-aminobutyrate (GABA) and glycine, two other amino acids strongly implicated in cochlear nucleus synaptic transmission. We mapped the effects of cochlear ablation on the levels of amino acids, including glutamate, GABA, glycine, aspartate, glutamine, taurine, serine, threonine, and arginine, in microscopic subregions of the rat cochlear nucleus. Submicrogram-size samples microdissected from freeze-dried brainstem sections were assayed for amino acid levels by high performance liquid chromatography. After cochlear ablation, glutamate and aspartate levels decreased by 2 days in regions receiving relatively dense innervation from the auditory nerve, whereas the levels of most other amino acids increased. The results are consistent with a close association of glutamate and aspartate with auditory nerve fibers and of other amino acids with other neurons and glia in the cochlear nucleus. A consistent decrease of GABA level in the lateral superior olive could be consistent with a role in some lateral olivocochlear neurons. The results are compared with those obtained with the same methods for the rat vestibular nerve root and nuclei after vestibular ganglionectomy.

  1. The Genome Sequence of the Highly Acetic Acid-Tolerant Zygosaccharomyces bailii-Derived Interspecies Hybrid Strain ISA1307, Isolated From a Sparkling Wine Plant

    PubMed Central

    Mira, Nuno P.; Münsterkötter, Martin; Dias-Valada, Filipa; Santos, Júlia; Palma, Margarida; Roque, Filipa C.; Guerreiro, Joana F.; Rodrigues, Fernando; Sousa, Maria João; Leão, Cecília; Güldener, Ulrich; Sá-Correia, Isabel

    2014-01-01

    In this work, it is described the sequencing and annotation of the genome of the yeast strain ISA1307, isolated from a sparkling wine continuous production plant. This strain, formerly considered of the Zygosaccharomyces bailii species, has been used to study Z. bailii physiology, in particular, its extreme tolerance to acetic acid stress at low pH. The analysis of the genome sequence described in this work indicates that strain ISA1307 is an interspecies hybrid between Z. bailii and a closely related species. The genome sequence of ISA1307 is distributed through 154 scaffolds and has a size of around 21.2 Mb, corresponding to 96% of the genome size estimated by flow cytometry. Annotation of ISA1307 genome includes 4385 duplicated genes (∼90% of the total number of predicted genes) and 1155 predicted single-copy genes. The functional categories including a higher number of genes are ‘Metabolism and generation of energy’, ‘Protein folding, modification and targeting’ and ‘Biogenesis of cellular components’. The knowledge of the genome sequence of the ISA1307 strain is expected to contribute to accelerate systems-level understanding of stress resistance mechanisms in Z. bailii and to inspire and guide novel biotechnological applications of this yeast species/strain in fermentation processes, given its high resilience to acidic stress. The availability of the ISA1307 genome sequence also paves the way to a better understanding of the genetic mechanisms underlying the generation and selection of more robust hybrid yeast strains in the stressful environment of wine fermentations. PMID:24453040

  2. Dietary docosahexaenoic acid and docosapentaenoic acid ameliorate amyloid-beta and tau pathology via a mechanism involving presenilin 1 levels.

    PubMed

    Green, Kim N; Martinez-Coria, Hilda; Khashwji, Hasan; Hall, Eileen B; Yurko-Mauro, Karin A; Ellis, Lorie; LaFerla, Frank M

    2007-04-18

    The underlying cause of sporadic Alzheimer disease (AD) is unknown, but a number of environmental and genetic factors are likely to be involved. One environmental factor that is increasingly being recognized as contributing to brain aging is diet, which has evolved markedly over modern history. Here we show that dietary supplementation with docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, in the 3xTg-AD mouse model of AD reduced the intraneuronal accumulation of both amyloid-beta (Abeta) and tau. In contrast, combining DHA with n-6 fatty acids, either arachidonic acid or docosapentaenoic acid (DPAn-6), diminished the efficacy of DHA over a 12 month period. Here we report the novel finding that the mechanism accounting for the reduction in soluble Abeta was attributable to a decrease in steady-state levels of presenilin 1, and not to altered processing of the amyloid precursor protein by either the alpha- or beta-secretase. Furthermore, the presence of DPAn-6 in the diet reduced levels of early-stage phospho-tau epitopes, which correlated with a reduction in phosphorylated c-Jun N-terminal kinase, a putative tau kinase. Collectively, these results suggest that DHA and DPAn-6 supplementations could be a beneficial natural therapy for AD.

  3. Amino acid limitation induces down-regulation of WNT5a at transcriptional level

    SciTech Connect

    Wang Zuguang; Chen Hong

    2009-01-23

    An aberrant WNT signaling contributes to the development and progression of multiple cancers. WNT5a is one of the WNT signaling molecules. This study was designed to test the hypothesis that amino acid deprivation induces changes in the WNT signaling pathway in colon cancer cells. Results showed that targets of the amino acid response pathway, ATF3 and p21, were induced in the human colon cancer cell line SW480 during amino acid limitation. There was a significant decrease in the WNT5a mRNA level following amino acid deprivation. The down-regulation of WNT5a mRNA by amino acid deprivation is not due to mRNA destabilization. There is a reduction of nuclear {beta}-catenin protein level by amino acid limitation. Under amino acid limitation, phosphorylation of ERK1/2 was increased and the blockage of ERK1/2 by the inhibitor U0126 partially restored WNT5a mRNA level. In conclusion, amino acid limitation in colon cancer cells induces phosphorylation of ERK1/2, which then down-regulates WNT5a expression.

  4. Solvent extraction in the treatment of acidic high-level liquid waste : where do we stand?

    SciTech Connect

    Horwitz, E. P.; Schulz, W. W.

    1998-06-18

    During the last 15 years, a number of solvent extraction/recovery processes have been developed for the removal of the transuranic elements, {sup 90}Sr and {sup 137}Cs from acidic high-level liquid waste. These processes are based on the use of a variety of both acidic and neutral extractants. This chapter will present an overview and analysis of the various extractants and flowsheets developed to treat acidic high-level liquid waste streams. The advantages and disadvantages of each extractant along with comparisons of the individual systems are discussed.

  5. Evaluation of circulating levels and renal clearance of natural amino acids in patients with Cushing's disease.

    PubMed

    Faggiano, A; Pivonello, R; Melis, D; Alfieri, R; Filippella, M; Spagnuolo, G; Salvatore, F; Lombardi, G; Colao, A

    2002-02-01

    Although the hypercortisolism-induced impairment of protein homeostasis is object of several studies, a detailed evaluation of the complete amino acid profile of patients with Cushing's syndrome (CS) has never been performed. The aim of the current open transversal controlled study was to evaluate serum and urinary concentrations as well as renal clearance of the complete series of natural amino acids and their relationship with glucose tolerance in patients with Cushing's disease (CD). Twenty patients with CD (10 active and 10 cured) and 20 sex- and age-matched healthy controls entered the study. Measurement of serum and urinary levels of the complete series of natural amino acids was performed in all patients analyzed by cationic exchange high performance liquid cromatography (HPLC) after 2 weeks of a standardized protein intake regimen. The renal clearance (renal excretion rate) of each amino acid was calculated on the basis of the serum and urinary concentrations of creatinine and the specific amino acid. Fasting glucose and insulin levels, glucose and insulin response to standard glucose load, insulinogenic and homeostasis model insulin resistance (Homa-R) indexes were also evaluated and correlated to the circulating levels and renal clearances of each amino acid. Significantly higher serum (p<0.01) and urinary (p<0.05) levels of alanine and cystine, lower serum and higher urinary levels of leucine, isoleucine and valine (p<0.05) and higher renal excretion rates of leucine, isoleucine and valine (p<0.01) were found in patients with active CD than in patients cured from the disease and in controls. No difference was found between cured patients and controls. Creatinine clearance was similar in active and cured patients and in controls. In patients with active CD, urinary cortisol levels were significantly correlated to urinary cystine levels (r=0.85; p<0.01) and renal excretion rate of leucine (r=-0.76; p<0.05), isoleucine (r=-0.76; p<0.05) and valine (r=-0

  6. Genetic loci associated with circulating levels of very long-chain saturated fatty acids[S

    PubMed Central

    Lemaitre, Rozenn N.; King, Irena B.; Kabagambe, Edmond K.; Wu, Jason H. Y.; McKnight, Barbara; Manichaikul, Ani; Guan, Weihua; Sun, Qi; Chasman, Daniel I.; Foy, Millennia; Wang, Lu; Zhu, Jingwen; Siscovick, David S.; Tsai, Michael Y.; Arnett, Donna K.; Psaty, Bruce M.; Djousse, Luc; Chen, Yii-Der I.; Tang, Weihong; Weng, Lu-Chen; Wu, Hongyu; Jensen, Majken K.; Chu, Audrey Y.; Jacobs, David R.; Rich, Stephen S.; Mozaffarian, Dariush; Steffen, Lyn; Rimm, Eric B.; Hu, Frank B.; Ridker, Paul M.; Fornage, Myriam; Friedlander, Yechiel

    2015-01-01

    Very long-chain saturated fatty acids (VLSFAs) are saturated fatty acids with 20 or more carbons. In contrast to the more abundant saturated fatty acids, such as palmitic acid, there is growing evidence that circulating VLSFAs may have beneficial biological properties. Whether genetic factors influence circulating levels of VLSFAs is not known. We investigated the association of common genetic variation with plasma phospholipid/erythrocyte levels of three VLSFAs by performing genome-wide association studies in seven population-based cohorts comprising 10,129 subjects of European ancestry. We observed associations of circulating VLSFA concentrations with common variants in two genes, serine palmitoyl-transferase long-chain base subunit 3 (SPTLC3), a gene involved in the rate-limiting step of de novo sphingolipid synthesis, and ceramide synthase 4 (CERS4). The SPTLC3 variant at rs680379 was associated with higher arachidic acid (20:0 , P = 5.81 × 10−13). The CERS4 variant at rs2100944 was associated with higher levels of 20:0 (P = 2.65 × 10−40) and in analyses that adjusted for 20:0, with lower levels of behenic acid (P = 4.22 × 10−26) and lignoceric acid (P = 3.20 × 10−21). These novel associations suggest an inter-relationship of circulating VLSFAs and sphingolipid synthesis. PMID:25378659

  7. A knowledge engineering approach to recognizing and extracting sequences of nucleic acids from scientific literature.

    PubMed

    García-Remesal, Miguel; Maojo, Victor; Crespo, José

    2010-01-01

    In this paper we present a knowledge engineering approach to automatically recognize and extract genetic sequences from scientific articles. To carry out this task, we use a preliminary recognizer based on a finite state machine to extract all candidate DNA/RNA sequences. The latter are then fed into a knowledge-based system that automatically discards false positives and refines noisy and incorrectly merged sequences. We created the knowledge base by manually analyzing different manuscripts containing genetic sequences. Our approach was evaluated using a test set of 211 full-text articles in PDF format containing 3134 genetic sequences. For such set, we achieved 87.76% precision and 97.70% recall respectively. This method can facilitate different research tasks. These include text mining, information extraction, and information retrieval research dealing with large collections of documents containing genetic sequences.

  8. A knowledge engineering approach to recognizing and extracting sequences of nucleic acids from scientific literature.

    PubMed

    García-Remesal, Miguel; Maojo, Victor; Crespo, José

    2010-01-01

    In this paper we present a knowledge engineering approach to automatically recognize and extract genetic sequences from scientific articles. To carry out this task, we use a preliminary recognizer based on a finite state machine to extract all candidate DNA/RNA sequences. The latter are then fed into a knowledge-based system that automatically discards false positives and refines noisy and incorrectly merged sequences. We created the knowledge base by manually analyzing different manuscripts containing genetic sequences. Our approach was evaluated using a test set of 211 full-text articles in PDF format containing 3134 genetic sequences. For such set, we achieved 87.76% precision and 97.70% recall respectively. This method can facilitate different research tasks. These include text mining, information extraction, and information retrieval research dealing with large collections of documents containing genetic sequences. PMID:21096556

  9. Identification of novel rice low phytic acid mutations via TILLING by sequencing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate or InsP6) accounts for 75-85% of the total phosphorus in seeds. Low phytic acid (lpa) mutants exhibit decreases in seed InsP6 with corresponding increases in inorganic P which, unlike phytic acid P, is readily utilized by humans and monogastric ...

  10. 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.

  11. Comparative Indole-3-Acetic Acid Levels in the Slender Pea and Other Pea Phenotypes 1

    PubMed Central

    Law, David M.; Davies, Peter J.

    1990-01-01

    Free indole-3-acetic acid levels were measured by gas chromatography-mass spectrometry in three ultra-tall `slender' Pisum sativum L. lines differing in gibberellin content. Measurements were made for apices and stem elongation zones of light-grown plants and values were compared with wild-type, dwarf, and nana phenotypes in which internode length is genetically regulated, purportedly via the gibberellin level. Indole-3-acetic acid levels of growing stems paralleled growth rates in all lines, and were high in all three slender genotypes. Growth was inhibited by p-chlorophenoxyisobutyric acid, demonstrating the requirement of auxin activity for stem elongation, and also by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. It is concluded that the slender phenotype may arise from constant activation of a gibberellin receptor or transduction chain event leading directly or indirectly to elevated levels of indole-3-acetic acid, and that increased indole-3-acetic acid levels are a significant factor in the promotion of stem elongation. PMID:16667653

  12. Evaluation of a novel food composition database that includes glutamine and other amino acids derived from gene sequencing data

    PubMed Central

    Lenders, CM; Liu, S; Wilmore, DW; Sampson, L; Dougherty, LW; Spiegelman, D; Willett, WC

    2011-01-01

    Objectives To determine the content of glutamine in major food proteins. Subjects/Methods We used a validated 131-food item food frequency questionnaire (FFQ) to identify the foods that contributed the most to protein intake among 70 356 women in the Nurses’ Health Study (NHS, 1984). The content of glutamine and other amino acids in foods was calculated based on protein fractions generated from gene sequencing methods (Swiss Institute of Bioinformatics) and compared with data from conventional (USDA) and modified biochemical (Khun) methods. Pearson correlation coefficients were used to compare the participants’ dietary intakes of amino acids by sequencing and USDA methods. Results The glutamine content varied from 0.01 to to 9.49 g/100 g of food and contributed from 1 to to 33% of total protein for all FFQ foods with protein. When comparing the sequencing and Kuhn’s methods, the proportion of glutamine in meat was 4.8 vs 4.4%. Among NHS participants, mean glutamine intake was 6.84 (s.d.=2.19) g/day and correlation coefficients for amino acid between intakes assessed by sequencing and USDA methods ranged from 0.94 to 0.99 for absolute intake, −0.08 to 0.90 after adjusting for 100 g of protein, and 0.88 to 0.99 after adjusting for 1000 kcal. The between-person coefficient of variation of energy-adjusted intake of glutamine was 16%. Conclusions These data suggest that (1) glutamine content can be estimated from gene sequencing methods and (2) there is a reasonably wide variation in energy-adjusted glutamine intake, allowing for exploration of glutamine consumption and disease. PMID:19756030

  13. The amino acid sequences of the cytochromes c553 from Porphyridium cruentum and Aphanizomenon flos-aquae.

    PubMed

    Sprinkle, J R; Hermodson, M; Krogmann, D W

    1986-01-01

    The amino acid sequences of cytochrome c553 from the eukaryotic red alga Porphyridium cruentum and from the prokaryotic cyanobacterium Aphanizomenon flos-aquae have been determined from the tryptic and cyanogen bromide peptides. The results indicate that a charged region of these proteins has evolved with special rapidity to accomodate a rapid evolution of a binding site in the P700 electron acceptor complex.

  14. Untangling the Effect of Fatty Acid Addition at Species Level Revealed Different Transcriptional Responses of the Biogas Microbial Community Members.

    PubMed

    Treu, Laura; Campanaro, Stefano; Kougias, Panagiotis G; Zhu, Xinyu; Angelidaki, Irini

    2016-06-01

    In the present study, RNA-sequencing was used to elucidate the change of anaerobic digestion metatranscriptome after long chain fatty acids (oleate) exposure. To explore the general transcriptional behavior of the microbiome, the analysis was first performed on shotgun reads without considering a reference metagenome. As a second step, RNA reads were aligned on the genes encoded by the microbial community, revealing the expression of more than 51 000 different transcripts. The present study is the first research which was able to dissect the transcriptional behavior at a single species level by considering the 106 microbial genomes previously identified. The exploration of the metabolic pathways confirmed the importance of Syntrophomonas species in fatty acids degradation, and also highlighted the presence of protective mechanisms toward the long chain fatty acid effects in bacteria belonging to Clostridiales, Rykenellaceae, and in species of the genera Halothermothrix and Anaerobaculum. Additionally, an interesting transcriptional activation of the chemotaxis genes was evidenced in seven species belonging to Clostridia, Halothermothrix, and Tepidanaerobacter. Surprisingly, methanogens revealed a very versatile behavior different from each other, even among similar species of the Methanoculleus genus, while a strong increase of the expression level in Methanosarcina sp. was evidenced after oleate addition.

  15. Uric Acid Level and Erectile Dysfunction In Patients With Coronary Artery Disease

    PubMed Central

    Solak, Yalcin; Akilli, Hakan; Kayrak, Mehmet; Aribas, Alpay; Gaipov, Abduzhappar; Turk, Suleyman; Perez-Pozo, Santos E.; Covic, Adrian; McFann, Kim; Johnson, Richard J.; Kanbay, Mehmet

    2013-01-01

    Introduction Erectile dysfunction (ED) is a frequent complaint of elderly subjects, and is closely associated with endothelial dysfunction and cardiovascular disease. Uric acid is also associated with endothelial dysfunction, oxidative stress and cardiovascular disease, raising the hypothesis that an increased serum uric acid might predict erectile dysfunction in patients who are at risk for coronary artery disease. Aim To evaluate the association of serum uric acid levels with presence and severity of ED in patients presenting with chest pain of presumed cardiac origin. Methods This is a cross-sectional study of 312 adult male patients with suspected coronary artery disease who underwent exercise stress test (EST) for workup of chest pain and completed a sexual health inventory for men (SHIM) survey form to determine the presence and severity of ED. Routine serum biochemistry (and uric acid levels) were measured. Logistic regression analysis was used to assess risk factors for ED. Main Outcome Measures The short version of the international index of erectile function (IIEF-5) questionnaire diagnosed ED (cutoff score ≤21). Serum Uric acid levels were determined. Patients with chest pain of suspected cardiac origin underwent an exercise stress test. Results 149 of 312 (47.7%) male subjects had ED by survey criteria. Patients with ED were older and had more frequent CAD, hypertension, diabetes, and impaired renal function, and also had significantly higher levels of uric acid, fibrinogen, glucose, CRP, triglycerides compared with patients without ED. Uric acid levels were associated with ED by univariate analysis (OR = 1.36, p = 0.002); however, this association was not observed in multivariate analysis adjusted for eGFR. Conclusion Subjects presenting with chest pain of presumed cardiac origin are more likely to have ED if they have elevated uric acid levels. PMID:24433559

  16. Effects of aspartame and carbohydrate administration on human and rat plasma large neutral amino acid levels and rat brain amino acid and monoamine levels.

    PubMed

    Romano, M; Casacci, F; De Marchi, F; Pacei, T; Esteve, A; Lomuscio, G; Mennini, T; Salmona, M

    1989-01-01

    Thirty fasted human volunteers were given 0.83 and 8.3 mg aspartame/kg body weight alone, as part of a basal low carbohydrate meal (648 kcal, 10% carbohydrate) or as part of a high energy carbohydrate-rich meal (1290 kcal, 34% carbohydrate). Amino acid concentrations in plasma were determined before and 30, 60 and 180 min after the consumption of aspartame. Under these conditions, which mimic realistic aspartame consumption, aspartame had no significant effect on plasma concentration of any amino acid. In addition, the effect of aspartame alone or with carbohydrates on plasma and brain amino acid levels was studied in rats after acute or subacute (14 d) oral treatment. In subacute dosing experiments aspartame was included in the diet. Brain monoamine concentrations were also measured in the same animals. Plasma concentrations of large neutral amino acids were modified under acute conditions. In contrast, after subacute treatment no significant differences in plasma or brain amino acid concentrations or in brain monoamine concentrations were observed.

  17. First draft genome sequencing of indole acetic acid producing and plant growth promoting fungus Preussia sp. BSL10.

    PubMed

    Khan, Abdul Latif; Asaf, Sajjad; Khan, Abdur Rahim; Al-Harrasi, Ahmed; Al-Rawahi, Ahmed; Lee, In-Jung

    2016-05-10

    Preussia sp. BSL10, family Sporormiaceae, was actively producing phytohormone (indole-3-acetic acid) and extra-cellular enzymes (phosphatases and glucosidases). The fungus was also promoting the growth of arid-land tree-Boswellia sacra. Looking at such prospects of this fungus, we sequenced its draft genome for the first time. The Illumina based sequence analysis reveals an approximate genome size of 31.4Mbp for Preussia sp. BSL10. Based on ab initio gene prediction, total 32,312 coding sequences were annotated consisting of 11,967 coding genes, pseudogenes, and 221 tRNA genes. Furthermore, 321 carbohydrate-active enzymes were predicted and classified into many functional families. PMID:26995610

  18. The amino acid sequence of GTP:AMP phosphotransferase from beef-heart mitochondria. Extensive homology with cytosolic adenylate kinase.

    PubMed

    Wieland, B; Tomasselli, A G; Noda, L H; Frank, R; Schulz, G E

    1984-09-01

    The amino acid sequence of GTP:AMP phosphotransferase (AK3) from beef-heart mitochondria has been determined, except for one segment of about 33 residues in the middle of the polypeptide chain. The established sequence has been unambiguously aligned to the sequence of cytosolic ATP:AMP phosphotransferase (AK1) from pig muscle, allowing for six insertions and deletions. With 30% of all aligned residues being identical, the homology between AK3 and AK1 is well established. As derived from the known three-dimensional structure of AK1, the missing segment is localized at a small surface area of the molecule, far apart from the active center. The pattern of conserved residues demonstrates that earlier views on substrate binding have to be modified. The observation of three different consecutive N-termini indicates enzyme processing.

  19. Fusion protein predicted amino acid sequence of the first US avian pneumovirus isolate and lack of heterogeneity among other US isolates.

    PubMed

    Seal, B S; Sellers, H S; Meinersmann, R J

    2000-02-01

    Avian pneumovirus (APV) was first isolated from turkeys in the west-central US following emergence of turkey rhinotracheitis (TRT) during 1996. Subsequently, several APV isolates were obtained from the north-central US. Matrix (M) and fusion (F) protein genes of these isolates were examined for sequence heterogeneity and compared with European APV subtypes A and B. Among US isolates the M gene shared greater than 98% nucleotide sequence identity with only one nonsynonymous change occurring in a single US isolate. Although the F gene among US APV isolates shared 98% nucleotide sequence identity, nine conserved substitutions were detected in the predicted amino acid sequence. The predicted amino acid sequence of the US APV isolate's F protein had 72% sequence identity to the F protein of APV subtype A and 71% sequence identity to the F protein of APV subtype B. This compares with 83% sequence identity between the APV subtype A and B predicted amino acid sequences of the F protein. The US isolates were phylogenetically distinguishable from their European counterparts based on F gene nucleotide or predicted amino acid sequences. Lack of sequence heterogeneity among US APV subtypes indicates these viruses have maintained a relatively stable population since the first outbreak of TRT. Phylogenetic analysis of the F protein among APV isolates supports classification of US isolates as a new APV subtype C.

  20. Amino acid sequence and glycosylation of functional unit RtH2-e from Rapana thomasiana (gastropod) hemocyanin.

    PubMed

    Stoeva, Stanka; Idakieva, Krasimira; Betzel, Christian; Genov, Nicolay; Voelter, Wolfgang

    2002-03-15

    The complete amino acid sequence of Rapana thomasiana hemocyanin functional unit RtH2-e was determined by direct sequencing and matrix-assisted laser desorption ionization mass spectrometry of peptides obtained by cleavage with EndoLysC proteinase, chymotrypsin, and trypsin. The single-polypeptide chain of RtH2-e consists of 413 amino acid residues and contains two consensus sequences NXS/T (positions 11-19 and 127-129), potential sites for N-glycosylation. Monosaccharide analysis of RtH2-e revealed a carbohydrate content of about 1.1% and the presence of xylose, fucose, mannose, and N-acetylglucosamine, demonstrating that only N-linked carbohydrate chains of high-mannose type seem to be present. On basis of the monosaccharide composition and MALDI-MS analysis of native and PNGase-F-treated chymotryptic glycopeptide fragment of RtH2-e the oligosaccharide Man(5)GlcNAc(2), attached to Asn(127), is suggested. Multiple sequence alignments with other molluscan hemocyanin e functional units revealed an identity of 63% to the cephalopod Octopus dofleini and of 69% to the gastropod Haliotis tuberculata. The present results are discussed in view of the recently determined X-ray structure of the functional unit g of the O. dofleini hemocyanin. PMID:11888200

  1. Amino acid sequence and domain structure of entactin. Homology with epidermal growth factor precursor and low density lipoprotein receptor

    PubMed Central

    1988-01-01

    Entactin (nidogen), a 150-kD sulfated glycoprotein, is a major component of basement membranes and forms a highly stable noncovalent complex with laminin. The complete amino acid sequence of mouse entactin has been derived from sequencing of cDNA clones. The 5.9-kb cDNA contains a 3,735-bp open reading frame followed by a 3'- untranslated region of 2.2 kb. The open reading frame encodes a 1,245- residue polypeptide with an unglycosylated Mr of 136,500, a 28-residue signal peptide, two Asn-linked glycosylation sites, and two potential Ca2+-binding sites. Analysis of the deduced amino acid sequence predicts that the molecule consists of two globular domains of 70 and 36 kD separated by a cysteine-rich domain of 28 kD. The COOH-terminal globular domain shows homology to the EGF precursor and the low density lipoprotein receptor. Entactin contains six EGF-type cysteine-rich repeat units and one copy of a cysteine-repeat motif found in thyroglobulin. The Arg-Gly-Asp cell recognition sequence is present in one of the EGF-type repeats, and a synthetic peptide from the putative cell-binding site of entactin was found to promote the attachment of mouse mammary tumor cells. PMID:3264556

  2. Amino acid sequences of lysozymes newly purified from invertebrates imply wide distribution of a novel class in the lysozyme family.

    PubMed

    Ito, Y; Yoshikawa, A; Hotani, T; Fukuda, S; Sugimura, K; Imoto, T

    1999-01-01

    Lysozymes were purified from three invertebrates: a marine bivalve, a marine conch, and an earthworm. The purified lysozymes all showed a similar molecular weight of 13 kDa on SDS/PAGE. Their N-terminal sequences up to the 33rd residue determined here were apparently homologous among them; in addition, they had a homology with a partial sequence of a starfish lysozyme which had been reported before. The complete sequence of the bivalve lysozyme was determined by peptide mapping and subsequent sequence analysis. This was composed of 123 amino acids including as many as 14 cysteine residues and did not show a clear homology with the known types of lysozymes. However, the homology search of this protein on the protein or nucleic acid database revealed two homologous proteins. One of them was a gene product, CELF22 A3.6 of C. elegans, which was a functionally unknown protein. The other was an isopeptidase of a medicinal leech, named destabilase. Thus, a new type of lysozyme found in at least four species across the three classes of the invertebrates demonstrates a novel class of protein/lysozyme family in invertebrates. The bivalve lysozyme, first characterized here, showed extremely high protein stability and hen lysozyme-like enzymatic features.

  3. Amino acid sequences of lysozymes newly purified from invertebrates imply wide distribution of a novel class in the lysozyme family.

    PubMed

    Ito, Y; Yoshikawa, A; Hotani, T; Fukuda, S; Sugimura, K; Imoto, T

    1999-01-01

    Lysozymes were purified from three invertebrates: a marine bivalve, a marine conch, and an earthworm. The purified lysozymes all showed a similar molecular weight of 13 kDa on SDS/PAGE. Their N-terminal sequences up to the 33rd residue determined here were apparently homologous among them; in addition, they had a homology with a partial sequence of a starfish lysozyme which had been reported before. The complete sequence of the bivalve lysozyme was determined by peptide mapping and subsequent sequence analysis. This was composed of 123 amino acids including as many as 14 cysteine residues and did not show a clear homology with the known types of lysozymes. However, the homology search of this protein on the protein or nucleic acid database revealed two homologous proteins. One of them was a gene product, CELF22 A3.6 of C. elegans, which was a functionally unknown protein. The other was an isopeptidase of a medicinal leech, named destabilase. Thus, a new type of lysozyme found in at least four species across the three classes of the invertebrates demonstrates a novel class of protein/lysozyme family in invertebrates. The bivalve lysozyme, first characterized here, showed extremely high protein stability and hen lysozyme-like enzymatic features. PMID:9914527

  4. Preterm Birth is Associated with Higher Uric Acid Levels in Adolescents

    PubMed Central

    Washburn, Lisa K.; Nixon, Patricia A.; Russell, Gregory B.; Snively, Beverly M.; O’Shea, T. Michael

    2015-01-01

    Objective To compare serum uric acid levels in adolescents born prematurely and adolescents born at term and to assess the correlation between serum uric acid and blood pressure in those born prematurely. Study design In this observational cohort study, 124 adolescents born prematurely and 44 adolescents born at term were studied at 14 years of age. Multivariate analyses were used to describe the relationship of premature birth to serum uric acid, while adjusting for confounding variables. Pearson correlation was used to describe the relationship between uric acid and systolic blood pressure among those born prematurely. Results Adjusting for race, sex, maternal hypertension and fetal growth, preterm adolescents had higher serum uric acid levels than adolescents born at term [adjusted mean difference (95% confidence interval: 0.46 (0.10, 0.81) mg/dL, 27.4 (6, 48.2) μmol/L, p=0.012]. Among those born prematurely, uric acid was positively correlated with systolic blood pressure [Pearson correlation coefficient: 0.29 (0.12, 0.44; p= 0.0013). Conclusions Serum uric acid levels are higher in adolescents born prematurely than in those born at term and this difference could contribute to higher blood pressure among individuals born prematurely. PMID:25868431

  5. Two Levels of Caffeine Ingestion on Blood Lactate and Free Fatty Acid Responses during Incremental Exercise.

    ERIC Educational Resources Information Center

    McNaughton, Lars

    1987-01-01

    Research was conducted to determine the effects of two doses of caffeine on the lactate threshold and also to examine the effects on substrate utilization during incremental cycle ergometry. Results found that caffeine increased heart rates and free fatty acid levels for all workloads and decreased blood lactate levels at some of the workloads.…

  6. Serum homocysteine, vitamin B12, folic acid levels and methylenetetrahydrofolate reductase (MTHFR) gene polymorphism in vitiligo.

    PubMed

    Yasar, Ali; Gunduz, Kamer; Onur, Ece; Calkan, Mehmet

    2012-01-01

    The aim of this study was to determine serum vitamin B12, folic acid and homocysteine (Hcy) levels as well as MTHFR (C677, A1298C) gene polymorphisms in patients with vitiligo, and to compare the results with healthy controls. Forty patients with vitiligo and 40 age and sex matched healthy subjects were studied. Serum vitamin B12 and folate levels were determined by enzyme-linked immunosorbent assay. Plasma Hcy levels and MTHFR polymorphisms were determined by chemiluminescence and real time PCR methods, respectively. Mean serum vitamin B12 and Hcy levels were not significantly different while folic acid levels were significantly lower in the control group. There was no significant relationship between disease activity and vitamin B12, folic acid and homocystein levels. No significant difference in C677T gene polymorphism was detected. Heterozygote A1298C gene polymorphism in the patient group was statistically higher than the control group. There was no significant relationship between MTHFR gene polymorphisms and vitamin B12, folic acid and homocysteine levels. In conclusion, vitamin B12, folate and Hcy levels are not altered in vitiligo and MTHFR gene mutations (C677T and A1298C) do not seem to create susceptibility for vitiligo. PMID:22846211

  7. Relationship between body weight and level of fat supplementation on fatty acid digestion in feedlot cattle.

    PubMed

    Plascencia, A; Mendoza, G D; Vásquez, C; Zinn, R A

    2003-11-01

    Eight Holstein steers with cannulas in the rumen and proximal duodenum were used in a split-plot design experiment to evaluate the interaction of body weight (175 vs. 370 kg) and level of fat supplementation (0, 3, 6, and 9% yellow grease) on characteristics of digestion and feeding value of fat in finishing diets. Dry matter intake was restricted to 2% of BW. There were no interactions between BW and level of fat supplementation (P > 0.10) on ruminal or total-tract digestion. Level of supplemental fat decreased (linear, P < 0.01) ruminal digestion of OM and NDF, and increased (linear, P < 0.05) ruminal N efficiency. There were no treatment effects (P > 0.10) on postruminal digestion of OM, NDF, and N. There tended to be an interaction (P < 0.10) between BW and level of fat supplementation on postruminal starch digestion. Increasing level of fat supplementation increased postruminal digestion of starch in heavier steers but did not affect starch digestion in lighter steers. There were no interactions (P > 0.10) between BW and level of fat supplementation on postruminal fatty acid digestion. Increasing level of fat supplementation decreased (linear, P < 0.01) postruminal fatty acid digestion, which was due to a decreased (linear, P < 0.01) postruminal digestion of C16:0 and C18:0. Supplemental fat decreased (linear, P < 0.01) total-tract digestion of OM and NDF. The estimated NEm (Mcal/kg) of yellow grease averaged (linear, P < 0.01) 6.02, 5.70, and 5.06 for the 3, 6, and 9% of level supplementation, respectively. We conclude that intestinal fatty acid digestion (FAD, %) is a predictable function (r2 = 0.89; P < 0.01) of total fatty acid intake per unit body weight (FAI, g/kg BW): FAD = 87.560 - 8.591FAI. Depressions in fatty acid digestion with increasing level of intake were due primarily to decreased intestinal absorption of palmitic and stearic acid. Level of fatty acids intake did not appreciably affect intestinal absorption of unsaturated fatty acid. Changes

  8. Drilling and dating New Jersey oligocene-miocene sequences: Ice volume, global sea level, and Exxon records

    SciTech Connect

    Miller, K.G.; Mountain, G.S.

    1996-02-23

    Oligocene to middle Miocene sequence boundaries on the New Jersey coastal plain (Ocean Drilling Project Leg 150X) and continental slope (Ocean Drilling Project Leg 150) were dated by integrating strontium isotopic stratigraphy, magnetostratigraphy, and biostratigraphy (planktonic foraminifera, nannofossils, dinocysts, and diatoms). The ages of coastal plain unconformities and slope seismic reflectors (unconformities or stratal breaks with no discernible hiatuses) match the ages of global {delta}{sup 18}O increases (inferred glacioeustatic lowerings) measured in deep-sea sites. These correlations confirm a causal link between coastal plain and slope sequence boundaries: both formed during global sea-level lowerings. The ages of New Jersey sequence boundaries and global {delta}{sup 18}O increases also correlate well within the Exxon Production Research sea-level records of Haq et al. and Vail et al., validating and refining their compilations. 33 refs., 2 figs., 1 tab.

  9. Evaluation of serum sialic acid, fucose levels and their ratio in oral squamous cell carcinoma

    PubMed Central

    Chinnannavar, Sangamesh Ningappa; Ashok, Lingappa; Vidya, Kodige Chandrashekhar; Setty, Sunil Mysore Kantharaja; Narasimha, Guru Eraiah; Garg, Ranjana

    2015-01-01

    Background: Detection of cancer at the early stage is of utmost importance to decrease the morbidity and mortality of the disease. Apart from the conventional biopsy, minimally invasive methods like serum evaluation are used for screening large populations. Thus, this study aimed to estimate serum levels of sialic acid and fucose and their ratio in oral cancer patients and in healthy control group to evaluate their role in diagnosis. Materials and Methods: Serum samples were collected from 52 healthy controls (group I) and 52 squamous cell carcinoma patients (group II). Estimation of serum levels of sialic acid and fucose and their ratio was performed. This was correlated histopathologically with the grades of carcinoma. Statistical analysis was done by using analysis of variance (ANOVA) test and unpaired “t” test. Results: Results showed that serum levels of sialic acid and fucose were significantly higher in oral cancer patients compared to normal healthy controls (P < 0.001). The sialic acid to fucose ratio was significantly lower in cancer patients than in normal controls (P < 0.01). However, comparison with histological grading, habits, gender, and age group did not show any significant result. Conclusion: The mean serum sialic acid and fucose levels showed an increasing trend from controls to malignant group and their corresponding ratio showed decreasing trend from controls to malignant group. The ratio of sialic acid to fucose can be a useful diagnostic aid for oral cancer patients. PMID:26759796

  10. Buffalo (Bubalus bubalis) interleukin-2: sequence analysis reveals high nucleotide and amino acid identity with interleukin-2 of cattle and other ruminants.

    PubMed

    Sreekumar, E; Premraj, A; Saravanakumar, M; Rasool, T J

    2002-08-01

    A 4400-bp genomic sequence and a 332-bp truncated cDNA sequence of the interleukin-2 (IL-2) gene of Indian water buffalo (Bubalus bubalis) were amplified by polymerase chain reaction and cloned. The coding sequence of the buffalo IL-2 gene was assembled from the 5' end of the genomic clone and the truncated cDNA clone. This sequence had 98.5% nucleotide identity and 98% amino acid identity with cattle IL-2. Three amino acid substitutions were observed at positions 63, 124 and 135. Comparison of the predicted protein structure of buffalo IL-2 with that of human and cattle IL-2 did not reveal significant differences. The putative amino acids responsible for IL-2 receptor binding were conserved in buffalo, cattle and human IL-2. The amino acid sequence of buffalo IL-2 also showed very high identity with that of other ruminants, indicating functional cross-reactivity.

  11. 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.

  12. Amino acid sequence of the alpha subunit of human leukocyte adhesion receptor Mo1 (complement receptor type 3)

    PubMed Central

    1988-01-01

    Mo1 (complement receptor type 3, CR3; CD11b/CD18) is an adhesion- promoting human leukocyte surface membrane heterodimer (alpha subunit 155 kD [CD11b] noncovalently linked to a beta subunit of 95 kD [CD18]). The complete amino acid sequence deduced from cDNA of the human alpha subunit is reported. The protein consists of 1,136 amino acids with a long amino-terminal extracytoplasmic domain, a 26-amino acid hydrophobic transmembrane segment, and a 19-carboxyl-terminal cytoplasmic domain. The extracytoplasmic region has three putative Ca2+- binding domains with good homology and one with weak homology to the "lock washer" Ca2+-binding consensus sequence. These metal-binding domains explain the divalent cation-dependent functions mediated by Mo1. The alpha subunit is highly homologous to the alpha subunit of leukocyte p150,95 and to a lesser extent, to the alpha subunit of other "integrin" receptors such as fibronectin, vitronectin, and platelet IIb/IIIa receptors in humans and position-specific antigen-2 (PS2) in Drosophila. Mo1 alpha, like p150, contains a unique 187-amino acid stretch NH2-terminal to the metal-binding domains. This region could be involved in some of the specific functions mediated by these leukocyte glycoproteins. PMID:2454931

  13. Childrens' learning and behaviour and the association with cheek cell polyunsaturated fatty acid levels.

    PubMed

    Kirby, A; Woodward, A; Jackson, S; Wang, Y; Crawford, M A

    2010-01-01

    Increasing interest in the role of omega-3 fatty acids in relation to neurodevelopmental disorders (e.g. ADHD, dyslexia, autism) has occurred as a consequence of some international studies highlighting this link. In particular, some studies have shown that children with ADHD may have lower concentrations of polyunsaturated fatty acids (PUFAs), particularly omega-3, in their red blood cells and plasma, and that supplementation with omega-3 fatty acids may alleviate behavioural symptoms in this population. However, in order to compare levels it seems appropriate to establish fatty acid levels in a mainstream school aged population and if levels relate to learning and behaviour. To date no study has established this. For this study, cheek cell samples from 411 typically developing school children were collected and analysed for PUFA content, in order to establish the range in this population. In addition, measures of general classroom attention and behaviour were assessed in these children by teachers and parents. Cognitive performance tests were also administered in order to explore whether an association between behaviour and/or cognitive performance and PUFA levels exists. Relationships between PUFA levels and socio-economic status were also explored. Measures of reading, spelling and intelligence did not show any association with PUFA levels, but some associations were noted with the level of omega-3 fatty acids and teacher and parental reports of behaviour, with some evidence that higher omega-3 levels were associated with decreased levels of inattention, hyperactivity, emotional and conduct difficulties and increased levels of prosocial behaviour. These findings are discussed in relation to previous findings from omega-3 supplementation studies with children. PMID:20172688

  14. Evaluation of Hanford high level waste vitrification chemistry for an NCAW simulant -- FY 1994: Potential exothermic reactions in the presence of formic acid, glycolic acid, and oxalic acid

    SciTech Connect

    Sills, J.A.

    1995-07-01

    A potential for an uncontrollable exothermic reaction between nitrate and organic salts during preparation of a high level waste melter feed has been identified. In order to examine this potential more closely, the thermal behavior of simulated neutralized current acid waste (NCAW) treated with various organic reductants was studied. Differential scanning calorimetry (DSC) measurements were collected on simulated waste samples and their supernates treated with organics. Organic reductants used were formic acid, glycolic acid, and oxalic acid. For comparison, samples of untreated simulant and untreated simulant with added noble metals were tested. When heated, untreated simulant samples both with and without noble metals showed no exothermic behavior. All of the treated waste simulant samples showed exothermic behavior. Onset temperatures of exothermic reactions were 120 C to 210 C. Many onset temperatures, particularly those for formic acid treated samples, are well below 181 C, the estimated maximum steam coil temperature (considered to be a worst case maximum temperature for chemical process tank contents). The enthalpies of the reactions were {minus}180 {times} 10{sup {minus}3} J/Kg supernate ({minus}181 J/g) for the oxalic acid treated simulant supernate to {minus}1,150 {times} 10{sup {minus}3} J/Kg supernate ({minus}1,153 J/g) for the formic acid treated simulant supernate.

  15. Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids

    PubMed Central

    Kim, Ki-Hyun; Nielsen, Peter E.; Glazer, Peter M.

    2007-01-01

    Sequence-specific DNA-binding molecules such as triple helix-forming oligonucleotides (TFOs) provide a means for inducing site-specific mutagenesis and recombination at chromosomal sites in mammalian cells. However, the utility of TFOs is limited by the requirement for homopurine stretches in the target duplex DNA. Here, we report the use of pseudo-complementary peptide nucleic acids (pcPNAs) for intracellular gene targeting at mixed sequence sites. Due to steric hindrance, pcPNAs are unable to form pcPNA–pcPNA duplexes but can bind to complementary DNA sequences by Watson–Crick pairing via double duplex-invasion complex formation. We show that psoralen-conjugated pcPNAs can deliver site-specific photoadducts and mediate targeted gene modification within both episomal and chromosomal DNA in mammalian cells without detectable off-target effects. Most of the induced psoralen-pcPNA mutations were single-base substitutions and deletions at the predicted pcPNA-binding sites. The pcPNA-directed mutagenesis was found to be dependent on PNA concentration and UVA dose and required matched pairs of pcPNAs. Neither of the individual pcPNAs alone had any effect nor did complementary PNA pairs of the same sequence. These results identify pcPNAs as new tools for site-specific gene modification in mammalian cells without purine sequence restriction, thereby providing a general strategy for designing gene targeting molecules. PMID:17977869

  16. Acid mine drainage neutralization in a pilot sequencing batch reactor using limestone from a paper and pulp industry.

    PubMed

    Vadapalli, V R K; Zvimba, J N; Mathye, M; Fischer, H; Bologo, L

    2015-01-01

    This study investigated the implications of using two grades of limestone from a paper and pulp industry for neutralization of acid mine drainage (AMD) in a pilot sequencing batch reactor (SBR). In this regard, two grades of calcium carbonate were used to neutralize AMD in a SBR with a hydraulic retention time (including settling) of 100 min and a sludge retention time of 360 min, by simultaneously monitoring the Fe(II) removal kinetics and overall assessment of the AMD after treatment. The Fe(II) kinetics removal and overall AMD treatment were observed to be highly dependent on the limestone grade used, with Fe(II) completely removed to levels lower than 50 mg/L in cycle 1 after 30 min using high quality or pure paper and pulp limestone. On the contrary, the other grade limestone, namely waste limestone, could only achieve a similar Fe(II) removal efficiency after four cycles. It was also noticed that suspended solids concentration plays a significant role in Fe(II) removal kinetics. In this regard, using pure limestone from the paper and pulp industry will have advantages compared with waste limestone for AMD neutralization. It has significant process impacts for the SBR configuration as it allows one cycle treatment resulting in a significant reduction of the feed stock, with subsequent generation of less sludge during AMD neutralization. However, the use of waste calcium carbonate from the paper and pulp industry as a feed stock during AMD neutralization can achieve significant cost savings as it is cheaper than the pure limestone and can achieve the same removal efficiency after four cycles.

  17. Amino acid sequence homology among the 2-hydroxy acid dehydrogenases: mitochondrial and cytoplasmic malate dehydrogenases form a homologous system with lactate dehydrogenase.

    PubMed Central

    Birktoft, J J; Fernley, R T; Bradshaw, R A; Banaszak, L J

    1982-01-01

    The amino acid sequence of porcine heart mitochondrial malate dehydrogenase (mMDH; L-malate: NAD+ oxidoreductase, EC 1.1.1.37) has been compared with the sequences of six different lactate dehydrogenases (LDH; L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) and with the "x-ray" sequence of cytoplasmic malate dehydrogenase (sMDH). The main points are that (i) all three enzymes are homologous; (ii) invariant residues in the catalytic center of these enzymes include a histidine and an internally located aspartate that function as a proton relay system; (iii) numerous residues important to coenzyme binding are conserved, including several glycines and charged residues; and (iv) amino acid side chains present in the subunit interface common to the MDHs and LDHs appear to be better conserved than those in the protein interior. It is concluded that LDH, sMDH, and mMDH are derived from a common ancestral gene and probably have similar catalytic mechanisms. PMID:6959107

  18. Enzymatic generation of peptides flanked by basic amino acids to obtain MS/MS spectra with 2× sequence coverage

    PubMed Central

    Ebhardt, H Alexander; Nan, Jie; Chaulk, Steven G; Fahlman, Richard P; Aebersold, Ruedi

    2014-01-01

    RATIONALE Tandem mass (MS/MS) spectra generated by collision-induced dissociation (CID) typically lack redundant peptide sequence information in the form of e.g. b- and y-ion series due to frequent use of sequence-specific endopeptidases cleaving C- or N-terminal to Arg or Lys residues. METHODS Here we introduce arginyl-tRNA protein transferase (ATE, EC 2.3.2.8) for proteomics. ATE recognizes acidic amino acids or oxidized Cys at the N-terminus of a substrate peptide and conjugates an arginine from an aminoacylated tRNAArg onto the N-terminus of the substrate peptide. This enzymatic reaction is carried out under physiological conditions and, in combination with Lys-C/Asp-N double digest, results in arginylated peptides with basic amino acids on both termini. RESULTS We demonstrate that in vitro arginylation of peptides using yeast arginyl tRNA protein transferase 1 (yATE1) is a robust enzymatic reaction, specific to only modifying N-terminal acidic amino acids. Precursors originating from arginylated peptides generally have an increased protonation state compared with their non-arginylated forms. Furthermore, the product ion spectra of arginylated peptides show near complete 2× fragment ladders within the same MS/MS spectrum using commonly available electrospray ionization peptide fragmentation modes. Unexpectedly, arginylated peptides generate complete y- and c-ion series using electron transfer dissociation (ETD) despite having an internal proline residue. CONCLUSIONS We introduce a rapid enzymatic method to generate peptides flanked on either terminus by basic amino acids, resulting in a rich, redundant MS/MS fragment pattern. © 2014 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd. PMID:25380496

  19. Inverse Association Between Serum Uric Acid Levels and Alzheimer's Disease Risk.

    PubMed

    Du, Na; Xu, Donghua; Hou, Xu; Song, Xuejia; Liu, Cancan; Chen, Ying; Wang, Yangang; Li, Xin

    2016-05-01

    The association between Alzheimer's disease and uric acid levels had gained great interest in recent years, but there was still lack of definite evidence. A systematic review and meta-analysis of relevant studies was performed to comprehensively estimate the association. Relevant studies published before October 26, 2014, were searched in PubMed, Embase, and China Biology Medicine (CBM) databases. Study-specific data were combined using random-effects or fixed-effects models of meta-analysis according to between-study heterogeneity. Twenty-four studies (21 case-control and 3 cohort studies) were finally included into the meta-analysis. Those 21 case-control studies included a total of 1128 cases of Alzheimer's disease and 2498 controls without Alzheimer's disease. Those 3 cohort studies included a total of 7327 participants. Meta-analysis showed that patients with Alzheimer's disease had lower levels of uric acid than healthy controls (weighted mean difference (WMD) = -0.77 mg/dl, 95% CI -2.28 to -0.36, P = 0.0002). High serum uric acid levels were significantly associated with decreased risk of Alzheimer's disease (risk ratio (RR) = 0.66, 95% CI 0.52-0.85, P = 0.001). There was low risk of publication bias in the meta-analysis. There is an inverse association between serum uric acid levels and Alzheimer's disease. High serum uric acid level is a protective factor of Alzheimer's disease.

  20. Cenozoic global sea level, sequences, and the New Jersey transect: Results from coastal plain and continental slope drilling

    USGS Publications Warehouse

    Miller, K.G.; Mountain, Gregory S.; Browning, J.V.; Kominz, M.; Sugarman, P.J.; Christie-Blick, N.; Katz, M.E.; Wright, J.D.

    1998-01-01

    The New Jersey Sea Level Transect was designed to evaluate the relationships among global sea level (eustatic) change, unconformity-bounded sequences, and variations in subsidence, sediment supply, and climate on a passive continental margin. By sampling and dating Cenozoic strata from coastal plain and continental slope locations, we show that sequence boundaries correlate (within ??0.5 myr) regionally (onshore-offshore) and interregionally (New Jersey-Alabama-Bahamas), implicating a global cause. Sequence boundaries correlate with ??18O increases for at least the past 42 myr, consistent with an ice volume (glacioeustatic) control, although a causal relationship is not required because of uncertainties in ages and correlations. Evidence for a causal connection is provided by preliminary Miocene data from slope Site 904 that directly link ??18O increases with sequence boundaries. We conclude that variation in the size of ice sheets has been a primary control on the formation of sequence boundaries since ~42 Ma. We speculate that prior to this, the growth and decay of small ice sheets caused small-amplitude sea level changes (<20 m) in this supposedly ice-free world because Eocene sequence boundaries also appear to correlate with minor ??18O increases. Subsidence estimates (backstripping) indicate amplitudes of short-term (million-year scale) lowerings that are consistent with estimates derived from ??18O studies (25-50 m in the Oligocene-middle Miocene and 10-20 m in the Eocene) and a long-term lowering of 150-200 m over the past 65 myr, consistent with estimates derived from volume changes on mid-ocean ridges. Although our results are consistent with the general number and timing of Paleocene to middle Miocene sequences published by workers at Exxon Production Research Company, our estimates of sea level amplitudes are substantially lower than theirs. Lithofacies patterns within sequences follow repetitive, predictable patterns: (1) coastal plain sequences consist

  1. Phylogenetic analysis of evolutionary relationships of the planctomycete division of the domain bacteria based on amino acid sequences of elongation factor Tu.

    PubMed

    Jenkins, C; Fuerst, J A

    2001-05-01

    Sequences from the tuf gene coding for the elongation factor EF-Tu were amplified and sequenced from the genomic DNA of Pirellula marina and Isosphaera pallida, two species of bacteria within the order Planctomycetales. A near-complete (1140-bp) sequence was obtained from Pi. marina and a partial (759-bp) sequence was obtained for I. pallida. Alignment of the deduced Pi. marina EF-Tu amino acid sequence against reference sequences demonstrated the presence of a unique 11-amino acid sequence motif not present in any other division of the domain Bacteria. Pi. marina shared the highest percentage amino acid sequence identity with I. pallida but showed only a low percentage identity with other members of the domain Bacteria. This is consistent with the concept of the planctomycetes as a unique division of the Bacteria. Neither primary sequence comparison of EF-Tu nor phylogenetic analysis supports any close relationship between planctomycetes and the chlamydiae, which has previously been postulated on the basis of 16S rRNA. Phylogenetic analysis of aligned EF-Tu amino acid sequences performed using distance, maximum-parsimony, and maximum-likelihood approaches yielded contradictory results with respect to the position of planctomycetes relative to other bacteria. It is hypothesized that long-branch attraction effects due to unequal evolutionary rates and mutational saturation effects may account for some of the contradictions. PMID:11443344

  2. The hybridization-stabilization assay: a solution-based isothermal method for rapid screening and determination of sequence preference of ligands that bind to duplexed nucleic acids.

    PubMed

    Gonzalez, C; Moore, M; Ribeiro, S; Schmitz, U; Schroth, G P; Turin, L; Bruice, T W

    2001-08-15

    The gene-to-drug quest will be most directly served by the discovery and development of small molecules that bind to nucleic acids and modulate gene expression at the level of transcription and/or inhibit replication of infectious agents. Full realization of this potential will require implementation of a complete suite of modern drug discovery technologies. Towards this end, here we describe our initial results with a new assay for identification and characterization of novel nucleic acid binding ligands. It is based on the well recognized property of stabilization of hybridization of complementary oligonucleotides by groove and/or intercalation binding ligands. Unlike traditional thermal melt methodologies, this assay is isothermal and, unlike gel-based footprinting techniques, the assay also is performed in solution and detection can be by any number of highly sensitive, non-radioisotopic modalities, such as fluorescence resonance energy transfer, described herein. Thus, the assay is simple to perform, versatile in design and amenable to miniaturization and high throughput automation. Assay validation was performed using various permutations of direct and competitive binding formats and previously well studied ligands, including pyrrole polyamide and intercalator natural products, designed hairpin pyrrole-imidazole polyamides and furan-based non-polyamide dications. DNA specific ligands were identified and their DNA binding site size and sequence preference profiles were determined. A systematic approach to studying the relationship of binding sequence specificity with variation in ligand structure was demonstrated, and preferred binding sites in longer DNA sequences were found by pseudo-footprinting, with results that are in accord with established findings. This assay methodology should promote a more rapid discovery of novel nucleic acid ligands and potential drug candidates.

  3. The evaluation for generic-level monophyly of Ancyrocephalinae (Monogenea, Dactylogyridae) using ribosomal DNA sequence data.

    PubMed

    Wu, Xiang-Yun; Zhu, Xing-Quan; Xie, Ming-Quan; Li, An-Xing

    2007-08-01

    To overcome the limited regional and taxonomic coverage in previous studies of the Ancyrocephalinae (Monogenea, Dactylogyridae), we present further findings on the molecular systematics and phylogeny of a broader selection of specimens, aiming to build a molecular phylogeny of the Ancyrocephalinae, and to assess the monophyly of each available genus, using D1-D2 domain of LSU rDNA and the combined LSU and partial sequence of SSU rDNA data sets. Our studies showed that 18 Haliotrema species were highly dispersive to form several clades with species from ten closely related genera. The host range of Euryhaliotrematoides species was not only restricted in butterfly fishes (Chaetodontidae), but widen to include the Lutjanidae. Euryhaliotrema was phylogenetically more closely related to Aliatrema than to Euryhaliotrematoides. Given that the species Haliotrema kurodai, Haliotrema spirotubiforum and Haliotrema anguiformis had a funnel-shaped base of the coiled male copulatory organ (MCO) lacking an accessory piece, and our molecular evidence, we proposed to transfer the three species to the Aliatrema as new combinations. We proposed to combine Bravohollisia and Caballeria into one genus, mainly based on molecular evidence and their similar MCO characters. Using SSU+ITS1 data set, Scutogyrus was robustly resolved as a polyphyletic group and its status should be questioned. Based on the present molecular evidence and their similar MCO characters, we proposed to combine Cichlidogyrus and Scutogyrus into one genus, Cichlidogyrus, by treating Scutogyrus as the synonym of Cichlidogyrus. Generally, the present study indicated that the vagina can make little contribution for understanding the generic-level monophyly, due to its high variability even among phylogenetically very closely related species, but it was useful for species determination. Given that phylogenetically closely related species from the same or closely related host species may have similar MCO characters but

  4. Species-level identification of staphylococci isolated from bovine mastitis in Brazil using partial 16S rRNA sequencing.

    PubMed

    Lange, Carla C; Brito, Maria A V P; Reis, Daniele R L; Machado, Marco A; Guimarães, Alessandro S; Azevedo, Ana L S; Salles, Érica B; Alvim, Mariana C T; Silva, Fabiana S; Meurer, Igor R

    2015-04-17

    Staphylococci isolated from bovine milk and not classified as Staphylococcus aureus represent a heterogeneous group of microorganisms that are frequently associated with bovine mastitis. The identification of these microorganisms is important, although it is difficult and relatively costly. Genotypic methods add precision in the identification of Staphylococcus species. In the present study, partial 16S rRNA sequencing was used for the species identification of coagulase-positive and coagulase-negative staphylococci isolated from bovine mastitis. Two hundred and two (95%) of the 213 isolates were successfully identified at the species level. The assigning of an isolate to a particular species was based on ≥99% identity with 16S rRNA sequences deposited in GenBank. The identified isolates belonged to 13 different Staphylococcus species; Staphylococcus chromogenes, S. aureus and Staphylococcus epidermidis were the most frequently identified species. Eight isolates could not be assigned to a single species, as the obtained sequences showed 99% or 100% similarity to sequences from two or three different Staphylococcus species. The relatedness of these isolates with the other isolates and reference strains was visualized using a cladogram. In conclusion, 16S rRNA sequencing was an objective and accurate method for the proper identification of Staphylococcus species isolated from bovine mastitis. Additional target genes could be used in non-conclusive cases for the species-level identification of these microorganisms.

  5. Higher-level phylogeny of new world vireos (aves: vireonidae) based on sequences of multiple mitochondrial DNA genes.

    PubMed

    Cicero, C; Johnson, N K

    2001-07-01

    Interfamilial relationships of the New World songbird family Vireonidae are uncertain. Thus, we sequenced 3069 bp of four mitochondrial genes (cyt b, ND2, ND3, COI) from 19 taxa in five families and two outgroups, to examine higher-level alliances with proposed relatives. We also sequenced cyt b and ND2 from an additional five vireonids to examine intergeneric relationships within the Vireonidae and incorporated 14 sequences of cyt b from GenBank to test the effects of taxon sampling on gene tree resolution. Families appeared monophyletic in all analyses, and the affinity of vireonids to Old World corvoids was corroborated. However, relationships among the Vireonidae and other families were not resolved. Sequences of vireonids revealed high levels of divergence within and between genera, with either Cyclarhis or Vireolanius positioned basally, depending on the analysis. On the basis of mitochondrial DNA and biogeographic evidence, vireonids represent a deep lineage derived from an Old World ancestor that colonized the New World, most likely via Beringia, with subsequent radiation in the Middle American tropics. We hypothesize postcolonization dispersal of the ancestor into Middle America, followed by extinction of the ancestor in North America. This extinction event left the North Temperate Zone unoccupied by any vireonid until northward reinvasion by some species of Vireo. Although the closest living relative of vireonids remains unidentified, broad-scale sequencing of additional extant corvoids with multiple molecular markers should further elucidate Old World alliances. PMID:11421646

  6. Species-level identification of staphylococci isolated from bovine mastitis in Brazil using partial 16S rRNA sequencing.

    PubMed

    Lange, Carla C; Brito, Maria A V P; Reis, Daniele R L; Machado, Marco A; Guimarães, Alessandro S; Azevedo, Ana L S; Salles, Érica B; Alvim, Mariana C T; Silva, Fabiana S; Meurer, Igor R

    2015-04-17

    Staphylococci isolated from bovine milk and not classified as Staphylococcus aureus represent a heterogeneous group of microorganisms that are frequently associated with bovine mastitis. The identification of these microorganisms is important, although it is difficult and relatively costly. Genotypic methods add precision in the identification of Staphylococcus species. In the present study, partial 16S rRNA sequencing was used for the species identification of coagulase-positive and coagulase-negative staphylococci isolated from bovine mastitis. Two hundred and two (95%) of the 213 isolates were successfully identified at the species level. The assigning of an isolate to a particular species was based on ≥99% identity with 16S rRNA sequences deposited in GenBank. The identified isolates belonged to 13 different Staphylococcus species; Staphylococcus chromogenes, S. aureus and Staphylococcus epidermidis were the most frequently identified species. Eight isolates could not be assigned to a single species, as the obtained sequences showed 99% or 100% similarity to sequences from two or three different Staphylococcus species. The relatedness of these isolates with the other isolates and reference strains was visualized using a cladogram. In conclusion, 16S rRNA sequencing was an objective and accurate method for the proper identification of Staphylococcus species isolated from bovine mastitis. Additional target genes could be used in non-conclusive cases for the species-level identification of these microorganisms. PMID:25704228

  7. Complete amino acid sequence of human plasma Zn-. cap alpha. /sub 2/-glycoprotein and its homology to histocompatibility antigens

    SciTech Connect

    Araki, T.; Gejyo, F.; Takagaki, K.; Haupt, H.; Schwick, H.G.; Buergi, W.; Marti, T.; Schaller, J.; Rickli, E.; Brossmer, R.

    1988-02-01

    In the present study the complete amino acid sequence of human plasma Zn-..cap alpha../sub 2/-glycoprotein was determined. This protein whose biological function is unknown consists of a single polypeptide chain of 276 amino acid residues including 8 tryptophan residues and has a pyroglutamyl residue at the amino terminus. The location of the two disulfide bonds in the polypeptide chain was also established. The three glycans, whose structure was elucidated with the aid of 500 MHz /sup 1/H NMR spectroscopy, were sialylated N-biantennas. The molecular weight calculated from the polypeptide and carbohydrate structure is 38,478, which is close to the reported value of approx. = 41,000 based on physicochemical measurements. The predicted secondary structure appeared to comprised of 23% ..cap alpha..-helix, 27% ..beta..-sheet, and 22% ..beta..-turns. The three N-glycans were found to be located in ..beta..-turn regions. An unexpected finding was made by computer analysis of the sequence data; this revealed that Zn-..cap alpha../sub 2/-glycoprotein is closely related to antigens of the major histocompatibility complex in amino acid sequence and in domain structure. There was an unusually high degree of sequence homology with the ..cap alpha.. chains of class I histocompatibility antigens. Moreover, this plasma protein was shown to be a member of the immunoglobulin gene superfamily. Zn-..cap alpha../sub 2/-glycoprotein appears to be truncated secretory major histocompatibility complex-related molecule, and it may have a role in the expression of the immune response.

  8. Spectra, energy levels, and energy transition of lanthanide complexes with cinnamic acid and its derivatives

    NASA Astrophysics Data System (ADS)

    Zhou, Kaining; Feng, Zhongshan; Shen, Jun; Wu, Bing; Luo, Xiaobing; Jiang, Sha; Li, Li; Zhou, Xianju

    2016-04-01

    High resolution spectra and luminescent lifetimes of 6 europium(III)-cinnamic acid complex {[Eu2L6(DMF)(H2O)]·nDMF·H2O}m (L = cinnamic acid I, 4-methyl-cinnamic acid II, 4-chloro-cinnamic acid III, 4-methoxy-cinnamic acid IV, 4-hydroxy-cinnamic acid V, 4-nitro-cinnamic acid VI; DMF = N, N-dimethylformamide, C3H7NO) were recorded from 8 K to room temperature. The energy levels of Eu3 + in these 6 complexes are obtained from the spectra analysis. It is found that the energy levels of the central Eu3 + ions are influenced by the nephelauxetic effect, while the triplet state of ligand is lowered by the p-π conjugation effect of the para-substituted functional groups. The best energy matching between the ligand triplet state and the central ion excited state is found in complex I. While the other complexes show poorer matching because the gap of 5D0 and triplet state contracts.

  9. Spectra, energy levels, and energy transition of lanthanide complexes with cinnamic acid and its derivatives.

    PubMed

    Zhou, Kaining; Feng, Zhongshan; Shen, Jun; Wu, Bing; Luo, Xiaobing; Jiang, Sha; Li, Li; Zhou, Xianju

    2016-04-01

    High resolution spectra and luminescent lifetimes of 6 europium(III)-cinnamic acid complex {[Eu2L6(DMF)(H2O)]·nDMF·H2O}m (L=cinnamic acid I, 4-methyl-cinnamic acid II, 4-chloro-cinnamic acid III, 4-methoxy-cinnamic acid IV, 4-hydroxy-cinnamic acid V, 4-nitro-cinnamic acid VI; DMF=N, N-dimethylformamide, C3H7NO) were recorded from 8 K to room temperature. The energy levels of Eu(3+) in these 6 complexes are obtained from the spectra analysis. It is found that the energy levels of the central Eu(3+) ions are influenced by the nephelauxetic effect, while the triplet state of ligand is lowered by the p-π conjugation effect of the para-substituted functional groups. The best energy matching between the ligand triplet state and the central ion excited state is found in complex I. While the other complexes show poorer matching because the gap of (5)D0 and triplet state contracts.

  10. Spectra, energy levels, and energy transition of lanthanide complexes with cinnamic acid and its derivatives.

    PubMed

    Zhou, Kaining; Feng, Zhongshan; Shen, Jun; Wu, Bing; Luo, Xiaobing; Jiang, Sha; Li, Li; Zhou, Xianju

    2016-04-01

    High resolution spectra and luminescent lifetimes of 6 europium(III)-cinnamic acid complex {[Eu2L6(DMF)(H2O)]·nDMF·H2O}m (L=cinnamic acid I, 4-methyl-cinnamic acid II, 4-chloro-cinnamic acid III, 4-methoxy-cinnamic acid IV, 4-hydroxy-cinnamic acid V, 4-nitro-cinnamic acid VI; DMF=N, N-dimethylformamide, C3H7NO) were recorded from 8 K to room temperature. The energy levels of Eu(3+) in these 6 complexes are obtained from the spectra analysis. It is found that the energy levels of the central Eu(3+) ions are influenced by the nephelauxetic effect, while the triplet state of ligand is lowered by the p-π conjugation effect of the para-substituted functional groups. The best energy matching between the ligand triplet state and the central ion excited state is found in complex I. While the other complexes show poorer matching because the gap of (5)D0 and triplet state contracts. PMID:26802538

  11. Whole genome sequencing reveals extensive community-level transmission of group A Streptococcus in remote communities.

    PubMed

    Bowen, A C; Harris, T; Holt, D C; Giffard, P M; Carapetis, J R; Campbell, P T; McVERNON, J; Tong, S Y C

    2016-07-01

    Impetigo is common in remote Indigenous children of northern Australia, with the primary driver in this context being Streptococcus pyogenes [or group A Streptococcus (GAS)]. To reduce the high burden of impetigo, the transmission dynamics of GAS must be more clearly elucidated. We performed whole genome sequencing on 31 GAS isolates collected in a single community from children in 11 households with ⩾2 GAS-infected children. We aimed to determine whether transmission was occurring principally within households or across the community. The 31 isolates were represented by nine multilocus sequence types and isolates within each sequence type differed from one another by only 0-3 single nucleotide polymorphisms. There was evidence of extensive transmission both within households and across the community. Our findings suggest that strategies to reduce the burden of impetigo in this setting will need to extend beyond individual households, and incorporate multi-faceted, community-wide approaches. PMID:26833141

  12. Genome-wide estimation of linkage disequilibrium from population-level high-throughput sequencing data.

    PubMed

    Maruki, Takahiro; Lynch, Michael

    2014-08-01

    Rapidly improving sequencing technologies provide unprecedented opportunities for analyzing genome-wide patterns of polymorphisms. In particular, they have great potential for linkage-disequilibrium analyses on both global and local genetic scales, which will substantially improve our ability to derive evolutionary inferences. However, there are some difficulties with analyzing high-throughput sequencing data, including high error rates associated with base reads and complications from the random sampling of sequenced chromosomes in diploid organisms. To overcome these difficulties, we developed a maximum-likelihood estimator of linkage disequilibrium for use with error-prone sampling data. Computer simulations indicate that the estimator is nearly unbiased with a sampling variance at high coverage asymptotically approaching the value expected when all relevant information is accurately estimated. The estimator does not require phasing of haplotypes and enables the estimation of linkage disequilibrium even when all individual reads cover just single polymorphic sites.

  13. Whole genome sequencing reveals extensive community-level transmission of group A Streptococcus in remote communities.

    PubMed

    Bowen, A C; Harris, T; Holt, D C; Giffard, P M; Carapetis, J R; Campbell, P T; McVERNON, J; Tong, S Y C

    2016-07-01

    Impetigo is common in remote Indigenous children of northern Australia, with the primary driver in this context being Streptococcus pyogenes [or group A Streptococcus (GAS)]. To reduce the high burden of impetigo, the transmission dynamics of GAS must be more clearly elucidated. We performed whole genome sequencing on 31 GAS isolates collected in a single community from children in 11 households with ⩾2 GAS-infected children. We aimed to determine whether transmission was occurring principally within households or across the community. The 31 isolates were represented by nine multilocus sequence types and isolates within each sequence type differed from one another by only 0-3 single nucleotide polymorphisms. There was evidence of extensive transmission both within households and across the community. Our findings suggest that strategies to reduce the burden of impetigo in this setting will need to extend beyond individual households, and incorporate multi-faceted, community-wide approaches.

  14. Amino acid sequences in the alpha 1 domain and not glycosylation are important in HLA-A2/beta 2-microglobulin association and cell surface expression.

    PubMed Central

    Santos-Aguado, J; Biro, P A; Fuhrmann, U; Strominger, J L; Barbosa, J A

    1987-01-01

    The role of the single carbohydrate moiety present on the HLA-A2 molecule was studied by introducing several amino acid substitutions (by site-directed mutagenesis of the HLA-A2 gene) in the consensus glycosylation sequence Asn-X-Ser. Two different amino acid substitutions of the asparagine residue at position 86 (glutamine and aspartic acid) resulted in the synthesis of ca. 39,000-molecular-weight nonglycosylated heavy chains that were detected in the cytoplasm but not on the surface of mouse L-cell transfectants. However, a low level of surface expression was detected following transfection of human (rhabdomyosarcoma) cells or mouse L cells containing human beta 2-microglobulin. The defect in surface expression was not due to the absence of the glycan moiety, since the substitution of a glycine for a serine at amino acid 88 did not have the same drastic effect in the presence of human beta 2-microglobulin. These and other data suggest that the asparagine residue may play a critical role in the conformation of the HLA heavy chain and its interaction with beta 2-microglobulin. Immunofluorescence microscopy following permeabilization of the transfectants demonstrated that the unglycosylated HLA heavy chains are sequestered in an unidentified cellular compartment that is different from the Golgi structure. Images PMID:3550437

  15. Autistic Children Exhibit Decreased Levels of Essential Fatty Acids in Red Blood Cells

    PubMed Central

    Brigandi, Sarah A.; Shao, Hong; Qian, Steven Y.; Shen, Yiping; Wu, Bai-Lin; Kang, Jing X.

    2015-01-01

    Omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids (PUFA) are essential nutrients for brain development and function. However, whether or not the levels of these fatty acids are altered in individuals with autism remains debatable. In this study, we compared the fatty acid contents between 121 autistic patients and 110 non-autistic, non-developmentally delayed controls, aged 3–17. Analysis of the fatty acid composition of red blood cell (RBC) membrane phospholipids showed that the percentage of total PUFA was lower in autistic patients than in controls; levels of n-6 arachidonic acid (AA) and n-3 docosahexaenoic acid (DHA) were particularly decreased (p < 0.001). In addition, plasma levels of the pro-inflammatory AA metabolite prostaglandin E2 (PGE2) were higher in a subset of the autistic participants (n = 20) compared to controls. Our study demonstrates an alteration in the PUFA profile and increased production of a PUFA-derived metabolite in autistic patients, supporting the hypothesis that abnormal lipid metabolism is implicated in autism. PMID:25946342

  16. Uric acid levels predict future blood pressure and new onset hypertension in the general Japanese population.

    PubMed

    Takase, H; Kimura, G; Dohi, Y

    2014-09-01

    We tested the hypothesis that uric acid levels predict new-onset hypertension in the Japanese general population. Normotensive individuals who visited our hospital for a yearly health checkup (n=8157, men=61.0% and age=50.7±12.2 years) were enrolled in the present study. After baseline evaluation, participants were followed up for a median of 48.3 months (range 4.9-101.0 months), with the endpoint being the development of hypertension, defined as systolic blood pressure (BP) > or = 140 mm Hg, diastolic BP > or = 90 mm Hg or the use of antihypertensive medication. The impact of uric acid and other cardiovascular risk factors at baseline on future BP and development of hypertension was assessed. During follow-up, 19.0% of women (n=605) and 29.5% of men (n=1469) participants developed hypertension. Incident hypertension was increased across the quartiles for baseline uric acid levels (P<0.0001), and multivariate Cox proportional hazards analysis revealed a significant and independent association between the uric acid level and the onset of hypertension in both men and women participants (P<0.05). Furthermore, uric acid was independently and positively correlated with future BP (P<0.05). Thus, uric acid is an independent predictor of new-onset hypertension in both women and men.

  17. The mechanism of low levels of nitrogen dioxide reaction with unsaturated fatty acid esters

    SciTech Connect

    Gallon, A.A.

    1990-01-01

    Nitrogen dioxide is a toxic air pollutant that exists at less than 0.5 ppm in the atmosphere. This toxic compound is known to initiate autoxidation of unsaturated fatty acids both in vivo and in vitro. When autoxidation occurs in vivo, membrane damage that can lead to cell death can occur. Low concentrations of nitrogen dioxide were shown to react with the polyunsaturated fatty acid esters, methyl linoleate and methyl linoleate, by a H-abstraction mechanism. However, methyl oleate, an unsaturated fatty acid ester, was demonstrated to react with a low concentration of nitrogen dioxide by only an addition mechanism. Although methyl oleate reacts by an addition mechanism, a 50:50 molar solution of methyl oleate and methyl linoleate reacted with a low level of nitrogen dioxide exclusively by a H-abstraction mechanism. Therefore, low levels of nitrogen dioxide will probably react with polyunsaturated fatty acid components of pulmonary lipids by a H-abstraction mechanism forming nitrous acid directly in the cell membrane. Vitamin E was demonstrated to be able to act as a preventative antioxidant in the nitrogen dioxide and methyl linoleate reactions; but vitamin C could not prevent nitrogen dioxide from reacting with methyl linoleate by a H-abstraction mechanism. These results suggest that low levels of nitrogen dioxide will react with polyunsaturated fatty acids by a H-abstraction mechanism and that vitamin E can be used to prevent the reaction from occurring.

  18. ENTPRISE: An Algorithm for Predicting Human Disease-Associated Amino Acid Substitutions from Sequence Entropy and Predicted Protein Structures

    PubMed Central

    Zhou, Hongyi; Gao, Mu; Skolnick, Jeffrey

    2016-01-01

    The advance of next-generation sequencing technologies has made exome sequencing rapid and relatively inexpensive. A major application of exome sequencing is the identification of genetic variations likely to cause Mendelian diseases. This requires processing large amounts of sequence information and therefore computational approaches that can accurately and efficiently identify the subset of disease-associated variations are needed. The accuracy and high false positive rates of existing computational tools leave much room for improvement. Here, we develop a boosted tree regression machine-learning approach to predict human disease-associated amino acid variations by utilizing a comprehensive combination of protein sequence and structure features. On comparing our method, ENTPRISE, to the state-of-the-art methods SIFT, PolyPhen-2, MUTATIONASSESSOR, MUTATIONTASTER, FATHMM, ENTPRISE exhibits significant improvement. In particular, on a testing dataset consisting of only proteins with balanced disease-associated and neutral variations defined as having the ratio of neutral/disease-associated variations between 0.3 and 3, the Mathews Correlation Coefficient by ENTPRISE is 0.493 as compared to 0.432 by PPH2-HumVar, 0.406 by SIFT, 0.403 by MUTATIONASSESSOR, 0.402 by PPH2-HumDiv, 0.305 by MUTATIONTASTER, and 0.181 by FATHMM. ENTPRISE is then applied to nucleic acid binding proteins in the human proteome. Disease-associated predictions are shown to be highly correlated with the number of protein-protein interactions. Both these predictions and the ENTPRISE server are freely available for academic users as a web service at http://cssb.biology.gatech.edu/entprise/. PMID:26982818

  19. ENTPRISE: An Algorithm for Predicting Human Disease-Associated Amino Acid Substitutions from Sequence Entropy and Predicted Protein Structures.

    PubMed

    Zhou, Hongyi; Gao, Mu; Skolnick, Jeffrey

    2016-01-01

    The advance of next-generation sequencing technologies has made exome sequencing rapid and relatively inexpensive. A major application of exome sequencing is the identification of genetic variations likely to cause Mendelian diseases. This requires processing large amounts of sequence information and therefore computational approaches that can accurately and efficiently identify the subset of disease-associated variations are needed. The accuracy and high false positive rates of existing computational tools leave much room for improvement. Here, we develop a boosted tree regression machine-learning approach to predict human disease-associated amino acid variations by utilizing a comprehensive combination of protein sequence and structure features. On comparing our method, ENTPRISE, to the state-of-the-art methods SIFT, PolyPhen-2, MUTATIONASSESSOR, MUTATIONTASTER, FATHMM, ENTPRISE exhibits significant improvement. In particular, on a testing dataset consisting of only proteins with balanced disease-associated and neutral variations defined as having the ratio of neutral/disease-associated variations between 0.3 and 3, the Mathews Correlation Coefficient by ENTPRISE is 0.493 as compared to 0.432 by PPH2-HumVar, 0.406 by SIFT, 0.403 by MUTATIONASSESSOR, 0.402 by PPH2-HumDiv, 0.305 by MUTATIONTASTER, and 0.181 by FATHMM. ENTPRISE is then applied to nucleic acid binding proteins in the human proteome. Disease-associated predictions are shown to be highly correlated with the number of protein-protein interactions. Both these predictions and the ENTPRISE server are freely available for academic users as a web service at http://cssb.biology.gatech.edu/entprise/.

  20. Infantile Refsum Disease: Influence of Dietary Treatment on Plasma Phytanic Acid Levels.

    PubMed

    Sá, Maria João Nabais; Rocha, Júlio C; Almeida, Manuela F; Carmona, Carla; Martins, Esmeralda; Miranda, Vasco; Coutinho, Miguel; Ferreira, Rita; Pacheco, Sara; Laranjeira, Francisco; Ribeiro, Isaura; Fortuna, Ana Maria; Lacerda, Lúcia

    2016-01-01

    Infantile Refsum disease (IRD) is one of the less severe of Zellweger spectrum disorders (ZSDs), a group of peroxisomal biogenesis disorders resulting from a generalized peroxisomal function impairment. Increased plasma levels of very long chain fatty acids (VLCFA) and phytanic acid are biomarkers used in IRD diagnosis. Furthermore, an increased plasma level of phytanic acid is known to be associated with neurologic damage. Treatment of IRD is symptomatic and multidisciplinary.The authors report a 3-year-old child, born from consanguineous parents, who presented with developmental delay, retinitis pigmentosa, sensorineural deafness and craniofacial dysmorphisms. While the relative level of plasma C26:0 was slightly increased, other VLCFA were normal. Thus, a detailed characterization of the phenotype was essential to point to a ZSD. Repeatedly increased levels of plasma VLCFA, along with phytanic acid and pristanic acid, deficient dihydroxyacetone phosphate acyltransferase activity in fibroblasts and identification of the homozygous pathogenic mutation c.2528G>A (p.Gly843Asp) in the PEX1 gene, confirmed this diagnosis. Nutritional advice and follow-up was proposed aiming phytanic acid dietary intake reduction. During dietary treatment, plasma levels of phytanic acid decreased to normal, and the patient's development evaluation showed slow progressive acquisition of new competences.This case report highlights the relevance of considering a ZSD in any child with developmental delay who manifests hearing and visual impairment and of performing a systematic biochemical investigation, when plasma VLCFA are mildly increased. During dietary intervention, a biochemical improvement was observed, and the long-term clinical effect of this approach needs to be evaluated.

  1. The sequence diversity and expression among genes of the folic acid biosynthesis pathway in industrial Saccharomyces strains.

    PubMed

    Goncerzewicz, Anna; Misiewicz, Anna

    2015-01-01

    Folic acid is an important vitamin in human nutrition and its deficiency in pregnant women's diets results in neural tube defects and other neurological damage to the fetus. Additionally, DNA synthesis, cell division and intestinal absorption are inhibited in case of adults. Since this discovery, governments and health organizations worldwide have made recommendations concerning folic acid supplementation of food for women planning to become pregnant. In many countries this has led to the introduction of fortifications, where synthetic folic acid is added to flour. It is known that Saccharomyces strains (brewing and bakers' yeast) are one of the main producers of folic acid and they can be used as a natural source of this vitamin. Proper selection of the most efficient strains may enhance the folate content in bread, fermented vegetables, dairy products and beer by 100% and may be used in the food industry. The objective of this study was to select the optimal producing yeast strain by determining the differences in nucleotide sequences in the FOL2, FOL3 and DFR1 genes of folic acid biosynthesis pathway. The Multitemperature Single Strand Conformation Polymorphism (MSSCP) method and further nucleotide sequencing for selected strains were applied to indicate SNPs in selected gene fragments. The RT qPCR technique was also applied to examine relative expression of the FOL3 gene. Furthermore, this is the first time ever that industrial yeast strains were analysed regarding genes of the folic acid biosynthesis pathway. It was observed that a correlation exists between the folic acid amount produced by industrial yeast strains and changes in the nucleotide sequence of adequate genes. The most significant changes occur in the DFR1 gene, mostly in the first part, which causes major protein structure modifications in KKP 232, KKP 222 and KKP 277 strains. Our study shows that the large amount of SNP contributes to impairment of the selected enzymes and S. cerevisiae and S

  2. The sequence diversity and expression among genes of the folic acid biosynthesis pathway in industrial Saccharomyces strains.

    PubMed

    Goncerzewicz, Anna; Misiewicz, Anna

    2015-01-01

    Folic acid is an important vitamin in human nutrition and its deficiency in pregnant women's diets results in neural tube defects and other neurological damage to the fetus. Additionally, DNA synthesis, cell division and intestinal absorption are inhibited in case of adults. Since this discovery, governments and health organizations worldwide have made recommendations concerning folic acid supplementation of food for women planning to become pregnant. In many countries this has led to the introduction of fortifications, where synthetic folic acid is added to flour. It is known that Saccharomyces strains (brewing and bakers' yeast) are one of the main producers of folic acid and they can be used as a natural source of this vitamin. Proper selection of the most efficient strains may enhance the folate content in bread, fermented vegetables, dairy products and beer by 100% and may be used in the food industry. The objective of this study was to select the optimal producing yeast strain by determining the differences in nucleotide sequences in the FOL2, FOL3 and DFR1 genes of folic acid biosynthesis pathway. The Multitemperature Single Strand Conformation Polymorphism (MSSCP) method and further nucleotide sequencing for selected strains were applied to indicate SNPs in selected gene fragments. The RT qPCR technique was also applied to examine relative expression of the FOL3 gene. Furthermore, this is the first time ever that industrial yeast strains were analysed regarding genes of the folic acid biosynthesis pathway. It was observed that a correlation exists between the folic acid amount produced by industrial yeast strains and changes in the nucleotide sequence of adequate genes. The most significant changes occur in the DFR1 gene, mostly in the first part, which causes major protein structure modifications in KKP 232, KKP 222 and KKP 277 strains. Our study shows that the large amount of SNP contributes to impairment of the selected enzymes and S. cerevisiae and S

  3. Alterations in the levels of plasma amino acids in polycystic ovary syndrome- A pilot study

    PubMed Central

    Unni, C. Sumithra N.; Lakshman, Lakshmi R.; Vaidyanathan, Kannan; Subhakumari, K.N.; Menon, N. Leela

    2015-01-01

    Background & objectives: Plasma amino acid levels are known to be altered in conditions like sepsis and burns which are situations of metabolic stress. Polycystic ovary syndrome (PCOS), a condition which affects a woman throughout her life, is said to be associated with metabolic stress. This study was undertaken to assess if there were significant alterations in the levels of plasma amino acids in women with PCOS. Methods: Sixty five women with PCOS along with the similar number of age matched normal controls were included in this study. Levels of 14 amino acids were determined using reverse phase high performance liquid chromatography. Results: The levels of methionine, cystine, isoleucine, phenylalanine, valine, tyrosine, proline, glycine, lysine and histidine were found to be significantly (P<0.001) lower in cases than in controls. Arginine and alanine levels were found to be significantly (P<0.001) higher in cases compared with controls. Interpretation & conclusions: Our findings showed significant derangement in the levels of plasma amino acids in women with PCOS which might be due to the oxidative and metabolic stress associated with it. Further studies need to be done to confirm the findings. PMID:26658589

  4. Enhanced level of n-3 fatty acid in membrane phospholipids induces lipid peroxidation in rats fed dietary docosahexaenoic acid oil.

    PubMed

    Song, J H; Miyazawa, T

    2001-03-01

    The effect of dietary docosahexaenoic acid (DHA, 22:6n-3) oil with different lipid types on lipid peroxidation was studied in rats. Each group of male Sprague-Dawley rats was pair fed 15% (w/w) of either DHA-triglycerides (DHA-TG), DHA-ethyl esters (DHA-EE) or DHA-phospholipids (DHA-PL) for up to 3 weeks. The palm oil (supplemented with 20% soybean oil) diet without DHA was fed as the control. Dietary DHA oils lowered plasma triglyceride concentrations in rats fed DHA-TG (by 30%), DHA-EE (by 45%) and DHA-PL (by 27%), compared to control. The incorporation of dietary DHA into plasma and liver phospholipids was more pronounced in the DHA-TG and DHA-EE group than in the DHA-PL group. However, DHA oil intake negatively influenced lipid peroxidation in both plasma and liver. Phospholipid peroxidation in plasma and liver was significantly higher than control in rats fed DHA-TG or DHA-EE, but not DHA-PL. These results are consistent with increased thiobarbituric acid reactive substances (TBARS) and decreased alpha-tocopherol levels in plasma and liver. In addition, liver microsomes from rats of each group were exposed to a mixture of chelated iron (Fe(3+)/ADP) and NADPH to determine the rate of peroxidative damage. During NADPH-dependent peroxidation of microsomes, the accumulation of phospholipid hydroperoxides, as well as TBARS, were elevated and alpha-tocopherol levels were significantly exhausted in DHA-TG and DHA-EE groups. During microsomal lipid peroxidation, there was a greater loss of n-3 fatty acids (mainly DHA) than of n-6 fatty acids, including arachidonic acid (20:4n-6). These results indicate that polyunsaturation of n-3 fatty acids is the most important target for lipid peroxidation. This suggests that the ingestion of large amounts of DHA oil enhances lipid peroxidation in the target membranes where greater amounts of n-3 fatty acids are incorporated, thereby increasing the peroxidizability and possibly accelerating the atherosclerotic process.

  5. Decreased docosahexaenoic acid levels in retina and pigment epithelium of frogs fed crickets.

    PubMed

    Chen, H; Wiegand, R D; Anderson, R E

    1992-06-01

    Whole retina, rod outer segments, and retinal pigment epithelium of frogs (Rana pipiens) fed crickets for more than 1 year had significantly lower levels of docosahexaenoic acid (22: 6n-3) than the same tissues of frogs fed crickets for less than 1 month. Decreases in 22:6n-3 levels in these tissues were compensated for by increases in the n-6 polyunsaturated fatty acids (PUFAs), primarily 22:5n-6. There were no changes in the levels of saturated, monoenoic, or dienoic acids. Analysis of diacyl phospholipid molecular species (PLMS) revealed decreases in both the 22:6(n-3)-containing dipolyenoic molecular species in phosphatidylethanolamine and phosphatidylserine, and the monopolyenoic molecular species in phosphatidylcholine. These PLMS were replaced by species containing 22:5n-6 or other n-6 PUFAs. Examination of fatty acid methyl esters of total lipids extracted from crickets revealed that less than 1 mol% fatty acids were of the n-3 family, while more than 30 mol% were of the n-6 family. Thus, frogs raised on an n-3-deficient diet have reduced levels of n-3 PUFA in their retinas, rod outer segments, and retinal pigment epithelium. Although such changes have been reported for mammals, this is the first report of the effects of n-3 deficiency on the lipids of amphibians.

  6. Uric acid levels in subjects with bipolar disorder: A comparative meta-analysis.

    PubMed

    Bartoli, Francesco; Crocamo, Cristina; Mazza, Mario Gennaro; Clerici, Massimo; Carrà, Giuseppe

    2016-10-01

    Previous research has hypothesised increased uric acid levels, possibly because of an amplified purinergic metabolism and a reduced adenosine activity, in subjects with bipolar disorder. This systematic review and meta-analysis aimed at estimating if individuals with bipolar disorder had uric acid levels higher than both healthy controls and subjects with major depression (trait marker hypothesis). It also tested if uric acid levels could differ in different phases of bipolar disorder (state marker hypothesis). Meta-analyses were carried out generating pooled standardized mean differences (SMDs), using random-effects models. Heterogeneity between studies was estimated using the I(2) index. Relevant sensitivity and meta-regression analyses were conducted. We searched main Electronic Databases, identifying twelve studies that met our inclusion criteria. Meta-analyses showed increased uric acid levels in individuals with bipolar disorder as compared with both healthy controls (SMD = 0.65, p < 0.001, I(2) = 82.9%) and those with major depression (SMD = 0.46, p < 0.001; I(2) = 68.7%). However, meta-regression analyses confirmed this association only as compared with healthy controls. Finally, though uric acid levels were higher in manic/mixed phases as compared with depressive ones (SMD = 0.34; p = 0.04, I(2) = 58.8%), a sensitivity analysis did not confirm the association. In sum, our meta-analysis shows that subjects with bipolar disorder have uric acid levels higher than healthy controls. The potential role of factors that might clarify the nature of this association deserves additional research.

  7. "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.

  8. "De-novo" amino acid sequence elucidation of protein G'e by combined "top-down" and "bottom-up" mass spectrometry.

    PubMed

    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 G´ 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. PMID:25560987

  9. Different Levels of Learning Interact to Shape the Congruency Sequence Effect

    ERIC Educational Resources Information Center

    Weissman, Daniel H.; Hawks, Zoë W.; Egner, Tobias

    2016-01-01

    The congruency effect in distracter interference tasks is often reduced after incongruent relative to congruent trials. Moreover, this "congruency sequence effect" (CSE) is influenced by learning related to concrete stimulus and response features as well as by learning related to abstract cognitive control processes. There is an ongoing…

  10. JRC GMO-Amplicons: a collection of nucleic acid sequences related to genetically modified organisms.

    PubMed

    Petrillo, Mauro; Angers-Loustau, Alexandre; Henriksson, Peter; Bonfini, Laura; Patak, Alex; Kreysa, Joachim

    2015-01-01

    The DNA target sequence is the key element in designing detection methods for genetically modified organisms (GMOs). Unfortunately this information is frequently lacking, especially for unauthorized GMOs. In addition, patent sequences are generally poorly annotated, buried in complex and extensive documentation and hard to link to the corresponding GM event. Here, we present the JRC GMO-Amplicons, a database of amplicons collected by screening public nucleotide sequence databanks by in silico determination of PCR amplification with reference methods for GMO analysis. The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) provides these methods in the GMOMETHODS database to support enforcement of EU legislation and GM food/feed control. The JRC GMO-Amplicons database is composed of more than 240 000 amplicons, which can be easily accessed and screened through a web interface. To our knowledge, this is the first attempt at pooling and collecting publicly available sequences related to GMOs in food and feed. The JRC GMO-Amplicons supports control laboratories in the design and assessment of GMO methods, providing inter-alia in silico prediction of primers specificity and GM targets coverage. The new tool can assist the laboratories in the analysis of complex issues, such as the detection and identification of unauthorized GMOs. Notably, the JRC GMO-Amplicons database allows the retrieval and characterization of GMO-related sequences included in patents documentation. Finally, it can help annotating poorly described GM sequences and identifying new relevant GMO-related sequences in public databases. The JRC GMO-Amplicons is freely accessible through a web-based portal that is hosted on the EU-RL GMFF website. Database URL: http://gmo-crl.jrc.ec.europa.eu/jrcgmoamplicons/. PMID:26424080

  11. JRC GMO-Amplicons: a collection of nucleic acid sequences related to genetically modified organisms

    PubMed Central

    Petrillo, Mauro; Angers-Loustau, Alexandre; Henriksson, Peter; Bonfini, Laura; Patak, Alex; Kreysa, Joachim

    2015-01-01

    The DNA target sequence is the key element in designing detection methods for genetically modified organisms (GMOs). Unfortunately this information is frequently lacking, especially for unauthorized GMOs. In addition, patent sequences are generally poorly annotated, buried in complex and extensive documentation and hard to link to the corresponding GM event. Here, we present the JRC GMO-Amplicons, a database of amplicons collected by screening public nucleotide sequence databanks by in silico determination of PCR amplification with reference methods for GMO analysis. The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) provides these methods in the GMOMETHODS database to support enforcement of EU legislation and GM food/feed control. The JRC GMO-Amplicons database is composed of more than 240 000 amplicons, which can be easily accessed and screened through a web interface. To our knowledge, this is the first attempt at pooling and collecting publicly available sequences related to GMOs in food and feed. The JRC GMO-Amplicons supports control laboratories in the design and assessment of GMO methods, providing inter-alia in silico prediction of primers specificity and GM targets coverage. The new tool can assist the laboratories in the analysis of complex issues, such as the detection and identification of unauthorized GMOs. Notably, the JRC GMO-Amplicons database allows the retrieval and characterization of GMO-related sequences included in patents documentation. Finally, it can help annotating poorly described GM sequences and identifying new relevant GMO-related sequences in public databases. The JRC GMO-Amplicons is freely accessible through a web-based portal that is hosted on the EU-RL GMFF website. Database URL: http://gmo-crl.jrc.ec.europa.eu/jrcgmoamplicons/ PMID:26424080

  12. JRC GMO-Amplicons: a collection of nucleic acid sequences related to genetically modified organisms.

    PubMed

    Petrillo, Mauro; Angers-Loustau, Alexandre; Henriksson, Peter; Bonfini, Laura; Patak, Alex; Kreysa, Joachim

    2015-01-01

    The DNA target sequence is the key element in designing detection methods for genetically modified organisms (GMOs). Unfortunately this information is frequently lacking, especially for unauthorized GMOs. In addition, patent sequences are generally poorly annotated, buried in complex and extensive documentation and hard to link to the corresponding GM event. Here, we present the JRC GMO-Amplicons, a database of amplicons collected by screening public nucleotide sequence databanks by in silico determination of PCR amplification with reference methods for GMO analysis. The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) provides these methods in the GMOMETHODS database to support enforcement of EU legislation and GM food/feed control. The JRC GMO-Amplicons database is composed of more than 240 000 amplicons, which can be easily accessed and screened through a web interface. To our knowledge, this is the first attempt at pooling and collecting publicly available sequences related to GMOs in food and feed. The JRC GMO-Amplicons supports control laboratories in the design and assessment of GMO methods, providing inter-alia in silico prediction of primers specificity and GM targets coverage. The new tool can assist the laboratories in the analysis of complex issues, such as the detection and identification of unauthorized GMOs. Notably, the JRC GMO-Amplicons database allows the retrieval and characterization of GMO-related sequences included in patents documentation. Finally, it can help annotating poorly described GM sequences and identifying new relevant GMO-related sequences in public databases. The JRC GMO-Amplicons is freely accessible through a web-based portal that is hosted on the EU-RL GMFF website. Database URL: http://gmo-crl.jrc.ec.europa.eu/jrcgmoamplicons/.

  13. Serology in the Digital Age: Using Long Synthetic Peptides Created from Nucleic Acid Sequences as Antigens in Microarrays

    PubMed Central

    Rizwan, Muhammad; Rönnberg, Bengt; Cistjakovs, Maksims; Lundkvist, Åke; Pipkorn, Rudiger; Blomberg, Jonas

    2016-01-01

    Background: Antibodies to microbes, or to autoantigens, are important markers of disease. Antibody detection (serology) can reveal both past and recent infections. There is a great need for development of rational ways of detecting and quantifying antibodies, both for humans and animals. Traditionally, serology using synthetic antigens covers linear epitopes using up to 30 amino acid peptides. Methods: We here report that peptides of 100 amino acids or longer (“megapeptides”), designed and synthesized for optimal serological performance, can successfully be used as detection antigens in a suspension multiplex immunoassay (SMIA). Megapeptides can quickly be created just from pathogen sequences. A combination of rational sequencing and bioinformatic routines for definition of diagnostically-relevant antigens can, thus, rapidly yield efficient serological diagnostic tools for an emerging infectious pathogen. Results: We designed megapeptides using bioinformatics and viral genome sequences. These long peptides were tested as antigens for the presence of antibodies in human serum to the filo-, herpes-, and polyoma virus families in a multiplex microarray system. All of these virus families contain recently discovered or emerging infectious viruses. Conclusion: Long synthetic peptides can be useful as serological diagnostic antigens, serving as biomarkers, in suspension microarrays. PMID:27600087

  14. The amino acid sequence of protein SCMK-B2C from the high-sulphur fraction of wool keratin

    PubMed Central

    Elleman, T. C.

    1972-01-01

    1. The amino acid sequence of a protein from the reduced and carboxymethylated high-sulphur fraction of wool has been determined. 2. The sequence of this S-carboxymethylkerateine (SCMK-B2C) of 151 amino acid residues displays much internal homology and an unusual residue distribution. Thus a ten-residue sequence occurs four times near the N-terminus and five times near the C-terminus with few changes. These regions contain much of the molecule's half-cystine, whereas between them there is a region of 19 residues that are mainly small and devoid of cystine and proline. 3. Certain models of the wool fibre based on its mechanical and physical properties propose a matrix of small compact globular units linked together to form beaded chains. The unusual distribution of the component residues of protein SCMK-B2C suggests structures in the wool-fibre matrix compatible with certain features of the proposed models. PMID:4678578

  15. Plasma levels of acetylsalicylic acid and salicylic acid after oral ingestion of plain and buffered acetylsalicylic acid in relation to bleeding time and thrombocyte function.