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Sample records for acid mature peptide

  1. Effects of L- and D-REKR amino acid-containing peptides on HIV and SIV envelope glycoprotein precursor maturation and HIV and SIV replication.

    PubMed Central

    Bahbouhi, Bouchaib; Chazal, Nathalie; Seidah, Nabil Georges; Chiva, Cristina; Kogan, Marcelo; Albericio, Fernando; Giralt, Ernest; Bahraoui, Elmostafa

    2002-01-01

    The aim of the present study was to evaluate the capacity of synthetic l- and d-peptides encompassing the HIV-1(BRU) gp160 REKR cleavage site to interfere with HIV and simian immuno-deficiency virus (SIV) replication and maturation of the envelope glycoprotein (Env) precursors. To facilitate their penetration into cells, a decanoyl (dec) group was added at the N-terminus. The sequences synthesized included dec5d or dec5l (decREKRV), dec9d or dec9l (decRVVQREKRV) and dec14d or dec14l (TKAKRRVVQREKRV). The peptide dec14d was also prepared with a chloromethane (cmk) group as C-terminus. Because l-peptides exhibit significant cytotoxicity starting at 35 microM, further characterization was conducted mostly with d-peptides, which exhibited no cytotoxicity at concentrations higher than 70 microM. The data show that only dec14d and dec14dcmk could inhibit HIV-1(BRU), HIV-2(ROD) and SIV(mac251) replication and their syncytium-inducing capacities. Whereas peptides dec5d and dec9d were inactive, dec14dcmk was at least twice as active as peptide dec14d. At the molecular level, our data show a direct correlation between anti-viral activity and the ability of the peptides to interfere with maturation of the Env precursors. Furthermore, we show that when tested in vitro the dec14d peptide inhibited PC7 with an inhibition constant K(i)=4.6 microM, whereas the peptide dec14l preferentially inhibited furin with a K(i)=28 microM. The fact that PC7 and furin are the major prohormone convertases reported to be expressed in T4 lymphocytes, the principal cell targets of HIV, suggests that they are involved in the maturation of HIV and SIV Env precursors. PMID:12071862

  2. Maturation processes and structures of small secreted peptides in plants

    PubMed Central

    Tabata, Ryo; Sawa, Shinichiro

    2014-01-01

    In the past decade, small secreted peptides have proven to be essential for various aspects of plant growth and development, including the maintenance of certain stem cell populations. Most small secreted peptides identified in plants to date are recognized by membrane-localized receptor kinases, the largest family of receptor proteins in the plant genome. This peptide-receptor interaction is essential for initiating intracellular signaling cascades. Small secreted peptides often undergo post-translational modifications and proteolytic processing to generate the mature peptides. Recent studies suggest that, in contrast to the situation in mammals, the proteolytic processing of plant peptides involves a number of complex steps. Furthermore, NMR-based structural analysis demonstrated that post-translational modifications induce the conformational changes needed for full activity. In this mini review, we summarize recent advances in our understanding of how small secreted peptides are modified and processed into biologically active peptides and describe the mature structures of small secreted peptides in plants. PMID:25071794

  3. PLAP efficiently generates mature antigenic peptides in vitro but in patterns distinct from ERAP11

    PubMed Central

    Georgiadou, Dimitra; Hearn, Arron; Evnouchidou, Irini; Chroni, Angeliki; Leondiadis, Leondios; York, Ian A.; Rock, Kenneth L.; Stratikos, Efstratios

    2010-01-01

    All three members of the oxytocinase sub-family of M1 aminopeptidases, ERAP1 (ERAAP), ERAP2 and PLAP (IRAP), have been implicated in the generation of MHC class I-presented peptides. ERAP1 and 2 trim peptides in the endoplasmic reticulum for direct presentation whereas PLAP has been recently implicated in cross presentation. The best characterized member of the family, ERAP1, has unique enzymatic properties that fit well with its role in antigen processing. ERAP1 can trim a large variety of long peptide sequences and efficiently accumulate mature antigenic epitopes of 8–9 amino acids long. In this study we evaluate the ability of PLAP to process antigenic peptide precursors in vitro and compare it to ERAP1. We find that, similarly to ERAP1, PLAP can trim a variety of long peptide sequences efficiently and, in most cases, accumulates appreciable amounts of correct length mature antigenic epitope. Again, similarly to ERAP1, PLAP continued trimming some of the epitopes tested and accumulated smaller products effectively destroying the epitope. However, the intermediate accumulation properties of ERAP1 and PLAP are distinct and epitope dependent, suggesting that these two enzymes may impose different selective pressures on epitope generation. Overall, although PLAP has the necessary enzymatic properties to participate in generating or destroying MHC class I presented peptides, its trimming behavior is distinct from that of ERAP1, something that supports a separate role for these two enzymes in antigen processing. PMID:20592285

  4. [Molecular cloning of activin betaA subunit mature peptide from peafowl and its application in taxonomy and phylogeny].

    PubMed

    Zou, Fang-Dong; Tong, Xin-Xin; Yue, Bi-Song

    2005-03-01

    The sequences of activin gene betaA subunit mature peptide have been amplified from white peafowl, blue peafowl (pavo cristatus) and green peafowl (pavo muticus) genomic DNA by polymerase chain reaction (PCR) with a pair of degenerate primers. The target fragments were cloned into the vector pMD18-T and sequenced. The length of activin gene betaA subunit mature peptide is 345bp, which encoded a peptide of 115 amino acid residues. Sequence analysis of activin gene betaA subunit mature peptide demonstrated that the identity of nucleotide is 98.0% between blue peaflowl and green peafowl, and the identity of that is 98.8% between blue peaflowl and white peafow. Sequences comparison in NCBI revealed that the sequences of activin gene betaA subunit mature peptides of different species are highly conserved during evolution process. In addition, the restriction enzyme map of activins is high similar between white peafowl and blue peafowl. Phylogenetic tree was constructed with Mega 2 and Clustalxldx software. The result showed that white peafowl has a closer relationship to blue peafowl than to green peafowl. Considered the nucleotide differences of peafowls' activin gene betaA subunit mature peptides, a highly conserved region, we supported that white peafowl was derived from blue peafowl, and it is more possible the hybrid but just the product of color mutation, or maybe as a subspecies of Pavo genus. PMID:15843351

  5. Occurrence and function of D-amino acid-containing peptides and proteins: antimicrobial peptides.

    PubMed

    Mignogna, G; Simmaco, M; Barra, D

    1998-01-01

    Antimicrobial peptides are widely distributed in living organisms, where they represent a constitutive defence system acting as a first line of response against infections. The number of such peptides discovered has increased rapidly in the last few years, and more than 100 have been described from different sources. So far, antimicrobial peptides containing a D-amino acid have only been found in the skin secretions of frogs belonging to the genus Bombina. In the second position of the sequence of the mature peptides either D-alloisoleucine or D-leucine were detected. The D-amino acids are derived from the corresponding L forms by an as yet unknown posttranslational reaction. PMID:9949866

  6. Roles of the signal peptide and mature domains in the secretion and maturation of the neutral metalloprotease from Streptomyces cacaoi.

    PubMed Central

    Chang, S C; Su, M H; Lee, Y H

    1997-01-01

    The neutral metalloprotease (Npr) of Streptomyces cacaoi is synthesized as a prepro-Npr precursor form consisting of a secretory signal peptide, a propeptide and the mature metalloprotease. The maturation of Npr occurs extracellularly via an autoproteolytic processing of the secreted pro-Npr. The integrity of the propeptide is essential for the formation of mature active Npr but not for its secretion [Chang, Chang and Lee (1994) J. Biol. Chem. 269, 3548-3554]. In this study we investigated whether the secretion and maturation of Npr require the integrity of its signal peptide region and mature protease domain. Five signal peptide mutants were generated, including the substitution mutations at the positively charged region (mutant IR6LE), the central hydrophobic region (mutants GI19EL and G19N), the boundary of the hydrophobic core-cleavage region (mutant P30L) and at the residues adjacent to the signal peptidase cleavage site (mutant YA33SM). All these lesions delayed the export of Npr to the growth medium and also resulted in a 2-10-fold decrease in Npr export. The most severe effect was noted in mutants GI19EL and P30L. When these signal peptide mutations were fused separately with the propeptide lacking the Npr mature domain, the secretory defect on the propeptide was also observed, and this impairment was again more severely expressed in mutants GI19EL and P30L. Thus the Npr signal peptide seems to have more constraints on the hydrophobic core region and at the proline residue within the boundary of the hydrophobic core-cleavage site. Deletion mutations within the C-terminal mature protease domain that left its active site intact still blocked the proteolytic processing of mutant precursor forms of pro-Npr, although their secretions were unaffected. These results, together with our previous findings, strongly suggest that the signal peptide of Npr plays a pivotal role in the secretion of both Npr and the propeptide, but not in the maturation of Npr. On the

  7. A relaxin-like peptide purified from radial nerves induces oocyte maturation and ovulation in the starfish, Asterina pectinifera

    PubMed Central

    Mita, Masatoshi; Yoshikuni, Michiyasu; Ohno, Kaoru; Shibata, Yasushi; Paul-Prasanth, Bindhu; Pitchayawasin, Suthasinee; Isobe, Minoru; Nagahama, Yoshitaka

    2009-01-01

    Gonad-stimulating substance (GSS) of starfish is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to the vertebrate luteinizing hormone (LH). Here, we purified GSS of starfish, Asterina pectinifera, from radial nerves and determined its amino acid sequence. The purified GSS was a heterodimer composed of 2 different peptides, A and B chains, with disulfide cross-linkages. Based on its cysteine motif, starfish GSS was classified as a member of the insulin/insulin-like growth factor (IGF)/relaxin superfamily. The cDNA of GSS encodes a preprohormone sequence with a C peptide between the A and B chains. Phylogenetic analyses revealed that starfish GSS was a relaxin-like peptide. Chemically synthesized GSS induced not only oocyte maturation and ovulation in isolated ovarian fragments, but also unique spawning behavior, followed by release of gametes shortly after the injection. Importantly, the action of the synthetic GSS on oocyte maturation and ovulation was mediated through the production of cAMP by isolated ovarian follicle cells, thereby producing the maturation-inducing hormone of this species, 1-methyladenine. In situ hybridization showed the transcription of GSS to occur in the periphery of radial nerves at the side of tube feet. Together, the structure, sequence, and mode of signal transduction strongly suggest that GSS is closely related to the vertebrate relaxin. PMID:19470645

  8. AFM-based force spectroscopy measurements of mature amyloid fibrils of the peptide glucagon

    NASA Astrophysics Data System (ADS)

    Dong, Mingdong; Bruun Hovgaard, Mads; Mamdouh, Wael; Xu, Sailong; Otzen, Daniel Erik; Besenbacher, Flemming

    2008-09-01

    We report on the mechanical characterization of individual mature amyloid fibrils by atomic force microscopy (AFM) and AFM-based single-molecule force spectroscopy (SMFS). These self-assembling materials, formed from the 29-residue amphiphatic peptide hormone glucagon, were found to display a reversible elastic behaviour. Based on AFM morphology and SMFS studies, we suggest that the observed elasticity is due to a force-induced conformational transition which is reversible due to the β-helical conformation of protofibrils, allowing a high degree of extension. The elastic properties of such mature fibrils contribute to their high stability, suggesting that the internal hydrophobic interactions of amyloid fibrils are likely to be of fundamental importance in the assembly of amyloid fibrils and therefore for the understanding of the progression of their associated pathogenic disorders. In addition, such biological amyloid fibril structures with highly stable mechanical properties can potentially be used to produce nanofibres (nanowires) that may be suitable for nanotechnological applications.

  9. A Novel Mechanism of Lysosomal Acid Sphingomyelinase Maturation

    PubMed Central

    Jenkins, Russell W.; Idkowiak-Baldys, Jolanta; Simbari, Fabio; Canals, Daniel; Roddy, Patrick; Riner, Clarke D.; Clarke, Christopher J.; Hannun, Yusuf A.

    2011-01-01

    Acid sphingomyelinase (aSMase) catalyzes the hydrolysis of sphingomyelin (SM) to form the bioactive lipid ceramide (Cer). Notably, aSMase exists in two forms: a zinc (Zn2+)-independent lysosomal aSMase (L-SMase) and a Zn2+-dependent secreted aSMase (S-SMase) that arise from alternative trafficking of a single protein precursor. Despite extensive investigation into the maturation and trafficking of aSMase, the exact identity of mature L-SMase has remained unclear. Here, we describe a novel mechanism of aSMase maturation involving C-terminal proteolytic processing within, or in close proximity to, endolysosomes. Using two different C-terminal-tagged constructs of aSMase (V5, DsRed), we demonstrate that aSMase is processed from a 75-kDa, Zn2+-activated proenzyme to a mature 65 kDa, Zn2+-independent L-SMase. L-SMase is recognized by a polyclonal Ab to aSMase, but not by anti-V5 or anti-DsRed antibodies, suggesting that the C-terminal tag is lost during maturation. Furthermore, indirect immunofluorescence staining demonstrated that mature L-SMase colocalized with the lysosomal marker LAMP1, whereas V5-aSMase localized to the Golgi secretory pathway. Moreover, V5-aSMase possessed Zn2+-dependent activity suggesting it may represent the common protein precursor of S-SMase and L-SMase. Importantly, the 65-kDa L-SMase, but not V5-aSMase, was sensitive to the lysosomotropic inhibitor desipramine, co-fractionated with lysosomes, and migrated at the same Mr as partially purified human aSMase. Finally, three aSMase mutants containing C-terminal Niemann-Pick mutations (R600H, R600P, ΔR608) exhibited defective proteolytic maturation. Taken together, these results demonstrate that mature L-SMase arises from C-terminal proteolytic processing of pro-aSMase and suggest that impaired C-terminal proteolysis may lead to severe defects in L-SMase function. PMID:21098024

  10. Solid Phase Synthesis of C-Terminal Boronic Acid Peptides.

    PubMed

    Behnam, Mira A M; Sundermann, Tom R; Klein, Christian D

    2016-05-01

    Peptides and peptidomimetics with a C-terminal boronic acid group have prolific applications in numerous fields of research, but their synthetic accessibility remains problematic. A convenient, high yield synthesis of peptide-boronic acids on a solid support is described here, using commercially available 1-glycerol polystyrene resin. The method is compatible with Fmoc chemistry and offers a versatile approach to aryl and alkyl aminoboronic acids without additional purification steps. PMID:27104613

  11. Ribosomal Synthesis of Peptides with Multiple β-Amino Acids.

    PubMed

    Fujino, Tomoshige; Goto, Yuki; Suga, Hiroaki; Murakami, Hiroshi

    2016-02-17

    The compatibility of β-amino acids with ribosomal translation was studied for decades, but it has been still unclear whether the ribosome can accept various β-amino acids, and whether the ribosome can introduce multiple β-amino acids in a peptide. In the present study, by using the Escherichia coli reconstituted cell-free translation system with a reprogramed genetic code, we screened β-amino acids that give high single incorporation efficiency and used them to synthesize peptides containing multiple β-amino acids. The experiments of single β-amino acid incorporation into a peptide revealed that 13 β-amino acids are compatible with ribosomal translation. Six of the tested β-amino acids (βhGly, l-βhAla, l-βhGln, l-βhPhg, l-βhMet, and d-βhPhg) showed high incorporation efficiencies, and seven (l-βhLeu, l-βhIle, l-βhAsn, l-βhPhe, l-βhLys, d-βhAla, and d-βhLeu) showed moderate incorporation efficiencies; whereas no full-length peptide was produced using other β-amino acids (l-βhPro, l-βhTrp, and l-βhGlu). Subsequent double-incorporation experiments using β-amino acids with high single incorporation efficiency revealed that elongation of peptides with successive β-amino acids is prohibited. Efficiency of the double-incorporation of the β-amino acids was restored by the insertion of Tyr or Ile between the two β-amino acids. On the basis of these experiments, we also designed mRNA sequences of peptides, and demonstrated the ribosomal synthesis of peptides containing different types of β-amino acids at multiple positions. PMID:26807980

  12. Designing amino acids to determine the local conformations of peptides.

    PubMed Central

    Burgess, A W

    1994-01-01

    The local conformations of proteins and peptides are determined by the amino acid sequence. However, the 20 amino acids encoded by the genome allow the peptide backbone to fold into many conformations, so that even for a small peptide it becomes very difficult to predict the three-dimensional structure. By using empirical conformational energy calculations, a set of amino acids has been designed that would be expected to constrain the conformation of a peptide or a protein to one or two local minima. Most of these amino acids are based on asymmetric substitutions at the C alpha atom of each residue. The H alpha atom of alanine was replaced by various groups: -OCH3, -NCH3, -SCH3, -CONH2, -CONHCH3, -CON(CH3)2, -NH.CO.CH3, -phenyl, or -o-(OCH3)phenyl. Several of these new amino acids are predicted to fold into unique peptide conformations such as right-handed alpha-helical, left-handed alpha-helical, or extended. In an attempt to produce an amino acid that favored the C(eq)7 conformation (torsion angles: phi = -70 degrees and psi = +70 degrees), an extra amide group was added to the C beta atom of the asparagine side chain. Conformationally restricted amino acids of this type could prove useful for developing new peptide pharmaceuticals, catalysts, or polymers. Images PMID:8146170

  13. Design and characterization of an acid-activated antimicrobial peptide.

    PubMed

    Li, Lina; He, Jian; Eckert, Randal; Yarbrough, Daniel; Lux, Renate; Anderson, Maxwell; Shi, Wenyuan

    2010-01-01

    Dental caries is a microbial biofilm infection in which the metabolic activities of plaque bacteria result in a dramatic pH decrease and shift the demineralization/remineralization equilibrium on the tooth surface towards demineralization. In addition to causing a net loss in tooth minerals, creation of an acidic environment favors growth of acid-enduring and acid-generating species, which causes further reduction in the plaque pH. In this study, we developed a prototype antimicrobial peptide capable of achieving high activity exclusively at low environmental pH to target bacterial species like Streptococcus mutans that produce acid and thrive under the low pH conditions detrimental for tooth integrity. The features of clavanin A, a naturally occurring peptide rich in histidine and phenylalanine residues with pH-dependent antimicrobial activity, served as a design basis for these prototype 'acid-activated peptides' (AAPs). Employing the major cariogenic species S. mutans as a model system, the two AAPs characterized in this study exhibited a striking pH-dependent antimicrobial activity, which correlated well with the calculated charge distribution. This type of peptide represents a potential new way to combat dental caries. PMID:19878192

  14. Heat capacities of amino acids, peptides and proteins.

    PubMed

    Makhatadze, G I

    1998-04-20

    The heat capacity is one of the fundamental parameters describing thermodynamic properties of a system. It has wide applications in a number of areas such as polymer chemistry, protein folding and DNA stability. To aid the scientific community in the analysis of such data, I have compiled a database on the experimentally measured heat capacities of amino acids, polyamino acids, peptides, and proteins in solid state and in aqueous solutions. PMID:9648205

  15. Changes in physicochemical characteristics and free amino acids of hawthorn (Crataegus pinnatifida) fruits during maturation.

    PubMed

    Li, Wei-Qin; Hu, Qing-Ping; Xu, Jian-Guo

    2015-05-15

    In this study, changes in physicochemical characteristics associated with fruit quality and free amino acids were investigated during maturation of hawthorn fruits. Significant differences in these parameters were found during maturation. The color turned progressively from mature green to semi-red, to reach bright red; the shape changed gradually from oval to round or approached round; the size, weight, and edible part (flesh/core ratio) of hawthorns increased while the density of intact fruits did not change. The content of moisture, total soluble sugars, soluble pectin, reduced ascorbic acid, total ascorbic acid, fructose, and sucrose increased while crude protein content decreased significantly. The levels of starch, sucrose, titratable acidity, protopectin, pectin, total free amino acids, and total essential amino acids initially increased and then decreased gradually during maturation. The outcomes of this study provide additional and useful information for fresh consumption and processing as well as utilization of dropped unripe hawthorn fruits. PMID:25577050

  16. Nutritional value of D-amino acids, D-peptides, and amino acid derivatives in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This paper describes a method for determining the nutritional value of D-amino acids, D-peptides, and amino acid derivatives using a growth assay in mice fed a synthetic all-amino acid diet. A large number of experiments were carried out in which a molar equivalent of the test compound replaced a n...

  17. Behavior of amino acids and peptides exposed in Earth orbit

    NASA Astrophysics Data System (ADS)

    Barbier, Bernard; Boillot, François; Chabin, Annie; Venet, Michel; Bure, Corinne; Jacquet, Romain; Bertrand-Urbaniak, Marylène; Brack, André

    2001-08-01

    In order to understand the chemical comportment of organic molecules of prebiotic interest when exposed to space conditions, amino acids, derivatives and peptides where exposed in Earth orbit during the CNES "Perseus-Exobiologie" mission. Dry films of samples were exposed free or associated with mineral powders to vacuum and to solar light down to 120 nm during three months outside the MIR station. After the mission, the remaining products were analyzed with respect of chemical degradation, racemization and polymerization. The analyses revealed a higher sensitivity of amino acids comparatively to peptides. The identification of by-products has allowed determining some photolysis pathways where decarboxylation and decarbonylation were found to be the major chemical reactions for amino acids and peptides, respectively. The study of associated minerals have shown that meteoritic powder was the most efficient to protect samples against UV light. The exposure of different peptides associated to meteorite powder of various thickness have allowed to determine that 5μm films were at least necessary to protect associated organics. Implications for the exogenous origin of organics are discussed.

  18. How Amino Acids and Peptides Shaped the RNA World

    PubMed Central

    van der Gulik, Peter T.S.; Speijer, Dave

    2015-01-01

    The “RNA world” hypothesis is seen as one of the main contenders for a viable theory on the origin of life. Relatively small RNAs have catalytic power, RNA is everywhere in present-day life, the ribosome is seen as a ribozyme, and rRNA and tRNA are crucial for modern protein synthesis. However, this view is incomplete at best. The modern protein-RNA ribosome most probably is not a distorted form of a “pure RNA ribosome” evolution started out with. Though the oldest center of the ribosome seems “RNA only”, we cannot conclude from this that it ever functioned in an environment without amino acids and/or peptides. Very small RNAs (versatile and stable due to basepairing) and amino acids, as well as dipeptides, coevolved. Remember, it is the amino group of aminoacylated tRNA that attacks peptidyl-tRNA, destroying the bond between peptide and tRNA. This activity of the amino acid part of aminoacyl-tRNA illustrates the centrality of amino acids in life. With the rise of the “RNA world” view of early life, the pendulum seems to have swung too much towards the ribozymatic part of early biochemistry. The necessary presence and activity of amino acids and peptides is in need of highlighting. In this article, we try to bring the role of the peptide component of early life back into focus. We argue that an RNA world completely independent of amino acids never existed. PMID:25607813

  19. Recent Developments in Peptide-Based Nucleic Acid Delivery

    PubMed Central

    Veldhoen, Sandra; Laufer, Sandra D.; Restle, Tobias

    2008-01-01

    Despite the fact that non-viral nucleic acid delivery systems are generally considered to be less efficient than viral vectors, they have gained much interest in recent years due to their superior safety profile compared to their viral counterpart. Among these synthetic vectors are cationic polymers, branched dendrimers, cationic liposomes and cell-penetrating peptides (CPPs). The latter represent an assortment of fairly unrelated sequences essentially characterised by a high content of basic amino acids and a length of 10–30 residues. CPPs are capable of mediating the cellular uptake of hydrophilic macromolecules like peptides and nucleic acids (e.g. siRNAs, aptamers and antisense-oligonucleotides), which are internalised by cells at a very low rate when applied alone. Up to now, numerous sequences have been reported to show cell-penetrating properties and many of them have been used to successfully transport a variety of different cargos into mammalian cells. In recent years, it has become apparent that endocytosis is a major route of internalisation even though the mechanisms underlying the cellular translocation of CPPs are poorly understood and still subject to controversial discussions. In this review, we will summarise the latest developments in peptide-based cellular delivery of nucleic acid cargos. We will discuss different mechanisms of entry, the intracellular fate of the cargo, correlation studies of uptake versus biological activity of the cargo as well as technical problems and pitfalls. PMID:19325804

  20. Recent developments in peptide-based nucleic acid delivery.

    PubMed

    Veldhoen, Sandra; Laufer, Sandra D; Restle, Tobias

    2008-06-01

    Despite the fact that non-viral nucleic acid delivery systems are generally considered to be less efficient than viral vectors, they have gained much interest in recent years due to their superior safety profile compared to their viral counterpart. Among these synthetic vectors are cationic polymers, branched dendrimers, cationic liposomes and cell-penetrating peptides (CPPs). The latter represent an assortment of fairly unrelated sequences essentially characterised by a high content of basic amino acids and a length of 10-30 residues. CPPs are capable of mediating the cellular uptake of hydrophilic macromolecules like peptides and nucleic acids (e.g. siRNAs, aptamers and antisense-oligonucleotides), which are internalised by cells at a very low rate when applied alone. Up to now, numerous sequences have been reported to show cell-penetrating properties and many of them have been used to successfully transport a variety of different cargos into mammalian cells. In recent years, it has become apparent that endocytosis is a major route of internalisation even though the mechanisms underlying the cellular translocation of CPPs are poorly understood and still subject to controversial discussions. In this review, we will summarise the latest developments in peptide-based cellular delivery of nucleic acid cargos. We will discuss different mechanisms of entry, the intracellular fate of the cargo, correlation studies of uptake versus biological activity of the cargo as well as technical problems and pitfalls. PMID:19325804

  1. [Molecular cloning of the DNA sequence of activin beta A subunit gene mature peptides from panda and related species and its application in the research of phylogeny and taxonomy].

    PubMed

    Wang, Xiao-Jing; Wang, Xiao-Xing; Wang, Ya-Jun; Wang, Xi-Zhong; He, Guang-Xin; Chen, Hong-Wei; Fei, Li-Song

    2002-09-01

    Activin, which is included in the transforming growth factor-beta (TGF beta) superfamily of proteins and receptors, is known to have broad-ranging effects in the creatures. The mature peptide of beta A subunit of this gene, one of the most highly conserved sequence, can elevate the basal secretion of follicle-stimulating hormone (FSH) in the pituitary and FSH is pivotal to organism's reproduction. Reproduction block is one of the main reasons which cause giant panda to extinct. The sequence of Activin beta A subunit gene mature peptides has been successfully amplified from giant panda, red panda and malayan sun bear's genomic DNA by using polymerase chain reaction (PCR) with a pair of degenerate primers. The PCR products were cloned into the vector pBlueScript+ of Esherichia coli. Sequence analysis of Activin beta A subunit gene mature peptides shows that the length of this gene segment is the same (359 bp) and there is no intron in all three species. The sequence encodes a peptide of 119 amino acid residues. The homology comparison demonstrates 93.9% DNA homology and 99% homology in amino acid among these three species. Both GenBank blast search result and restriction enzyme map reveal that the sequences of Activin beta A subunit gene mature peptides of different species are highly conserved during the evolution process. Phylogeny analysis is performed with PHYLIP software package. A consistent phylogeny tree has been drawn with three different methods. The software analysis outcome accords with the academic view that giant panda has a closer relationship to the malayan sun bear than the red panda. Giant panda should be grouped into the bear family (Uersidae) with the malayan sun bear. As to the red panda, it would be better that this animal be grouped into the unique family (red panda family) because of great difference between the red panda and the bears (Uersidae). PMID:12561224

  2. Pulse-chase analysis for studies of MHC class II biosynthesis, maturation, and peptide loading

    PubMed Central

    Hou, Tieying; Rinderknecht, Cornelia H; Hadjinicolaou, Andreas V; Busch, Robert; Mellins, Elizabeth

    2014-01-01

    Pulse-chase analysis is a commonly used technique for studying the synthesis, processing and transport of proteins. Cultured cells expressing proteins of interest are allowed to take up radioactively labeled amino acids for a brief interval (“pulse”), during which all newly synthesized proteins incorporate the label. The cells are then returned to non-radioactive culture medium for various times (“chase”), during which proteins may undergo conformational changes, trafficking, or degradation. Proteins of interest are isolated (usually by immunoprecipitation) and resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the fate of radiolabeled molecules is examined by autoradiography. This chapter describes a pulse-chase protocol suitable for studies of major histocompatibility complex (MHC) class II biosynthesis and maturation. We discuss how results are affected by the recognition by certain anti-class II antibodies of distinct class II conformations associated with particular biosynthetic states. Our protocol can be adapted to follow the fate of many other endogenously synthesized proteins, including viral or transfected gene products, in cultured cells. PMID:23329504

  3. Regulation of peptide YY homeostasis by gastric acid and gastrin.

    PubMed

    Gomez, G; Padilla, L; Udupi, V; Tarasova, N; Sundler, F; Townsend, C M; Thompson, J C; Greeley, G H

    1996-04-01

    Peptide YY (PYY) is a gut hormone localized primarily in the distal bowel. Because circulating PYY inhibits gastric acid secretion, we investigated the effects of gastric acid secretion and gastrin on gene expression and secretion of PYY. In conscious dogs, PYY release in response to oral food was inhibited (P < 0.05) by pharmacologic inhibition of gastric acid secretion (omeprazole, famotidine). In rats, omeprazole treatment resulted in a significant elevation in serum gastrin concentrations and a simultaneous decrease in PYY messenger RNA (mRNA) and peptide levels in the colon; administration of a gastrin receptor antagonist (L365, 260) prevented the inhibitory actions of omeprazole on colonic PYY mRNA levels. In athymic-nude mice, implantation of a human gastrinoma resulted in an elevation of serum gastrin concentrations and a concomitant depression of colonic PYY mRNA levels. We conclude that endogenous gastric acid secretion up-regulates PYY release and PYY mRNA expression. Circulating gastrin acts to down-regulate PYY release and PYY mRNA expression. This study provides evidence that foregut functions (i.e., gastric acid secretion and gastrin release) exert control over an antiacid signal (e.g. PYY release) emanating from the hindgut. PMID:8625912

  4. Photosensitizing effect of cations on amino acids and peptides.

    PubMed

    Bogdanova, N P; Khenokh, M A

    1969-01-01

    In connection with a study of the chemical evolution of abiogenically synthesized organic compounds on primitive Earth and the physical conditions of other planets, this paper reports the experimental results obtained by the photolysis of solutions of aliphatic amino acids (glycine, alanine, valine, leucine, n. leucine) and peptides in the atmosphere of the air, N2, Ar and CO2 in the presence of the most simple photocatalyzers-cations of sulphates. The evidence shows that the photochemical conversion of NH2 acids depends on the content of the atmosphere. The decay of NH2-group is most active in air. N2 and Ar exert no significant influence on deamination, whereas in the atmosphere of CO2 the formation of ammonia in valine, for example, was only 29 per cent of its total amount during photolysis in the air. Cu2+ and Fe2+ catalyzed while Al3+ inhibited the ammonia excretion. The formation of acetaldehyde during alanine photolysis was actually independent from the atmosphere of N2 and was inhibited in Ar and CO2. Oxydative processes inducing the formation of glyoxalic acid and formaldehyde were sharply inhibited in Ar, N2 and CO2. Under the influence of ultraviolet light of the decay of NH2-acids is also accompanied by the formation of new NH2-acids. The photosensitizing effect of cations induces a rupture of -CO-NH-bonds in peptides and, provided heavy radiation doses, prevents the formation of new NH2-acids. The longer the dipeptide chain, the more significant the quantum yield of its decomposition. The photolysis of dipeptides, leading to their decay, does not necessarily induce a hydrolytic rupture of -CO-NH-bonds resulting in the formation of three amino acids. The results obtained permit approaching problems concerning the effect of the gas content of the atmosphere and various cations (photocatalyzers) on photolytic conversion of abiogenically synthesized and biogenically significant substances, amino acids for example, at the action of ultraviolet light. PMID

  5. Neonatal skin maturation--vernix caseosa and free amino acids.

    PubMed

    Visscher, Marty O; Utturkar, Radhika; Pickens, William L; LaRuffa, Angela A; Robinson, Marisa; Wickett, R Randell; Narendran, Vivek; Hoath, Steven B

    2011-01-01

    Neonatal skin hydration decreases rapidly postnatally and then increases, indicating adaptive changes in stratum corneum water handling properties. Transition from high to low humidity at birth may initiate filaggrin proteolysis to free amino acids. Neonatal skin with vernix caseosa retained is more hydrated than skin with vernix removed. This study examines the potential roles of free amino acids and vernix in postnatal adaptation of infant stratum corneum in vivo. Specifically, the ontogeny of free amino acid generation in neonatal stratum corneum and the role of vernix caseosa in postnatal adaptation were examined using high performance liquid chromatography. Free amino acids were quantified for infant skin samples collected at (i) birth and 1 month and (ii) birth and 24 hours after vernix caseosa retention or removal and compared to neonatal foreskin, vernix caseosa, and adult stratum corneum using t-tests, analysis of variance, or univariate procedures. Free amino acids were extremely low at birth, significantly higher 1 month later but lower than in adults. Vernix caseosa retention led to significantly higher free amino acids 24 hours after birth compared to infants with vernix caseosa removed, and it paralleled the higher stratum corneum hydration of vernix caseosa-retained skin. Vernix caseosa contained free amino acids, with glutamic acid and histidine levels higher than in infants. Free amino acids in vernix caseosa-retained skin appear to originate from vernix caseosa. Free amino acids were lower in neonatal foreskin than adult forearm stratum corneum. Arginine was higher than citrulline at birth, but levels were comparable in older infants. The free amino acid increase at 1 month may be initiated by the humidity transition at birth and supports results in animals. The findings have implications for infant skin care practices. PMID:21504444

  6. A nine-country study of the protein content and amino acid composition of mature human milk

    PubMed Central

    Feng, Ping; Gao, Ming; Burgher, Anita; Zhou, Tian Hui; Pramuk, Kathryn

    2016-01-01

    Background Numerous studies have evaluated protein and amino acid levels in human milk. However, research in this area has been limited by small sample sizes and study populations with little ethnic or racial diversity. Objective Evaluate the protein and amino acid composition of mature (≥30 days) human milk samples collected from a large, multinational study using highly standardized methods for sample collection, storage, and analysis. Design Using a single, centralized laboratory, human milk samples from 220 women (30–188 days postpartum) from nine countries were analyzed for amino acid composition using Waters AccQ-Tag high-performance liquid chromatography and total nitrogen content using the LECO FP-528 nitrogen analyzer. Total protein was calculated as total nitrogen×6.25. True protein, which includes protein, free amino acids, and peptides, was calculated from the total amino acids. Results Mean total protein from individual countries (standard deviation [SD]) ranged from 1,133 (125.5) to 1,366 (341.4) mg/dL; the mean across all countries (SD) was 1,192 (200.9) mg/dL. Total protein, true protein, and amino acid composition were not significantly different across countries except Chile, which had higher total and true protein. Amino acid profiles (percent of total amino acids) did not differ across countries. Total and true protein concentrations and 16 of 18 amino acid concentrations declined with the stage of lactation. Conclusions Total protein, true protein, and individual amino acid concentrations in human milk steadily decline from 30 to 151 days of lactation, and are significantly higher in the second month of lactation compared with the following 4 months. There is a high level of consistency in the protein content and amino acid composition of human milk across geographic locations. The size and diversity of the study population and highly standardized procedures for the collection, storage, and analysis of human milk support the validity and

  7. Role of hyaluronic acid in maturation and further early embryo development of bovine oocytes.

    PubMed

    Marei, W F; Ghafari, F; Fouladi-Nashta, A A

    2012-08-01

    Hyaluronic acid (HA), an important component of the extracellular matrix, plays a crucial role for cumulus cell expansion. Genes and proteins involved in HA synthesis and its receptor CD44 are expressed in cumulus oocyte complexes (COCs) in different animal species and increase during maturation. Hyaluronidase enzymes (Hyal) degrade HA into smaller biologically active HA fragments. To investigate the effects of the molecular size and concentration of HA on oocyte maturation and further embryo development, bovine oocytes were matured in vitro in the presence or absence of HA, Hyal-2 or 4-methylumbelliferone (4-MU); an HA synthesis inhibitor. The rates of oocyte nuclear maturation to metaphase II stage and development of embryos to blastocyst stage and blastocyst quality were recorded. Hyal-2 inhibited cumulus cell expansion without affecting oocyte maturation and further embryo development. Whereas, 4-MU at 1 mm reduced cumulus cell expansion, oocyte maturation rate and further embryo development; an effect which was partially abrogated by exogenous HA supplementation. These data suggest that HA production by cumulus cells during maturation is essential not only for cumulus cell expansion, but also for oocyte maturation and further embryo development. This effect is not affected by HA-degradation by Hyal-2. PMID:22541325

  8. ACID AIR AND AEROBIOLOGY RELATED TO THE MATURING HUMAN LUNG

    EPA Science Inventory

    The effect of 'acid air' on human health was studied by considering the effects of hygroscopicity upon aerosol deposition in the lung as a function of human subject age. Children are a critical sub-population to be incorporated into health effects analyses following ambient expos...

  9. The nature of peptide interactions with acid end-group PLGAs and facile aqueous-based microencapsulation of therapeutic peptides

    PubMed Central

    Sophocleous, Andreas M.; Desai, Kashappa-Goud H.; Mazzara, J. Maxwell; Tong, Ling; Cheng, Ji-Xin; Olsen, Karl F.; Schwendeman, Steven P.

    2013-01-01

    An important poorly understood phenomenon in controlled-release depots involves the strong interaction between common cationic peptides and low Mw free acid end-group poly(lactic-co-glycolic acids) (PLGAs) used to achieve continuous peptide release kinetics. The kinetics of peptide sorption to PLGA was examined by incubating peptide solutions of 0.2-4 mM octreotide or leuprolide acetate salts in 0.1 M HEPES buffer, pH 7.4, with polymer particles or films at 4-37 °C for 24 h. The extent of absorption/loading of peptides in PLGA particles/films was assayed by two-phase extraction and amino acid analysis. Confocal Raman microspectroscopy and stimulated Raman scattering (SRS) and laser scanning confocal imaging techniques were used to examine peptide penetration in the polymer phase. The release of sorbed peptide from leuprolide-PLGA particles was evaluated both in vitro (PBST + 0.02% sodium azide, 37 °C) and in vivo (male Sprague-Dawley rats). We found that when the PLGA-COOH chains are sufficiently mobilized, therapeutic peptides not only bind at the surface, a common belief to date, but can also internalized and distributed throughout the polymer phase at physiological temperature forming a salt with low-molecular weight PLGA-COOH. Importantly, absorption of leuprolide into low MW PLGA-COOH particles yielded ~17 wt% leuprolide loading in the polymer (i.e., ~70% of PLGA-COOH acids occupied), and the absorbed peptide was released from the polymer for > 2 weeks in a controlled fashion in vitro and as indicated by sustained testosterone suppression in male Sprague-Dawley rats. This new approach, which bypasses the traditional encapsulation method and associated production cost, opens up the potential for facile production of low-cost controlled-release injectable depots for leuprolide and related peptides. PMID:24021356

  10. AN INSULIN-LIKE PEPTIDE REGULATES EGG MATURATION AND METABOLISM IN THE MOSQUITO AEDES AEGYPTI

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ingestion of vertebrate blood is essential for egg maturation and transmission of disease-causing parasites by female mosquitoes. Prior studies with the yellow fever mosquito, Aedes aegypti, indicated blood feeding stimulates egg production by triggering the release of hormones from MNCs in the mosq...

  11. Cyclolization of D-lysergic acid alkaloid peptides.

    PubMed

    Havemann, Judith; Vogel, Dominik; Loll, Bernhard; Keller, Ullrich

    2014-01-16

    The tripeptide chains of the ergopeptines, a class of pharmacologically important D-lysergic acid alkaloid peptides, are arranged in a unique bicyclic cyclol based on an amino-terminal α-hydroxyamino acid and a terminal orthostructure. D-lysergyl-tripeptides are assembled by the nonribosomal peptide synthetases LPS1 and LPS2 of the ergot fungus Claviceps purpurea and released as N-(D-lysergyl-aminoacyl)-lactams. We show total enzymatic synthesis of ergopeptines catalyzed by a Fe²⁺/2-ketoglutarate-dependent dioxygenase (EasH) in conjunction with LPS1/LPS2. Analysis of the reaction indicated that EasH introduces a hydroxyl group into N-(D-lysergyl-aminoacyl)-lactam at α-C of the aminoacyl residue followed by spontaneous condensation with the terminal lactam carbonyl group. Sequence analysis revealed that EasH belongs to the wide and diverse family of the phytanoyl coenzyme A hydroxylases. We provide a high-resolution crystal structure of EasH that is most similar to that of phytanoyl coenzyme A hydroxylase, PhyH, from human. PMID:24361048

  12. Identification of protein N-termini in Cyanophora paradoxa cyanelles: transit peptide composition and sequence determinants for precursor maturation

    PubMed Central

    Köhler, Daniel; Dobritzsch, Dirk; Hoehenwarter, Wolfgang; Helm, Stefan; Steiner, Jürgen M.; Baginsky, Sacha

    2015-01-01

    Glaucophyta, rhodophyta, and chloroplastida represent the three main evolutionary lineages that diverged from a common ancestor after primary endosymbiosis. Comparative analyses between members of these three lineages are a rich source of information on ancestral plastid features. We analyzed the composition and the cleavage site of cyanelle transit peptides from the glaucophyte Cyanophora paradoxa by terminal amine labeling of substrates (TAILS), and compared their characteristics to those of representatives of the chloroplastida. Our data show that transit peptide architecture is similar between members of these two lineages. This entails a comparable modular structure, an overrepresentation of serine or alanine and similarities in the amino acid composition around the processing peptidase cleavage site. The most distinctive difference is the overrepresentation of phenylalanine in the N-terminal 1–10 amino acids of cyanelle transit peptides. A quantitative proteome analysis with periplasm-free cyanelles identified 42 out of 262 proteins without the N-terminal phenylalanine, suggesting that the requirement for phenylalanine in the N-terminal region is not absolute. Proteins in this set are on average of low abundance, suggesting that either alternative import pathways are operating specifically for low abundance proteins or that the gene model annotation is incorrect for proteins with fewer EST sequences. We discuss these two possibilities and provide examples for both interpretations. PMID:26257763

  13. Targeting DNA G-Quadruplex Structures with Peptide Nucleic Acids

    PubMed Central

    Panyutin, Igor G.; Onyshchenko, Mykola I.; Englund, Ethan A.; Appella, Daniel H.; Neumann, Ronald D.

    2012-01-01

    Regulation of genetic functions based on targeting DNA or RNA sequences with complementary oligonucleotides is especially attractive in the post-genome era. Oligonucleotides can be rationally designed to bind their targets based on simple nucleic acid base pairing rules. However, the use of natural DNA and RNA oligonucleotides as targeting probes can cause numerous off-target effects. In addition, natural nucleic acids are prone to degradation in vivo by various nucleases. To address these problems, nucleic acid mimics such as peptide nucleic acids (PNA) have been developed. They are more stable, show less off-target effects, and, in general, have better binding affinity to their targets. However, their high affinity to DNA can reduce their sequence-specificity. The formation of alternative DNA secondary structures, such as the G-quadruplex, provides an extra level of specificity as targets for PNA oligomers. PNA probes can target the loops of G-quadruplex, invade the core by forming PNA-DNA guanine-tetrads, or bind to the open bases on the complementary cytosine-rich strand. Not only could the development of such G-quadruplex-specific probes allow regulation of gene expression, but it will also provide a means to clarify the biological roles G-quadruplex structures may possess. PMID:22376112

  14. Di-heterometalation of thiol-functionalized peptide nucleic acids

    PubMed Central

    Joshi, Tanmaya; Patra, Malay; Spiccia, Leone; Gasser, Gilles

    2013-01-01

    As a proof-of-principle, two hetero-bimetallic PNA oligomers containing a ruthenium(II) polypyridyl and a cyclopentadienyl manganese tricarbonyl complex have been prepared by serial combination of solid-phase peptide coupling and in-solution thiol chemistry. Solid-phase N-terminus attachment of Ru(II)-polypyridyl carboxylic acid derivative, C1, onto the thiol-functionalized PNA backbone (H-a-a-g-t-c-t-g-c-linker-cys-NH2) has been performed by standard peptide coupling method. As two parallel approaches, the strong affinity of thiols for maleimide and haloacetyl group has been exploited for subsequent post-SPPS addition of cymantrene-based organometallic cores, C2 and C3. Michael-like addition and thioether ligation of thiol functionalized PNA1 (H-gly-a-a-g-t-c-t-g-c-linker-cys-NH2) and PNA2 (C1-a-a-g-t-c-t-g-c-linker-cys-NH2) to cymantrene maleimide and chloroacetyl derivatives, C2 and C3, respectively, has been performed. The synthesized ruthenium(II)-cymantrenyl PNA oligomers have been characterized by mass spectrometry (ESI-MS) and IR spectroscopy. The distinct Mn-CO vibrational IR stretches, between 1,924–2,074 cm−1, have been used as markers to confirm the presence of cymantrenyl units in the PNA sequences and the purity of the HPLC-purified PNA thioethers assessed using LC-MS. PMID:23422249

  15. Intrinsic Structural Heterogeneity and Long-Term Maturation of Amyloid β Peptide Fibrils

    PubMed Central

    2013-01-01

    Amyloid β peptides form fibrils that are commonly assumed to have a dry, homogeneous, and static internal structure. To examine these assumptions, fibrils under various conditions and different ages have been examined with multidimensional infrared spectroscopy. Each peptide in the fibril had a 13C=18O label in the backbone of one residue to disinguish the amide I′ absorption due to that residue from the amide I′ absorption of other residues. Fibrils examined soon after they formed, and reexamined after 1 year in the dry state, exhibited spectral changes confirming that structurally significant water molecules were present in the freshly formed fibrils. Results from fibrils incubated in solution for 4 years indicate that water molecules remained trapped within fibrils and mobile over the 4 year time span. These water molecules are structurally significant because they perturb the parallel β-sheet hydrogen bonding pattern at frequent intervals and at multiple points within individual fibrils, creating structural heterogeneity along the length of a fibril. These results show that the interface between β-sheets in an amyloid fibril is not a “dry zipper”, and that the internal structure of a fibril evolves while it remains in a fibrillar state. These features, water trapping, structural heterogeneity, and structural evolution within a fibril over time, must be accommodated in models of amyloid fibril structure and formation. PMID:23701594

  16. Response of soil respiration to acid rain in forests of different maturity in southern China.

    PubMed

    Liang, Guohua; Liu, Xingzhao; Chen, Xiaomei; Qiu, Qingyan; Zhang, Deqiang; Chu, Guowei; Liu, Juxiu; Liu, Shizhong; Zhou, Guoyi

    2013-01-01

    The response of soil respiration to acid rain in forests, especially in forests of different maturity, is poorly understood in southern China despite the fact that acid rain has become a serious environmental threat in this region in recent years. Here, we investigated this issue in three subtropical forests of different maturity [i.e. a young pine forest (PF), a transitional mixed conifer and broadleaf forest (MF) and an old-growth broadleaved forest (BF)] in southern China. Soil respiration was measured over two years under four simulated acid rain (SAR) treatments (CK, the local lake water, pH 4.5; T1, water pH 4.0; T2, water pH 3.5; and T3, water pH 3.0). Results indicated that SAR did not significantly affect soil respiration in the PF, whereas it significantly reduced soil respiration in the MF and the BF. The depressed effects on both forests occurred mostly in the warm-wet seasons and were correlated with a decrease in soil microbial activity and in fine root biomass caused by soil acidification under SAR. The sensitivity of the response of soil respiration to SAR showed an increasing trend with the progressive maturity of the three forests, which may result from their differences in acid buffering ability in soil and in litter layer. These results indicated that the depressed effect of acid rain on soil respiration in southern China may be more pronounced in the future in light of the projected change in forest maturity. However, due to the nature of this field study with chronosequence design and the related pseudoreplication for forest types, this inference should be read with caution. Further studies are needed to draw rigorous conclusions regarding the response differences among forests of different maturity using replicated forest types. PMID:23626790

  17. Response of Soil Respiration to Acid Rain in Forests of Different Maturity in Southern China

    PubMed Central

    Chen, Xiaomei; Qiu, Qingyan; Zhang, Deqiang; Chu, Guowei; Liu, Juxiu; Liu, Shizhong; Zhou, Guoyi

    2013-01-01

    The response of soil respiration to acid rain in forests, especially in forests of different maturity, is poorly understood in southern China despite the fact that acid rain has become a serious environmental threat in this region in recent years. Here, we investigated this issue in three subtropical forests of different maturity [i.e. a young pine forest (PF), a transitional mixed conifer and broadleaf forest (MF) and an old-growth broadleaved forest (BF)] in southern China. Soil respiration was measured over two years under four simulated acid rain (SAR) treatments (CK, the local lake water, pH 4.5; T1, water pH 4.0; T2, water pH 3.5; and T3, water pH 3.0). Results indicated that SAR did not significantly affect soil respiration in the PF, whereas it significantly reduced soil respiration in the MF and the BF. The depressed effects on both forests occurred mostly in the warm-wet seasons and were correlated with a decrease in soil microbial activity and in fine root biomass caused by soil acidification under SAR. The sensitivity of the response of soil respiration to SAR showed an increasing trend with the progressive maturity of the three forests, which may result from their differences in acid buffering ability in soil and in litter layer. These results indicated that the depressed effect of acid rain on soil respiration in southern China may be more pronounced in the future in light of the projected change in forest maturity. However, due to the nature of this field study with chronosequence design and the related pseudoreplication for forest types, this inference should be read with caution. Further studies are needed to draw rigorous conclusions regarding the response differences among forests of different maturity using replicated forest types. PMID:23626790

  18. A novel glucokinase gene mutation and its effect on glycemic/C-peptide fluctuations in a patient with maturity-onset diabetes of the young type 2.

    PubMed

    Loomba-Albrecht, Lindsey A; Jame, Maryam; Bremer, Andrew A

    2010-03-01

    Maturity-onset diabetes of the young (MODY) is a group of disorders accounting for 2-5% of diabetes; MODY2 is caused by inactivating GCK mutations. We report a case of MODY2 caused by a novel GCK mutation and demonstrate differential glycemic/C-peptide responses to treatment with insulin, no medication, and an oral sulfonylurea. PMID:20015564

  19. Proteins, Peptides and Amino Acids: Role in Infant Nutrition.

    PubMed

    Nutten, Sophie

    2016-01-01

    Proteins are polymers composed of 30 or more amino acids; some of them are essential dietary components, since they are not synthetized by human metabolic processes. They are crucial for healthy growth and development and influence major functions of the body. The infant's first year is a critical time of rapid growth and development, which must be supported by a high rate of protein synthesis. Breast milk, as a single specific food source in the first months of life, is providing the total protein and essential amino acids required. Infant formulas have been designed for infants who cannot be breastfed. They should be similar to breast milk in their composition and their functional outcomes, insuring appropriate growth, optimal development, maturation of the immune system, easy digestion and healthy metabolic programming. By modifying their protein components, specific infant formulas have also been developed for specific needs. For example, partially hydrolyzed (prevention of atopic dermatitis) and extensively hydrolyzed or amino-acid-based infant formulas (reduction in allergy symptoms) have been designed for the management of cow's milk protein allergy. In conclusion, proteins provided via breast milk or infant formula are essential components of the infant's diet; therefore, the specific quality, quantity and conformation of proteins are of utmost importance for healthy growth and development. PMID:27336588

  20. Expression of genes associated with fatty acid metabolism during maturation in diploid and triploid female rainbow trout

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To study effects of sexual maturation on fatty acid metabolism in fish on a high nutritional plane, expression of thirty-five genes involved in fatty acid metabolism was determined in sexually maturing diploid (2N; fertile) and triploid (3N; sterile) female rainbow trout. Gene expression was assesse...

  1. Linoleic acid stimulates neutral lipid accumulation in lipid droplets of maturing bovine oocytes.

    PubMed

    Carro, M; Buschiazzo, J; Ríos, G L; Oresti, G M; Alberio, R H

    2013-03-01

    Linoleic acid (LA) is a polyunsaturated fatty acid present in high concentrations in bovine follicular fluid; when added to maturation culture media, it affects oocyte competence (depending on the type and concentration of LA used). To date, little is known about the effective level of incorporation of LA and there is apparently no information regarding its esterification into various lipid fractions of the oocyte and its effect on neutral lipid storage. Therefore, the objective was to assess the uptake and subcellular lipid distribution of LA by analyzing incorporation of radiolabeled LA into oocyte polar and neutral lipid classes. The effects of various concentrations of LA on the nuclear status and cytoplasmic lipid content of bovine oocytes matured in vitro was also analyzed, with particular emphasis on intermediate concentrations of LA. Neutral lipids stored in lipid droplets were quantified with a fluorescence approach. Linoleic acid at 9 and 43 μM did not affect the nuclear status of oocytes matured in vitro, and 100 μM LA inhibited germinal vesicle breakdown, resulting in a higher percentage of oocytes arrested at the germinal state (43.5 vs. 3.0 in controls; P < 0.05). Bovine oocytes actively incorporated LA from the maturation medium (83.4 pmol LA per 100 oocytes at 22 hours of incubation; P < 0.05) and metabolized it mainly into major lipid classes, e.g., triacylglycerols and phospholipids (61.1% and 29.3%, respectively). Supplementation of the maturation medium with LA increased triacylglycerol accumulation in cytoplasmic lipid droplets at all concentrations assayed (P < 0.05). In conclusion, LA added to a defined maturation medium at concentrations that did not alter the nuclear status of bovine oocytes matured in vitro (9 and 43 μM) improved their quality by increasing the content of neutral lipids stored in lipid droplets. By directing the free fatty acid (LA) to triacylglycerol synthesis pathways and increasing the degree of unsaturation of

  2. A requirement for fatty acid oxidation in the hormone-induced meiotic maturation of mouse oocytes.

    PubMed

    Valsangkar, Deepa; Downs, Stephen M

    2013-08-01

    We have previously shown that fatty acid oxidation (FAO) is required for AMP-activated protein kinase (PRKA)-induced maturation in vitro. In the present study, we have further investigated the role of this metabolic pathway in hormone-induced meiotic maturation. Incorporating an assay with (3)H-palmitic acid as the substrate, we first examined the effect of PRKA activators on FAO levels. There was a significant stimulation of FAO in cumulus cell-enclosed oocytes (CEO) treated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and RSVA405. In denuded oocytes (DO), AICAR stimulated FAO only in the presence of carnitine, the molecule that facilitates fatty acyl CoA entry into the mitochondria. The carnitine palmitoyltransferase 1 activator C75 successfully stimulated FAO in CEO. All three of these activators trigger germinal vesicle breakdown. Meiotic resumption induced by follicle-stimulating hormone (FSH) or amphiregulin was completely inhibited by the FAO inhibitors etomoxir, mercaptoacetate, and malonyl CoA. Importantly, FAO was increased in CEO stimulated by FSH and epidermal growth factor, and this increase was blocked by FAO inhibitors. Moreover, compound C, a PRKA inhibitor, prevented the FSH-induced increase in FAO. Both carnitine and palmitic acid augmented hormonal induction of maturation. In a more physiological setting, etomoxir eliminated human chorionic gonadotropin (hCG)-induced maturation in follicle-enclosed oocytes. In addition, CEO and DO from hCG-treated mice displayed an etomoxir-sensitive increase in FAO, indicating that this pathway was stimulated during in vivo meiotic resumption. Taken together, our data indicate that hormone-induced maturation in mice requires a PRKA-dependent increase in FAO. PMID:23863407

  3. Distribution of CART (cocaine- and amphetamine-regulated transcript) peptide in mature and developing marsupial brain.

    PubMed

    Ashwell, K W S; Mai, J K

    2010-01-01

    CART (cocaine- and amphetamine-regulated transcript) is a neuromodulator involved in feeding, drug reward, stress and cardiovascular function. We have immunohistochemically studied the distribution of the CART peptide in the brains of two adult marsupial species: the brown antechinus (Antechinus stuartii) as a representative of polyprotodont marsupials and the tammar wallaby (Macropus eugenii) as a representative of diprotodont marsupials. We have also examined the distribution of CART during postnatal development in the tammar wallaby. There were similarities and differences both between the two marsupial species and between the marsupials and eutherians in CART distribution. Both marsupials showed immunoreactivity to CART in the olfactory bulb, piriform cortex, extended amygdala, the supraoptic, paraventricular and arcuate nuclei of the hypothalamus, somatosensory and auditory nuclei of the brainstem, vagal/solitary complex, raphe obscurus and raphe pallidus and presumptive presympathetic neurons of the ventrolateral medulla, as has been seen in eutherians. On the other hand, immunoreactivity to CART was weak in or absent from isocortical areas, and immunoreactivity to CART was poor or minimal in the ventral tegmental area and nucleus accumbens of both species; regions where immunoreactivity to CART is very strong in the brains of eutherians. During development, CART was present at birth (P0) in the lateral trigeminal ganglion, spinal trigeminal tract and the vagal sensorimotor complex, but did not appear in mid- or forebrain regions until much later (from P37). These anatomical findings indicate that although CART is likely to serve very similar functions in both eutherians and marsupials, there are potentially functionally significant differences between the two mammalian groups. PMID:20881368

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

    PubMed

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

    2002-09-16

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

  5. Elevated non-esterified fatty acid concentrations during bovine oocyte maturation compromise early embryo physiology.

    PubMed

    Van Hoeck, Veerle; Sturmey, Roger G; Bermejo-Alvarez, Pablo; Rizos, Dimitrios; Gutierrez-Adan, Alfonso; Leese, Henry J; Bols, Peter E J; Leroy, Jo L M R

    2011-01-01

    Elevated concentrations of serum non-esterified fatty acids (NEFA), associated with maternal disorders such as obesity and type II diabetes, alter the ovarian follicular micro-environment and have been associated with subfertility arising from reduced oocyte developmental competence. We have asked whether elevated NEFA concentrations during oocyte maturation affect the development and physiology of zygotes formed from such oocytes, using the cow as a model. The zygotes were grown to blastocysts, which were evaluated for their quality in terms of cell number, apoptosis, expression of key genes, amino acid turnover and oxidative metabolism. Oocyte maturation under elevated NEFA concentrations resulted in blastocysts with significantly lower cell number, increased apoptotic cell ratio and altered mRNA abundance of DNMT3A, IGF2R and SLC2A1. In addition, the blastocysts displayed reduced oxygen, pyruvate and glucose consumption, up-regulated lactate consumption and higher amino acid metabolism. These data indicate that exposure of maturing oocytes to elevated NEFA concentrations has a negative impact on fertility not only through a reduction in oocyte developmental capacity but through compromised early embryo quality, viability and metabolism. PMID:21858021

  6. Amino acid sequence of a vitamin K-dependent Ca2+-binding peptide from bovine prothrombin.

    PubMed

    Howard, J B; Fausch, M D

    1975-08-10

    The amino acid sequence of a 31-residue peptide from bovine prothrombin has been determined. This peptide has been shown to contain the vitamin K-dependent modification required for Ca2+ binding (Nelsestuen, G. L., and Suttie, J. W. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 3366-3370) and the modified amino acid, gamma-carboxyglutamic acid (Nelsestuen, G. L., Zytkovicz, T., and Howard, J. B. (1974) J. Biol. Chem. 249, 6347-6350). The peptide was shown to correspond to residues 12 to 42 of prothrombin. PMID:807581

  7. Supramolecular control of self-assembling terthiophene-peptide conjugates through the amino acid side chain

    SciTech Connect

    Lehrman, Jessica A.; Cui, Honggang; Tsai, Wei-Wen; Moyer, Tyson J.; Stupp, Samuel I.

    2013-07-30

    The self-assembly of oligothiophene–peptide conjugates can be directed through the systematic variation of the peptide sequence into different nanostructures, including flat spicules, nanotubes, spiral sheets, and giant, flat sheets. Furthermore, the assembly of these molecules is not controlled by steric interactions between the amino acid side chains.

  8. A peptide & peptide nucleic acid synthesis technology for transporter molecules and theranostics--the SPPS.

    PubMed

    Pipkorn, Ruediger; Braun, Klaus; Wiessler, Manfred; Waldeck, Waldemar; Schrenk, Hans-Hermann; Koch, Mario; Semmler, Wolfhard; Komljenovic, Dorde

    2014-01-01

    Advances in imaging diagnostics using magnetic resonance tomography (MRT), positron emission tomography (PET) and fluorescence imaging including near infrared (NIR) imaging methods are facilitated by constant improvement of the concepts of peptide synthesis. Feasible patient-specific theranostic platforms in the personalized medicine are particularly dependent on efficient and clinically applicable peptide constructs. The role of peptides in the interrelations between the structure and function of proteins is widely investigated, especially by using computer-assisted methods. Nowadays the solid phase synthesis (SPPS) chemistry emerges as a key technology and is considered as a promising methodology to design peptides for the investigation of molecular pharmacological processes at the transcriptional level. SPPS syntheses could be carried out in core facilities producing peptides for large-scale scientific implementations as presented here. PMID:24843319

  9. A Peptide & Peptide Nucleic Acid Synthesis Technology for Transporter Molecules and Theranostics - The SPPS

    PubMed Central

    Pipkorn, Ruediger; Braun, Klaus; Wiessler, Manfred; Waldeck, Waldemar; Schrenk, Hans-Hermann; Koch, Mario; Semmler, Wolfhard; Komljenovic, Dorde

    2014-01-01

    Advances in imaging diagnostics using magnetic resonance tomography (MRT), positron emission tomography (PET) and fluorescence imaging including near infrared (NIR) imaging methods are facilitated by constant improvement of the concepts of peptide synthesis. Feasible patient-specific theranostic platforms in the personalized medicine are particularly dependent on efficient and clinically applicable peptide constructs. The role of peptides in the interrelations between the structure and function of proteins is widely investigated, especially by using computer-assisted methods. Nowadays the solid phase synthesis (SPPS) chemistry emerges as a key technology and is considered as a promising methodology to design peptides for the investigation of molecular pharmacological processes at the transcriptional level. SPPS syntheses could be carried out in core facilities producing peptides for large-scale scientific implementations as presented here. PMID:24843319

  10. Peptides inhibitors of acid-sensing ion channels.

    PubMed

    Diochot, S; Salinas, M; Baron, A; Escoubas, P; Lazdunski, M

    2007-02-01

    Acid-sensing ion channels (ASICs) channels are proton-gated cationic channels mainly expressed in central and peripheric nervous system and related to the epithelial amiloride-sensitive Na(+) channels and to the degenerin family of ion channels. ASICs comprise four proteins forming functional channel subunits (ASIC1a, ASIC1b, ASIC2a, and ASIC3) and two proteins (ASIC2b and ASIC4) without yet known activators. Functional channels are activated by external pH variations ranging from pH(0.5) 6.8 to 4.0 and currents are characterized by either rapid kinetics of inactivation (ASIC1a, ASIC1b, ASIC3) or slow kinetics of inactivation (ASIC2a) and sometimes the presence of a plateau phase (ASIC3). ASIC1a and ASIC3, which are expressed in nociceptive neurons, have been implicated in inflammation and knockout mice studies support the role of ASIC3 in various pain processes. ASIC1a seems more related to synaptic plasticity, memory, learning and fear conditioning in the CNS. ASIC2a contributes to hearing in the cochlea, sour taste sensation, and visual transduction in the retina. The pharmacology of ASICs is limited to rather nonselective drugs such as amiloride, nonsteroid anti-inflammatory drugs, and neuropeptides. Recently, two peptides, PcTx1 and APETx2, isolated from a spider and a sea anemone, have been characterized as selective and high-affinity inhibitors for ASIC1a and ASIC3 channels, respectively. PcTx1 inhibits ASIC1a homomers with an affinity of 0.7 nM (IC(50)) without any effect on ASIC1a containing heteromers and thus helped to characterize ASIC1a homomeric channels in peripheric and central neurons. PcTx1 acts as a gating modifier since it shifts the channel from the resting to an inactivated state by increasing its affinity for H(+). APETx2 is less selective since it inhibits several ASIC3-containing channels (IC(50) from 63 nM to 2 microM) and to date its mode of action is unknown. Nevertheless, APETx2 structure is related to other sea anemone peptides, which

  11. Amino acid and peptide absorption from partial digests of proteins in isolated rat small intestine.

    PubMed Central

    Gardner, M L

    1978-01-01

    1. Absorption of each of sixteen amino acids, free and peptide-bound, has been measured in isolated rat small intestine perfused with five partial digests of proteins. 2. At low concentrations net absorption of each amino acid was proportional to its luminal concentration and independent of the nature of the amino acid. 3. A series of first-order multiple regressions was found to describe well the characteristics of absorption. 4. Rate constants for disappearance of free and peptide-bound amino acids from the lumen were closely similar. However, substantial back-flux occurred of amino acids derived from peptide hydrolysis. Hence 60-70% of the amino-N entering the serosal tissue fluid probably had left the lumen as free amino acids. 5. Intact peptides crossed the mucosa during absorption from a soy bean hydrolysate and in substantial quantities during absorption from one casein digest but not from another. With other hydrolysates there was no evidence for passage of peptides to the serosa. 6. In several cases there was a serious discrepancy between the amount of amino-N absorbed from the lumen and the amount accounted for as peptide or free amino acid in the serosal secretion. 7. The characteristics of absorption were similar (apart from the exceptions in 5 above) for all the digests studied except for soy bean hydrolysate. PMID:731590

  12. Aqueous Synthesis of Peptide Thioesters from Amino Acids and a Thiol Using 1,1‧-Carbonyldiimidazole

    NASA Astrophysics Data System (ADS)

    Weber, Arthur L.

    2005-10-01

    A new method was developed for the synthesis of peptide thioesters from free amino acids and thiols in water. This one-pot simple method involves two steps: (1) activation in water of an amino acid presumably as its N-carboxyanhydride (NCA) using 1,1‧-carbonyldiimidazole (CDI), and (2) subsequent condensation of the activated amino acid-NCA in the presence of a thiol. With this method citrulline peptide thioesters containing up to 10 amino acid residues were prepared in a single reaction. This aqueous synthetic method provides a simple way to prepare peptide thioesters for studies of peptide replication involving ligation of peptide thioesters on peptide templates. The relevance of peptide replication to the origin-of-life process is supported by previous studies showing that amino acid thioesters (peptide thioester precursors) can be synthesized under prebiotic conditions by reaction of small sugars with ammonia and a thiol.

  13. Cyclic Sulfamidate Enabled Syntheses of Amino Acids, Peptides, Carbohydrates, and Natural Products

    EPA Science Inventory

    This article reviews the emergence of cyclic sulfamidates as versatile intermediatesfor the synthesis of unnatural amino acids, chalcogen peptides, modified sugars, drugs and drug candidates, and important natural products.

  14. Tetrazine-Containing Amino Acid for Peptide Modification and Live Cell Labeling

    PubMed Central

    Ni, Zhongqiu; Zhou, Lanxia; Li, Xu; Zhang, Jing; Dong, Shouliang

    2015-01-01

    A novel amino acid derivative 3-(4-(1, 2, 4, 5-tetrazine-3-yl) phenyl)-2-aminopropanoic acid was synthesized in this study. The compound possessed better water-solubility and was synthesized more easily compared with the well-known and commercially available 3-(p-benzylamino)-1, 2, 4, 5-tetrazine. Tetrazine-containing amino acid showed excellent stability in biological media and might be used for cancer cell labeling. Moreover, the compound remained relatively stable in 50% TFA/DCM with little decomposition after prolonged exposure at room temperature. The compound could be utilized as phenylalanine or tyrosine analogue in peptide modification, and the tetrazine-containing peptide demonstrated more significant biological activity than that of the parent peptide. The combination of tetrazine group and amino acid offered broad development prospects of the bioorthogonal labeling and peptide synthesis. PMID:26536589

  15. Stimulation of Lysine Decarboxylase Production in Escherichia coli by Amino Acids and Peptides1

    PubMed Central

    Cascieri, T.; Mallette, M. F.

    1973-01-01

    A commercial hydrolysate of casein stimulated production of lysine decarboxylase (EC 4.1.1.18) by Escherichia coli B. Cellulose and gel chromatography of this hydrolysate yielded peptides which were variably effective in this stimulation. Replacement of individual, stimulatory peptides by equivalent amino acids duplicated the enzyme levels attained with those peptides. There was no indication of specific stimulation by any peptide. The peptides were probably taken up by the oligopeptide transport system of E. coli and hydrolyzed intracellularly by peptidases to their constituent amino acids for use in enzyme synthesis. Single omission of amino acids from mixtures was used to screen them for their relative lysine decarboxylase stimulating abilities. Over 100 different mixtures were evaluated in establishing the total amino acid requirements for maximal synthesis of lysine decarboxylase by E. coli B. A mixture containing all of the common amino acids except glutamic acid, aspartic acid, and alanine increased lysine decarboxylase threefold over an equivalent weight of casein hydrolysate. The nine most stimulatory amino acids were methionine, arginine, cystine, leucine, isoleucine, glutamine, threonine, tyrosine, and asparagine. Methionine and arginine quantitatively were the most important. A mixture of these nine was 87% as effective as the complete mixture. Several amino acids were inhibitory at moderate concentrations, and alanine (2.53 mM) was the most effective. Added pyridoxine increased lysine decarboxylase activity 30%, whereas other B vitamins and cyclic adenosine 5′-monophosphate had no effect. PMID:4588201

  16. Efficacy of peptide nucleic acid and selected conjugates against specific cellular pathologies of amyotrophic lateral sclerosis.

    PubMed

    Browne, Elisse C; Parakh, Sonam; Duncan, Luke F; Langford, Steven J; Atkin, Julie D; Abbott, Belinda M

    2016-04-01

    Cellular studies have been undertaken on a nonamer peptide nucleic acid (PNA) sequence, which binds to mRNA encoding superoxide dismutase 1, and a series of peptide nucleic acids conjugated to synthetic lipophilic vitamin analogs including a recently prepared menadione (vitamin K) analog. Reduction of both mutant superoxide dismutase 1 inclusion formation and endoplasmic reticulum stress, two of the key cellular pathological hallmarks in amyotrophic lateral sclerosis, by two of the prepared PNA oligomers is reported for the first time. PMID:26935939

  17. Increasing abscisic acid levels by immunomodulation in barley grains induces precocious maturation without changing grain composition.

    PubMed

    Staroske, Nicole; Conrad, Udo; Kumlehn, Jochen; Hensel, Götz; Radchuk, Ruslana; Erban, Alexander; Kopka, Joachim; Weschke, Winfriede; Weber, Hans

    2016-04-01

    Abscisic acid (ABA) accumulates in seeds during the transition to the seed filling phase. ABA triggers seed maturation, storage activity, and stress signalling and tolerance. Immunomodulation was used to alter the ABA status in barley grains, with the resulting transgenic caryopses responding to the anti-ABA antibody gene expression with increased accumulation of ABA. Calculation of free versus antibody-bound ABA reveals large excess of free ABA, increasing signficantly in caryopses from 10 days after fertilization. Metabolite and transcript profiling in anti-ABA grains expose triggered and enhanced ABA-functions such as transcriptional up-regulation of sucrose-to-starch metabolism, storage protein synthesis and ABA-related signal transduction. Thus, enhanced ABA during transition phases induces precocious maturation but negatively interferes with growth and development. Anti-ABA grains display broad constitutive gene induction related to biotic and abiotic stresses. Most of these genes are ABA- and/or stress-inducible, including alcohol and aldehyde dehydrogenases, peroxidases, chaperones, glutathione-S-transferase, drought- and salt-inducible proteins. Conclusively, ABA immunomodulation results in precocious ABA accumulation that generates an integrated response of stress and maturation. Repression of ABA signalling, occurring in anti-ABA grains, potentially antagonizes effects caused by overshooting production. Finally, mature grain weight and composition are unchanged in anti-ABA plants, although germination is somewhat delayed. This indicates that anti-ABA caryopses induce specific mechanisms to desensitize ABA signalling efficiently, which finally yields mature grains with nearly unchanged dry weight and composition. Such compensation implicates the enormous physiological and metabolic flexibilities of barley grains to adjust effects of unnaturally high ABA amounts in order to ensure and maintain proper grain development. PMID:26951372

  18. Increasing abscisic acid levels by immunomodulation in barley grains induces precocious maturation without changing grain composition

    PubMed Central

    Staroske, Nicole; Conrad, Udo; Kumlehn, Jochen; Hensel, Götz; Radchuk, Ruslana; Erban, Alexander; Kopka, Joachim; Weschke, Winfriede; Weber, Hans

    2016-01-01

    Abscisic acid (ABA) accumulates in seeds during the transition to the seed filling phase. ABA triggers seed maturation, storage activity, and stress signalling and tolerance. Immunomodulation was used to alter the ABA status in barley grains, with the resulting transgenic caryopses responding to the anti-ABA antibody gene expression with increased accumulation of ABA. Calculation of free versus antibody-bound ABA reveals large excess of free ABA, increasing signficantly in caryopses from 10 days after fertilization. Metabolite and transcript profiling in anti-ABA grains expose triggered and enhanced ABA-functions such as transcriptional up-regulation of sucrose-to-starch metabolism, storage protein synthesis and ABA-related signal transduction. Thus, enhanced ABA during transition phases induces precocious maturation but negatively interferes with growth and development. Anti-ABA grains display broad constitutive gene induction related to biotic and abiotic stresses. Most of these genes are ABA- and/or stress-inducible, including alcohol and aldehyde dehydrogenases, peroxidases, chaperones, glutathione-S-transferase, drought- and salt-inducible proteins. Conclusively, ABA immunomodulation results in precocious ABA accumulation that generates an integrated response of stress and maturation. Repression of ABA signalling, occurring in anti-ABA grains, potentially antagonizes effects caused by overshooting production. Finally, mature grain weight and composition are unchanged in anti-ABA plants, although germination is somewhat delayed. This indicates that anti-ABA caryopses induce specific mechanisms to desensitize ABA signalling efficiently, which finally yields mature grains with nearly unchanged dry weight and composition. Such compensation implicates the enormous physiological and metabolic flexibilities of barley grains to adjust effects of unnaturally high ABA amounts in order to ensure and maintain proper grain development. PMID:26951372

  19. Rational Evolution of Antimicrobial Peptides Containing Unnatural Amino Acids to Combat Burn Wound Infections.

    PubMed

    Xiong, Meng; Chen, Ming; Zhang, Jue

    2016-09-01

    Antimicrobial peptides have long been raised as a promising strategy to combat bacterial infection in burn wounds. Here, we attempted to rationally design small antimicrobial peptides containing unnatural amino acids by integrating in silico analysis and in vitro assay. Predictive quantitative sequence-activity models were established and validated rigorously based on a large panel of nonamer antimicrobial peptides with known antibacterial activity. The best quantitative sequence-activity model predictor was employed to guide genetic evolution of a peptide population. In the evolution procedure, a number of unnatural amino acids with desired physicochemical properties were introduced, resulting in a genetic evolution-improved population, from which seven peptide candidates with top scores, containing 1-3 unnatural amino acids, and having diverse structures were successfully identified, and their antibacterial potencies against two antibiotic-resistant bacterial strains isolated from infected burn wounds were measured using in vitro susceptibility test. Consequently, four (WL-Orn-LARKIV-NH2 , ARKRWF-Dab-FL-NH2 , KFI-Hag-IWR-Orn-R-NH2 and YW-Hag-R-Cit-RF-Orn-N-NH2 ) of the seven tested peptides were found to be more potent than reference Bac2A, the smallest naturally occurring broad spectrum antimicrobial peptide. Molecular dynamics simulations revealed that the designed peptides can fold into amphipathic helical structure that allows them to interact directly with microbial membranes. PMID:27062533

  20. Installing amino acids and peptides on N-heterocycles under visible-light assistance

    PubMed Central

    Jin, Yunhe; Jiang, Min; Wang, Hui; Fu, Hua

    2016-01-01

    Readily available natural α-amino acids are one of nature’s most attractive and versatile building blocks in synthesis of natural products and biomolecules. Peptides and N-heterocycles exhibit various biological and pharmaceutical functions. Conjugation of amino acids or peptides with N-heterocycles provides boundless potentiality for screening and discovery of diverse biologically active molecules. However, it is a great challenge to install amino acids or peptides on N-heterocycles through formation of carbon-carbon bonds under mild conditions. In this article, eighteen N-protected α-amino acids and three peptides were well assembled on phenanthridine derivatives via couplings of N-protected α-amino acid and peptide active esters with substituted 2-isocyanobiphenyls at room temperature under visible-light assistance. Furthermore, N-Boc-proline residue was successfully conjugated with oxindole derivatives using similar procedures. The simple protocol, mild reaction conditions, fast reaction, and high efficiency of this method make it an important strategy for synthesis of diverse molecules containing amino acid and peptide fragments. PMID:26830014

  1. Installing amino acids and peptides on N-heterocycles under visible-light assistance.

    PubMed

    Jin, Yunhe; Jiang, Min; Wang, Hui; Fu, Hua

    2016-01-01

    Readily available natural α-amino acids are one of nature's most attractive and versatile building blocks in synthesis of natural products and biomolecules. Peptides and N-heterocycles exhibit various biological and pharmaceutical functions. Conjugation of amino acids or peptides with N-heterocycles provides boundless potentiality for screening and discovery of diverse biologically active molecules. However, it is a great challenge to install amino acids or peptides on N-heterocycles through formation of carbon-carbon bonds under mild conditions. In this article, eighteen N-protected α-amino acids and three peptides were well assembled on phenanthridine derivatives via couplings of N-protected α-amino acid and peptide active esters with substituted 2-isocyanobiphenyls at room temperature under visible-light assistance. Furthermore, N-Boc-proline residue was successfully conjugated with oxindole derivatives using similar procedures. The simple protocol, mild reaction conditions, fast reaction, and high efficiency of this method make it an important strategy for synthesis of diverse molecules containing amino acid and peptide fragments. PMID:26830014

  2. Rapid complexing of oxoacylglycerols with amino acids, peptides and aminophospholipids.

    PubMed

    Kurvinen, J P; Kuksis, A; Ravandi, A; Sjövall, O; Kallio, H

    1999-03-01

    We prepared model Schiff bases from 2-[9-oxo]nonanoyl glycerol (2-MAG-ALD) and various amino compounds. 2-MAG-ALD was obtained by pancreatic lipase hydrolysis of trioleoyl glycerol and reductive ozonolysis of the resulting 2-monooleoyl glycerol. The reaction products were purified by thin-layer chromatography. Schiff bases were synthesized in greater than 50% yield by reacting 2-MAG-ALD with twofold molar excess of valine, Nalpha-acetyl-L-lysine methyl ester and the tripeptides glycyl-glycyl-glycine, glycyl-glycyl-histidine, and glycyl-histidyl-lysine in aqueous methanol and with 1-palmitoyl-2-stearoyl glycerophosphoethanolamine (PE) in chloroform/methanol for 16 h at room temperature. Prior to analysis the bases were reduced with sodium cyanoborohydride in methanol for 30 min at 4 degrees C. Reaction products were analyzed by high-performance liquid chromatography/electrospray ionization/mass spectrometry (HPLC/ESI/MS). Reduced Schiff bases of 2-MAG-ALD with PE and amino acids were analyzed by normal-phase HPLC/ESI/MS and those with peptides by reversed-phase HPLC/ESI/MS. Single adducts were obtained in all cases and both the alpha-amino group of valine and the epsilon-amino group of Nalpha-acetyl-L-lysine methyl ester were reactive. Molecular ions of reaction products were the only detected ions in the negative ionization mode, whereas in the positive ion mode sodiated molecular ions were also detected. The present study suggests that 2-MAG-ALD may form Schiff base adducts with amino compounds in other aqueous media, such as the intestinal lumen and in the hydrophobic environment of cell membranes. PMID:10230725

  3. The impact of α-hydrazino acids embedded in short fluorescent peptides on peptide interactions with DNA and RNA.

    PubMed

    Suć, Josipa; Tumir, Lidija-Marija; Glavaš-Obrovac, Ljubica; Jukić, Marijana; Piantanida, Ivo; Jerić, Ivanka

    2016-06-01

    A series of novel hydrazino-based peptidomimetics and analogues comprising N-terminal lysine and C-terminal phenanthridinyl-l-alanine were prepared. The presented results demonstrate the up to now unknown possibility to finely modulate peptide interactions with DNA/RNA by α-hydrazino group insertion and how the different positioning of two α-hydrazino groups in peptides controls binding to various double stranded and single stranded DNA and RNA. All peptidomimetics bind with 1-10 micromolar affinity to ds-DNA/RNA, whereby the binding mode is a combination of electrostatic interactions and hydrophobic interactions within DNA/RNA grooves. Insertion of the α-hydrazino group into the peptide systematically decreased its fluorimetric response to DNA/RNA binding in the order: mono-hydrazino < alternating-hydrazino < sequential-hydrazino group. Binding studies of ss-polynucleotides suggest intercalation of phenanthridine between polynucleotide bases, whereby affinity and fluorimetric response decrease with the number of α-hydrazino groups in the peptide sequence. Particularly interesting was the interaction of two sequential α-hydrazino acids-peptidomimetic with poly rG, characterised by a specific strong increase of CD bands, while all other peptide/ssRNA combinations gave only a CD-band decrease. All mentioned interactions could also be reversibly controlled by adjusting the pH, due to the protonation of the fluorophore. PMID:27161341

  4. Affinity maturation of T-cell receptor-like antibodies for Wilms tumor 1 peptide greatly enhances therapeutic potential

    PubMed Central

    Zhao, Qi; Ahmed, Mahiuddin; Tassev, Dimiter V.; Hasan, Aisha; Kuo, Tzu-Yun; Guo, Hong-fen; O’Reilly, Richard J.; Cheung, Nai-Kong V.

    2016-01-01

    WT1126 (RMFPNAPYL) is a human leukocyte antigen-A2 (HLA-A2) restricted peptide derived from Wilms tumor protein (WT1), which is widely expressed in a broad spectrum of leukemias, lymphomas and solid tumors. A novel T-cell-receptor (TCR)-like single chain variable fragment (scFv) antibody specific for the T cell epitope consisting of the WT1/HLA-A2 complex was isolated from a human scFv phage library. This scFv was affinity-matured by mutagenesis combined with yeast display, and structurally analyzed using a homology model. This monovalent scFv showed a 100-fold affinity improvement (dissociation constant [KD]= 3nM) and exquisite specificity towards its targeted epitope or HLA-A2+/WT1+ tumor cells. Bivalent scFv-huIgG1-Fc fusion protein demonstrated an even higher avidity (KD = 2pM) binding to the T cell epitope and to tumor targets, and was capable of mediating antibody-dependent cell-mediated cytotoxicity or tumor lysis by chimeric antigen receptor (CAR)-expressing human T or NK-92-MI transfected cells. This antibody demonstrated specific and potent cytotoxicity in vivo towards WT1-positive leukemia xenograft that was HLA-A2 restricted. In summary, T cell epitopes can provide novel targets for antibody-based therapeutics. By combining phage and yeast displays and scFv-Fc fusion platforms, a strategy for developing high affinity TCR-like antibodies could be rapidly explored for potential clinical development. PMID:25987253

  5. Formation of Amino Acid Thioesters for Prebiotic Peptide Synthesis: Catalysis By Amino Acid Products

    NASA Technical Reports Server (NTRS)

    Weber, Arthur L.; DeVincenzi, Donald L. (Technical Monitor)

    1999-01-01

    The origin of life can be described as a series of events in which a prebiotic chemical process came increasingly under the control of its catalytic products. In our search for this prebiotic process that yielded catalytic takeover products (such as polypeptides), we have been investigating a reaction system that generates peptide-forming amino acid thioesters from formaldehyde, glycolaldehyde, and ammonia in the presence of thiols. As shown below, this model process begins by aldol condensation of formaldehyde and glycolaldehyde to give trioses and releases. These sugars then undergo beta-dehydration yielding their respective alpha-ketoaldehydes. Addition of ammonia to the alpha-ketoaldehydes yields imines which can either: (a) rearrange in the presence of thesis to give amino acid thioesters or (be react with another molecule of aldehyde to give imidazoles. This 'one-pot' reaction system operates under mild aqueous conditions, and like modem amino acid biosynthesis, uses sugar intermediates which are converted to products by energy-yielding redox reactions. Recently, we discovered that amino acids, such as the alanine reaction product, catalyze the first and second steps of the process. In the presence of ammonia the process also generates other synthetically useful products, like the important biochemical -- pyruvic acid.

  6. Growth response of Pinus ponderosa seedlings and mature tree branches to acid rain and ozone exposure

    SciTech Connect

    Anderson, P.D.; Houpis, J.L.J.; Helms, J.A.

    1994-10-01

    Forests of the central and southern Sierra Nevada in California have been subjected to chronic damage by ozone and other atmospheric pollutants for the past several decades. Until recently, pollutant exposure of northern Sierra Nevada forests has been mild but increasing population and changes in land use throughout the Sacramento Valley and Sierra Nevada foothills may lead to increased pollutant damage in these forests. Although, better documented in other regions of the United States, little is known regarding the potential for acidic precipitation damage to Sierra Nevada forests. Only recently have studies directed towards understanding the potential interactive effects of ozone and acidic precipitation been undertaken. A key issue in resolving potential regional impacts of pollutants on forests is the extent to which research results can be scaled across genotypes and life-stages. Most of the pollution research to date has been performed using seedlings with varying degrees of genetic control. It is important to determine if the results obtained in such studies can be extrapolated to mature trees and to different genetic sources. In this paper, we present results from a one-year study examining the interactive effects of foliar exposure to acidic rain and ozone on the growth of ponderosa pine (Pinus ponderosa), a conifer known to be sensitive to ozone. The response to pollutants is characterized for both seedlings and mature tree branches of three genotypes grown in a common environment.

  7. Molecular mechanics and dynamics studies on the interaction of gallic acid with collagen-like peptides

    NASA Astrophysics Data System (ADS)

    Madhan, B.; Thanikaivelan, P.; Subramanian, V.; Raghava Rao, J.; Unni Nair, Balachandran; Ramasami, T.

    2001-10-01

    Molecular modelling approaches have been used to understand the interaction of collagen-like peptides with gallic acid, which mimic vegetable tanning processes involved in protein stabilization. Several interaction sites have been identified and the binding energies of the complexes have been calculated. The calculated binding energies for various geometries are in the range 6-13 kcal/mol. It is found that some complexes exhibit hydrogen bonding, and electrostatic interaction plays a dominant role in the stabilization of the peptide by gallic acid. The π-OH type of interaction is also observed in the peptide stabilization. Molecular dynamics (MD) simulation for 600 ps revealed the possibility of hydrogen bonding between the collagen-like peptide and gallic acid.

  8. Peptides containing β-amino acid patterns: challenges and successes in medicinal chemistry.

    PubMed

    Cabrele, Chiara; Martinek, Tamás A; Reiser, Oliver; Berlicki, Łukasz

    2014-12-11

    The construction of bioactive peptides using β-amino acid-containing sequence patterns is a very promising strategy to obtain analogues that exhibit properties of high interest for medicinal chemistry applications. β-Amino acids have been shown to modulate the conformation, dynamics, and proteolytic susceptibility of native peptides. They can be either combined with α-amino acids by following specific patterns, which results in backbone architectures with well-defined orientations of the side chain functional groups, or assembled in de novo-designed bioactive β- or α,β-peptidic sequences. Such peptides display various biological functions, including antimicrobial activity, inhibition of protein-protein interactions, agonism/antagonism of GPCR ligands, and anti-angiogenic activity. PMID:25207470

  9. Effect of In Vitro Maturation Technique and Alpha Lipoic Acid Supplementation on Oocyte Maturation Rate: Focus on Oxidative Status of Oocytes

    PubMed Central

    Zavareh, Saeed; Karimi, Isaac; Salehnia, Mojdeh; Rahnama, Ali

    2016-01-01

    Background Therapeutic potential of in vitro maturation (IVM) in infertility is growing with great promise. Although significant progress is obtained in recent years, existing IVM protocols are far from favorable results. The first aim of this study was to investigate whether two step IVM manner change reactive oxygen species (ROS) and total anti- oxidant capacity (TAC) levels. The second aim was to find the effect of alpha lipoic acid (ALA) supplementation on oocyte maturation rate and on ROS/TAC levels during IVM. Materials and Methods In this experimental study, mouse germinal vesicle (GV) oocytes divided into cumulus denuded oocytes (DOs) and cumulus oocyte complexes (COCs) groups. GVs were matured in vitro in the presence or absence of ALA only for 18 hours (control) or with pre-culture of forskolin plus cilostamide for an additional 18 hours. Matured oocytes obtained following 18 and 36 hours based on experimental design. In parallel, the ROS and TAC levels were measured at different time (0, 18 and 36 hours) by 2',7'-dichlorodihydrofluorescein (DCFH) probe and ferric reducing/antioxidant power (FRAP) assay, respectively. Results Maturation rate of COCs was significantly higher than DOs in control group (P<0.05), while there was no significant difference between COCs and DOs when were pre-cultured with forskolin plus cilostamide. ROS and TAC levels was increased and decreased respectively in DOs after 18 hours while in COCs did not change at 18 hours and showed a significant increase and decrease respectively at 36 hours (P<0.05). ROS and TAC levels in the presence of ALA were significantly decreased and increased respectively after 36 hours (P<0.05) whereas, maturation rates of COCs and DOs were similar to their corresponding control groups. Conclusion ALA decreased ROS and increased TAC but could not affect maturation rate of both COCs and DOs in one or two step IVM manner. PMID:26985332

  10. Relationship between cadmium, zinc, Cd-peptide, and organic acid in tobacco suspension cells

    SciTech Connect

    Krotz, R.M.; Evangelou, B.P.; Wagner, G.J. )

    1989-10-01

    Responses of tobacco (Nicotiana tabacum) suspension cells to Cd and Zn were studied in the presence and absence of ligand of Cd-peptide in order to understand the role of this peptide versus other mechanisms in Cd and Zn accumulation and accommodation in plants. With 45 micromolar Cd and 300 micromolar Zn (non-growth-inhibiting levels), metals appeared rapidly within cells, and intracellular Cd and Zn reached medium concentrations after 6 to 10 hours. Cd-peptide was observed in response to Cd after 2 hours, but this form only accounted for {approximately}30% of soluble Cd after 24 hours. Peptide was not observed in cells exposed to 300 micromolar Zn for up to 7 days. Organic acid-to-metal stoichiometry indicated that endogenous organic acid content of cells was more than sufficient to complex absorbed metals and no evidence was found for stimulation of organic acid biosynthesis by Cd or Zn. Metal-complexing potential of organic acids for Cd and Zn versus endogenous cations is discussed as is vacuolar-extravacuolar distribution of metals. The absence of Cd-peptide does not limit Cd-accumulation in the system studied. Results suggest that tobacco suspension cells accommodte the presence of non-growth-inhibiting and growth-inhibiting levels of Cd and Zn by sequestration in the vacuole as complexes with endogenous organic acids and that this may be a principal means for accommodation of Cd as well as Zn in the presence and absence of Cd-peptide.

  11. Synthesis and biological properties of amino acids and peptides containing a tetrazolyl moiety

    NASA Astrophysics Data System (ADS)

    Popova, E. A.; Trifonov, R. E.

    2015-09-01

    Literature data published mainly in the last 15 years on the synthesis and biological properties of amino acid analogues and derivatives containing tetrazolyl moieties are analyzed. Tetrazolyl analogues and derivatives of amino acids and peptides are shown to be promising for medicinal chemistry. Being polynitrogen heterocyclic systems comprising four endocyclic nitrogen atoms, tetrazoles can behave as acids and bases and form strong hydrogen bonds with proton donors (more rarely, with acceptors). They have high metabolic stability and are able to penetrate biological membranes. The review also considers the synthesis and properties of linear and cyclic peptides based on modified amino acids incorporating a tetrazolyl moiety. A special issue is the discussion of the biological properties of tetrazole-containing amino acids and peptides, which exhibit high biological activity and can be used to design new drugs. The bibliography includes 200 references.

  12. Thermal stability of ascorbic acid and ascorbic acid oxidase in african cowpea leaves ( Vigna unguiculata ) of different maturities.

    PubMed

    Wawire, Michael; Oey, Indrawati; Mathooko, Francis; Njoroge, Charles; Shitanda, Douglas; Hendrickx, Marc

    2011-03-01

    Cowpea, an African leafy vegetable ( Vigna unguiculata ), contains a high level of vitamin C. The leaves harvested at 4-9 weeks are highly prone to vitamin C losses during handling and processing. Therefore, the purpose of this research was to study the effect of thermal treatment on the stability of ascorbic acid oxidase (AAO), total vitamin C content (l-ascorbic acid, l-AA), and dehydroascorbic acid (DHAA) and l-AA/DHAA ratio in cowpea leaves harvested at different maturities (4, 6, and 8 weeks old). The results showed that AAO activity, total vitamin C content, and l-AA/DHAA ratio in cowpea leaves increased with increasing maturity (up to 8 weeks). Eight-week-old leaves were the best source of total vitamin C and showed a high ratio of l-AA/DHAA (4:1). Thermal inactivation of AAO followed first-order reaction kinetics. Heating at temperatures above 90 °C for short times resulted in a complete AAO inactivation, resulting in a protective effect of l-AA toward enzyme-catalyzed oxidation. Total vitamin C in young leaves (harvested at 4 and 6 weeks) was predominantly in the form of DHAA, and therefore temperature treatment at 30-90 °C for 10 min decreased the total vitamin C content, whereas total vitamin C in 8-week-old cowpea leaves was more than 80% in the form of l-AA, so that a high retention of the total vitamin C can be obtained even after heating and/or reheating (30-90 °C for 10 min) before consumption. The results indicated that the stability of total vitamin C in situ was strongly dependent on the plant maturity stage and the processing conditions applied. PMID:21309563

  13. Endogenous flow of amino acids in the avian ileum as influenced by increasing dietary peptide concentrations.

    PubMed

    Ravindran, Velmurugu; Morel, Patrick C H; Rutherfurd, Shane M; Thomas, Donald V

    2009-03-01

    The aim of the present study was to establish whether feeding broiler chickens with diets containing increasing dietary peptide concentrations would cause increases in ileal endogenous amino acid flow. The flow of N and most amino acids increased quadratically (P < 0.05 to 0.001) with increasing dietary concentrations of peptides. The exceptions were the flow of threonine, serine, glycine, tyrosine and cystine, which increased linearly (P < 0.001) with dietary peptide levels. Another notable exception to the general trend was the flow of proline, which was significantly higher (P < 0.01) in birds fed the protein-free diet. The amino acid profile of endogenous protein, expressed as proportion of crude protein, indicated that the ratios of threonine, glutamic acid, proline, glycine, leucine, histidine, arginine and cystine were influenced (P < 0.05) with increasing dietary peptide concentrations. In general, compared with the protein-free diet, the ratios of threonine and arginine in endogenous protein were lower (P < 0.05) and those of glutamic acid, glycine and histidine were greater (P < 0.05) in diets with high concentrations of peptides. The ratio of proline was found to decrease (P < 0.05) with increasing dietary peptide concentrations. These changes in the amino acid profile of endogenous protein are probably reflective of changes in the output of one or more of the components of endogenous protein. Overall, the present results demonstrated that increasing dietary peptide concentrations increased the flow of endogenous amino acid flow at the terminal ileum of broiler chickens in a dose-dependent manner and also caused changes in the composition of endogenous protein. The observed changes in endogenous amino flow will influence the maintenance requirements for amino acids and also have implications for the calculation of true digestibility coefficient of feedstuffs. PMID:18662428

  14. A toy model of prebiotic peptide evolution: the possible role of relative amino acid abundances.

    PubMed

    Polanco, Carlos; Buhse, Thomas; Samaniego, José Lino; Castañón González, Jorge Alberto

    2013-01-01

    This paper presents a mathematical-computational toy model based on the assumed dynamic principles of prebiotic peptide evolution. Starting from a pool of amino acid monomers, the model describes in a generalized manner the generation of peptides and their sequential information. The model integrates the intrinsic and dynamic key elements of the initiation of biopolymerization, such as the relative amino acid abundances and polarities, as well as the oligomer reversibility, i.e. fragmentation and recombination, and peptide self-replication. Our modeling results suggest that the relative amino acid abundances, as indicated by Miller-Urey type electric discharge experiments, played a principal role in the early sequential information of peptide profiles. Moreover, the computed profiles display an astonishing similarity to peptide profiles observed in so-called biological common ancestors found in the following three microorganisms; E. coli, M. jannaschii, and S. cereviasiae. The prebiotic peptide fingerprint was obtained by the so-called polarity index method that was earlier reported as a tool for the identification of cationic amphipathic antibacterial short peptides. PMID:23741717

  15. Therapeutic alteration of insulin-dependent diabetes mellitus progression by T cell tolerance to glutamic acid decarboxylase 65 peptides in vitro and in vivo.

    PubMed

    Wilson, S S; White, T C; DeLuca, D

    2001-07-01

    We have reported previously that nonobese diabetic (NOD) fetal pancreas organ cultures lose the ability to produce insulin when maintained in contact with NOD fetal thymus organ cultures (FTOC). Initial studies indicated that exposure to glutamic acid decarboxylase (GAD65) peptides in utero resulted in delay or transient protection from insulin-dependent diabetes mellitus (IDDM) in NOD mice. We also found that exposure of young adult NOD mice to the same peptides could result in acceleration of the disease. To more closely examine the effects of early and late exposure to diabetogenic Ags on T cells, we applied peptides derived from GAD65 (GAD AA 246-266, 509-528, and 524-543), to our "in vitro IDDM" (ivIDDM) model. T cells derived from NOD FTOC primed during the latter stages of organ culture, when mature T cell phenotypes are present, had the ability to proliferate to GAD peptides. ivIDDM was exacerbated under these conditions, suggesting that GAD responsiveness correlates with the ivIDDM phenotype, and parallels the acceleration of IDDM we had seen in young adult NOD mice. When GAD peptides were present during the initiation of FTOC, GAD proliferative responses were inhibited, and ivIDDM was reduced. This result suggests that tolerance to GAD peptides may reduce the production of diabetogenic T cells or their capacity to respond, as suggested by the in utero therapies studied in NOD mice. PMID:11418696

  16. Formulation of a Peptide Nucleic Acid Based Nucleic Acid Delivery Construct

    PubMed Central

    Millili, Peter G.; Yin, Daniel H.; Fan, Haihong; Naik, Ulhas P.; Sullivan, Millicent O.

    2010-01-01

    Gene delivery biomaterials need to be designed to efficiently achieve nuclear delivery of plasmid DNA. Polycations have been used to package DNA and other nucleic acids within sub-micron sized particles, offering protection from shear-induced or enzymatic degradation. However, cytotoxicity issues coupled with limited in vivo transfection efficiencies minimize the effectiveness of this approach. In an effort to improve upon existing technologies aimed at delivering nucleic acids, an alternative approach to DNA packaging was explored. Peptide nucleic acids (PNAs) were used to directly functionalize DNA with poly(ethylene glycol) (PEG) chains that provide a steric layer and inhibit multimolecular aggregation during complexation. DNA prePEGylation by this strategy was predicted to enable the formation of more homogeneous and efficiently packaged polyplexes. In this work, DNA-PNA-peptide-PEG (DP3) conjugates were synthesized and self-assembled with 25 kDa poly(ethylenimine) (PEI). Complexes with small standard deviations and average diameters ranging from 30 – 50 nm were created, with minimal dependence of complex size on N:P ratio (PEI amines to DNA phosphates). Furthermore, PEI-DNA interactions were altered by the derivitization strategy, resulting in tighter compaction of the PEI-DP3 complexes in comparison with PEI-DNA complexes. Transfection experiments in Chinese Hamster Ovary (CHO) cells revealed comparable transfection efficiencies but reduced cytotoxicities of the PEI-DP3 complexes relative to PEI-DNA complexes. The enhanced cellular activities of the PEI-DP3 complexes were maintained following the removal of free PEI from the PEI-DP3 formulations, whereas the cellular activity of the conventional PEI-DNA formulations was reduced by free PEI removal. These findings suggest that DNA prePEGylation by the PNA-based strategy might provide a way to circumvent cytotoxicity and formulation issues related to the use of PEI for in vivo gene delivery. PMID:20131756

  17. Analysis of Endogenous D-Amino Acid-Containing Peptides in Metazoa

    PubMed Central

    Bai, Lu; Sheeley, Sarah; Sweedler, Jonathan V.

    2010-01-01

    Peptides are chiral molecules with their structure determined by the composition and configuration of their amino acid building blocks. The naturally occurring amino acids, except glycine, possess two chiral forms. This allows the formation of multiple peptide diastereomers that have the same sequence. Although living organisms use L-amino acids to make proteins, a group of D-amino acid-containing peptides (DAACPs) has been discovered in animals that have at least one of their residues isomerized to the D-form via an enzyme-catalyzed process. In many cases, the biological functions of these peptides are enhanced due to this structural conversion. These DAACPs are different from those known to occur in bacterial cell wall and antibiotic peptides, the latter of which are synthesized in a ribosome-independent manner. DAACPs have now also been identified in a number of distinct groups throughout the Metazoa. Their serendipitous discovery has often resulted from discrepancies observed in bioassays or in chromatographic behavior between natural peptide fractions and peptides synthesized according to a presumed all-L sequence. Because this L-to-D post-translational modification is subtle and not detectable by most sequence determination approaches, it is reasonable to suspect that many studies have overlooked this change; accordingly, DAACPs may be more prevalent than currently thought. Although diastereomer separation techniques developed with synthetic peptides in recent years have greatly aided in the discovery of natural DAACPs, there is a need for new, more robust methods for naturally complex samples. In this review, a brief history of DAACPs in animals is presented, followed by discussion of a variety of analytical methods that have been used for diastereomeric separation and detection of peptides. PMID:20490347

  18. The Prebiotic Synthesis of Ethylenediamine Monoacetic Acid, The Repeating Unit of Peptide Nucleic Acids

    NASA Technical Reports Server (NTRS)

    Nelson, Kevin E.; Miller, Stanley L.

    1992-01-01

    The polymerization of ribonucleic acids or their precursors constitutes an important event in prebiotic chemistry. The various problems using ribonucleotides to make RNA suggest that there may have been a precursor. An attractive possibility are the peptide nucleic acids (PNA). PNAs are nucleotide analogs that make use of a polymer of ethylenediamine monoacetic acid (EDMA or 2-amninoethyl glycine) with the bases attached by an acetic acid. EDMA is an especially attractive alternative to the ribose phosphate or deoxyribose phosphate backbone because it contains no chiral centers and is potentially prebiotic, but there is no reported prebiotic synthesis. We have synthesized both EDMA and ethylenediamine diacetic acid (EDDA) from the prebiotic compounds ethylenediamine, formaldehyde, and hydrogen cyanide. The yields of EDMA range from 11 to 79% along with some sEDDA and uEDDA. These reactions work with concentrations of 10(exp -1)M and as low as 10(exp -4)M, and the reaction is likely to be effective at even lower concentrations. Ethylenediamine is a likely prebiotic compound, but it has not yet been demonstrated, although compounds such as ethanolamine and cysteamine have been proven to be prebiotic. Under neutral pH and heating at l00 C, EDMA is converted to the lactam, monoketopiperazine (MKP). The cyclization occurs and has an approximate ratio of MKP/EDMA = 3 at equilibrium. We have measured the solubilities of EDMA center dot H20 as 6.4 m, EDMA center dot HCl center dot H20 as 13.7 m, and EDMA center dot 2HCl center dot H20 as 3.4 m. These syntheses together with the high solubility of EDMA suggest that EDMA would concentrate in drying lagoons and might efficiently form polymers. Given the instability of ribose and the poor polymerizability of nucleotides, the prebiotic presence of EDMA and the possibility of its polymerization raises the possibility that PNAs are the progenitors of present day nucleic acids. A pre-RNA world may have existed in which PNAs or

  19. Effect of fatty acids isolated from edible oils like mustard, linseed or coconut on astrocytes maturation.

    PubMed

    Joardar, Anindita; Das, Sumantra

    2007-12-01

    The omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA, 22:6n-3) has been previously shown to facilitate some of the vital functions of astrocytes. Since some dietary oils contain alpha-linolenic acid (ALA, 18:3n-3), which is a precursor of DHA, we examined their effect on astrocyte development. Fatty acids (FAs) were isolated from commonly used oils and their compositions were determined by GLC. FAs from three oils, viz. coconut, mustard and linseed were studied for their effect on astrocyte morphology. Parallel studies were conducted with FAs from the same oils after heating for 72 h. Unlike coconut oil, FAs from mustard and linseed, both heated and raw, caused significant morphogenesis of astrocytes in culture. ss-AR binding was also substantially increased in astrocytes treated with FAs from raw mustard and linseed oils as compared to astrocytes grown in normal medium. The expression profile of the isoforms of GFAP showed that astrocyte maturation by FAs of mustard and linseed oil was associated with appearance of acidic variants of GFAP and disappearance of some neutral isoforms similar to that observed in cultures grown in serum containing medium or in the presence of DHA. Taken together, the study highlights the contribution of specific dietary oils in facilitating astrocyte development that can have potential impact on human health. PMID:17823864

  20. Prolonged acid rain facilitates soil organic carbon accumulation in a mature forest in Southern China.

    PubMed

    Wu, Jianping; Liang, Guohua; Hui, Dafeng; Deng, Qi; Xiong, Xin; Qiu, Qingyan; Liu, Juxiu; Chu, Guowei; Zhou, Guoyi; Zhang, Deqiang

    2016-02-15

    With the continuing increase in anthropogenic activities, acid rain remains a serious environmental threat, especially in the fast developing areas such as southern China. To detect how prolonged deposition of acid rain would influence soil organic carbon accumulation in mature subtropical forests, we conducted a field experiment with simulated acid rain (SAR) treatments in a monsoon evergreen broadleaf forest at Dinghushan National Nature Reserve in southern China. Four levels of SAR treatments were set by irrigating plants with water of different pH values: CK (the control, local lake water, pH ≈ 4.5), T1 (water pH=4.0), T2 (water pH=3.5), and T3 (water pH=3.0). Results showed reduced pH measurements in the topsoil exposed to simulated acid rains due to soil acidification. Soil respiration, soil microbial biomass and litter decomposition rates were significantly decreased by the SAR treatments. As a result, T3 treatment significantly increased the total organic carbon by 24.5% in the topsoil compared to the control. Furthermore, surface soil became more stable as more recalcitrant organic matter was generated under the SAR treatments. Our results suggest that prolonged acid rain exposure may have the potential to facilitate soil organic carbon accumulation in the subtropical forest in southern China. PMID:26657252

  1. Enamel proteins mitigate mechanical and structural degradations in mature human enamel during acid attack

    NASA Astrophysics Data System (ADS)

    Lubarsky, Gennady V.; Lemoine, Patrick; Meenan, Brian J.; Deb, Sanjukta; Mutreja, Isha; Carolan, Patrick; Petkov, Nikolay

    2014-04-01

    A hydrazine deproteination process was used to investigate the role of enamel proteins in the acid erosion of mature human dental enamel. Bright field high resolution transmission electron micrographs and x-ray diffraction analysis show no crystallographic changes after the hydrazine treatment with similar nanoscale hydroxyapatite crystallite size and orientation for sound and de-proteinated enamel. However, the presence of enamel proteins reduces the erosion depth, the loss of hardness and the loss of structural order in enamel, following exposure to citric acid. Nanoindentation creep is larger for sound enamel than for deproteinated enamel but it reduces in sound enamel after acid attack. These novel results are consistent with calcium ion-mediated visco-elasticty in enamel matrix proteins as described previously for nacre, bone and dental proteins. They are also in good agreement with a previous double layer force spectroscopy study by the authors which found that the proteins electrochemically buffer enamel against acid attack. Finally, this suggests that acid attack, and more specifically dental erosion, is influenced by ionic permeation through the enamel layer and that it is mitigated by the enamel protein matrix.

  2. Resistance to benzalkonium chloride, peracetic acid and nisin during formation of mature biofilms by Listeria monocytogenes.

    PubMed

    Saá Ibusquiza, P; Herrera, J J R; Cabo, M L

    2011-05-01

    Increase of resistance to the application of benzalkonium chloride (BAC), peracetic acid (PA) and nisin during biofilm formation at 25 °C by three strains of Listeria monocytogenes (CECT 911, CECT 4032, CECT 5873 and BAC-adapted CECT 5873) in different scenarios was compared. For this purpose, resistance after 4 and 11-days of biofilm formation was quantified in terms of lethal dose 90% values (LD(90)), determined according with a dose-response logistic mathematical model. Microscopic analyses after 4 and 11-days of L. monocytogenes biofilm formation were also carried out. Results demonstrated a relation between the microscopic structure and the resistance to the assayed biocides in matured biofilms. The worst cases being biofilms formed by the strain 4032 (in both stainless steel and polypropylene), which showed a complex "cloud-type" structure that correlates with the highest resistance of this strain against the three biocides during biofilm maturation. However, that increase in resistance and complexity appeared not to be dependent on initial bacterial adherence, thus indicating mature biofilms rather than planctonic cells or early-stage biofilms must be considered when disinfection protocols have to be optimized. PA seemed to be the most effective of the three disinfectants used for biofilms. We hypothesized both its high oxidizing capacity and low molecular size could suppose an advantage for its penetration inside the biofilm. We also demonstrated that organic material counteract with the biocides, thus indicating the importance of improving cleaning protocols. Finally, by comparing strains 5873 and 5873 adapted to BAC, several adaptative cross-responses between BAC and nisin or peracetic acid were identified. PMID:21356446

  3. Gas-phase ion/ion reactions of peptides and proteins: acid/base, redox, and covalent chemistries

    PubMed Central

    Prentice, Boone M.

    2013-01-01

    Gas-phase ion/ion reactions are emerging as useful and flexible means for the manipulation and characterization of peptide and protein biopolymers. Acid/base-like chemical reactions (i.e., proton transfer reactions) and reduction/oxidation (redox) reactions (i.e., electron transfer reactions) represent relatively mature classes of gas-phase chemical reactions. Even so, especially in regards to redox chemistry, the widespread utility of these two types of chemistries is undergoing rapid growth and development. Additionally, a relatively new class of gas-phase ion/ion transformations is emerging which involves the selective formation of functional-group-specific covalent bonds. This feature details our current work and perspective on the developments and current capabilities of these three areas of ion/ion chemistry with an eye towards possible future directions of the field. PMID:23257901

  4. A method for the 32P labeling of peptides or peptide nucleic acid oligomers

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

    A novel approach to the radioactive labeling of peptides and PNA oligomers is described. It is based on the conjugation of a deoxynucleoside 3'-phosphate with the terminal amine of the substrate, followed by phosphorylation of the 5'-hydroxyl group of the nucleotide using T4 polynucleotide kinase and [gamma-32P]ATP.

  5. Neurokinin B-related Peptide Suppresses the Expression of GnRH I, Kiss2 and tac3 in the Brain of Mature Female Nile tilapia Oreochromis niloticus

    PubMed Central

    Jin, Ye Hwa; Park, Jin Woo; Kim, Jung-Hyun; Kwon, Joon Yeong

    2016-01-01

    Neurokinin B (NKB) and neurokinin B related peptide (NKBRP) belong to tachykinin peptide family. Theyact as a neurotransmitter and/or neuromodulator. Mutation of NKB and/or its cognate receptor, NK3R resulted in hypogonadotropic hypogonadism in mammals, implying a strong involvement of NKB/NK3R system in controlling mammalian reproduction. Teleosts possess NKBRP as well as NKB, but their roles in fish reproduction need to be clarified. In this study, NKB and NKBRP coding gene (tac3) was cloned from Nile tilapia and sequenced. Based on the sequence, Nile tilapia NKB and NKBRP peptide were synthesized and their biological potencies were tested in vitro pituitary culture. The synthetic NKBRP showed direct inhibitory effect on the expression of GTH subunits at the pituitary level. This inhibitory effect was confirmed in vivo by means of intraperitoneal (ip) injection of synthetic NKB and NKBRP to mature female tilapia (20 pmol/g body weight [BW]). Both NKB and NKBRP had no effect on the plasma level of sex steroids, E2 and 11-KT. However, NKBRP caused declines of expression level of GnRH I, Kiss2 and tac3 mRNAs in the brain while NKB seemed to have no distinct effect. These results indicate some inhibitory roles of NKBRP in reproduction of mature female Nile tilapia, although their exact functions are not clear at the moment. PMID:27294210

  6. Neurokinin B-related Peptide Suppresses the Expression of GnRH I, Kiss2 and tac3 in the Brain of Mature Female Nile tilapia Oreochromis niloticus.

    PubMed

    Jin, Ye Hwa; Park, Jin Woo; Kim, Jung-Hyun; Kwon, Joon Yeong

    2016-03-01

    Neurokinin B (NKB) and neurokinin B related peptide (NKBRP) belong to tachykinin peptide family. Theyact as a neurotransmitter and/or neuromodulator. Mutation of NKB and/or its cognate receptor, NK3R resulted in hypogonadotropic hypogonadism in mammals, implying a strong involvement of NKB/NK3R system in controlling mammalian reproduction. Teleosts possess NKBRP as well as NKB, but their roles in fish reproduction need to be clarified. In this study, NKB and NKBRP coding gene (tac3) was cloned from Nile tilapia and sequenced. Based on the sequence, Nile tilapia NKB and NKBRP peptide were synthesized and their biological potencies were tested in vitro pituitary culture. The synthetic NKBRP showed direct inhibitory effect on the expression of GTH subunits at the pituitary level. This inhibitory effect was confirmed in vivo by means of intraperitoneal (ip) injection of synthetic NKB and NKBRP to mature female tilapia (20 pmol/g body weight [BW]). Both NKB and NKBRP had no effect on the plasma level of sex steroids, E2 and 11-KT. However, NKBRP caused declines of expression level of GnRH I, Kiss2 and tac3 mRNAs in the brain while NKB seemed to have no distinct effect. These results indicate some inhibitory roles of NKBRP in reproduction of mature female Nile tilapia, although their exact functions are not clear at the moment. PMID:27294210

  7. The cumulus cell layer protects the bovine maturing oocyte against fatty acid-induced lipotoxicity.

    PubMed

    Lolicato, Francesca; Brouwers, Jos F; de Lest, Chris H A van; Wubbolts, Richard; Aardema, Hilde; Priore, Paola; Roelen, Bernard A J; Helms, J Bernd; Gadella, Bart M

    2015-01-01

    Mobilization of fatty acids from adipose tissue during metabolic stress increases the amount of free fatty acids in blood and follicular fluid and is associated with impaired female fertility. In a previous report, we described the effects of the three predominant fatty acids in follicular fluid (saturated palmitate and stearate and unsaturated oleate) on oocyte maturation and quality. In the current study, the effects of elevated fatty acid levels on cumulus cells were investigated. In a dose-dependent manner, the three fatty acids induced lipid storage in cumulus cells accompanied by an enhanced immune labeling of perilipin-2, a marker for lipid droplets. Lipidomic analysis confirmed incorporation of the administered fatty acids into triglyceride, resulting in a 3- to 6-fold increase of triglyceride content. In addition, palmitate selectively induced ceramide formation, which has been implicated in apoptosis. Indeed, of the three fatty acids tested, palmitate induced reactive oxygen species formation, caspase 3 activation, and mitochondria deterioration, leading to degeneration of the cumulus cell layers. This effect could be mimicked by addition of the ceramide-C2 analog and could be inhibited by the ceramide synthase inhibitor fumonisin-B1. Interfering with the intactness of the cumulus cell layers, either by mechanical force or by palmitate treatment, resulted in enhanced uptake of lipids in the oocyte and increased radical formation. Our results show that cumulus cells act as a barrier, protecting oocytes from in vitro induced lipotoxic effects. We suggest that this protective function of the cumulus cell layers is important for the developmental competence of the oocyte. The relevance of our findings for assisted reproduction technologies is discussed. PMID:25297544

  8. CD10/neutral endopeptidase 24.11 regulates fetal lung growth and maturation in utero by potentiating endogenous bombesin-like peptides.

    PubMed Central

    King, K A; Hua, J; Torday, J S; Drazen, J M; Graham, S A; Shipp, M A; Sunday, M E

    1993-01-01

    Bombesin-like peptides (BLPs) are mitogens for bronchial epithelial cells and small cell lung carcinomas, and increase fetal lung growth and maturation in utero and in organ cultures. BLPs are hydrolyzed by the enzyme CD10/neutral endopeptidase 24.11 (CD10/NEP) which is expressed in bronchial epithelium and functions to inhibit BLP-mediated growth of small cell lung carcinomas. To determine whether CD10/NEP regulates peptide-mediated lung development, we administered a specific CD10/NEP inhibitor, SCH32615, to fetal mice in utero from gestational days e15-17. Fetal lung tissues were evaluated on e18 for: (a) growth using [3H]thymidine incorporation into nuclear DNA; and (b) maturation using: [3H]-choline incorporation into surfactant phospholipids, electron microscopy for type II pneumocytes, and Northern blot analyses for surfactant apoproteins A, B, and C. Inhibition of CD10/NEP stimulated [3H]thymidine incorporation into DNA (70% above baseline, P < 0.005), [3H]choline incorporation into surfactant phospholipids (38% above baseline, P < 0.005), increased numbers of type II pneumocytes (36% above baseline, P = 0.07), and fivefold higher surfactant protein A transcripts (P < 0.05). CD10/NEP-mediated effects were completely blocked by the specific bombesin receptor antagonist, [D-Phe12, Leu14]bombesin. These observations suggest that CD10/NEP regulates fetal lung growth and maturation mediated by endogenous BLPs. Images PMID:8486767

  9. Changes in brain seabream GnRH mRNA and pituitary seabream GnRH peptide levels during ovarian maturation in female barfin flounder.

    PubMed

    Amano, Masafumi; Pham, Ky Xuan; Amiya, Noriko; Yamanome, Takeshi; Yamamori, Kunio

    2008-09-01

    The pleuronectid barfin flounder Verasper moseri expresses three forms of gonadotropin-releasing hormones (GnRHs), i.e., seabream GnRH (sbGnRH), salmon GnRH, and chicken GnRH-II. Among these, sbGnRH is the dominant form in the pituitary, indicating that sbGnRH regulates gonadal maturation. In order to clarify the physiological roles of sbGnRH during ovarian maturation in reared female barfin flounder, the changes in brain sbGnRH mRNA levels and pituitary sbGnRH peptide levels were examined by real-time quantitative PCR and time-resolved fluoroimmunoassay, respectively. The fish hatched in April 2002. The gonadosomatic index remained low until August 2004 and increased thereafter until April 2005 when the fish began to ovulate. The sbGnRH mRNA levels per brain increased significantly from April 2004 to April 2005. Pituitary sbGnRH peptide levels also increased significantly during this period. These results indicate that sbGnRH is involved in ovarian maturation and ovulation in the barfin flounder. PMID:18662692

  10. Site-Specific Characterization of d-Amino Acid Containing Peptide Epimers by Ion Mobility Spectrometry

    PubMed Central

    2013-01-01

    Traditionally, the d-amino acid containing peptide (DAACP) candidate can be discovered by observing the differences of biological activity and chromatographic retention time between the synthetic peptides and naturally occurring peptides. However, it is difficult to determine the exact position of d-amino acid in the DAACP candidates. Herein, we developed a novel site-specific strategy to rapidly and precisely localize d-amino acids in peptides by ion mobility spectrometry (IMS) analysis of mass spectrometry (MS)-generated epimeric fragment ions. Briefly, the d/l-peptide epimers were separated by online reversed-phase liquid chromatography and fragmented by collision-induced dissociation (CID), followed by IMS analysis. The epimeric fragment ions resulting from d/l-peptide epimers exhibit conformational differences, thus showing different mobilities in IMS. The arrival time shift between the epimeric fragment ions was used as criteria to localize the d-amino acid substitution. The utility of this strategy was demonstrated by analysis of peptide epimers with different molecular sizes, [d-Trp]-melanocyte-stimulating hormone, [d-Ala]-deltorphin, [d-Phe]-achatin-I, and their counterparts that contain all-l amino acids. Furthermore, the crustacean hyperglycemia hormones (CHHs, 8.5 kDa) were isolated from the American lobster Homarus americanus and identified by integration of MS-based bottom-up and top-down sequencing approaches. The IMS data acquired using our novel site-specific strategy localized the site of isomerization of l- to d-Phe at the third residue of the CHHs from the N-terminus. Collectively, this study demonstrates a new method for discovery of DAACPs using IMS technique with the ability to localize d-amino acid residues. PMID:24328107

  11. Targeting lipopolyplexes using bifunctional peptides incorporating hydrophobic spacer amino acids: synthesis, transfection, and biophysical studies.

    PubMed

    Pilkington-Miksa, Michael A; Writer, Michele J; Sarkar, Supti; Meng, Qing-Hai; Barker, Suzie E; Shamlou, Parviz Ayazi; Hailes, Helen C; Hart, Stephen L; Tabor, Alethea B

    2007-01-01

    We have developed efficient synthetic routes to two hydrophobic amino acids, suitably protected for solid-phase peptide synthesis, and have successfully synthesized peptides containing these or other hydrophobic amino acids as spacers between a Lys16 moiety and an integrin-targeting motif. These peptides have in turn been used to formulate a range of lipopolyplex vectors with Lipofectin and plasmid DNA. The transfection efficiencies of these vectors and their aggregation behavior in buffers and in serum have been studied. We have shown that vectors containing peptides incorporating long linkers that are entirely hydrophobic are less efficient transfection agents. However, linkers of equivalent length that are in part hydrophobic show improved transfection properties, which is probably due to the improved accessibility of the integrin-binding motif. PMID:17915956

  12. Incorporation of N-amidino-pyroglutamic acid into peptides using intramolecular cyclization of alpha-guanidinoglutaric acid.

    PubMed

    Burov, Sergey; Moskalenko, Yulia; Dorosh, Marina; Shkarubskaya, Zoya; Panarin, Evgeny

    2009-11-01

    N-terminal modification of peptides by unnatural amino acids significantly affects their enzymatic stability, conformational properties and biological activity. Application of N-amidino-amino acids, positively charged under physiological conditions, can change peptide conformation and its affinity to the corresponding receptor. In this article, we describe synthesis of short peptides, containing a new building block-N-amidino-pyroglutamic acid. Although direct guanidinylation of pyroglutamic acid and oxidation of N-amidino-proline using RuO(4) did not produce positive results, N-amidino-Glp-Phe-OH was synthesized on Wang polymer by cyclization of alpha-guanidinoglutaric acid residue. In the course of synthesis, it was found that literature procedure of selective Boc deprotection using TMSOTf/TEA reagent is accompanied by concomitant side reaction of triethylamine alkylation by polymer linker fragment. It should be mentioned that independently from cyclization time and coupling agent (DIC or HCTU), the lactam formation was incomplete. Separation of the cyclic product from the linear precursor was achieved by HPLC in ammonium formate buffer at pH 6. HPLC analysis showed N-amidino-Glp-Phe-OH stability at acidic and physiological pH and fast ring opening in water solution at pH 9. The suggested method of N-amidino-Glp residue formation can be applied in the case of short peptide chains, whereas synthesis of longer ones will require fragment condensation approach. PMID:19739127

  13. Interaction of linear cationic peptides with phospholipid membranes and polymers of sialic acid.

    PubMed

    Kuznetsov, A S; Dubovskii, P V; Vorontsova, O V; Feofanov, A V; Efremov, R G

    2014-05-01

    Polysialic acid (PSA) is a natural anionic polymer typically occurring on the outer surface of cell membranes. PSA is involved in cell signaling and intermolecular interactions with proteins and peptides. The antimicrobial potential of peptides is usually evaluated in model membranes consisting of lipid bilayers but devoid of either PSA or its analogs. The goal of this work was to investigate the possible effect of PSA on the structure of melittin (Mlt) and latarcins Ltc1K, Ltc2a, and the activity of these peptides with respect to model membranes. These peptides are linear cationic ones derived from the venom of bee (Mlt) and spider (both latarcins). The length of each of the peptides is 26 amino acid residues, and they all have antimicrobial activity. However, they differ with respect to conformational mobility, hydrophobic characteristics, and overall charge. In this work, using circular dichroism spectroscopy, we show that the peptides adopt an α-helical conformation upon interaction with either PSA or phospholipid liposomes formed of either zwitterionic or anionic phospholipids or their mixtures. The extent of helicity depends on the amino acid sequence and properties of the medium. Based on small angle X-ray scattering data and the analysis of the fluorescence spectrum of the Trp residue in Mlt, we conclude that the peptide forms an oligomeric complex consisting of α-helical Mlt and several PSA molecules. Both latarcins, unlike Mlt, the most hydrophobic of the peptides, interact weakly with zwitterionic liposomes. However, they bind anionic liposomes or those composed of anionic/zwitterionic lipid mixtures. Latarcin Ltc1K forms associates on liposomes composed of zwitterionic/anionic lipid mixture. The structure of the peptide associates is either disordered or of β-sheet conformation. In all other cases the studied peptides adopt predominately α-helical conformation. In addition, we demonstrate that PSA inhibits membranolytic activity of Mlt and latarcin

  14. 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. PMID:27010507

  15. Stem growth reduction in mature Sitka spruce trees exposed to acid mist.

    PubMed

    Crossley, A; Sheppard, L J; Cape, J N; Smith, R I; Harvey, F J

    1997-01-01

    An eighteen-year-old clone of Sitka spruce (Picea sitchensis (Bong.) Carr) growing in the field was used to evaluate the whole tree response of 'mature' Sitka spruce to acid mist treatment. The mist, an equimolar mixture of H(2)SO(4) and NH(4)NO(3) at pH 2.5 with or without particles (soda glass ballotini < 20 microm diameter), was applied twice weekly (equivalent to 4 mm precipitation week(-1)) throughout the growing season, May-November 1990-1992. The annual dose of S, N, H applied as mist (at 51, 48 and 3.3 kg ha(-1), respectively) was 2.5 times that measured in the Scottish uplands. Throughout the experiment there was no evidence of visible injury symptoms, yet there was a highly significant reduction (p < 0.02) in the stem-area increment relative to the stem area at the start, measured using vernier dendrometer bands. There was no significant difference between the (acid mist + particle) and the acid mist only treatments. The mean relative stem-area increment over two complete growing seasons (1991-1992) was 65% for control trees, but only 53% for acid-misted trees. PMID:15093418

  16. Biomimetic vaterite formation at surfaces structurally templated by oligo(glutamic acid) peptides.

    PubMed

    Lu, Hao; Hood, Matthew A; Mauri, Sergio; Baio, Joe E; Bonn, Mischa; Muñoz-Espí, Rafael; Weidner, Tobias

    2015-11-14

    Previous studies have reported that the metastable vaterite phase of calcium carbonate can be stabilized in solution by acidic additives. Here we demonstrate that vaterite can also be stabilized directly at surfaces by engineered peptides. Our data show that the mineralisation occurs in a 'self-templating' process where calcium ions restructure the peptide backbone, which in turn allows for effective vaterite precipitation. PMID:26376942

  17. Self-assembly pathway of peptide nanotubes formed by a glutamatic acid-based bolaamphiphile.

    PubMed

    da Silva, Emerson Rodrigo; Alves, Wendel Andrade; Castelletto, Valeria; Reza, Mehedi; Ruokolainen, Janne; Hussain, Rohanah; Hamley, Ian William

    2015-07-25

    The self-assembly of peptide nanotubes formed by an L-glutamic acid-based bolaamphiphile is shown to proceed via a remarkable mechanism where the peptide conformation changes from β-sheet to unordered. The kinetics of this process are elucidated via X-ray scattering and UV circular dichroism methods. The reverse transition from "unordered" to β-sheet structures is triggered by UV radiation. PMID:26094619

  18. Predicting Three-Dimensional Conformations of Peptides Constructed of Only Glycine, Alanine, Aspartic Acid, and Valine

    NASA Astrophysics Data System (ADS)

    Oda, Akifumi; Fukuyoshi, Shuichi

    2015-06-01

    The GADV hypothesis is a form of the protein world hypothesis, which suggests that life originated from proteins (Lacey et al. 1999; Ikehara 2002; Andras 2006). In the GADV hypothesis, life is thought to have originated from primitive proteins constructed of only glycine, alanine, aspartic acid, and valine ([GADV]-proteins). In this study, the three-dimensional (3D) conformations of randomly generated short [GADV]-peptides were computationally investigated using replica-exchange molecular dynamics (REMD) simulations (Sugita and Okamoto 1999). Because the peptides used in this study consisted of only 20 residues each, they could not form certain 3D structures. However, the conformational tendencies of the peptides were elucidated by analyzing the conformational ensembles generated by REMD simulations. The results indicate that secondary structures can be formed in several randomly generated [GADV]-peptides. A long helical structure was found in one of the hydrophobic peptides, supporting the conjecture of the GADV hypothesis that many peptides aggregated to form peptide multimers with enzymatic activity in the primordial soup. In addition, these results indicate that REMD simulations can be used for the structural investigation of short peptides.

  19. C-3 benzoic acid derivatives of C-3 deoxybetulinic acid and deoxybetulin as HIV-1 maturation inhibitors.

    PubMed

    Liu, Zheng; Swidorski, Jacob J; Nowicka-Sans, Beata; Terry, Brian; Protack, Tricia; Lin, Zeyu; Samanta, Himadri; Zhang, Sharon; Li, Zhufang; Parker, Dawn D; Rahematpura, Sandhya; Jenkins, Susan; Beno, Brett R; Krystal, Mark; Meanwell, Nicholas A; Dicker, Ira B; Regueiro-Ren, Alicia

    2016-04-15

    A series of C-3 phenyl- and heterocycle-substituted derivatives of C-3 deoxybetulinic acid and C-3 deoxybetulin was designed and synthesized as HIV-1 maturation inhibitors (MIs) and evaluated for their antiviral activity and cytotoxicity in cell culture. A 4-subsituted benzoic acid moiety was identified as an advantageous replacement for the 3'3'-dimethylsuccinate moiety present in previously disclosed MIs that illuminates new aspects of the topography of the pharmacophore. The new analogs exhibit excellent in vitro antiviral activity against wild-type (wt) virus and a lower serum shift when compared with the prototypical HIV-1 MI bevirimat (1, BVM), the first MI to be evaluated in clinical studies. Compound 9a exhibits comparable cell culture potency toward wt virus as 1 (WT EC50=16nM for 9a compared to 10nM for 1). However, the potency of 9a is less affected by the presence of human serum, while the compound displays a similar pharmacokinetic profile in rats to 1. Hence 9a, the 4-benzoic acid derivative of deoxybetulinic acid, represents a new starting point from which to explore the design of a 2nd generation MI. PMID:26968652

  20. Incorporation of Noncanonical Amino Acids into Rosetta and Use in Computational Protein-Peptide Interface Design

    PubMed Central

    Renfrew, P. Douglas; Choi, Eun Jung; Bonneau, Richard; Kuhlman, Brian

    2012-01-01

    Noncanonical amino acids (NCAAs) can be used in a variety of protein design contexts. For example, they can be used in place of the canonical amino acids (CAAs) to improve the biophysical properties of peptides that target protein interfaces. We describe the incorporation of 114 NCAAs into the protein-modeling suite Rosetta. We describe our methods for building backbone dependent rotamer libraries and the parameterization and construction of a scoring function that can be used to score NCAA containing peptides and proteins. We validate these additions to Rosetta and our NCAA-rotamer libraries by showing that we can improve the binding of a calpastatin derived peptides to calpain-1 by substituting NCAAs for native amino acids using Rosetta. Rosetta (executables and source), auxiliary scripts and code, and documentation can be found at (http://www.rosettacommons.org/). PMID:22431978

  1. Thioglycolic acid inhibits mouse oocyte maturation and affects chromosomal arrangement and spindle configuration.

    PubMed

    Hou, S Y; Zhang, L; Wu, K; Xia, L

    2008-05-01

    Previous studies have shown that thioglycolic acid (TGA) leads to potential reproductive toxicology. To clarify the exact effects of this compound on reproduction, mice oocytes were treated with different TGA doses. At the end of the culture period, the nuclear status of mice oocytes was assessed under an inverted microscope. After immunofluorescence staining, the chromosomal arrangement and spindle configuration of oocytes were evaluated. The results indicated that TGA decreases the percentage of first polar body formation but does not influence that of germinal vesicle breakdown. TGA induces abnormal chromosomal arrangement and spindle elongation. In conclusion, TGA inhibits in-vitro maturation of mice oocytes and affects chromosomal arrangement and spindle configuration. Furthermore, it probably interferes with biochemical changes that occur during meiosis, resulting in aberrant development. PMID:19022875

  2. Calcium Binding to Amino Acids and Small Glycine Peptides in Aqueous Solution: Toward Peptide Design for Better Calcium Bioavailability.

    PubMed

    Tang, Ning; Skibsted, Leif H

    2016-06-01

    Deprotonation of amino acids as occurs during transfer from stomach to intestines during food digestion was found by comparison of complex formation constants as determined electrochemically for increasing pH to increase calcium binding (i) by a factor of around 6 for the neutral amino acids, (ii) by a factor of around 4 for anions of the acidic amino acids aspartic and glutamic acid, and (iii) by a factor of around 5.5 for basic amino acids. Optimized structures of the 1:1 complexes and ΔHbinding for calcium binding as calculated by density functional theory (DFT) confirmed in all complexes a stronger calcium binding and shorter calcium-oxygen bond length in the deprotonated form. In addition, the stronger calcium binding was also accompanied by a binding site shift from carboxylate binding to chelation by α-amino group and carboxylate oxygen for leucine, aspartate, glutamate, alanine, and asparagine. For binary amino acid mixtures, the calcium-binding constant was close to the predicted geometric mean of the individual amino acid binding constants indicating separate binding of calcium to two amino acids when present together in solution. At high pH, corresponding to conditions for calcium absorption, the binding affinity increased in the order Lys < Arg < Cys < Gln < Gly ∼ Ala < Asn < His < Leu < Glu< Asp. In a series of glycine peptides, calcium-binding affinity was found to increase in the order Gly-Leu ∼ Gly-Gly < Ala-Gly < Gly-His ∼ Gly-Lys-Gly < Glu-Cys-Gly < Gly-Glu, an ordering confirmed by DFT calculations for the dipeptides and which also accounted for large synergistic effects in calcium binding for up to 6 kJ/mol when compared to the corresponding amino acid mixtures. PMID:27159329

  3. [Amino acid composition and peptide maps of udder and serum albumins in lactating and nonlactating cows].

    PubMed

    Lagodiuk, P Z; Klos, Iu S; Charkin, V A; Kisil', I O

    1983-01-01

    Amino acids and peptides of albumin hydrolyzates from the mammary gland and blood serum were studied for lactating and nonlactating (dry, pregnant 1-4.5 and 4.5-9 months) black-and-white cows. Most pronounced difference between the content of certain amino acids of the mammary gland and blood serum albumins are established for lactating cows and least pronounced for nonlactating dry cows. Dactylography detected 55-57 fragments of products resulted from trypsin hydrolysis of the mammary gland and blood serum albumins of the animals under study. Differences are found in the content and mobility of certain peptides. PMID:6829076

  4. Extracellular matrix-like surfactant polymers containing arginine-glycine-aspartic acid (RGD) peptides.

    PubMed

    Anderson, Eric H; Ruegsegger, Mark A; Murugesan, Gurunathan; Kottke-Marchant, Kandice; Marchant, Roger E

    2004-08-01

    We report on a novel series of biomimetic polymers exhibiting interfacial properties similar to the extracellular matrix. A series of well-defined surfactant polymers were synthesized by simultaneously incorporating arginine-glycine-aspartic acid (RGD) peptide, dextran oligosaccharide, and hexyl ligands with controlled feed ratios onto a poly(vinyl amine) (PVAm) backbone. The peptide sequence was H-GSSSGRGDSPA-NH(2) (Pep) having a hydrophilic extender at the amino terminus and capped carboxy terminus. The peptide-to-dextran (Pep:Dex) ratios were varied to create surfactants having 0, 25, 50, 75, and 100 mol-% peptide relative to dextran. The surfactants were characterized by IR, NMR and atomic force microscopy (AFM) for composition and surface active properties. AFM confirmed full surface coverage of PVAm(Pep)(100%) on graphite, and supported the mechanism of interdigitation of hexyl ligands between surfactant molecules within a specified range of hexyl chain densities. the attachment and growth of human pulmonary artery endothelial cells on the PVAm(Pep)(100%) surface was identical to the fibronectin positive control. Cell adhesion decreased dramatically with decreasing peptide density on the surfactant polymers. Molecular model of a peptide surfactant polymer, consisting of poly(vinyl amine) backbone with peptide, dextran oligosaccharide and hexyl branches coupled to the polymer chain. PMID:15468270

  5. Peptide Synthesis through Cell-Free Expression of Fusion Proteins Incorporating Modified Amino Acids as Latent Cleavage Sites for Peptide Release.

    PubMed

    Liutkus, Mantas; Fraser, Samuel A; Caron, Karine; Stigers, Dannon J; Easton, Christopher J

    2016-05-17

    Chlorinated analogues of Leu and Ile are incorporated during cell-free expression of peptides fused to protein, by exploiting the promiscuity of the natural biosynthetic machinery. They then act as sites for clean and efficient release of the peptides simply by brief heat treatment. Dehydro analogues of Leu and Ile are similarly incorporated as latent sites for peptide release through treatment with iodine under cold conditions. These protocols complement enzyme-catalyzed methods and have been used to prepare calcitonin, gastrin-releasing peptide, cholecystokinin-7, and prolactin-releasing peptide prohormones, as well as analogues substituted with unusual amino acids, thus illustrating their practical utility as alternatives to more traditional chemical peptide synthesis. PMID:26918308

  6. THE AMPHOTERIC PROPERTIES OF SOME AMINO-ACIDS AND PEPTIDES.

    PubMed

    Eckweiler, H; Noyes, H M; Falk, K G

    1921-01-20

    The titration curves of solutions of glycine, alanine, alpha-ammo-butyric acid, leucine, glycyl-glycine, alanyl-glycine, alanyl-alanine, acetone, acetamide, urea, acetic acid, and aceturic acid were determined and some of the relations as dependent upon the chemical structures discussed. The isoelectric points of some of the amphoteric electrolytes were found experimentally. The definition of isoelectric point, its theoretical significance, and method of calculation were considered in some detail. PMID:19871865

  7. Characterization of bioactive RGD peptide immobilized onto poly(acrylic acid) thin films by plasma polymerization

    NASA Astrophysics Data System (ADS)

    Seo, Hyun Suk; Ko, Yeong Mu; Shim, Jae Won; Lim, Yun Kyong; Kook, Joong-Ki; Cho, Dong-Lyun; Kim, Byung Hoon

    2010-11-01

    Plasma surface modification can be used to improve the surface properties of commercial pure Ti by creating functional groups to produce bioactive materials with different surface topography. In this study, a titanium surface was modified with acrylic acid (AA) using a plasma treatment and immobilized with bioactive arginine-glycine-aspartic acid (RGD) peptide, which may accelerate the tissue integration of bone implants. Both terminals containing the -NH2 of RGD peptide sequence and -COOH of poly(acrylic acid) (PAA) thin film were combined with a covalent bond in the presence of 1-ethyl-3-3-dimethylaminopropyl carbodiimide (EDC). The chemical structure and morphology of AA film and RGD immobilized surface were investigated by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR), atomic force microscopy (AFM), and scanning electron microscopy (SEM). All chemical analysis showed full coverage of the Ti substrate with the PAA thin film containing COOH groups and the RGD peptide. The MC3T3-E1 cells were cultured on each specimen, and the cell alkaline phosphatase (ALP) activity were examined. The surface-immobilized RGD peptide has a significantly increased the ALP activity of MC3T3-E1 cells. These results suggest that the RGD peptide immobilization on the titanium surface has an effect on osteoblastic differentiation of MC3T3-E1 cells and potential use in osteo-conductive bone implants.

  8. Lactobacillus gasseri requires peptides, not proteins or free amino acids, for growth in milk.

    PubMed

    Arakawa, K; Matsunaga, K; Takihiro, S; Moritoki, A; Ryuto, S; Kawai, Y; Masuda, T; Miyamoto, T

    2015-03-01

    Lactobacillus gasseri is a widespread commensal lactic acid bacterium inhabiting human mucosal niches and has many beneficial effects as a probiotic. However, L. gasseri is difficult to grow in milk, which hurts usability for the food industry. It had been previously reported that supplementation with yeast extract or proteose peptone, including peptides, enables L. gasseri to grow well in milk. In this study, our objective was to confirm peptide requirement of L. gasseri and evaluate efficacy of peptide release by enzymatic proteolysis on growth of L. gassei in milk. Three strains of L. gasseri did not grow well in modified DeMan, Rogosa, Sharpe broth without any nitrogen sources (MRS-N), but addition of a casein-derived peptide mixture, tryptone, promoted growth. In contrast, little effect was observed after adding casein or a casein-derived amino acid mixture, casamino acids. These results indicate that L. gasseri requires peptides, not proteins or free amino acids, among milk-derived nitrogen sources for growth. Lactobacillus gasseri JCM 1131T hardly had growth capacity in 6 kinds of milk-based media: bovine milk, human milk, skim milk, cheese whey, modified MRS-N (MRSL-N) supplemented with acid whey, and MRSL-N supplemented with casein. Moreover, treatment with digestive proteases, particularly pepsin, to release peptides made it grow well in each milk-based medium. The pepsin treatment was the most effective for growth of strain JCM 1131T in skim milk among the tested food-grade proteases such as trypsin, α-chymotrypsin, calf rennet, ficin, bromelain, and papain. As well as strain JCM 1131T, pepsinolysis of milk improved growth of other L. gasseri strains and some strains of enteric lactobacilli such as Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus johnsonii, and Lactobacillus reuteri. These results suggest that some relatives of L. gasseri also use peptides as desirable nitrogen sources, and that milk may be a good supplier of nutritious

  9. p-Nitromandelic acid as a highly acid-stable safety-catch linker for solid-phase synthesis of peptide and depsipeptide acids.

    PubMed

    Isidro-Llobet, Albert; Alvarez, Mercedes; Burger, Klaus; Spengler, Jan; Albericio, Fernando

    2007-04-12

    [reaction: see text] p-Nitromandelic acid as a safety-catch linker for Boc/Bzl-SPPS of base-labile compounds like peptides and depsipeptides is described. This linker permits acidic removal of side-chain protection groups from the resin. For cleavage from the solid support, the p-nitro group was reduced with tin(II) chloride. After washing off the reducing agents, the (depsi)peptide acids with or without the side-chain protection schemes were obtained by microwave irradiation at 50 degrees C with 5% TFA in dioxane. PMID:17367151

  10. Bile acid-FXRα pathways regulate male sexual maturation in mice.

    PubMed

    Baptissart, Marine; Martinot, Emmanuelle; Vega, Aurélie; Sédes, Lauriane; Rouaisnel, Betty; de Haze, Angélique; Baron, Silvère; Schoonjans, Kristina; Caira, Françoise; Volle, David H

    2016-04-12

    The bile acid receptor Farnesol-X-Receptor alpha (FRXα) is a member of the nuclear receptor superfamily. FRXα is expressed in the interstitial compartment of the adult testes, which contain the Leydig cells. In adult, short term treatment (12 hours) with FRXα agonist inhibits the expression of steroidogenic genes via the induction of the Small heterodimer partner (SHP). However the consequences of FRXα activation on testicular pathophysiology have never been evaluated. We demonstrate here that mice fed a diet supplemented with bile acid during pubertal age show increased incidence of infertility. This is associated with altered differentiation and increase apoptosis of germ cells due to lower testosterone levels. At the molecular level, next to the repression of basal steroidogenesis via the induction expression of Shp and Dax-1, two repressors of steroidogenesis, the main action of the BA-FRXα signaling is through lowering the Leydig cell sensitivity to the hypothalamo-pituitary axis, the main regulator of testicular endocrine function. In conclusion, BA-FRXα signaling is a critical actor during sexual maturation. PMID:26848619

  11. Bile acid-FXRα pathways regulate male sexual maturation in mice

    PubMed Central

    Vega, Aurélie; Sédes, Lauriane; Rouaisnel, Betty; de Haze, Angélique; Baron, Silvère; Schoonjans, Kristina; Caira, Françoise; Volle, David H.

    2016-01-01

    The bile acid receptor Farnesol-X-Receptor alpha (FRXα) is a member of the nuclear receptor superfamily. FRXα is expressed in the interstitial compartment of the adult testes, which contain the Leydig cells. In adult, short term treatment (12 hours) with FRXα agonist inhibits the expression of steroidogenic genes via the induction of the Small heterodimer partner (SHP). However the consequences of FRXα activation on testicular pathophysiology have never been evaluated. We demonstrate here that mice fed a diet supplemented with bile acid during pubertal age show increased incidence of infertility. This is associated with altered differentiation and increase apoptosis of germ cells due to lower testosterone levels. At the molecular level, next to the repression of basal steroidogenesis via the induction expression of Shp and Dax-1, two repressors of steroidogenesis, the main action of the BA-FRXα signaling is through lowering the Leydig cell sensitivity to the hypothalamo-pituitary axis, the main regulator of testicular endocrine function. In conclusion, BA-FRXα signaling is a critical actor during sexual maturation. PMID:26848619

  12. Small acidic peptides from wheat germ chromatin. I. Isolation and biochemical characterization.

    PubMed

    Mancinelli, L; Castigli, E; Qualadrucci, P; Gianfranceschi, G L; Bramucci, M; Miano, A; Amici, D

    1992-01-01

    RNA synthesis in cell and cell-free systems is inhibited by a family of acidic, low molecular weight chromatin peptides (CPs). These peptides were extracted from deproteinized DNA of prokaryotic and eukaryotic cells, but the low yield of purified material by this procedure hinders efforts aimed at understanding their action mechanism in gene regulation. In this report we describe two purification methods of CPs from an easily available source, wheat germ. A comparison is made between the method starting from deproteinized DNA and the method from purified chromatin. The biological effects (inhibition of L1210 cell growth and DNA in vitro transcription) of CPs from wheat germ together with their chemical characteristics (molecular weight, amino acid composition and presence of phosphoserine) show strong homology with those of CPs from other sources. These results suggest a possible role of these chromatin peptides in controlling gene expression. PMID:1508994

  13. Synthesis of peptides from amino acids and ATP with lysine-rich proteinoid

    NASA Technical Reports Server (NTRS)

    Nakashima, T.; Fox, S. W.

    1980-01-01

    The paper examines the synthesis of peptides from aminoacids and ATP with a lysine-rich protenoid. The latter in aqueous solution catalyzes the formation of peptides from free amino acids and ATP; this catalytic activity is not found in acidic protenoids, even though the latter contain a basic aminoacid. The pH optimum for the synthesis is about 11, but it is appreciable below 8 and above 13. Temperature data indicate an optimum at 20 C or above, with little increase in rate up to 60 C. Pyrophosphate can be used instead of ATP, but the yields are lower. The ATP-aided syntheses of peptides in aqueous solution occur with several types of proteinous aminoacids.

  14. Role of the transmembrane domain in SNARE protein mediated membrane fusion: peptide nucleic acid/peptide model systems.

    PubMed

    Wehland, Jan-Dirk; Lygina, Antonina S; Kumar, Pawan; Guha, Samit; Hubrich, Barbara E; Jahn, Reinhard; Diederichsen, Ulf

    2016-08-16

    Fusion of synaptic vesicles with the presynaptic plasma membrane is mediated by Soluble NSF (N-ethylmaleimide-sensitive factor) Attachment Protein Receptor proteins also known as SNAREs. The backbone of this essential process is the assembly of SNAREs from opposite membranes into tight four helix bundles forcing membranes in close proximity. With model systems resembling SNAREs with reduced complexity we aim to understand how these proteins work at the molecular level. Here, peptide nucleic acids (PNAs) are used as excellent candidates for mimicking the SNARE recognition motif by forming well-characterized duplex structures. Hybridization between complementary PNA strands anchored in liposomes through native transmembrane domains (TMDs) induces the merger of the outer leaflets of the participating vesicles but not of the inner leaflets. A series of PNA/peptide hybrids differing in the length of TMDs and charges at the C-terminal end is presented. Interestingly, mixing of both outer and inner leaflets is seen for TMDs containing an amide in place of the natural carboxylic acid at the C-terminal end. Charged side chains at the C-terminal end of the TMDs are shown to have a negative impact on the mixing of liposomes. The length of the TMDs is vital for fusion as with the use of shortened TMDs, fusion was completely prevented. PMID:27345759

  15. 2-Chlorotrityl chloride resin. Studies on anchoring of Fmoc-amino acids and peptide cleavage.

    PubMed

    Barlos, K; Chatzi, O; Gatos, D; Stavropoulos, G

    1991-06-01

    The esterification of 2-chlorotrityl chloride resin with Fmoc-amino acids in the presence of DIEA is studied under various conditions. High esterification yields are obtained using 0.6 equiv. Fmoc-amino acid/mmol resin in DCM or DCE, in 25 min, at room temperature. The reaction proceeds without by product formation even in the case of Fmoc-Asn and Fmoc-Gln. The quantitative and easy cleavage of amino acids and peptides from 2-chlorotrityl resin, by using AcOH/TFE/DCM mixtures, is accomplished within 15-60 min at room temperature, while t-butyl type protecting groups remain unaffected. Under these exceptionally mild conditions 2-chlorotrityl cations generated during the cleavage of amino acids and peptides from resin do not attack the nucleophilic side chains of Trp, Met, and Tyr. PMID:1917309

  16. Binding of small basic peptides to membranes containing acidic lipids: theoretical models and experimental results.

    PubMed Central

    Ben-Tal, N; Honig, B; Peitzsch, R M; Denisov, G; McLaughlin, S

    1996-01-01

    We measured directly the binding of Lys3, Lys5, and Lys7 to vesicles containing acidic phospholipids. When the vesicles contain 33% acidic lipids and the aqueous solution contains 100 mM monovalent salt, the standard Gibbs free energy for the binding of these peptides is 3, 5, and 7 kcal/mol, respectively. The binding energies decrease as the mol% of acidic lipids in the membrane decreases and/or as the salt concentration increases. Several lines of evidence suggest that these hydrophilic peptides do not penetrate the polar headgroup region of the membrane and that the binding is mainly due to electrostatic interactions. To calculate the binding energies from classical electrostatics, we applied the nonlinear Poisson-Boltzmann equation to atomic models of the phospholipid bilayers and the basic peptides in aqueous solution. The electrostatic free energy of interaction, which arises from both a long-range coulombic attraction between the positively charged peptide and the negatively charged lipid bilayer, and a short-range Born or image charge repulsion, is a minimum when approximately 2.5 A (i.e., one layer of water) exists between the van der Waals surfaces of the peptide and the lipid bilayer. The calculated molar association constants, K, agree well with the measured values: K is typically about 10-fold smaller than the experimental value (i.e., a difference of about 1.5 kcal/mol in the free energy of binding). The predicted dependence of K (or the binding free energies) on the ionic strength of the solution, the mol% of acidic lipids in the membrane, and the number of basic residues in the peptide agree very well with the experimental measurements. These calculations are relevant to the membrane binding of a number of important proteins that contain clusters of basic residues. Images FIGURE 2 FIGURE 3 PMID:8842196

  17. 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.

  18. Cholera Toxin B Subunit Linked to Glutamic Acid Decarboxylase Suppresses Dendritic Cell Maturation and Function

    PubMed Central

    Odumosu, Oludare; Nicholas, Dequina; Payne, Kimberly; Langridge, William

    2012-01-01

    Dendritic cells are the largest population of antigen presenting cells in the body. One of their main functions is to regulate the delicate balance between immunity and tolerance responsible for maintenance of immunological homeostasis. Disruption of this delicate balance often results in chronic inflammation responsible for initiation of organ specific autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and type I diabetes. The cholera toxin B subunit (CTB) is a weak mucosal adjuvant known for its ability to stimulate immunity to antigenic proteins. However, conjugation of CTB to many autoantigens can induce immunological tolerance resulting in suppression of autoimmunity. In this study, we examined whether linkage of CTB to a 5 kDa C-terminal protein fragment of the major diabetes autoantigen glutamic acid decarboxylase (GAD35), can block dendritic cell (DC) functions such as biosynthesis of co-stimulatory factor proteins CD86, CD83, CD80 and CD40 and secretion of inflammatory cytokines. The results of human umbilical cord blood monocyte-derived DC - GAD35 autoantigen incubation experiments showed that inoculation of immature DCs (iDCs), with CTB-GAD35 protein dramatically suppressed levels of CD86, CD83, CD80 and CD40 co-stimulatory factor protein biosynthesis in comparison with GAD35 alone inoculated iDCs. Surprisingly, incubation of iDCs in the presence of the CTB-autoantigen and the strong immunostimulatory molecules PMA and Ionomycin revealed that CTB-GAD35 was capable of arresting PMA + Ionomycin induced DC maturation. Consistant with this finding, CTB-GAD35 mediated suppression of DC maturation was accompanied by a dramatic decrease in the secretion of the pro-inflammatory cytokines IL-12/23p40 and IL-6 and a significant increase in secretion of the immunosuppressive cytokine IL-10. Taken together, our experimental data suggest that linkage of the weak adjuvant CTB to the dominant type 1 diabetes autoantigen GAD strongly inhibits DC

  19. Phytochemicals that modulate amino acid and peptide catabolism by caprine rumen microbes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Microbe-derived ionophores and macrolide antibiotics are often added to ruminant diets, and growth promotion and feed efficiency are among the benefits. One mechanism is inhibition of microbes that catabolize amino acids or peptides and produce ammonia. Plants also produce antimicrobial ...

  20. Spectral Transition in Bio-Inspired Self-Assembled Peptide Nucleic Acid Photonic Crystals.

    PubMed

    Berger, Or; Yoskovitz, Eyal; Adler-Abramovich, Lihi; Gazit, Ehud

    2016-03-01

    The self-assembly of guanine-based peptide nucleic acid monomers into photonic crystals is described. A highly reflective lattice of guanine nanocrystals is found in the skin and ocular tissues of different species providing vivid structural colors. The fabricated guanine-based supramolecular structures respond to changes in osmolarity similar to the active spectral change mechanism employed by chameleons. PMID:26779770

  1. One-pot nanoparticulation of potentially bioactive peptides and gallic acid encapsulation.

    PubMed

    Nourbakhsh, Himan; Madadlou, Ashkan; Emam-Djomeh, Zahra; Wang, Yi-Cheng; Gunasekaran, Sundaram

    2016-11-01

    Whey protein isolate was hydrolyzed to an in vitro antioxidative hydrolysate, followed by transglutaminase-induced cross-linking and microemulsification in an oil phase. The obtained microemulsion was then dispersed in a gallic acid-rich model wastewater which caused gallic acid transportation into internal nanodroplets. Whey peptides were consequently gelled, yielding nanoparticles. Electrophoresis showed that β-lactoglobulin and low molecular weight peptides were cross-linked by transglutaminase. Protein hydrolysis and subsequent enzymatic cross-linking increased the ζ-potential value. Microscopic investigation indicated that most particles were non-spherical. Non-cross-linked and cross-linked peptides underwent a form of heat-triggered self-assembly in the dry state, while nanoparticles did not show such behavior. Peptide crystallites size was increased by cross-linking and acid-induced particle formation. The latter also caused a reduction in intensity of C-H stretching and C-N bending peaks in infra-red spectrum. Gallic acid release from particles to simulated gastrointestinal fluids was through diffusion from swollen particles, and reached almost 70% release. PMID:27211653

  2. Variant fatty acid-like molecules Conjugation, novel approaches for extending the stability of therapeutic peptides

    PubMed Central

    Li, Ying; Wang, Yuli; Wei, Qunchao; Zheng, Xuemin; Tang, Lida; Kong, Dexin; Gong, Min

    2015-01-01

    The multiple physiological properties of glucagon-like peptide-1 (GLP-1) make it a promising drug candidate for the treatment of type 2 diabetes. However, the in vivo half-life of GLP-1 is short due to rapid degradation by dipeptidyl peptidase-IV (DPP-IV) and renal clearance. The poor stability of GLP-1 has significantly limited its clinical utility; however, many studies are focused on extending its stability. Fatty acid conjugation is a traditional approach for extending the stability of therapeutic peptides because of the high binding affinity of human serum albumin for fatty acids. However, the conjugate requires a complex synthetic approach, usually involving Lys and occasionally involving a linker. In the current study, we conjugated the GLP-1 molecule with fatty acid derivatives to simplify the synthesis steps. Human serum albumin binding assays indicated that the retained carboxyl groups of the fatty acids helped maintain a tight affinity to HSA. The conjugation of fatty acid-like molecules improved the stability and increased the binding affinity of GLP-1 to HSA. The use of fatty acid-like molecules as conjugating components allowed variant conjugation positions and freed carboxyl groups for other potential uses. This may be a novel, long-acting strategy for the development of therapeutic peptides. PMID:26658631

  3. Variant fatty acid-like molecules Conjugation, novel approaches for extending the stability of therapeutic peptides.

    PubMed

    Li, Ying; Wang, Yuli; Wei, Qunchao; Zheng, Xuemin; Tang, Lida; Kong, Dexin; Gong, Min

    2015-01-01

    The multiple physiological properties of glucagon-like peptide-1 (GLP-1) make it a promising drug candidate for the treatment of type 2 diabetes. However, the in vivo half-life of GLP-1 is short due to rapid degradation by dipeptidyl peptidase-IV (DPP-IV) and renal clearance. The poor stability of GLP-1 has significantly limited its clinical utility; however, many studies are focused on extending its stability. Fatty acid conjugation is a traditional approach for extending the stability of therapeutic peptides because of the high binding affinity of human serum albumin for fatty acids. However, the conjugate requires a complex synthetic approach, usually involving Lys and occasionally involving a linker. In the current study, we conjugated the GLP-1 molecule with fatty acid derivatives to simplify the synthesis steps. Human serum albumin binding assays indicated that the retained carboxyl groups of the fatty acids helped maintain a tight affinity to HSA. The conjugation of fatty acid-like molecules improved the stability and increased the binding affinity of GLP-1 to HSA. The use of fatty acid-like molecules as conjugating components allowed variant conjugation positions and freed carboxyl groups for other potential uses. This may be a novel, long-acting strategy for the development of therapeutic peptides. PMID:26658631

  4. Laser ion beam photodissociation studies of model amino acids and peptides

    SciTech Connect

    Techlenburg, R.E. Jr.; Miller, M.N.; Russell, D.H. )

    1989-02-15

    Visible (458-514.5 nm) and uv (333-385 nm) photodissociation of the (M + H){sup +} ions of dinitrophenyl (DNP) derivatized amino acids and peptides is reported. Photoexcitation of the DNP peptides by a visible proton results in fragmentation of the peptide chain with little fragmentation within the chromophore. Conversely, uv photoexcitation of the DNP peptides results in fragmentation of the chromophore as well as the peptide chain, but loss of NO or NO{sub 2} (within the chromophore) often dominates the photofragment ion spectrum. These results are rationalized with particular emphasis on energy-selective dissociation channels of large ionic systems. DNP-leucine and DNP-isoleucine (M + H){sup +} can be differentiated on the basis of photodissociation reactions which yield distonic radical cations. The rate of dissociation of photoexcited ions of DNP peptides is shown to decrease with increasing molecular weight (degrees of freedom). Lastly, comparisons between photodissociation and collision-induced dissociation as a structural probe are presented. 55 refs., 8 figs., 3 tabs.

  5. Effect of acid predissolution on fibril size and fibril flexibility of synthetic beta-amyloid peptide.

    PubMed Central

    Shen, C L; Fitzgerald, M C; Murphy, R M

    1994-01-01

    beta-amyloid peptide (A beta) is the major protein component of senile plaques and cerebrovascular amyloid deposits in Alzheimer's patients. Several researchers have demonstrated that A beta is neurotoxic in in vitro and in vivo systems. Peptide aggregation state and/or conformation might play a significant role in determining the toxicity of the peptide. The size and flexibility of fibrils formed from the synthetic peptide beta (1-39), corresponding to the first 39 residues of A beta, were determined. Samples were prepared either directly from lyophilized peptide or diluted from a 10 mg/ml stock solution in 0.1% trifluoroacetic acid (TFA). All samples had a final peptide concentration of 0.5 mg/ml, a final pH of 7.4, and a final NaCl concentration of 0.14 M. The molecular weight and linear density of the fibrils increased with increasing pre-incubation time in TFA, based on static light scattering measurements. Analysis of the angular dependence of the intensity of scattered light indicated that the fibrils were semi-flexible chains and that the fibril flexibility decreased with increasing pre-incubation time in TFA. There was a concomitant change in phase behavior from precipitation to gelation with the decrease in fibril flexibility. Images FIGURE 3 PMID:7811938

  6. Novel amino acids: synthesis of furoxan and sydnonimine containing amino acids and peptides as potential nitric oxide releasing motifs.

    PubMed

    Nortcliffe, Andrew; Botting, Nigel P; O'Hagan, David

    2013-07-28

    The incorporation of furoxan and sydnonimine ring systems into amino acid side chains is demonstrated with the preparation of four novel amino acids which carry these nitric oxide-releasing motifs. N-((4-Nitrophenoxy)carbonyl)-3-phenylsydnonimine 9 and bis(phenylsulfonyl)furoxan 10 are the key intermediates for introducing the heterocycle side chains onto appropriate amine and alcohol functionalities respectively. Furoxan 5 and 7 both displayed NO release based on determination of nitrite production. Orthogonal amino acid protecting group strategies were deployed to demonstrate that the amino acids could be incorporated into peptide frameworks. By way of demonstration the amino acids were placed centrally into several tripeptide motifs. Griess test assays showed that these amino acids released NO in the presence of γ-glutathione (GST). PMID:23753002

  7. Stable Isotope Peptide Mass Spectrometry To Decipher Amino Acid Metabolism in Dehalococcoides Strain CBDB1

    PubMed Central

    Marco-Urrea, Ernest; Seifert, Jana; von Bergen, Martin

    2012-01-01

    Dehalococcoides species are key players in the anaerobic transformation of halogenated solvents at contaminated sites. Here, we analyze isotopologue distributions in amino acid pools from peptides of Dehalococcoides strain CBDB1 after incubation with 13C-labeled acetate or bicarbonate as a carbon source. The resulting data were interpreted with regard to genome annotations to identify amino acid biosynthesis pathways. In addition to using gas chromatography-mass spectrometry (GC-MS) for analyzing derivatized amino acids after protein hydrolysis, we introduce a second, much milder method, in which we directly analyze peptide masses after tryptic digest and peptide fragments by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS). With this method, we identify isotope incorporation patterns for 17 proteinaceous amino acids, including proline, cysteine, lysine, and arginine, which escaped previous analyses in Dehalococcoides. Our results confirmed lysine biosynthesis via the α-aminoadipate pathway, precluding lysine formation from aspartate. Similarly, the isotopologue pattern obtained for arginine provided biochemical evidence of its synthesis from glutamate. Direct peptide MS/MS analysis of the labeling patterns of glutamine and asparagine, which were converted to glutamate and aspartate during protein hydrolysis, gave biochemical evidence of their precursors and confirmed glutamate biosynthesis via a Re-specific citrate synthase. By addition of unlabeled free amino acids to labeled cells, we show that in strain CBDB1 none of the 17 tested amino acids was incorporated into cell mass, indicating that they are all synthesized de novo. Our approach is widely applicable and provides a means to analyze amino acid metabolism by studying specific proteins even in mixed consortia. PMID:22661690

  8. Question 1: Peptide Nucleic Acids and the Origin and Homochirality of Life

    NASA Astrophysics Data System (ADS)

    Nielsen, Peter E.

    2007-10-01

    The possibilities of pseudo peptide DNA mimics like PNA (peptide nucleic acid) having a role for the prebiotic origin of life prior to an RNA world is discussed. In particular a scenario is proposed in which protocells with an achiral genetic material through several generations stepwise is converted into a chiral genetic material, e.g., by incorporation of RNA units. Provided that a sufficiently large sequence space is occupied, a selection process based on catalytic function in which a single cell (first common ancestor) has a definite evolutionary advantage, selection of this cell would by contingency also lock it into homochirality.

  9. Amino acids and peptides activate at least five members of the human bitter taste receptor family.

    PubMed

    Kohl, Susann; Behrens, Maik; Dunkel, Andreas; Hofmann, Thomas; Meyerhof, Wolfgang

    2013-01-01

    Amino acids and peptides represent important flavor molecules eliciting various taste sensations. Here, we present a comprehensive assessment of the interaction of various peptides and all proteinogenic amino acids with the 25 human TAS2Rs expressed in cell lines. L-Phenylalanine and L-tryptophan activate TAS2R1 and TAS2R4, respectively, whereas TAS2R4 and TAS2R39 responded to D-tryptophan. Structure-function analysis uncovered the basis for the lack of stereoselectivity of TAS2R4. The same three TAS2Rs or subsets thereof were also sensitive to various dipeptides containing L-tryptophan, L-phenylalanine, or L-leucine and to Trp-Trp-Trp, whereas Leu-Leu-Leu specifically activated TAS2R4. Trp-Trp-Trp also activated TAS2R46 and TAS2R14. Two key bitter peptides from Gouda cheese, namely, Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn-Ser and Leu-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn, both activated TAS2R1 and TAS2R39. Thus, the data demonstrate that the bitterness of amino acids and peptides is not mediated by specifically tuned TAS2Rs but rather is brought about by an unexpectedly complex pattern of sensitive TAS2Rs. PMID:23214402

  10. Acidity-Mediated, Electrostatic Tuning of Asymmetrically Charged Peptides Interactions with Protein Nanopores.

    PubMed

    Asandei, Alina; Chinappi, Mauro; Kang, Hee-Kyoung; Seo, Chang Ho; Mereuta, Loredana; Park, Yoonkyung; Luchian, Tudor

    2015-08-01

    Despite success in probing chemical reactions and dynamics of macromolecules on submillisecond time and nanometer length scales, a major impasse faced by nanopore technology is the need to cheaply and controllably modulate macromolecule capture and trafficking across the nanopore. We demonstrate herein that tunable charge separation engineered at the both ends of a macromolecule very efficiently modulates the dynamics of macromolecules capture and traffic through a nanometer-size pore. In the proof-of-principle approach, we employed a 36 amino acids long peptide containing at the N- and C-termini uniform patches of glutamic acids and arginines, flanking a central segment of asparagines, and we studied its capture by the α-hemolysin (α-HL) and the mean residence time inside the pore in the presence of a pH gradient across the protein. We propose a solution to effectively control the dynamics of peptide interaction with the nanopore, with both association and dissociation reaction rates of peptide-α-HL interactions spanning orders of magnitude depending upon solution acidity on the peptide addition side and the transmembrane electric potential, while preserving the amplitude of the blockade current signature. PMID:26144534

  11. Complete amino acid analysis of peptides and proteins after hydrolysis by a mixture of Sepharose-bound peptidases

    PubMed Central

    Bennett, H. P. J.; Elliott, D. F.; Evans, B. E.; Lowry, P. J.; McMartin, C.

    1972-01-01

    Incubation with a mixture of Sepharose-bound peptidases was shown to result in the quantitative release of amino acids from certain peptides and S-aminoethylated proteins. Subtraction of the low background values of amino acids generated by the enzymes enables amino acid ratios of corticotrophin-(1–24)-tetracosapeptide to be determined with a standard deviation on repeat digestions of 3–5%. Good values were obtained for amino acids that are completely or partially destroyed on acid hydrolysis, i.e. tryptophan, tyrosine, serine, asparagine and glutamine. Experiments with peptides containing d-amino acids showed that the enzyme mixture is stereospecific and could therefore be used to detect the presence of d-residues in peptides. The enzyme mixture completely hydrolyses peptide fragments obtained after Edman degradation and should therefore be useful for determining sequences of peptides containing acid-labile amino acid residues. The activities of the bound enzymes were unaltered over a period of 7 months and they provide a simple, reproducible procedure for the quantitative determination of amino acids in peptides and proteins containing l-amino acids. PMID:4349115

  12. Effect of linoleic acid supplementation on in vitro maturation, embryo development and apoptotic related gene expression in ovine

    PubMed Central

    Amini, Ebrahim; Asadpour, Reza; Roshangar, Leila; Jafari-Joozani, Razi

    2016-01-01

    Background: Linoleic acid (LA) is a polyunsaturated fatty acid present in high concentrations in follicular fluid, when added to maturation culture media, it affects oocyte competence. Objective: In the present study, we investigated effect of linoleic acid supplementation on in vitro maturation, embryo development and apoptotic related gene expression in ovine Materials and Methods: The experiments conducted on 450 ovine Cumulus-oocyte complexes (COCs) with homogenous ooplasm and more than two compact layers of cumulus cells. For in vitro maturation COCs were randomly allocated into four treatment groups for 24 hr period. Treatment groups were as follow: control maturation media, 0 µM LA, 50 µM LA, 100 µM LA and 200 µM LA. The cumulus cell expansion and blastocysts rates were recorded. Total RNA was isolated from embryo pools, reverse transcribed into cDNA, and subjected to apoptotic gene expression by real-time PCR. Results: Highest concentration (200 µM/mL) of LA significantly decreased the rate of fully expanded cumulus cells 24 hr after in vitro maturation (IVM) and the percentage of blastocyste rate compared with the control (p<0.05). These inhibitory effects were associated with an increased in relative mRNA expression of Bax (Bcl-2- associated X) gene compared with controls. Conclusion: Data obtained in present study suggest that low concentration of LA used for maturation had no deleterious effect on subsequent embryonic development compared to high concentration of LA. Relative expression of Bcl-2 (B-cell lymphoma 2) and Bax in embryos seems to be associated with LA concentration. PMID:27351027

  13. Inspiration from the mirror: D-amino acid containing peptides in biomedical approaches.

    PubMed

    Feng, Zhaoqianqi; Xu, Bing

    2016-06-01

    D-amino acids, the enantiomers of naturally abundant L-amino acids, bear unique stereochemistry properties that lead to the resistance towards most of the endogenous enzymes. Previous works have demonstrated applications of D-amino acids in therapeutic development with the aid of mirror-image phage display and retro-inverso peptide synthesis. In this review, we highlight the recent progress and challenges in the exploration of D-amino acids at the interface of chemistry and life science. First, we will introduce some progress made in traditional application of D-amino acids to enhance biostability of peptide therapeutics. Then, we discuss some works that explore the relatively underexplored interactions between the enzyme and D-amino acids and enzymatic reactions of D-amino acids. To highlight the enzymatic reactions of D-amino acids, we will describe several emerging works on the enzyme-instructed self-assembly (EISA) and their potential application in selective anti-inflammatory or anticancer therapies. At the end, we briefly mention the challenges and possible future directions. PMID:27159920

  14. Mass spectral study of hybrid peptides derived from (R)-aminoxy ester and [beta]-amino acids: The influence of aminoxy peptide bond (CO-NH-O) on peptide fragmentation under electrospray ionization conditions

    NASA Astrophysics Data System (ADS)

    Ramesh, V.; Ramesh, M.; Srinivas, R.; Sharma, G. V. M.; Manohar, V.

    2009-04-01

    A new class of Boc-protected aminoxy hybrid peptides containing repeats of [beta]-hAla-(R)-Ama-, and [beta]-Caa-(R)-Ama- ([beta]-hAla = [beta]3-(S)-hAlanine, (R)-Ama = (R)-aminoxy ester, and [beta]-Caa = (R)-C-linked carbo-[beta]3-amino acid) have been studied by electrospray ionization (ESI) ion-trap and quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) of their protonated, cationized, and negative ions. MS3 CID of protonated aminoxy peptides of [beta]-hAla-(R)-Ama- yield intense [beta]-amino acid characteristic retro-Mannich fragmentation. The bn+ and [bn-methyl imine]+ (n = 3, 5) ions formed by cleavage of aminoxy peptide bond (CO-NH-O) are more intense than bn+ (n = 2, 4) formed by that of peptide bond (CO-NH-C) cleavage. Another characteristic ion observed is due to loss of H3NO from yn+ ions. The cationized (Li+, and Na+) peptides dissociate differently compared to protonated peptides. Intense cationized cn and zn ions are formed due to the cleavage of N-O bond. The deprotonated peptides also show abundant cn- and zn- ions (n = 1, 3, 5) and do not form any yn- ions. All these results clearly indicate the influence of aminoxy peptide bond on fragmentation of these hybrid peptides.

  15. A novel sea anemone peptide that inhibits acid-sensing ion channels.

    PubMed

    Rodríguez, Armando Alexei; Salceda, Emilio; Garateix, Anoland Georgina; Zaharenko, André Junqueira; Peigneur, Steve; López, Omar; Pons, Tirso; Richardson, Michael; Díaz, Maylín; Hernández, Yasnay; Ständker, Ludger; Tytgat, Jan; Soto, Enrique

    2014-03-01

    Sea anemones produce ion channels peptide toxins of pharmacological and biomedical interest. However, peptides acting on ligand-gated ion channels, including acid-sensing ion channel (ASIC) toxins, remain poorly explored. PhcrTx1 is the first compound characterized from the sea anemone Phymanthus crucifer, and it constitutes a novel ASIC inhibitor. This peptide was purified by gel filtration, ion-exchange and reversed-phase chromatography followed by biological evaluation on ion channels of isolated rat dorsal root ganglia (DRG) neurons using patch clamp techniques. PhcrTx1 partially inhibited ASIC currents (IC50∼100 nM), and also voltage-gated K(+) currents but the effects on the peak and on the steady state currents were lower than 20% in DRG neurons, at concentrations in the micromolar range. No significant effect was observed on Na(+) voltage-gated currents in DRG neurons. The N-terminal sequencing yielded 32 amino acid residues, with a molecular mass of 3477 Da by mass spectrometry. No sequence identity to other sea anemone peptides was found. Interestingly, the bioinformatic analysis of Cys-pattern and secondary structure arrangement suggested that this peptide presents an Inhibitor Cystine Knot (ICK) scaffold, which has been found in other venomous organisms such as spider, scorpions and cone snails. Our results show that PhcrTx1 represents the first member of a new structural group of sea anemones toxins acting on ASIC and, with much lower potency, on Kv channels. Moreover, this is the first report of an ICK peptide in cnidarians, suggesting that the occurrence of this motif in venomous animals is more ancient than expected. PMID:23764262

  16. Biological Activity of Aminophosphonic Acids and Their Short Peptides

    NASA Astrophysics Data System (ADS)

    Lejczak, Barbara; Kafarski, Pawel

    The biological activity and natural occurrence of the aminophosphonic acids were described half a century ago. Since then the chemistry and biology of this class of compounds have developed into the separate field of phosphorus chemistry. Today it is well acknowledged that these compounds possess a wide variety of promising, and in some cases commercially useful, physiological activities. Thus, they have found applications ranging from agrochemical (with the herbicides glyphosate and bialaphos being the most prominent examples) to medicinal (with the potent antihypertensive fosinopril and antiosteoporetic bisphosphonates being examples).

  17. Amino Acid Metaclusters: Implications of Growth Trends on Peptide Self-Assembly and Structure.

    PubMed

    Do, Thanh D; de Almeida, Natália E C; LaPointe, Nichole E; Chamas, Ali; Feinstein, Stuart C; Bowers, Michael T

    2016-01-01

    Ion-mobility mass spectrometry is utilized to examine the metacluster formation of serine, asparagine, isoleucine, and tryptophan. These amino acids are representative of different classes of noncharged amino acids. We show that they can form relatively large metaclusters in solution that are difficult or impossible to observe by traditional solution techniques. We further demonstrate, as an example, that the formation of Ser metaclusters is not an ESI artifact because large metaclusters can be detected in negative polarity and low concentration with similar cross sections to those measured in positive polarity and higher concentration. The growth trends of tryptophan and isoleucine metaclusters, along with serine, asparagine, and the previously studied phenylalanine, are balanced among various intrinsic properties of individual amino acids (e.g., hydrophobicity, size, and shape). The metacluster cross sections of hydrophilic residues (Ser, Asn, Trp) tend to stay on or fall below the isotropic model trend lines whereas those of hydrophobic amino acids (Ile, Phe) deviate positively from the isotropic trend lines. The growth trends correlate well to the predicted aggregation propensity of individual amino acids. From the metacluster data, we introduce a novel approach to score and predict aggregation propensity of peptides, which can offer a significant improvement over the existing methods in terms of accuracy. Using a set of hexapeptides, we show that the strong negative deviations of Ser metaclusters from the isotropic model leads a prediction of microcrystalline formation for the SFSFSF peptide, whereas the strong positive deviation of Ile leads to prediction or fibril formation for the NININI peptide. Both predictions are confirmed experimentally using ion mobility and TEM measurements. The peptide SISISI is predicted to only weakly aggregate, a prediction confirmed by TEM. PMID:26632663

  18. Peptide sequencing by using a combination of partial acid hydrolysis and fast-atom-bombardment mass spectrometry.

    PubMed Central

    De Angelis, F; Botta, M; Ceccarelli, S; Nicoletti, R

    1986-01-01

    To overcome the limit of the intensity of ions carrying sequence information in structural determinations of peptides by fast-atom-bombardment m.s., we have developed a method that consists in taking spectra of the peptide acid hydrolysates at different hydrolysis times. Peaks correspond to the oligomers arising from the peptide partial hydrolysis. The sequence can then be identified from the structurally overlapping fragments. PMID:2428356

  19. Nucleic Acid-Peptide Complex Phase Controlled by DNA Hybridization

    NASA Astrophysics Data System (ADS)

    Vieregg, Jeffrey; Lueckheide, Michael; Leon, Lorraine; Marciel, Amanda; Tirrell, Matthew

    When polyanions and polycations are mixed, counterion release drives formation of polymer-rich complexes that can either be solid (precipitates) or liquid (coacervates) depending on the properties of the polyelectrolytes. These complexes are important in many fields, from encapsulation of industrial polymers to membrane-free segregation of biomolecules such as nucleic acids and proteins. Condensation of long double-stranded DNA has been studied for several decades, but comparatively little attention has been paid to the polyelectrolyte behavior of oligonucleotides. We report here studies of DNA oligonucleotides (10 - 88 nt) complexed with polylysine (10 - 100 aa). Unexpectedly, we find that the phase of the resulting complexes is controlled by the hybridization state of the nucleic acid, with double-stranded DNA forming precipitates and single-stranded DNA forming coacervates. Stability increases with polyelectrolyte length and decreases with solution salt concentration, with complexes of the longer double-stranded polymers undergoing precipitate/coacervate/soluble transitions as ionic strength is increased. Mixing coacervates formed by complementary single-stranded oligonucleotides results in precipitate formation, raising the possibility of stimulus-responsive material design.

  20. Heteropoly acids triggered self-assembly of cationic peptides into photo- and electro-chromic gels.

    PubMed

    Li, Jingfang; Xu, Jing; Li, Xiaodong; Gao, Wenmei; Wang, Liyan; Wu, Lixin; Lee, Myongsoo; Li, Wen

    2016-07-01

    A series of cationic peptides with alternating lysines and hydrophobic residues were designed and synthesized. These kinds of short peptides with protonated lysines can complex with anionic heteropoly acids (HPAs) to form a stable gel in water/ethanol mixed solution. Circular dichroism spectroscopy showed that the short peptides adopted a mixed conformation (β-sheet and random-coil) within the gel matrix. Scanning and transmission electron microscopy revealed that the heteropoly acids, acting as nanosized cross-linkers, first initiated the self-assembly of the cationic peptides into spherical nanostructures. Then these nanospheres accumulated with each other through hydrogen bonds and hydrophobic interactions to form large sheet-like assemblies, which further interconnected with each other forming continuous 3D network structures. Fourier-transform infrared spectroscopy showed that the structural integrity of the HPAs was maintained during the gelation process. The resultant hybrid gels showed reversible photo- and elecrtro-chromic properties. X-ray photoelectron spectroscopy revealed that the hybrid gels, capable of persistent and reversible changes of their colour, are attributed to the intervalence charge-transfer transition of the HPAs. Reversible information writing and erasing were demonstrated through a repeated photo-lithograph or electric stimuli without significant loss of the gel performance. PMID:27240759

  1. Fatty acid composition modulates sensitivity of Legionella pneumophila to warnericin RK, an antimicrobial peptide.

    PubMed

    Verdon, Julien; Labanowski, Jérome; Sahr, Tobias; Ferreira, Thierry; Lacombe, Christian; Buchrieser, Carmen; Berjeaud, Jean-Marc; Héchard, Yann

    2011-04-01

    Warnericin RK is an antimicrobial peptide, produced by a Staphyloccocus warneri strain, described to be specifically active against Legionella, the pathogenic bacteria responsible for Legionnaires' disease. Warnericin RK is an amphiphilic alpha-helical peptide, which possesses a detergent-like mode of action. Two others peptides, δ-hemolysin I and II, produced by the same S. warneri strain, are highly similar to S. aureus δ-hemolysin and also display anti-Legionella activity. It has been recently reported that S. aureus δ-hemolysin activity on vesicles is likewise related to phospholipid acyl-chain structure, such as chain length and saturation. As staphylococcal δ-hemolysins were highly similar, we thus hypothesized that fatty acid composition of Legionella's membrane might influence the sensitivity of the bacteria to warnericin RK. Relationship between sensitivity to the peptide and fatty acid composition was then followed in various conditions. Cells in stationary phase, which were already described as less resistant than cells in exponential phase, displayed higher amounts of branched-chain fatty acids (BCFA) and short chain fatty acids. An adapted strain, able to grow at a concentration 33 fold higher than minimal inhibitory concentration of the wild type (i.e. 1μM), was isolated after repeated transfers of L. pneumophila in the presence of increased concentrations of warnericin RK. The amount of BCFA was significantly higher in the adapted strain than in the wild type strain. Also, a transcriptomic analysis of the wild type and adapted strains showed that two genes involved in fatty acid biosynthesis were repressed in the adapted strain. These genes encode enzymes involved in desaturation and elongation of fatty acids respectively. Their repression was in agreement with the decrease of unsaturated fatty acids and fatty acid chain length in the adapted strain. Conclusively, our results indicate that the increase of BCFA and the decrease of fatty acid

  2. Effect of Maturity on Phenolics (Phenolic Acids and Flavonoids) Profile of Strawberry Cultivars and Mulberry Species from Pakistan

    PubMed Central

    Mahmood, Tahir; Anwar, Farooq; Abbas, Mateen; Saari, Nazamid

    2012-01-01

    In this study, we investigated how the extent of ripeness affects the yield of extract, total phenolics, total flavonoids, individual flavonols and phenolic acids in strawberry and mulberry cultivars from Pakistan. In strawberry, the yield of extract (%), total phenolics (TPC) and total flavonoids (TFC) ranged from 8.5–53.3%, 491–1884 mg gallic acid equivalents (GAE)/100 g DW and 83–327 mg catechin equivalents (CE)/100 g DW, respectively. For the different species of mulberry the yield of extract (%), total phenolics and total flavonoids of 6.9–54.0%, 201–2287 mg GAE/100 g DW and 110–1021 mg CE/100 g DW, respectively, varied significantly as fruit maturity progressed. The amounts of individual flavonols and phenolic acid in selected berry fruits were analyzed by RP-HPLC. Among the flavonols, the content of myricetin was found to be high in Morus alba (88 mg/100 g DW), the amount of quercetin as high in Morus laevigata (145 mg/100 g DW) while kaempferol was highest in the Korona strawberry (98 mg/100 g DW) at fully ripened stage. Of the six phenolic acids detected, p-hydroxybenzoic and p-coumaric acid were the major compounds in the strawberry. M. laevigata and M. nigra contained p-coumaric acid and vanillic acid while M. macroura and M. alba contained p-hydroxy-benzoic acid and chlorogenic acid as the major phenolic acids. Overall, a trend to an increase in the percentage of extraction yield, TPC, TFC, flavonols and phenolic acids was observed as maturity progressed from un-ripened to fully-ripened stages. PMID:22605997

  3. Self-Assembled Antibody Multimers through Peptide Nucleic Acid Conjugation

    PubMed Central

    Kazane, Stephanie A.; Axup, Jun Y; Kim, Chan Hyuk; Ciobanu, Mihai; Wold, Erik D.; Barluenga, Sofia; Hutchins, Benjamin A.; Schultz, Peter G.; Winssinger, Nicolas; Smider, Vaughn V.

    2013-01-01

    With the recent clinical success of bispecific antibodies, a strategy to rapidly synthesize and evaluate bispecific or higher order multispecific molecules could facilitate the discovery of new therapeutic agents. Here we show that unnatural amino acids (UAAs) with orthogonal chemical reactivity can be used to generate site-specific antibody-oligonucleotide conjugates. These constructs can then be self-assembled into multimeric complexes with defined composition, valency and geometry. Using this approach, we generated potent bispecific antibodies that recruit cytotoxic T lymphocytes to Her2 and CD20 positive cancer cells, as well as multimeric antibody fragments with enhanced activity. This strategy should accelerate the synthesis and in vitro characterization of antibody constructs with unique specificities and molecular architectures. PMID:23210862

  4. Cation-halide transport through peptide pores containing aminopicolinic acid.

    PubMed

    Basak, Debajyoti; Sridhar, Sucheta; Bera, Amal K; Madhavan, Nandita

    2016-05-18

    Synthetic pores that selectively transport ions of biological significance through membranes could be potentially used in medical diagnostics or therapeutics. Herein, we report cation-selective octapeptide pores derived from alanine and aminopicolinic acid. The ion transport mechanism through the pores has been established to be a cation-chloride symport. The cation-chloride co-transport is biologically essential for the efficient functioning of the central nervous system and has been implicated in diseases such as epilepsy. The pores formed in synthetic lipid bilayers do not exhibit any closing events. The ease of synthesis as well as infinite lifetimes of these pores provides scope for modifying their transport behaviour to develop sensors. PMID:27137995

  5. Amyloid-beta peptide 1-42 causes microtubule deregulation through N-methyl-D-aspartate receptors in mature hippocampal cultures.

    PubMed

    Mota, Sandra I; Ferreira, Ildete L; Pereira, Claudia; Oliveira, Catarina R; Rego, A Cristina

    2012-09-01

    Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder among the elderly. Nmethyl- D-aspartate receptor (NMDAR) overactivation has been implicated in early synaptic dysfunction that precedes late neurodegeneration in AD. Moreover, oligomers of amyloid-beta peptide (Aβ) 1-42 are considered the most synaptotoxic forms, responsible for early cognitive deficits in AD. In this work we evaluate the role of NMDARs on Aβ-evoked neuronal dysfunction and cell death through changes in microtubule polymerization in mature hippocampal cultures. Exposure to Aβ 1-42 caused a decrease in total and polymerized levels of beta-III tubulin and polymerized alpha-tubulin, suggesting microtubule disassembly. Moreover, Aβ induced DNA fragmentation in both neuronal and non-neuronal cells. Indeed, the effects of Aβ on beta-III tubulin polymerization were significantly correlated with reduced neurite length and neuronal DNA fragmentation. Interestingly, these effects were prevented by MK-801 and memantine, suggesting a role for extrasynaptic NMDARs in Aβ toxicity, and by ifenprodil, further indicating the involvement of GluN2B-containing NMDARs. Nevertheless, exposure to Aβ did not potentiate the effects caused by selective activation of NMDARs. Data largely suggest that Aβ-induced hippocampal neuronal dysfunction occurs through NMDAR-dependent microtubule disassembly associated to neurite retraction and DNA fragmentation in mature hippocampal cells. PMID:22631440

  6. Phenotypic and Genotypic Characterization of Non-Starter Lactic Acid Bacteria in Mature Cheddar Cheese

    PubMed Central

    Fitzsimons, N. A.; Cogan, T. M.; Condon, S.; Beresford, T.

    1999-01-01

    Non-starter lactic acid bacteria were isolated from 14 premium-quality and 3 sensorially defective mature Irish Cheddar cheeses, obtained from six manufacturers. From countable plates of Lactobacillus-selective agar, 20 single isolated colonies were randomly picked per cheese. All 331 viable isolates were biochemically characterized as mesophilic (i.e., group II) Lactobacillus spp. Phenotypically, the isolates comprised 96.4% L. paracasei, 2.1% L. plantarum, 0.3% L. curvatus, 0.3% L. brevis, and 0.9% unidentified species. Randomly amplified polymorphic DNA (RAPD) analysis was used to rapidly identify the dominant strain groups in nine cheeses from three of the factories, and through clustering by the unweighted pair group method with arithmetic averages, an average of seven strains were found per cheese. In general, strains isolated from cheese produced at the same factory clustered together. The majority of isolates associated with premium-quality cheese grouped together and apart from clusters of strains from defective-quality cheese. No correlation was found between the isomer of lactate produced and RAPD profiles, although isolates which did not ferment ribose clustered together. The phenotypic and genotypic methods employed were validated with a selection of 31 type and reference strains of mesophilic Lactobacillus spp. commonly found in Cheddar cheese. RAPD analysis was found to be a useful and rapid method for identifying isolates to the species level. The low homology exhibited between RAPD banding profiles for cheese isolates and collection strains demonstrated the heterogeneity of the L. paracasei complex. PMID:10427029

  7. 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

  8. Scanning of 16S Ribosomal RNA for Peptide Nucleic Acid Targets.

    PubMed

    Górska, Anna; Markowska-Zagrajek, Agnieszka; Równicki, Marcin; Trylska, Joanna

    2016-08-25

    We have designed a protocol and server to aid in the search for putative binding sites in 16S rRNA that could be targeted by peptide nucleic acid oligomers. Various features of 16S rRNA were considered to score its regions as potential targets for sequence-specific binding that could result in inhibition of ribosome function. Specifically, apart from the functional importance of a particular rRNA region, we calculated its accessibility, flexibility, energetics of strand invasion by an oligomer, as well as similarity to human rRNA. To determine 16S rRNA flexibility in the ribosome context, we performed all-atom molecular dynamics simulations of the 30S subunit in explicit solvent. We proposed a few 16S RNA target sites, and one of them was tested experimentally to verify inhibition of bacterial growth by a peptide nucleic acid oligomer. PMID:27105576

  9. Efficient Eradication of Mature Pseudomonas aeruginosa Biofilm via Controlled Delivery of Nitric Oxide Combined with Antimicrobial Peptide and Antibiotics.

    PubMed

    Ren, Hang; Wu, Jianfeng; Colletta, Alessandro; Meyerhoff, Mark E; Xi, Chuanwu

    2016-01-01

    Fast eradication of mature biofilms is the 'holy grail' in the clinical management of device-related infections. Endogenous nitric oxide (NO) produced by macrophages plays an important role in host defense against intracellular pathogens, and NO is a promising agent in preventing biofilms formation in vitro. However, the rate of delivery of NO by various NO donors (e.g., diazeniumdiolates, S-nitrosothiols, etc.) is difficult to control, which hinders fundamental studies aimed at understanding the role of NO in biofilm control. In this study, by using a novel precisely controlled electrochemical NO releasing catheter device, we examine the effect of physiological levels of NO on eradicating mature Pseudomonas aeruginosa biofilm (7 days), as well as the potential application of the combination of NO with antimicrobial agents. It is shown that physiological levels of NO exhibit mixed effects of killing bacteria and dispersing ambient biofilm. The overall biofilm-eradicating effect of NO is quite efficient in a dose-dependent manner over a 3 h period of NO treatment. Moreover, NO also greatly enhances the efficacy of antimicrobial agents, including human beta-defensin 2 (BD-2) and several antibiotics, in eradicating biofilm and its detached cells, which otherwise exhibited high recalcitrance to these antimicrobial agents. The electrochemical NO release technology offers a powerful tool in evaluating the role of NO in biofilm control as well as a promising approach when combined with antimicrobial agents to treat biofilm-associated infections in hospital settings, especially infections resulting from intravascular catheters. PMID:27582732

  10. Investigating the inclusion properties of aromatic amino acids complexing beta-cyclodextrins in model peptides.

    PubMed

    Caso, Jolanda Valentina; Russo, Luigi; Palmieri, Maddalena; Malgieri, Gaetano; Galdiero, Stefania; Falanga, Annarita; Isernia, Carla; Iacovino, Rosa

    2015-10-01

    Cyclodextrins are commonly used as complexing agents in biological, pharmaceutical, and industrial applications since they have an effect on protein thermal and proteolytic stability, refolding yields, solubility, and taste masking. β-cyclodextrins (β-CD), because of their cavity size are a perfectly suited complexing agent for many common guest moieties. In the case of peptide-cyclodextrin and protein-cyclodextrin host-guest complexes the aromatic amino acids are reported to be the principal responsible of the interaction. For these reasons, we have investigated the inclusion properties of nine designed tripeptides, obtained permuting the position of two L-alanines (Ala, A) with that of one L-tryptophan (Trp, W), L-phenylalanine (Phe, F), or L-tyrosine (Tyr, Y), respectively. Interestingly, the position of the aromatic side-chain in the sequence appears to modulate the β-CD:peptide binding constants, determined via UV-Vis and NMR spectroscopy, which in turn assumes values higher than those reported for the single amino acid. The tripeptides containing a tyrosine showed the highest binding constants, with the central position in the Ac-AYA-NH2 peptide becoming the most favorite for the interaction. A combined NMR and Molecular Docking approach permitted to build detailed complex models, highlighting the stabilizing interactions of the neighboring amino acids backbone atoms with the upper rim of the β-CD. PMID:25985927

  11. Remote Enantioselection Transmitted by an Achiral Peptide Nucleic Acid Backbone

    NASA Technical Reports Server (NTRS)

    Kozlov, Igor A.; Orgel, Leslie E.; Nielsen, Peter E.

    2000-01-01

    short homochiral segment of DNA into a PNA helix could have guaranteed that the next short segment of DNA to be incorporated would have the same handedness as the first. Once two segments of the same handedness were present, the probability that a third segment would have the same handedness would increase, and so on. Evolution could then slowly dilute out the PNA part. This scenario would ultimately allow the formation of a chiral oligonucleotide by processes that are largely resistant to enantiomeric crossinhibition. It is important to note that the ligation of homochiral dinucleotides on a nucleic acid template would probably be at least as enantiospecific as the reaction that we have studied. The disadvantage of using chiral monomers as components of a replicating system arises from the difficulty of generating a first long homochiral template from a racemic mixture of monomers, although results of experiments designed to overcome this difficulty by employing homochiral tetramers have been reported.l l The probability of obtaining a homochiral n-mer from achiral substrates is approximately 1P-I if the nontemplate-directed extension of the primer is not enantioselective. Hence, it would be very hard to get started with a homochiral 40-mer, for example. No such difficulty exists in a scenario that originates with an achiral genetic material and in which the incorporation of very few chiral monomers in this achiral background gradually progresses towards homochirality. It seems possible that some PNA sequences could act as catalysts, analogous to ribozymes, even though PNA lacks clear metal binding sites. Although such catalysts could not be enantioselective, the incorporation of as few as two chiral nucleotides could then impose chiral specificity on the system. Furthermore, such patch chimeras could help to bridge the gap in catalytic potential between PNA and RNA, while guaranteeing enantioselectivity.

  12. Synthesis of all nineteen appropriately protected chiral alpha-hydroxy acid equivalents of the alpha-amino acids for Boc solid-phase depsi-peptide synthesis.

    PubMed

    Deechongkit, Songpon; You, Shu-Li; Kelly, Jeffery W

    2004-02-19

    [reaction: see text] The preparation of depsi-peptides, amide-to-ester-substituted peptides used to probe the role of hydrogen bonding in protein folding energetics, is accomplished by replacing specific l-alpha-amino acid residues by their alpha-hydroxy acid counterparts in a solid-phase synthesis employing a t-Boc strategy. Herein we describe the efficient stereoselective synthesis of all 19 appropriately protected alpha-hydroxy acid equivalents of the l-alpha-amino acids, employing commercially available materials, expanding the number of available alpha-hydroxy acids from 9 to 19. PMID:14961607

  13. Role of perinatal long-chain omega-3 fatty acids in cortical circuit maturation: Mechanisms and implications for psychopathology.

    PubMed

    McNamara, Robert K; Vannest, Jennifer J; Valentine, Christina J

    2015-03-22

    Accumulating translational evidence suggests that the long-chain omega-3 fatty acid docosahexaenoic acid (DHA) plays a role in the maturation and stability of cortical circuits that are impaired in different recurrent psychiatric disorders. Specifically, rodent and cell culture studies find that DHA preferentially accumulates in synaptic and growth cone membranes and promotes neurite outgrowth, dendritic spine stability, and synaptogenesis. Additional evidence suggests that DHA may play a role in microglia-mediated synaptic pruning, as well as myelin development and resilience. In non-human primates n-3 fatty acid insufficiency during perinatal development leads to widespread deficits in functional connectivity in adult frontal cortical networks compared to primates raised on DHA-fortified diet. Preterm delivery in non-human primates and humans is associated with early deficits in cortical DHA accrual. Human preterm birth is associated with long-standing deficits in myelin integrity and cortical circuit connectivity and increased risk for attention deficit/hyperactivity disorder (ADHD), mood, and psychotic disorders. In general, ADHD and mood and psychotic disorders initially emerge during rapid periods of cortical circuit maturation and are characterized by DHA deficits, myelin pathology, and impaired cortical circuit connectivity. Together these associations suggest that early and uncorrected deficits in fetal brain DHA accrual may represent a modifiable risk factor for cortical circuit maturation deficits in psychiatric disorders, and could therefore have significant implications for informing early intervention and prevention strategies. PMID:25815252

  14. Role of perinatal long-chain omega-3 fatty acids in cortical circuit maturation: Mechanisms and implications for psychopathology

    PubMed Central

    McNamara, Robert K; Vannest, Jennifer J; Valentine, Christina J

    2015-01-01

    Accumulating translational evidence suggests that the long-chain omega-3 fatty acid docosahexaenoic acid (DHA) plays a role in the maturation and stability of cortical circuits that are impaired in different recurrent psychiatric disorders. Specifically, rodent and cell culture studies find that DHA preferentially accumulates in synaptic and growth cone membranes and promotes neurite outgrowth, dendritic spine stability, and synaptogenesis. Additional evidence suggests that DHA may play a role in microglia-mediated synaptic pruning, as well as myelin development and resilience. In non-human primates n-3 fatty acid insufficiency during perinatal development leads to widespread deficits in functional connectivity in adult frontal cortical networks compared to primates raised on DHA-fortified diet. Preterm delivery in non-human primates and humans is associated with early deficits in cortical DHA accrual. Human preterm birth is associated with long-standing deficits in myelin integrity and cortical circuit connectivity and increased risk for attention deficit/hyperactivity disorder (ADHD), mood, and psychotic disorders. In general, ADHD and mood and psychotic disorders initially emerge during rapid periods of cortical circuit maturation and are characterized by DHA deficits, myelin pathology, and impaired cortical circuit connectivity. Together these associations suggest that early and uncorrected deficits in fetal brain DHA accrual may represent a modifiable risk factor for cortical circuit maturation deficits in psychiatric disorders, and could therefore have significant implications for informing early intervention and prevention strategies. PMID:25815252

  15. Efficient Eradication of Mature Pseudomonas aeruginosa Biofilm via Controlled Delivery of Nitric Oxide Combined with Antimicrobial Peptide and Antibiotics

    PubMed Central

    Ren, Hang; Wu, Jianfeng; Colletta, Alessandro; Meyerhoff, Mark E.; Xi, Chuanwu

    2016-01-01

    Fast eradication of mature biofilms is the ‘holy grail’ in the clinical management of device-related infections. Endogenous nitric oxide (NO) produced by macrophages plays an important role in host defense against intracellular pathogens, and NO is a promising agent in preventing biofilms formation in vitro. However, the rate of delivery of NO by various NO donors (e.g., diazeniumdiolates, S-nitrosothiols, etc.) is difficult to control, which hinders fundamental studies aimed at understanding the role of NO in biofilm control. In this study, by using a novel precisely controlled electrochemical NO releasing catheter device, we examine the effect of physiological levels of NO on eradicating mature Pseudomonas aeruginosa biofilm (7 days), as well as the potential application of the combination of NO with antimicrobial agents. It is shown that physiological levels of NO exhibit mixed effects of killing bacteria and dispersing ambient biofilm. The overall biofilm-eradicating effect of NO is quite efficient in a dose-dependent manner over a 3 h period of NO treatment. Moreover, NO also greatly enhances the efficacy of antimicrobial agents, including human beta-defensin 2 (BD-2) and several antibiotics, in eradicating biofilm and its detached cells, which otherwise exhibited high recalcitrance to these antimicrobial agents. The electrochemical NO release technology offers a powerful tool in evaluating the role of NO in biofilm control as well as a promising approach when combined with antimicrobial agents to treat biofilm-associated infections in hospital settings, especially infections resulting from intravascular catheters. PMID:27582732

  16. Absorption of amino acids and peptides from a complex mixture in the isolated small intestine of the rat.

    PubMed Central

    Gardner, M L

    1975-01-01

    Amino acid and peptide absorption from a pancreatic digest of casein at low concentration by an isolated preparation of perfused rat small intestine has been measured. 2. The rate of absorption of each amino acid (free or peptide-bound) is closely proportional to its concentration in the perfusate; this implies a constant Vmax/Km ration for all amino acids in the mixture. 3. There is a high correlation between the compositions of luminal perfusate and secretion into the tissue fluid (apart from the content of glutamic and aspartic acids and alanine). 4. The concentrations of each free amino acid are, on average, 9 times as great in secretion as in lumen; the total peptide-N concentration in secretion is approximately 4 times that in the lumen. 5. The rate of absorption of each free amino acid is highly negatively dependent on the rate of absorption of that amino acid in peptide-bound form, in addition to being positively dependent on the perfusate concentration of free amino acid. 6. While peptide-bound proline appears to be well absorbed, free proline liberated by hydrolysis appears to pass back into the lumen as well as into the tissue fluid. Substantial back flux of hydrolysis products may occur for all amino acids. 7. About one-third of the amino acids appearing in the secretion on the serosal surface are peptide-bound. 8. The rate of absorption of peptides appears to determine the rate of their hydrolysis which probably occurs mainly after entry into the mucosal cells. PMID:1204629

  17. Amino acid absorption and homeostasis in mice lacking the intestinal peptide transporter PEPT1.

    PubMed

    Nässl, Anna-Maria; Rubio-Aliaga, Isabel; Fenselau, Henning; Marth, Mena Katharina; Kottra, Gabor; Daniel, Hannelore

    2011-07-01

    The intestinal peptide transporter PEPT1 mediates the uptake of di- and tripeptides derived from dietary protein breakdown into epithelial cells. Whereas the transporter appears to be essential to compensate for the reduced amino acid delivery in patients with mutations in amino acid transporter genes, such as in cystinuria or Hartnup disease, its physiological role in overall amino acid absorption is still not known. To assess the quantitative importance of PEPT1 in overall amino acid absorption and metabolism, PEPT1-deficient mice were studied by using brush border membrane vesicles, everted gut sacs, and Ussing chambers, as well as by transcriptome and proteome analysis of intestinal tissue samples. Neither gene expression nor proteome profiling nor functional analysis revealed evidence for any compensatory changes in the levels and/or function of transporters for free amino acids in the intestine. However, most plasma amino acid levels were increased in Pept1(-/-) compared with Pept1(+/+) animals, suggesting that amino acid handling is altered. Plasma appearance rates of (15)N-labeled amino acids determined after intragastric administration of a low dose of protein remained unchanged, whereas administration of a large protein load via gavage revealed marked differences in plasma appearance of selected amino acids. PEPT1 seems, therefore, important for overall amino acid absorption only after high dietary protein intake when amino acid transport processes are saturated and PEPT1 can provide additional absorption capacity. Since renal amino acid excretion remained unchanged, elevated basal concentrations of plasma amino acids in PEPT1-deficient animals seem to arise mainly from alterations in hepatic amino acid metabolism. PMID:21350187

  18. Effect of Maturation Degree on Composition of Fatty Acids and Tocopherols of Fruit Oil from Pistacia atlantica Growing Wild in Algeria.

    PubMed

    Guenane, Hamid; Bombarda, Isabelle; OuldElhadj, Mohamed Didi; Yousfi, Mohamed

    2015-10-01

    Pistacia atlantica fruit oil has been used for a long time by local populations for culinary and medicinal purposes. In this study, the fatty acid composition and tocopherol content were determined in twelve samples of P. atlantica fruit oil at three stages of maturation (immature, intermediate maturity and mature) collected in three different sites from the region of Laghouat. The results indicated a significant difference between the oil of mature fruits (green and black) and the immature ones (light red), which were distinguished by richness in unsaturated fatty acids and tocopherols. The oil from fruits of intermediate maturity (dark red) seems to combine these properties with those of the mature group, including oil yields. Such data emphasize the value of this oil, which needs further investigation. PMID:26669112

  19. Negative electron-transfer dissociation nLC-MS/MS facilitates the analysis of thousands of acidic peptides

    PubMed Central

    McAlister, Graeme C.; Russell, Jason D.; Rumachik, Neil G.; Hebert, Alexander S.; Syka, John E. P.; Geer, Lewis Y.; Westphall, Michael S.; Pagliarini, David J.; Coon, Joshua J.

    2012-01-01

    We describe the first implementation of negative electron-transfer dissociation (NETD) on a hybrid ion trap-orbitrap mass spectrometer and its application to high-throughput sequencing of peptide anions. NETD – coupled with high pH separations, negative electrospray ionization (ESI), and an NETD compatible version of OMSSA – is part of a complete workflow that includes the formation, interrogation and sequencing of peptide anions. Together these interlocking pieces facilitated the identification of more than 2,000 unique peptides from Saccharomyces cerevisiae representing the most comprehensive analysis of peptide anions by tandem mass spectrometry to date. The same S. cerevisiae samples were interrogated using traditional, positive modes of peptide LC-MS/MS analysis (e.g., acidic LC separations, positive ESI, and collision activated dissociation), and the resulting peptide identifications of the different workflows were compared. Due to a decreased flux of peptide anions, and a tendency to produced lowly charged precursors, the NETD-based LC-MS/MS workflow was not as sensitive as the positive mode methods. However, the use of NETD readily permits access to underrepresented acidic portions of the proteome by identifying peptides that tended to have lower pI values. As such NETD improves sequence coverage, filling out the acidic portions of proteins that are often overlooked by the other methods. PMID:22335612

  20. Receptor Binding by Cholera Toxin B-Subunit and Amino Acid Modification Improves Minimal Peptide Immunogenicity

    PubMed Central

    Boberg, Andreas; Stålnacke, Alexandra; Bråve, Andreas; Hinkula, Jorma; Wahren, Britta; Carlin, Nils

    2012-01-01

    We increase our understanding of augmenting a cellular immune response, by using an HIV-1 protease-derived epitope (PR75–84), and variants thereof, coupled to the C-terminal, of the B subunit of cholera toxin (CTB). Fusion proteins were used for immunizations of HLA-A0201 transgenic C57BL/6 mice. We observed different capacities to elicit a cellular immune response by peptides with additions of five to ten amino acids to the PR epitope. There was a positive correlation between the magnitude of the elicited cellular immune response and the capacity of the fusion protein to bind GM-1. This binding capacity is affected by its ability to form natural pentamers of CTB. Our results suggest that functional CTB pentamers containing a foreign amino acid-modified epitope is a novel way to overcome the limited cellular immunogenicity of minimal peptide antigens. This way of using a functional assay as readout for improved cellular immunogenicity might become highly valuable for difficult immunogens such as short peptides (epitopes).

  1. Slow peptide bond formation by proline and other N-alkylamino acids in translation.

    PubMed

    Pavlov, Michael Y; Watts, Richard E; Tan, Zhongping; Cornish, Virginia W; Ehrenberg, Måns; Forster, Anthony C

    2009-01-01

    Proteins are made from 19 aa and, curiously, one N-alkylamino acid ("imino acid"), proline (Pro). Pro is thought to be incorporated by the translation apparatus at the same rate as the 19 aa, even though the alkyl group in Pro resides directly on the nitrogen nucleophile involved in peptide bond formation. Here, by combining quench-flow kinetics and charging of tRNAs with cognate and noncognate amino acids, we find that Pro incorporates in translation significantly more slowly than Phe or Ala and that other N-alkylamino acids incorporate much more slowly. Our results show that the slowest step in incorporation of N-alkylamino acids is accommodation/peptidyl transfer after GTP hydrolysis on EF-Tu. The relative incorporation rates correlate with expectations from organic chemistry, suggesting that amino acid sterics and basicities affect translation rates at the peptidyl transfer step. Cognate isoacceptor tRNAs speed Pro incorporation to rates compatible with in vivo, although still 3-6 times slower than Phe incorporation from Phe-tRNA(Phe) depending on the Pro codon. Results suggest that Pro is the only N-alkylamino acid in the genetic code because it has a privileged cyclic structure that is more reactive than other N-alkylamino acids. Our data on the variation of the rate of incorporation of Pro from native Pro-tRNA(Pro) isoacceptors at 4 different Pro codons help explain codon bias not accounted for by the "tRNA abundance" hypothesis. PMID:19104062

  2. Programmable Multivalent Display of Receptor Ligands using Peptide Nucleic Acid Nanoscaffolds

    PubMed Central

    Englund, Ethan A.; Wang, Deyun; Fujigaki, Hidetsugu; Sakai, Hiroyasu; Micklitsch, Christopher M.; Ghirlando, Rodolfo; Martin-Manso, Gema; Pendrak, Michael L.; Roberts, David D.; Durell, Stewart R.; Appella, Daniel H.

    2012-01-01

    Multivalent effects dictate the binding affinity of multiple ligands on one molecular entity to receptors. Integrins are receptors that mediate cell attachment through multivalent binding to peptide sequences within the extracellular matrix, and overexpression promotes the metastasis of some cancers. Multivalent display of integrin antagonists enhances their efficacy, but current scaffolds have limited ranges and precision for the display of ligands. Here we present an approach to study multivalent effects across wide ranges of ligand number, density, and three-dimensional arrangement. Using L-lysine γ-substituted peptide nucleic acids, the multivalent effects of an integrin antagonist were examined over a range of 1 to 45 ligands. The optimal construct improves the inhibitory activity of the antagonist by two orders of magnitude against the binding of melanoma cells to the extracellular matrix in both in vitro and in vivo models. PMID:22233624

  3. Formation of peptides from amino acids by single or multiple additions of ATP to suspensions of nucleoproteinoid microparticles

    NASA Technical Reports Server (NTRS)

    Nakashima, T.; Fox, S. W.

    1981-01-01

    The synthesis of peptides from individual amino acids or pairs of amino acids and ATP in the presence of catalysis by nucleoproteinoid microparticles is investigated. Experiments were performed with suspensions formed from the condensation of lysine-rich and acidic proteinoids with polyadenylic acid, to which were added glycine, phenylalanine, proline, lysine or glycine-phenylalanine mixtures, and ATP either at once or serially. Peptide yields are found to be greatest for equal amounts of acidic and basic proteinoids. The addition of imidazole is found to alter the preference of glycine-phenylalanine mixtures to form mixed heteropeptides rather than homopeptides. A rapid ATP decay in the peptide synthesis reaction is observed, and a greater yield is obtained for repeated small additions than for a single addition of ATP. The experimental system has properties similar to modern cells, and represents an organizational unit ready for the evolution of associated biochemical pathways.

  4. Solvation thermodynamics of amino acid side chains on a short peptide backbone

    SciTech Connect

    Hajari, Timir; Vegt, Nico F. A. van der

    2015-04-14

    The hydration process of side chain analogue molecules differs from that of the actual amino acid side chains in peptides and proteins owing to the effects of the peptide backbone on the aqueous solvent environment. A recent molecular simulation study has provided evidence that all nonpolar side chains, attached to a short peptide backbone, are considerably less hydrophobic than the free side chain analogue molecules. In contrast to this, the hydrophilicity of the polar side chains is hardly affected by the backbone. To analyze the origin of these observations, we here present a molecular simulation study on temperature dependent solvation free energies of nonpolar and polar side chains attached to a short peptide backbone. The estimated solvation entropies and enthalpies of the various amino acid side chains are compared with existing side chain analogue data. The solvation entropies and enthalpies of the polar side chains are negative, but in absolute magnitude smaller compared with the corresponding analogue data. The observed differences are large; however, owing to a nearly perfect enthalpy-entropy compensation, the solvation free energies of polar side chains remain largely unaffected by the peptide backbone. We find that a similar compensation does not apply to the nonpolar side chains; while the backbone greatly reduces the unfavorable solvation entropies, the solvation enthalpies are either more favorable or only marginally affected. This results in a very small unfavorable free energy cost, or even free energy gain, of solvating the nonpolar side chains in strong contrast to solvation of small hydrophobic or nonpolar molecules in bulk water. The solvation free energies of nonpolar side chains have been furthermore decomposed into a repulsive cavity formation contribution and an attractive dispersion free energy contribution. We find that cavity formation next to the peptide backbone is entropically favored over formation of similar sized nonpolar side

  5. Spectroscopic and structural elucidation of amino acid derivatives and small peptides: experimental and theoretical tools.

    PubMed

    Kolev, Tsonko; Spiteller, Michael; Koleva, Bojidarka

    2010-01-01

    This mini review deals with the modern aspects of the spectroscopy and structural elucidation of amino acid derivatives and small biologically active compounds. Free peptide bond rotation in these systems yields various conformers, which possess differing biological activities. Another phenomenon is the intermolecular or intramolecular stacking observed in aromatic small peptides. Specifically, the main aim is to illustrate the successful application of the "complex tool", consisting of a combination of the theoretical approximation methods with experimental linear polarized infrared (IR-LD) and/or Raman spectroscopy of oriented colloid suspensions in a nematic host. The possibilities and limitations of the approach for detailed vibrational assignment and structural elucidation of small peptides are discussed. Having in mind that physical and chemical properties of these systems can be precisely calculated by means of ab initio and DFT methods at Hartee-Fock, MP2 and B3LYP level of theory, varying basis sets, the results obtained allow a precise assignment of many vibrational bands to the corresponding normal modes, electronic structures and conformational state. The validity of the conclusions about the structure or vibrational properties of these systems have been supported, compared and/or additionally proved by the results from independent physical methods. In this respect (1)H and (13)C-NMR, single crystal X-ray diffraction, HPLC tandem mass spectrometry as well as thermal methods are all employed. A well ordered crystal must first be grown in order to determine the molecular structure by the absolute method of single crystal X-ray diffraction. Although the 3D structures of peptides have been determined over the past decades, peptide crystallization is still a major obstacle to X-ray diffraction work, the presence of chiral centre/s makes for this difficulty. For this reason the "complex tool" presented can be regarded as an alternative method for obtaining of

  6. Expression pattern of peptide and amino acid genes in digestive tract of transporter juvenile turbot ( Scophthalmus maximus L.)

    NASA Astrophysics Data System (ADS)

    Xu, Dandan; He, Gen; Mai, Kangsen; Zhou, Huihui; Xu, Wei; Song, Fei

    2016-04-01

    Turbot ( Scophthalmus maximus L.), a carnivorous fish species with high dietary protein requirement, was chosen to examine the expression pattern of peptide and amino acid transporter genes along its digestive tract which was divided into six segments including stomach, pyloric caeca, rectum, and three equal parts of the remainder of the intestine. The results showed that the expression of two peptide and eleven amino acid transporters genes exhibited distinct patterns. Peptide transporter 1 (PepT1) was rich in proximal intestine while peptide transporter 2 (PepT2) was abundant in distal intestine. A number of neutral and cationic amino acid transporters expressed richly in whole intestine including B0-type amino acid transporter 1 (B0AT1), L-type amino acid transporter 2 (LAT2), T-type amino acid transporter 1 (TAT1), proton-coupled amino acid transporter 1 (PAT1), y+L-type amino acid transporter 1 (y+LAT1), and cationic amino acid transporter 2 (CAT2) while ASC amino acid transporter 2 (ASCT2), sodium-coupled neutral amino acid transporter 2 (SNAT2), and y+L-type amino acid transporter 2 (y+LAT2) abundantly expressed in stomach. In addition, system b0,+ transporters (rBAT and b0,+AT) existed richly in distal intestine. These findings comprehensively characterized the distribution of solute carrier family proteins, which revealed the relative importance of peptide and amino acid absorption through luminal membrane. Our findings are helpful to understand the mechanism of the utilization of dietary protein in fish with a short digestive tract.

  7. Protective immunogenicity of two synthetic peptides selected from the amino acid sequence of Bordetella pertussis toxin subunit S1.

    PubMed Central

    Askelöf, P; Rodmalm, K; Wrangsell, G; Larsson, U; Svenson, S B; Cowell, J L; Undén, A; Bartfai, T

    1990-01-01

    Two peptides, corresponding to amino acids 1-17 and 169-186 of the amino acid sequence of pertussis toxin (PT) subunit S1, were synthesized and coupled to the diphtheria toxin cross-reactive mutant protein CRM 197 and evaluated for immunogenicity and protective capacity against PT challenge in vivo. The peptide-CRM conjugates induced high antibody titers against native toxin in mice (BALB/c, C57/Black, and outbred NMRI) as measured by ELISA. Upon PT challenge (0.5 microgram of toxin) of the NMRI mice, the CRM conjugates of peptides 1-17 and 169-186 fully protected the mice from PT-induced leukocytosis. Immunization with the corresponding bovine serum albumin conjugates of these two peptides also fully protected mice. Rabbit antiserum to the peptide 1-17-CRM conjugate was highly efficient in inhibiting the ADP-ribosylating activity of PT but did not neutralize the clustering effect of PT on Chinese hamster ovary cells. In contrast, the rabbit antiserum raised against the peptide 169-186-CRM conjugate neutralized the clustering effect of PT on Chinese hamster ovary cells but did not inhibit the enzymatic activity of PT. Peptide 169-186-CRM conjugates mimic the immunoglobulin binding properties of PT and also cause clustering of Chinese hamster ovary cells. The CRM conjugates of these two peptides constitute a synthetic pertussis vaccine candidate with the ability to provide a chemically well-defined, safe, and efficient pertussis vaccine. Images PMID:2304902

  8. Quantitative analysis of single amino acid variant peptides associated with pancreatic cancer in serum by an isobaric labeling quantitative method.

    PubMed

    Nie, Song; Yin, Haidi; Tan, Zhijing; Anderson, Michelle A; Ruffin, Mack T; Simeone, Diane M; Lubman, David M

    2014-12-01

    Single amino acid variations are highly associated with many human diseases. The direct detection of peptides containing single amino acid variants (SAAVs) derived from nonsynonymous single nucleotide polymorphisms (SNPs) in serum can provide unique opportunities for SAAV associated biomarker discovery. In the present study, an isobaric labeling quantitative strategy was applied to identify and quantify variant peptides in serum samples of pancreatic cancer patients and other benign controls. The largest number of SAAV peptides to date in serum including 96 unique variant peptides were quantified in this quantitative analysis, of which five variant peptides showed a statistically significant difference between pancreatic cancer and other controls (p-value < 0.05). Significant differences in the variant peptide SDNCEDTPEAGYFAVAVVK from serotransferrin were detected between pancreatic cancer and controls, which was further validated by selected reaction monitoring (SRM) analysis. The novel biomarker panel obtained by combining α-1-antichymotrypsin (AACT), Thrombospondin-1 (THBS1) and this variant peptide showed an excellent diagnostic performance in discriminating pancreatic cancer from healthy controls (AUC = 0.98) and chronic pancreatitis (AUC = 0.90). These results suggest that large-scale analysis of SAAV peptides in serum may provide a new direction for biomarker discovery research. PMID:25393578

  9. Templated synthesis of peptide nucleic acids via sequence-selective base-filling reactions.

    PubMed

    Heemstra, Jennifer M; Liu, David R

    2009-08-19

    The templated synthesis of nucleic acids has previously been achieved through the backbone ligation of preformed nucleotide monomers or oligomers. In contrast, here we demonstrate templated nucleic acid synthesis using a base-filling approach in which individual bases are added to abasic sites of a peptide nucleic acid (PNA). Because nucleobase substrates in this approach are not self-reactive, a base-filling approach may reduce the formation of nontemplated reaction products. Using either reductive amination or amine acylation chemistries, we observed efficient and selective addition of each of the four nucleobases to an abasic site in the middle of the PNA strand. We also describe the addition of single nucleobases to the end of a PNA strand through base filling, as well as the tandem addition of two bases to the middle of the PNA strand. These findings represent an experimental foundation for nonenzymatic information transfer through base filling. PMID:19722647

  10. Templated Synthesis of Peptide Nucleic Acids via Sequence-Selective Base-Filling Reactions

    PubMed Central

    2009-01-01

    The templated synthesis of nucleic acids has previously been achieved through the backbone ligation of preformed nucleotide monomers or oligomers. In contrast, here we demonstrate templated nucleic acid synthesis using a base-filling approach in which individual bases are added to abasic sites of a peptide nucleic acid (PNA). Because nucleobase substrates in this approach are not self-reactive, a base-filling approach may reduce the formation of nontemplated reaction products. Using either reductive amination or amine acylation chemistries, we observed efficient and selective addition of each of the four nucleobases to an abasic site in the middle of the PNA strand. We also describe the addition of single nucleobases to the end of a PNA strand through base filling, as well as the tandem addition of two bases to the middle of the PNA strand. These findings represent an experimental foundation for nonenzymatic information transfer through base filling. PMID:19722647

  11. Highly efficient peptide formation from N-acetylaminoacyl-AMP anhydride and free amino acid

    NASA Technical Reports Server (NTRS)

    Mullins, D. W., Jr.; Lacey, J. C., Jr.

    1983-01-01

    The kinetics of formation of the N-blocked dipeptide, N-acetylglycylglycine, from N-acetylglycyl adenylate anhydride and glycine in aqueous solution at 25 C, and at various PH's are reported. The reaction is of interest in that over a physiologically relevant pH range (6-8), peptide synthesis proceeds more rapidly than hydrolysis, even at those pH's at which this compound becomes increasingly susceptible to base-catalyzed hydrolysis. Under similar conditions, the corresponding unblocked aminoacyl adenylate anhydrides are considerably more unstable, and undergo appreciable hydrlysis in the presence of free amino acid. Because N-blocked aminoacyl adenylate anhydrides serve as model compounds of peptidyl adenylate anhydrides, these results suggest that primitive amino acid polymerization systems may have operated by cyclic reactivation of the peptidyl carboxyl group, rather than that of the incoming amino acid.

  12. Wide range of racemization of amino acids in peptides from human fossil bone and its implications for amino acid racemization dating

    SciTech Connect

    Kimber, R.W.L.; Hare, P.E. )

    1992-02-01

    Aspartic acid from an HCl hydrolyzed portion of 20-25th Dynasty Egyptian bone gave a D/L value of 0.28. Various peptide and molecular weight fractions separated before hydrolysis from another aliquot of the same bone portion yielded D/L aspartic acid values ranging from 0.09 to 0.68. Higher molecular weight and higher content of hydrophobic amino acids are factors leading to lower D/L aspartic acid values. Insoluble, high molecular weight polypeptide residues showed very low D/L aspartic acid values of 0.09. The authors propose that particularly stable peptides be isolated and characterized and then used for comparison with similarly isolated peptides from other fossil bone samples for purposes of age estimation.

  13. Effects of 8-mer acidic peptide concentration on the morphology and photoluminescence of synthesized ZnO nanomaterials

    NASA Astrophysics Data System (ADS)

    Moon, Chung Hee; Tousi, Marzieh; Cheeney, Joseph; Ngo-Duc, Tam-Triet; Zuo, Zheng; Liu, Jianlin; Haberer, Elaine D.

    2015-11-01

    An 8-mer ZnO-binding peptide, VPGAAEHT, was identified using a M13 pVIII phage display library and employed as an additive during aqueous-based ZnO synthesis at 65 °C. Unlike most other well-studied ZnO-binding sequences which are strongly basic (pI > pH 7), the 8-mer peptide was overall acidic (pI < pH 7) in character, including only a single basic residue. The selected peptide strongly influenced ZnO nanostructure formation. Morphology and optical emission properties were found to be dependent on the concentration of peptide additive. Using lower peptide concentrations (<0.1 mM), single crystal hexagonal rods and platelets were produced, and using higher peptide concentrations (≥0.1 mM), polycrystalline layered platelets, yarn-like structures, and microspheres were assembled. Photoluminescence analysis revealed a characteristic ZnO band-edge peak, as well as sub-bandgap emission peaks. Defect-related green emission, typically associated with surface-related oxygen and zinc vacancies, was significantly reduced by the peptide additive, while blue emission, attributable to oxygen and zinc interstitials, emerged with increased peptide concentrations. Peptide-directed synthesis of ZnO materials may be useful for gas sensing and photocatalytic applications in which properly engineered morphology and defect levels have demonstrated enhanced performance.

  14. Targeting Multidrug-resistant Staphylococci with an anti-rpoA Peptide Nucleic Acid Conjugated to the HIV-1 TAT Cell Penetrating Peptide.

    PubMed

    Abushahba, Mostafa Fn; Mohammad, Haroon; Seleem, Mohamed N

    2016-01-01

    Staphylococcus aureus infections present a serious challenge to healthcare practitioners due to the emergence of resistance to numerous conventional antibiotics. Due to their unique mode of action, peptide nucleic acids are novel alternatives to traditional antibiotics to tackle the issue of bacterial multidrug resistance. In this study, we designed a peptide nucleic acid covalently conjugated to the HIV-TAT cell penetrating peptide (GRKKKRRQRRRYK) in order to target the RNA polymerase α subunit gene (rpoA) required for bacterial genes transcription. We explored the antimicrobial activity of the anti-rpoA construct (peptide nucleic acid-TAT) against methicillin-resistant S. aureus, vancomycin-intermediate S. aureus, vancomycin-resistant S. aureus, linezolid-resistant S. aureus, and methicillin-resistant S. epidermidis in pure culture, infected mammalian cell culture, and in an in vivo Caenorhabditis elegans infection model. The anti-rpoA construct led to a concentration-dependent inhibition of bacterial growth (at micromolar concentrations) in vitro and in both infected cell culture and in vivo in C. elegans. Moreover, rpoA gene silencing resulted in suppression of its message as well as reduced expression of two important methicillin-resistant S. aureus USA300 toxins (α-hemolysin and Panton-Valentine leukocidin). This study confirms that rpoA gene is a potential target for development of novel antisense therapeutics to treat infections caused by methicillin-resistant S. aureus. PMID:27434684

  15. The chain length of biologically produced (R)-3-hydroxyalkanoic acid affects biological activity and structure of anti-cancer peptides.

    PubMed

    Szwej, Emilia; Devocelle, Marc; Kenny, Shane; Guzik, Maciej; O'Connor, Stephen; Nikodinovic-Runic, Jasmina; Radivojevic, Jelena; Maslak, Veselin; Byrne, Annete T; Gallagher, William M; Zulian, Qun Ren; Zinn, Manfred; O'Connor, Kevin E

    2015-06-20

    Conjugation of DP18L peptide with (R)-3-hydroxydecanoic acid, derived from the biopolymer polyhydroxyalkanoate, enhances its anti-cancer activity (O'Connor et al., 2013. Biomaterials 34, 2710-2718). However, it is unknown if other (R)-3-hydroxyalkanoic acids (R3HAs) can enhance peptide activity, if chain length affects enhancement, and what effect R3HAs have on peptide structure. Here we show that the degree of enhancement of peptide (DP18L) anti-cancer activity by R3HAs is carbon chain length dependent. In all but one example the R3HA conjugated peptides were more active against cancer cells than the unconjugated peptides. However, R3HAs with 9 and 10 carbons were most effective at improving DP18L activity. DP18L peptide variant DP17L, missing a hydrophobic amino acid (leucine residue 4) exhibited lower efficacy against MiaPaCa cells. Circular dichroism analysis showed DP17L had a lower alpha helix content and the conjugation of any R3HA ((R)-3-hydroxyhexanoic acid to (R)-3-hydroxydodecanoic acid) to DP17L returned the helix content back to levels of DP18L. However (R)-3-hydroxyhexanoic did not enhance the anti-cancer activity of DP17L and at least 7 carbons were needed in the R3HA to enhance activity of D17L. DP17L needs a longer chain R3HA to achieve the same activity as DP18L conjugated to an R3HA. As a first step to assess the synthetic potential of polyhydroxyalkanoate derived R3HAs, (R)-3-hydroxydecanoic acid was synthetically converted to (±)3-chlorodecanoic acid, which when conjugated to DP18L improved its antiproliferative activity against MiaPaCa cells. PMID:25820126

  16. Antitumor Effects of EGFR Antisense Guanidine-Based Peptide Nucleic Acids in Cancer Models

    PubMed Central

    Thomas, Sufi M.; Sahu, Bichismita; Rapireddy, Srinivas; Bahal, Raman; Wheeler, Sarah E.; Procopio, Eva M.; Kim, Joseph; Joyce, Sonali C.; Contrucci, Sarah; Wang, Yun; Chiosea, Simion I.; Lathrop, Kira L.; Watkins, Simon; Grandis, Jennifer R.; Armitage, Bruce A.; Ly, Danith H.

    2013-01-01

    Peptide nucleic acids have emerged over the past two decades as a promising class of nucleic acid mimics because of their strong binding affinity and sequence selectivity toward DNA and RNA, and resistance to enzymatic degradation by proteases and nucleases. While they have been shown to be effective in regulation of gene expression in vitro, and to a small extent in vivo, their full potential for molecular therapy has not yet been fully realized due to poor cellular uptake. Herein, we report the development of cell-permeable, guanidine-based peptide nucleic acids targeting the epidermal growth factor receptor (EGFR) in preclinical models as therapeutic modality for head and neck squamous cell carcinoma (HNSCC) and nonsmall cell lung cancer (NSCLC). A GPNA oligomer, 16 nucleotides in length, designed to bind to EGFR gene transcript elicited potent antisense effects in HNSCC and NSCLC cells in preclinical models. When administered intraperitoneally in mice, EGFRAS-GPNA was taken-up by several tissues including the xenograft tumor. Systemic administration of EGFRAS-GPNA induced antitumor effects in HNSCC xenografts, with similar efficacies as the FDA-approved EGFR inhibitors: cetuximab and erlotinib. In addition to targeting wild-type EGFR, EGFRAS-GPNA is effective against the constitutively active EGFR vIII mutant implicated in cetuximab resistance. Our data reveals that GPNA is just as effective as a molecular platform for treating cetuximab resistant cells, demonstrating its utility in the treatment of cancer. PMID:23113581

  17. Diverse HLA-I Peptide Repertoires of the APC Lines MUTZ3-Derived Immature and Mature Dendritic Cells and THP1-Derived Macrophages.

    PubMed

    Nyambura, Lydon Wainaina; Jarmalavicius, Saulius; Baleeiro, Renato Brito; Walden, Peter

    2016-09-15

    Dendritic cells (DCs) and macrophages are specialized APCs that process and present self-Ags for induction of tolerance and foreign Ags to initiate T cell-mediated immunity. Related to differentiation states they have specific phenotypes and functions. However, the impact of these differentiations on Ag processing and presentation remains poorly defined. To gain insight into this, we analyzed and compared the HLA-I peptidomes of MUTZ3-derived human immature and mature DC lines and THP1-derived macrophages by liquid chromatography tandem mass spectrometry. We found that the HLA-I peptidomes were heterogeneous and individualized and were dominated by nonapeptides with similar HLA-I binding affinities and anchor residues. MUTZ3-derived DCs and THP1-derived macrophages were able to sample peptides from source proteins of almost all subcellular locations and were involved in various cellular functions in similar proportion, with preference to proteins involved in cell communication, signal transduction, protein metabolism, and transcription factor/regulator activity. PMID:27543614

  18. Oxidative decarboxylation of free and peptide-linked amino acids in phagocytizing guinea pig granulocytes.

    PubMed Central

    Adeniyi-Jones, S K; Karnovsky, M L

    1981-01-01

    The oxidative decarboxylation of amino acids by a system consisting of myeloperoxidase-hydrogen peroxide-chloride has been demonstrated previously by others and the process has been considered to be part of the microbicidal armamentarium of some phagocytic leukocytes. We were able to translate these earlier observations, made on model systems, to intact guinea pig granulocytes. We could demonstrate differences in the cellular handling of peptide-linked amino acids as particles, compared with free amino acids. Specific inhibitors were used to explore two routes of oxidative decarboxylation: (a) the myeloperoxidase-catalyzed direct decarboxylation-deamination reaction, and (b) oxidation of alpha-keto acids after transamination of amino acids. These inhibitors were cyanide, azide, and tapazole for the former pathway, and amino-oxyacetate for the latter. Amino-oxyacetate profoundly inhibited the decarboxylation of free 14C-amino acids (alanine and aspartate) in both resting and stimulated cells, but had only a minimal effect on 14CO2 production from ingested insoluble 14C-protein. On the other hand, the peroxidase inhibitors cyanide, azide, and tapazole dramatically inhibited the production of 14CO2 from ingested particulate 14C-protein, but had only small effects on the decarboxylation of free amino acid. Soluble, uniformly labeled 14C-protein was not significantly converted to 14CO2 even in the presence of phagocytizable polystyrene beads. These observation suggest that the amino acids taken up by phagocytosis (e.g., as denatured protein particles) are oxidatively decarboxylated and deaminated in the phagosomes by the myeloperoxidase-hydrogen peroxide-chloride system; soluble free amino acids that enter the cytoplasm by diffusion or transport are oxidatively decarboxylated after transamination by the normal cellular amino acid oxidative pathway. PMID:6267101

  19. Aliphatic acid-conjugated antimicrobial peptides--potential agents with anti-tumor, multidrug resistance-reversing activity and enhanced stability.

    PubMed

    Deng, Xin; Qiu, Qianqian; Ma, Ke; Wang, Xuekun; Huang, Wenlong; Qian, Hai

    2015-07-28

    Compared with traditional therapeutics, antimicrobial peptides as novel anti-tumor agents have prominent advantages of higher specificity and circumvention of multi-drug resistance. In a previous study, we found that B1, an antimicrobial peptide derived from Cathelicidin-BF15, presented specific anti-tumor activity against several tumor cells. Since aliphatic chain-conjugated peptides have shown ameliorative activity and stability, we conjugated aliphatic acids with different lengths to the amino terminal of B1. All the conjugated peptides exhibited improved anti-tumor activity over B1. Further investigations revealed that the peptides were capable of disrupting the cell membrane, stimulating cytochrome c release into the cytosol, which results in apoptosis. The peptides also acted against multidrug resistant cells and had multidrug resistance-reversing effects. Additionally, conjugation of aliphatic acid enhanced the peptide stability in plasma. In summary, aliphatic acid-modified peptides might be promising anti-tumor agents in the future. PMID:26083110

  20. Convenient and Scalable Synthesis of Fmoc-Protected Peptide Nucleic Acid Backbone

    PubMed Central

    Feagin, Trevor A.; Shah, Nirmal I.; Heemstra, Jennifer M.

    2012-01-01

    The peptide nucleic acid backbone Fmoc-AEG-OBn has been synthesized via a scalable and cost-effective route. Ethylenediamine is mono-Boc protected, then alkylated with benzyl bromoacetate. The Boc group is removed and replaced with an Fmoc group. The synthesis was performed starting with 50 g of Boc anhydride to give 31 g of product in 32% overall yield. The Fmoc-protected PNA backbone is a key intermediate in the synthesis of nucleobase-modified PNA monomers. Thus, improved access to this molecule is anticipated to facilitate future investigations into the chemical properties and applications of nucleobase-modified PNA. PMID:22848796

  1. Convenient and scalable synthesis of fmoc-protected Peptide nucleic Acid backbone.

    PubMed

    Feagin, Trevor A; Shah, Nirmal I; Heemstra, Jennifer M

    2012-01-01

    The peptide nucleic acid backbone Fmoc-AEG-OBn has been synthesized via a scalable and cost-effective route. Ethylenediamine is mono-Boc protected, then alkylated with benzyl bromoacetate. The Boc group is removed and replaced with an Fmoc group. The synthesis was performed starting with 50 g of Boc anhydride to give 31 g of product in 32% overall yield. The Fmoc-protected PNA backbone is a key intermediate in the synthesis of nucleobase-modified PNA monomers. Thus, improved access to this molecule is anticipated to facilitate future investigations into the chemical properties and applications of nucleobase-modified PNA. PMID:22848796

  2. Incorporation of Naked Peptide Nucleic Acids into Liposomes Leads to Fast and Efficient Delivery.

    PubMed

    Avitabile, Concetta; Accardo, Antonella; Ringhieri, Paola; Morelli, Giancarlo; Saviano, Michele; Montagner, Giulia; Fabbri, Enrica; Gallerani, Eleonora; Gambari, Roberto; Romanelli, Alessandra

    2015-08-19

    The delivery of peptide nucleic acids (PNAs) to cells is a very challenging task. We report here that a liposomal formulation composed of egg PC/cholesterol/DSPE-PEG2000 can be loaded, according to different encapsulation techniques, with PNA or fluorescent PNA oligomers. PNA loaded liposomes efficiently and quickly promote the uptake of a PNA targeting the microRNA miR-210 in human erythroleukemic K562 cells. By using this innovative delivery system for PNA, down-regulation of miR-210 is achieved at a low PNA concentration. PMID:26176882

  3. Topical Delivery of Hyaluronic Acid into Skin using SPACE-peptide Carriers

    PubMed Central

    Chen, Ming; Gupta, Vivek; Anselmo, Aaron C.; Muraski, John A.; Mitragotri, Samir

    2014-01-01

    Topical penetration of macromolecules into skin is limited by their low permeability. Here, we report the use of a skin penetrating peptide, SPACE peptide, to enhance topical delivery of a macromolecule, hyaluronic acid (HA, MW: 200–325 kDa). The peptide was conjugated to phospholipids and used to prepare an ethosomal carrier system (~110 nm diameter), encapsulating HA. The SPACE-ethosomal system (SES) enhanced HA penetration into porcine skin in vitro by 7.8+/−1.1-fold compared to PBS. The system also enhanced penetration of HA in human skin in vitro, penetrating deep into the epidermis and dermis in skin of both species. In vivo experiments performed using SKH1 hairless mice also confirmed increased dermal penetration of HA using the delivery system; a 5-fold enhancement in penetration was found compared to PBS control. Concentrations of HA in skin were about 1000-fold higher than those in blood; confirming the localized nature of HA delivery into skin. The SPACE-ethosomal delivery system provides a formulation for topical delivery of macromolecules that are otherwise difficult to deliver into skin. PMID:24129342

  4. Enhanced Epimerization of Glycosylated Amino Acids During Solid Phase Peptide Synthesis

    PubMed Central

    Zhang, Yalong; Muthana, Saddam M.; Farnsworth, David; Ludek, Olaf; Adams, Kristie; Barchi, Joseph J.; Gildersleeve, Jeffrey C.

    2012-01-01

    Glycopeptides are extremely useful for basic research and clinical applications, but access to structurally-defined glycopeptides is limited by the difficulties in synthesizing this class of compounds. In this study, we demonstrate that many common peptide coupling conditions used to prepare O-linked glycopeptides result in substantial amounts of epimerization at the alpha position. In fact, epimerization resulted in up to 80% of the non-natural epimer, indicating that it can be the major product in some reactions. Through a series of mechanistic studies, we demonstrate that the enhanced epimerization relative to non-glycosylated amino acids is due to a combination of factors, including a faster rate of epimerization, an energetic preference for the unnatural epimer over the natural epimer, and a slower overall rate of peptide coupling. In addition, we demonstrate that use of 2,4,6-trimethylpyridine (TMP) as the base in peptide couplings produces glycopeptides with high efficiency and low epimerization. The information and improved reaction conditions will facilitate the preparation of glycopeptides as therapeutic compounds and vaccine antigens. PMID:22390544

  5. Black mamba venom peptides target acid-sensing ion channels to abolish pain.

    PubMed

    Diochot, Sylvie; Baron, Anne; Salinas, Miguel; Douguet, Dominique; Scarzello, Sabine; Dabert-Gay, Anne-Sophie; Debayle, Delphine; Friend, Valérie; Alloui, Abdelkrim; Lazdunski, Michel; Lingueglia, Eric

    2012-10-25

    Polypeptide toxins have played a central part in understanding physiological and physiopathological functions of ion channels. In the field of pain, they led to important advances in basic research and even to clinical applications. Acid-sensing ion channels (ASICs) are generally considered principal players in the pain pathway, including in humans. A snake toxin activating peripheral ASICs in nociceptive neurons has been recently shown to evoke pain. Here we show that a new class of three-finger peptides from another snake, the black mamba, is able to abolish pain through inhibition of ASICs expressed either in central or peripheral neurons. These peptides, which we call mambalgins, are not toxic in mice but show a potent analgesic effect upon central and peripheral injection that can be as strong as morphine. This effect is, however, resistant to naloxone, and mambalgins cause much less tolerance than morphine and no respiratory distress. Pharmacological inhibition by mambalgins combined with the use of knockdown and knockout animals indicates that blockade of heteromeric channels made of ASIC1a and ASIC2a subunits in central neurons and of ASIC1b-containing channels in nociceptors is involved in the analgesic effect of mambalgins. These findings identify new potential therapeutic targets for pain and introduce natural peptides that block them to produce a potent analgesia. PMID:23034652

  6. Self-assembling peptide of D-amino acids boosts selectivity and antitumor efficacy of 10-hydroxycamptothecin.

    PubMed

    Liu, Jianfeng; Liu, Jinjian; Chu, Liping; Zhang, Yumin; Xu, Hongyan; Kong, Deling; Yang, Zhimou; Yang, Cuihong; Ding, Dan

    2014-04-23

    D-peptides, which consist of D-amino acids and can resist the hydrolysis catalyzed by endogenous peptidases, are one of the promising candidates for construction of peptide materials with enhanced biostability in vivo. In this paper, we report on a self-assembling supramolecular nanostructure of D-amino acid-based peptide Nap-G(D)F(D)F(D)YGRGD (D-fiber, (D)F meant D-phenylalanine, (D)Y meant D-tyrosine), which were used as carriers for 10-hydroxycamptothecin (HCPT). Transmission electron microscopy observations demonstrated the filamentous morphology of the HCPT-loaded peptides (d-fiber-HCPT). The better selectivity and antitumor activity of D-fiber-HCPT than L-fiber-HCPT were found in the in vitro and in vivo antitumor studies. These results highlight that this model D-fiber system holds great promise as vehicles of hydrophobic drugs for cancer therapy. PMID:24660962

  7. Some things get better with age: differences in salicylic acid accumulation and defense signaling in young and mature Arabidopsis

    PubMed Central

    Carella, Philip; Wilson, Daniel C.; Cameron, Robin K.

    2015-01-01

    In Arabidopsis, much of what we know about the phytohormone salicylic acid (SA) and its role in plant defense comes from experiments using young plants. We are interested in understanding why young plants are susceptible to virulent strains of Pseudomonas syringae, while mature plants exhibit a robust defense response known as age-related resistance (ARR). SA-mediated signaling is important for defense in young plants, however, ARR occurs independently of the defense regulators NPR1 and WHY1. Furthermore, intercellular SA accumulation is an important component of ARR, and intercellular washing fluids from ARR-competent plants exhibit antibacterial activity, suggesting that SA acts as an antimicrobial agent in the intercellular space. Young plants accumulate both intracellular and intercellular SA during PAMP- and effector-triggered immunity, however, virulent P. syringae promotes susceptibility by suppressing SA accumulation using the phytotoxin coronatine. Here we outline the hypothesis that mature, ARR-competent Arabidopsis alleviates coronatine-mediated suppression of SA accumulation. We also explore the role of SA in other mature-plant processes such as flowering and senescence, and discuss their potential impact on ARR. PMID:25620972

  8. Development of a method for environmentally friendly chemical peptide synthesis in water using water-dispersible amino acid nanoparticles

    PubMed Central

    2011-01-01

    Due to the vast importance of peptides in biological processes, there is an escalating need for synthetic peptides to be used in a wide variety of applications. However, the consumption of organic solvent is extremely large in chemical peptide syntheses because of the multiple condensation steps in organic solvents. That is, the current synthesis method is not environmentally friendly. From the viewpoint of green sustainable chemistry, we focused on developing an organic solvent-free synthetic method using water, an environmentally friendly solvent. Here we described in-water synthesis technology using water-dispersible protected amino acids. PMID:21867548

  9. Characterization and in vitro activity of a branched peptide boronic acid that interacts with HIV-1 RRE RNA.

    PubMed

    Wynn, Jessica E; Zhang, Wenyu; Tebit, Denis M; Gray, Laurie R; Hammarskjold, Marie-Louise; Rekosh, David; Santos, Webster L

    2016-09-01

    A branched peptide containing multiple boronic acids was found to bind RRE IIB selectively and inhibit HIV-1 p24 capsid production in a dose-dependent manner. Structure-activity relationship studies revealed that branching in the peptide is crucial for the low micromolar binding towards RRE IIB, and the peptide demonstrates selectivity towards RRE IIB in the presence of tRNA. Footprinting studies suggest a binding site on the upper stem and internal loop regions of the RNA, which induces enzymatic cleavage of the internal loops of RRE IIB upon binding. PMID:27091070

  10. Comparison of amino acid v peptide based enteral diets in active Crohn's disease: clinical and nutritional outcome.

    PubMed Central

    Royall, D; Jeejeebhoy, K N; Baker, J P; Allard, J P; Habal, F M; Cunnane, S C; Greenberg, G R

    1994-01-01

    Elemental diets are considered an effective primary treatment for active Crohn's disease. This study examined the hypothesis that improvement occurs because of the presence of amino acids or the low fat content, or both. A randomised, controlled trial was undertaken in 40 patients with active Crohn's disease to evaluate clinical and nutritional outcomes after an amino acid based diet containing 3% fat was given by a feeding tube compared with a peptide based diet containing 33% fat. After three weeks' treatment, clinical remission occurred in 84% of patients who were given the amino acid diet and 75% of patients who received the peptide diet (p = 0.38). Plasma linoleic acid concentration was reduced after the amino acid but not the peptide diet. An increase in total body nitrogen was associated with the magnitude of nutritional depletion before treatment and at six months' follow up, only patients who showed gains in total body nitrogen after enteral nutrition had a sustained clinical remission. This study shows that peptide based high fat diets are as effective as amino acid low fat diets for achieving clinical remission in active Crohn's disease. Improved total body protein stores but not essential fatty acid depletion may be an important indicator of a sustained remission. PMID:8020806

  11. Adapting and testing a portable Raman spectrometer for SERS analysis of amino acids and small peptides

    NASA Astrophysics Data System (ADS)

    Brambilla, A.; Philippidis, A.; Nevin, A.; Comelli, D.; Valentini, G.; Anglos, D.

    2013-07-01

    Surface-Enhanced Raman Spectroscopy (SERS), a powerful spectrochemical technique enabling highly sensitive analysis of organic and biological materials, is investigated for applications in the analysis of archaeological materials including in situ screening. In this work, a compact mobile Raman spectrometer is employed for acquiring Surface-Enhanced Raman spectra from natural amino acids (L-Arg, L-Phe, L-Met) and a tripeptide (Glutathione), adsorbed on silver colloids. The detection limits of the portable Raman spectrometer, together with an optimization of sample preparation and experimental parameters, are reported. The collection and interpretation of SER spectra of amino acids and peptides is a starting point for the optimization of the instrumentation and its application in the study of more complex biological molecules in the context of detection and analysis of archaeological materials and residues.

  12. Information transfer from peptide nucleic acids to RNA by template-directed syntheses

    NASA Technical Reports Server (NTRS)

    Schmidt, J. G.; Nielsen, P. E.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1997-01-01

    Peptide nucleic acids (PNAs) are uncharged analogs of DNA and RNA in which the ribose-phosphate backbone is substituted by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. A PNA C10 oligomer has been shown to act as template for efficient formation of oligoguanylates from activated guanosine ribonucleotides. In a previous paper we used heterosequences of DNA as templates in sequence-dependent polymerization of PNA dimers. In this paper we show that information can be transferred from PNA to RNA. We describe the reactions of activated mononucleotides on heterosequences of PNA. Adenylic, cytidylic and guanylic acids were incorporated into the products opposite their complement on PNA, although less efficiently than on DNA templates.

  13. Bioactive Molecules Released in Food by Lactic Acid Bacteria: Encrypted Peptides and Biogenic Amines

    PubMed Central

    Pessione, Enrica; Cirrincione, Simona

    2016-01-01

    Lactic acid bacteria (LAB) can produce a huge amount of bioactive compounds. Since their elective habitat is food, especially dairy but also vegetal food, it is frequent to find bioactive molecules in fermented products. Sometimes these compounds can have adverse effects on human health such as biogenic amines (tyramine and histamine), causing allergies, hypertensive crises, and headache. However, some LAB products also display benefits for the consumers. In the present review article, the main nitrogen compounds produced by LAB are considered. Besides biogenic amines derived from the amino acids tyrosine, histidine, phenylalanine, lysine, ornithine, and glutamate by decarboxylation, interesting peptides can be decrypted by the proteolytic activity of LAB. LAB proteolytic system is very efficient in releasing encrypted molecules from several proteins present in different food matrices. Alpha and beta-caseins, albumin and globulin from milk and dairy products, rubisco from spinach, beta-conglycinin from soy and gluten from cereals constitute a good source of important bioactive compounds. These encrypted peptides are able to control nutrition (mineral absorption and oxidative stress protection), metabolism (blood glucose and cholesterol lowering) cardiovascular function (antithrombotic and hypotensive action), infection (microbial inhibition and immunomodulation) and gut-brain axis (opioids and anti-opioids controlling mood and food intake). Very recent results underline the role of food-encrypted peptides in protein folding (chaperone-like molecules) as well as in cell cycle and apoptosis control, suggesting new and positive aspects of fermented food, still unexplored. In this context, the detailed (transcriptomic, proteomic, and metabolomic) characterization of LAB of food interest (as starters, biocontrol agents, nutraceuticals, and probiotics) can supply a solid evidence-based science to support beneficial effects and it is a promising approach as well to obtain

  14. Bioactive Molecules Released in Food by Lactic Acid Bacteria: Encrypted Peptides and Biogenic Amines.

    PubMed

    Pessione, Enrica; Cirrincione, Simona

    2016-01-01

    Lactic acid bacteria (LAB) can produce a huge amount of bioactive compounds. Since their elective habitat is food, especially dairy but also vegetal food, it is frequent to find bioactive molecules in fermented products. Sometimes these compounds can have adverse effects on human health such as biogenic amines (tyramine and histamine), causing allergies, hypertensive crises, and headache. However, some LAB products also display benefits for the consumers. In the present review article, the main nitrogen compounds produced by LAB are considered. Besides biogenic amines derived from the amino acids tyrosine, histidine, phenylalanine, lysine, ornithine, and glutamate by decarboxylation, interesting peptides can be decrypted by the proteolytic activity of LAB. LAB proteolytic system is very efficient in releasing encrypted molecules from several proteins present in different food matrices. Alpha and beta-caseins, albumin and globulin from milk and dairy products, rubisco from spinach, beta-conglycinin from soy and gluten from cereals constitute a good source of important bioactive compounds. These encrypted peptides are able to control nutrition (mineral absorption and oxidative stress protection), metabolism (blood glucose and cholesterol lowering) cardiovascular function (antithrombotic and hypotensive action), infection (microbial inhibition and immunomodulation) and gut-brain axis (opioids and anti-opioids controlling mood and food intake). Very recent results underline the role of food-encrypted peptides in protein folding (chaperone-like molecules) as well as in cell cycle and apoptosis control, suggesting new and positive aspects of fermented food, still unexplored. In this context, the detailed (transcriptomic, proteomic, and metabolomic) characterization of LAB of food interest (as starters, biocontrol agents, nutraceuticals, and probiotics) can supply a solid evidence-based science to support beneficial effects and it is a promising approach as well to obtain

  15. Peptide nucleic acids rather than RNA may have been the first genetic molecule

    NASA Technical Reports Server (NTRS)

    Nelson, K. E.; Levy, M.; Miller, S. L.

    2000-01-01

    Numerous problems exist with the current thinking of RNA as the first genetic material. No plausible prebiotic processes have yet been demonstrated to produce the nucleosides or nucleotides or for efficient two-way nonenzymatic replication. Peptide nucleic acid (PNA) is a promising precursor to RNA, consisting of N-(2-aminoethyl)glycine (AEG) and the adenine, uracil, guanine, and cytosine-N-acetic acids. However, PNA has not yet been demonstrated to be prebiotic. We show here that AEG is produced directly in electric discharge reactions from CH(4), N(2), NH(3), and H(2)O. Electric discharges also produce ethylenediamine, as do NH(4)CN polymerizations. AEG is produced from the robust Strecker synthesis with ethylenediamine. The NH(4)CN polymerization in the presence of glycine leads to the adenine and guanine-N(9)-acetic acids, and the cytosine and uracil-N(1)-acetic acids are produced in high yield from the reaction of cyanoacetaldehyde with hydantoic acid, rather than urea. Preliminary experiments suggest that AEG may polymerize rapidly at 100 degrees C to give the polypeptide backbone of PNA. The ease of synthesis of the components of PNA and possibility of polymerization of AEG reinforce the possibility that PNA may have been the first genetic material.

  16. Amino acid and peptide absorption in patients with coeliac disease and dermatitis herpetiformis

    PubMed Central

    Silk, D. B. A.; Kumar, Parveen J.; Perrett, D.; Clark, M. L.; Dawson, A. M.

    1974-01-01

    A double-lumen perfusion technique has been used to study amino acid and peptide absorption in eight normal control subjects, 13 patients with untreated adult coeliac disease, and 16 patients with dermatitis herpetiformis who had varying morphological abnormalities of the small bowel. All subjects were perfused with isotonic solutions containing 10 mM glycyl-L-alanine and 10 mM glycine + 10 mM L-alanine. Patients with adult coeliac disease had impaired absorption of glycine (p < 0·01) and L-alanine (p < 0·05) from the amino acid solution compared with the control subjects. Amino acid uptake from the dipeptide solution was not significantly impaired, although four individual patients had impaired uptake of both amino acids. In contrast to these findings, very few patients with dermatitis herpetiformis had impaired amino acid absorption from either solution. Sodium absorption was impaired from both solutions when the groups of patients with adult coeliac disease and dermatitis herpetiformis with subtotal villous atrophy and partial villous atrophy were studied, and there were patients in each group who secreted sodium and water. The results suggest that malabsorption of dietary protein is unlikely to occur in dermatitis herpetiformis but may occur and contribute to protein deficiency seen in some severe cases of adult coeliac disease. The impairment of sodium and water absorption provides evidence that there may be functional impairment of the jejunal mucosa in dermatitis herpetiformis as well as in adult coeliac disease. PMID:4820629

  17. Potent Antibacterial Antisense Peptide–Peptide Nucleic Acid Conjugates Against Pseudomonas aeruginosa

    PubMed Central

    Ghosal, Anubrata

    2012-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections in hospital settings, especially with immune compromised patients, and the increasing prevalence of multidrug resistant strains urges search for new drugs with novel mechanisms of action. In this study we introduce antisense peptide–peptide nucleic acid (PNA) conjugates as antibacterial agents against P. aeruginosa. We have designed and optimized antisense peptide–PNA conjugates targeting the translation initiation region of the ftsZ gene (an essential bacterial gene involved in cell division) or the acpP gene (an essential bacterial gene involved in fatty acid synthesis) of P. aeruginosa (PA01) and characterized these compounds according to their antimicrobial activity and mode of action. Four antisense PNA oligomers conjugated to the H-(R-Ahx-R)4-Ahx-βala or the H-(R-Ahx)6-βala peptide exhibited complete growth inhibition of P. aeruginosa strains PA01, PA14, and LESB58 at 1–2 μM concentrations without any indication of bacterial membrane disruption (even at 20 μM), and resulted in specific reduction of the targeted mRNA levels. One of the four compounds showed clear bactericidal activity while the other significantly reduced bacterial survival. These results open the possibility of development of antisense antibacterials for treatment of Pseudomonas infections. PMID:23030590

  18. Template tailoring: Accurate determination of heterozygous alleles using peptide nucleic acid and dideoxyNTP

    PubMed Central

    Tariq, Muhammad Akram; Pourmand, Nader

    2010-01-01

    Measurement of the length of DNA fragments plays a pivotal role in genetic mapping, disease diagnostics, human identification and forensic applications. PCR followed by electrophoresis is used for DNA length measurement of STRs, a process that requires labeled primers and allelic ladders as standards to avoid machine error. Sequencing-based approaches can be used for STR analysis to eliminate the requirement of labeled primers and allelic ladder. However, the limiting factor with this approach is unsynchronized polymerization in heterozygous sample analysis, in which alleles with different lengths can lead to imbalanced heterozygote peak height ratios. We have developed a rapid DNA length measurement method using peptide nucleic acid and dideoxy dNTPs to “tailor” DNA templates for accurate sequencing to overcome this hurdle. We also devised an accelerated “dyad” pyrosequencing strategy, such that the combined approach can be used as a faster, more accurate alternative to de novo sequencing. Dyad sequencing interrogates two bases at a time by allowing the polymerase to incorporate two nucleotides to DNA template, cutting the analysis time in half. In addition, for the first time, we show the effect of peptide nucleic acid as a blocking probe to stop polymerization, which is essential to analyze the heterozygous samples by sequencing. This approach provides a new platform for rapid and cost-effective DNA length measurement for STRs and resequencing of small DNA fragments. PMID:20408144

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

    PubMed

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

    1997-01-01

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

  20. Oligosaccharide structure and amino acid sequence of the major glycopeptides of mature human. beta. -hexosaminidase

    SciTech Connect

    O'Dowd, B.F.; Cumming, D.A.; Gravel, R.A.; Mahuran, D.

    1988-07-12

    Human ..beta..-hexosaminidase is a lysosomal enzyme that hydrolyzes terminal N-acetylhexosamines from GM/sub 2/ ganglioside, oligosaccharides, and other carbohydrate-containing macromolecules. There are two major forms of hexosaminidase: hexosaminidase A, with the structure ..cap alpha..(..beta../sub a/..beta../sub b/), and hexosaminidase B, 2(..beta../sub a/..beta../sub b/). Like other lysosomal proteins, hexosaminidase is targeted to its destination via glycosylation and processing in the rough endoplasmic reticulum and Golgi apparatus. Phosphorylation of specific mannose residues allows binding of the protein to the phosphomannosyl receptor and transfer to the lysosome. In order to define the structure and placement of the oligosaccharides in mature hexosaminidase and thus identify candidate mannose 6-phosphate recipient sites, the major tryptic/chymotryptic glycopeptides from each isozyme were purified by reverse-phase high-performance liquid chromatography. Two major concanavalin A binding glycopeptides, localized to the ..beta../sub b/f chain, and one non concanavalin A binding glycopeptide, localized to the ..beta../sub a/ chain, were found associated with the ..beta..-subunit in both hexosaminidase A and hexosaminidase B. The oligosaccharide structures were determined by nuclear magnetic resonance spectrometry. The unique glycopeptide associated with the ..beta../sub a/ chain contained a single GlcNAc residue. Thus all three mature polypeptides comprising the ..cap alpha.. and ..beta.. subunits of hexosaminidase contain carbohydrate, the structures of which have the appearance of being partially degraded in the lysosome. In the ..cap alpha.. chain they found only one possible site for in vivo phosphorylation. In the ..beta.. it is unclear if only one or all three of the sites could have contained phosphate. However, mature placental hexosaminidase A and B can be rephosphorylated in vitro. This requires the presence of an oligosaccharide containing an ..cap

  1. Role of SbmA in the uptake of peptide nucleic acid (PNA)-peptide conjugates in E. coli.

    PubMed

    Ghosal, Anubrata; Vitali, Ally; Stach, James E M; Nielsen, Peter E

    2013-02-15

    Antisense PNA oligomers targeting essential genes (acpP or ftsZ) and conjugated to the delivery peptide L((KFF)(3)K) show complete growth inhibition of wild type E. coli strain (MG1655) with submicromolar MIC. In this study we show that resistant mutants generated against such PNA-peptide conjugates had disruptions in the region of sbmA, a gene encoding an inner membrane peptide transporter. The wild type sensitivity to the PNA conjugates was re-established in the resistance mutants by complementation with sbmA. Furthermore, deletion of sbmA in E. coli AS19, a strain that is sensitive to unmodified PNA, resulted in resistance to PNA. Finally, PNA conjugated with the corresponding non-biological H-D((KFF)(3)K) peptide retained antibacterial activity in sbmA deletion strains, whereas the same conjugate with a protease-sensitive linker did not. These results clearly identify SbmA as a carrier of naked PNA over the inner bacterial membrane and thereby infer that the peptide is transporting the PNA conjugates over the outer membrane. Strains lacking SbmA were used to screen novel peptide-PNA carriers that were SbmA-independent. Four such PNA-peptide conjugates, H-D((KFF)(3)K), H-(RFR)(4)-Ahx-βAla, H-(R-Ahx-R)(4)-Ahx-βAla, and H-(R-Ahx)(6)-βAla, were identified that utilize an alternative uptake mechanism but retain their antimicrobial potency. In addition SbmA is the first protein identified to recognize PNA. PMID:23138594

  2. Chemiluminescence detection of amino acids, peptides, and proteins using tris-2,2 prime -bipyridine ruthenium(III)

    SciTech Connect

    Li He; Cox, K.A.; Danielson, N.D. )

    1990-01-01

    The feasibility of using the tris-2-2{prime}-bipyridine ruthenium(III) chemiluminescent (CL) reaction for the detection of amino acids, peptides, and proteins has been studied. Detection limits of the amino acids as determined by flow injection analysis (FIA) ranged from 20 pmol of proline to 50 nmol of asparagine. In general, amino acids containing secondary amine groups yielded the strongest responses. A reaction mechanism for Ru(bpy){sub 3}{sup 3+} chemiluminescence of aliphatic amines has been proposed. Studies of peptide molecules and poly-prolines showed that the peptide bond barely contributes to the detection signals. The separation of hydroxyproline and proline in synthetic collagen by HPLC with Ru(bpy){sub 3}{sup 3+} chemiluminescence detection has been shown to be possible.

  3. Fatty Acid Composition of Developing Sea Buckthorn (Hippophae rhamnoides L.) Berry and the Transcriptome of the Mature Seed

    PubMed Central

    Fatima, Tahira; Snyder, Crystal L.; Schroeder, William R.; Cram, Dustin; Datla, Raju; Wishart, David; Weselake, Randall J.; Krishna, Priti

    2012-01-01

    Background Sea buckthorn (Hippophae rhamnoides L.) is a hardy, fruit-producing plant known historically for its medicinal and nutraceutical properties. The most recognized product of sea buckthorn is its fruit oil, composed of seed oil that is rich in essential fatty acids, linoleic (18∶2ω-6) and α-linolenic (18∶3ω-3) acids, and pulp oil that contains high levels of monounsaturated palmitoleic acid (16∶1ω-7). Sea buckthorn is fast gaining popularity as a source of functional food and nutraceuticals, but currently has few genomic resources; therefore, we explored the fatty acid composition of Canadian-grown cultivars (ssp. mongolica) and the sea buckthorn seed transcriptome using the 454 GS FLX sequencing technology. Results GC-MS profiling of fatty acids in seeds and pulp of berries indicated that the seed oil contained linoleic and α-linolenic acids at 33–36% and 30–36%, respectively, while the pulp oil contained palmitoleic acid at 32–42%. 454 sequencing of sea buckthorn cDNA collections from mature seeds yielded 500,392 sequence reads, which identified 89,141 putative unigenes represented by 37,482 contigs and 51,659 singletons. Functional annotation by Gene Ontology and computational prediction of metabolic pathways indicated that primary metabolism (protein>nucleic acid>carbohydrate>lipid) and fatty acid and lipid biosynthesis pathways were highly represented categories. Sea buckthorn sequences related to fatty acid biosynthesis genes in Arabidopsis were identified, and a subset of these was examined for transcript expression at four developing stages of the berry. Conclusion This study provides the first comprehensive genomic resources represented by expressed sequences for sea buckthorn, and demonstrates that the seed oil of Canadian-grown sea buckthorn cultivars contains high levels of linoleic acid and α-linolenic acid in a close to 1∶1 ratio, which is beneficial for human health. These data provide the foundation for further studies on

  4. A conjugate of the lytic peptide Hecate and gallic acid: structure, activity against cervical cancer, and toxicity.

    PubMed

    Sanches, Paulo R S; Carneiro, Bruno M; Batista, Mariana N; Braga, Ana Cláudia S; Lorenzón, Esteban N; Rahal, Paula; Cilli, Eduardo Maffud

    2015-07-01

    Conjugate compounds constitute a new class of molecules of important biological interest mainly for the treatment of diseases such as cancer. The N-terminus region of cationic peptides has been described as important for their biological activity. The aim of this study was to evaluate the lytic peptide Hecate (FALALKALKKALKKLKKALKKAL) and the effect of conjugating this macromolecule with gallic acid (C7H6O5) in terms of structure, anti-cancer activity, and toxicity. An N-terminus GA-Hecate peptide conjugate was synthesized to provide information regarding the relationship between the amino-terminal region and its charge and the secondary structure and biological activity of the peptide; and the effects of gallic acid on these parameters. Peptide secondary structure was confirmed using circular dichroism (CD). The CD measurements showed that the peptide has a high incidence of α-helical structures in the presence of SDS and LPC, while GA-Hecate presented lower incidence of α-helical structures in the same chemical environment. An evaluation of the anti-cancer activity in HeLa cancer cells indicated that both peptides are active, but that coupling gallic acid at the N-terminus decreased the activity of the free peptide. GA-Hecate showed lower activity in non-tumor keratinocyte cells but higher hemolytic activity. Our findings suggest that the N-terminus of Hecate plays an important role in its activity against cervical cancer by affecting it secondary structure, toxicity, and hemolytic activity. This study highlights the importance of the N-terminus in antitumor activity and could provide an important tool for developing new anti-cancer drugs. PMID:25868656

  5. Conformations of helical Aib peptides containing a pair of L-amino acid and D-amino acid.

    PubMed

    Demizu, Yosuke; Yabuki, Yu-U; Doi, Mitsunobu; Sato, Yukiko; Tanaka, Masakazu; Kurihara, Masaaki

    2012-07-01

    A pair of L-leucine (L-Leu) and D-leucine (D-Leu) was incorporated into a-aminoisobutyric acid (Aib) peptide segments. Thedominant conformations of four hexapeptides, Boc-L-Leu-Aib-Aib-Aib-Aib-L-Leu-OMe (1a), Boc-D-Leu-Aib-Aib-Aib-Aib-L-Leu-OMe(1b), Boc-Aib-Aib-L-Leu-L-Leu-Aib-Aib-OMe (2a), and Boc-Aib-Aib-D-Leu-L-Leu-Aib-Aib-OMe (2b), were investigated by IR,¹H NMR, CD spectra, and X-ray crystallographic analysis. All peptides 1a,b and 2a,b formed 3₁₀-helical structures in solution. X-ray crystallographic analysis revealed that right-handed (P) 3₁₀-helices were present in 1a and 1b and a mixture of right-handed(P) and left-handed (M) 3₁₀-helices was present in 2b in their crystalline states. PMID:22619002

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

    SciTech Connect

    Xu,G.; Chance, M.

    2005-01-01

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

  7. Slow peptide bond formation by proline and other N-alkylamino acids in translation

    PubMed Central

    Pavlov, Michael Y.; Watts, Richard E.; Tan, Zhongping; Cornish, Virginia W.; Ehrenberg, Måns; Forster, Anthony C.

    2009-01-01

    Proteins are made from 19 aa and, curiously, one N-alkylamino acid (“imino acid”), proline (Pro). Pro is thought to be incorporated by the translation apparatus at the same rate as the 19 aa, even though the alkyl group in Pro resides directly on the nitrogen nucleophile involved in peptide bond formation. Here, by combining quench-flow kinetics and charging of tRNAs with cognate and noncognate amino acids, we find that Pro incorporates in translation significantly more slowly than Phe or Ala and that other N-alkylamino acids incorporate much more slowly. Our results show that the slowest step in incorporation of N-alkylamino acids is accommodation/peptidyl transfer after GTP hydrolysis on EF-Tu. The relative incorporation rates correlate with expectations from organic chemistry, suggesting that amino acid sterics and basicities affect translation rates at the peptidyl transfer step. Cognate isoacceptor tRNAs speed Pro incorporation to rates compatible with in vivo, although still 3–6 times slower than Phe incorporation from Phe-tRNAPhe depending on the Pro codon. Results suggest that Pro is the only N-alkylamino acid in the genetic code because it has a privileged cyclic structure that is more reactive than other N-alkylamino acids. Our data on the variation of the rate of incorporation of Pro from native Pro-tRNAPro isoacceptors at 4 different Pro codons help explain codon bias not accounted for by the “tRNA abundance” hypothesis. PMID:19104062

  8. Reversible Major Histocompatibility Complex I-Peptide Multimers Containing Ni2+-Nitrilotriacetic Acid Peptides and Histidine Tags Improve Analysis and Sorting of CD8+ T Cells*

    PubMed Central

    Schmidt, Julien; Guillaume, Philippe; Irving, Melita; Baumgaertner, Petra; Speiser, Daniel; Luescher, Immanuel F.

    2011-01-01

    MHC-peptide multimers containing biotinylated MHC-peptide complexes bound to phycoerythrin (PE) streptavidin (SA) are widely used for analyzing and sorting antigen-specific T cells. Here we describe alternative T cell-staining reagents that are superior to conventional reagents. They are built on reversible chelate complexes of Ni2+-nitrilotriacetic acid (NTA) with oligohistidines. We synthesized biotinylated linear mono-, di-, and tetra-NTA compounds using conventional solid phase peptide chemistry and studied their interaction with HLA-A*0201-peptide complexes containing a His6, His12, or 2×His6 tag by surface plasmon resonance on SA-coated sensor chips and equilibrium dialysis. The binding avidity increased in the order His6 < His12 < 2×His6 and NTA1 < NTA2 < NTA4, respectively, depending on the configuration of the NTA moieties and increased to picomolar KD for the combination of a 2×His6 tag and a 2×Ni2+-NTA2. We demonstrate that HLA-A2–2×His6-peptide multimers containing either Ni2+-NTA4-biotin and PE-SA- or PE-NTA4-stained influenza and Melan A-specific CD8+ T cells equal or better than conventional multimers. Although these complexes were highly stable, they very rapidly dissociated in the presence of imidazole, which allowed sorting of bona fide antigen-specific CD8+ T cells without inducing T cell death as well as assessment of HLA-A2-peptide monomer dissociation kinetics on CD8+ T cells. PMID:21990358

  9. Vibrational analysis of amino acids and short peptides in hydrated media. II. Role of KLLL repeats to induce helical conformations in minimalist LK-peptides.

    PubMed

    Guiffo-Soh, Guy; Hernandez, Belén; Coïc, Yves-Marie; Boukhalfa-Heniche, Fatima-Zohra; Ghomi, Mahmoud

    2007-11-01

    Aqueous solution secondary structures of minimalist LK-peptides, with the generic sequence defined as KLL(KLLL)nKLLK, have been analyzed by means of circular dichroism (CD) and Raman scattering techniques. Our discussion in the present paper is mainly focused on four synthetic peptides (from 5 to 19 amino acids), KLLLK, KLLKLLLKLLK, KLLKLLLKLLLKLLK, and KLLKLLLKLLLKLLLKLLK, corresponding to the repeat unit, and to the peptide chains with the values of n = 1-3, respectively. CD and Raman spectra were analyzed in order to study both structural features of the peptide chains and their capability to form aggregates. On the basis of the obtained results it was concluded that the conformational flexibility of the shortest peptides (5-mer and 11-mer) is high enough to adopt random, beta-type, and helical chains in aqueous solution. However, the 11-mer shows a clear tendency to form beta-strands in phosphate buffer. The conformational equilibrium can be completely shifted to beta-type structures upon increasing ionic strength, i.e., in PBS and tris buffers. This equilibrium can also be shifted toward helical chains in the presence of methanol. Finally, the longest peptides (15-mer and 19-mer) are shown to form alpha-helical chains with an amphipathic character in aqueous solution. The possibility of bundle formation between helical chains is discussed over the temperature-dependent H-D exchange on labile hydrogens and particularly by considering the particular behavior of an intense Raman mode at 1127 cm-1 originating from the leucine residue side chain. The conformational dependence of this mode observed upon selective deuteration has never been documented up to now. PMID:17918991

  10. Hydration studies of electrospray ions from amino acids and small peptides

    NASA Astrophysics Data System (ADS)

    Nguyen, Chuong (Steve)

    This project was undertaken to gain a better understanding of the hydration behaviors of gas phase ions from solutions containing amino acids and peptides. In order to characterize their hydration behavior, the molecules of interest in solutions were first converted into gas phase ions by electrospray ionization (ESI). The completely desolvated ions were then deliberately dispersed into an inert bath gas, usually nitrogen, containing accurately known concentrations of solvent vapor. The resulting mixtures of ions and bath gas were subsequently passed into a vacuum chamber by way of an adiabatic supersonic free jet expansion. The cooling during that expansion caused solvation of the ions, the extent of which was determined by a quadrupole mass analyzer. Mass analysis of the solute ions in the absence of vapor showed peaks with the mass to charge ratios corresponding to the desolvated ions. On the other hand, mass spectrometric analyses of ions in the presence of solvent vapor showed sequences of peaks corresponding to the solvated ions with varying numbers of water molecules. The extent of the ion solvation was controlled by varying the concentration of solvent vapor in the bath gas. Two different scales were proposed for the evaluation of the relative affinities of amino acids for water molecules. One was based primarily on the assumption that the affinities of amino acids for water molecules are directly proportional to their gas phase solvation rate constants ( k). An alternative approach produced an affinity scale based on the extent of ion hydration occurred during the free jet expansion. It was found that the addition of a polar solvent vapor to the bath gas at low concentrations substantially enhanced the production of the bare solute ions from the evaporating charged droplets. This remarkable result not only provided a means to increase the ion production and thus detection sensitivity of mass spectrometric analyses, but also yielded important information

  11. Extended neocortical maturation time encompasses speciation, fatty acid and lateralization theories of the evolution of schizophrenia and creativity.

    PubMed

    Seldon, H Lee

    2007-01-01

    I suggest that the extended maturation time of some regions of the human neocortex is the uniquely human factor which allows the development of language, creativity and madness. The genetic event or events which contribute to the long delay to final maturation occurred at or after the speciation of Homo sapiens sapiens. Neocortical growth may follow the previously detailed "balloon model", which suggests that intra- and subcortical myelin production during development physically stretches each local area of the cortex tangentially to the pial surface, thereby causing neuronal columns to become more disjoint and more functionally independent, thereby increasing the functional capacity of the area [Seldon HL. Does brain white matter growth expand the cortex like a balloon? Hypothesis and consequences. Laterality 2005;10(1):81-95]. This occurs in addition to Hebbian synaptic modeling. Therefore, the size and functional capacity of each cytoarchitectonic area of each individual adult neocortex are the outcomes of partly deterministic (e.g., genetic) and partly statistical growth processes with numerous factors including environmental stimuli and fatty acid content in diets. The possible functional capacity and variation among growth outcomes increase with the length of time allowed to "finalize" synaptic weights, myelination and other plastic processes. For example, acquisition of quite differing linguistic skills becomes possible only in Homo sapiens because of the extended, decades-long plasticity of temporal lobe areas; in contrast, tactile skills vary little among human races and cultures, or even among higher primates, because of the faster maturation of the somatomotor areas. Some of the statistically extreme variations of the neocortical growth processes lead to abnormal cognition and behavior called "madness" or "genius". This maturation hypothesis overcomes some problems with those based purely on fatty acid metabolism or on functional asymmetry (non

  12. Self-assembly of short peptides composed of only aliphatic amino acids and a combination of aromatic and aliphatic amino acids.

    PubMed

    Subbalakshmi, Chilukuri; Manorama, Sunkara V; Nagaraj, Ramakrishnan

    2012-05-01

    The morphology of structures formed by the self-assembly of short N-terminal t-butyloxycarbonyl (Boc) and C-terminal methyl ester (OMe) protected and Boc-deprotected hydrophobic peptide esters was investigated. We have observed that Boc-protected peptide esters composed of either only aliphatic hydrophobic amino acids or aliphatic hydrophobic amino acids in combination with aromatic amino acids, formed highly organized structures, when dried from methanol solutions. Transmission and scanning electron microscopic images of the peptides Boc-Ile-Ile-OMe, Boc-Phe-Phe-Phe-Ile-Ile-OMe and Boc-Trp-Ile-Ile-OMe showed nanotubular structures. Removal of the Boc group resulted in disruption of the ability to form tubular structures though spherical aggregates were formed. Both Boc-Leu-Ile-Ile-OMe and H-Leu-Ile-Ile-OMe formed only spherical nanostructures. Dynamic light scattering studies showed that aggregates of varying dimensions were present in solution suggesting that self-assembly into ordered structures is facilitated by aggregation in solution. Fourier transform infrared spectroscopy and circular dichroism spectroscopy data show that although all four of the protected peptides adopt well-defined tertiary structures, upon removal of the Boc group, only H-Phe-Phe-Phe-Ile-Ile-OMe had the ability to adopt β-structure. Our results indicate that hydrophobic interaction is a very important determinant for self-assembly and presence of charged and aromatic amino acids in a peptide is not necessary for self-assembly. PMID:22431418

  13. Acidity and metal (Mg2+, Ca2+, Zn2+) affinity of L-γ-carboxyglutamic acid and its peptide analog

    NASA Astrophysics Data System (ADS)

    Remko, Milan; Broer, Ria; Remková, Anna; Van Duijnen, Piet Th.

    2014-10-01

    Density functional theory methods with the B3LYP and B97D functionals with triple-zeta 6-311++G(d,p) basis set have been used to study the acidity, basicity and metal affinity of L-γ-carboxyglutamic acid (GLA) and its peptide derivative [2-acetylamino-3-(methylamino)-3-oxopropyl]malonic acid (AMD-GLA). The Gibbs interaction energies of the GLA2-…M2+ and AMD-GLA2-…M2+ (M = Mg, Ca, Zn) complexes show an increasing binding affinity in the order Ca2+ < Mg2+ < Zn2+ The transition metal Zn2+ is most effectively recognized by the dianions of GLA and AMD-GLA. Of the dianions studied the AMD-GLA dianion is the strongest Lewis base. Computations that include the effect of solvation showed that in water the relative stability of GLA2-…M2+ and AMD-GLA2-…M2+ ionic bonds is rapidly diminished. The computed interaction Gibbs energy in water is small and negative.

  14. Theoretical study on application of peptide nanoring to chiral recognition of amino acid

    NASA Astrophysics Data System (ADS)

    Takeuchi, Jo; Takeda, Kyozaburo

    2016-03-01

    Chiral recognition of a guest amino acid by a host peptide nanoring (PNR) is studied by ab initio calculations. The intermolecular hydrogen bonds (HBs) between the present host and the guest force the guest amino acid to land into the host PNR, and also the side chain of the host PNR to hold the guest amino acid. Thus, the host PNR captures the guest amino acid and gives a distinct energy difference in accordance with the chirality of the captured guest d/l-SerH+. The calculated energy difference of 18 kJ/mol in Gibbs free energy is comparable to that by the host crown ether, by which Moreno et al. have succeeded in experimentally recognizing the chirality of the guest d/l-SerH+ [J. R. A. Moreno, M. M. Q. Moreno, J. J. L. González, and B. M. Haya, J. Phys. Chem. B 117, 9362 (2013)]. The PNRs could be unique biomolecular materials from the perspective that the chirality of an “amino acid” is recognized by the PNR consisting of “amino acid” residues.

  15. Absorption of Amino Acids and Peptides in a Child with a Variant of Hartnup Disease and Coexistent Coeliac Disease

    PubMed Central

    Tarlow, M. J.; Seakins, J. W. T.; Lloyd, June K.; Matthews, D. M.; Cheng, B.; Thomas, A. J.

    1972-01-01

    A child with a variant of Hartnup disease and co-existent coeliac disease is described. Oral tolerance tests with L-histidine, L-tyrosine, and glycyl-L-tyrosine, and in vitro uptake studies on a small intestinal biopsy with L-histidine and glycyl-L-histidine, showed impaired absorption of the free amino acids, and showed that absorption of tyrosine and mucosal uptake of histidine was better from the dipeptides than from the free amino acids. This supports the hypothesis that the intestinal mucosa can take up small peptides intact, and that the peptide uptake mechanism is not involved in the intestinal defect of Hartnup disease. PMID:5086513

  16. The fsr Quorum-Sensing System and Cognate Gelatinase Orchestrate the Expression and Processing of Proprotein EF_1097 into the Mature Antimicrobial Peptide Enterocin O16

    PubMed Central

    Dundar, Halil; Brede, Dag A.; La Rosa, Sabina Leanti; El-Gendy, Ahmed Osama; Diep, Dzung B.

    2015-01-01

    ABSTRACT A novel antimicrobial peptide designated enterocin O16 was purified from Enterococcus faecalis. Mass spectrometry showed a monoisotopic mass of 7,231 Da, and N-terminal Edman degradation identified a 29-amino-acid sequence corresponding to residues 90 to 119 of the EF_1097 protein. Bioinformatic analysis showed that enterocin O16 is composed of the 68 most C-terminal residues of the EF_1097 protein. Introduction of an in-frame isogenic deletion in the ef1097 gene abolished the production of enterocin O16. Enterocin O16 has a narrow inhibitory spectrum, as it inhibits mostly lactobacilli. Apparently, E. faecalis is intrinsically resistant to the antimicrobial peptide, as no immunity connected to the production of enterocin O16 could be identified. ef1097 has previously been identified as one of three loci regulated by the fsr quorum-sensing system. The introduction of a nonsense mutation into fsrB consistently impaired enterocin O16 production, but externally added gelatinase biosynthesis-activating pheromone restored the antimicrobial activity. Functional genetic analysis showed that the EF_1097 proprotein is processed extracellularly into enterocin O16 by the metalloprotease GelE. Thus, it is evident that the fsr quorum-sensing system constitutes the regulatory unit that controls the expression of the EF_1097 precursor protein and the protease GelE and that the latter is required for the formation of enterocin O16. On the basis of these results, this study identified antibacterial antagonism as a novel aspect related to the function of fsr and provides a rationale for why ef1097 is part of the fsr regulon. IMPORTANCE The fsr quorum-sensing system modulates important physiological functions in E. faecalis via the activity of GelE. The present study presents a new facet of fsr signaling. The system controls the expression of three primary target operons (fsrABCD, gelE-sprE, and ef1097-ef1097b). We demonstrate that the concerted expression of these operons

  17. Penetration depth of surfactant peptide KL4 into membranes is determined by fatty acid saturation.

    PubMed

    Antharam, Vijay C; Elliott, Douglas W; Mills, Frank D; Farver, R Suzanne; Sternin, Edward; Long, Joanna R

    2009-05-20

    KL(4) is a 21-residue functional peptide mimic of lung surfactant protein B, an essential protein for lowering surface tension in the alveoli. Its ability to modify lipid properties and restore lung compliance was investigated with circular dichroism, differential scanning calorimetry, and solid-state NMR spectroscopy. KL(4) binds fluid lamellar phase PC/PG lipid membranes and forms an amphipathic helix that alters lipid organization and acyl chain dynamics. The binding and helicity of KL(4) is dependent on the level of monounsaturation in the fatty acid chains. At physiologic temperatures, KL(4) is more peripheral and dynamic in fluid phase POPC/POPG MLVs but is deeply inserted into fluid phase DPPC/POPG vesicles, resulting in immobilization of the peptide. Substantial increases in the acyl chain order are observed in DPPC/POPG lipid vesicles with increasing levels of KL(4), and POPC/POPG lipid vesicles show small decreases in the acyl chain order parameters on addition of KL(4). Additionally, a clear effect of KL(4) on the orientation of the fluid phase PG headgroups is observed, with similar changes in both lipid environments. Near the phase transition temperature of the DPPC/POPG lipid mixtures, which is just below the physiologic temperature of lung surfactant, KL(4) causes phase separation with the DPPC remaining in a gel phase and the POPG partitioned between gel and fluid phases. The ability of KL(4) to differentially partition into lipid lamellae containing varying levels of monounsaturation and subsequent changes in curvature strain suggest a mechanism for peptide-mediated lipid organization and trafficking within the dynamic lung environment. PMID:19450480

  18. Penetration Depth of Surfactant Peptide KL4 into Membranes Is Determined by Fatty Acid Saturation

    PubMed Central

    Antharam, Vijay C.; Elliott, Douglas W.; Mills, Frank D.; Farver, R. Suzanne; Sternin, Edward; Long, Joanna R.

    2009-01-01

    KL4 is a 21-residue functional peptide mimic of lung surfactant protein B, an essential protein for lowering surface tension in the alveoli. Its ability to modify lipid properties and restore lung compliance was investigated with circular dichroism, differential scanning calorimetry, and solid-state NMR spectroscopy. KL4 binds fluid lamellar phase PC/PG lipid membranes and forms an amphipathic helix that alters lipid organization and acyl chain dynamics. The binding and helicity of KL4 is dependent on the level of monounsaturation in the fatty acid chains. At physiologic temperatures, KL4 is more peripheral and dynamic in fluid phase POPC/POPG MLVs but is deeply inserted into fluid phase DPPC/POPG vesicles, resulting in immobilization of the peptide. Substantial increases in the acyl chain order are observed in DPPC/POPG lipid vesicles with increasing levels of KL4, and POPC/POPG lipid vesicles show small decreases in the acyl chain order parameters on addition of KL4. Additionally, a clear effect of KL4 on the orientation of the fluid phase PG headgroups is observed, with similar changes in both lipid environments. Near the phase transition temperature of the DPPC/POPG lipid mixtures, which is just below the physiologic temperature of lung surfactant, KL4 causes phase separation with the DPPC remaining in a gel phase and the POPG partitioned between gel and fluid phases. The ability of KL4 to differentially partition into lipid lamellae containing varying levels of monounsaturation and subsequent changes in curvature strain suggest a mechanism for peptide-mediated lipid organization and trafficking within the dynamic lung environment. PMID:19450480

  19. Composition of free and peptide-bound amino acids in beef chuck, loin, and round cuts.

    PubMed

    Wu, G; Cross, H R; Gehring, K B; Savell, J W; Arnold, A N; McNeill, S H

    2016-06-01

    Meat is a food for humans. However, beef consumption in the United States has steadily declined by >14% over the past decade due to a variety of factors, including insufficient knowledge of animal protein. This study quantified all proteinogenic AA as well as nutritionally and physiologically significant nonproteinogenic AA and small peptides in beef cuts from 3 subprimals (chuck, round, and loin). Beef carcasses ( = 10) were selected at 3 commercial packing plants in the United States. Retail-cut samples were analyzed for the nitrogenous substances after acid, alkaline, or enzymatic hydrolysis and after deproteinization. In these chuck, round, and loin cuts, total amounts of glutamate (free plus peptide bound) were the highest (69-75 mg/g dry weight) followed by lysine, leucine, arginine, and glutamine in descending order. This is the first study to determine aspartate, asparagine, glutamate, and glutamine in meat proteins of any animal species. In all the beef samples evaluated, glutamine was the most abundant free AA (4.0-5.7 mg/g dry weight) followed by taurine, alanine, glutamate, and β-alanine. Additionally, samples from all beef cuts had high concentrations of anserine, carnosine, and glutathione, which were 2.8 to 3.7, 15.2 to 24.2, and 0.68 to 0.79 mg/g dry weight, respectively. Beef top loin steaks appear to provide higher protein nutrition values than top round steaks and under blade roasts, but all are excellent sources of proteinogenic AA as well as antioxidant AA and peptides to improve human growth, development, and health. Our findings may help guide future decisions regarding human and animal nutrition. PMID:27285936

  20. Kinetic properties of the binding of alpha-lytic protease to peptide boronic acids.

    PubMed

    Kettner, C A; Bone, R; Agard, D A; Bachovchin, W W

    1988-10-01

    The kinetic parameters for peptide boronic acids in their interaction with alpha-lytic protease were determined and found to be similar to those of other serine proteases [Kettner, C., & Shenvi, A. B. (1984) J. Biol. Chem. 259, 15106-15114]. alpha-Lytic protease hydrolyzes substrates with either alanine or valine in the P1 site and has a preference for substrate with a P1 alanine. The most effective inhibitors are tri- and tetrapeptide analogues that have a -boroVal-OH residue in the P1 site. At pH 7.5, MeOSuc-Ala-Ala-Pro-boroVal-OH has a Ki of 6.4 nM and Boc-Ala-Pro-boroVal-OH has a Ki of 0.35 nM. Ac-boroVal-OH and Ac-Pro-boroVal-OH are 220,000- and 500-fold less effective, respectively, than the tetrapeptide analogue. The kinetic properties of the tri- and tetrapeptide analogues are consistent with the mechanism for slow-binding inhibition, E + I in equilibrium EI in equilibrium EI*, while the less effective inhibitors are simple competitive inhibitors. MeO-Suc-Ala-Ala-Pro-boroAla-OH is a simple competitive inhibitor with a Ki of 67 nM at pH 7.5. Other peptide boronic acids, which are analogues of nonsubstrates, are less effective than substrate analogues but still are effective competitive inhibitors. For example, MeOSuc-Ala-Ala-Pro-boroPhe-OH has a Ki of 0.54 microM although substrates with a phenylalanine in the P1 position are not hydrolyzed. Binding for boronic acid analogues of both substrate and nonsubstrate analogues is pH dependent with higher affinity near pH 7.5. Similar binding properties have been observed for pancreatic elastase. Both enzymes have almost identical requirements for an extended peptide inhibitor sequence in order to exhibit highly effective binding and slow-binding characteristics.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3207699

  1. Effect of the amino acid composition of cyclic peptides on their self-assembly in lipid bilayers.

    PubMed

    Danial, Maarten; Perrier, Sébastien; Jolliffe, Katrina A

    2015-02-28

    The effect of amino acid composition on the formation of transmembrane channels in lipid bilayers upon self-assembly of alt-(L,D)-α-cyclic octapeptides has been investigated. Cyclic peptides comprising D-leucine, alternating with different combinations of L-azidolysine, L-lysine(Alloc), L-lysine and L-tryptophan were synthesized and the size of pores formed via self-assembly of these molecules in lipid bilayers was elucidated using large unilamellar vesicle fluorescence assays and dynamic light scattering. Pore formation was examined in large unilamellar vesicles made up of egg yolk phosphatidylcholine or Escherichia coli total lipid extract. From these analyses, we have established that cyclic peptides with charged side chains form large pores while those with neutral side chains form unimeric pores. Furthermore, the cyclic peptides that consist of non-symmetric amino acid configurations possess a higher membrane activity than the cyclic peptides with a symmetric amino acid configuration. In addition, we have found that peptide amphiphilicity plays a vital role in selective partitioning between bilayers that consist of egg yolk phosphatidylcholine and those comprised of E. coli total lipid extract. These results suggest that selective transbilayer channel formation via self-assembly may be a viable alternative for many applications that currently use more expensive, multistep synthesis methods. PMID:25566760

  2. Increased yield of high purity recombinant human brain natriuretic peptide by acid hydrolysis of short fusion partner in Escherichia coli.

    PubMed

    Kanumuri, Radha Madhavi; Bajji, Chitra; Tummuru, Rajesh R; Tatireddigari, Venkat R R Arva; Mangamoori, Lakshmi Narasu; Panati, Kalpana; Narala, Venkata Ramireddy

    2015-07-01

    Recombinant human B-type natriuretic peptide (rhBNP) is a 32-amino acid peptide used to treat congestive heart failure. In this paper, we report a method for the increased production of rhBNP in Escherichia coli with high purity. hBNP was cloned with a short growth hormone fusion partner coupled with a unique acid-labile dipeptide linker to cleave the fusion protein to release the rhBNP. The recombinant fusion protein was expressed as an inclusion body (IB) and the fermentation process was optimized to produce on large scale. The IBs were recovered by cell lysis, and the pure IBs were directly treated with diluted acid to get the target peptide from the fusion protein and the resultant peptide was purified by reversed phase chromatography. The final purity of the rhBNP was more than 99% with yield of 50mg per liter of culture, which is ten times higher than the previous reports. The purified rhBNP exhibited specific biological activity similar to the standard peptide in producing cyclic-guanosine monophosphate. PMID:25823948

  3. Anti-biofilm activity of ultrashort cinnamic acid peptide derivatives against medical device-related pathogens.

    PubMed

    Laverty, Garry; McCloskey, Alice P; Gorman, Sean P; Gilmore, Brendan F

    2015-10-01

    The threat of antimicrobial resistance has placed increasing emphasis on the development of innovative approaches to eradicate multidrug-resistant pathogens. Biofilm-forming microorganisms, for example, Staphylococcus epidermidis and Staphylococcus aureus, are responsible for increased incidence of biomaterial infection, extended hospital stays and patient morbidity and mortality. This paper highlights the potential of ultrashort tetra-peptide conjugated to hydrophobic cinnamic acid derivatives. These peptidomimetic molecules demonstrate selective and highly potent activity against resistant biofilm forms of Gram-positive medical device-related pathogens. 3-(4-Hydroxyphenyl)propionic)-Orn-Orn-Trp-Trp-NH2 displays particular promise with minimum biofilm eradication concentration (MBEC) values of 125 µg/ml against methicillin sensitive (ATCC 29213) and resistant (ATCC 43300) S. aureus and activity shown against biofilm forms of Escherichia coli (MBEC: 1000 µg/ml). Kill kinetics confirms complete eradication of established 24-h biofilms at MBEC with 6-h exposure. Reduced cell cytotoxicity, relative to Gram-positive pathogens, was proven via tissue culture (HaCaT) and haemolysis assays (equine erythrocytes). Existing in nature as part of the immune response, antimicrobial peptides display great promise for exploitation by the pharmaceutical industry in order to increase the library of available therapeutic molecules. Ultrashort variants are particularly promising for translation as clinical therapeutics as they are more cost-effective, easier to synthesise and can be tailored to specific functional requirements based on the primary sequence allowing factors such as spectrum of activity to be varied. PMID:26310860

  4. Fasciola gigantica cathepsin B5 is an acidic endo- and exopeptidase of the immature and mature parasite.

    PubMed

    Siricoon, Sinee; Vichasri Grams, Suksiri; Lertwongvisarn, Kittisak; Abdullohfakeeyah, Muntana; Smooker, Peter M; Grams, Rudi

    2015-12-01

    Cysteine proteases of the liver fluke Fasciola have been described as essential molecules in the infection process of the mammalian host. Destinct cathepsin Bs, which are already expressed in the metacercarial stage and released by the newly excysted juvenile are major actors in this process. Following infection their expression is stopped and the proteins will not be detectable any longer after the first month of development. On the contrary, the novel cathepsin B5 of Fasciola gigantica (FgCB5) described in this work was also found expressed in later juvenile stages and the mature worm. Like all previously described Fasciola family members it was located in the cecal epithelium of the parasite. Western blot analysis of adult antigen preparations detected procathepsin B5 in crude worm extract and in small amounts in the ES product. In support of these data, the sera of infected rabbits and mice were reactive with recombinant FgCB5 in Western blot and ELISA. Biochemical analysis of yeast-expressed FgCB5 revealed that it has properties of a lysosomal hydrolase optimized for activity at acid pH and that it is able to efficiently digest a broad spectrum of host proteins. Unlike previously characterized Fasciola family members FgCB5 carries a histidine doublet in the occluding loop equivalent to residues His110 and His111 of human mature cathepsin B and consequently showed substantial carboxydipeptidyl activity which depends on these two residues. PMID:26453811

  5. Microbial degradation rates of small peptides and amino acids in the oxygen minimum zone of Chilean coastal waters

    NASA Astrophysics Data System (ADS)

    Pantoja, Silvio; Rossel, Pamela; Castro, Rodrigo; Cuevas, L. Antonio; Daneri, Giovanni; Córdova, Candy

    2009-07-01

    We found similar microbial degradation rates of labile dissolved organic matter in oxic and suboxic waters off northern Chile. Rates of peptide hydrolysis and amino acid uptake in unconcentrated water samples were not low in the water column where oxygen concentration was depleted. Hydrolysis rates ranged from 65 to 160 nmol peptide L -1 h -1 in the top 20 m, 8-28 nmol peptide L -1 h -1 between 100 and 300 m (O 2-depleted zone), and 14-19 nmol peptide L -1 h -1 between 600 and 800 m. Dissolved free amino acid uptake rates were 9-26, 3-17, and 6 nmol L -1 h -1 at similar depth intervals. Since these findings are consistent with a model of comparable potential activity of microbes in degrading labile substrates of planktonic origin, we suggest, as do other authors, that differences in decomposition rates with high and low oxygen concentrations may be a matter of substrate lability. The comparison between hydrolysis and uptake rates indicates that microbial peptide hydrolysis occurs at similar or faster rates than amino acid uptake in the water column, and that the hydrolysis of peptides is not a rate-limiting step for the complete remineralization of labile macromolecules. Low O 2 waters process about 10 tons of peptide carbon per h, double the amount processed in surface-oxygenated water. In the oxygen minimum zone, we suggest that the C balance may be affected by the low lability of the dissolved organic matter when this is upwelled to the surface. An important fraction of dissolved organic matter is processed in the oxygen minimum layer, a prominent feature of the coastal ocean in the highly productive Humboldt Current System.

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

    PubMed

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

    2005-10-15

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

  7. Selective labeling of a membrane peptide with 15N-amino acids using cells grown in rich medium.

    PubMed

    Englander, Jacqueline; Cohen, Leah; Arshava, Boris; Estephan, Racha; Becker, Jeffrey M; Naider, Fred

    2006-01-01

    Nuclear magnetic resonance spectra of membrane proteins containing multiple transmembrane helices have proven difficult to resolve due to the redundancy of aliphatic and Ser/Thr residues in transmembrane domains and the low chemical shift dispersity exhibited by residues in alpha-helical structures. Although (13)C- and (15)N-labeling are useful tools in the biophysical analysis of proteins, selective labeling of individual amino acids has been used to help elucidate more complete structures and to probe ligand-protein interactions. In general, selective labeling has been performed in Escherichia coli expression systems using minimal media supplemented with a single labeled amino acid and nineteen other unlabeled amino acids and/or by using auxotrophs for specific amino acids. Growth in minimal media often results in low yields of cells or expression products. We demonstrate a method in which one labeled amino acid is added to a rich medium. These conditions resulted in high expression (> or =100 mg/L) of a test fusion protein and milligram quantities of the selectively labeled membrane peptide after cyanogen bromide cleavage to release the peptide from the fusion protein. High levels of (15)N incorporation and acceptable levels of cross-labeling into other amino acid residues of the peptide were achieved. Growth in rich media is a simple and convenient alternative to growth in supplemented minimal media and is readily applicable to the expression of proteins selectively labeled with specific amino acids. PMID:16741986

  8. The world of beta- and gamma-peptides comprised of homologated proteinogenic amino acids and other components.

    PubMed

    Seebach, Dieter; Beck, Albert K; Bierbaum, Daniel J

    2004-08-01

    The origins of our nearly ten-year research program of chemical and biological investigations into peptides based on homologated proteinogenic amino acids are described. The road from the biopolymer poly[ethyl (R)-3-hydroxybutanoate] to the beta-peptides was primarily a step from organic synthesis methodology (the preparation of enantiomerically pure compounds (EPCs)) to supramolecular chemistry (higher-order structures maintained through non-covalent interactions). The performing of biochemical and biological tests on the beta- and gamma-peptides, which differ from natural peptides/proteins by a single or two additional CH(2) groups per amino acid, then led into bioorganic chemistry and medicinal chemistry. The individual chapters of this review article begin with descriptions of work on beta-amino acids, beta-peptides, and polymers (Nylon-3) that dates back to the 1960s, even to the times of Emil Fischer, but did not yield insights into structures or biological properties. The numerous, often highly physiologically active, or even toxic, natural products containing beta- and gamma-amino acid moieties are then presented. Chapters on the preparation of homologated amino acids with proteinogenic side chains, their coupling to provide the corresponding peptides, both in solution (including thioligation) and on the solid phase, their isolation by preparative HPLC, and their characterization by mass spectrometry (HR-MS and MS sequencing) follow. After that, their structures, predominantly determined by NMR spectroscopy in methanolic solution, are described: helices, pleated sheets, and turns, together with stack-, crankshaft-, paddlewheel-, and staircase-like patterns. The presence of the additional C--C bonds in the backbones of the new peptides did not give rise to a chaotic increase in their secondary structures as many protein specialists might have expected: while there are indeed more structure types than are observed in the alpha-peptide realm - three different

  9. Site-Selective Binding of Nanoparticles to Double-Stranded DNA via Peptide Nucleic Acid "Invasion"

    SciTech Connect

    Stadler, A.L.; van der Lelie, D.; Sun, D.; Maye, M. M.; Gang, O.

    2011-04-01

    We demonstrate a novel method for by-design placement of nano-objects along double-stranded (ds) DNA. A molecular intercalator, designed as a peptide nucleic acid (PNA)-DNA chimera, is able to invade dsDNA at the PNA-side due to the hybridization specificity between PNA and one of the duplex strands. At the same time, the single-stranded (ss) DNA tail of the chimera, allows for anchoring of nano-objects that have been functionalized with complementary ssDNA. The developed method is applied for interparticle attachment and for the fabrication of particle clusters using a dsDNA template. This method significantly broadens the molecular toolbox for constructing nanoscale systems by including the most conventional not yet utilized DNA motif, double helix DNA.

  10. Plasmid DNA delivery by arginine-rich cell-penetrating peptides containing unnatural amino acids.

    PubMed

    Kato, Takuma; Yamashita, Hiroko; Misawa, Takashi; Nishida, Koyo; Kurihara, Masaaki; Tanaka, Masakazu; Demizu, Yosuke; Oba, Makoto

    2016-06-15

    Cell-penetrating peptides (CPPs) have been developed as drug, protein, and gene delivery tools. In the present study, arginine (Arg)-rich CPPs containing unnatural amino acids were designed to deliver plasmid DNA (pDNA). The transfection ability of one of the Arg-rich CPPs examined here was more effective than that of the Arg nonapeptide, which is the most frequently used CPP. The transfection efficiencies of Arg-rich CPPs increased with longer post-incubation times and were significantly higher at 48-h and 72-h post-incubation than that of the commercially available transfection reagent TurboFect. These Arg-rich CPPs were complexed with pDNA for a long time in cells and effectively escaped from the late endosomes/lysosomes into the cytoplasm. These results will be helpful for designing novel CPPs for pDNA delivery. PMID:27132868

  11. Roles of d-Amino Acids on the Bioactivity of Host Defense Peptides.

    PubMed

    Li, Hao; Anuwongcharoen, Nuttapat; Malik, Aijaz Ahmad; Prachayasittikul, Virapong; Wikberg, Jarl E S; Nantasenamat, Chanin

    2016-01-01

    Host defense peptides (HDPs) are positively-charged and amphipathic components of the innate immune system that have demonstrated great potential to become the next generation of broad spectrum therapeutic agents effective against a vast array of pathogens and tumor. As such, many approaches have been taken to improve the therapeutic efficacy of HDPs. Amongst these methods, the incorporation of d-amino acids (d-AA) is an approach that has demonstrated consistent success in improving HDPs. Although, virtually all HDP review articles briefly mentioned about the role of d-AA, however it is rather surprising that no systematic review specifically dedicated to this topic exists. Given the impact that d-AA incorporation has on HDPs, this review aims to fill that void with a systematic discussion of the impact of d-AA on HDPs. PMID:27376281

  12. Roles of d-Amino Acids on the Bioactivity of Host Defense Peptides

    PubMed Central

    Li, Hao; Anuwongcharoen, Nuttapat; Malik, Aijaz Ahmad; Prachayasittikul, Virapong; Wikberg, Jarl E. S.; Nantasenamat, Chanin

    2016-01-01

    Host defense peptides (HDPs) are positively-charged and amphipathic components of the innate immune system that have demonstrated great potential to become the next generation of broad spectrum therapeutic agents effective against a vast array of pathogens and tumor. As such, many approaches have been taken to improve the therapeutic efficacy of HDPs. Amongst these methods, the incorporation of d-amino acids (d-AA) is an approach that has demonstrated consistent success in improving HDPs. Although, virtually all HDP review articles briefly mentioned about the role of d-AA, however it is rather surprising that no systematic review specifically dedicated to this topic exists. Given the impact that d-AA incorporation has on HDPs, this review aims to fill that void with a systematic discussion of the impact of d-AA on HDPs. PMID:27376281

  13. Enhanced lubrication on tissue and biomaterial surfaces through peptide-mediated binding of hyaluronic acid

    NASA Astrophysics Data System (ADS)

    Singh, Anirudha; Corvelli, Michael; Unterman, Shimon A.; Wepasnick, Kevin A.; McDonnell, Peter; Elisseeff, Jennifer H.

    2014-10-01

    Lubrication is key for the efficient function of devices and tissues with moving surfaces, such as articulating joints, ocular surfaces and the lungs. Indeed, lubrication dysfunction leads to increased friction and degeneration of these systems. Here, we present a polymer-peptide surface coating platform to non-covalently bind hyaluronic acid (HA), a natural lubricant in the body. Tissue surfaces treated with the HA-binding system exhibited higher lubricity values, and in vivo were able to retain HA in the articular joint and to bind ocular tissue surfaces. Biomaterials-mediated strategies that locally bind and concentrate HA could provide physical and biological benefits when used to treat tissue-lubricating dysfunction and to coat medical devices.

  14. Fluorescent 2-Aminopyridine Nucleobases for Triplex-Forming Peptide Nucleic Acids.

    PubMed

    Cheruiyot, Samwel K; Rozners, Eriks

    2016-08-17

    Development of new fluorescent peptide nucleic acids (PNAs) is important for fundamental research and practical applications. The goal of this study was the design of fluorogenic nucleobases for incorporation in triplex-forming PNAs. The underlying design principle was the use of a protonation event that accompanied binding of a 2-aminopyridine (M) nucleobase to a G-C base pair as an on switch for a fluorescence signal. Two fluorogenic nucleobases, 3-(1-phenylethynyl)-M and phenylpyrrolo-M, were designed, synthesized and studied. The new M derivatives provided modest enhancement of fluorescence upon protonation but showed reduced RNA binding affinity and quenching of fluorescence signal upon triple-helix formation with cognate double-stranded RNA. Our study illustrates the principal challenges of design and provides guidelines for future improvement of fluorogenic PNA nucleobases. The 3-(1-phenylethynyl)-M may be used as a fluorescent nucleobase to study PNA-RNA triple-helix formation. PMID:27223320

  15. Metallamacrocycle formation through dimerization of metal bioconjugates derived from amino acids and peptides.

    PubMed

    Álvarez, Celedonio M; García-Rodríguez, Raúl; Miguel, Daniel

    2016-01-21

    Metallamacrocycles of 12, 16, and 22 members are obtained by deprotonation of the carboxylic group of the side chain of iminopyridine complexes derived from the amino acid β-alanine, and the peptides Gly-Gly and Gly-Gly-Gly. Instead of the expected intramolecular attack to give tridentate (N,N,O) ligands, the deprotonated carboxylate attacks in an inter-molecular manner to give dimers in which the ligand acts as a bridge bonded in a κ(2)(N,N') chelating fashion to one metal and as κ(O) to the other metal. The formation of the dimers is supported by NMR spectroscopy, mass spectrometry and X-ray crystallography. PMID:26645303

  16. Adsorption of peptide nucleic acid and DNA decamers at electrically charged surfaces.

    PubMed Central

    Fojta, M; Vetterl, V; Tomschik, M; Jelen, F; Nielsen, P; Wang, J; Palecek, E

    1997-01-01

    Adsorption behavior of peptide nucleic acid (PNA) and DNA decamers (GTAGATCACT and the complementary sequence) on a mercury surface was studied by means of AC impedance measurements at a hanging mercury drop electrode. The nucleic acid was first attached to the electrode by adsorption from a 5-microliter drop of PNA (or DNA) solution, and the electrode with the adsorbed nucleic acid layer was then washed and immersed in the blank background electrolyte where the differential capacity C of the electrode double layer was measured as a function of the applied potential E. It was found that the adsorption behavior of the PNA with an electrically neutral backbone differs greatly from that of the DNA (with a negatively charged backbone), whereas the DNA-PNA hybrid shows intermediate behavior. At higher surface coverage PNA molecules associate at the surface, and the minimum value of C is shifted to negative potentials because of intermolecular interactions of PNA at the surface. Prolonged exposure of PNA to highly negative potentials does not result in PNA desorption, whereas almost all of the DNA is removed from the surface at these potentials. Adsorption of PNA decreases with increasing NaCl concentration in the range from 0 to 50 mM NaCl, in contrast to DNA, the adsorption of which increases under the same conditions. PMID:9129832

  17. Branched-chain amino acid transport regulation in mutants blocked in tRNA maturation and transcriptional termination.

    PubMed Central

    Quay, S C; Lawther, R P; Hatfield, G W; Oxender, D L

    1978-01-01

    The regulation of branched-chain amino acid transport and binding protein biosynthesis was studied in Escherichia coli strains containing hisT (the structural gene for pseudouridine synthetase) and rho (the structural gene for the mRNA transcriptional termination factor rho) mutations. The results indicate that the hisT strain cannot be fully derepressed for transport and that the hisT rho double mutant is partially derepressed under excess leucine conditions, but cannot be further derepressed by leucine deprivation. These data are consistent with a model in which fully mature tRNALeu is required for derepression and in which rho interacts with tRNALeu in regulating transport by terminating transcription, especially in excess-leucine growth conditions. PMID:350834

  18. Determination of the gas-phase acidities of cysteine-polyalanine peptides using the extended kinetic method.

    PubMed

    Tan, John P; Ren, Jianhua

    2007-02-01

    We determined the gas-phase acidities of two cysteine-polyalanine peptides, HSCA3 and HSCA4, using a triple-quadrupole mass spectrometer through application of the extended kinetic method with full entropy analysis. Five halogenated carboxylic acids were used as the reference acids. The negatively charged proton-bound dimers of the deprotonated peptides with the conjugate bases of the reference acids were generated by electrospray ionization. Collision-induced dissociation (CID) experiments were carried out at three collision energies. The enthalpies of deprotonation (Delta(acid)H) of the peptides were derived according to the linear relationship between the logarithms of the CID product ion branching ratios and the differences of the gas-phase acidities. The values were determined to be Delta(acid)H(HSCA3) = 317.3 +/- 2.4 kcal/mol and Delta(acid)H (HSCA4) = 316.2 +/- 3.9 kcal/mol. Large entropy effects (Delta(DeltaS) = 13-16 cal/mol K) were observed for these systems. Combining the enthalpies of deprotonation with the entropy term yielded the apparent gas-phase acidities (Delta(acid)G(app)) of 322.1 +/- 2.4 kcal/mol (HSCA3) and 320.1 +/- 3.9 kcal/mol (HSCA4), in agreement with the results obtained from the CID-bracketing experiments. Compared with that in the isolated cysteine residue, the thiol group in HSCA3,4 has a stronger gas-phase acidity by about 20 kcal/mol. This increased acidity is likely due to the stabilization of the negatively charged thiolate group through internal solvation. PMID:17067812

  19. Specificity and formation of unusual amino acids of an amide ligation strategy for unprotected peptides.

    PubMed

    Tam, J P; Rao, C; Liu, C F; Shao, J

    1995-03-01

    An important step in the recently developed ligation strategy known as domain ligation strategy to link unprotected peptide segments without activation is the ring formation between the C-terminal ester aldehyde and the N-terminal amino acid bearing a beta-thiol or beta-hydroxide. A new method was developed to define the specificity of this reaction using a dye-labeled alanyl ester aldehyde to react with libraries of 400 dipeptides which contained all dipeptide combinations of the 20 genetically coded amino acids. Three different ester aldehydes of the dye-labeled alanine: alpha-formylmethyl (FM), beta-formylethyl (FE), and beta,beta,beta-dimethyl and formylethyl esters (DFE), were examined. The DFE ester was overly hindered and reacted with N-terminal Cys dipeptides (Cys-X). Interestingly, it also reacted slowly with the sequences of X-Gly where Gly was the second amino acid and the X-Gly amide bond participated in the ring formation. Although the FE ester reacted similarly as the FM ester in the ring formation, the subsequent O,N-acyl transfer was at least 30-fold slower than those of the FM-ester. The FM alpha-formyl methyl ester was the most suitable ester and was reactive with dipeptides of six N-terminal amino acids: Cys, Thr, Trp, Ser, His and Asn. The order and extent of their reactivity were highly dependent on pH, solvent and neighboring participation by the adjacent amino acid. In general, they could be divided into three categories. (1) N-Terminal Cys and Thr were the most reactive.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7775013

  20. Electrostatic binding and hydrophobic collapse of peptide-nucleic acid aggregates quantified using force spectroscopy.

    PubMed

    Camunas-Soler, Joan; Frutos, Silvia; Bizarro, Cristiano V; de Lorenzo, Sara; Fuentes-Perez, Maria Eugenia; Ramsch, Roland; Vilchez, Susana; Solans, Conxita; Moreno-Herrero, Fernando; Albericio, Fernando; Eritja, Ramón; Giralt, Ernest; Dev, Sukhendu B; Ritort, Felix

    2013-06-25

    Knowledge of the mechanisms of interaction between self-aggregating peptides and nucleic acids or other polyanions is key to the understanding of many aggregation processes underlying several human diseases (e.g., Alzheimer's and Parkinson's diseases). Determining the affinity and kinetic steps of such interactions is challenging due to the competition between hydrophobic self-aggregating forces and electrostatic binding forces. Kahalalide F (KF) is an anticancer hydrophobic peptide that contains a single positive charge that confers strong aggregative properties with polyanions. This makes KF an ideal model to elucidate the mechanisms by which self-aggregation competes with binding to a strongly charged polyelectrolyte such as DNA. We use optical tweezers to apply mechanical forces to single DNA molecules and show that KF and DNA interact in a two-step kinetic process promoted by the electrostatic binding of DNA to the aggregate surface followed by the stabilization of the complex due to hydrophobic interactions. From the measured pulling curves we determine the spectrum of binding affinities, kinetic barriers, and lengths of DNA segments sequestered within the KF-DNA complex. We find there is a capture distance beyond which the complex collapses into compact aggregates stabilized by strong hydrophobic forces and discuss how the bending rigidity of the nucleic acid affects this process. We hypothesize that within an in vivo context, the enhanced electrostatic interaction of KF due to its aggregation might mediate the binding to other polyanions. The proposed methodology should be useful to quantitatively characterize other compounds or proteins in which the formation of aggregates is relevant. PMID:23706043

  1. Global analysis of myocardial peptides containing cysteines with irreversible sulfinic and sulfonic acid post-translational modifications.

    PubMed

    Paulech, Jana; Liddy, Kiersten A; Engholm-Keller, Kasper; White, Melanie Y; Cordwell, Stuart J

    2015-03-01

    Cysteine (Cys) oxidation is a crucial post-translational modification (PTM) associated with redox signaling and oxidative stress. As Cys is highly reactive to oxidants it forms a range of post-translational modifications, some that are biologically reversible (e.g. disulfides, Cys sulfenic acid) and others (Cys sulfinic [Cys-SO2H] and sulfonic [Cys-SO3H] acids) that are considered "irreversible." We developed an enrichment method to isolate Cys-SO2H/SO3H-containing peptides from complex tissue lysates that is compatible with tandem mass spectrometry (MS/MS). The acidity of these post-translational modification (pKa Cys-SO3H < 0) creates a unique charge distribution when localized on tryptic peptides at acidic pH that can be utilized for their purification. The method is based on electrostatic repulsion of Cys-SO2H/SO3H-containing peptides from cationic resins (i.e. "negative" selection) followed by "positive" selection using hydrophilic interaction liquid chromatography. Modification of strong cation exchange protocols decreased the complexity of initial flowthrough fractions by allowing for hydrophobic retention of neutral peptides. Coupling of strong cation exchange and hydrophilic interaction liquid chromatography allowed for increased enrichment of Cys-SO2H/SO3H (up to 80%) from other modified peptides. We identified 181 Cys-SO2H/SO3H sites from rat myocardial tissue subjected to physiologically relevant concentrations of H2O2 (<100 μm) or to ischemia/reperfusion (I/R) injury via Langendorff perfusion. I/R significantly increased Cys-SO2H/SO3H-modified peptides from proteins involved in energy utilization and contractility, as well as those involved in oxidative damage and repair. PMID:25561502

  2. A bottom-up approach to build the hyperpolarizability of peptides and proteins from their amino acids.

    PubMed

    Duboisset, Julien; Deniset-Besseau, Ariane; Benichou, Emmanuel; Russier-Antoine, Isabelle; Lascoux, Noelle; Jonin, Christian; Hache, François; Schanne-Klein, Marie-Claire; Brevet, Pierre-François

    2013-08-29

    We experimentally demonstrate that some peptides and proteins lend themselves to an elementary analysis where their first hyperpolarizability can be decomposed into the coherent superposition of the first hyperpolarizability of their elementary units. We then show that those elementary units can be associated with the amino acids themselves in the case of nonaromatic amino acids and nonresonant second harmonic generation. As a case study, this work investigates the experimentally determined first hyperpolarizability of rat tail Type I collagen and compares it to that of the shorter peptide [(PPG)10]3, where P and G are the one-letter code for Proline and Glycine, respectively, and that of the triamino acid peptides PPG and GGG. An absolute value of (0.16 ± 0.01) × 10(-30) esu for the first hyperpolarizability of nonaromatic amino acids is then obtained by using the newly defined 0.087 × 10(-30) esu reference value for water. By using a collagen like model, the microscopic hyperpolarizability along the peptide bond can be evaluated at (0.7 ± 0.1) × 10(-30) esu. PMID:23879840

  3. Formulation of pH responsive peptides as inhalable dry powders for pulmonary delivery of nucleic acids

    PubMed Central

    Liang, Wanling; Kwok, Philip C.L.; Chow, Michael Y.T.; Tang, Patricia; Mason, A. James; Chan, Hak-Kim; Lam, Jenny. K.W.

    2013-01-01

    Nucleic acids have the potential to be used as therapies or vaccines for many different types of disease but delivery remains the most significant challenge to their clinical adoption. pH responsive peptides containing either histidine or derivatives of 2,3-diaminopropionic acid (Dap) can mediate effective DNA transfection in lung epithelial cells with the latter remaining effective even in the presence of lung surfactant containing bronchoalveolar fluid (BALF), making this class of peptides attractive candidates for delivering nucleic acids to lung tissues. To further assess the suitability of pH responsive peptides for pulmonary delivery by inhalation, dry powder formulations of pH responsive peptides and plasmid DNA, with mannitol as carrier, were produced by either spray drying (SD) or spray freeze drying (SFD). The properties of the two types of powders were characterised and compared using scanning electron microscopy (SEM), next generation impaction (NGI), gel retardation and in vitro transfection via a twin-stage impinger (TSI) following aerosolisation by a dry powder inhaler (Osmohaler™). Although the aerodynamic performance and transfection efficacy of both powders were good, the overall performance revealed SD powders to have a number of advantages over SFD powders and are the more effective formulation with potential for efficient nucleic acid delivery through inhalation. PMID:23702276

  4. Impact of the N-terminal amino acid on the formation of pyrazines from peptides in Maillard model systems.

    PubMed

    Van Lancker, Fien; Adams, An; De Kimpe, Norbert

    2012-05-01

    Only a minor part of Maillard reaction studies in the literature focused on the reaction between carbohydrates and peptides. Therefore, in continuation of a previous study in which the influence of the peptide C-terminal amino acid was investigated, this study focused on the influence of the peptide N-terminal amino acid on the production of pyrazines in model reactions of glucose, methylglyoxal, or glyoxal. Nine different dipeptides and three tripeptides were selected. It was shown that the structure of the N-terminal amino acid is determinative for the overall pyrazine production. Especially, the production of 2,5(6)-dimethylpyrazine and trimethylpyrazine was low in the case of proline, valine, or leucine at the N-terminus, whereas it was very high for glycine, alanine, or serine. In contrast to the alkyl-substituted pyrazines, unsubstituted pyrazine was always produced more in the case of experiments with free amino acids. It is clear that different mechanisms must be responsible for this observation. This study clearly illustrates the capability of peptides to produce flavor compounds such as pyrazines. PMID:22463717

  5. Bile acids induce glucagon-like peptide 2 secretion with limited effects on intestinal adaptation in early weaned pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Early weaning is a stressful event characterized by a transient period of intestinal atrophy that may be mediated by reduced secretion of glucagon-like peptide (GLP) 2. We tested whether enterally fed bile acids or plant sterols could increase nutrient-dependent GLP-2 secretion and improve intestina...

  6. In Vitro and In Vivo Activities of Antimicrobial Peptides Developed Using an Amino Acid-Based Activity Prediction Method

    PubMed Central

    Wu, Xiaozhe; Wang, Zhenling; Li, Xiaolu; Fan, Yingzi; He, Gu; Wan, Yang; Yu, Chaoheng; Tang, Jianying; Li, Meng; Zhang, Xian; Zhang, Hailong; Xiang, Rong; Pan, Ying; Liu, Yan; Lu, Lian

    2014-01-01

    To design and discover new antimicrobial peptides (AMPs) with high levels of antimicrobial activity, a number of machine-learning methods and prediction methods have been developed. Here, we present a new prediction method that can identify novel AMPs that are highly similar in sequence to known peptides but offer improved antimicrobial activity along with lower host cytotoxicity. Using previously generated AMP amino acid substitution data, we developed an amino acid activity contribution matrix that contained an activity contribution value for each amino acid in each position of the model peptide. A series of AMPs were designed with this method. After evaluating the antimicrobial activities of these novel AMPs against both Gram-positive and Gram-negative bacterial strains, DP7 was chosen for further analysis. Compared to the parent peptide HH2, this novel AMP showed broad-spectrum, improved antimicrobial activity, and in a cytotoxicity assay it showed lower toxicity against human cells. The in vivo antimicrobial activity of DP7 was tested in a Staphylococcus aureus infection murine model. When inoculated and treated via intraperitoneal injection, DP7 reduced the bacterial load in the peritoneal lavage solution. Electron microscope imaging and the results indicated disruption of the S. aureus outer membrane by DP7. Our new prediction method can therefore be employed to identify AMPs possessing minor amino acid differences with improved antimicrobial activities, potentially increasing the therapeutic agents available to combat multidrug-resistant infections. PMID:24982064

  7. 9-cis Retinoic acid inhibits cumulus cell apoptosis during the maturation of bovine cumulus-oocyte-complexes.

    PubMed

    Deb, G K; Dey, S R; Bang, J I; Lee, J G; Kong, I K

    2012-06-01

    Cumulus cell (CC) apoptosis is inversely correlated with embryonic development in vitro. Therefore, inhibition of CC apoptosis is important for proper embryonic development and quality. Retinoic acids (all-transRA and 9-cisRA) are natural components of retinoids, and 9-cisRA is the physiologically active metabolite of retinoic acid in vitro. During in vitro maturation, 9-cisRA enhances oocyte competence through multiple mechanisms affecting the oocyte and preimplantation embryo; however, the effect of 9-cisRA on CC apoptosis has yet to be elucidated. The aim of the present study was to evaluate the effect of 9-cisRA on CC apoptosis and to identify the molecular mechanism underlying that effect. Bovine slaughterhouse cumulus-oocyte complexes (COC) were matured in vitro in the absence or presence of 5 nM 9-cisRA. Cumulus cells were collected from immature and matured COC for the detection of apoptosis and gene expression analysis. Results showed that 9-cisRA reduced the number of apoptotic CC by about 2.7 fold (P < 0.023), compared with control. However, apoptosis is rare in CC of immature COC (0.01% ± 0.001). Transcripts involved in the caspase cascade were down-regulated upon exposure to 9-cisRA, including tumor necrosis factor alpha (TNF-α, 11.1 fold, P < 0.001), tumor necrosis factor alpha receptor 1 (TNFR1, 2.3 fold, P < 0.01), caspase 9 (CASP9, 2.0 fold, P < 0.031), caspase 8 (CASP8, 2.2 fold, P < 0.012), and caspase 3 (CASP3, 2.1 fold, P < 0.006), while antiapoptotic B-cell lymphoma 2 (BCL2) transcript was increased (3.1 fold, P < 0.004), compared with control. In addition, 9-cisRA inhibited mitogen activated protein kinase mRNA expression in CC, including extracellular signal-regulated kinase 1/2 (ERK1, 2.7 fold, P < 0.02; ERK2, 2.7 fold, P < 0.03), and c-Jun N-terminal kinase (JNK, 1.6 fold, P < 0.044), as well as the activator protein-1 (AP1) family members c-jun (1.6 fold, P < 0.041) and c-fos (2.0 fold, P < 0.06). The transcript abundances of TNF

  8. Effects of sexual maturation and feeding level on fatty acid metabolism gene expression in muscle, liver, and visceral adipose tissue of diploid and triploid rainbow trout, Oncorhynchus mykiss.

    PubMed

    Manor, Meghan L; Cleveland, Beth M; Weber, Gregory M; Kenney, P Brett

    2015-01-01

    In many cultured fish species, such as salmonids, gonadal development occurs at the expense of stored energy and nutrients, including lipids. However, mechanisms regulating nutrient repartitioning during sexual maturation are not well understood. This study compared sexually maturing diploid (2N) and sterile triploid (3N) female rainbow trout to investigate effects of sexual maturation on expression of 35 genes involved in fatty acid metabolism, including genes within fatty acid synthesis, β-oxidation, and cofactors of the mTOR and PPAR signaling pathways, in liver, white muscle, and visceral adipose tissue. Diploid fish were fed at different rations (0.25% and 0.50% tank biomass, and satiation) to determine effects of ration on gene expression. Gene expression was affected by ration level only in white muscle; erk and acat2 had higher expression in fish fed higher rations. On the other hand, sexual maturation affected gene expression across all three tissue types. Data indicate 2N fish have higher expression of β-oxidation genes within white muscle and within visceral adipose tissue. These findings support enhanced fatty acid mobilization within these tissues during sexual maturation. Higher expression of fatty acid synthesis genes in 3N female liver is associated with higher expression of mTOR cofactors and pparγ, which reflects continued deposition of lipids in these fish. Furthermore, greater expression of genes involved in β-oxidation pathways across ration levels in 2N females suggests that sexual maturation and the associated maturation-related signals are stronger regulators of lipid metabolism-related genes rather the rations applied in the current study. PMID:25242626

  9. Enzymatic recycling of ascorbic acid from dehydroascorbic acid by glutathione-like peptides in the extracellular loops of aminergic G-protein coupled receptors.

    PubMed

    Root-Bernstein, Robert; Fewins, Jenna; Rhinesmith, Tyler; Koch, Ariana; Dillon, Patrick F

    2016-07-01

    The intracellular recycling of ascorbic acid from dehydroascorbic acid by the glutathione-glutathione reductase system has been well-characterized. We propose that extracellular recycling of ascorbic acid is performed in a similar manner by cysteine-rich, glutathione-like regions of the first and second extracellular loops of some aminergic receptors including adrenergic, histaminergic, and dopaminergic receptors. Previous research in our laboratory demonstrated that ascorbic acid binds to these receptors at a site on their first or second extracellular loops, significantly enhancing ligand activity, and apparently recycling hundreds of times their own concentration of ascorbate in an enzymatic fashion. In this study, we have synthesized 25 peptides from the first and second extracellular loops of aminergic and insulin receptors and compared them directly to glutathione for their ability to prevent the oxidation of ascorbate and to regenerate ascorbate from dehydroascorbic acid. Peptide sequences that mimic glutathione in containing a cysteine and a glutamic acid-like amino acid also mimic glutathione activity in effects and in kinetics. Some (but not all) peptide sequences that contain one or more methionines instead of cysteine can significantly retard the oxidation of ascorbic acid but do not recycle it from dehydroascorbate into ascorbate. Peptides lacking both cysteines and methionines uniformly failed to alter significantly ascorbate or dehydroascorbate oxidation or reduction. We believe that this is the first proof that receptors may carry out both ligand binding and enzymatic activity extracellularly. Our results suggest the existence of a previously unknown extracellular system for recycling ascorbate. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26749062

  10. L-amino acid ligase from Pseudomonas syringae producing tabtoxin can be used for enzymatic synthesis of various functional peptides.

    PubMed

    Arai, Toshinobu; Arimura, Yasuhiro; Ishikura, Shun; Kino, Kuniki

    2013-08-01

    Functional peptides are expected to be beneficial compounds that improve our quality of life. To address the growing need for functional peptides, we have examined peptide synthesis by using microbial enzymes. l-Amino acid ligase (Lal) catalyzes the condensation of unprotected amino acids in an ATP-dependent manner and is applicable to fermentative production. Hence, Lal is a promising enzyme to achieve cost-effective synthesis. To obtain a Lal with novel substrate specificity, we focused on the putative Lal involved in the biosynthesis of the dipeptidic phytotoxin designated tabtoxin. The tabS gene was cloned from Pseudomonas syringae NBRC14081 and overexpressed in Escherichia coli cells. The recombinant TabS protein produced showed the broadest substrate specificity of any known Lal; it detected 136 of 231 combinations of amino acid substrates when dipeptide synthesis was examined. In addition, some new substrate specificities were identified and unusual amino acids, e.g., l-pipecolic acid, hydroxy-l-proline, and β-alanine, were found to be acceptable substrates. Furthermore, kinetic analysis and monitoring of the reactions over a short time revealed that TabS showed distinct substrate selectivity at the N and C termini, which made it possible to specifically synthesize a peptide without by-products such as homopeptides and heteropeptides with the reverse sequence. TabS specifically synthesized the following functional peptides, including their precursors: l-arginyl-l-phenylalanine (antihypertensive effect; yield, 62%), l-leucyl-l-isoleucine (antidepressive effect; yield, 77%), l-glutaminyl-l-tryptophan (precursor of l-glutamyl-l-tryptophan, which has antiangiogenic activity; yield, 54%), l-leucyl-l-serine (enhances saltiness; yield, 83%), and l-glutaminyl-l-threonine (precursor of l-glutamyl-l-threonine, which enhances saltiness; yield, 96%). Furthermore, our results also provide new insights into tabtoxin biosynthesis. PMID:23770908

  11. Molecular mechanisms underlying bile acid-stimulated glucagon-like peptide-1 secretion

    PubMed Central

    Parker, HE; Wallis, K; le Roux, CW; Wong, KY; Reimann, F; Gribble, FM

    2012-01-01

    BACKGROUND AND PURPOSE The glucagon-like peptides GLP-1 and GLP-2 are secreted from enteroendocrine L-cells following nutrient ingestion. Drugs that increase activity of the GLP-1 axis are highly successful therapies for type 2 diabetes, and boosting L-cell secretion is a potential strategy for future diabetes treatment. The aim of the present study was to further our understanding of the bile acid receptor GPBA (TGR5), an L-cell target currently under therapeutic exploration. EXPERIMENTAL APPROACH GLUTag cells and mixed primary murine intestinal cultures were exposed to bile acids and a specific agonist, GPBAR-A. Secretion was measured using hormone assays and intracellular calcium and cAMP responses were monitored using real-time imaging techniques. KEY RESULTS Bile acid-triggered GLP-1 secretion from GLUTag cells was GPBA-dependent, as demonstrated by its abolition following tgr5 siRNA transfection. Bile acids and GPBAR-A increased GLP-1 secretion from intestinal cultures, with evidence for synergy between the effects of glucose and GPBA activation. Elevation of cAMP was observed following GPBA activation in individual GLUTag cells. Direct calcium responses to GPBAR-A were small, but in the presence of the agonist, a subpopulation of cells that was previously poorly glucose-responsive exhibited robust glucose responses. In vivo, increased delivery of bile to more distal regions of the ileum augmented L-cell stimulation. CONCLUSIONS AND IMPLICATIONS GPBA signalling in L-cells involves rapid elevation of cAMP, and enhanced calcium and secretory responses to glucose. Modulation of this receptor therapeutically may be an attractive strategy to enhance GLP-1 secretion and achieve better glycaemic control in diabetic patients. PMID:21718300

  12. Nisin-induced expression of a recombinant antihypertensive peptide in dairy lactic acid bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Peptides with antihypertensive activity have been identified from the enzymatic hydrolysis of bovine milk proteins. A 12-residue peptide (FFVAPFPEVFGK) shown to inhibit the angiotensin I-converting enzyme is released from the enzymatic breakdown of aS1-casein. A synthetic gene encoding this peptid...

  13. Serine protease EpiP from Staphylococcus epidermidis catalyzes the processing of the epidermin precursor peptide.

    PubMed Central

    Geissler, S; Götz, F; Kupke, T

    1996-01-01

    The function of serine protease EpiP in epidermin biosynthesis was investigated. Epidermin is synthesized as a 52-amino-acid precursor peptide, EpiA, which is posttranslationally modified and processed to the mature 22-amino-acid peptide antibiotic. epiP was expressed in Staphylococcus carnosus with xylose-regulated expression vector pCX15. The cleavage of the unmodified EpiA precursor peptide to leader peptide and proepidermin by EpiP-containing culture filtrates of S. carnosus (pCX15epiP) was followed by reversed-phase chromatography and subsequent electrospray mass spectrometry. PMID:8550430

  14. Single Molecule Spectroscopy of Amino Acids and Peptides by Recognition Tunneling

    PubMed Central

    Zhao, Yanan; Ashcroft, Brian; Zhang, Peiming; Liu, Hao; Sen, Suman; Song, Weisi; Im, JongOne; Gyarfas, Brett; Manna, Saikat; Biswas, Sovan; Borges, Chad; Lindsay, Stuart

    2014-01-01

    The human proteome has millions of protein variants due to alternative RNA splicing and post-translational modifications, and variants that are related to diseases are frequently present in minute concentrations. For DNA and RNA, low concentrations can be amplified using the polymerase chain reaction, but there is no such reaction for proteins. Therefore, the development of single molecule protein sequencing is a critical step in the search for protein biomarkers. Here we show that single amino acids can be identified by trapping the molecules between two electrodes that are coated with a layer of recognition molecules and measuring the electron tunneling current across the junction. A given molecule can bind in more than one way in the junction, and we therefore use a machine-learning algorithm to distinguish between the sets of electronic ‘fingerprints’ associated with each binding motif. With this recognition tunneling technique, we are able to identify D, L enantiomers, a methylated amino acid, isobaric isomers, and short peptides. The results suggest that direct electronic sequencing of single proteins could be possible by sequentially measuring the products of processive exopeptidase digestion, or by using a molecular motor to pull proteins through a tunnel junction integrated with a nanopore. PMID:24705512

  15. Single-molecule spectroscopy of amino acids and peptides by recognition tunnelling.

    PubMed

    Zhao, Yanan; Ashcroft, Brian; Zhang, Peiming; Liu, Hao; Sen, Suman; Song, Weisi; Im, JongOne; Gyarfas, Brett; Manna, Saikat; Biswas, Sovan; Borges, Chad; Lindsay, Stuart

    2014-06-01

    The human proteome has millions of protein variants due to alternative RNA splicing and post-translational modifications, and variants that are related to diseases are frequently present in minute concentrations. For DNA and RNA, low concentrations can be amplified using the polymerase chain reaction, but there is no such reaction for proteins. Therefore, the development of single-molecule protein sequencing is a critical step in the search for protein biomarkers. Here, we show that single amino acids can be identified by trapping the molecules between two electrodes that are coated with a layer of recognition molecules, then measuring the electron tunnelling current across the junction. A given molecule can bind in more than one way in the junction, and we therefore use a machine-learning algorithm to distinguish between the sets of electronic 'fingerprints' associated with each binding motif. With this recognition tunnelling technique, we are able to identify D and L enantiomers, a methylated amino acid, isobaric isomers and short peptides. The results suggest that direct electronic sequencing of single proteins could be possible by sequentially measuring the products of processive exopeptidase digestion, or by using a molecular motor to pull proteins through a tunnel junction integrated with a nanopore. PMID:24705512

  16. Disrupting Protein Expression with Peptide Nucleic Acids Reduces Infection by Obligate Intracellular Rickettsia

    PubMed Central

    Pelc, Rebecca S.; McClure, Jennifer C.; Kaur, Simran J.; Sears, Khandra T.; Rahman, M. Sayeedur; Ceraul, Shane M.

    2015-01-01

    Peptide Nucleic Acids (PNAs) are single-stranded synthetic nucleic acids with a pseudopeptide backbone in lieu of the phosphodiester linked sugar and phosphate found in traditional oligos. PNA designed complementary to the bacterial Shine-Dalgarno or start codon regions of mRNA disrupts translation resulting in the transient reduction in protein expression. This study examines the use of PNA technology to interrupt protein expression in obligate intracellular Rickettsia sp. Their historically intractable genetic system limits characterization of protein function. We designed PNA targeting mRNA for rOmpB from Rickettsia typhi and rickA from Rickettsia montanensis, ubiquitous factors important for infection. Using an in vitro translation system and competitive binding assays, we determined that our PNAs bind target regions. Electroporation of R. typhi and R. montanensis with PNA specific to rOmpB and rickA, respectively, reduced the bacteria’s ability to infect host cells. These studies open the possibility of using PNA to suppress protein synthesis in obligate intracellular bacteria. PMID:25781160

  17. Single-molecule spectroscopy of amino acids and peptides by recognition tunnelling

    NASA Astrophysics Data System (ADS)

    Zhao, Yanan; Ashcroft, Brian; Zhang, Peiming; Liu, Hao; Sen, Suman; Song, Weisi; Im, Jongone; Gyarfas, Brett; Manna, Saikat; Biswas, Sovan; Borges, Chad; Lindsay, Stuart

    2014-06-01

    The human proteome has millions of protein variants due to alternative RNA splicing and post-translational modifications, and variants that are related to diseases are frequently present in minute concentrations. For DNA and RNA, low concentrations can be amplified using the polymerase chain reaction, but there is no such reaction for proteins. Therefore, the development of single-molecule protein sequencing is a critical step in the search for protein biomarkers. Here, we show that single amino acids can be identified by trapping the molecules between two electrodes that are coated with a layer of recognition molecules, then measuring the electron tunnelling current across the junction. A given molecule can bind in more than one way in the junction, and we therefore use a machine-learning algorithm to distinguish between the sets of electronic `fingerprints' associated with each binding motif. With this recognition tunnelling technique, we are able to identify D and L enantiomers, a methylated amino acid, isobaric isomers and short peptides. The results suggest that direct electronic sequencing of single proteins could be possible by sequentially measuring the products of processive exopeptidase digestion, or by using a molecular motor to pull proteins through a tunnel junction integrated with a nanopore.

  18. Information transfer from DNA to peptide nucleic acids by template-directed syntheses

    NASA Technical Reports Server (NTRS)

    Schmidt, J. G.; Christensen, L.; Nielsen, P. E.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1997-01-01

    Peptide nucleic acids (PNAs) are analogs of nucleic acids in which the ribose-phosphate backbone is replaced by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. Oligocytidylates have been shown to act as templates for formation of longer oligomers of G from PNA G2 dimers. In this paper we show that information can be transferred from DNA to PNA. DNA C4T2C4 is an efficient template for synthesis of PNA G4A2G4 using G2 and A2 units as substrates. The corresponding synthesis of PNA G4C2G4 on DNA C4G2C4 is less efficient. Incorporation of PNA T2 into PNA products on DNA C4A2C4 is the least efficient of the three reactions. These results, obtained using PNA dimers as substrates, parallel those obtained using monomeric activated nucleotides.

  19. Progress in nanoparticulate systems for peptide, proteins and nucleic acid drug delivery.

    PubMed

    Slomkowski, Stanislaw; Gosecki, Mateusz

    2011-11-01

    Progress in many therapies, in particular in the therapies based on peptides, proteins and nucleic acids used as bioactive compounds, strongly depends on development of appropriate carriers which would be suitable for controlled delivery of the intact abovementioned compounds to required tissues, cells and intracellular compartments. This review presents last ten years' achievements and problems in development and application of synthetic polymer nanoparticulate carriers for oral, pulmonary and nasal delivery routes of oligopeptides and proteins. Whereas some traditional synthetic polymer carriers are only briefly recalled the main attention is concentrated on nanoparticles produced from functional copolymers mostly with hydroxyl, carboxyl and amino groups, suitable for immobilization of targeting moieties and for assuring prolonged circulation of nanoparticles in blood. Formulations of various nanoparticulate systems are described, including solid particles, polymer micelles, nanovesicles and nanogels, especially systems allowing drug release induced by external stimuli. Discussed are properties of these species, in particular stability in buffers and models of body fluids, loading with drugs and with drug models, drug release processes and results of biological studies. There are also discussed systems for gene delivery with special attention devoted to polymers suitable for compacting nucleic acids into nanoparticles as well as the relations between chemical structure of polymer carriers and ability of the latter for crossing cell membranes and for endosomal escape. PMID:21902630

  20. Incorporation of Acid-Labile Masking Groups for the Traceless Synthesis of C-Terminal Peptide α-Ketoacids.

    PubMed

    Thuaud, Frédéric; Rohrbacher, Florian; Zwicky, André; Bode, Jeffrey W

    2016-08-01

    An optimized protocol for the masking of α-ketoacids with acid-labile cyclic acetal protecting groups is reported. Unlike prior approaches, these new conditions allow the synthesis of protected α-ketoacids bearing aromatic, hindered alkyl, and protected polar side chains. Attachment to a Wang-type linker and solid support provides a resin that delivers fully unprotected C-terminal peptide α-ketoacids upon resin cleavage. These peptides are the key starting materials for chemical protein synthesis using the α-ketoacid-hydroxylamine ligation. PMID:27439001

  1. Identification and binding mechanism of phage displayed peptides with specific affinity to acid-alkali treated titanium.

    PubMed

    Sun, Yuhua; Tan, Jing; Wu, Baohua; Wang, Jianxin; Qu, Shuxin; Weng, Jie; Feng, Bo

    2016-10-01

    Acid-alkali treatment is one of means widely used for preparing bioactive titanium surfaces. Peptides with specific affinity to titanium surface modified by acid-alkali two-steps treatment were obtained via phage display technology. Out of the eight new unique peptides, titanium-binding peptide 54 displayed by monoclonal M13 phage at its pIII coat protein (TBP54-M13 phage) was proved to have higher binding affinity to the substrate. The binding interaction occurred at the domain from phenylalanine at position 1 to arginine at position 6 in the sequences of TBP54 (FAETHRGFHFSF) mainly via the reaction of these residues with the Ti surface. Together the coordination and electrostatic interactions controlled the specific binding of the phage to the substrate. The binding affinity was dependent on the surface basic hydroxyl group content. In addition, the phage showed a different interaction way with the Ti surface without acid-alkali treatment along with an impaired affinity. This study could provide more understanding of the interaction mechanism between the selected peptide and its specific substrate, and develop a promising method for the biofunctionalization of titanium. PMID:27371890

  2. Antioxidant capacity of hydrolyzed animal by-products and relation to amino acid composition and peptide size distribution.

    PubMed

    Damgaard, Trine; Lametsch, René; Otte, Jeanette

    2015-10-01

    The antioxidative capacity of six different tissue hydrolysates (porcine colon, heart and neck and bovine lung, kidney and pancreas) were tested by three different assays monitoring iron chelation, ABTS radical scavenging and inhibition of lipid oxidation in emulsions, respectively. The hydrolysates were also investigated with respect to amino acid composition and peptide size distribution. The hydrolysates contained peptides ranging from 20 kDa to below 100 Da with a predominance of peptides with low molecular weight (53.8 to 89.0 % below 3 kDa). All hydrolysates exhibited antioxidant activity as assessed with all three methods; inhibition of lipid oxidation ranging from 72 to 88 % (at a final protein concentration of 7 mg/mL), iron chelation capacity from 23 to 63 % (at 1.1 mg/mL), and ABTS radical scavenging from 38 to 50 % (at 10 μg /mL). The antioxidant activity did not correlate with the proportion of low molecular weight peptides in the hydrolysed tissues, but with the content of specific amino acid residues. The ABTS radical scavenging capacity of the tissues was found to correlate with the content of Trp, Tyr, Met and Arg, whereas the ability to inhibit the oxidation of lineoleic acid correlated with the content of Glu and His. The chosen animal by-products thus represent a natural source of antioxidants with potential for food application. PMID:26396396

  3. Tuning One-Dimensional Nanostructures of Bola-Like Peptide Amphiphiles by Varying the Hydrophilic Amino Acids.

    PubMed

    Zhao, Yurong; Deng, Li; Yang, Wei; Wang, Dong; Pambou, Elias; Lu, Zhiming; Li, Zongyi; Wang, Jiqian; King, Stephen; Rogers, Sarah; Xu, Hai; Lu, Jian R

    2016-08-01

    By combining experimental measurements and computer simulations, we here show that for the bola-like peptide amphiphiles XI4 X, where X=K, R, and H, the hydrophilic amino acid substitutions have little effect on the β-sheet hydrogen-bonding between peptide backbones. Whereas all of the peptides self-assemble into one dimensional (1D) nanostructures with completely different morphologies, that is, nanotubes and helical nanoribbons for KI4 K, flat and multilayered nanoribbons for HI4 H, and twisted and bilayered nanoribbons for RI4 R. These different 1D morphologies can be explained by the distinct stacking degrees and modes of the three peptide β-sheets along the x-direction (width) and the z-direction (height), which microscopically originate from the hydrogen-bonding ability of the sheets to solvent molecules and the pairing of hydrophilic amino acid side chains between β-sheet monolayers through stacking interactions and hydrogen bonding. These different 1D nanostructures have distinct surface chemistry and functions, with great potential in various applications exploiting the respective properties of these hydrophilic amino acids. PMID:27362441

  4. Effect of organic acids on calcium phosphate nucleation and osteogenic differentiation of human mesenchymal stem cells on peptide functionalized nanofibers.

    PubMed

    Barati, Danial; Walters, Joshua D; Shariati, Seyed Ramin Pajoum; Moeinzadeh, Seyedsina; Jabbari, Esmaiel

    2015-05-12

    Carboxylate-rich organic acids play an important role in controlling the growth of apatite crystals and the extent of mineralization in the natural bone. The objective of this work was to investigate the effect of organic acids on calcium phosphate (CaP) nucleation on nanofiber microsheets functionalized with a glutamic acid peptide and osteogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on the CaP-nucleated microsheets. High molecular weight poly(dl-lactide) (DL-PLA) was mixed with low molecular weight L-PLA conjugated with Glu-Glu-Gly-Gly-Cys peptide, and the mixture was electrospun to generate aligned nanofiber microsheets. The nanofiber microsheets were incubated in a modified simulated body fluid (mSBF) supplemented with different organic acids for nucleation and growth of CaP crystals on the nanofibers. Organic acids included citric acid (CA), hydroxycitric acid (HCA), tartaric acid (TART), malic acid (MA), ascorbic acid (AsA), and salicylic acid (SalA). HCA microsheets had the highest CaP content at 240 ± 10% followed by TART and CA with 225 ± 8% and 225 ± 10%, respectively. The Ca/P ratio and percent crystallinity of the nucleated CaP in TART microsheets was closest to that of stoichiometric hydroxyapatite. The extent of CaP nucleation and growth on the nanofiber microsheets depended on the acidic strength and number of hydrogen-bonding hydroxyl groups of the organic acids. Compressive modulus and degradation of the CaP nucleated microsheets were related to percent crystallinity and CaP content. Osteogenic differentiation of hMSCs seeded on the microsheets and cultured in osteogenic medium increased only for those microsheets nucleated with CaP by incubation in CA or AsA-supplemented mSBF. Further, only CA microsheets stimulated bone nodule formation by the seeded hMSCs. PMID:25879768

  5. Separation of proteins and peptides by capillary electrophoresis in acid buffers containing high concentrations of surfactants.

    PubMed

    Miksík, I; Deyl, Z

    1999-08-01

    Separations of proteins at acid pH in the presence of a high concentration of surfactant [sodium laurylsulfate (SDS), 50 mmol/l] was investigated. The purpose of using high concentrations of SDS as background electrolyte modifier was threefold: First, the surfactant exerts a washing effect upon the capillary wall thus preventing binding of analytes and possible clogging of the capillary. Second, it was revealed that even under very acid conditions (below pH 3) the surfactant is capable of forming associates with protein analytes which still bear considerable negative charge and can be separated on this basis. Third, the system can be applied not only for protein mixtures sufficiently soluble in neutral to alkaline media (leukocyte lysates, standard proteins), but it can be used also with proteins, that are under such conditions virtually insoluble and their solubilization is possible in acid buffers only (eggshell proteins or collagen CNBr fragments). The result was that adsorption to the capillary wall was minimized and the analytes were separated as negatively charged associates with high efficiency. With collagen fragments partition was possible on the affinity differences of the peptides to the surfactant micelles and inner wall of the capillary. Theoretical plate counts approaching 100,000 were easily achieved even with proteins which under the more conventional operation conditions exhibit considerable sticking to the capillary wall. The other feature of this system is that the associates move very rapidly to the anode. Owing to the low pH, endoosmotic flow is negligible, and therefore the system has to be operated at reversed polarity. PMID:10480258

  6. A comparison of the use of BrimA versus soluble solids/titratable acidity ratio as a maturity standard for navel oranges

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maturity and time of harvest for California navel oranges is primarily determined by the ratio of the soluble solids concentration (SSC) to titratable acidity (TA). This standard has been used in the industry for decades but its usefulness and relationship to flavor have often been questioned. In re...

  7. Effects of feeding level and sexual maturation on fatty acid composition of energy stores in diploid and triploid rainbow trout (Oncorhynchus mykiss)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sexual maturation is an energy demanding, physiological process that alters growth efficiency and compromises muscle quality in many food-fish species. Lipid mobilization supplies energy required for this process. To study the effect of ration level on fatty acid mobilization, diploid (2N) rainbow t...

  8. Detection of antimicrobial (poly)peptides with acid urea polyacrylamide gel electrophoresis followed by Western immunoblot.

    PubMed

    Porter, Edith; Valore, Erika V; Anouseyan, Rabin; Salzman, Nita H

    2015-01-01

    Antimicrobial (poly)peptides (AMPs) are ancient key effector molecules of innate host defense and have been identified in mammals, insects, plants, and even fungi (Nakatsuji and Gallo, J Invest Dermatol, 132: 887-895, 2012). They exhibit a cationic net charge at physiological pH and are rich in hydrophobic amino acids (Dufourc et al., Curr Protein Pept Sci, 13: 620-631, 2012). Their mode of action has been best investigated in bacteria. When assuming secondary structure the cationic and hydrophobic amino acids are sequestered creating a bipartitioned molecule in which the cationic amino acids mediate initial electrostatic interaction with the negatively charged bacterial surface and the hydrophobic amino acids mediate embedding into the bacterial membranes followed by a multitude of effects interfering with bacterial viability (Nicolas, FEBS J, 276: 6483-6496, 2009; Padovan et al., Curr Protein Pept Sci, 11: 210-219, 2010). However, immunomodulatory, antitumor, and other effects have been added to the ever increasing list of AMP functions (Pushpanathan et al., Int J Pept, 2013: 675391, 2013). Several classes of AMPs have been distinguished based on structure, namely anti-parallel beta-sheet, alpha-helical, circular, as well as disulfide bridge connectivity (Bond and Khalid, Protein Pept Lett, 17: 1313-1327, 2010). Many of the AMPs undergo posttranslational modification including further proteolysis. Biochemical analysis at the protein level is of great interest for a wide range of scientists and important when studying host-pathogen interaction, for example Salmonella invasion of the small intestine. Acid-urea polyacrylamide gel electrophoresis (AU-PAGE) followed by Western immunoblotting is an important tool for the identification and quantification of cationic AMPs. The protocol for these procedures outlined here describes, in detail, the necessary steps; including pouring the AU-gels, preparing the test samples, performing the electrophoretic separation and

  9. Nanoparticle Formation from Hybrid, Multiblock Copolymers of Poly(Acrylic Acid) and VPGVG Peptide

    PubMed Central

    Grieshaber, Sarah E.; Paik, Bradford A.; Bai, Shi; Kiick, Kristi L.; Jia, Xinqiao

    2012-01-01

    Elastin-mimetic hybrid copolymers with an alternating molecular architecture were synthesized via the step growth polymerization of azide-functionalized, telechelic poly(tert-butyl acrylate) (PtBA) and an alkyne-terminated, valine and glycine-rich peptide with a sequence of (VPGVG)2 (VG2). The resultant hybrid copolymer, [PtBA-VG2]n, contains up to six constituent building blocks and has a polydispersity index (PDI) of ~1.9. Trifluoroacetic acid (TFA) treatment of [PtBA-VG2]n gave rise to an alternating copolymer of poly(acrylic acid) (PAA) and VG2 ([PAA-VG2]n). The modular design permits facile adjustment of the copolymer composition by varying the molecular weight of PAA (22 and 63 repeat units). Characterization by dynamic light scattering indicated that the multiblock copolymers formed discrete nanoparticles at room temperature in aqueous solution at pH 3.8, with an average diameter of 250-270 nm and a particle size distribution of 0.34 for multiblock copolymers containing PAA22 and 0.17 for those containing PAA63. Upon increasing the pH to 7.4, both types of particles were able to swell without being disintegrated, reaching an average diameter of 285-300 nm for [PAA22-VG2]n and 330-350 nm for [PAA63-VG2]n, respectively. The nanoparticles were not dissociated upon the addition of urea, further confirming their unusual stability. The nanoparticles were capable of sequestering a hydrophobic fluorescent dye (pyrene), and the critical aggregation concentration (CAC) was determined to be 1.09 × 10-2 or 1.05 × 10-2 mg/mL for [PAA22-VG2]n and [PAA63-VG2]n, respectively. We suggest that the multiblock copolymers form through collective H-bonding and hydrophobic interactions between the PAA and VG2 peptide units, and that the unusual stability of the multiblock nanoparticles is conferred by the multiblock architecture. These hybrid multiblock copolymers are potentially useful as pH-responsive drug delivery vehicles, with the possibility of drug loading through

  10. Peptides Composed of Alternating L- and D-Amino Acids Inhibit Amyloidogenesis in Three Distinct Amyloid Systems Independent of Sequence.

    PubMed

    Kellock, Jackson; Hopping, Gene; Caughey, Byron; Daggett, Valerie

    2016-06-01

    There is now substantial evidence that soluble oligomers are primary toxic agents in amyloid diseases. The development of an antibody recognizing the toxic soluble oligomeric forms of different and unrelated amyloid species suggests a common conformational intermediate during amyloidogenesis. We previously observed a common occurrence of a novel secondary structure element, which we call α-sheet, in molecular dynamics (MD) simulations of various amyloidogenic proteins, and we hypothesized that the toxic conformer is composed of α-sheet structure. As such, α-sheet may represent a conformational signature of the misfolded intermediates of amyloidogenesis and a potential unique binding target for peptide inhibitors. Recently, we reported the design and characterization of a novel hairpin peptide (α1 or AP90) that adopts stable α-sheet structure and inhibits the aggregation of the β-Amyloid Peptide Aβ42 and transthyretin. AP90 is a 23-residue hairpin peptide featuring alternating D- and L-amino acids with favorable conformational propensities for α-sheet formation, and a designed turn. For this study, we reverse engineered AP90 to identify which of its design features is most responsible for conferring α-sheet stability and inhibitory activity. We present experimental characterization (CD and FTIR) of seven peptides designed to accomplish this. In addition, we measured their ability to inhibit aggregation in three unrelated amyloid species: Aβ42, transthyretin, and human islet amylin polypeptide. We found that a hairpin peptide featuring alternating L- and D-amino acids, independent of sequence, is sufficient for conferring α-sheet structure and inhibition of aggregation. Additionally, we show a correlation between α-sheet structural stability and inhibitory activity. PMID:27012425

  11. Structural Dynamic of a Self-Assembling Peptide d-EAK16 Made of Only D-Amino Acids

    PubMed Central

    Luo, Zhongli; Zhao, Xiaojun; Zhang, Shuguang

    2008-01-01

    We here report systematic study of structural dynamics of a 16-residue self-assembling peptide d-EAK16 made of only D-amino acids. We compare these results with its chiral counterpart L-form, l-EAK16. Circular dichroism was used to follow the structural dynamics under various temperature and pH conditions. At 25°C the d-EAK16 peptide displayed a typical beta-sheet spectrum. Upon increasing the temperature above 70°C, there was a spectrum shift as the 218 nm valley widens toward 210 nm. Above 80°C, the d-EAK16 peptide transformed into a typical alpha-helix CD spectrum without going through a detectable random-coil intermediate. When increasing the temperature from 4°C to 110°C then cooling back from 110°C to 4°C, there was a hysteresis: the secondary structure from beta-sheet to alpha-helix and then from alpha-helix to beta-sheet occurred. d-EAK16 formed an alpha-helical conformation at pH0.76 and pH12 but formed a beta-sheet at neutral pH. The effects of various pH conditions, ionic strength and denaturing agents were also noted. Since D-form peptides are resistant to natural enzyme degradation, such drastic structural changes may be exploited for fabricating molecular sensors to detect minute environmental changes. This provides insight into the behaviors of self-assembling peptides made of D-amino acids and points the way to designing new peptide materials for biomedical engineering and nanobiotechnology. PMID:18509542

  12. Effects of Thioglycolic Acid on in vivo Oocytes Maturation in Mice

    PubMed Central

    Ren, Xiaomei; Wang, Zhuoran

    2011-01-01

    Background Thioglycolic acid (TGA) is widely used in the hairdressing industry, which mostly caters to women. Recently, TGA has been reported to impair several organs, especially reproductive ones such as testes and ovaries. The reproductive toxicity of TGA on females has become an issue that cannot be neglected. Methodology/Principal Findings In the present work, superovulated female mice were percutaneously treated with different doses of TGA (37.81, 75.62, and 151.25 mg/kg). The mice were sacrificed to collect ovulated oocytes, whose numbers were counted and compared. Immunofluorescence-stained oocytes were observed under a confocal microscope to investigate the effects of TGA on spindle morphology, distribution of cortical granules (CGs), and parthenogenetic activation. The number of ovulated oocytes was decreased by TGA. The ovulated oocytes in the 151.25 mg/kg TGA group were significantly less than in the control and in the 37.81 mg/kg TGA groups. The ovulated oocytes in the 75.62 mg/kg TGA group were less than in the 37.81 mg/kg dose group. Abnormal spindle configuration in vivo was also induced by TGA. The spindle areas in the 75.62 and 151.25 mg/kg TGA groups were significantly larger than in the control and 37.81 mg/kg TGA groups. The parthenogenetic activation of ovulated oocytes in vitro was inhibited as well. The percentage of activated oocytes in the 75.62 and 151.25 mg/kg TGA groups was significantly lower than in the control and 37.81 mg/kg TGA groups. The percentage in the 151.25 mg/kg TGA group was also less than in the 75.62 mg/kg group. CG distribution was not affected by TGA. Conclusion Mice were percutaneously treated with TGA. Consequently, the number of ovulated oocytes decreased, abnormal spindle configurations were induced, and the parthenogenetic activation of ovulated oocytes was inhibited. CG distribution was not affected. PMID:21909408

  13. Peptide agonists of the thrombopoietin receptor.

    PubMed

    Dower, W J; Cwirla, S E; Balasubramanian, P; Schatz, P J; Baccanari, D P; Barrett, R W

    1998-01-01

    We have screened a variety of L-amino acid peptide libraries against the extracellular domain of the human thrombopoietin (HuTPO) receptor, c-Mpl. A large number of peptide ligands were recovered and categorized into two families. Peptides from each family compete with the binding of HuTPO and with the binding of peptides from the other familiy. Representative peptides were synthesized and found to activate the full-length HuTPO receptor expressed in Ba/F3 cells to promote proliferation. These peptide families show no apparent homology to the primary sequence of TPO. We have focused our optimization efforts on one of the peptides, a linear 14-mer (IEGPTLRQWLAARA) with an IC50 of 2 nM in a competition binding assay and an EC50 of 400 nM in the proliferation assay. In order to enhance the potency of the compound, we constructed dimeric peptides by linking the carboxy-termini of the 14-mers to a lysine branch. These molecules exhibited slightly higher affinity (0.5 nM) and greatly increased potency (0.1 nM). The EC50 of the dimeric peptide was equivalent to that of the 332 aa form of baculovirus-expressed recombinant HuTPO. As previously shown for the erythropoietin-mimetic peptides, the TPO-mimetic peptides probably activate the TPO receptor by binding and inducing receptor dimerization. This supposition is supported by the observation that covalent dimerization of the peptide enhances its potency by 4,000-fold over that of the monomer. The peptide dimer is also active in stimulating in vitro proliferation of progenitors and maturation of megakaryocytes from human bone marrow, and in promoting an increase in platelet count when administered to normal mice. PMID:11012174

  14. An equation to estimate the difference between theoretically predicted and SDS PAGE-displayed molecular weights for an acidic peptide.

    PubMed

    Guan, Yihong; Zhu, Qinfang; Huang, Delai; Zhao, Shuyi; Jan Lo, Li; Peng, Jinrong

    2015-01-01

    The molecular weight (MW) of a protein can be predicted based on its amino acids (AA) composition. However, in many cases a non-chemically modified protein shows an SDS PAGE-displayed MW larger than its predicted size. Some reports linked this fact to high content of acidic AA in the protein. However, the exact relationship between the acidic AA composition and the SDS PAGE-displayed MW is not established. Zebrafish nucleolar protein Def is composed of 753 AA and shows an SDS PAGE-displayed MW approximately 13 kDa larger than its predicted MW. The first 188 AA in Def is defined by a glutamate-rich region containing ~35.6% of acidic AA. In this report, we analyzed the relationship between the SDS PAGE-displayed MW of thirteen peptides derived from Def and the AA composition in each peptide. We found that the difference between the predicted and SDS PAGE-displayed MW showed a linear correlation with the percentage of acidic AA that fits the equation y = 276.5x - 31.33 (x represents the percentage of acidic AA, 11.4% ≤ x ≤ 51.1%; y represents the average ΔMW per AA). We demonstrated that this equation could be applied to predict the SDS PAGE-displayed MW for thirteen different natural acidic proteins. PMID:26311515

  15. Efficient 18F-Labeling of Large 37-Amino Acid pHLIP Peptide Analogues and their Biological Evaluation

    PubMed Central

    Daumar, Pierre; Wanger-Baumann, Cindy A.; Pillarsetty, NagaVaraKishore; Fabrizio, Laura; Carlin, Sean D.; Andreev, Oleg A.; Reshetnyak, Yana K.; Lewis, Jason S.

    2012-01-01

    Solid tumors often develop an acidic microenvironment, which plays a critical role in tumor progression and is associated with increased level of invasion and metastasis. The 37-residue pH (low) insertion peptide (pHLIP®) is under study as an imaging platform because of its unique ability to insert into cell membranes at a low extracellular pH (pHe<7). Labeling of peptides with [18F]-fluorine is usually performed via prosthetic groups using chemoselective coupling reactions. One of the most successful procedures involves the alkyne-azide copper(I) catalyzed cycloaddition (CuAAC). However, none of the known “click” methods have been applied to peptides as large as pHLIP. We designed a novel prosthetic group and extended the use of the CuAAC “click chemistry” for the simple and efficient 18F-labeling of large peptides. For the evaluation of this labeling approach, a D-amino acid analogue of WT-pHLIP and a L-amino acid control peptide K-pHLIP, both functionalized at the N-terminus with 6-azidohexanoic acid, were used. The novel 6-[18F]fluoro-2-ethynylpyridine prosthetic group, was obtained via nucleophilic substitution on the corresponding bromo-precursor after 10 min at 130 °C with a radiochemical yield of 27.5 ± 6.6% (decay corrected) with high radiochemical purity ≥ 98%. The subsequent CuI catalyzed “click” reaction with the azido functionalized pHLIP peptides was quantitative within 5 min at 70 °C in a mixture of water and ethanol using Cu-acetate and sodium L-ascorbate. [18F]-D-WT-pHLIP and [18F]-L-K-pHLIP were obtained with total radiochemical yields of 5–20% after HPLC purification. The total reaction time was only 85 min including formulation. In vitro stability tests revealed high stability of the [18F]-D-WT-pHLIP in human and mouse plasma after 120 min, with the parent tracer remaining intact at 65 and 85%, respectively. PET imaging and biodistribution studies in LNCaP and PC-3 xenografted mice with the [18F]-D-WT-pHLIP and the negative

  16. Highly sensitive detection of influenza virus by boron-doped diamond electrode terminated with sialic acid-mimic peptide.

    PubMed

    Matsubara, Teruhiko; Ujie, Michiko; Yamamoto, Takashi; Akahori, Miku; Einaga, Yasuaki; Sato, Toshinori

    2016-08-01

    The progression of influenza varies according to age and the presence of an underlying disease; appropriate treatment is therefore required to prevent severe disease. Anti-influenza therapy, such as with neuraminidase inhibitors, is effective, but diagnosis at an early phase of infection before viral propagation is critical. Here, we show that several dozen plaque-forming units (pfu) of influenza virus (IFV) can be detected using a boron-doped diamond (BDD) electrode terminated with a sialic acid-mimic peptide. The peptide was used instead of the sialyloligosaccharide receptor, which is the common receptor of influenza A and B viruses required during the early phase of infection, to capture IFV particles. The peptide, which was previously identified by phage-display technology, was immobilized by click chemistry on the BDD electrode, which has excellent electrochemical characteristics such as low background current and weak adsorption of biomolecules. Electrochemical impedance spectroscopy revealed that H1N1 and H3N2 IFVs were detectable in the range of 20-500 pfu by using the peptide-terminated BDD electrode. Our results demonstrate that the BDD device integrated with the receptor-mimic peptide has high sensitivity for detection of a low number of virus particles in the early phase of infection. PMID:27457924

  17. Primary structure of a histidine-rich proteolytic fragment of human ceruloplasmin. II. Amino acid sequence of the tryptic peptides.

    PubMed

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

    1980-04-10

    Amino acid sequence studies of tryptic peptides isolated from a histidine-rich fragment (Cp F5) of human ceruloplasmin are described. Nineteen tryptic peptides were isolated from unmodified Cp F5 and five tryptic peptides were isolated from citraconylated Cp F5. These peptides, together with the cyanogen bromide fragments reported previously, allowed the assembly of the complete sequence of Cp F5. The fragment has 159 residues and a molecular weight of 18,650; it lacks carbohydrate, is rich in histidine, and contains 1 free cysteine that may be part of a copper-binding site. Human ceruloplasmin is a single polypeptide chain with a molecular weight of about 130,000 that is readily cleaved to large fragments by proteolytic enzymes; the relationships of Cp F5 to intact ceruloplasmin and to structural subunits earlier proposed is described. Cp F5 probably is an intact globular domain that is attached to the COOH-terminal end of ceruloplasmin by a labile interdomain peptide bond. PMID:6987230

  18. Lipid remodelling during epididymal maturation of rat spermatozoa. Enrichment in plasmenylcholines containing long-chain polyenoic fatty acids of the n-9 series.

    PubMed Central

    Aveldaño, M I; Rotstein, N P; Vermouth, N T

    1992-01-01

    In their transit from the caput to the cauda segments of the epididymis, rat spermatozoa undergo significant modifications in lipid content and composition. The amount of lipid phosphorus per cell decreases, and most lipid classes show specific changes in their constituent fatty acids. A depletion of phosphatidylcholine and phosphatidylethanolamine, concomitant with a virtually unchanged amount of the corresponding plasmalogens, are the major alterations, plasmenylcholine thereby becoming the major phospholipid. Diphosphatidylglycerol, sphingomyelin and the phosphoinositides decrease to a lesser extent or do not change at all, also resulting in relative increases with sperm maturation. Concerning the fatty acids, the proportions of oleate (C18:1, n-9) and linoleate (C18:2, n-6) in most lipids decrease on movement of sperm from caput to cauda, augmenting in turn the proportions of longer-chain (C20 to C24) and more unsaturated fatty acids. Docosapentaenoate (C22:5, n-6) is a major acyl chain present in all lipids at both stages, but uncommon long-chain polyenoic fatty acids of the n-9 series are also present, being almost exclusively found in the choline glycerophospholipids. These fatty acids are found to undergo the most significant changes during sperm maturation. They are minor components of plasmenylcholine in immature spermatozoa, but increase severalfold on maturation, representing more than half of the acyl chains of this major lipid in cells from the cauda. The high concentration of n-9 polyenes in mature sperm plasmenylcholine raises intriguing questions on the possible role epididymal cells may play in providing spermatozoa with such an unusual phospholipid. These plasmenylcholines could contribute to the characteristic lipid domain organization of the mature spermatozoa plasma membrane. PMID:1567371

  19. Lipid remodelling during epididymal maturation of rat spermatozoa. Enrichment in plasmenylcholines containing long-chain polyenoic fatty acids of the n-9 series.

    PubMed

    Aveldaño, M I; Rotstein, N P; Vermouth, N T

    1992-04-01

    In their transit from the caput to the cauda segments of the epididymis, rat spermatozoa undergo significant modifications in lipid content and composition. The amount of lipid phosphorus per cell decreases, and most lipid classes show specific changes in their constituent fatty acids. A depletion of phosphatidylcholine and phosphatidylethanolamine, concomitant with a virtually unchanged amount of the corresponding plasmalogens, are the major alterations, plasmenylcholine thereby becoming the major phospholipid. Diphosphatidylglycerol, sphingomyelin and the phosphoinositides decrease to a lesser extent or do not change at all, also resulting in relative increases with sperm maturation. Concerning the fatty acids, the proportions of oleate (C18:1, n-9) and linoleate (C18:2, n-6) in most lipids decrease on movement of sperm from caput to cauda, augmenting in turn the proportions of longer-chain (C20 to C24) and more unsaturated fatty acids. Docosapentaenoate (C22:5, n-6) is a major acyl chain present in all lipids at both stages, but uncommon long-chain polyenoic fatty acids of the n-9 series are also present, being almost exclusively found in the choline glycerophospholipids. These fatty acids are found to undergo the most significant changes during sperm maturation. They are minor components of plasmenylcholine in immature spermatozoa, but increase severalfold on maturation, representing more than half of the acyl chains of this major lipid in cells from the cauda. The high concentration of n-9 polyenes in mature sperm plasmenylcholine raises intriguing questions on the possible role epididymal cells may play in providing spermatozoa with such an unusual phospholipid. These plasmenylcholines could contribute to the characteristic lipid domain organization of the mature spermatozoa plasma membrane. PMID:1567371

  20. Phage Selection of Chemically Stabilized α-Helical Peptide Ligands.

    PubMed

    Diderich, Philippe; Bertoldo, Davide; Dessen, Pierre; Khan, Maola M; Pizzitola, Irene; Held, Werner; Huelsken, Joerg; Heinis, Christian

    2016-05-20

    Short α-helical peptides stabilized by linkages between constituent amino acids offer an attractive format for ligand development. In recent years, a range of excellent ligands based on stabilized α-helices were generated by rational design using α-helical peptides of natural proteins as templates. Herein, we developed a method to engineer chemically stabilized α-helical ligands in a combinatorial fashion. In brief, peptides containing cysteines in position i and i + 4 are genetically encoded by phage display, the cysteines are modified with chemical bridges to impose α-helical conformations, and binders are isolated by affinity selection. We applied the strategy to affinity mature an α-helical peptide binding β-catenin. We succeeded in developing ligands with Kd's as low as 5.2 nM, having >200-fold improved affinity. The strategy is generally applicable for affinity maturation of any α-helical peptide. Compared to hydrocarbon stapled peptides, the herein evolved thioether-bridged peptide ligands can be synthesized more easily, as no unnatural amino acids are required and the cyclization reaction is more efficient and yields no stereoisomers. A further advantage of the thioether-bridged peptide ligands is that they can be expressed recombinantly as fusion proteins. PMID:26929989

  1. MALDI TOF/TOF-Based Approach for the Identification of d- Amino Acids in Biologically Active Peptides and Proteins.

    PubMed

    Koehbach, Johannes; Gruber, Christian W; Becker, Christian; Kreil, David P; Jilek, Alexander

    2016-05-01

    Several biologically active peptides contain a d- amino acid in a well-defined position, which is position 2 in all peptide epimers isolated to date from vertebrates and also some from invertebrates. The detection of such D- residues by standard analytical techniques is challenging. In tandem mass spectrometric (MS) analysis, although fragment masses are the same for all stereoisomers, peak intensities are known to depend on chirality. Here, we observe that the effect of a d- amino acid in the second N-terminal position on the fragmentation pattern in matrix assisted laser desorption time-of-flight spectrometry (MALDI-TOF/TOF MS) strongly depends on the peptide sequence. Stereosensitive fragmentation (SF) is correlated to a neighborhood effect, but the d- residue also exerts an overall effect influencing distant bonds. In a fingerprint analysis, multiple peaks can thus serve to identify the chirality of a sample in short time and potentially high throughput. Problematic variations between individual spots could be successfully suppressed by cospotting deuterated analogues of the epimers. By identifying the [d-Leu2] isomer of the predicted peptide GH-2 (gene derived bombininH) in skin secretions of the toad Bombina orientalis, we demonstrated the analytical power of SF-MALDI-TOF/TOF measurements. In conclusion, SF-MALDI-TOF/TOF MS combines high sensitivity, versatility, and the ability to complement other methods. PMID:26985971

  2. Killing of Mycobacterium avium by Lactoferricin Peptides: Improved Activity of Arginine- and d-Amino-Acid-Containing Molecules

    PubMed Central

    Silva, Tânia; Magalhães, Bárbara; Maia, Sílvia; Gomes, Paula; Nazmi, Kamran; Bolscher, Jan G. M.; Rodrigues, Pedro N.; Bastos, Margarida

    2014-01-01

    Mycobacterium avium causes respiratory disease in susceptible individuals, as well as disseminated infections in immunocompromised hosts, being an important cause of morbidity and mortality among these populations. Current therapies consist of a combination of antibiotics taken for at least 6 months, with no more than 60% overall clinical success. Furthermore, mycobacterial antibiotic resistance is increasing worldwide, urging the need to develop novel classes of antimicrobial drugs. One potential and interesting alternative strategy is the use of antimicrobial peptides (AMP). These are present in almost all living organisms as part of their immune system, acting as a first barrier against invading pathogens. In this context, we investigated the effect of several lactoferrin-derived AMP against M. avium. Short peptide sequences from both human and bovine lactoferricins, namely, hLFcin1-11 and LFcin17-30, as well as variants obtained by specific amino acid substitutions, were evaluated. All tested peptides significantly inhibited the axenic growth of M. avium, the bovine peptides being more active than the human. Arginine residues were found to be crucial for the display of antimycobacterial activity, whereas the all-d-amino-acid analogue of the bovine sequence displayed the highest mycobactericidal activity. These findings reveal the promising potential of lactoferricins against mycobacteria, thus opening the way for further research on their development and use as a new weapon against mycobacterial infections. PMID:24709266

  3. MALDI TOF/TOF-Based Approach for the Identification of d- Amino Acids in Biologically Active Peptides and Proteins

    PubMed Central

    2016-01-01

    Several biologically active peptides contain a d- amino acid in a well-defined position, which is position 2 in all peptide epimers isolated to date from vertebrates and also some from invertebrates. The detection of such D- residues by standard analytical techniques is challenging. In tandem mass spectrometric (MS) analysis, although fragment masses are the same for all stereoisomers, peak intensities are known to depend on chirality. Here, we observe that the effect of a d- amino acid in the second N-terminal position on the fragmentation pattern in matrix assisted laser desorption time-of-flight spectrometry (MALDI-TOF/TOF MS) strongly depends on the peptide sequence. Stereosensitive fragmentation (SF) is correlated to a neighborhood effect, but the d- residue also exerts an overall effect influencing distant bonds. In a fingerprint analysis, multiple peaks can thus serve to identify the chirality of a sample in short time and potentially high throughput. Problematic variations between individual spots could be successfully suppressed by cospotting deuterated analogues of the epimers. By identifying the [d-Leu2] isomer of the predicted peptide GH-2 (gene derived bombininH) in skin secretions of the toad Bombina orientalis, we demonstrated the analytical power of SF-MALDI-TOF/TOF measurements. In conclusion, SF-MALDI-TOF/TOF MS combines high sensitivity, versatility, and the ability to complement other methods. PMID:26985971

  4. Analysis of peptide-protein binding using amino acid descriptors: prediction and experimental verification for human histocompatibility complex HLA-A0201.

    PubMed

    Guan, Pingping; Doytchinova, Irini A; Walshe, Valerie A; Borrow, Persephone; Flower, Darren R

    2005-11-17

    Amino acid descriptors are often used in quantitative structure-activity relationship (QSAR) analysis of proteins and peptides. In the present study, descriptors were used to characterize peptides binding to the human MHC allele HLA-A0201. Two sets of amino acid descriptors were chosen: 93 descriptors taken from the amino acid descriptor database AAindex and the z descriptors defined by Wold and Sandberg. Variable selection techniques (SIMCA, genetic algorithm, and GOLPE) were applied to remove redundant descriptors. Our results indicate that QSAR models generated using five z descriptors had the highest predictivity and explained variance (q2 between 0.6 and 0.7 and r2 between 0.6 and 0.9). Further to the QSAR analysis, 15 peptides were synthesized and tested using a T2 stabilization assay. All peptides bound to HLA-A0201 well, and four peptides were identified as high-affinity binders. PMID:16279801

  5. 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.

  6. Cell Penetrating Peptide Conjugated Chitosan for Enhanced Delivery of Nucleic Acid

    PubMed Central

    Layek, Buddhadev; Lipp, Lindsey; Singh, Jagdish

    2015-01-01

    Gene therapy is an emerging therapeutic strategy for the cure or treatment of a spectrum of genetic disorders. Nevertheless, advances in gene therapy are immensely reliant upon design of an efficient gene carrier that can deliver genetic cargoes into the desired cell populations. Among various nonviral gene delivery systems, chitosan-based carriers have gained increasing attention because of their high cationic charge density, excellent biocompatibility, nearly nonexistent cytotoxicity, negligible immune response, and ideal ability to undergo chemical conjugation. However, a major shortcoming of chitosan-based carriers is their poor cellular uptake, leading to inadequate transfection efficiency. The intrinsic feature of cell penetrating peptides (CPPs) for transporting diverse cargoes into multiple cell and tissue types in a safe manner suggests that they can be conjugated to chitosan for improving its transfection efficiency. In this review, we briefly discuss CPPs and their classification, and also the major mechanisms contributing to the cellular uptake of CPPs and cargo conjugates. We also discuss immense improvements for the delivery of nucleic acids using CPP-conjugated chitosan-based carriers with special emphasis on plasmid DNA and small interfering RNA. PMID:26690119

  7. Peptide Nucleic Acid Promotes Systemic Dystrophin Expression and Functional Rescue in Dystrophin-deficient mdx Mice

    PubMed Central

    Gao, Xianjun; Shen, Xiaoyong; Dong, Xue; Ran, Ning; Han, Gang; Cao, Limin; Gu, Ben; Yin, HaiFang

    2015-01-01

    Antisense oligonucleotide (AO)-mediated exon-skipping therapeutics shows great promise for Duchenne muscular dystrophy (DMD) patients. However, recent failure with drisapersen, an AO candidate drug in phase 3 trial, highlights the importance of exploring other effective AO chemistries for DMD. Previously, we demonstrated the appreciable biological activity of peptide nucleic acid (PNA) AOs in restoring dystrophin expression in dystrophin-deficient mdx mice intramuscularly. Here, we further explore the systemic potential and feasibility of PNA AOs in mediating exon skipping in mdx mice as a comprehensive systemic evaluation remains lacking. Systemic delivery of PNA AOs resulted in therapeutic level of dystrophin expression in body-wide peripheral muscles and improved dystrophic pathology in mdx mice without any detectable toxicity. Up to 40% of dystrophin restoration was achieved in gastrocnemius, to a less extent with other skeletal muscles, with no dystrophin in heart. Notably, comparable systemic activity was obtained between PNA AOs and phosphorodiamidate morpholino oligomer, a DMD AO chemistry in phase 3 clinical trial, under an identical dosing regimen. Overall, our data demonstrate that PNA is viable for DMD exon-skipping therapeutics with 20 mer showing the best combination of activity, solubility, and safety and further modifications to increase PNA aqueous solubility can enable longer, more effective therapeutics without the associated toxicity. PMID:26440599

  8. Targeted gene correction using psoralen, chlorambucil and camptothecin conjugates of triplex forming peptide nucleic acid (PNA)

    PubMed Central

    Birkedal, Henrik

    2011-01-01

    Gene correction activation effects of a small series of triplex forming peptide nucleic acid (PNA) covalently conjugated to the DNA interacting ligands psoralen, chlorambucil and camptothecin targeted proximal to a stop codon mutation in an EGFP reporter gene were studied. A 15-mer homopyrimidine PNA conjugated to the topoisomerase I inhibitor camptothecin was found to increase the frequency of repair domain mediated gene correctional events of the EGFP reporter in an in vitro HeLa cell nuclear extract assay, whereas PNA psoralen or chlorambucil conjugates both of which form covalent and also interstrand crosslinked adducts with dsDNA dramatically decreased the frequency of targeted repair/correction. The PNA conjugates were also studied in mammalian cell lines upon transfection of PNA bound EGFP reporter vector and scoring repair of the EGFP gene by FACS analysis of functional EGFP expression. Consistent with the extract experiments, treatment with adduct forming PNA conjugates (psoralen and chlorambucil) resulted in a decrease in background correction frequencies in transiently transfected cells, whereas unmodified PNA or the PNA-camptothecin conjugate had little or no effect. These results suggest that simple triplex forming PNAs have little effect on proximal gene correctional events whereas PNA conjugates capable of forming DNA adducts and interstrand crosslinks are strong inhibitors. Most interestingly the PNA conjugated to the topoisomerase inhibitor, camptothecin enhanced repair in nuclear extract. Thus the effects and use of camptothecin conjugates in gene targeted repair merit further studies. PMID:21686249

  9. Hybrid polymeric hydrogels via peptide nucleic acid (PNA)/DNA complexation.

    PubMed

    Chu, Te-Wei; Feng, Jiayue; Yang, Jiyuan; Kopeček, Jindřich

    2015-12-28

    This work presents a new concept in hybrid hydrogel design. Synthetic water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) polymers grafted with multiple peptide nucleic acids (PNAs) are crosslinked upon addition of the linker DNA. The self-assembly is mediated by the PNA-DNA complexation, which results in the formation of hydrophilic polymer networks. We show that the hydrogels can be produced through two different types of complexations. Type I hydrogel is formed via the PNA/DNA double-helix hybridization. Type II hydrogel utilizes a unique "P-form" oligonucleotide triple-helix that comprises two PNA sequences and one DNA. Microrheology studies confirm the respective gelation processes and disclose a higher critical gelation concentration for the type I gel when compared to the type II design. Scanning electron microscopy reveals the interconnected microporous structure of both types of hydrogels. Type I double-helix hydrogel exhibits larger pore sizes than type II triple-helix gel. The latter apparently contains denser structure and displays greater elasticity as well. The designed hybrid hydrogels have potential as novel biomaterials for pharmaceutical and biomedical applications. PMID:26394062

  10. Fluorescence imaging of siRNA delivery by peptide nucleic acid-based probe.

    PubMed

    Sato, Takaya; Sato, Yusuke; Iwai, Kenta; Kuge, Shusuke; Teramae, Norio; Nishizawa, Seiichi

    2015-01-01

    We report on the use of a peptide nucleic acid (PNA)-based fluorescent probe for the analysis of siRNA delivery to living cells. The probe, Py-AA-TO, possesses thiazole orange (TO) and pyrene moieties in the C- and N-termini of PNA, and can function as a light-up probe capable of selective binding to 3'-overhanging nucleotides of target siRNAs. The affinity-labeling of the siRNAs with Py-AA-TO facilitates fluorescence imaging of cellular uptake of polymer-based carriers encapsulating the siRNAs (polyplexes) through endocytosis and subsequent sequestration into lysosome. In addition, flow cytometric measurements reveal that the monitoring of Py-AA-TO fluorescence inside the cells is successfully applicable to the analysis of the polyplex disassembly. These promising functions of Py-AA-TO are presented and discussed as a basis for the design of molecular probes for fluorescent imaging and quantitative analysis of the siRNA delivery process. PMID:25864675

  11. Isodesmic reaction for accurate theoretical pKa calculations of amino acids and peptides.

    PubMed

    Sastre, S; Casasnovas, R; Muñoz, F; Frau, J

    2016-04-20

    Theoretical and quantitative prediction of pKa values at low computational cost is a current challenge in computational chemistry. We report that the isodesmic reaction scheme provides semi-quantitative predictions (i.e. mean absolute errors of 0.5-1.0 pKa unit) for the pKa1 (α-carboxyl), pKa2 (α-amino) and pKa3 (sidechain groups) of a broad set of amino acids and peptides. This method fills the gaps of thermodynamic cycles for the computational pKa calculation of molecules that are unstable in the gas phase or undergo proton transfer reactions or large conformational changes from solution to the gas phase. We also report the key criteria to choose a reference species to make accurate predictions. This method is computationally inexpensive and makes use of standard density functional theory (DFT) and continuum solvent models. It is also conceptually simple and easy to use for researchers not specialized in theoretical chemistry methods. PMID:27052591

  12. Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence in situ hybridization method

    PubMed Central

    Machado, António; Castro, Joana; Cereija, Tatiana; Almeida, Carina

    2015-01-01

    Bacterial vaginosis (BV) is one of most common vaginal infections. However, its diagnosis by classical methods reveals low specificity. Our goal was to evaluate the accuracy diagnosis of 150 vaginal samples with research gold standard methods and our Peptide Nucleic Acid (PNA) probes by Fluorescence in situ Hybridization (FISH) methodology. Also, we described the first PNA-FISH methodology for BV diagnosis, which provides results in approximately 3 h. The results showed a sensitivity of 84.6% (95% confidence interval (CI), from 64.3 to 95.0%) and a specificity of 97.6% (95% CI [92.6–99.4%]), demonstrating the higher specificity of the PNA-FISH method and showing false positive results in BV diagnosis commonly obtained by the classical methods. This methodology combines the specificity of PNA probes for Lactobacillus species and G. vaginalis visualization and the calculation of the microscopic field by Nugent score, allowing a trustful evaluation of the bacteria present in vaginal microflora and avoiding the occurrence of misleading diagnostics. Therefore, the PNA-FISH methodology represents a valuable alternative for BV diagnosis. PMID:25737820

  13. A Sensitive Peptide Nucleic Acid Probe Assay for Detection of BRAF V600 Mutations in Melanoma.

    PubMed

    Chen, Tai-Long; Chang, John Wen-Cheng; Hsieh, Jia-Juan; Cheng, Hsin-Yi; Chiou, Chiuan-Chian

    Mutated v-Raf murine sarcoma viral oncogene homolog B (BRAF) is an important biomarker for the prediction of therapeutic efficacy of several anticancer drugs. The detection of BRAF mutation faces two challenges: Firstly, there are multiple types of mutations, and secondly, tumor samples usually contain various amounts of wild-type, normal tissues. Here, we describe a newly established method for sensitive detection of multiple types of BRAF V600 mutations in excess wild-type background. The method introduced a fluorophore-tagged peptide nucleic acid (PNA) to serve as both polymerase chain reaction (PCR) clamp and sensor probe, which inhibited the amplification of wild-type templates during PCR and revealed multiple types of mutant signals during melting analysis. We demonstrated the design and optimization process of the method, and applied it in the detection of BRAF mutations in 49 melanoma samples. This PNA probe assay method detected three types of mutations in 17 samples, and was much more sensitive than conventional PCR plus Sanger sequencing. PMID:27566656

  14. Caffeic acid phenethyl ester (CAPE), an active component of propolis, inhibits Helicobacter pylori peptide deformylase activity.

    PubMed

    Cui, Kunqiang; Lu, Weiqiang; Zhu, Lili; Shen, Xu; Huang, Jin

    2013-05-31

    Helicobacter pylori (H. pylori) is a major causative factor for gastrointestinal illnesses, H. pylori peptide deformylase (HpPDF) catalyzes the removal of formyl group from the N-terminus of nascent polypeptide chains, which is essential for H. pylori survival and is considered as a promising drug target for anti-H. pylori therapy. Propolis, a natural antibiotic from honeybees, is reported to have an inhibitory effect on the growth of H. pylori in vitro. In addition, previous studies suggest that the main active constituents in the propolis are phenolic compounds. Therefore, we evaluated a collection of phenolic compounds derived from propolis for enzyme inhibition against HpPDF. Our study results show that Caffeic acid phenethyl ester (CAPE), one of the main medicinal components of propolis, is a competitive inhibitor against HpPDF, with an IC50 value of 4.02 μM. Furthermore, absorption spectra and crystal structural characterization revealed that different from most well known PDF inhibitors, CAPE block the substrate entrance, preventing substrate from approaching the active site, but CAPE does not have chelate interaction with HpPDF and does not disrupt the metal-dependent catalysis. Our study provides valuable information for understanding the potential anti-H. pylori mechanism of propolis, and CAPE could be served as a lead compound for further anti-H. pylori drug discovery. PMID:23611786

  15. Peptide Nucleic Acid Promotes Systemic Dystrophin Expression and Functional Rescue in Dystrophin-deficient mdx Mice.

    PubMed

    Gao, Xianjun; Shen, Xiaoyong; Dong, Xue; Ran, Ning; Han, Gang; Cao, Limin; Gu, Ben; Yin, HaiFang

    2015-01-01

    Antisense oligonucleotide (AO)-mediated exon-skipping therapeutics shows great promise for Duchenne muscular dystrophy (DMD) patients. However, recent failure with drisapersen, an AO candidate drug in phase 3 trial, highlights the importance of exploring other effective AO chemistries for DMD. Previously, we demonstrated the appreciable biological activity of peptide nucleic acid (PNA) AOs in restoring dystrophin expression in dystrophin-deficient mdx mice intramuscularly. Here, we further explore the systemic potential and feasibility of PNA AOs in mediating exon skipping in mdx mice as a comprehensive systemic evaluation remains lacking. Systemic delivery of PNA AOs resulted in therapeutic level of dystrophin expression in body-wide peripheral muscles and improved dystrophic pathology in mdx mice without any detectable toxicity. Up to 40% of dystrophin restoration was achieved in gastrocnemius, to a less extent with other skeletal muscles, with no dystrophin in heart. Notably, comparable systemic activity was obtained between PNA AOs and phosphorodiamidate morpholino oligomer, a DMD AO chemistry in phase 3 clinical trial, under an identical dosing regimen. Overall, our data demonstrate that PNA is viable for DMD exon-skipping therapeutics with 20 mer showing the best combination of activity, solubility, and safety and further modifications to increase PNA aqueous solubility can enable longer, more effective therapeutics without the associated toxicity. PMID:26440599

  16. Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence in situ hybridization method.

    PubMed

    Machado, António; Castro, Joana; Cereija, Tatiana; Almeida, Carina; Cerca, Nuno

    2015-01-01

    Bacterial vaginosis (BV) is one of most common vaginal infections. However, its diagnosis by classical methods reveals low specificity. Our goal was to evaluate the accuracy diagnosis of 150 vaginal samples with research gold standard methods and our Peptide Nucleic Acid (PNA) probes by Fluorescence in situ Hybridization (FISH) methodology. Also, we described the first PNA-FISH methodology for BV diagnosis, which provides results in approximately 3 h. The results showed a sensitivity of 84.6% (95% confidence interval (CI), from 64.3 to 95.0%) and a specificity of 97.6% (95% CI [92.6-99.4%]), demonstrating the higher specificity of the PNA-FISH method and showing false positive results in BV diagnosis commonly obtained by the classical methods. This methodology combines the specificity of PNA probes for Lactobacillus species and G. vaginalis visualization and the calculation of the microscopic field by Nugent score, allowing a trustful evaluation of the bacteria present in vaginal microflora and avoiding the occurrence of misleading diagnostics. Therefore, the PNA-FISH methodology represents a valuable alternative for BV diagnosis. PMID:25737820

  17. A peptide nucleic acid targeting nuclear RAD51 sensitizes multiple myeloma cells to melphalan treatment.

    PubMed

    Alagpulinsa, David Abasiwani; Yaccoby, Shmuel; Ayyadevara, Srinivas; Shmookler Reis, Robert Joseph

    2015-01-01

    RAD51-mediated recombinational repair is elevated in multiple myeloma (MM) and predicts poor prognosis. RAD51 has been targeted to selectively sensitize and/or kill tumor cells. Here, we employed a peptide nucleic acid (PNA) to inhibit RAD51 expression in MM cells. We constructed a PNA complementary to a unique segment of the RAD51 gene promoter, spanning the transcription start site, and conjugated it to a nuclear localization signal (PKKKRKV) to enhance cellular uptake and nuclear delivery without transfection reagents. This synthetic construct, (PNArad51_nls), significantly reduced RAD51 transcripts in MM cells, and markedly reduced the number and intensity of de novo and melphalan-induced nuclear RAD51 foci, while increasing the level of melphalan-induced γH2AX foci. Melphalan alone markedly induced the expression of 5 other genes involved in homologous-recombination repair, yet suppression of RAD51 by PNArad51_nls was sufficient to synergize with melphalan, producing significant synthetic lethality of MM cells in vitro. In a SCID-rab mouse model mimicking the MM bone marrow microenvironment, treatment with PNArad51_nls ± melphalan significantly suppressed tumor growth after 2 weeks, whereas melphalan plus control PNArad4µ_nls was ineffectual. This study highlights the importance of RAD51 in myeloma growth and is the first to demonstrate that anti-RAD51 PNA can potentiate conventional MM chemotherapy. PMID:25996477

  18. Boswellic acids target the human immune system-modulating antimicrobial peptide LL-37.

    PubMed

    Henkel, Arne; Tausch, Lars; Pillong, Max; Jauch, Johann; Karas, Michael; Schneider, Gisbert; Werz, Oliver

    2015-12-01

    The antimicrobial peptide LL-37 is the sole member of the human cathelicidin family with immune system-modulating properties and roles in autoimmune disease development. Small molecules able to interact with LL-37 and to modulate its functions have not been described yet. Boswellic acids (BAs) are pentacyclic triterpene acids that are bioactive principles of frankincense extracts used as anti-inflammatory remedies. Although various anti-inflammatory modes of action have been proposed for BAs, the pharmacological profile of these compounds is still incompletely understood. Here, we describe the identification of human LL-37 as functional target of BAs. In unbiased target fishing experiments using immobilized BAs as bait and human neutrophils as target source, LL-37 was identified as binding partner assisted by MALDI-TOF mass spectrometry. Thermal stability experiments using circular dichroism spectroscopy confirm direct interaction between BAs and LL-37. Of interest, this binding of BAs resulted in an inhibition of the functionality of LL-37. Thus, the LPS-neutralizing properties of isolated LL-37 were inhibited by 3-O-acetyl-β-BA (Aβ-BA) and 3-O-acetyl-11-keto-β-BA (AKβ-BA) in a cell-free limulus amoebocyte lysate assay with EC50=0.2 and 0.8 μM, respectively. Also, LL-37 activity was inhibited by these BAs in LL-37-enriched supernatants of stimulated neutrophils or human plasma derived from stimulated human whole blood. Together, we reveal BAs as inhibitors of LL-37, which might be a relevant mechanism underlying the anti-inflammatory properties of BAs and suggests BAs as suitable chemical tools or potential agents for intervention with LL-37 and related disorders. PMID:26361729

  19. California Hass Avocado: Profiling of Carotenoids, tocopherol, fatty acid, and fat content during maturation and from different growing areas

    PubMed Central

    Lu, Qing-Yi; Zhang, Yanjun; Wang, Yue; Wang, David; Lee, Ru-po; Gao, Kun; Byrns, Russell; Heber, David

    2009-01-01

    The California Hass avocado (Persea Americana) is an example of a domesticated berry fruit that matures on the tree during its growing season but ripens only after being harvested. Avocados are typically harvested multiple times during the growing season in California. Previous research has demonstrated potential health benefits of avocados and extracts of avocado against inflammation and cancer cell growth, but seasonal variations in the phytochemical profile of the fruits being studied may affect the results obtained in future research. Therefore in the present study, avocados were harvested in January, April, July and September 2008 from four different growing locations in California (San Luis Obispo, Ventura, Riverside and San Diego), and analyzed fortotal fat content, fatty acid profile, carotenoids and vitamin E. A significant increase in total carotenoid and fat content of avocados from all regions was noted as the season progressed from January to September. Four carotenoids not previously described in the avocado were quantified. The total content of carotenoids was highly correlated with total fat content (r=0.99, p<0.001) demonstrating a remarkable degree of constancy of carotenoid intake per gram of fat content in the California Hass avocado.. Future clinical research on the health benefits of the avocado should specify the time of harvest, degree of ripening, growing area and the total phytochemical profile of the fruit or extract being studied. These steps will enable researchers to account for potential nutrient-nutrient interactions that might affect the research outcomes. PMID:19813713

  20. California Hass avocado: profiling of carotenoids, tocopherol, fatty acid, and fat content during maturation and from different growing areas.

    PubMed

    Lu, Qing-Yi; Zhang, Yanjun; Wang, Yue; Wang, David; Lee, Ru-po; Gao, Kun; Byrns, Russell; Heber, David

    2009-11-11

    The California Hass avocado ( Persea americana ) is an example of a domesticated berry fruit that matures on the tree during its growing season but ripens only after being harvested. Avocados are typically harvested multiple times during the growing season in California. Previous research has demonstrated potential health benefits of avocados and extracts of avocado against inflammation and cancer cell growth, but seasonal variations in the phytochemical profile of the fruits being studied may affect the results obtained in future research. Therefore, in the present study, avocados were harvested in January, April, July, and September, 2008, from four different growing locations in California (San Luis Obispo, Ventura, Riverside, and San Diego) and analyzed for total fat content, fatty acid profile, carotenoids, and vitamin E. A significant increase in total carotenoid and fat content of avocados from all regions was noted as the season progressed from January to September. Four carotenoids not previously described in the avocado were quantified. The total content of carotenoids was highly correlated with the total fat content (r = 0.99, p < 0.001) demonstrating a remarkable degree of constancy of carotenoid intake per gram of fat content in the California Hass avocado. Future clinical research on the health benefits of the avocado should specify the time of harvest, degree of ripening, growing area, and the total phytochemical profile of the fruit or extract being studied. These steps will enable researchers to account for potential nutrient-nutrient interactions that might affect the research outcomes. PMID:19813713

  1. Isolation and properties of the acid site-specific endonuclease from mature eggs of the sea urchin Strongylocentrotus intermedius

    SciTech Connect

    Sibirtsev, Yu.T.; Konechnyi, A.A.; Rasskazov, V.A.

    1986-01-10

    An acid site-specific endonuclease has been detected in mature sea urchin eggs and cells of embryos at early stages of differentiation. Fractionation with ammonium sulfate, followed by chromatography on columns with DEAE, phosphocellulose, and hydroxyapatite resulted in an 18,000-fold purification. The molecular weight of the enzyme was determined at approx. 29,000, the optimum pH 5.5. The activity of the enzyme does not depend on divalent metal ions, EDTA, ATP, and tRNA, but it is modulated to a substantial degree by NaCl. The maximum rate of cleavage of the DNA supercoil (form I) is observed at 100 mM NaCl. Increasing the NaCl concentration to 350 mM only slightly lowers the rate of cleavage of form I, yielding form II, but entirely suppresses the accumulation of form III. Restriction analysis of the products of enzymatic hydrolysis of Co1E1 and pBR322 DNA showed that at the early stages of hydrolysis the enzyme exhibits pronounced specificity for definite sites, the number of which is 12 for Co1 E1 DNA and 8 sites for pBR322 DNA.

  2. Grain Development Mutants of Barley ([alpha]-Amylase Production during Grain Maturation and Its Relation to Endogenous Gibberellic Acid Content).

    PubMed Central

    Green, L. S.; Faergestad, E. M.; Poole, A.; Chandler, P. M.

    1997-01-01

    Barley (Hordeum vulgare L. Himalaya) mutants with altered grain morphology were isolated to investigate whether defects in grain development, possibly involving gibberellins (GAs) and abscisic acid, would lead to altered patterns of [alpha]-amylase gene expression. Following treatment with sodium azide, 75 mutants, typically showing grain shriveling, were identified. At grain maturity 15 of the 75 mutants had higher [alpha]-amylase activities in shriveled grains compared with either phenotypically normal grains that developed on the same heterozygous plant or with grains of cv Himalaya. Studies of four of these mutants demonstrated increased levels of both high- and low-isoelectric point [alpha]-amylase isozymes midway through grain development. This category of mutant has been designated pga, for premature grain [alpha]-amylase. One such mutant (M326) showed an endosperm-determined inheritance pattern. When crossed into a (GA-deficient) dwarfing background there was a 10- to 20-fold reduction in [alpha]-amylase activity, suggesting a requirement for GA biosynthesis. Endogenous GAs and abscisic acid were quantified by combined gas chromatography-specific ion monitoring in normal and mutant grains of heterozygous M326 plants during the period of [alpha]-amylase accumulation. Mutant grains had significantly higher (5.8-fold) levels of the bioactive GA1 compared with normal grains but much lower (approximately 10-fold) levels of the 2[beta]-hydroxylated ("inactive") GAs, typical of developing barley grains (e.g. GA8, GA34, GA48). We propose that a reduced extent of 2[beta]-hydroxylation in the mutant grains results in an increased level of GA1, which is responsible for premature [alpha]-amylase gene expression. PMID:12223700

  3. Role of arachidonic acid and protein kinase C during maturation-inducing hormone-dependent meiotic resumption and ovulation in ovarian follicles of Atlantic croaker

    USGS Publications Warehouse

    Patino, R.; Yoshizaki, G.; Bolamba, D.; Thomas, P.

    2003-01-01

    The roles of arachidonic acid (AA) and protein kinase C (PKC) during in vitro maturation-inducing hormone (MIH)-dependent meiotic resumption (maturation) and ovulation were studied in ovarian follicles of Atlantic croaker (Micropogonias undulatus). The requirement for cyclooxygenase (COX) metabolites of AA was examined using a nonspecific COX inhibitor, indomethacin (IM), as well as two COX products, prostaglandin (PG) F2?? and PGE2, whereas the role of lipoxygenase (LOX) was investigated using a specific LOX inhibitor, nordihydroguaiaretic acid (NDGA). The involvement of PKC was examined using phorbol 12-myristate 13-acetate (PMA), a PKC activator, as well as GF109203X (GF), a specific inhibitor of PKC and 1-(5-isoquin- olinesulfonyl)-2-methylpiperazine (H7), nonspecific inhibitor of protein kinases. Genomic mechanisms were examined with the transcription-inhibitor actinomycin D (ActD) and the functionality of heterologous (oocyte-granulosa) gap junctions (GJ) with a dye transfer assay. The AA (100 ??M) and PGF2?? (5 ??M) did not induce maturation, and NDGA (10 ??M) did not affect MIH-dependent maturation. However, IM (100 ??M) partially inhibited MIH-dependent maturation. Conversely, AA and both PGs induced, and IM and NDGA inhibited, MIH-dependent ovulation in matured follicles. The PMA (1 ??g/ml) did not induce maturation but caused ovulation in matured follicles, whereas PKC inhibitors (GF, 5 ??M; H7, 50??M) did not affect MIH-dependent maturation but inhibited MIH- and PMA-dependent ovulation. The PMA-dependent ovulation was inhibited by IM but not by NDGA. In addition, ActD (5 ??M) blocked MIH-dependent, but not PMA-dependent, ovulation, and PGF2?? restored MIH-dependent ovulation in ActD-blocked follicles. The AA and PGs did not induce, and GF did not inhibit, MIH-dependent heterologous GJ uncoupling. In conclusion, AA and PKC mediate MIH-dependent ovulation but not meiotic resumption or heterologous GJ uncoupling in croaker follicles, but a permissive role

  4. Folic Acid Inhibits Amyloid β-Peptide Production through Modulating DNA Methyltransferase Activity in N2a-APP Cells

    PubMed Central

    Li, Wen; Jiang, Mingyue; Zhao, Shijing; Liu, Huan; Zhang, Xumei; Wilson, John X.; Huang, Guowei

    2015-01-01

    Alzheimer’s disease (AD) is a common neurodegenerative disease resulting in progressive dementia, and is a principal cause of dementia among older adults. Folate acts through one-carbon metabolism to support the methylation of multiple substrates. We hypothesized that folic acid supplementation modulates DNA methyltransferase (DNMT) activity and may alter amyloid β-peptide (Aβ) production in AD. Mouse Neuro-2a cells expressing human APP695 were incubated with folic acid (2.8–40 μmol/L), and with or without zebularine (the DNMT inhibitor). DNMT activity, cell viability, Aβ and DNMTs expression were then examined. The results showed that folic acid stimulated DNMT gene and protein expression, and DNMT activity. Furthermore, folic acid decreased Aβ protein production, whereas inhibition of DNMT activity by zebularine increased Aβ production. The results indicate that folic acid induces methylation potential-dependent DNMT enzymes, thereby attenuating Aβ production. PMID:26492244

  5. Folic Acid Inhibits Amyloid β-Peptide Production through Modulating DNA Methyltransferase Activity in N2a-APP Cells.

    PubMed

    Li, Wen; Jiang, Mingyue; Zhao, Shijing; Liu, Huan; Zhang, Xumei; Wilson, John X; Huang, Guowei

    2015-01-01

    Alzheimer's disease (AD) is a common neurodegenerative disease resulting in progressive dementia, and is a principal cause of dementia among older adults. Folate acts through one-carbon metabolism to support the methylation of multiple substrates. We hypothesized that folic acid supplementation modulates DNA methyltransferase (DNMT) activity and may alter amyloid β-peptide (Aβ) production in AD. Mouse Neuro-2a cells expressing human APP695 were incubated with folic acid (2.8-40 μmol/L), and with or without zebularine (the DNMT inhibitor). DNMT activity, cell viability, Aβ and DNMTs expression were then examined. The results showed that folic acid stimulated DNMT gene and protein expression, and DNMT activity. Furthermore, folic acid decreased Aβ protein production, whereas inhibition of DNMT activity by zebularine increased Aβ production. The results indicate that folic acid induces methylation potential-dependent DNMT enzymes, thereby attenuating Aβ production. PMID:26492244

  6. Short communication: Measuring the angiotensin-converting enzyme inhibitory activity of an 8-amino acid (8mer) fragment of the C12 antihypertensive peptide

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An eight amino acid fragment (PFPEVFGK) of a known milk protein-derived antihypertensive peptide was synthesized by microwave-assisted solid phase peptide synthesis and purified by reverse phase HPLC. Its ability to inhibit the angiotensin-converting enzyme was assessed and compared to that of the ...

  7. Radiation chemistry of amino acids, peptides and proteins in relation to the radiation sterilization of high-protein foods

    SciTech Connect

    Garrison, W. M.

    1981-12-01

    An important source of information on the question of whether or not toxic or other deleterious substances are formed in the radiation sterilization of foods is the chemical study of reaction products and reaction mechanisms in the radiolysis of individual food components. The present evaluation of the radiation chemistry of amino acids, peptides, and proteins outlines the various radiation-induced processes which lead to amino acid degradation and to the synthesis of amino acid derivatives of higher molecular weight. Among the latter are the ..cap alpha..,..cap alpha..'-diamino dicarboxylic acids which are formed as major products in the radiolysis of peptides both in aqueous solution and in the solid state. The ..cap alpha..,..cap alpha..'-diamino acids are of particular interest as irradiation products because they represent a class of compounds not normally encountered in plant and animal protein sources. Such compounds have, however, been isolated from certain types of bacteria and bacterial products. All of the available data strongly suggest that the ..cap alpha..,..cap alpha..'-diamino acids are produced in significant yield in the radiation sterilization of high protein foods. The importance of initiating extensive chemical and biological studies of these and of other high molecular weight products in irradiated food is emphasized.

  8. Synthesis and structural characterization of monomeric and dimeric peptide nucleic acids prepared by using microwave-promoted multicomponent reactions.

    PubMed

    Ovadia, Reuben; Lebrun, Aurélien; Barvik, Ivan; Vasseur, Jean-Jacques; Baraguey, Carine; Alvarez, Karine

    2015-12-01

    A solution phase synthesis of peptide nucleic acid monomers and dimers was developed by using microwave-promoted Ugi multicomponent reactions. A mixture of a functionalized amine, a carboxymethyl nucleobase, paraformaldehyde and an isocyanide as building blocks generates PNA monomers which are then partially deprotected and used in a second Ugi 4CC reaction, leading to PNA dimers. Conformational rotamers were identified by using NMR and MD simulations. PMID:26394794

  9. Visualization of the mycelia of wood-rotting fungi by fluorescence in situ hybridization using a peptide nucleic acid probe.

    PubMed

    Nakada, Yuji; Nakaba, Satoshi; Matsunaga, Hiroshi; Funada, Ryo; Yoshida, Makoto

    2013-01-01

    White rot fungus, Phanerochaete chrysosporium, and brown rot fungus, Postia placenta, grown on agar plates, were visualized by fluorescence in situ hybridization (FISH) using a peptide nucleic acid (PNA) probe. Mycelia grown on wood chips were also clearly detected by PNA-FISH following blocking treatment. To the best of our knowledge, this is the first report on the visualization of fungi in wood by FISH. PMID:23391931

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

    PubMed

    Topham, Christopher M; Smith, Jeremy C

    2015-02-01

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

  11. Dual Peptide Nucleic Acid- and Peptide-functionalized Shell Crosslinked Nanoparticles Designed to Target mRNA toward the Diagnosis and Treatment of Acute Lung Injury

    PubMed Central

    Shrestha, Ritu; Shen, Yuefei; Pollack, Kevin A.; Taylor, John-Stephen A.; Wooley, Karen L.

    2012-01-01

    In this work, multi-functional bio-synthetic hybrid nanostructures were prepared and studied for their potential utility in the recognition and inhibition of mRNA sequences for inducible nitric oxide synthase (iNOS), which are overexpressed at sites of inflammation, such as in cases of acute lung injury. Shell crosslinked knedel-like polymer nanoparticles (SCKs) that present peptide nucleic acids, for binding to complementary mRNAs, and cell penetrating peptides (CPPs), to gain cell entry, along with fluorescent labels and sites for radiolabeling, were prepared by a series of robust, efficient and versatile synthetic steps that proceeded from monomers to polymers to functional nanoparticles. Amphiphilic block graft copolymers having combinations of methoxy- and thioacetyl-terminated poly(ethylene glycol) (PEG) and DOTA-lysine units grafted from the backbone of poly(acrylic acid) (PAA) and extending with a backbone segment of poly(octadecyl acrylate-co-decyl acrylate) (P(ODA-co-DA)) were prepared by a combination of reversible addition-fragmentation chain transfer (RAFT) polymerization and chemical modification reactions, which were then used as the building blocks for the formation of well-defined SCKs decorated with reactive thiols accessible to the surface. Fluorescent labeling with Alexa Fluor 633 hydrazide was then accomplished by amidation with residual acrylic acid residues within the SCK shells. Finally, the PNAs and CPP units were covalently conjugated to the SCKs via Michael addition of thiols on the SCKs to maleimide units on the termini of PNAs and CPPs. Confirmation of the ability of the PNAs to bind selectively to the target iNOS mRNAs when tethered to the SCK nanoparticles was determined by in vitro competition experiments. When attached to the SCKs having a hydrodynamic diameter of 60 ± 16 nm, the Kd values of the PNAs were ca. an order of magnitude greater than the free PNAs, while the mismatched PNA showed no significant binding. PMID:22372643

  12. Molecular mechanism of acid-catalyzed hydrolysis of peptide bonds using a model compound.

    PubMed

    Pan, Bin; Ricci, Margaret S; Trout, Bernhardt L

    2010-04-01

    The stability of peptide bonds is a critical aspect of biological chemistry and therapeutic protein applications. Recent studies found elevated nonenzymatic hydrolysis in the hinge region of antibody molecules, but no mechanism was identified. As a first step in providing a mechanistic interpretation, this computational study examines the rate-determining step of the hydrolytic reaction of a peptide bond under acidic pH by a path sampling technique using a model compound N-MAA. Most previous computational studies did not include explicit water molecules, whose effects are significant in solution chemistry, nor did they provide a dynamic picture for the reaction process in aqueous conditions. Because no single trajectory can be used to describe the reaction dynamics due to fluctuations at finite temperatures, a variant version of the transition path sampling technique, the aimless shooting algorithm, was used to sample dynamic trajectories and to generate an ensemble of transition trajectories according to their statistical weights in the trajectory space. Each trajectory was computed as the time evolution of the molecular system using the Car-Parrinello molecular dynamics technique. The likelihood maximization procedure and its modification were used in extracting dynamically relevant degrees of freedom in the system, and approximations of the reaction coordinate were compared. Its low log-likelihood score and poor p(B) histogram indicate that the C-O distance previously assumed as the reaction coordinate for the rate-determining step is inadequate in describing the dynamics of the reaction. More than one order parameter in a candidate set including millions of geometric quantities was required to produce a convergent reaction coordinate model; its involvement of many degrees of freedom suggests that this hydrolytic reaction step is very complex. In addition to affecting atoms directly involved in bond-making and -breaking processes, the water network also has

  13. Enrichment of ACE inhibitory peptides in navy bean (Phaseolus vulgaris) using lactic acid bacteria.

    PubMed

    Rui, Xin; Wen, Delan; Li, Wei; Chen, Xiaohong; Jiang, Mei; Dong, Mingsheng

    2015-02-01

    The present study was conducted to explore a novel strategy to enhance angiotensin I-converting enzyme (ACE) inhibitory activities of navy bean by preparation of navy bean milk (NBM) which was then subjected to fermentation of four lactic acid bacteria (LAB) strains, namely, Lactobacillus bulgaricus, Lactobacillus helveticus MB2-1, Lactobacillus plantarum B1-6, and Lactobacillus plantarum 70810. With the exception of L. helveticus MB2-1, the other three selected strains had good growth performances in NBM with viable counts increased to log 8.30-8.39 cfu ml(-1) during 6 h of fermentation, and thus were selected for the following investigations. Protein contents of NBM significantly reduced when treated with L. bulgaricus and L. plantarum B1-6, and the electrophoresis patterns showed the preferable proteins for LAB strains to hydrolyze were α- and β-type phaseolins, whereas γ-type phaseolin was resistant to hydrolysis. RP-HPLC analysis demonstrated all fermented NBM had higher intensities of peaks with retention times between 2.5 and 3.5 min indicative of formation of small peptides. All fermented NBM showed higher ACE inhibitory activity compared to the unfermented ones, for which 2 h, 3 h, and 5 h were found to be the optimum fermentation periods for respectively L. plantarum 70810, L. plantarum B1-6 and L. bulgaricus, with IC50 values of 109 ± 5.1, 108 ± 1.1, and 101 ± 2.2 μg protein ml(-1). The subsequent in vitro gastrointestinal simulation afforded all fermented extracts reduced IC50 values and the extracts fermented by L. plantarum B1-6 exerted the lowest IC50 value of 21 ± 2.1 μg protein ml(-1). The research has broadened our knowledge bases on the effect of LAB fermentation on the degradation of navy bean proteins and the capacity to release ACE inhibitory peptides. The approach was promising to obtain probiotic products with potential to serve as functional ingredients targeting hypertension. PMID:25536445

  14. The Polyunsaturated Fatty Acids Arachidonic Acid and Docosahexaenoic Acid Induce Mouse Dendritic Cells Maturation but Reduce T-Cell Responses In Vitro

    PubMed Central

    Carlsson, Johan A.; Wold, Agnes E.; Sandberg, Ann-Sofie; Östman, Sofia M.

    2015-01-01

    Long-chain polyunsaturated fatty acids (PUFAs) might regulate T-cell activation and lineage commitment. Here, we measured the effects of omega-3 (n-3), n-6 and n-9 fatty acids on the interaction between dendritic cells (DCs) and naïve T cells. Spleen DCs from BALB/c mice were cultured in vitro with ovalbumin (OVA) with 50 μM fatty acids; α-linolenic acid, arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid or oleic acid and thereafter OVA-specific DO11.10 T cells were added to the cultures. Fatty acids were taken up by the DCs, as shown by gas chromatography analysis. After culture with arachidonic acid or DHA CD11c+ CD11b+ and CD11c+ CD11bneg DCs expressed more CD40, CD80, CD83, CD86 and PDL-1, while IAd remained unchanged. However, fewer T cells co-cultured with these DCs proliferated (CellTrace Violetlow) and expressed CD69 or CD25, while more were necrotic (7AAD+). We noted an increased proportion of T cells with a regulatory T cell (Treg) phenotype, i.e., when gating on CD4+ FoxP3+ CTLA-4+, CD4+ FoxP3+ Helios+ or CD4+ FoxP3+ PD-1+, in co-cultures with arachidonic acid- or DHA-primed DCs relative to control cultures. The proportion of putative Tregs was inversely correlated to T-cell proliferation, indicating a suppressive function of these cells. With arachidonic acid DCs produced higher levels of prostaglandin E2 while T cells produced lower amounts of IL-10 and IFNγ. In conclusion arachidonic acid and DHA induced up-regulation of activation markers on DCs. However arachidonic acid- and DHA-primed DCs reduced T-cell proliferation and increased the proportion of T cells expressing FoxP3, indicating that these fatty acids can promote induction of regulatory T cells. PMID:26619195

  15. Inhibition of IgA1 proteinases from Neisseria gonorrhoeae and Hemophilus influenzae by peptide prolyl boronic acids.

    PubMed

    Bachovchin, W W; Plaut, A G; Flentke, G R; Lynch, M; Kettner, C A

    1990-03-01

    The alpha-aminoboronic acid analog of proline has been synthesized and incorporated into a number of peptides as the COOH-terminal residue. These peptide prolyl boronic acids are potent inhibitors of both the type 1 and type 2 IgA proteinases from Neisseria gonorrhoeae and Hemophilus influenzae, but not of the functionally similar IgA proteinase from Streptococcus sanguis. The best inhibitors synthesized thus far have Ki values in the nanomolar range (4.0 to 60 nM). These results indicate that the N. gonorrhoeae and the H. influenzae enzymes belong to the serine protease family of proteolytic enzymes while that from S. sanguis does not. As a group, the IgA proteinases have been noted for their remarkable specificity; thus, the peptide prolyl boronic acids reported here are the first small synthetic molecules to exhibit a relatively high affinity for the active site of an IgA proteinase and are therefore the first to yield some insight into the active site structure and specificity requirements of these enzymes. PMID:2105953

  16. Antioxidative Peptides Derived from Enzyme Hydrolysis of Bone Collagen after Microwave Assisted Acid Pre-Treatment and Nitrogen Protection

    PubMed Central

    Lin, Yun-Jian; Le, Guo-Wei; Wang, Jie-Yun; Li, Ya-Xin; Shi, Yong-Hui; Sun, Jin

    2010-01-01

    This study focused on the preparation method of antioxidant peptides by enzymatic hydrolysis of bone collagen after microwave assisted acid pre-treatment and nitrogen protection. Phosphoric acid showed the highest ability of hydrolysis among the four other acids tested (hydrochloric acid, sulfuric acid and/or citric acid). The highest degree of hydrolysis (DH) was 9.5% using 4 mol/L phosphoric acid with a ratio of 1:6 under a microwave intensity of 510 W for 240 s. Neutral proteinase gave higher DH among the four protease tested (Acid protease, neutral protease, Alcalase and papain), with an optimum condition of: (1) ratio of enzyme and substrate, 4760 U/g; (2) concentration of substrate, 4%; (3) reaction temperature, 55 °C and (4) pH 7.0. At 4 h, DH increased significantly (P < 0.01) under nitrogen protection compared with normal microwave assisted acid pre-treatment hydrolysis conditions. The antioxidant ability of the hydrolysate increased and reached its maximum value at 3 h; however DH decreased dramatically after 3 h. Microwave assisted acid pre-treatment and nitrogen protection could be a quick preparatory method for hydrolyzing bone collagen. PMID:21151439

  17. The delta EEG (sleep)-inducing peptide (DSIP). XI. Amino-acid analysis, sequence, synthesis and activity of the nonapeptide.

    PubMed

    Schoenenberger, G A; Maier, P F; Tobler, H J; Wilson, K; Monnier, M

    1978-09-01

    A peptide which induces slow-wave EEG (sleep) after intraventricular infusion into the brain has been isolated from the extracorporeal dialysate of cerebral venous blood in rabbits submitted to hypnogenic electrical stimulation of the intralaminar thalamic area. It was shown by amino-acid analysis and sequence determination to be Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu and named "Delta Sleep-Inducing Peptide" (DSIP). This compound was synthesized as well as 5 possible metabolic products (1--8, 2--9, 2--8, 1--4 and 5--9), 2 nonapeptide analogues (with one and two amino-acids exchanged) and a related tripeptide (Trp-Ser-Glu). All 9 synthetic peptides were infused intraventricularly in rabbits (6 nmol/kg in 0.05 ml of CSF-like solution over 3.5 min) and tested under double-blind conditions. A total of 61 rabbits including controls were used. The EEG from the frontal neocortex and the limbic archicortex were subjected to direct fast-Fourier transformation and analyzed by an 1108 computer system. A highly specific delta and spindle EEG-enhancing effect of the synthetic DSIP could be demonstrated. The mean increase of EEG delta activity reached 35% in the neocortex and limbic cortex as compared to control animals receiving CSF-like solution or any of the other 8 peptides. The final chemical characterization of the synthetic DSIP revealed that only the pure alpha-aspartyl peptide is highly active in contrast to its beta-Asp isomer. A neurohumoral modulating and programming activity was suggested. PMID:568769

  18. An investigation of the effects of simulated acid rain and elevated ozone on the physiology of ponderosa pine (Pinus ponderosa Dougl. ex Laws.) seedlings and mature trees

    SciTech Connect

    Momen, B.

    1993-12-31

    This study investigated the combined effects of simulated acid rain and ozone on foliar water relations, carbon and nitrogen contents, gas exchange, and respiration of ponderosa pine seedlings and mature trees grown in the field at the USDA Forest Service Tree Improvement Center in Chico, California. Acid rain levels (pH 5.1 and 3) were applied weekly on foliage only, from January to April, 1992. Plants were exposed to ozone levels (ambient and twice ambient) during the day only, from August to December, 1990, and from September to November, 1992. Results suggested that elevated ozone, particularly in combination with strong acid, caused osmotic adjustment that may benefit plants during drought. The observed effects of pollutants are similar to the reported effects of drought on plant water relations. Elevated ozone decreased foliar nitrogen content and thus increased the C:N ratio, particularly in seedlings. Stomatal conductance was not affected by pollutants but net photosynthesis was decreased by elevated ozone, especially in mature trees. The greater sensitivity of net photosynthesis of mature trees to elevated ozone was contrary to all other plant characteristics investigated. Elevated ozone increased seedling respiration. Under controlled, temperature, light, and vapor pressure deficit conditions, net photosynthesis responded positively to increases in plant age, light intensity, and rain pH, but negatively to increases in tissue age, heat, and ozone concentration. Overall results indicated that acid rain and elevated ozone declined the carbon pool of ponderosa pine due to increased respiration and decreased net photosynthesis. Pollutant effects were more profound in mid-summer when ozone concentrations were highest. On many occasions the effects of acid rain and ozone levels interacted. Seedlings were more sensitive to pollutants than mature trees.

  19. Ribosome-mediated incorporation of a non-standard amino acid into a peptide through expansion of the genetic code.

    PubMed

    Bain, J D; Switzer, C; Chamberlin, A R; Benner, S A

    1992-04-01

    One serious limitation facing protein engineers is the availability of only 20 'proteinogenic' amino acids encoded by natural messenger RNA. The lack of structural diversity among these amino acids restricts the mechanistic and structural issues that can be addressed by site-directed mutagenesis. Here we describe a new technology for incorporating non-standard amino acids into polypeptides by ribosome-based translation. In this technology, the genetic code is expanded through the creation of a 65th codon-anticodon pair from unnatural nucleoside bases having non-standard hydrogen-bonding patterns. This new codon-anticodon pair efficiently supports translation in vitro to yield peptides containing a non-standard amino acid. The versatility of the ribosome as a synthetic tool offers new possibilities for protein engineering, and compares favourably with another recently described approach in which the genetic code is simply rearranged to recruit stop codons to play a coding role. PMID:1560827

  20. Isoxazole-Derived Amino Acids are Bromodomain-Binding Acetyl-Lysine Mimics: Incorporation into Histone H4 Peptides and Histone H3.

    PubMed

    Sekirnik Née Measures, Angelina R; Hewings, David S; Theodoulou, Natalie H; Jursins, Lukass; Lewendon, Katie R; Jennings, Laura E; Rooney, Timothy P C; Heightman, Tom D; Conway, Stuart J

    2016-07-11

    A range of isoxazole-containing amino acids was synthesized that displaced acetyl-lysine-containing peptides from the BAZ2A, BRD4(1), and BRD9 bromodomains. Three of these amino acids were incorporated into a histone H4-mimicking peptide and their affinity for BRD4(1) was assessed. Affinities of the isoxazole-containing peptides are comparable to those of a hyperacetylated histone H4-mimicking cognate peptide, and demonstrated a dependence on the position at which the unnatural residue was incorporated. An isoxazole-based alkylating agent was developed to selectively alkylate cysteine residues in situ. Selective monoalkylation of a histone H4-mimicking peptide, containing a lysine to cysteine residue substitution (K12C), resulted in acetyl-lysine mimic incorporation, with high affinity for the BRD4 bromodomain. The same technology was used to alkylate a K18C mutant of histone H3. PMID:27264992

  1. Solid-Phase Synthesis with Attachment of Peptide to Resin through an Amino Acid Side Chain: [8-Lysine]-Vasopressin

    PubMed Central

    Meienhofer, Johannes; Trzeciak, Arnold

    1971-01-01

    It is proposed that the scope of solid-phase peptide synthesis could be considerably broadened by attaching peptides to the solid-phase through functional side-chain groups rather than through the commonly used α-carboxyl groups. Side-chain attachment offers the use of a large variety of chemical linkages to solid supports. Attachment through the ε-amino group of the lysine residue to a polystyrene resin has been applied to a solid-phase synthesis of lysine-vasopressin. Nα-tert-butyl-oxycarbonyl-L-lysyl-glycinamide was condensed with chloroformoxymethyl polystyrene-2% divinylbenzene resin. After removal of the Nα-protecting tert-butyloxycarbonyl group, the peptide chain was elongated by standard Merrifield procedures to give Tos-Cys(Bzl)-Tyr-Phe-Glu-(NH2) - Asp(NH2) - Cys(Bzl) - Pro - Lys(Z - resin) - Gly-NH2. Cleavage from the resin with HBr in dioxane or trifluoroacetic acid gave a partially protected nonapeptide hydrobromide. For purification, it was converted into a fully protected peptide by treatment with benzyl p-nitro-phenyl carbonate and crystallized. Deprotection by sodium in liquid ammonia, oxidative cyclization, IRC-50 desalting, and ion-exchange chromatography gave lysinevasopressin with high potency in a rat-pressor assay. PMID:5280519

  2. Evolutionary Importance of the Intramolecular Pathways of Hydrolysis of Phosphate Ester Mixed Anhydrides with Amino Acids and Peptides

    NASA Astrophysics Data System (ADS)

    Liu, Ziwei; Beaufils, Damien; Rossi, Jean-Christophe; Pascal, Robert

    2014-12-01

    Aminoacyl adenylates (aa-AMPs) constitute essential intermediates of protein biosynthesis. Their polymerization in aqueous solution has often been claimed as a potential route to abiotic peptides in spite of a highly efficient CO2-promoted pathway of hydrolysis. Here we investigate the efficiency and relevance of this frequently overlooked pathway from model amino acid phosphate mixed anhydrides including aa-AMPs. Its predominance was demonstrated at CO2 concentrations matching that of physiological fluids or that of the present-day ocean, making a direct polymerization pathway unlikely. By contrast, the occurrence of the CO2-promoted pathway was observed to increase the efficiency of peptide bond formation owing to the high reactivity of the N-carboxyanhydride (NCA) intermediate. Even considering CO2 concentrations in early Earth liquid environments equivalent to present levels, mixed anhydrides would have polymerized predominantly through NCAs. The issue of a potential involvement of NCAs as biochemical metabolites could even be raised. The formation of peptide-phosphate mixed anhydrides from 5(4H)-oxazolones (transiently formed through prebiotically relevant peptide activation pathways) was also observed as well as the occurrence of the reverse cyclization process in the reactions of these mixed anhydrides. These processes constitute the core of a reaction network that could potentially have evolved towards the emergence of translation.

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

    PubMed Central

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

    2015-01-01

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

  4. Design and evaluation of peptide nucleic acid probes for specific identification of Candida albicans.

    PubMed

    Kim, Hyun-Joong; Brehm-Stecher, Byron F

    2015-02-01

    Candida albicans is an important cause of systemic fungal infections, and rapid diagnostics for identifying and differentiating C. albicans from other Candida species are critical for the timely application of appropriate antimicrobial therapy, improved patient outcomes, and pharmaceutical cost savings. In this work, two 28S rRNA-directed peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) probes, P-Ca726 (targeting a novel region of the ribosome) and P-CalB2208 (targeting a previously reported region), were evaluated. Hybridization conditions were optimized by using both fluorescence microscopy (FM) and flow cytometry (FCM), and probes were screened for specificity and discriminative ability against a panel of C. albicans and various nontarget Candida spp. The performance of these PNA probes was compared quantitatively against that of DNA probes or DNA probe/helper combinations directed against the same target regions. Ratiometric analyses of FCM results indicated that both the hybridization quality and yield of the PNA probes were higher than those of the DNA probes. In FCM-based comparisons of the PNA probes, P-Ca726 was found to be highly specific, showing 2.5- to 5.5-fold-higher discriminatory power for C. albicans than P-CalB2208. The use of formamide further improved the performance of the new probe. Our results reinforce the significant practical and diagnostic advantages of PNA probes over their DNA counterparts for FISH and indicate that P-Ca726 may be used advantageously for the rapid and specific identification of C. albicans in clinical and related applications, especially when combined with FCM. PMID:25428160

  5. Use of a peptide rather than free amino acid nitrogen source in chemically defined "elemental" diets.

    PubMed

    Silk, D B; Fairclough, P D; Clark, M L; Hegarty, J E; Marrs, T C; Addison, J M; Burston, D; Clegg, K M; Matthews, D M

    1980-01-01

    Previous studies have shown that amino acid (AA) residues are absorbed more rapidly from di- tripeptides than from free AA. In the present study, an intestinal perfusion technique has been used in normal human subjects to compare absorption of AA residues and total alpha-amino nitrogen (N) from 4 partial enzymic hydrolysates of protein (50--80% of the N contents present as small peptides) and their respective equimolar free AA mixtures. alpha-Amino N absorption was greater from 2 casein hydrolytes and a lactalbumin hydrolysate than from the respective free AA mixtures but similar to that from a fish protein hydrolysate and its AA mixture. The considerable variation in absorption of individual AA residues from the AA mixtures was much reduced when the protein hydrolysates were perfused, as a number of AA which were poorly absorbed from the AA mixtures were absorbed to a greater extent from the protein hydrolysates. The casein and lactalbumin hydrolysates had a stimulatory effect on jejunal absorption of water and electrolytes. In contrast, the fish protein hydrolysate appeared to cause a mean net secretion of fluid and electrolytes. The findings indicate that when absorption is limited by diminished luminal hydrolysis or absorptive capacity, serious consideration might be given to using partial enzymic hydrolysates of whole protein rather than free AA mixtures as the N source in "elemental" diets. Care should be taken, however, in ensuring that the preparation of choice does not promote a net secretion of fluid and electrolytes for such a property could have a deleterious effect in the clinical setting. PMID:6780707

  6. Altering behavioral responses and dopamine transporter protein with antisense peptide nucleic acids.

    PubMed

    Tyler-McMahon, B M; Stewart, J A; Jackson, J; Bitner, M D; Fauq, A; McCormick, D J; Richelson, E

    2001-10-01

    The dopamine transporter (DAT) plays a role in locomotion and is an obligatory target for amphetamines. We designed and synthesized an antisense peptide nucleic acid (PNA) to rat DAT to examine the effect of this antisense molecule on locomotion and on responsiveness to amphetamines. Rats were injected intraperitoneally daily for 9 days with either saline, an antisense DAT PNA, a scrambled DAT PNA, or a mismatch DAT PNA. On days 7 and 9 after initial motility measurements were taken, the animals were challenged with 10 mg/kg of amphetamine and scored for motility. On day 7, there was no significant difference between the baseline levels of activity of any of the groups or their responses to amphetamine. On day 9, the antisense PNA-treated rats showed a statistically significant increase in their resting motility (P < 0.01). When these rats were challenged with amphetamine, motility of the saline-, scrambled PNA-, and mismatch PNA-treated animals showed increases of 31-, 36-, and 20-fold, respectively, while the antisense PNA-treated animals showed increases of only 3.4-fold (P < 0.01). ELISA results revealed a 32% decrease in striatal DAT in antisense PNA-treated rats compared with the saline, scrambled PNA, and mismatch PNA controls (P < 0.001). These results extend our previous findings that brain proteins can be knocked down in a specific manner by antisense molecules administered extracranially. Additionally, these results suggest some novel approaches for the treatment of diseases dependent upon the function of the dopamine transporter. PMID:11543728

  7. Design and Evaluation of Peptide Nucleic Acid Probes for Specific Identification of Candida albicans

    PubMed Central

    Kim, Hyun-Joong

    2014-01-01

    Candida albicans is an important cause of systemic fungal infections, and rapid diagnostics for identifying and differentiating C. albicans from other Candida species are critical for the timely application of appropriate antimicrobial therapy, improved patient outcomes, and pharmaceutical cost savings. In this work, two 28S rRNA-directed peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) probes, P-Ca726 (targeting a novel region of the ribosome) and P-CalB2208 (targeting a previously reported region), were evaluated. Hybridization conditions were optimized by using both fluorescence microscopy (FM) and flow cytometry (FCM), and probes were screened for specificity and discriminative ability against a panel of C. albicans and various nontarget Candida spp. The performance of these PNA probes was compared quantitatively against that of DNA probes or DNA probe/helper combinations directed against the same target regions. Ratiometric analyses of FCM results indicated that both the hybridization quality and yield of the PNA probes were higher than those of the DNA probes. In FCM-based comparisons of the PNA probes, P-Ca726 was found to be highly specific, showing 2.5- to 5.5-fold-higher discriminatory power for C. albicans than P-CalB2208. The use of formamide further improved the performance of the new probe. Our results reinforce the significant practical and diagnostic advantages of PNA probes over their DNA counterparts for FISH and indicate that P-Ca726 may be used advantageously for the rapid and specific identification of C. albicans in clinical and related applications, especially when combined with FCM. PMID:25428160

  8. A Prosaposin-Derived Peptide Alleviates Kainic Acid-Induced Brain Injury

    PubMed Central

    Nabeka, Hiroaki; Shimokawa, Tetsuya; Doihara, Takuya; Saito, Shouichiro; Wakisaka, Hiroyuki; Hamada, Fumihiko; Kobayashi, Naoto; Matsuda, Seiji

    2015-01-01

    Four sphingolipid activator proteins (i.e., saposins A–D) are synthesized from a single precursor protein, prosaposin (PS), which exerts exogenous neurotrophic effects in vivo and in vitro. Kainic acid (KA) injection in rodents is a good model in which to study neurotrophic factor elevation; PS and its mRNA are increased in neurons and the choroid plexus in this animal model. An 18-mer peptide (LSELIINNATEELLIKGL; PS18) derived from the PS neurotrophic region prevents neuronal damage after ischemia, and PS18 is a potent candidate molecule for use in alleviating ischemia-induced learning disabilities and neuronal loss. KA is a glutamate analog that stimulates excitatory neurotransmitter release and induces ischemia-like neuronal degeneration; it has been used to define mechanisms involved in neurodegeneration and neuroprotection. In the present study, we demonstrate that a subcutaneous injection of 0.2 and 2.0 mg/kg PS18 significantly improved behavioral deficits of Wistar rats (n = 6 per group), and enhanced the survival of hippocampal and cortical neurons against neurotoxicity induced by 12 mg/kg KA compared with control animals. PS18 significantly protected hippocampal synapses against KA-induced destruction. To evaluate the extent of PS18- and KA-induced effects in these hippocampal regions, we performed histological evaluations using semithin sections stained with toluidine blue, as well as ordinal sections stained with hematoxylin and eosin. We revealed a distinctive feature of KA-induced brain injury, which reportedly mimics ischemia, but affects a much wider area than ischemia-induced injury: KA induced neuronal degeneration not only in the CA1 region, where neurons degenerate following ischemia, but also in the CA2, CA3, and CA4 hippocampal regions. PMID:25993033

  9. Construction of a novel peptide nucleic acid piezoelectric gene sensor microarray detection system.

    PubMed

    Chen, Ming; Liu, Minghua; Yu, Lili; Cai, Guoru; Chen, Qinghai; Wu, Rong; Wang, Feng; Zhang, Bo; Jiang, Tianlun; Fu, Welling

    2005-08-01

    A novel 2 x 5 clamped style piezoelectric gene sensor microarray has been successfully constructed. Every crystal unit of the fabricated gene sensor can oscillate independently without interfering with each other. The bis-peptide nucleic acid (bis-PNA) probe, which can combine with target DNA or RNA sequences more effectively and specifically than a DNA probe, was designed and immobilized on the surface of the gene sensor microarray to substitute the conventional DNA probe for direct detection of the hepatitis B virus (HBV) genomic DNA. Detection conditions were then explored and optimized. Results showed that PBS buffer of pH 6.8, an ion concentration of 20 mmol/liter, and a probe concentration of 1.5 micromol/liter were optimal for the detection system. Under such optimized experimental conditions, the specificity of bis-PNA was proved much higher than that of DNA probe. The relationship between quantity of target and decrease of frequency showed a typical saturation curve when concentrations of target HBV DNA varied from 10 pg/liter to 100 microg/liter, and 10 microg/liter was the watershed, with a statistic linear regression equation of I gC = -2.7455 + 0.0691 deltaF and the correlating coefficient of 0.9923. Fortunately, this is exactly the most ordinary variant range of the HBV virus concentration in clinical hepatitis samples. So, a good technical platform is successfully constructed and it will be applied to detect HBV quantitatively in clinical samples. PMID:16193990

  10. 2D-Qsar for 450 types of amino acid induction peptides with a novel substructure pair descriptor having wider scope

    PubMed Central

    2011-01-01

    Background Quantitative structure-activity relationships (QSAR) analysis of peptides is helpful for designing various types of drugs such as kinase inhibitor or antigen. Capturing various properties of peptides is essential for analyzing two-dimensional QSAR. A descriptor of peptides is an important element for capturing properties. The atom pair holographic (APH) code is designed for the description of peptides and it represents peptides as the combination of thirty-six types of key atoms and their intermediate binding between two key atoms. Results The substructure pair descriptor (SPAD) represents peptides as the combination of forty-nine types of key substructures and the sequence of amino acid residues between two substructures. The size of the key substructures is larger and the length of the sequence is longer than traditional descriptors. Similarity searches on C5a inhibitor data set and kinase inhibitor data set showed that order of inhibitors become three times higher by representing peptides with SPAD, respectively. Comparing scope of each descriptor shows that SPAD captures different properties from APH. Conclusion QSAR/QSPR for peptides is helpful for designing various types of drugs such as kinase inhibitor and antigen. SPAD is a novel and powerful descriptor for various types of peptides. Accuracy of QSAR/QSPR becomes higher by describing peptides with SPAD. PMID:22047717

  11. Peptide-Modulated Activity Enhancement of Acidic Protease Cathepsin E at Neutral pH

    PubMed Central

    Komatsu, Masayuki; Biyani, Madhu; Ghimire Gautam, Sunita; Nishigaki, Koichi

    2012-01-01

    Enzymes are regulated by their activation and inhibition. Enzyme activators can often be effective tools for scientific and medical purposes, although they are more difficult to obtain than inhibitors. Here, using the paired peptide method, we report on protease-cathepsin-E-activating peptides that are obtained at neutral pH. These selected peptides also underwent molecular evolution, after which their cathepsin E activation capability improved. Thus, the activators we obtained could enhance cathepsin-E-induced cancer cell apoptosis, which indicated their potential as cancer drug precursors. PMID:23365585

  12. Permeation of membranes by the neutral form of amino acids and peptides: relevance to the origin of peptide translocation

    NASA Technical Reports Server (NTRS)

    Chakrabarti, A. C.; Deamer, D. W.; Miller, S. L. (Principal Investigator)

    1994-01-01

    The flux of amino acids and other nutrient solutes such as phosphate across lipid bilayers (liposomes) is 10(5) slower than facilitated inward transport across biological membranes. This suggest that primitive cells lacking highly evolved transport systems would have difficulty transporting sufficient nutrients for cell growth to occur. There are two possible ways by which early life may have overcome this difficulty: (1) The membranes of the earliest cellular life-forms may have been intrinsically more permeable to solutes; or (2) some transport mechanism may have been available to facilitate transbilayer movement of solutes essential for cell survival and growth prior to the evolution of membrane transport proteins. Translocation of neutral species represents one such mechanism. The neutral forms of amino acids modified by methylation (creating protonated weak bases) permeate membranes up to 10(10) times faster than charged forms. This increased permeability when coupled to a transmembrane pH gradient can result in significantly increased rates of net unidirectional transport. Such pH gradients can be generated in vesicles used to model protocells that preceded and were presumably ancestral to early forms of life. This transport mechanism may still play a role in some protein translocation processes (e.g. for certain signal sequences, toxins and thylakoid proteins) in vivo.

  13. Light-emitting self-assembled peptide nucleic acids exhibit both stacking interactions and Watson-Crick base pairing

    NASA Astrophysics Data System (ADS)

    Berger, Or; Adler-Abramovich, Lihi; Levy-Sakin, Michal; Grunwald, Assaf; Liebes-Peer, Yael; Bachar, Mor; Buzhansky, Ludmila; Mossou, Estelle; Forsyth, V. Trevor; Schwartz, Tal; Ebenstein, Yuval; Frolow, Felix; Shimon, Linda J. W.; Patolsky, Fernando; Gazit, Ehud

    2015-05-01

    The two main branches of bionanotechnology involve the self-assembly of either peptides or DNA. Peptide scaffolds offer chemical versatility, architectural flexibility and structural complexity, but they lack the precise base pairing and molecular recognition available with nucleic acid assemblies. Here, inspired by the ability of aromatic dipeptides to form ordered nanostructures with unique physical properties, we explore the assembly of peptide nucleic acids (PNAs), which are short DNA mimics that have an amide backbone. All 16 combinations of the very short di-PNA building blocks were synthesized and assayed for their ability to self-associate. Only three guanine-containing di-PNAs—CG, GC and GG—could form ordered assemblies, as observed by electron microscopy, and these di-PNAs efficiently assembled into discrete architectures within a few minutes. The X-ray crystal structure of the GC di-PNA showed the occurrence of both stacking interactions and Watson-Crick base pairing. The assemblies were also found to exhibit optical properties including voltage-dependent electroluminescence and wide-range excitation-dependent fluorescence in the visible region.

  14. Conjugates of amino acids and peptides with 5-o-mycaminosyltylonolide and their interaction with the ribosomal exit tunnel.

    PubMed

    Shishkina, Anna; Makarov, Gennady; Tereshchenkov, Andrey; Korshunova, Galina; Sumbatyan, Nataliya; Golovin, Andrey; Svetlov, Maxim; Bogdanov, Alexey

    2013-11-20

    During protein synthesis the nascent polypeptide chain (NC) extends through the ribosomal exit tunnel (NPET). Also, the large group of macrolide antibiotics binds in the nascent peptide exit tunnel. In some cases interaction of NC with NPET leads to the ribosome stalling, a significant event in regulation of translation. In other cases NC-ribosome interactions lead to pauses in translation that play an important role in cotranslational folding of polypeptides emerging from the ribosome. The precise mechanism of NC recognition in NPET as well as factors that determine NC conformation in the ribosomal tunnel are unknown. A number of derivatives of the macrolide antibiotic 5-O-mycaminosyltylonolide (OMT) containing N-acylated amino acid or peptide residues were synthesized in order to study potential sites of NC-NPET interactions. The target compounds were prepared by conjugation of protected amino acids and peptides with the C23 hydroxyl group of the macrolide. These OMT derivatives showed high although varying abilities to inhibit the firefly luciferase synthesis in vitro. Three glycil-containing derivatives appeared to be strong inhibitors of translation, more potent than parental OMT. Molecular dynamics (MD) simulation of complexes of tylosin, OMT, and some of OMT derivatives with the large ribosomal subunit of E. coli illuminated a plausible reason for the high inhibitory activity of Boc-Gly-OMT. In addition, the MD study detected a new putative site of interaction of the nascent polypeptide chain with the NPET walls. PMID:24090034

  15. Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells.

    PubMed

    Haryadi, Ryan; Ho, Steven; Kok, Yee Jiun; Pu, Helen X; Zheng, Lu; Pereira, Natasha A; Li, Bin; Bi, Xuezhi; Goh, Lin-Tang; Yang, Yuansheng; Song, Zhiwei

    2015-01-01

    Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells. PMID:25706993

  16. Optimization of Heavy Chain and Light Chain Signal Peptides for High Level Expression of Therapeutic Antibodies in CHO Cells

    PubMed Central

    Haryadi, Ryan; Ho, Steven; Kok, Yee Jiun; Pu, Helen X.; Zheng, Lu; Pereira, Natasha A.; Li, Bin; Bi, Xuezhi; Goh, Lin-Tang; Yang, Yuansheng; Song, Zhiwei

    2015-01-01

    Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells. PMID:25706993

  17. A spliced antigenic peptide comprising a single spliced amino acid is produced in the proteasome by reverse splicing of a longer peptide fragment followed by trimming.

    PubMed

    Michaux, Alexandre; Larrieu, Pierre; Stroobant, Vincent; Fonteneau, Jean-François; Jotereau, Francine; Van den Eynde, Benoît J; Moreau-Aubry, Agnès; Vigneron, Nathalie

    2014-02-15

    Peptide splicing is a novel mechanism of production of peptides relying on the proteasome and involving the linkage of fragments originally distant in the parental protein. Peptides produced by splicing can be presented on class I molecules of the MHC and recognized by CTLs. In this study, we describe a new antigenic peptide, which is presented by HLA-A3 and comprises two noncontiguous fragments of the melanoma differentiation Ag gp100(PMEL17) spliced together in the reverse order to that in which they appear in the parental protein. Contrary to the previously described spliced peptides, which are produced by the association of fragments of 3-6 aa, the peptide described in this work results from the ultimate association of an 8-aa fragment with a single arginine residue. As described before, peptide splicing takes place in the proteasome by transpeptidation involving an acyl-enzyme intermediate linking one of the peptide fragment to a catalytic subunit of the proteasome. Interestingly, we observe that the peptide causing the nucleophilic attack on the acyl-enzyme intermediate must be at least 3 aa long to give rise to a spliced peptide. The spliced peptide produced from this reaction therefore bears an extended C terminus that needs to be further trimmed to produce the final antigenic peptide. We show that the proteasome is able to perform the final trimming step required to produce the antigenic peptide described in this work. PMID:24453253

  18. Peptide and amino acid metabolism is controlled by an OmpR-family response regulator in Lactobacillus casei.

    PubMed

    Alcántara, Cristina; Bäuerl, Christine; Revilla-Guarinos, Ainhoa; Pérez-Martínez, Gaspar; Monedero, Vicente; Zúñiga, Manuel

    2016-04-01

    A Lactobacillus casei BL23 strain defective in an OmpR-family response regulator encoded by LCABL_18980 (PrcR, RR11), showed enhanced proteolytic activity caused by overexpression of the gene encoding the proteinase PrtP. Transcriptomic analysis revealed that, in addition to prtP expression, PrcR regulates genes encoding peptide and amino acid transporters, intracellular peptidases and amino acid biosynthetic pathways, among others. Binding of PrcR to twelve promoter regions of both upregulated and downregulated genes, including its own promoter, was demonstrated by electrophoretic mobility shift assays showing that PrcR can act as a transcriptional repressor or activator. Phosphorylation of PrcR increased its DNA binding activity and this effect was abolished after replacement of the phosphorylatable residue Asp-52 by alanine. Comparison of the transcript levels in cells grown in the presence or absence of tryptone in the growth medium revealed that PrcR activity responded to the presence of a complex amino acid source in the growth medium. We conclude that the PrcR plays a major role in the control of the peptide and amino acid metabolism in L. casei BL23. Orthologous prcR genes are present in most members of the Lactobacillaceae and Leuconostocaceae families. We hypothesize that they play a similar role in these bacterial groups. PMID:26711440

  19. Diamond nanowires modified with poly[3-(pyrrolyl)carboxylic acid] for the immobilization of histidine-tagged peptides.

    PubMed

    Subramanian, Palaniappan; Mazurenko, Ievgen; Zaitsev, Vladimir; Coffinier, Yannick; Boukherroub, Rabah; Szunerits, Sabine

    2014-09-01

    Coating boron-doped diamond nanowires (BDD NWs) with a conducting polymer, poly[3-(pyrrolyl)carboxylic acid], has been reported. Polymer coating was achieved through electropolymerization of 3-(pyrrolyl)carboxylic acid at the electrode interface by amperometrically biasing the BDD NWs interface until a predefined charge has passed. The poly[3-(pyrrolyl)carboxylic acid] modified BDD NWs (PPA-BDD NWs) were characterized by scanning electron microscopy (SEM) and cyclic voltammetry (CV). Using a deposition charge of 11 mC cm(-2) resulted in a thin polymer film deposition. The availability of the carboxylic groups of the polymer coated BDD NWs electrode was demonstrated through copper ion (Cu(2+)) chelation. The resulting complex was successfully used for the site-specific immobilization of histidine-tagged peptides. The binding process was followed by electrochemical impedance spectroscopy (EIS). The Cu(2+)-chelated PPA-BDD NWs interface showed peptide loading capability comparable to those of commercially available interfaces and can be easily regenerated several times using ethylenediaminetetraacetic acid (EDTA). PMID:25009833

  20. ST-scale as a novel amino acid descriptor and its application in QSAM of peptides and analogues.

    PubMed

    Yang, Li; Shu, Mao; Ma, Kaiwang; Mei, Hu; Jiang, Yongjun; Li, Zhiliang

    2010-03-01

    In this study, structural topology scale (ST-scale) was recruited as a novel structural topological descriptor derived from principal component analysis on 827 structural variables of 167 amino acids. By using partial least squares (PLS), we applied ST-scale for the study of quantitative sequence-activity models (QSAMs) on three peptide datasets (58 angiotensin-converting enzyme (ACE) inhibitors, 34 antimicrobial peptides (AMPs) and 89 elastase substrates (ES)). The results of QSAMs were superior to that of the earlier studies, with determination coefficient (r(2)) and cross-validated (q(2)) equal to 0.855, 0.774; 0.79, 0.371 (OSC-PLS: 0.995, 0.848) and 0.846, 0.747, respectively. Therefore, ST-scale descriptors were considered to be competent to extract information from 827 structural variables and relate with their bioactivities. PMID:19373543

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

    PubMed

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

    2002-07-31

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

  2. Structure-based rational design of peptide hydroxamic acid inhibitors to target tumor necrosis factor-α converting enzyme as potential therapeutics for hepatitis.

    PubMed

    Wu, Dan; Gu, Qiuhong; Zhao, Ning; Xia, Fei; Li, Zhiwei

    2015-12-01

    The human tumor necrosis factor-α converting enzyme (TACE) has recently been raised as a new and promising therapeutic target of hepatitis and other inflammatory diseases. Here, we reported a successful application of the solved crystal structure of TACE complex with a peptide-like ligand INN for rational design of novel peptide hydroxamic acid inhibitors with high potency and selectivity to target and inhibit TACE. First, the intermolecular interactions between TACE catalytic domain and INN were characterized through an integrated bioinformatics approach, with which the key substructures of INN that dominate ligand binding were identified. Subsequently, the INN molecular structure was simplified to a chemical sketch of peptide hydroxamic acid compound, which can be regarded as a linear tripeptide capped by a N-terminal carboxybenzyl group (chemically protective group) and a C-terminal hydroxamate moiety (coordinated to the Zn(2+) at TACE active site). Based on the sketch, a virtual combinatorial library containing 180 peptide hydroxamic acids was generated, from which seven samples were identified as promising candidates by using a knowledge-based protein-peptide affinity predictor and were then tested in vitro with a standard TACE activity assay protocol. Consequently, three designed peptide hydroxamic acids, i.e. Cbz-Pro-Ile-Gln-hydroxamic acid, Cbz-Leu-Ile-Val-hydroxamic acid and Cbz-Phe-Val-Met-hydroxamic acid, exhibited moderate or high inhibitory activity against TACE, with inhibition constants Ki of 36 ± 5, 510 ± 46 and 320 ± 26 nM, respectively. We also examined the structural basis and non-bonded profile of TACE interaction with a designed peptide hydroxamic acid inhibitor, and found that the inhibitor ligand is tightly buried in the active pocket of TACE, forming a number of hydrogen bonds, hydrophobic forces and van der Waals contacts at the interaction interface, conferring both stability and specificity for TACE-inhibitor complex

  3. The Effects of cAMP-elevating Agents and Alpha Lipoic Acid on In Vitro Maturation of Mouse Germinal Vesicle Oocytes

    PubMed Central

    Rahnama, Ali; Zavareh, Saeed; Ghorbanian, Mohammad Taghi; Karimi, Isaac

    2013-01-01

    Background In spite of extensive efforts to improve in vitro oocyte maturation, the obtained results are not very efficient. This study was conducted to assess impacts of cAMP elevating agents and alpha lipoic acid (ALA) on in vitro oocyte maturation and fertilization. Methods Mouse germinal vesicle (GV) oocytes were categorized into cumulus denuded oocytes (DOs; n=896) and cumulus oocyte complexes (COCs; n=1077) groups. GV oocytes were matured in vitro with or without ALA; (I) without the meiotic inhibitors; (II) supplemented with cilostamide; (III) supplemented with forskolin and (IV) supplemented with Forskolin plus cilostamide. The obtained metaphase II (MII) oocytes were subjected to in vitro fertilization. Independent-samples t-testand ANOVA were used for data analysis. A p-value less than 0.05 was considered to be statistically significant. Results The COCs maturation, fertilization and two cell embryo rates were higher than those of DOs in the control group, while no significant difference was observed between relevant COCs and DOs when they were cultured with cilostamide meiotic inhibitors in two step manner. Combined treatment of cilostamide and forskolin significantly elevated the developmental rates in both COCs and DOs as compared to other groups. The developmental rates of COCs and DOs in the presence of ALA were similar to their respective groups without ALA. Conclusion cAMP elevating agents were more effective on DOs than COCs with regard to rates of maturation and fertilization. However, ALA did not affect the developmental rates of both COCs and DOs in in vitro maturation in one or two step manner. PMID:24551571

  4. New Compstatin Peptides Containing N-Terminal Extensions and Non-Natural Amino Acids Exhibit Potent Complement Inhibition and Improved Solubility Characteristics

    PubMed Central

    2015-01-01

    Compstatin peptides are complement inhibitors that bind and inhibit cleavage of complement C3. Peptide binding is enhanced by hydrophobic interactions; however, poor solubility promotes aggregation in aqueous environments. We have designed new compstatin peptides derived from the W4A9 sequence (Ac-ICVWQDWGAHRCT-NH2, cyclized between C2 and C12), based on structural, computational, and experimental studies. Furthermore, we developed and utilized a computational framework for the design of peptides containing non-natural amino acids. These new compstatin peptides contain polar N-terminal extensions and non-natural amino acid substitutions at positions 4 and 9. Peptides with α-modified non-natural alanine analogs at position 9, as well as peptides containing only N-terminal polar extensions, exhibited similar activity compared to W4A9, as quantified via ELISA, hemolytic, and cell-based assays, and showed improved solubility, as measured by UV absorbance and reverse-phase HPLC experiments. Because of their potency and solubility, these peptides are promising candidates for therapeutic development in numerous complement-mediated diseases. PMID:25494040

  5. Interaction of Peptide Transporter 1 With D-Glucose and L-Glutamic Acid; Possible Involvement of Taste Receptors.

    PubMed

    Arakawa, Hiroshi; Ohmachi, Taichi; Ichiba, Kiko; Kamioka, Hiroki; Tomono, Takumi; Kanagawa, Masahiko; Idota, Yoko; Hatano, Yasuko; Yano, Kentaro; Morimoto, Kaori; Ogihara, Takuo

    2016-01-01

    We investigated the influence of sweet and umami (savory) tastants on the intestinal absorption of cephalexin (CEX), a substrate of peptide transporter 1 (PEPT1, SLC15A1) in rats. After oral administration of glucose or mannitol to rats, CEX was administered together with a second dose of glucose or mannitol. Western blot analysis indicated that expression of PEPT1 in rat jejunum membrane was decreased by glucose, compared to mannitol. Furthermore, the maximum plasma concentration (Cmax) of orally administered CEX was reduced by glucose compared to mannitol. The effect of glucose was diminished by nifedipine, a L-type Ca(2+) channel blocker. We also found that Cmax of orally administered CEX was reduced by treatment with L-glutamic acid, compared to D-glutamic acid. Thus, excessive intake of glucose and L-glutamic acid may impair oral absorption of PEPT1 substrates. PMID:26852864

  6. Self-assembled multicompartment liquid crystalline lipid carriers for protein, peptide, and nucleic acid drug delivery.

    PubMed

    Angelova, Angelina; Angelov, Borislav; Mutafchieva, Rada; Lesieur, Sylviane; Couvreur, Patrick

    2011-02-15

    Lipids and lipopolymers self-assembled into biocompatible nano- and mesostructured functional materials offer many potential applications in medicine and diagnostics. In this Account, we demonstrate how high-resolution structural investigations of bicontinuous cubic templates made from lyotropic thermosensitive liquid-crystalline (LC) materials have initiated the development of innovative lipidopolymeric self-assembled nanocarriers. Such structures have tunable nanochannel sizes, morphologies, and hierarchical inner organizations and provide potential vehicles for the predictable loading and release of therapeutic proteins, peptides, or nucleic acids. This Account shows that structural studies of swelling of bicontinuous cubic lipid/water phases are essential for overcoming the nanoscale constraints for encapsulation of large therapeutic molecules in multicompartment lipid carriers. For the systems described here, we have employed time-resolved small-angle X-ray scattering (SAXS) and high-resolution freeze-fracture electronic microscopy (FF-EM) to study the morphology and the dynamic topological transitions of these nanostructured multicomponent amphiphilic assemblies. Quasi-elastic light scattering and circular dichroism spectroscopy can provide additional information at the nanoscale about the behavior of lipid/protein self-assemblies under conditions that approximate physiological hydration. We wanted to generalize these findings to control the stability and the hydration of the water nanochannels in liquid-crystalline lipid nanovehicles and confine therapeutic biomolecules within these structures. Therefore we analyzed the influence of amphiphilic and soluble additives (e.g. poly(ethylene glycol)monooleate (MO-PEG), octyl glucoside (OG), proteins) on the nanochannels' size in a diamond (D)-type bicontinuous cubic phase of the lipid glycerol monooleate (MO). At body temperature, we can stabilize long-living swollen states, corresponding to a diamond cubic phase

  7. Effects of Ti surface treatments with silane and arginylglycylaspartic acid peptide on bone cell progenitors.

    PubMed

    Chen, Wen-Cheng; Lo, Yang; Chen, Hong-Sen

    2015-09-01

    Achieving optimal aesthetic appearance is a major objective in dental implant design, and the interaction between the materials and the bone cell progenitors is an important factor in the attainment of this objective. In this study, a novel concept was evaluated by varying the surface modifications on titanium (Ti). Different levels of roughness can be attained by machine grinding (M), sand blasting, and acid etching (SLA) of the samples. The behavior of bone cell progenitors (D1) on the surfaces of Ti disks with different surface modifications was investigated. The surfaces of M or SLA disks were silanized (MS or SLAS group) through treatment with silane/Gly-Arg-Gly-Asp-Ser (GRGDS) peptide (MSP or SLASP group) and anchored particles of tetracalcium phosphate (TTCP) on the specimen surfaces (SLA-TTCP group). Physicochemical analysis was performed by metallographic microscopy, scanning electron microscopy, and contact angle analysis. The proliferation and the quantitative alkaline phosphatase (ALP) production of D1 cells on the surface of different sample groups were determined. The SLASP group had a significantly larger D1 cell proliferation than the other groups after 4 and 7 d of incubation (p < 0.05). ALP expression was a very early marker of differentiation, and was the first indication of the increasing number of cells at 7 d of culture. Among the groups in the M substrate series (i.e., M, MS, and MSP) and in the SLA series (i.e., SLA, SLAS, and SLASP), the MSP and SLASP specimens exhibited superior differentiation abilities on respective cultures until day 7 and day 10. A high number of hydrophilic surfaces dominated cell proliferation in the early stage of cell attachment. However, factors affecting the pore structure and the surface morphology can improve cell proliferation and differentiation. According to analyses of proliferation and ALP expression of bone cell progenitors D1, the original SLA implant surface can be improved with surface treatment

  8. Antimicrobial and Immunomodulatory Activities of PR-39 Derived Peptides

    PubMed Central

    Veldhuizen, Edwin J. A.; Schneider, Viktoria A. F.; Agustiandari, Herfita; van Dijk, Albert; Tjeerdsma-van Bokhoven, Johanna L. M.; Bikker, Floris J.; Haagsman, Henk P.

    2014-01-01

    The porcine cathelicidin PR-39 is a host defence peptide that plays a pivotal role in the innate immune defence of the pig against infections. Besides direct antimicrobial activity, it is involved in immunomodulation, wound healing and several other biological processes. In this study, the antimicrobial- and immunomodulatory activity of PR-39, and N- and C-terminal derivatives of PR-39 were tested. PR-39 exhibited an unexpected broad antimicrobial spectrum including several Gram positive strains such as Bacillus globigii and Enterococcus faecalis. Of organisms tested, only Staphylococcus aureus was insensitive to PR-39. Truncation of PR-39 down to 15 (N-terminal) amino acids did not lead to major loss of activity, while peptides corresponding to the C-terminal part of PR-39 were hampered in their antimicrobial activity. However, shorter peptides were all much more sensitive to inhibition by salt. Active peptides induced ATP leakage and loss of membrane potential in Bacillus globigii and Escherichia coli, indicating a lytic mechanism of action for these peptides. Finally, only the mature peptide was able to induce IL-8 production in porcine macrophages, but some shorter peptides also had an effect on TNF-α production showing differential regulation of cytokine induction by PR-39 derived peptides. None of the active peptides showed high cytotoxicity highlighting the potential of these peptides for use as an alternative to antibiotics. PMID:24755622

  9. Role of Side Chains in β-Sheet Self-Assembly into Peptide Fibrils. IR and VCD Spectroscopic Studies of Glutamic Acid-Containing Peptides.

    PubMed

    Tobias, Fernando; Keiderling, Timothy A

    2016-05-10

    Poly(glutamic acid) at low pH self-assembles after incubation at higher temperature into fibrils composed of antiparallel sheets that are stacked in a β2-type structure whose amide carbonyls have bifurcated H-bonds involving the side chains from the next sheet. Oligomers of Glu can also form such structures, and isotope labeling has provided insight into their out-of-register antiparallel structure [ Biomacromolecules 2013 , 14 , 3880 - 3891 ]. In this paper we report IR and VCD spectra and transmission electron micrograph (TEM) images for a series of alternately sequenced oligomers, Lys-(Aaa-Glu)5-Lys-NH2, where Aaa was varied over a variety of polar, aliphatic, or aromatic residues. Their spectral and TEM data show that these oligopeptides self-assemble into different structures, both local and morphological, that are dependent on both the nature of the Aaa side chains and growth conditions employed. Such alternate peptides substituted with small or polar residues, Ala and Thr, do not yield fibrils; but with β-branched aliphatic residues, Val and Ile, that could potentially pack with Glu side chains, these oligopeptides do show evidence of β2-stacking. By contrast, for Leu, with longer side chains, only β1-stacking is seen while with even larger Phe side chains, either β-form can be detected separately, depending on preparation conditions. These structures are dependent on high temperature incubation after reducing the pH and in some cases after sonication of initial fibril forms and reincubation. Some of these fibrillar peptides, but not all, show enhanced VCD, which can offer evidence for formation of long, multistrand, often twisted structures. Substitution of Glu with residues having selected side chains yields a variety of morphologies, leading to both β1- and β2-structures, that overall suggests two different packing modes for the hydrophobic side chains depending on size and type. PMID:27099990

  10. Sum Frequency Generation Vibrational Spectroscopy of Adsorbed Amino Acids, Peptides and Proteins of Hydrophilic and Hydrophobic Solid-Water Interfaces

    SciTech Connect

    Holinga IV, George Joseph

    2010-09-01

    Sum frequency generation (SFG) vibrational spectroscopy was used to investigate the interfacial properties of several amino acids, peptides, and proteins adsorbed at the hydrophilic polystyrene solid-liquid and the hydrophobic silica solid-liquid interfaces. The influence of experimental geometry on the sensitivity and resolution of the SFG vibrational spectroscopy technique was investigated both theoretically and experimentally. SFG was implemented to investigate the adsorption and organization of eight individual amino acids at model hydrophilic and hydrophobic surfaces under physiological conditions. Biointerface studies were conducted using a combination of SFG and quartz crystal microbalance (QCM) comparing the interfacial structure and concentration of two amino acids and their corresponding homopeptides at two model liquid-solid interfaces as a function of their concentration in aqueous solutions. The influence of temperature, concentration, equilibration time, and electrical bias on the extent of adsorption and interfacial structure of biomolecules were explored at the liquid-solid interface via QCM and SFG. QCM was utilized to quantify the biological activity of heparin functionalized surfaces. A novel optical parametric amplifier was developed and utilized in SFG experiments to investigate the secondary structure of an adsorbed model peptide at the solid-liquid interface.

  11. {{text{C}}_{α }} - {text{C}} Bond Cleavage of the Peptide Backbone in MALDI In-Source Decay Using Salicylic Acid Derivative Matrices

    NASA Astrophysics Data System (ADS)

    Asakawa, Daiki; Takayama, Mitsuo

    2011-07-01

    The use of 5-formylsalicylic acid (5-FSA) and 5-nitrosalicylic acid (5-NSA) as novel matrices for in-source decay (ISD) of peptides in matrix-assisted laser desorption/ionization (MALDI) is described. The use of 5-FSA and 5-NSA generated a- and x-series ions accompanied by oxidized peptides [M - 2 H + H]+. The preferential formation of a- and x-series ions was found to be dependent on the hydrogen-accepting ability of matrix. The hydrogen-accepting ability estimated from the ratio of signal intensity of oxidized product [M - 2 H + H]+ to that of non-oxidized protonated molecule [M + H]+ of peptide was of the order 5-NSA > 5-FSA > 5-aminosalicylic acid (5-ASA) ≒ 2,5-dihydroxyl benzoic acid (2,5-DHB) ≒ 0. The results suggest that the hydrogen transfer reaction from peptide to 5-FSA and 5-NSA occurs during the MALDI-ISD processes. The hydrogen abstraction from peptides results in the formation of oxidized peptides containing a radical site on the amide nitrogen with subsequent radical-induced cleavage at the {{{C}}_{α }} - {{C}} bond, leading to the formation of a- and x-series ions. The most significant feature of MALDI-ISD with 5-FSA and 5-NSA is the specific cleavage of the {{{C}}_{α }} - {{C}} bond of the peptide backbone without degradation of side-chain and post-translational modifications (PTM). The matrix provides a useful complementary method to conventional MALDI-ISD for amino acid sequencing and site localization of PTMs in peptides.

  12. T-scale as a novel vector of topological descriptors for amino acids and its application in QSARs of peptides

    NASA Astrophysics Data System (ADS)

    Tian, Feifei; Zhou, Peng; Li, Zhiliang

    2007-03-01

    In this paper, a new topological descriptor T-scale is derived from principal component analysis (PCA) on the collected 67 kinds of structural and topological variables of 135 amino acids. Applying T-scale to three peptide panels as 58 angiotensin-converting enzyme (ACE) inhibitors, 20 thromboplastin inhibitors (TI) and 28 bovine lactoferricin-(17-31)-pentadecapeptides (LFB), the resulting QSAR models, constructed by partial least squares (PLS), are all superior to reference reports, with correlative coefficient r2 and cross-validated q2 of 0.845, 0.786; 0.996, 0.782 (0.988, 0.961); 0.760, 0.627, respectively.

  13. Cumulus expansion, nuclear maturation and connexin 43, cyclooxygenase-2 and FSH receptor mRNA expression in equine cumulus-oocyte complexes cultured in vitro in the presence of FSH and precursors for hyaluronic acid synthesis

    PubMed Central

    Dell'Aquila, Maria Elena; Caillaud, Maud; Maritato, Filippo; Martoriati, Alain; Gérard, Nadine; Aiudi, Giulio; Minoia, Paolo; Goudet, Ghylène

    2004-01-01

    The aim of this study was to investigate cumulus expansion, nuclear maturation and expression of connexin 43, cyclooxygenase-2 and FSH receptor transcripts in equine cumuli oophori during in vivo and in vitro maturation in the presence of equine FSH (eFSH) and precursors for hyaluronic acid synthesis. Equine cumulus-oocyte complexes (COC) were cultured in a control defined medium supplemented with eFSH (0 to 5 micrograms/ml), Fetal Calf Serum (FCS), precursors for hyaluronic acid synthesis or glutamine according to the experiments. After in vitro maturation, the cumulus expansion rate was increased with 1 microgram/ml eFSH, and was the highest with 20% FCS. It was not influenced by precursors for hyaluronic acid synthesis or glutamine. The expression of transcripts related to cumulus expansion was analyzed in equine cumulus cells before maturation, and after in vivo and in vitro maturation, by using reverse transcription-polymerase chain reaction (RT-PCR) with specific primers. Connexin 43, cyclooxygenase-2 (COX-2) and FSH receptor (FSHr) mRNA were detected in equine cumulus cells before and after maturation. Their level did not vary during in vivo or in vitro maturation and was influenced neither by FSH nor by precursors for hyaluronic acid synthesis. Results indicate that previously reported regulation of connexin 43 and COX-2 proteins during equine COC maturation may involve post-transcriptional mechanisms. PMID:15212696

  14. Influence of Amino Acid Composition and Phosphorylation on the Ion Yields of Peptides in MALDI-MS

    NASA Astrophysics Data System (ADS)

    Asakawa, Daiki; Moriguchi, Shohey; Takayama, Mitsuo

    2012-01-01

    The influence of arginine (Arg), lysine (Lys), and phenylalanine (Phe) residues and phosphorylation on the molecular ion yields of model peptides have been quantitatively studied using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry in both positive- and negative-ion mode. The results obtained from these experiments have been interpreted from the standpoint of two different components, namely, desorption and ionization, on the basis of the physicochemical properties of constituent amino acids of the model peptides. The presence of basic residues such as Arg and Lys enhanced the ion yields of protonated molecules [M + H]+. An N-terminal rather than a C-terminal Arg residue was advantageous for the formation of both [M + H]+ and [M - H]-. The presence of the Phe residue resulted in the increase of the ion yields of both [M + H]+ and [M - H]-. In contrast, the presence of phosphate group(s) contributed to the suppression of the yields of both [M + H]+ and [M - H]- due to the loss of phosphate group. The detection limits for both [M + H]+ and [M - H]- of model peptides have been evaluated.

  15. Evolutionary Importance of the Intramolecular Pathways of Hydrolysis of Phosphate Ester Mixed Anhydrides with Amino Acids and Peptides

    PubMed Central

    Liu, Ziwei; Beaufils, Damien; Rossi, Jean-Christophe; Pascal, Robert

    2014-01-01

    Aminoacyl adenylates (aa-AMPs) constitute essential intermediates of protein biosynthesis. Their polymerization in aqueous solution has often been claimed as a potential route to abiotic peptides in spite of a highly efficient CO2-promoted pathway of hydrolysis. Here we investigate the efficiency and relevance of this frequently overlooked pathway from model amino acid phosphate mixed anhydrides including aa-AMPs. Its predominance was demonstrated at CO2 concentrations matching that of physiological fluids or that of the present-day ocean, making a direct polymerization pathway unlikely. By contrast, the occurrence of the CO2-promoted pathway was observed to increase the efficiency of peptide bond formation owing to the high reactivity of the N-carboxyanhydride (NCA) intermediate. Even considering CO2 concentrations in early Earth liquid environments equivalent to present levels, mixed anhydrides would have polymerized predominantly through NCAs. The issue of a potential involvement of NCAs as biochemical metabolites could even be raised. The formation of peptide–phosphate mixed anhydrides from 5(4H)-oxazolones (transiently formed through prebiotically relevant peptide activation pathways) was also observed as well as the occurrence of the reverse cyclization process in the reactions of these mixed anhydrides. These processes constitute the core of a reaction network that could potentially have evolved towards the emergence of translation. PMID:25501391

  16. A Peptide Mimetic of 5-Acetylneuraminic Acid-Galactose Binds with High Avidity to Siglecs and NKG2D

    PubMed Central

    Eggink, Laura L.; Spyroulias, Georgios A.; Jones, Norman G.; Hanson, Carl V.; Hoober, J. Kenneth

    2015-01-01

    We previously identified several peptide sequences that mimicked the terminal sugars of complex glycans. Using plant lectins as analogs of lectin-type cell-surface receptors, a tetravalent form of a peptide with the sequence NPSHPLSG, designated svH1C, bound with high avidity to lectins specific for glycans with terminal 5-acetylneuraminic acid (Neu5Ac)-galactose (Gal)/N-acetylgalactosamine (GalNAc) sequences. In this report, we show by circular dichroism and NMR spectra that svH1C lacks an ordered structure and thus interacts with binding sites from a flexible conformation. The peptide binds with high avidity to several recombinant human siglec receptors that bind preferentially to Neu5Ac(α2,3)Gal, Neu5Ac(α2,6)GalNAc or Neu5Ac(α2,8)Neu5Ac ligands. In addition, the peptide bound the receptor NKG2D, which contains a lectin-like domain that binds Neu5Ac(α2,3)Gal. The peptide bound to these receptors with a KD in the range of 0.6 to 1 μM. Binding to these receptors was inhibited by the glycoprotein fetuin, which contains multiple glycans that terminate in Neu5Ac(α2,3)Gal or Neu5Ac(α2,6)Gal, and by sialyllactose. Binding of svH1C was not detected with CLEC9a, CLEC10a or DC-SIGN, which are lectin-type receptors specific for other sugars. Incubation of neuraminidase-treated human peripheral blood mononuclear cells with svH1C resulted in binding of the peptide to a subset of the CD14+ monocyte population. Tyrosine phosphorylation of siglecs decreased dramatically when peripheral blood mononuclear cells were treated with 100 nM svH1C. Subcutaneous, alternate-day injections of svH1C into mice induced several-fold increases in populations of several types of immune cells in the peritoneal cavity. These results support the conclusion that svH1C mimics Neu5Ac-containing sequences and interacts with cell-surface receptors with avidities sufficient to induce biological responses at low concentrations. The attenuation of inhibitory receptors suggests that svH1C has

  17. Amino acid sequences of peptides from a tryptic digest of a urea-soluble protein fraction (U.S.3) from oxidized wool

    PubMed Central

    Corfield, M. C.; Fletcher, J. C.; Robson, A.

    1967-01-01

    1. A tryptic digest of the protein fraction U.S.3 from oxidized wool has been separated into 32 peptide fractions by cation-exchange resin chromatography. 2. Most of these fractions have been resolved into their component peptides by a combination of the techniques of cation-exchange resin chromatography, paper chromatography and paper electrophoresis. 3. The amino acid compositions of 58 of the peptides in the digest present in the largest amounts have been determined. 4. The amino acid sequences of 38 of these have been completely elucidated and those of six others partially derived. 5. These findings indicate that the parent protein in wool from which the protein fraction U.S.3 is derived has a minimum molecular weight of 74000. 6. The structures of wool proteins are discussed in the light of the peptide sequences determined, and, in particular, of those sequences in fraction U.S.3 that could not be elucidated. PMID:16742497

  18. Enzymatic characterization of peptidic materials isolated from aqueous solutions of ammonium cyanide (pH 9) and hydrocyanic acid (pH 6) exposed to ionizing radiation.

    PubMed

    Niketic, V; Draganić, Z; Nesković, S; Draganić, I

    1982-01-01

    The enzymatic digestion of some radiolytically produced peptidic materials was examined. The substrates were compounds isolated from 0.1 molar solutions of NH4CN (pH 9) and HCN (pH 6), after their exposure to gamma rays from a 60Co source (15-20 Mrad doses). Commercial proteolytic enzymes pronase and aminopeptidase M were used. The examined materials were of composite nature and proteolytic action was systematically observed after their subsequent purification. In some fractions the effect was found to be positive with up to 30% of peptide bonds cleaved with respect to the amino acid content. These findings support our previous conclusions on the free radical induced formation of peptidic backbones without the intervention of amino acids. Some side effects were also noted which might be of interest in observations on enzymatic cleavage of other composite peptidic materials of abiotic origin. PMID:6124639

  19. Proinsulin C-peptide antagonizes the profibrotic effects of TGF-beta1 via up-regulation of retinoic acid and HGF-related signaling pathways.

    PubMed

    Hills, Claire E; Willars, Gary B; Brunskill, Nigel J

    2010-04-01

    Novel signaling roles for C-peptide have recently been discovered with evidence that it can ameliorate complications of type 1 diabetes. Here we sought to identify new pathways regulated by C-peptide of relevance to the pathophysiology of diabetic nephropathy. Microarray analysis was performed to identify genes regulated by either C-peptide and/or TGF-beta1 in a human proximal tubular cell line, HK-2. Expression of retinoic acid receptor beta (RARbeta), hepatocyte growth factor (HGF), cellular retinoic acid-binding protein II (CRABPII), vimentin, E-cadherin, Snail, and beta-catenin was assessed by immunoblotting. The cellular localization of vimentin and beta-catenin was determined by immunocytochemistry. Changes in cell morphology were assessed by phase contrast microscopy. Gene expression profiling demonstrated differential expression of 953 and 1458 genes after C-peptide exposure for 18 h or 48 h, respectively. From these, members of the antifibrotic retinoic acid (RA)- and HGF-signaling pathways were selected. Immunoblotting demonstrated that C-peptide increased RARbeta, CRABPII, and HGF. We confirmed a role for RA in reversal of TGF-beta1-induced changes associated with epithelial-mesenchymal transition, including expression changes in Snail, E-cadherin, vimetin, and redistribution of beta-catenin. Importantly, these TGF-beta1-induced changes were inhibited by C-peptide. Further, effects of TGF-beta1 on Snail and E-cadherin expression were blocked by HGF, and inhibitory effects of C-peptide were removed by blockade of HGF activity. This study identifies a novel role for HGF as an effector of C-peptide, possibly via an RA-signaling pathway, highlighting C-peptide as a potential therapy for diabetic nephropathy. PMID:20197308

  20. Understanding the effects of mature adipocytes and endothelial cells on fatty acid metabolism and vascular tone in physiological fatty tissue for vascularized adipose tissue engineering.

    PubMed

    Huber, Birgit; Volz, Ann-Cathrin; Kluger, Petra J

    2015-11-01

    Engineering of large vascularized adipose tissue constructs is still a challenge for the treatment of extensive high-graded burns or the replacement of tissue after tumor removal. Communication between mature adipocytes and endothelial cells is important for homeostasis and the maintenance of adipose tissue mass but, to date, is mainly neglected in tissue engineering strategies. Thus, new co-culture strategies are needed to integrate adipocytes and endothelial cells successfully into a functional construct. This review focuses on the cross-talk of mature adipocytes and endothelial cells and considers their influence on fatty acid metabolism and vascular tone. In addition, the properties and challenges with regard to these two cell types for vascularized tissue engineering are highlighted. PMID:26340984

  1. Stoichiometric inhibition of amyloid beta-protein aggregation with peptides containing alternating alpha,alpha-disubstituted amino acids.

    PubMed

    Etienne, Marcus A; Aucoin, Jed P; Fu, Yanwen; McCarley, Robin L; Hammer, Robert P

    2006-03-22

    We have prepared two peptides based on the hydrophobic core (Lys-Leu-Val-Phe-Phe) of amyloid beta-protein (Abeta) that contain alpha,alpha-disubstituted amino acids at alternating positions, but differ in the positioning of the oligolysine chain (AMY-1, C-terminus; AMY-2, N-terminus). We have studied the effects of AMY-1 and AMY-2 on the aggregation of Abeta and find that, at stoichiometric concentrations, both peptides completely stop Abeta fibril growth. Equimolar mixtures of AMY-1 and Abeta form only globular aggregates as imaged by scanning force microscopy and transmission electron microscopy. These samples show no signs of protofibrillar or fibrillar material even after prolonged periods of time (4.5 months). Also, 10 mol % of AMY-1 prevents Abeta self-assembly for long periods of time; aged samples (4.5 months) show only a few protofibrillar or fibrillar aggregates. Circular dichroism spectroscopy of equimolar mixtures of AMY-1 and Abeta show that the secondary structure of the mixture changes over time and progresses to a predominantly beta-sheet structure, which is consistent with the design of these inhibitors preferring a sheet-like conformation. Changing the position of the charged tail on the peptide, AMY-2 interacts with Abeta differently in that equimolar mixtures form large ( approximately 1 mum) globular aggregates which do not progress to fibrils, but precipitate out of solution. The differences in the aggregation mediated by the two peptides is discussed in terms of a model where the inhibitors act as cosurfactants that interfere with the native ability of Abeta to self-assemble by disrupting hydrophobic interactions either at the C-terminus or N-terminus of Abeta. PMID:16536517

  2. Calcium carbonate crystal growth beneath Langmuir monolayers of acidic β-hairpin peptides.

    PubMed

    Gong, Haofei; Yang, Yi; Pluntke, Manuela; Marti, Othmar; Majer, Zsuzsa; Sewald, Norbert; Volkmer, Dirk

    2014-11-28

    Four amphiphilic peptides with designed hairpin structure were synthesized and their monolayers were employed as model systems to study biologically inspired calcium carbonate crystallization. Langmuir monolayers of hairpin peptides were investigated by surface pressure area isotherms, surface potential isotherms, Brewster angle microscopy (BAM), atomic force microscopy (AFM) and Fourier transform infrared (FTIR) spectroscopy. A β-hairpin conformation was found for all peptides at the air-water interface although their packing arrangements seem to be different. Crystallization of calcium carbonate under these peptide monolayers was investigated at different surface pressures and growth times both by in situ optical microscopy, BAM and ex situ investigations such as scanning electron microscopy (SEM) and transmission electron microscopy (TEM). An amorphous calcium carbonate precursor was found at the initial crystallization stage. The crystallization process occurred in three stages. It starts from the nucleation of amorphous particles being a kinetically controlled process. Crystal nuclei subsequently aggregate to large particles and vaterite crystals start to form inside the amorphous layer, with the monolayer fluidity exerting an important role. The third process includes the re-crystallization of vaterite to calcite, which is thermodynamically controlled by monolayer structural factors including the monolayer flexibility and packing arrangement of the polar headgroups. Thus, the kinetic factors, monolayer fluidity and flexibility as well as structure factors govern the crystal morphology and polymorph distribution simultaneously and synergistically. PMID:25292256

  3. Sea anemone peptide with uncommon β-hairpin structure inhibits acid-sensing ion channel 3 (ASIC3) and reveals analgesic activity.

    PubMed

    Osmakov, Dmitry I; Kozlov, Sergey A; Andreev, Yaroslav A; Koshelev, Sergey G; Sanamyan, Nadezhda P; Sanamyan, Karen E; Dyachenko, Igor A; Bondarenko, Dmitry A; Murashev, Arkadii N; Mineev, Konstantin S; Arseniev, Alexander S; Grishin, Eugene V

    2013-08-01

    Three novel peptides were isolated from the venom of the sea anemone Urticina grebelnyi. All of them are 29 amino acid peptides cross-linked by two disulfide bridges, with a primary structure similar to other sea anemone peptides belonging to structural group 9a. The structure of the gene encoding the shared precursor protein of the identified peptides was determined. One peptide, π-AnmTX Ugr 9a-1 (short name Ugr 9-1), produced a reversible inhibition effect on both the transient and the sustained current of human ASIC3 channels expressed in Xenopus laevis oocytes. It completely blocked the transient component (IC50 10 ± 0.6 μM) and partially (48 ± 2%) inhibited the amplitude of the sustained component (IC50 1.44 ± 0.19 μM). Using in vivo tests in mice, Ugr 9-1 significantly reversed inflammatory and acid-induced pain. The other two novel peptides, AnmTX Ugr 9a-2 (Ugr 9-2) and AnmTX Ugr 9a-3 (Ugr 9-3), did not inhibit the ASIC3 current. NMR spectroscopy revealed that Ugr 9-1 has an uncommon spatial structure, stabilized by two S-S bridges, with three classical β-turns and twisted β-hairpin without interstrand disulfide bonds. This is a novel peptide spatial structure that we propose to name boundless β-hairpin. PMID:23801332

  4. Recognition of a single amino acid change on the surface of a major transplantation antigen is in the context of self peptide.

    PubMed

    Pullen, J K; Tallquist, M D; Melvold, R W; Pease, L R

    1994-04-01

    The transcripts encoding two strongly alloantigenic class I mutant molecules, Kdm4 and Kdm5, were characterized and found to encode products that differ from the parental Kd glycoprotein by single amino acid substitutions. The Kdm4 molecule has an amino acid change at position 114, an integral component of a beta-sheet associated with pockets D and E of the peptide binding site. The basis for strong alloantigenicity of the variant molecule can be attributed to differences in peptide binding that were visualized by HPLC analysis of eluted peptides. In contrast, the Kdm5 molecule differs from the parent at position 158, a component of the alpha-helix that is not associated with any of the pockets of the peptide binding site. No differences in peptide binding by Kdm5 in comparison with the parent Kd molecule were seen by HPLC, suggesting that the variant and parent molecules bind the same set of peptides. The ability of (dm4 x dm5) F1 hybrid mice to recognize and lyse BALB/c stimulator cells indicates that the alloantigenic properties determined by the 158 substitution result from the interactions of the alpha-helix regions (changed in dm5) with the pockets of the binding site (changed in dm4). We conclude that self peptides shared by the F1 hybrid and the BALB/c stimulator cells are recognized in the context of structural features of the helices of the Ag-presenting molecule as alloantigenic determinants. PMID:8144927

  5. Sea Anemone Peptide with Uncommon β-Hairpin Structure Inhibits Acid-sensing Ion Channel 3 (ASIC3) and Reveals Analgesic Activity*

    PubMed Central

    Osmakov, Dmitry I.; Kozlov, Sergey A.; Andreev, Yaroslav A.; Koshelev, Sergey G.; Sanamyan, Nadezhda P.; Sanamyan, Karen E.; Dyachenko, Igor A.; Bondarenko, Dmitry A.; Murashev, Arkadii N.; Mineev, Konstantin S.; Arseniev, Alexander S.; Grishin, Eugene V.

    2013-01-01

    Three novel peptides were isolated from the venom of the sea anemone Urticina grebelnyi. All of them are 29 amino acid peptides cross-linked by two disulfide bridges, with a primary structure similar to other sea anemone peptides belonging to structural group 9a. The structure of the gene encoding the shared precursor protein of the identified peptides was determined. One peptide, π-AnmTX Ugr 9a-1 (short name Ugr 9-1), produced a reversible inhibition effect on both the transient and the sustained current of human ASIC3 channels expressed in Xenopus laevis oocytes. It completely blocked the transient component (IC50 10 ± 0.6 μm) and partially (48 ± 2%) inhibited the amplitude of the sustained component (IC50 1.44 ± 0.19 μm). Using in vivo tests in mice, Ugr 9-1 significantly reversed inflammatory and acid-induced pain. The other two novel peptides, AnmTX Ugr 9a-2 (Ugr 9-2) and AnmTX Ugr 9a-3 (Ugr 9-3), did not inhibit the ASIC3 current. NMR spectroscopy revealed that Ugr 9-1 has an uncommon spatial structure, stabilized by two S-S bridges, with three classical β-turns and twisted β-hairpin without interstrand disulfide bonds. This is a novel peptide spatial structure that we propose to name boundless β-hairpin. PMID:23801332

  6. Inhibitors of HIV-1 maturation: Development of structure-activity relationship for C-28 amides based on C-3 benzoic acid-modified triterpenoids.

    PubMed

    Swidorski, Jacob J; Liu, Zheng; Sit, Sing-Yuen; Chen, Jie; Chen, Yan; Sin, Ny; Venables, Brian L; Parker, Dawn D; Nowicka-Sans, Beata; Terry, Brian J; Protack, Tricia; Rahematpura, Sandhya; Hanumegowda, Umesh; Jenkins, Susan; Krystal, Mark; Dicker, Ira B; Meanwell, Nicholas A; Regueiro-Ren, Alicia

    2016-04-15

    We have recently reported on the discovery of a C-3 benzoic acid (1) as a suitable replacement for the dimethyl succinate side chain of bevirimat (2), an HIV-1 maturation inhibitor that reached Phase II clinical trials before being discontinued. Recent SAR studies aimed at improving the antiviral properties of 2 have shown that the benzoic acid moiety conferred topographical constraint to the pharmacophore and was associated with a lower shift in potency in the presence of human serum albumin. In this manuscript, we describe efforts to improve the polymorphic coverage of the C-3 benzoic acid chemotype through modifications at the C-28 position of the triterpenoid core. The dimethylaminoethyl amides 17 and 23 delivered improved potency toward bevirimat-resistant viruses while increasing C24 in rat oral PK studies. PMID:26988305

  7. Fmoc-modified amino acids and short peptides: simple bio-inspired building blocks for the fabrication of functional materials.

    PubMed

    Tao, Kai; Levin, Aviad; Adler-Abramovich, Lihi; Gazit, Ehud

    2016-07-11

    Amino acids and short peptides modified with the 9-fluorenylmethyloxycarbonyl (Fmoc) group possess eminent self-assembly features and show distinct potential for applications due to the inherent hydrophobicity and aromaticity of the Fmoc moiety which can promote the association of building blocks. Given the extensive study and numerous publications in this field, it is necessary to summarize the recent progress concerning these important bio-inspired building blocks. Therefore, in this review, we explore the self-organization of this class of functional molecules from three aspects, i.e., Fmoc-modified individual amino acids, Fmoc-modified di- and tripeptides, and Fmoc-modified tetra- and pentapeptides. The relevant properties and applications related to cell cultivation, bio-templating, optical, drug delivery, catalytic, therapeutic and antibiotic properties are subsequently summarized. Finally, some existing questions impeding the development of Fmoc-modified simple biomolecules are discussed, and corresponding strategies and outlooks are suggested. PMID:27115033

  8. The permease homologue Ssy1p controls the expression of amino acid and peptide transporter genes in Saccharomyces cerevisiae.

    PubMed

    Didion, T; Regenberg, B; Jørgensen, M U; Kielland-Brandt, M C; Andersen, H A

    1998-02-01

    Amino acid transporters of the yeast plasma membrane (permeases) belong to a family of integral membrane proteins with pronounced structural similarity. We present evidence that a member of this family, encoded by the open reading frame (ORF) YDR160w (SSY1), is required for the expression of a set of transporter genes. Thus, deletion of the SSY1 gene causes loss of leucine-inducible transcription of the amino acid permease genes BAP2, TAT1 and BAP3 (ORF YDR046c) and the peptide transporter, PTR2. D-leucine can generate the signal without entering the cell. We propose that Ssy1p is situated in the plasma membrane and is involved in sensing leucine in the medium. PMID:9489675

  9. Anxiolytic-Like Effect of a Salmon Phospholipopeptidic Complex Composed of Polyunsaturated Fatty Acids and Bioactive Peptides

    PubMed Central

    Belhaj, Nabila; Desor, Frédéric; Gleizes, Céline; Denis, Frédéric M.; Arab-Tehrany, Elmira; Soulimani, Rachid; Linder, Michel

    2013-01-01

    A phospholipopeptidic complex obtained by the enzymatic hydrolysis of salmon heads in green conditions; exert anxiolytic-like effects in a time and dose-dependent manner, with no affection of locomotor activity. This study focused on the physico-chemical properties of the lipidic and peptidic fractions from this natural product. The characterization of mineral composition, amino acid and fatty acids was carried out. Stability of nanoemulsions allowed us to realize a behavioral study conducted with four different tests on 80 mice. This work highlighted the dose dependent effects of the natural complex and its various fractions over a period of 14 days compared to a conventional anxiolytic. The intracellular redox status of neural cells was evaluated in order to determine the free radicals scavenging potential of these products in the central nervous system (CNS), after mice sacrifice. The complex peptidic fraction showed a strong scavenging property and similar results were found for the complex as well as its lipidic fraction. For the first time, the results of this study showed the anxiolytic-like and neuroprotective properties of a phospholipopeptidic complex extracted from salmon head. The applications on anxiety disorders might be relevant, depending on the doses, the fraction used and the chronicity of the supplementation. PMID:24177675

  10. Antibacterial and anticancer activity of a series of novel peptides incorporating cyclic tetra-substituted C(α) amino acids.

    PubMed

    Hicks, Rickey P

    2016-09-15

    Eleven antimicrobial peptides (AMP) based on the incorporation of cyclic tetra substituted C(α) amino acids, as well as other unnatural amino acids were designed, synthesized and screened for in vitro activity against 18 strains of bacteria as well as 12 cancer cell lines. The AMPs discussed herein are derived from the following peptide sequence: Ac-GF(X)G(X)B(X)G(X)F(X)G(X)GB(X)BBBB-amide, X=any one of the following residues, A5c, A6c, Tic or Oic and B=any one of the following residues, Arg, Lys, Orn, Dpr or Dab. A diversity of in vitro inhibitory activity was observed for these AMPs. Several analogs exhibited single digit μM activity against drug resistant bacteria including; multiple drug resistant Mycobacterium tuberculosis, extremely drug resistant Mycobacterium tuberculosis and MRSA. The physicochemical properties of the basic amino acid residues incorporated into these AMPs seem to play a major role in defining antibacterial activity. Overall hydrophobicity seems to play a limited role in defining antibacterial activity. The ESKAPE pathogens were used to compare the activity of these AMPs to another family of synthetic AMPs incorporating the unnatural amino acids Tic and Oic. In most cases similarly substituted members of both families exhibited similar inhibitory activity against the ESKAPE pathogens. In specific cases differences in activity as high as 15 fold were observed between analogs. In addition four of these AMPs exhibited promising IC50 (<7.5μM) values against 12 different and diverse cancer cell lines. Five other AMPs exhibited promising IC50 (<7.5μM) values against selected cancer cell lines. PMID:27387357

  11. Conformational characterization of peptides rich in the cycloaliphatic C alpha,alpha-disubstituted glycine 1-aminocyclononane-1-carboxylic acid.

    PubMed

    Gatos, M; Formaggio, F; Crisma, M; Valle, G; Toniolo, C; Bonora, G M; Saviano, M; Iacovino, R; Menchise, V; Galdiero, S; Pedone, C; Benedetti, E

    1997-01-01

    A series of N- and C-protected, monodispersed homo-oligopeptides (to the pentamer level) from the cycloaliphatic C alpha,alpha-dialkylated glycine 1-aminocyclononane-1-carboxylic acid (Ac9c) and two Ala/Ac9c tripeptides have been synthesized by solution methods and fully characterized. The conformational preferences of all the model peptides were determined in deuterochloroform solution by FT-IR absorption and 1H-NMR. The molecular structures of the amino acid derivatives mCIAc-Ac9c-OH and Z-Ac9c-OtBu, the dipeptide pBrBz-(Ac9c)2-OtBu, the tetrapeptide Z-(Ac9c)4-OtBu, and the pentapeptide Z-(Ac9c)5-OtBu were determined in the crystal state by X-ray diffraction. Based on this information, the average geometry and the preferred conformation for the cyclononyl moiety of the Ac9c residue have been assessed. The backbone conformational data are strongly in favour of the conclusion that the Ac9c residue is a strong beta-turn and helix former. A comparison with the structural propensity of alpha-aminoisobutyric acid, the prototype of C alpha,alpha-dialkylated glycines, and the other extensively investigated members of the family of 1-aminocycloalkane-1-carboxylic acids (Acnc, with n = 3-8) is made and the implications for the use of the Ac9c residue in conformationally constrained analogues of bioactive peptides are briefly examined. PMID:9391912

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

    PubMed

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

    2015-06-15

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

  13. Lasso peptide, a highly stable structure and designable multifunctional backbone.

    PubMed

    Zhao, Ning; Pan, Yongxu; Cheng, Zhen; Liu, Hongguang

    2016-06-01

    Lasso peptide belongs to a new class of natural product with highly compact and stable structure. It has varieties of biological activities, among which the most important one is its antibacterial efficacy. Novel lasso peptides have been constantly discovered and analyzed by advanced techniques, and the biosynthesis or even chemical synthesis of lasso peptide has been studied after learning its constituent amino acids and maturation process. Structural identification of lasso peptide provides information for elucidating the mechanisms of its antibacterial activity and basis for further modifications. Ring of lasso peptide is the key to both its highly compact and stable structure and its intrinsic antibacterial property. The loop has been considered as suitable modification region of lasso peptide, such as V11-S18 of MccJ25 being modifiable without disrupting the lasso structure in biosynthesis. The tail is the immunity protein that can export lasso peptide out of its produced strain and serve as a self-protection mechanism at the same time. Most of currently known lasso peptides are non-pathogenic, which implies that the modified lasso peptides are promising candidates for medical applications. Arginine, glycine, and aspartic acid as a ligands of cancer-specific receptor have been grafted to the loop of lasso peptide without losing its bioactivity, and many other targets are expected to be used for lasso peptide modification. Multi-molecular modification and large-scale production need to be studied and solved in future for designing and using multifunctional lasso peptide based on its extraordinary stable structure. PMID:27074719

  14. Plane wave density functional theory studies of the structural and the electronic properties of amino acids attached to graphene oxide via peptide bonding

    NASA Astrophysics Data System (ADS)

    Min, Byeong June; Jeong, Hae Kyung; Lee, ChangWoo

    2015-08-01

    We studied via plane wave pseudopotential total-energy calculations within the local spin density approximation (LSDA) the electronic and the structural properties of amino acids (alanine, glycine, and histidine) attached to graphene oxide (GO) by peptide bonding. The HOMO-LUMO gap, the Hirshfeld charges, and the equilibrium geometrical structures exhibit distinctive variations that depend on the species of the attached amino acid. The GO-amino acid system appears to be a good candidate for a biosensor.

  15. Production of carrier-peptide conjugates using chemically reactive unnatural amino acids

    SciTech Connect

    Young, Travis; Schultz, Peter G

    2013-12-17

    Provided are methods of making carrier polypeptide that include incorporating a first unnatural amino acid into a carrier polypeptide variant, incorporating a second unnatural amino acid into a target polypeptide variant, and reacting the first and second unnatural amino acids to produce the conjugate. Conjugates produced using the provided methods are also provided. In addition, orthogonal translation systems in methylotrophic yeast and methods of using these systems to produce carrier and target polypeptide variants comprising unnatural amino acids are provided.

  16. Production of carrier-peptide conjugates using chemically reactive unnatural amino acids

    SciTech Connect

    Young, Travis; Schultz, Peter G

    2014-01-28

    Provided are methods of making carrier polypeptide that include incorporating a first unnatural amino acid into a carrier polypeptide variant, incorporating a second unnatural amino acid into a target polypeptide variant, and reacting the first and second unnatural amino acids to produce the conjugate. Conjugates produced using the provided methods are also provided. In addition, orthogonal translation systems in methylotrophic yeast and methods of using these systems to produce carrier and target polypeptide variants comprising unnatural amino acids are provided.

  17. Production of carrier-peptide conjugates using chemically reactive unnatural amino acids

    SciTech Connect

    Young, Travis; Schultz, Peter G.

    2015-08-18

    Provided are methods of making carrier polypeptide that include incorporating a first unnatural amino acid into a carrier polypeptide variant, incorporating a second unnatural amino acid into a target polypeptide variant, and reacting the first and second unnatural amino acids to produce the conjugate. Conjugates produced using the provided methods are also provided. In addition, orthogonal translation systems in methylotrophic yeast and methods of using these systems to produce carrier and target polypeptide variants comprising unnatural amino acids are provided.

  18. Single amino acid mutation in alpha-helical peptide affect second harmonic generation hyperpolarizability

    NASA Astrophysics Data System (ADS)

    Wei, Jing; Wang, Jin-Yun; Zhang, Min-Yi; Chai, Guo-Liang; Lin, Chen-Sheng; Cheng, Wen-Dan

    2013-01-01

    We investigate the effect of side chain on the first-order hyperpolarizability in α-helical polyalanine peptide with the 10th alanine mutation (Acetyl(ala)9X(ala)7NH2). Structures of various substituted peptides are optimized by ONIOM (DFT: AM1) scheme, and then linear and nonlinear optical properties are calculated by SOS//CIS/6-31G∗ method. The polarizability and first-order hyperpolarizability increase obviously only when 'X' represents phenylalanine, tyrosine and tryptophan. We also discuss the origin of nonlinear optical response and determine what caused the increase of first-order hyperpolarizability. Our results strongly suggest that side chains containing benzene, phenol and indole have important contributions to first-order hyperpolarizability.

  19. Quantitative structure-activity relationship study of antioxidative peptide by using different sets of amino acids descriptors

    NASA Astrophysics Data System (ADS)

    Li, Yao-Wang; Li, Bo; He, Jiguo; Qian, Ping

    2011-07-01

    by the hydrogen bond (positively to activity) of the center amino acid. The N-terminal amino acid should be a high hydrophobic and low electronic amino acid (such as Ala, Gly, Val, and Leu); the center amino acid would be an amino acid that possesses high hydrogen bond property (such as base amino acid Arg, Lys, and His). The structural characteristics of antioxidative peptide be found in this paper may contribute to the further research of antioxidative mechanism.

  20. Overcoming the Refractory Expression of Secreted Recombinant Proteins in Mammalian Cells through Modification of the Signal Peptide and Adjacent Amino Acids

    PubMed Central

    Güler-Gane, Gülin; Kidd, Sara; Sridharan, Sudharsan; Vaughan, Tristan J.; Wilkinson, Trevor C. I.

    2016-01-01

    The expression and subsequent purification of mammalian recombinant proteins is of critical importance to many areas of biological science. To maintain the appropriate tertiary structure and post-translational modifications of such proteins, transient mammalian expression systems are often adopted. The successful utilisation of these systems is, however, not always forthcoming and some recombinant proteins prove refractory to expression in mammalian hosts. In this study we focussed on the role of different N-terminal signal peptides and residues immediately downstream, in influencing the level of secreted recombinant protein obtained from suspension HEK293 cells. Using secreted alkaline phosphatase (SEAP) as a model protein, we identified that the +1/+2 downstream residues flanking a heterologous signal peptide significantly affect secreted levels. By incorporating these findings we conducted a comparison of different signal peptide sequences and identified the most productive as secrecon, a computationally-designed sequence. Importantly, in the context of the secrecon signal peptide and SEAP, we also demonstrated a clear preference for specific amino acid residues at the +1 position (e.g. alanine), and a detrimental effect of others (cysteine, proline, tyrosine and glutamine). When proteins that naturally contain these “undesirable” residues at the +1 position were expressed with their native signal peptide, the heterologous secrecon signal peptide, or secrecon with an additional alanine at the +1 or +1 and +2 position, the level of expression differed significantly and in an unpredictable manner. For each protein, however, at least one of the panel of signal peptide/adjacent amino acid combinations enabled successful recombinant expression. In this study, we highlight the important interplay between a signal peptide and its adjacent amino acids in enabling protein expression, and we describe a strategy that could enable recombinant proteins that have so far

  1. Overcoming the Refractory Expression of Secreted Recombinant Proteins in Mammalian Cells through Modification of the Signal Peptide and Adjacent Amino Acids.

    PubMed

    Güler-Gane, Gülin; Kidd, Sara; Sridharan, Sudharsan; Vaughan, Tristan J; Wilkinson, Trevor C I; Tigue, Natalie J

    2016-01-01

    The expression and subsequent purification of mammalian recombinant proteins is of critical importance to many areas of biological science. To maintain the appropriate tertiary structure and post-translational modifications of such proteins, transient mammalian expression systems are often adopted. The successful utilisation of these systems is, however, not always forthcoming and some recombinant proteins prove refractory to expression in mammalian hosts. In this study we focussed on the role of different N-terminal signal peptides and residues immediately downstream, in influencing the level of secreted recombinant protein obtained from suspension HEK293 cells. Using secreted alkaline phosphatase (SEAP) as a model protein, we identified that the +1/+2 downstream residues flanking a heterologous signal peptide significantly affect secreted levels. By incorporating these findings we conducted a comparison of different signal peptide sequences and identified the most productive as secrecon, a computationally-designed sequence. Importantly, in the context of the secrecon signal peptide and SEAP, we also demonstrated a clear preference for specific amino acid residues at the +1 position (e.g. alanine), and a detrimental effect of others (cysteine, proline, tyrosine and glutamine). When proteins that naturally contain these "undesirable" residues at the +1 position were expressed with their native signal peptide, the heterologous secrecon signal peptide, or secrecon with an additional alanine at the +1 or +1 and +2 position, the level of expression differed significantly and in an unpredictable manner. For each protein, however, at least one of the panel of signal peptide/adjacent amino acid combinations enabled successful recombinant expression. In this study, we highlight the important interplay between a signal peptide and its adjacent amino acids in enabling protein expression, and we describe a strategy that could enable recombinant proteins that have so far

  2. Effect of Diaminopropionic acid (Dap) on the Biophysical Properties of a Modified Synthetic Channel-Forming Peptide

    PubMed Central

    Bukovnik, Urska; Sala-Rabanal, Monica; Francis, Simonne; Frazier, Shawnalea J.; Schultz, Bruce D.; Nichols, Colin G.; Tomich, John M.

    2013-01-01

    Channel replacement therapy, based on synthetic channel-forming peptides (CFPs) with the ability to supersede defective endogenous ion channels, is a novel treatment modality that may augment existing interventions against multiple diseases. Previously, we derived CFPs from the second transmembrane segment of the α-subunit of the glycine receptor, M2GlyR, which forms chloride-selective channels in its native form. The best candidate, NK4-M2GlyR T19R, S22W (p22-T19R, S22W), was water-soluble, incorporated into cell membranes and was non-immunogenic, but lacked the structural properties for high conductance and anion selectivity when assembled into a pore. Further studies suggested that the threonine residues at positions 13, 17 and 20 line the pore of assembled p22-T19R, S22W, and here we used 2, 3-diaminopropionic acid (Dap) substitutions to introduce positive charges to the pore-lining interface of the predicted p22-T19R, S22W channel. Dap-substituted p22-T19R, S22W peptides retained the α-helical secondary structure characteristic of their parent peptide, and induced short-circuit transepithelial currents when exposed to the apical membrane of Madin-Darby canine kidney (MDCK) cells; the sequences containing multiple Dap-substituted residues induced larger currents than the peptides with single or no Dap-substitutions. To gain further insights into the effects of Dap residues on the properties of the putative pore, we performed two-electrode voltage clamp electrophysiology on Xenopus oocytes exposed to p22-T19R, S22W or its Dap-modified analogs. We observed that Dap-substituted peptides also induced significantly larger voltage-dependent currents than the parent compound, but there was no apparent change in reversal potential upon replacement of external Na+, Cl− or K+, indicating that these currents remained non-selective. These results suggest that the introduction of positively charged side chains in predicted pore-lining residues does not improve anion

  3. Glucose-dependent insulinotropic peptide: structure of the precursor and tissue-specific expression in rat.

    PubMed Central

    Tseng, C C; Jarboe, L A; Landau, S B; Williams, E K; Wolfe, M M

    1993-01-01

    Glucose-dependent insulinotropic peptide (GIP) is a 42-amino acid gastrointestinal regulatory peptide that stimulates insulin secretion from pancreatic beta cells in the presence of glucose. Approximately 7.8 x 10(5) recombinant clones of a neonatal rat intestinal cDNA library were screened by using plaque hybridization, and three clones were identified and sequenced with the dideoxynucleotide chain-termination method. The translated amino acid sequence deduced from the nucleotide sequence of the cDNA indicated that rat GIP was derived by proteolytic processing of a 144-amino acid precursor polypeptide. The mature peptide is flanked by a 43-amino acid NH2-terminal peptide that contains a 21-amino acid signal peptide and by a 59-amino acid COOH-terminal peptide. Analysis of the nucleotide and amino acid sequence of rat GIP revealed only two substitutions from the known human GIP peptide. The use of high-stringency RNA blot-hybridization analysis of total RNA extracted from various organs demonstrated expression of the GIP gene in the duodenum and jejunum and, to a lesser extent, in the ileum. In addition, expression of the GIP gene was observed in the submandibular salivary gland both by RNA analysis and RIA. In response to duodenal perfusion of a 20% Lipomul meal for 60 min, duodenal mucosal GIP mRNA concentrations increased by 42.8% and 48.2% at 30 and 60 min, respectively. Images Fig. 1 Fig. 4 PMID:8446620

  4. Solid-phase synthesis of amidine-substituted phenylbenzimidazoles and incorporation of this DNA binding and recognition motif into amino acid and peptide conjugates.

    PubMed

    Garner, Matthew L; Georgiadis, Taxiarchis M; Li, Jessica Bo; Wang, Tianxiu; Long, Eric C

    2014-05-01

    Amidine-substituted phenylbenzimidazoles are well-established DNA-binding structural motifs that have contributed to the development of diverse classes of DNA-targeted agents; this ring system not only assists in increasing the overall DNA affinity of an agent, but can also influence its site selectivity. Seeking a means to conveniently exploit these attributes, a protocol for the on-resin synthesis of amino acid- and peptide-phenylbenzimidazole-amidine conjugates was developed to facilitate installation of phenylbenzimidazole-amidines into peptide chains during the course of standard solid-phase syntheses. Building from a resin-bound amino acid or peptide on Rink amide resin, 4-formyl benzoic acid was coupled to the resin-bound free amine followed by introduction of 3,4-diamino-N'-hydroxybenzimidamide (in the presence of 1,4-benzoquinone) to construct the benzimidazole heterocycle. Finally, the resin-bound N'-hydroxybenzimidamide functionality was reduced to an amidine via 1 M SnCl2·2H2O in DMF prior to resin cleavage to release final product. This procedure permits the straightforward synthesis of amino acids or peptides that are N-terminally capped by a phenylbenzimidazole-amidine ring system. Employing this protocol, a series of amino acid-phenylbenzimidazole-amidine (Xaa-R) conjugates was synthesized as well as dipeptide conjugates of the general form Xaa-Gly-R (where R is the phenylbenzimidazole-amidine and Xaa is any amino acid). PMID:24562478

  5. Morphogenic Peptides in Regeneration of Load Bearing Tissues.

    PubMed

    Moeinzadeh, Seyedsina; Jabbari, Esmaiel

    2015-01-01

    Morphogenic proteins due to their short half-life require high doses of growth factors in regeneration of load bearing tissues which leads to undesirable side effects. These side effects include bone overgrowth, tumor formation and immune reaction. An alternative approach to reduce undesirable side effects of proteins in regenerative medicine is to use morphogenic peptides derived from the active domains of morphogenic proteins or soluble and insoluble components of the extracellular matrix of mineralized load bearing tissues to induce differentiation of progenitor cells, mineralization, maturation and bone formation. In that regard, many peptides with osteogenic activity have been discovered. These include peptides derived from bone morphogenic proteins (BMPs), those based on interaction with integrin and heparin-binding receptors, collagen derived peptides, peptides derived from other soluble ECM proteins such as bone sialoprotein and enamel matrix proteins, and those peptides derived from vasculoinductive and neuro-inductive proteins. Although these peptides show significant osteogenic activity in vitro and increase mineralization and bone formation in animal models, they are not widely used in clinical orthopedic applications as an alternative to morphogenic proteins. This is partly due to the limited availability of data on structure and function of morphogenic peptides in physiological medium, particularly in tissue engineered scaffolds. Due to their amphiphilic nature, peptides spontaneously self-assemble and aggregate into micellar structures in physiological medium. Aggregation alters the sequence of amino acids in morphogenic peptides that interact with cell surface receptors thus affecting osteogenic activity of the peptide. Aggregation and micelle formation can dramatically reduce the active concentration of morphogenic peptides with many-fold increase in peptide concentration in physiological medium. Other factors that affect bioactivity are the non

  6. PH dependent adhesive peptides

    DOEpatents

    Tomich, John; Iwamoto, Takeo; Shen, Xinchun; Sun, Xiuzhi Susan

    2010-06-29

    A novel peptide adhesive motif is described that requires no receptor or cross-links to achieve maximal adhesive strength. Several peptides with different degrees of adhesive strength have been designed and synthesized using solid phase chemistries. All peptides contain a common hydrophobic core sequence flanked by positively or negatively charged amino acids sequences.

  7. Protective Effect of Cod (Gadus macrocephalus) Skin Collagen Peptides on Acetic Acid-Induced Gastric Ulcer in Rats.

    PubMed

    Niu, Huina; Wang, Zhicong; Hou, Hu; Zhang, Zhaohui; Li, Bafang

    2016-07-01

    This research was performed to explore the protective effect of cod skin collagen peptides (CCP) on gastric ulcer induced by acetic acid. The CCP were fractionated into low molecular CCP (LMCCP, Mw < 3 kDa) and high molecular CCP (HMCCP, Mw > 3 kDa). In HMCCP and LMCCP, glycine of accounted for about one-third of the total amino acids without cysteine and tryptophan, and hydrophobic amino acids accounted for about 50%. After 21 d CCP treatment (60 or 300 mg/kg, p.o./daily), the healing effects on acetic acid-induced gastric ulcers were evaluated by macroscopic measure, microscopic measure, and immune histochemistry. Moreover, the expression levels of the growth factors, such as vascular endothelial growth factor, epidermal growth factor, transforming growth factor β1 (TGFβ1), and the heat shock protein 70 (HSP70) was detected. The results showed that both LMCCP and HMCCP could significantly decrease the ulcer areas and promote the healing of the lesions. They also could improve the levels of hexosamine, glutathione, superoxide dismutase, and glutathione peroxidase, and reduce the content of malondialdehyde and inducible nitric oxide synthase. In addition, the expression level of TGFβ1 gene and HSP70 mRNA was significantly improved by the treatment. It suggested that CCP could be able to improve symptoms of gastric ulcer and probably be used in the treatment of gastric ulcer. PMID:27219644

  8. α-Azido Acids in Solid-Phase Peptide Synthesis: Compatibility with Fmoc Chemistry and an Alternative Approach to the Solid Phase Synthesis of Daptomycin Analogs.

    PubMed

    Lohani, Chuda Raj; Rasera, Benjamin; Scott, Bradley; Palmer, Michael; Taylor, Scott D

    2016-03-18

    α-Azido acids have been used in solid phase peptide synthesis (SPPS) for almost 20 years. Here we report that peptides bearing an N-terminal α-azidoaspartate residue undergo elimination of an azide ion when treated with reagents that are commonly used for removing the Fmoc group during SPPS. We also report an alternative solid-phase route to the synthesis of an analog of daptomycin that uses a reduced number of α-azido amino acids and without elimination of an azide ion. PMID:26938305

  9. Novel Thiosemicarbazide Hybrids with Amino Acids and Peptides Against Hepatocellular Carcinoma: A Molecular Designing Approach Towards Multikinase Inhibitor.

    PubMed

    Chacko, Shinu; Samanta, Subir

    2015-01-01

    Hepatocellular Carcinoma is the most common primary malignant tumor of the liver. Development of multidrug resistance is the main obstacle to the success of anticancer drugs. In this study, designing and docking study of thiosemicarbazide hybrids with amino acids or peptides against hepatocellular carcinoma was performed since hybrids of biologically active compounds with amino acids or peptides may show target specificity and lower toxicity. All the structures were drawn in 2D platform and converted to the 3D platform using ChemDraw 10.0. Evaluations of ADME properties were done by using QikProp 3.0 to check for the possibility of oral delivery. In silico prediction of LD50 values were performed using Pro-Tox webserver. Interestingly, it was found that conjugation with amino acids decreases toxicity and increases the therapeutic index of thiosemicarbazide. Finally, all the compounds were docked to the crystal structure of the Vascular Endothelial Growth Factor Receptor-2 and Checkpoint kinase-1 utilizing Glide 5.0, Schrödinger 8.5, to understand the interaction of ligands with the receptor. A significant number of derivatives have been found active in both the receptors and also displayed multikinase inhibitory activity similar to Sorafenib, against hepatocellular carcinoma. Further, wet lab synthesis, in vitro ADMET and biological screening studies need to be performed to prove that designed compounds are effective against hepatocellular carcinoma as predicted by molecular modeling. However, as predicted by molecular modeling, the efficacy of designed compounds against hepatocellular carcinoma, needs to be confirmed by wet lab synthesis, in vitro ADMET and biological screening studies. PMID:26526710

  10. Peptide vaccine against canine parvovirus: identification of two neutralization subsites in the N terminus of VP2 and optimization of the amino acid sequence.

    PubMed

    Casal, J I; Langeveld, J P; Cortés, E; Schaaper, W W; van Dijk, E; Vela, C; Kamstrup, S; Meloen, R H

    1995-11-01

    The N-terminal domain of the major capsid protein VP2 of canine parvovirus was shown to be an excellent target for development of a synthetic peptide vaccine, but detailed information about number of epitopes, optimal length, sequence choice, and site of coupling to the carrier protein was lacking. Therefore, several overlapping peptides based on this N terminus were synthesized to establish conditions for optimal and reproducible induction of neutralizing antibodies in rabbits. The specificity and neutralizing ability of the antibody response for these peptides were determined. Within the N-terminal 23 residues of VP2, two subsites able to induce neutralizing antibodies and which overlapped by only two glycine residues at positions 10 and 11 could be discriminated. The shortest sequence sufficient for neutralization induction was nine residues. Peptides longer than 13 residues consistently induced neutralization, provided that their N termini were located between positions 1 and 11 of VP2. The orientation of the peptides at the carrier protein was also of importance, being more effective when coupled through the N terminus than through the C terminus to keyhole limpet hemocyanin. The results suggest that the presence of amino acid residues 2 to 21 (and probably 3 to 17) of VP2 in a single peptide is preferable for a synthetic peptide vaccine. PMID:7474152

  11. Conjugation of fatty acids with different lengths modulates the antibacterial and antifungal activity of a cationic biologically inactive peptide.

    PubMed

    Malina, Amir; Shai, Yechiel

    2005-09-15

    Many studies have shown that an amphipathic structure and a threshold of hydrophobicity of the peptidic chain are crucial for the biological function of AMPs (antimicrobial peptides). However, the factors that dictate their cell selectivity are not yet clear. In the present study, we show that the attachment of aliphatic acids with different lengths (10, 12, 14 or 16 carbon atoms) to the N-terminus of a biologically inactive cationic peptide is sufficient to endow the resulting lipopeptides with lytic activity against different cells. Mode-of-action studies were performed with model phospholipid membranes mimicking those of bacterial, mammalian and fungal cells. These include determination of the structure in solution and membranes by using CD and ATR-FTIR (attenuated total reflectance Fourier-transform infrared) spectroscopy, membrane leakage experiments and by visualizing bacterial and fungal damage via transmission electron microscopy. The results obtained reveal that: (i) the short lipopeptides (10 and 12 carbons atoms) are non-haemolytic, active towards both bacteria and fungi and monomeric in solution. (ii) The long lipopeptides (14 and 16 carbons atoms) are highly antifungal, haemolytic only at concentrations above their MIC (minimal inhibitory concentration) values and aggregate in solution. (iii) All the lipopeptides adopt a partial alpha-helical structure in 1% lysophosphatidylcholine and bacterial and mammalian model membranes. However, the two short lipopeptides contain a significant fraction of random coil in fungal membranes, in agreement with their reduced antifungal activity. (iv) All the lipopeptides have a membranolytic effect on all types of cells assayed. Overall, the results reveal that the length of the aliphatic chain is sufficient to control the pathogen specificity of the lipopeptides, most probably by controlling both the overall hydrophobicity and the oligomeric state of the lipopeptides in solution. Besides providing us with basic

  12. Hydrolysis of Sequenced β-Casein Peptides Provides New Insight into Peptidase Activity from Thermophilic Lactic Acid Bacteria and Highlights Intrinsic Resistance of Phosphopeptides

    PubMed Central

    Deutsch, Stéphanie-Marie; Molle, Daniel; Gagnaire, Valérie; Piot, Michel; Atlan, Danièle; Lortal, Sylvie

    2000-01-01

    The peptidases of thermophilic lactic acid bacteria have a key role in the proteolysis of Swiss cheeses during warm room ripening. To compare their peptidase activities toward a dairy substrate, a tryptic/chymotryptic hydrolysate of purified β-casein was used. Thirty-four peptides from 3 to 35 amino acids, including three phosphorylated peptides, constitute the β-casein hydrolysate, as shown by tandem mass spectrometry. Cell extracts prepared from Lactobacillus helveticus ITG LH1, ITG LH77, and CNRZ 32, Lactobacillus delbrueckii subsp. lactis ITG LL14 and ITG LL51, L. delbrueckii subsp. bulgaricus CNRZ 397 and NCDO 1489, and Streptococcus thermophilus CNRZ 385, CIP 102303, and TA 060 were standardized in protein. The peptidase activities were assessed with the β-casein hydrolysate as the substrate at pH 5.5 and 24°C (conditions of warm room ripening) by (i) free amino acid release, (ii) reverse-phase chromatography, and (iii) identification of undigested peptides by mass spectrometry. Regardless of strain, L. helveticus was the most efficient in hydrolyzing β-casein peptides. Interestingly, cell extracts of S. thermophilus were not able to release a significant level of free proline from the β-casein hydrolysate, which was consistent with the identification of numerous dipeptides containing proline. With the three lactic acid bacteria tested, the phosphorylated peptides remained undigested or weakly hydrolyzed indicating their high intrinsic resistance to peptidase activities. Finally, several sets of peptides differing by a single amino acid in a C-terminal position revealed the presence of at least one carboxypeptidase in the cell extracts of these species. PMID:11097915

  13. pACC1 peptide loaded chitosan nanoparticles induces apoptosis via reduced fatty acid synthesis in MDA-MB-231 cells

    NASA Astrophysics Data System (ADS)

    Kaliaperumal, Jagatheesh; Hari, Natarajan; Pavankumar, Padarthi; Elangovan, Namasivayam

    2015-06-01

    The development of formulations with therapeutic peptides has been restricted to poor cell penetration and in this attempt; we developed pACC1 peptide loaded chitosan nanoparticles. The prepared nanoparticles were characterized with FT-IR, XRD, SEM and TEM. In addition, the suitable formulation was evaluated for hemocompatibility, plasma stability and embryo toxicity using Danio rerio embryo model. The results showed that pACC1 peptide loaded chitosan nanoparticles were compatible with plasma. They possess sustained release pattern and also found to be safe up to 300 mg/L in embryo toxicity tests. Cytotoxicity assays with MDA-MB-231 cell lines suggested that, pACC1 peptide loaded chitosan nanoparticles were capable of enhanced cellular penetration and reduced palmitic acid content, which was confirmed by H1 NMR. Hence, these nanoparticles could be employed as excellent adjuvant therapeutics while treating solid tumors with multi-drug resistance.

  14. pACC1 peptide loaded chitosan nanoparticles induces apoptosis via reduced fatty acid synthesis in MDA-MB-231 cells

    NASA Astrophysics Data System (ADS)

    Kaliaperumal, Jagatheesh; Hari, Natarajan; Pavankumar, Padarthi; Elangovan, Namasivayam

    2016-06-01

    The development of formulations with therapeutic peptides has been restricted to poor cell penetration and in this attempt; we developed pACC1 peptide loaded chitosan nanoparticles. The prepared nanoparticles were characterized with FT-IR, XRD, SEM and TEM. In addition, the suitable formulation was evaluated for hemocompatibility, plasma stability and embryo toxicity using Danio rerio embryo model. The results showed that pACC1 peptide loaded chitosan nanoparticles were compatible with plasma. They possess sustained release pattern and also found to be safe up to 300 mg/L in embryo toxicity tests. Cytotoxicity assays with MDA-MB-231 cell lines suggested that, pACC1 peptide loaded chitosan nanoparticles were capable of enhanced cellular penetration and reduced palmitic acid content, which was confirmed by H1 NMR. Hence, these nanoparticles could be employed as excellent adjuvant therapeutics while treating solid tumors with multi-drug resistance.

  15. Enhanced visualization of small peptides absorbed in rat small intestine by phytic-acid-aided matrix-assisted laser desorption/ionization-imaging mass spectrometry.

    PubMed

    Hong, Seong-Min; Tanaka, Mitsuru; Yoshii, Saori; Mine, Yoshinori; Matsui, Toshiro

    2013-11-01

    Enhanced visualization of small peptides absorbed through a rat intestinal membrane was achieved by matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-IMS) with the aid of phytic acid as a matrix additive. Penetrants through intestinal peptide transporter 1, i.e., glycyl-sarcosine (Gly-Sar, 147.1 m/z) and antihypertensive dipeptide, Val-Tyr (281.2 m/z), were chosen for MALDI-IMS. The signal-to-noise (S/N) ratios of dipeptides Gly-Sar and Val-Tyr were seen to increase by 2.4- and 8.0-fold, respectively, when using a 2',4',6'-trihydroxyacetophenone (THAP) matrix containing 5.0 mM phytic acid, instead of the THAP matrix alone. Owing to the phytic-acid-aided MALDI-IMS method, Gly-Sar and Val-Tyr absorbed in the rat intestinal membrane were successfully visualized. The proposed imaging method also provided useful information on intestinal peptide absorption; to some extent, Val-Tyr was rapidly hydrolyzed to Tyr by peptidases located at the intestinal microvillus during the absorption process. In conclusion, the strongly acidic additive, phytic acid, is beneficial for enhancing the visualization of small peptides using MALDI-IMS, owing to the suppression of ionization-interfering salts in the tissue. PMID:24063774

  16. Pulmonary lung surfactant synthetic peptide concentration-dependent modulation of DPPC and POPG acyl chain order in a DPPC:POPG:palmitic acid lipid mixture.

    PubMed

    Krill, S L; Gupta, S L; Smith, T

    1994-05-01

    Lung surfactant-associated protein interaction with lipid matrices and the effects on lipid thermotropic phase behavior are areas of active research. Many studies limit the lipids to a single or two-component system. The current investigation utilizes a three-lipid component matrix (DPPC:POPG:palmitic acid) to investigate the impact of a synthetic surfactant protein B fragment (SP-B 53-78 DiACM) on the dynamic surface activity of the lipid admixture as measured by a Wilhelmy surface balance. Also, the modulation of the individual lipid acyl chain order by the peptide within the lipid matrix is studied through the use of thermal perturbation FTIR spectroscopy. The data clearly demonstrate a concentration-dependent effect of the peptide on the surface activity with an improvement in the dynamic surface tension diagram characteristics (decreased surface tension and increased collapse plateau) especially at low, 0.36 M%, peptide concentrations. These effects are diminished upon further addition of the peptide. FTIR spectral data demonstrate that the peptide addition results in a significant increase in the acyl chain order of the DPPC and POPG components as measured by the position of the methylene stretching vibrational bands. DPPC is most sensitive to the peptide presence, while the palmitic acid is least affected. The transition temperatures of the individual lipids are also increased with the addition of the peptide. The presence of POPG in the matrix achieves the surface activity similarly seen with natural lung surfactant relative to a DPPC/palmitic acid lipid matrix alone. Its presence increases the sensitivity of the DPPC acyl chains to the presence of the peptide. These effects on the chain order are most probably related to the increased acyl chain fluidity which POPG imparts to the lipid matrix because of the presence of the cis double bond. The phosphatidylglycerol headgroup also adds a negative charge to the lipid matrix which enhances the peptide

  17. Modeling of Peptide Adsorption Interactions with a Poly(lactic acid) Surface

    PubMed Central

    O'Brien, C. P.; Stuart, S. J.; Bruce, D. A.; Latour, R. A.

    2009-01-01

    The biocompatibility of implanted materials and devices is governed by the conformation, orientation, and composition of the layer of proteins that adsorb to the surface of the material immediately upon implantation, so an understanding of this adsorbed protein layer is essential to the rigorous and methodical design of implant materials. In this study, novel molecular dynamics techniques were employed in order to determine the change in free energy for the adsorption of a solvated nine-residue peptide (GGGG-K-GGGG) to a crystalline polylactide surface in an effort to elucidate the fundamental mechanisms that govern protein adsorption. This system, like many others, involves two distinct types of sampling problems: a spatial sampling problem, which arises due to entropic effects creating barriers in the free energy profile, and a conformational sampling problem, which occurs due to barriers in the potential energy landscape. In a two-step process that addresses each sampling problem in turn, the technique of biased replica exchange molecular dynamics was refined and applied in order to overcome these sampling problems and, using the information available at the atomic level of detail afforded by molecular simulation, both quantify and characterize the interactions between the peptide and a relevant biomaterial surface. The results from these simulations predict a fairly strong adsorption response with an adsorption free energy of -2.5 ± 0.6 kcal/mol (mean ±95% confidence interval), with adsorption primarily due to hydrophobic interactions between the nonpolar groups of the peptide and the PLA surface. As part of a larger and ongoing effort involving both simulation and experimental investigations, this work contributes to the goal of transforming the engineering of biomaterials from one dominated by trial-and-error to one which is guided by an atomic-level understanding of the interactions that occur at the tissue-biomaterial interface. PMID:19360943

  18. cis9, trans11-Conjugated Linoleic Acid Differentiates Mouse 3T3-L1 Preadipocytes into Mature Small Adipocytes through Induction of Peroxisome Proliferator-activated Receptor γ.

    PubMed

    Sakuma, Satoru; Nishioka, Yuki; Imanishi, Ryohta; Nishikawa, Kenji; Sakamoto, Hirotada; Fujisawa, Junji; Wada, Koichiro; Kamisaki, Yoshinori; Fujimoto, Yohko

    2010-09-01

    Dietary conjugated linoleic acid (CLA) has been reported to exhibit a number of therapeutic effects in animal models and patients, such as anti-hypertensive, anti-hyperlipidemic, anti-arteriosclerotic, anti-carcinogenic, and anti-diabetic effects. However, the underlying mechanism is not well-characterized. In the present study, the effects of cis(c)9, trans(t)11-CLA on the differentiation of mouse 3T3-L1 preadipocytes into mature adipocytes were examined. Treatment with c9, t11-CLA in the presence of insulin, dexamethasone, and 3-isobutyl-1-methyl-xanthine (differentiation cocktail) significantly stimulated the accumulation of triacylglycerol. The microscopic observation of cells stained by Oil Red O demonstrated that c9, t11-CLA increases the amount and proportion of small mature adipocytes secreting adiponectin, a benign adipocytokine, when compared to the differentiation cocktail alone. Furthermore, c9, t11-CLA increased bioactive peroxisome proliferator-activated receptor γ (PPARγ) levels in a nuclear extract of 3T3-L1 cells, suggesting the enhancing effect of this fatty acid on the nuclear transmission of PPARγ, a master regulator of adipocyte differentiation, in 3T3-L1 cells. These results suggest that the therapeutic effects of c9, t11-CLA on lifestyle-related diseases are partially due to the enhanced formation of small adipocytes from preadipocytes via PPARγ stimulation. PMID:20838573

  19. Biophysical Property and Broad Anti-HIV Activity of Albuvirtide, a 3-Maleimimidopropionic Acid-Modified Peptide Fusion Inhibitor

    PubMed Central

    Chong, Huihui; Yao, Xue; Zhang, Chao; Cai, Lifeng; Cui, Sheng; Wang, Youchun; He, Yuxian

    2012-01-01

    Albuvirtide (ABT) is a 3-maleimimidopropionic acid (MPA)-modified peptide HIV fusion inhibitor that can irreversibly conjugate to serum albumin. Previous studies demonstrated its in vivo long half-life and potent anti-HIV activity. Here, we focused to characterize its biophysical properties and evaluate its antiviral spectrum. In contrast to T20 (Enfuvirtide, Fuzeon), ABT was able to form a stable α-helical conformation with the target sequence and block the fusion-active six-helix bundle (6-HB) formation in a dominant-negative manner. It efficiently inhibited HIV-1 Env-mediated cell membrane fusion and virus entry. A large panel of 42 HIV-1 pseudoviruses with different genotypes were constructed and used for the antiviral evaluation. The results showed that ABT had potent inhibitory activity against the subtypes A, B and C that predominate the worldwide AIDS epidemics, and subtype B′, CRF07_BC and CRF01_AE recombinants that are currently circulating in China. Furthermore, ABT was also highly effective against HIV-1 variants resistant to T20. Taken together, our data indicate that the chemically modified peptide ABT can serve as an ideal HIV-1 fusion inhibitor. PMID:22403678

  20. Peptide-conjugated hyaluronic acid surface for the culture of human induced pluripotent stem cells under defined conditions.

    PubMed

    Zhang, Xiaohong; Zhou, Ping; Zhao, Yinghui; Wang, Mengke; Wei, Shicheng

    2016-01-20

    Hyaluronic acid (HA) has been cross-linked to form hydrogel for potential applications in the self-renewal and differentiation of human pluripotent stem cells (hPSCs) for years. However, HA hydrogel with improved residence time and mechanical integrity that allows the survival of hPSCs under defined conditions is still much needed for clinical applications. In this study, HA was modified with methacrylate functional groups (MeHA) and cross-linked by photo-crosslinking method. After subsequent conjugation with adhesive peptide, these MeHA surfaces demonstrated performance in facilitating human induced pluripotent stem cells (hiPSCs) proliferation, and good pluripotency maintenance of hiPSCs under defined conditions. Moreover, MeHA films on glass-slides exhibited long residence time and mechanical stability throughout hiPSC culture. Our photo-crosslinkable MeHA possesses great value in accelerating the application of HA hydrogel in hiPSCs proliferation and differentiation with the conjugation of adhesive peptides. PMID:26572447

  1. Spectrophotometric Determination and Removal of Unchelated Europium Ions from Solutions Containing Eu-Diethylenetriaminepentaacetic Acid Chelate-Peptide Conjugates1

    PubMed Central

    Dayan Elshan, N. G. R.; Patek, Renata; Vagner, Josef; Mash, Eugene A.

    2014-01-01

    Europium chelates conjugated with peptide ligands are routinely used as probes for conducting in vitro binding experiments. The presence of unchelated Eu ions in these formulations gives high background luminescence and can lead to poor results in binding assays. In our experience, the reported methods for purification of these probes do not achieve adequate removal of unchelated metal ions in a reliable manner. In this work, a xylenol orange-based assay for the quantification of unchelated metal ions was streamlined and used to determine levels of metal ion contamination, as well as the success of metal ion removal upon attempted purification. We compared the use of Empore™ chelating disks and Chelex® 100 resin for the selective removal of unchelated Eu ions from several Eu-diethylenetriaminepentaacetic acid chelate-peptide conjugates. Both purification methods gave complete and selective removal of the contaminant metal ions. However, Empore™ chelating disks were found to give much higher recoveries of the probes under the conditions utilized. Related to the issue of probe recovery, we also describe a significantly more efficient method for the synthesis of one such probe using Rink amide AM resin in place of Tentagel S resin. PMID:25058927

  2. Scorpion Venom Heat-Resistant Peptide Attenuates Glial Fibrillary Acidic Protein Expression via c-Jun/AP-1.

    PubMed

    Cao, Zhen; Wu, Xue-Fei; Peng, Yan; Zhang, Rui; Li, Na; Yang, Jin-Yi; Zhang, Shu-Qin; Zhang, Wan-Qin; Zhao, Jie; Li, Shao

    2015-11-01

    Scorpion venom has been used in the Orient to treat central nervous system diseases for many years, and the protein/peptide toxins in Buthus martensii Karsch (BmK) venom are believed to be the effective components. Scorpion venom heat-resistant peptide (SVHRP) is an active component of the scorpion venom extracted from BmK. In a previous study, we found that SVHRP could inhibit the formation of a glial scar, which is characterized by enhanced glial fibrillary acidic protein (GFAP) expression, in the epileptic hippocampus. However, the cellular and molecular mechanisms underlying this process remain to be clarified. The results of the present study indicate that endogenous GFAP expression in primary rat astrocytes was attenuated by SVHRP. We further demonstrate that the suppression of GFAP was primarily mediated by inhibiting both c-Jun expression and its binding with AP-1 DNA binding site and other factors at the GFAP promoter. These results support that SVHRP contributes to reducing GFAP at least in part by decreasing the activity of the transcription factor AP-1. In conclusion, the effects of SVHRP on astrocytes with respect to the c-Jun/AP-1 signaling pathway in vitro provide a practical basis for studying astrocyte activation and inhibition and a scientific basis for further studies of traditional medicine. PMID:26134308

  3. High efficacy of a 20 amino acid peptide of the acidic ribosomal protein P0 against the cattle tick, Rhipicephalus microplus.

    PubMed

    Rodríguez-Mallon, Alina; Encinosa, Pedro E; Méndez-Pérez, Lídice; Bello, Yamil; Rodríguez Fernández, Rafmary; Garay, Hilda; Cabrales, Ania; Méndez, Luis; Borroto, Carlos; Estrada, Mario Pablo

    2015-06-01

    Current strategies to control cattle ticks use integrated control programs (ICP) that include vaccination. Reduction in the use of chemicals and in the cost of tick control, the delay or elimination of acaricide resistance and the decreasing of environmental pollution are the advantages of using these programs. This integrated program is potentially applicable to all genotypes of chemical resistant ticks. However, the problem here is to improve the efficacy of anti-tick vaccines. The P0 protein is a structural component of the ribosome of all organisms. We have identified an immunogenic region of ribosomal protein P0 from Rhipicephalus spp. ticks that is not very conserved compared to the orthologous protein in their hosts. A synthetic 20 amino acid peptide from this sequence was effective as a vaccine against Rhipicephalus sanguineus infestations in an immunization and challenge experiment using rabbits. In this paper, the same peptide used as vaccine against the cattle tick Rhipicephalus Boophilus microplus shows a significant diminution in the number of engorged females recovered, in the weight of females and the weight of egg masses. The number of eggs hatched was also significantly reduced for the vaccinated group, with an overall effectivity for the antigen pP0 of 96%. These results, together with the conserved sequence of the P0 peptide among ticks, suggest that this antigen could be a good broad spectrum vaccine candidate. It would be expected to be active against many species of ticks and thus has promise in an ICP for effective control of ticks and thereby to improve the efficiency and productivity of the livestock industry. PMID:25958782

  4. Adsorption of biomedical coating molecules, amino acids, and short peptides on magnetite (110).

    PubMed

    Aschauer, Ulrich; Selloni, Annabella

    2015-07-28

    Superparamagnetic iron oxide nanoparticles for biomedical applications are usually coated with organic molecules to form a steric barrier against agglomeration. The stability of these coatings is well established in the synthesis medium but is more difficult to assess in physiological environment. To obtain a first theoretical estimate of their stability in such an environment, we perform density functional theory calculations of the adsorption of water, polyvinyl alcohol (PVA) and polyethylene glycol (PEG) coating molecules, as well as the monomer and dimer of glycine as a prototype short peptide, on the (110) surface of magnetite (Fe3O4) in vacuo. Our results show that PVA binds significantly stronger to the surface than both PEG and glycine, while the difference between the latter two is quite small. Depending on the coverage, the water adsorption strength is intermediate between PVA and glycine. Due to its strongly interacting OH side groups, PVA is likely to remain bound to the surface in the presence of short peptides. This stability will have to be further assessed by molecular dynamics in the solvated state for which the present work forms the basis. PMID:26233155

  5. Improved Synthesis Strategy for Peptide Nucleic Acids (PNA) appropriate for Cell-specific Fluorescence Imaging

    PubMed Central

    Pipkorn, Rüdiger; Wiessler, Manfred; Waldeck, Waldemar; Hennrich, Ute; Nokihara, Kiyoshi; Beining, Marcel; Braun, Klaus

    2012-01-01

    Progress in genomics and proteomics attended to the door for better understanding the recent rapid expanding complex research field of metabolomics. This trend in biomedical research increasingly focuses to the development of patient-specific therapeutic approaches with higher efficiency and sustainability. Simultaneously undesired adverse reactions are avoided. In parallel, the development of molecules for molecular imaging is required not only for the imaging of morphological structures but also for the imaging of metabolic processes like the aberrant expression of the cysteine protease cathepsin B (CtsB) gene and the activity of the resulting product associated with metastasis and invasiveness of malign tumors. Finally the objective is to merge imaging and therapy at the same level. The design of molecules which fulfil these responsibilities is pivotal and requires proper chemical methodologies. In this context our modified solid phase peptide chemistry using temperature shifts during synthesis is considered as an appropriate technology. We generated highly variable conjugates which consist of molecules useful as diagnostically and therapeutically active molecules. As an example the modular PNA products with the complementary sequence to the CtsB mRNA and additionally with a cathepsin B cleavage site had been prepared as functional modules for distinction of cell lines with different CtsB gene expression. After ligation to the modular peptide-based BioShuttle carrier, which was utilized to facilitate the delivery of the functional modules into the cells' cytoplasm, the modules were scrutinized. PMID:22211082

  6. Design of antimicrobial peptides conjugated biodegradable citric acid derived hydrogels for wound healing.

    PubMed

    Xie, Zhiwei; Aphale, Nikhil V; Kadapure, Tejaswi D; Wadajkar, Aniket S; Orr, Sara; Gyawali, Dipendra; Qian, Guoying; Nguyen, Kytai T; Yang, Jian

    2015-12-01

    Wound healing is usually facilitated by the use of a wound dressing that can be easily applied to cover the wound bed, maintain moisture, and avoid bacterial infection. In order to meet all of these requirements, we developed an in situ forming biodegradable hydrogel (iFBH) system composed of a newly developed combination of biodegradable poly(ethylene glycol) maleate citrate (PEGMC) and poly(ethylene glycol) diacrylate (PEGDA). The in situ forming hydrogel systems are able to conform to the wound shape in order to cover the wound completely and prevent bacterial invasion. A 2(k) factorial analysis was performed to examine the effects of polymer composition on specific properties, including the curing time, Young's modulus, swelling ratio, and degradation rate. An optimized iFBH formulation was achieved from the systematic factorial analysis. Further, in vitro biocompatibility studies using adult human dermal fibroblasts (HDFs) confirmed that the hydrogels and degradation products are not cytotoxic. The iFBH wound dressing was conjugated and functionalized with antimicrobial peptides as well. Evaluation against bacteria both in vitro and in vivo in rats demonstrated that the peptide-incorporated iFBH wound dressing offered excellent bacteria inhibition and promoted wound healing. These studies indicated that our in situ forming antimicrobial biodegradable hydrogel system is a promising candidate for wound treatment. PMID:26014899

  7. Peptide nucleic acids targeting β-globin mRNAs selectively inhibit hemoglobin production in murine erythroleukemia cells.

    PubMed

    Montagner, Giulia; Gemmo, Chiara; Fabbri, Enrica; Manicardi, Alex; Accardo, Igea; Bianchi, Nicoletta; Finotti, Alessia; Breveglieri, Giulia; Salvatori, Francesca; Borgatti, Monica; Lampronti, Ilaria; Bresciani, Alberto; Altamura, Sergio; Corradini, Roberto; Gambari, Roberto

    2015-01-01

    In the treatment of hemoglobinopathies, amending altered hemoglobins and/or globins produced in excess is an important part of therapeutic strategies and the selective inhibition of globin production may be clinically beneficial. Therefore the development of drug-based methods for the selective inhibition of globin accumulation is required. In this study, we employed peptide nucleic acids (PNAs) to alter globin gene expression. The main conclusion of the present study was that PNAs designed to target adult murine β-globin mRNA inhibit hemoglobin accumulation and erythroid differentiation of murine erythroleukemia (MEL) cells with high efficiency and fair selectivity. No major effects were observed on cell proliferation. Our study supports the concept that PNAs may be used to target mRNAs that, similar to globin mRNAs, are expressed at very high levels in differentiating erythroid cells. Our data suggest that PNAs inhibit the excess production of globins involved in the pathophysiology of hemoglobinopathies. PMID:25405921

  8. Peptide Nucleic Acid with a Lysine Side Chain at the β-Position: Synthesis and Application for DNA Cleavage.

    PubMed

    Sugiyama, Toru; Kuwata, Keiko; Imamura, Yasutada; Demizu, Yosuke; Kurihara, Masaaki; Takano, Masashi; Kittaka, Atsushi

    2016-01-01

    This paper reports the synthesis of new β-Lys peptide nucleic acid (PNA) monomers and their incorporation into a 10-residue PNA sequence. PNA containing β-Lys PNA units formed a stable hybrid duplex with DNA. However, incorporation of β-Lys PNA units caused destabilization of PNA-DNA duplexes to some extent. Electrostatic attractions between β-PNA and DNA could reduce this destabilization effect. Subsequently, bipyridine-conjugated β-Lys PNA was prepared and exhibited sequence selective cleavage of DNA. Based on the structures of the cleavage products and molecular modeling, we reasoned that bipyridine moiety locates within the minor groove of the PNA-DNA duplexes. The lysine side chain of β-PNA is a versatile handle for attaching various functional molecules. PMID:27373637

  9. Using Triple Helix Forming Peptide Nucleic Acids for Sequence-selective Recognition of Double-stranded RNA

    PubMed Central

    Hnedzko, Dziyana; Cheruiyot, Samwel K.; Rozners, Eriks

    2014-01-01

    Non-coding RNAs play important roles in regulation of gene expression. Specific recognition and inhibition of these biologically important RNAs that form complex double-helical structures will be highly useful for fundamental studies in biology and practical applications in medicine. This protocol describes a strategy developed in our laboratory for sequence-selective recognition of double-stranded RNA (dsRNA) using triple helix forming peptide nucleic acids (PNAs) that bind in the major grove of RNA helix. The strategy developed uses chemically modified nucleobases, such as 2-aminopyridine (M) that enables strong triple helical binding at physiologically relevant conditions, and 2-pyrimidinone (P) and 3-oxo-2,3-dihydropyridazine (E) that enable recognition of isolated pyrimidines in the purine rich strand of the RNA duplex. Detailed protocols for preparation of modified PNA monomers, solid-phase synthesis and HPLC purification of PNA oligomers, and measuring dsRNA binding affinity using isothermal titration calorimetry are included. PMID:25199637

  10. Hyaluronic acid hydrogels with IKVAV peptides for tissue repair and axonal regeneration in an injured rat brain

    NASA Astrophysics Data System (ADS)

    Wei, Y. T.; Tian, W. M.; Yu, X.; Cui, F. Z.; Hou, S. P.; Xu, Q. Y.; Lee, In-Seop

    2007-09-01

    A biocompatible hydrogel of hyaluronic acid with the neurite-promoting peptide sequence of IKVAV was synthesized. The characterization of the hydrogel shows an open porous structure and a large surface area available for cell interaction. Its ability to promote tissue repair and axonal regeneration in the lesioned rat cerebrum is also evaluated. After implantation, the polymer hydrogel repaired the tissue defect and formed a permissive interface with the host tissue. Axonal growth occurred within the microstructure of the network. Within 6 weeks the polymer implant was invaded by host-derived tissue, glial cells, blood vessels and axons. Such a hydrogel matrix showed the properties of neuron conduction. It has the potential to repair tissue defects in the central nervous system by promoting the formation of a tissue matrix and axonal growth by replacing the lost tissue.

  11. Duplex DNA-Invading γ-Modified Peptide Nucleic Acids Enable Rapid Identification of Bloodstream Infections in Whole Blood

    PubMed Central

    Nölling, Jörk; Rapireddy, Srinivas; Amburg, Joel I.; Crawford, Elizabeth M.; Prakash, Ranjit A.; Rabson, Arthur R.

    2016-01-01

    ABSTRACT Bloodstream infections are a leading cause of morbidity and mortality. Early and targeted antimicrobial intervention is lifesaving, yet current diagnostic approaches fail to provide actionable information within a clinically viable time frame due to their reliance on blood culturing. Here, we present a novel pathogen identification (PID) platform that features the use of duplex DNA-invading γ-modified peptide nucleic acids (γPNAs) for the rapid identification of bacterial and fungal pathogens directly from blood, without culturing. The PID platform provides species-level information in under 2.5 hours while reaching single-CFU-per-milliliter sensitivity across the entire 21-pathogen panel. The clinical utility of the PID platform was demonstrated through assessment of 61 clinical specimens, which showed >95% sensitivity and >90% overall correlation to blood culture findings. This rapid γPNA-based platform promises to improve patient care by enabling the administration of a targeted first-line antimicrobial intervention. PMID:27094328

  12. Nitrogen-15 NMR spectroscopy of the catalytic-triad histidine of a serine protease in peptide boronic acid inhibitor complexes

    SciTech Connect

    Bachovchin, W.W.; Wong, W.Y.L.; Farr-Jones, S. ); Shenvi, A.B.; Kettner, C.A. )

    1988-10-04

    {sup 15}N NMR spectroscopy was used to examine the active-site histidyl residue of {alpha}-lytic protease in peptide boronic acid inhibitor complexes. Two distinct types of complexes were observed: (1) Boronic acids that are analogues of substrates form complexes in which the active-site imidazole ring is protonated and both imidazole N-H protons are strongly hydrogen bonded. (2) Boronic acids that are not substrate analogues form complexes in which N{sup {epsilon}2} of the active-site histidine is covalently bonded to the boron atom of the inhibitor. The proton bound to N{sup {delta}1} of the histidine in these histidine-boronate adducts remains strongly hydrogen bonded, presumably to the active-site aspartate. In both types of complexes the N-H protons of His-57 exchange unusually slowly as evidenced by the room temperature visibility of the low-field {sup 1}H resonances and the {sup 15}N-H spin couplings. These results indicate that occupancy of the specificity subsites may be required to fully form the transition-state binding site. The significance of these findings for understanding inhibitor binding and the catalytic mechanism of serine proteases is discussed.

  13. Effect of lipid composition and amino acid sequence upon transmembrane peptide-accelerated lipid transleaflet diffusion (flip-flop).

    PubMed

    LeBarron, Jamie; London, Erwin

    2016-08-01

    We examined how hydrophobic peptide-accelerated transleaflet lipid movement (flip-flop) was affected by peptide sequence and vesicle composition and properties. A peptide with a completely hydrophobic sequence had little if any effect upon flip-flop. While peptides with a somewhat less hydrophobic sequence accelerated flip-flop, the half-time remained slow (hours) with substantial (0.5mol%) peptide in the membranes. It appears that peptide-accelerated lipid flip-flop involves a rare event that may reflect a rare state of the peptide or lipid bilayer. There was no simple relationship between peptide overall hydrophobicity and flip-flop. In addition, flip-flop was not closely linked to whether the peptides were in a transmembrane or non-transmembrane (interfacial) inserted state. Flip-flop was also not associated with peptide-induced pore formation. We found that peptide-accelerated flip-flop is initially faster in small (highly curved) unilamellar vesicles relative to that in large unilamellar vesicles. Peptide-accelerated flip-flop was also affected by lipid composition, being slowed in vesicles with thick bilayers or those containing 30% cholesterol. Interestingly, these factors also slow spontaneous lipid flip-flop in the absence of peptide. Combined with previous studies, the results are most consistent with acceleration of lipid flip-flop by peptide-induced thinning of bilayer width. PMID:27131444

  14. A scorpion venom peptide fraction induced prostaglandin biosynthesis in guinea pig kidneys: incorporation of 14C-linoleic acid.

    PubMed

    el-Saadani, Muhammad A

    2004-01-01

    A peptide fraction isolated from the venom of the Egyptian scorpion Buthus occitanus was proved to have a bradykinin- potentiating activity. In vivo and in vitro modes of action of the isolated bradykinin-potentiating peptide (BPP) on kidneys of guinea pigs were investigated. Animals received five successive i.p. doses of the scorpion BPP (1 microg/g body weight) at one-week intervals. The control animals were i.p. injected with saline solution only. In vivo experiments showed a significant increase in renal tissue PGE(2) content and lipid peroxides of the treated guinea pigs compared to the control animals (p < 0.05). Nonsignificant changes were detected in the levels of tissue c-AMP and 5-nucleotidase activity (p > 0.05) of the treated animals, while the changes in c-GMP and c-AMP/c-GMP ratio were both significant (p < 0.05). In vitro experiments demonstrated enhanced capacity of guinea pig-renal tissue to convert (14)C-linoleic acid to its metabolites, 6-keto-PGF(1)alpha, PGF(2)alpha, PGE(2), TxB(2), PGD(2), and arachidonic acid, in response to the added PBP (1 microg/ml) and bradykinin (1 microg/ml). This enhanced response was abolished upon the addition of 1 microg/ml of BK-inhibitor (D-Arg- [Hyp(3), Thi(5,6), Phe(7)]). The capacity for labeled metabolites recovery in BPP treated renal tissue was 19.78%, while it was 13.00% in the basal control. The total increase that evoked by BPP was 62.78%. The results clearly indicate that the isolated BPP induced prostaglandin biosynthesis, which may trigger enhanced glomerular filtration in guinea pigs. PMID:14999016

  15. Influence of Amino Acid Compositions and Peptide Profiles on Antioxidant Capacities of Two Protein Hydrolysates from Skipjack Tuna (Katsuwonus pelamis) Dark Muscle.

    PubMed

    Chi, Chang-Feng; Hu, Fa-Yuan; Wang, Bin; Li, Zhong-Rui; Luo, Hong-Yu

    2015-05-01

    Influence of amino acid compositions and peptide profiles on antioxidant capacities of two protein hydrolysates from skipjack tuna (Katsuwonus pelamis) dark muscle was investigated. Dark muscles from skipjack tuna were hydrolyzed using five separate proteases, including pepsin, trypsin, Neutrase, papain and Alcalase. Two hydrolysates, ATH and NTH, prepared using Alcalase and Neutrase, respectively, showed the strongest antioxidant capacities and were further fractionated using ultrafiltration and gel filtration chromatography. Two fractions, Fr.A3 and Fr.B2, isolated from ATH and NTH, respectively, showed strong radical scavenging activities toward 2,2-diphenyl-1-picrylhydrazyl radicals (EC50 1.08% ± 0.08% and 0.98% ± 0.07%), hydroxyl radicals (EC50 0.22% ± 0.03% and 0.48% ± 0.05%), and superoxide anion radicals (EC50 1.31% ± 0.11% and 1.56% ± 1.03%) and effectively inhibited lipid peroxidation. Eighteen peptides from Fr.A3 and 13 peptides from Fr.B2 were isolated by reversed-phase high performance liquid chromatography, and their amino acid sequences were determined. The elevated antioxidant activity of Fr.A3 might be due to its high content of hydrophobic and aromatic amino acid residues (181.1 and 469.9 residues/1000 residues, respectively), small molecular sizes (3-6 peptides), low molecular weights (524.78 kDa), and amino acid sequences (antioxidant score 6.11). This study confirmed that a smaller molecular size, the presence of hydrophobic and aromatic amino acid residues, and the amino acid sequences were the key factors that determined the antioxidant activities of the proteins, hydrolysates and peptides. The results also demonstrated that the derived hydrolysates and fractions from skipjack tuna (K. pelamis) dark muscles could prevent oxidative reactions and might be useful for food preservation and medicinal purposes. PMID:25923316

  16. Influence of Amino Acid Compositions and Peptide Profiles on Antioxidant Capacities of Two Protein Hydrolysates from Skipjack Tuna (Katsuwonus pelamis) Dark Muscle

    PubMed Central

    Chi, Chang-Feng; Hu, Fa-Yuan; Wang, Bin; Li, Zhong-Rui; Luo, Hong-Yu

    2015-01-01

    Influence of amino acid compositions and peptide profiles on antioxidant capacities of two protein hydrolysates from skipjack tuna (Katsuwonus pelamis) dark muscle was investigated. Dark muscles from skipjack tuna were hydrolyzed using five separate proteases, including pepsin, trypsin, Neutrase, papain and Alcalase. Two hydrolysates, ATH and NTH, prepared using Alcalase and Neutrase, respectively, showed the strongest antioxidant capacities and were further fractionated using ultrafiltration and gel filtration chromatography. Two fractions, Fr.A3 and Fr.B2, isolated from ATH and NTH, respectively, showed strong radical scavenging activities toward 2,2-diphenyl-1-picrylhydrazyl radicals (EC50 1.08% ± 0.08% and 0.98% ± 0.07%), hydroxyl radicals (EC50 0.22% ± 0.03% and 0.48% ± 0.05%), and superoxide anion radicals (EC50 1.31% ± 0.11% and 1.56% ± 1.03%) and effectively inhibited lipid peroxidation. Eighteen peptides from Fr.A3 and 13 peptides from Fr.B2 were isolated by reversed-phase high performance liquid chromatography, and their amino acid sequences were determined. The elevated antioxidant activity of Fr.A3 might be due to its high content of hydrophobic and aromatic amino acid residues (181.1 and 469.9 residues/1000 residues, respectively), small molecular sizes (3–6 peptides), low molecular weights (524.78 kDa), and amino acid sequences (antioxidant score 6.11). This study confirmed that a smaller molecular size, the presence of hydrophobic and aromatic amino acid residues, and the amino acid sequences were the key factors that determined the antioxidant activities of the proteins, hydrolysates and peptides. The results also demonstrated that the derived hydrolysates and fractions from skipjack tuna (K. pelamis) dark muscles could prevent oxidative reactions and might be useful for food preservation and medicinal purposes. PMID:25923316

  17. Quercetin, kaempferol, myricetin, and fatty acid content among several Hibiscus sabdariffa accession calyces based on maturity in a greenhouse

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flavonols including quercetin, kaempferol, myricetin, and fatty acids in plants have many useful health attributes including antioxidants, cholesterol lowering, and cancer prevention. Six accessions of roselle, Hibiscus sabdariffa calyces were evaluated for quercetin, kaempferol, and myricetin conte...

  18. Compositional Variation in Sugars and Organic Acids at Different Maturity Stages in Selected Small Fruits from Pakistan

    PubMed Central

    Mahmood, Tahir; Anwar, Farooq; Abbas, Mateen; Boyce, Mary C.; Saari, Nazamid

    2012-01-01

    Selected soluble sugars and organic acids were analyzed in strawberry, sweet cherry, and mulberry fruits at different ripening stages by HPLC. The amounts of fructose, glucose and sucrose were found to be: strawberry (1.79–2.86, 1.79–2.25 and 0.01–0.25 g/100 g FW), sweet cherry (0.76–2.35, 0.22–3.39 and 0.03–0.13 g/100 g) and mulberry (3.07–9.41, 1.53–4.95 and 0.01–0.25 g/100 g) at un-ripened to fully-ripened stages, respectively. The strawberry, sweet cherry and mulberry mainly contained tartaric, citric and ascorbic acids in the range of 16–55, 70–1934 and 11–132 mg/100 g; 2–8, 2–10 and 10–17 mg/100 g; 2–118, 139–987 and 2–305 mg/100 g at un-ripened to fully-ripened stages, respectively. Fructose and glucose were established to be the major sugars in all the tested fruit while citric and ascorbic acid were the predominant organic acids in strawberry and mulberry while tartaric acid was mainly present in sweet cherry. The tested fruits mostly showed an increase in the concentration of sugars and organic acids with ripening. PMID:22408396

  19. THE APPLICATION OF PEPTIDE NUCLEIC ACID PROBES FOR RAPID DETECTION AND ENUMERATION OF EUBACTERIA, STAPHYLOCOCCUS AUREUS AND PSEUDOMONAS AERUGINOSA IN RECREATIONAL BEACHES OF S. FLORIDA. (R828830)

    EPA Science Inventory

    A novel chemiluminescent in situ hybridization technique using peptide nucleic acids (PNA) was adapted for the detection of bacteria in beach sand and recreational waters in South Florida. The simultaneous detection and enumeration of eubacteria and the novel indicators, S...

  20. An acidic peptide sequence of nucleolin-related protein can mediate the attachment of calcium oxalate to renal tubule cells.

    PubMed

    Sorokina, Elena A; Wesson, Jeffrey A; Kleinman, Jack G

    2004-08-01

    Crystals that form in tubular fluid must be retained in the kidney to become stones. Nucleolin-related protein (NRP) is found on the surface of inner medullary collecting duct (IMCD) cells in culture (cIMCD) and selectively adsorbs to calcium oxalate (CaOx). We proposed that NRP mediates attachment to the renal tubular epithelium of Ca stone crystals through an electrostatic interaction with a highly acidic region (acidic fragment [AF]) similar to those of other proteins that have been reported to affect urinary crystal formation. The current studies demonstrate that nucleolin is expressed on both apical and basolateral cell surfaces of cIMCD, reaching a peak in the late stages of mitosis and gradually declining to undetectable levels with maturation of the polarized epithelium. Scraping areas of mature monolayers stimulated the cells surrounding the defects to migrate and proliferate so as to repair them, and these areas demonstrate surface NRP expression and enhanced attachment of CaOx monohydrate crystals. Surface expression of the NRP AF was produced by cloning the NRP AF into a display vector. Transfected cIMCD demonstrating copious surface expression of AF enhanced CaOx attachment 6.7-fold compared with control cIMCD, whereas cells transfected with a vector without the AF did not differ from control. AF was also cloned into a replication-deficient adenovirus and expressed in 293 cells, resulting in AF secretion into the nutrient medium. This medium inhibited CaOx attachment to cIMCD, compared with conditioned medium from cells infected with wild-type virus. These results demonstrate that surface-bound AF can mediate CaOx attachment and that secreted AF can inhibit attachment. These results support the notion that surface-associated NRP could mediate attachment of CaOx to the renal tubule epithelium, thereby causing retention of crystals that might eventually become kidney stones. PMID:15284292

  1. Heat-initiated prebiotic formation of peptides from glycine/aspartic acid and glycine/valine in aqueous environment and clay suspension

    NASA Astrophysics Data System (ADS)

    Pant, Chandra Kala; Lata, Hem; Pathak, Hari Datt; Mehata, Mohan Singh

    2009-04-01

    The effect of heat on the reaction system of glycine/aspartic acid and glycine/valine in the aqueous environment as well as in montmorillonite clay suspension with or without divalent cations (Ca2+, Mg2+ and Ni2+) has been investigated at 85°C±5°C for varying periods under prebiotic drying and wetting conditions. The resulting products were analysed and characterized by chromatographic and spectroscopic methods. Peptide formation appears to depend on the duration of heat effect, nature of reactant amino acids and, to some extent, on montmorillonite clay incorporated with divalent cations. In the glycine/aspartic acid system, oligomerization of glycine was limited up to trimer level (Gly)3 along with the formation of glycyl-aspartic acid, while linear and cyclic peptides of aspartic acid were not formed, whereas the glycine/valine system preferentially elongated homo-oligopeptide of glycine up to pentamer level (Gly)5 along with formation of hetero-peptides (Gly-Val and Val-Gly). These studies are relevant in the context of the prebiotic origin of proteins and the role of clay and metal ions in condensation and oligomerization of amino acids. The length of the bio-oligomer chain depends upon the reaction conditions. However, condensation into even a small length seems significant, as the same process would have taken millions of years in the primitive era of the Earth, leading to the first proteins.

  2. Pretreatment of cultured preadipocytes with arachidonic acid during the differentiation phase without a cAMP-elevating agent enhances fat storage after the maturation phase.

    PubMed

    Khan, Ferdous; Syeda, Pinky Karim; Nartey, Michael Nii N; Rahman, Mohammad Shahidur; Islam, Mohammad Safiqul; Nishimura, Kohji; Jisaka, Mitsuo; Shono, Fumiaki; Yokota, Kazushige

    2016-03-01

    Arachidonic acid (AA) and the related prostanoids exert complex effects on the adipocyte differentiation depending on the culture conditions and life stages. Here, we investigated the effect of the pretreatment of cultured 3T3-L1 preadipocytes with exogenous AA during the differentiation phase without 3-isobutyl-1-methylxanthine (IBMX), a cAMP-elevating agent, on the storage of fats after the maturation phase. This pretreatment with AA stimulated appreciably adipogenesis after the maturation phase as evident with the up-regulated gene expression of adipogenic markers. The stimulatory effect of the pretreatment with AA was attenuated by the co-incubation with each of cyclooxygenase (COX) inhibitors. Among exogenous prostanoids and related compounds, the pretreatment with MRE-269, a selective agonist of the IP receptor for prostaglandin (PG) I2, strikingly stimulated the storage of fats in adipocytes. The gene expression analysis of arachidonate COX pathway revealed that the transcript levels of inducible COX-2, membrane-bound PGE synthase-1, and PGF synthase declined more greatly in cultured preadipocytes treated with AA. By contrast, the expression levels of COX-1, cytosolic PGE synthase, and PGI synthase remained constitutive. The treatment of cultured preadipocytes with AA resulted in the decreased synthesis of PGE2 and PGF2α serving as anti-adipogenic PGs although the biosynthesis of pro-adipogenic PGI2 was up-regulated during the differentiation phase. Moreover, the gene expression levels of EP4 and FP, the respective prostanoid receptors for PGE2 and PGF2α, were gradually suppressed by the supplementation with AA, whereas that of IP for PGI2 remained relatively constant. Collectively, these results suggest the predominant role of endogenous PGI2 in the stimulatory effect of the pretreatment of cultured preadipoccytes with AA during the differentiation phase without IBMX on adipogenesis after the maturation phase. PMID:26928048

  3. Nitrogen-15 NMR spectroscopy of the catalytic-triad histidine of a serine protease in peptide boronic acid inhibitor complexes.

    PubMed

    Bachovchin, W W; Wong, W Y; Farr-Jones, S; Shenvi, A B; Kettner, C A

    1988-10-01

    15N NMR spectroscopy was used to examine the active-site histidyl residue of alpha-lytic protease in peptide boronic acid inhibitor complexes. Two distinct types of complexes were observed: (1) Boronic acids that are analogues of substrates form complexes in which the active-site imidazole ring is protonated and both imidazole N-H protons are strongly hydrogen bonded. With the better inhibitors of the class this arrangement is stable over the pH range 4.0-10.5. The results are consistent with a putative tetrahedral intermediate like complex involving a negatively charged, tetrahedral boron atom covalently bonded to O gamma of the active-site serine. (2) Boronic acids that are not substrate analogues form complexes in which N epsilon 2 of the active-site histidine is covalently bonded to the boron atom of the inhibitor. The proton bound to N delta 1 of the histidine in these histidine-boronate adducts remains strongly hydrogen bonded, presumably to the active-site aspartate. Benzeneboronic acid, which falls in this category, forms an adduct with histidine. In both types of complexes the N-H protons of His-57 exchange unusually slowly as evidenced by the room temperature visibility of the low-field 1H resonances and the 15N-H spin couplings. These results, coupled with the kinetic data of the preceding paper [Kettner, C. A., Bone, R., Agard, D. A., & Bachovchin, W. W. (1988) Biochemistry (preceding paper in this issue)], indicate that occupancy of the specificity subsites may be required to fully form the transition-state binding site. The significance of these findings for understanding inhibitor binding and the catalytic mechanism of serine proteases is discussed. PMID:3207700

  4. Peptide coupling between amino acids and the carboxylic acid of a functionalized chlorido-gold(I)-phosphane.

    PubMed

    Kriechbaum, Margit; List, Manuela; Himmelsbach, Markus; Redhammer, Günther J; Monkowius, Uwe

    2014-10-01

    We have developed a protocol for the direct coupling between methyl ester protected amino acids and the chlorido-gold(I)-phosphane (p-HOOC(C6H4)PPh2)AuCl. By applying the EDC·HCl/NHS strategy (EDC·HCl = N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide hydrochloride, NHS = N-hydroxysuccinimide), the methyl esters of l-phenylalanine, glycine, l-leucine, l-alanine, and l-methionine are coupled with the carboxylic acid of the gold complex in moderate to good yields (62-88%). All amino acid tagged gold complexes were characterized by (1)H and (13)C NMR spectroscopy and high-resolution mass spectrometry. As corroborated by measurement of the angle of optical rotation, no racemization occurred during the reaction. The molecular structure of the leucine derivative was determined by single-crystal X-ray diffraction. In the course of developing an efficient coupling protocol, the acyl chlorides (p-Cl(O)C(C6H4)PPh2)AuX (X = Cl, Br) were also prepared and characterized. PMID:25203269

  5. Synthesis of the cancer preventive peptide lunasin by lactic acid bacteria during sourdough fermentation.

    PubMed

    Rizzello, Carlo G; Nionelli, Luana; Coda, Rossana; Gobbetti, Marco

    2012-01-01

    This study aimed to exploit the potential of sourdough lactic acid bacteria to release lunasin during fermentation of cereal and nonconventional flours. The peptidase activities of a large number of sourdough lactic acid bacteria were screened using synthetic substrates. Selected lactic acid bacteria were used as sourdough starters to ferment wholemeal wheat, soybean, barley, amaranth, and rye flours. Proteinase activity during fermentation was characterized by SDS-PAGE analysis of the water-soluble extracts. Albumins having molecular masses of 18 to 22 kDa, which included the size of lunasin precursors, were markedly affected by proteolysis of lactic acid bacteria. After fermentation, lunasin from the water-soluble extracts was quantified, purified, and identified through RP-HPLC and nano-LC-ESI-MS analyses. Compared to control doughs, the concentration of lunasin increased up to 2-4 times during fermentation. Lactobacillus curvatus SAL33 and Lactobacillus brevis AM7 synthesized the highest concentrations of lunasin in all the flours. Besides the presence of the entire lunasin sequence, fragments containing the immunoreactive epitope RGDDDDDDDDD were also found. This study shows that fermentation by lactic acid bacteria increased the concentration of lunasin to levels that would suggest new possibilities for the biological synthesis and for the formulation of functional foods. PMID:22098174

  6. Investigating the effects of L- to D-amino acid substitution and deamidation on the activity and membrane interactions of antimicrobial peptide anoplin.

    PubMed

    Won, Amy; Khan, Mourin; Gustin, Sorin; Akpawu, Akuvi; Seebun, Deeptee; Avis, Tyler J; Leung, Bonnie O; Hitchcock, Adam P; Ianoul, Anatoli

    2011-06-01

    Isolated from the venom sac of solitary spider wasp, Anoplius samariensis, anoplin is the smallest linear α-helical antimicrobial peptide found naturally with broad spectrum activity against both Gram-positive and Gram-negative bacteria, and little hemolytic activity toward human erythrocytes. Deamidation was found to decrease the peptide's antibacterial properties. In the present work, interactions of amidated (Ano-NH2) and deamidated (Ano-OH) forms of anoplin as well as Ano-NH2 composed of all D-amino acids (D-Ano-NH2) with model cell membranes were investigated by means of Langmuir Blodgett (LB) technique, atomic force microscopy (AFM), X-ray photoemission electron microscopy (X-PEEM) and carboxyfluorescein leakage assay in order to gain a better understanding of the effect of these peptide modifications on membrane binding and lytic properties. According to LB, all three peptides form stable monolayers at the air/water interface with Ano-NH2 occupying a slightly greater area per molecule than Ano-OH. All three forms of the peptide interact preferentially with anionic 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), rather than zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid monolayer. Peptides form nanoscale clusters in zwitterionic but not in anionic monolayers. Finally, membrane lytic activity of all derivatives was found to depend strongly on membrane composition and lipid/peptide ratio. The results suggest that amidated forms of peptides are likely to possess higher membrane binding affinity due to the increased charge. PMID:21078293

  7. Frequency Patterns of T-Cell Exposed Amino Acid Motifs in Immunoglobulin Heavy Chain Peptides Presented by MHCs.

    PubMed

    Bremel, Robert D; Homan, E Jane

    2014-01-01

    Immunoglobulins are highly diverse protein sequences that are processed and presented to T-cells by B-cells and other antigen presenting cells. We examined a large dataset of immunoglobulin heavy chain variable regions (IGHV) to assess the diversity of T-cell exposed motifs (TCEMs). TCEM comprise those amino acids in a MHC-bound peptide, which face outwards, surrounded by the MHC histotope, and which engage the T-cell receptor. Within IGHV there is a distinct pattern of predicted MHC class II binding and a very high frequency of re-use of the TCEMs. The re-use frequency indicates that only a limited number of different cognate T-cells are required to engage many different clonal B-cells. The amino acids in each outward-facing TCEM are intercalated with the amino acids of inward-facing MHC groove-exposed motifs (GEM). Different GEM may have differing, allele-specific, MHC binding affinities. The intercalation of TCEM and GEM in a peptide allows for a vast combinatorial repertoire of epitopes, each eliciting a different response. Outcome of T-cell receptor binding is determined by overall signal strength, which is a function of the number of responding T-cells and the duration of engagement. Hence, the frequency of TCEM re-use appears to be an important determinant of whether a T-cell response is stimulatory or suppressive. The frequency distribution of TCEMs implies that somatic hypermutation is followed by T-cell clonal expansion that develops along repeated pathways. The observations of TCEM and GEM derived from immunoglobulins suggest a relatively simple, yet powerful, mechanism to correlate T-cell polyspecificity, through re-use of TCEMs, with a very high degree of specificity achieved by combination with a diversity of GEMs. The frequency profile of TCEMs also points to an economical mechanism for maintaining T-cell memory, recall, and self-discrimination based on an endogenously generated profile of motifs. PMID:25389426

  8. Development of novel radiogallium-labeled bone imaging agents using oligo-aspartic acid peptides as carriers.

    PubMed

    Ogawa, Kazuma; Ishizaki, Atsushi; Takai, Kenichiro; Kitamura, Yoji; Kiwada, Tatsuto; Shiba, Kazuhiro; Odani, Akira

    2013-01-01

    (68)Ga (T 1/2 = 68 min, a generator-produced nuclide) has great potential as a radionuclide for clinical positron emission tomography (PET). Because poly-glutamic and poly-aspartic acids have high affinity for hydroxyapatite, to develop new bone targeting (68)Ga-labeled bone imaging agents for PET, we used 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as a chelating site and conjugated aspartic acid peptides of varying lengths. Subsequently, we compared Ga complexes, Ga-DOTA-(Asp)n (n = 2, 5, 8, 11, or 14) with easy-to-handle (67)Ga, with the previously described (67)Ga-DOTA complex conjugated bisphosphonate, (67)Ga-DOTA-Bn-SCN-HBP. After synthesizing DOTA-(Asp)n by a Fmoc-based solid-phase method, complexes were formed with (67)Ga, resulting in (67)Ga-DOTA-(Asp)n with a radiochemical purity of over 95% after HPLC purification. In hydroxyapatite binding assays, the binding rate of (67)Ga-DOTA-(Asp)n increased with the increase in the length of the conjugated aspartate peptide. Moreover, in biodistribution experiments, (67)Ga-DOTA-(Asp)8, (67)Ga-DOTA-(Asp)11, and (67)Ga-DOTA-(Asp)14 showed high accumulation in bone (10.5 ± 1.5, 15.1 ± 2.6, and 12.8 ± 1.7% ID/g, respectively) but were barely observed in other tissues at 60 min after injection. Although bone accumulation of (67)Ga-DOTA-(Asp)n was lower than that of (67)Ga-DOTA-Bn-SCN-HBP, blood clearance of (67)Ga-DOTA-(Asp)n was more rapid. Accordingly, the bone/blood ratios of (67)Ga-DOTA-(Asp)11 and (67)Ga-DOTA-(Asp)14 were comparable with those of (67)Ga-DOTA-Bn-SCN-HBP. In conclusion, these data provide useful insights into the drug design of (68)Ga-PET tracers for the diagnosis of bone disorders, such as bone metastases. PMID:24391942

  9. Chemical structure requirements and cellular targeting of microRNA-122 by peptide nucleic acids anti-miRs

    PubMed Central

    Torres, Adrian G.; Fabani, Martin M.; Vigorito, Elena; Williams, Donna; Al-Obaidi, Naowras; Wojciechowski, Filip; Hudson, Robert H. E.; Seitz, Oliver; Gait, Michael J.

    2012-01-01

    Anti-miRs are oligonucleotide inhibitors complementary to miRNAs that have been used extensively as tools to gain understanding of specific miRNA functions and as potential therapeutics. We showed previously that peptide nucleic acid (PNA) anti-miRs containing a few attached Lys residues were potent miRNA inhibitors. Using miR-122 as an example, we report here the PNA sequence and attached amino acid requirements for efficient miRNA targeting and show that anti-miR activity is enhanced substantially by the presence of a terminal-free thiol group, such as a Cys residue, primarily due to better cellular uptake. We show that anti-miR activity of a Cys-containing PNA is achieved by cell uptake through both clathrin-dependent and independent routes. With the aid of two PNA analogues having intrinsic fluorescence, thiazole orange (TO)-PNA and [bis-o-(aminoethoxy)phenyl]pyrrolocytosine (BoPhpC)-PNA, we explored the subcellular localization of PNA anti-miRs and our data suggest that anti-miR targeting of miR-122 may take place in or associated with endosomal compartments. Our findings are valuable for further design of PNAs and other oligonucleotides as potent anti-miR agents. PMID:22070883

  10. Improved detection of multi-phosphorylated peptides in the presence of phosphoric acid in liquid chromatography/mass spectrometry

    SciTech Connect

    Kim, Jeongkwon; Camp, David G.; Smith, Richard D.

    2004-02-18

    In contrast to lower phosphorylation states (e.g., the tryptic monophosphopeptide FQpSEEQQQTEDELQDK from bovine -casein), the specific detection of multi-phosphorylated peptides (e.g. the tetraphosphopeptide RELEELNVPGEIVEpSLpSpSpSEESITR from tryptic digestion of bovine -casein) has often been problematic for liquid chromatography-mass spectrometry analysis due to their high affinity for adsorption to exposed surfaces. We observed an enhancement in the overall detection of phosphopeptides upon addition of phosphoric acid (0.1% to 1.0%) to the sample solution; a 10-fold increase in sensitivity was measured for the detection of two tryptic phosphopeptides as well as a significant improvement in the detection of the tetraphosphopeptide. Using capillary LC with an ion trap tandem mass spectrometer for detection and identification, the achievable detection limits were 50 fmol and 50 pmol for the monophosphopeptide and the tetraphosphopeptide, respectively. Phosphoric acid is believed to act as a blocking agent to available silanol groups on both the silica capillary surface and the C-18-bonded silica surface.

  11. Vibrational analysis of amino acids and short peptides in aqueous media. V. The effect of the disulfide bridge on the structural features of the peptide hormone somatostatin-14.

    PubMed

    Hernández, Belén; Carelli, Claude; Coïc, Yves-Marie; De Coninck, Joël; Ghomi, Mahmoud

    2009-09-24

    To emphasize the role played by the S-S bridge in the structural features of somatostatin-14 (SST-14), newly recorded CD and Raman spectra of this cyclic peptide and its open analogue obtained by Cys-->Ser substitution are presented. CD spectra of both peptides recorded in aqueous solutions in the 100-500 microM concentration range are strikingly similar. They reveal principally that random conformers constitute the major population in both peptides. Consequently, the S-S bridge has no structuring effect at submillimolar concentrations. In methanol, the CD spectrum of somatostatin-14 keeps globally the same spectral shape as that observed in water, whereas its open analogue presents a major population of helical conformers. Raman spectra recorded as a function of peptide concentration (5-20 mM) and also in the presence of 150 mM NaCl provide valuable conformational information. All Raman spectra present a mixture of random and beta-hairpin structures for both cyclic and open peptides. More importantly, the presence or the absence of the disulfide bridge does not seem to influence considerably different populations of secondary structures within this range of concentrations. CD and Raman data obtained in the submillimolar and millimolar ranges of concentrations, respectively, lead us to accept the idea that SST-14 monomers aggregate upon increasing concentration, thus stabilizing beta-hairpin conformations in solution. However, even at high concentrations, random conformers do not disappear. Raman spectra of SST-14 also reveal a concentration effect on the flexibility of the S-S linkage and consequently on that of its cyclic part. In conclusion, although the disulfide linkage does not seem to markedly influence the SST-14 conformational features in aqueous solutions, its presence seems to be necessary to ensure the flexibility of the cyclic part of this peptide and to maintain its closed structure in lower dielectric constant environments. PMID:19708669

  12. Replacement of the N-terminal tyrosine residue in opioid peptides with 3-(2,6-dimethyl-4-carbamoylphenyl)propanoic acid (Dcp) results in novel opioid antagonists.

    PubMed

    Lu, Yixin; Lum, Tze Keong; Leow Augustine, Yoon Wui; Weltrowska, Grazyna; Nguyen, Thi M-D; Lemieux, Carole; Chung, Nga N; Schiller, Peter W

    2006-08-24

    3-(2,6-Dimethyl-4-carbamoylphenyl)propanoic acid (Dcp), a 2',6'-dimethyltyrosine analogue containing a carbamoyl group in place of the hydroxyl function and lacking the amino group, was synthesized. The replacement of Tyr1 in an enkephalin analogue and in dynorphin A(1-11)-NH2 with Dcp resulted in the first opioid peptide-derived antagonists that do not contain a phenolic hydroxyl group at the 1-position residue. The cyclic peptide Dcp-c[D-Cys-Gly-Phe(pNO2)-D-Cys]NH2 represents a novel, potent mu opioid antagonist. PMID:16913729

  13. Manipulating the pH response of 2,3-diaminopropionic acid rich peptides to mediate highly effective gene silencing with low-toxicity☆

    PubMed Central

    Abbate, Vincenzo; Liang, Wanling; Patel, Jayneil; Lan, Yun; Capriotti, Luigi; Iacobucci, Valentina; Bui, Tam T.; Chaudhuri, Poulami; Kudsiova, Laila; Vermeer, Louic S.; Chan, Patrick F.L.; Kong, Xiaole; Drake, Alex F.; Lam, Jenny K.W.; Bansal, Sukhvinder S.; Mason, A. James

    2013-01-01

    Cationic amphipathic pH responsive peptides possess high in vitro and in vivo nucleic acid delivery capabilities and function by forming a non-covalent complex with cargo, protecting it from nucleases, facilitating uptake via endocytosis and responding to endosomal acidification by being released from the complex and inserting into and disordering endosomal membranes. We have designed and synthesised peptides to show how Coulombic interactions between ionizable 2,3-diaminopropionic acid (Dap) side chains can be manipulated to tune the functional pH response of the peptides to afford optimal nucleic acid transfer and have modified the hydrogen bonding capabilities of the Dap side chains in order to reduce cytotoxicity. When compared with benchmark delivery compounds, the peptides are shown to have low toxicity and are highly effective at mediating gene silencing in adherent MCF-7 and A549 cell lines, primary human umbilical vein endothelial cells and both differentiated macrophage-like and suspension monocyte-like THP-1 cells. PMID:24144917

  14. Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

    SciTech Connect

    Fernández-Sainz, I.J.; Largo, E.; Gladue, D.P.; Fletcher, P.; O’Donnell, V.; Holinka, L.G.; Carey, L.B.; Lu, X.; Nieva, J.L.; Borca, M.V.

    2014-05-15

    E2, along with E{sup rns} and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, {sup 818}CPIGWTGVIEC{sup 828}, containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a β-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP {sup 818}CPIGWTGVIEC{sup 828} indicates a membrane fusion activity and a critical role in virus replication. - Highlights: • A putative fusion peptide (FP) region in CSFV E2 protein was shown to be critical for virus growth. • Synthetic FPs were shown to efficiently penetrate into lipid membranes using an in vitro model. • Individual residues in the FP affecting virus replication were identified by reverse genetics. • The same FP residues are also responsible for mediating membrane fusion.

  15. Vector-Mediated Delivery of a Polyamide ("Peptide") Nucleic Acid Analogue through the Blood-Brain Barrier in vivo

    NASA Astrophysics Data System (ADS)

    Pardridge, William M.; Boado, Ruben J.; Kang, Young-Sook

    1995-06-01

    Polyamide ("peptide") nucleic acids (PNAs) are molecules with antigene and antisense effects that may prove to be effective neuropharmaceuticals if these molecules are enabled to undergo transport through the brain capillary endothelial wall, which makes up the blood-brain barrier in vivo. The model PNA used in the present studies is an 18-mer that is antisense to the rev gene of human immunodeficiency virus type 1 and is biotinylated at the amino terminus and iodinated at a tyrosine residue near the carboxyl terminus. The biotinylated PNA was linked to a conjugate of streptavidin (SA) and the OX26 murine monoclonal antibody to the rat transferrin receptor. The blood-brain barrier is endowed with high transferrin receptor concentrations, enabling the OX26-SA conjugate to deliver the biotinylated PNA to the brain. Although the brain uptake of the free PNA was negligible following intravenous administration, the brain uptake of the PNA was increased at least 28-fold when the PNA was bound to the OX26-SA vector. The brain uptake of the PNA bound to the OX26-SA vector was 0.1% of the injected dose per gram of brain at 60 min after an intravenous injection, approximating the brain uptake of intravenously injected morphine. The PNA bound to the OX26-SA vector retained the ability to bind to synthetic rev mRNA as shown by RNase protection assays. In summary, the present studies show that while the transport of PNAs across the blood-brain barrier is negligible, delivery of these potential neuropharmaceutical drugs to the brain may be achieved by coupling them to vector-mediated peptide-drug delivery systems.

  16. L-Rhamnosylation of Listeria monocytogenes Wall Teichoic Acids Promotes Resistance to Antimicrobial Peptides by Delaying Interaction with the Membrane

    PubMed Central

    Carvalho, Filipe; Atilano, Magda L.; Pombinho, Rita; Covas, Gonçalo; Gallo, Richard L.; Filipe, Sérgio R.; Sousa, Sandra; Cabanes, Didier

    2015-01-01

    Listeria monocytogenes is an opportunistic Gram-positive bacterial pathogen responsible for listeriosis, a human foodborne disease. Its cell wall is densely decorated with wall teichoic acids (WTAs), a class of anionic glycopolymers that play key roles in bacterial physiology, including protection against the activity of antimicrobial peptides (AMPs). In other Gram-positive pathogens, WTA modification by amine-containing groups such as D-alanine was largely correlated with resistance to AMPs. However, in L. monocytogenes, where WTA modification is achieved solely via glycosylation, WTA-associated mechanisms of AMP resistance were unknown. Here, we show that the L-rhamnosylation of L. monocytogenes WTAs relies not only on the rmlACBD locus, which encodes the biosynthetic pathway for L-rhamnose, but also on rmlT encoding a putative rhamnosyltransferase. We demonstrate that this WTA tailoring mechanism promotes resistance to AMPs, unveiling a novel link between WTA glycosylation and bacterial resistance to host defense peptides. Using in vitro binding assays, fluorescence-based techniques and electron microscopy, we show that the presence of L-rhamnosylated WTAs at the surface of L. monocytogenes delays the crossing of the cell wall by AMPs and postpones their contact with the listerial membrane. We propose that WTA L-rhamnosylation promotes L. monocytogenes survival by decreasing the cell wall permeability to AMPs, thus hindering their access and detrimental interaction with the plasma membrane. Strikingly, we reveal a key contribution of WTA L-rhamnosylation for L. monocytogenes virulence in a mouse model of infection. PMID:26001194

  17. Efficacy of a Complex of 5-Aminolevulinic Acid and Glycyl-Histidyl-Lysine Peptide on Hair Growth

    PubMed Central

    Sim, Hyun Bo; Jang, Yong Hyun; Lee, Seok-Jong; Kim, Do Won; Yim, Soon-Ho

    2016-01-01

    Background Pattern hair loss is a very common problem. Although effective therapeutics for the treatment of pattern hair loss have been used, novel therapeutic modalities are still required to enhance hair growth. Objective We investigated the efficacy and safety of a complex (ALAVAX) of 5-aminolevulinic acid (5-ALA) and glycyl-histidyl-lysine (GHK) peptide for the treatment of pattern hair loss. Methods Forty-five patients with male pattern hair loss were treated with ALAVAX 100 mg/ml (group A), ALAVAX 50 mg/ml (group B) or placebo (group C) once a day for 6 months. Total hair count, hair length, hair thickness, patient's assessment and adverse events were evaluated at month 1, 3, and 6. Results An increase in hair count for 6 months was 52.6 (p<0.05) in group A, 71.5 (p<0.05) in group B, and 9.6 in group C. The ratio of changes in hair count between group B (2.38) and group C (1.21) at 6 months showed a statistically significant difference (p<0.05). The proportion above good satisfaction was higher in group A (26.7%) than in the other groups (group B: 14.3%, group C: 7.1%). There was no statistically significant difference in hair length and hair thickness among 3 groups at 6 months. There was no adverse event in 3 groups. Conclusion Our study showed that a complex of 5-ALA and GHK peptide may be considered as one of the complementary agents for the treatment of male pattern hair loss. PMID:27489425

  18. Cyanobacteria Produce N-(2-Aminoethyl)Glycine, a Backbone for Peptide Nucleic Acids Which May Have Been the First Genetic Molecules for Life on Earth

    PubMed Central

    Banack, Sandra Anne; Metcalf, James S.; Jiang, Liying; Craighead, Derek; Ilag, Leopold L.; Cox, Paul Alan

    2012-01-01

    Prior to the evolution of DNA-based organisms on earth over 3.5 billion years ago it is hypothesized that RNA was the primary genetic molecule. Before RNA-based organisms arose, peptide nucleic acids may have been used to transmit genetic information by the earliest forms of life on earth. We discovered that cyanobacteria produce N-(2-aminoethyl)glycine (AEG), a backbone for peptide nucleic acids. We detected AEG in axenic strains of cyanobacteria with an average concentration of 1 µg/g. We also detected AEG in environmental samples of cyanobacteria as both a free or weakly bound molecule and a tightly bound form released by acid hydrolysis, at concentrations ranging from not detected to 34 µg/g. The production of AEG by diverse taxa of cyanobacteria suggests that AEG may be a primitive feature which arose early in the evolution of life on earth. PMID:23145061

  19. Kainic acid as a tool to study the regulation and function of opioid peptides in the hippocampus.

    PubMed

    Hong, J S; Grimes, L; Kanamatsu, T; McGinty, J F

    1987-10-30

    Kainic acid (KA), an excitatory neurotoxin, was used as a tool to study the metabolism of hippocampal opioid peptides and their functional role in the expression of wet-dog shakes (WDS). A single intracerebral injection of KA (1 microgram/rat) caused recurrent motor seizures lasting 3-6 h. During the convulsive period, native Met5-enkephalin-like (ME-LI) and dynorphin A(1-8)-like (DYN-LI) immunoreactivities in hippocampus decreased by 31 and 63%, respectively. By 24 h after dosing, the hippocampal opioid peptides had returned to control levels, and by 48 h ME-LI had increased 270% and DYN-LI 150%. Immunocytochemical analysis revealed that ME-LI and Leu5-enkephalin-like (LE-LI) immunostaining in the mossy fibers of dentate granule cells and the perforant-temporoammonic pathway had decreased visibly by 6 h and had increased markedly by 48 h following KA. A visible decrease in DYN-LI in mossy fiber axons within 6 h was followed by a substantial increase at 48 h. To determine whether the increases in hippocampal ME-LI reflected changes in ME biosynthesis, levels of mRNA coding for preproenkephalin (mRNAenk) and cryptic ME-LI cleaved by enzyme digestion from preproenkephalin were measured. Following the convulsive period (6 h), mRNAenk was 400% of control, and by 24 h, cryptic ME-LI was 300% of control. Increases in native and cryptic ME-LI and in mRNAenk were also noted in entorhinal cortex, but not in hypothalamus or uninjected striatum. Our data suggest that KA-induced seizures cause an increase in ME release, followed by a compensatory increase in ME biosynthesis in the hippocampus and entorhinal cortex. Several lines of evidence from this study have suggested that hippocampal enkephalins are intimately related to KA-elicited WDS. The shaking behavior was attenuated by pretreatment with naloxone or antisera against [Met5]-enkephalin. We also observed that KA-induced WDS can be mimicked by intrahippocampal injection of enkephalin-related peptides. Furthermore, this

  20. Plant peptide hormone signalling.

    PubMed

    Motomitsu, Ayane; Sawa, Shinichiro; Ishida, Takashi

    2015-01-01

    The ligand-receptor-based cell-to-cell communication system is one of the most important molecular bases for the establishment of complex multicellular organisms. Plants have evolved highly complex intercellular communication systems. Historical studies have identified several molecules, designated phytohormones, that function in these processes. Recent advances in molecular biological analyses have identified phytohormone receptors and signalling mediators, and have led to the discovery of numerous peptide-based signalling molecules. Subsequent analyses have revealed the involvement in and contribution of these peptides to multiple aspects of the plant life cycle, including development and environmental responses, similar to the functions of canonical phytohormones. On the basis of this knowledge, the view that these peptide hormones are pivotal regulators in plants is becoming increasingly accepted. Peptide hormones are transcribed from the genome and translated into peptides. However, these peptides generally undergo further post-translational modifications to enable them to exert their function. Peptide hormones are expressed in and secreted from specific cells or tissues. Apoplastic peptides are perceived by specialized receptors that are located at the surface of target cells. Peptide hormone-receptor complexes activate intracellular signalling through downstream molecules, including kinases and transcription factors, which then trigger cellular events. In this chapter we provide a comprehensive summary of the biological functions of peptide hormones, focusing on how they mature and the ways in which they modulate plant functions. PMID:26374891

  1. Effects of maturity stage and lactic acid bacteria on the fermentation quality and aerobic stability of Siberian wildrye silage.

    PubMed

    Li, Ping; Bai, Shiqie; You, Minghong; Shen, Yixin

    2016-09-01

    It is difficult to make good quality of silage from alpine gramineous from the Qinghai Tibetan plateau. The effects of lactic acid bacteria (LAB) on the fermentation quality and aerobic stability of Siberian wildrye silage were studied in southeast of the Qinghai Tibetan plateau. Siberian wildrye materials were freshly cut at the sprouting stage, flowering stage, and milky stage. Silage was prepared by using a small-scale silage fermentation system (bag silos). Lactobacillus plantarum (LP, 5 × 10(8) cfu/kg FM), Lactobacillus buchneri (LB, 5 × 10(8) cfu/kg FM) and their mixture (LP+LB, 5 × 10(8) cfu/kg FM) as silage additives were separately added to ensiled forages, and no additive served as control (CK). These bag silos were kept at room temperature (<15°C), and the silage qualities were analyzed after 60 days of ensiling. The number of indigenous LAB on fresh materials was less than that of yeasts and molds, and LAB species showed specification adapted to low temperature. LAB inoculated silages had lower (P < 0.05) pH value, NH 3-N/TN and butyric acid content compared with control silage. Silage treated with LB had higher contents of acetic acid, propionic acid, WSC and CP. However, the aerobic stability of silages inoculated with LAB did not differ significantly between stages (P > 0.05). When fermentation characteristics, chemical composition, and aerobic stability were considered, treatment with L. plantarum resulted in high quality of Siberian wildrye silage harvested at the flowering stage in the alpine region. PMID:27625768

  2. Boron-11 pure quadrupole resonance investigation of peptide boronic acid inhibitors bound to alpha-lytic protease.

    PubMed

    Ivanov, Dmitri; Bachovchin, William W; Redfield, Alfred G

    2002-02-01

    Pure quadrupole resonance is a potentially useful spectroscopic approach to study the coordination of quadrupolar nuclei in biological systems. We used a field-cycling NMR method to observe boron pure quadrupole resonance of two peptide boronic acid inhibitors bound to alpha-lytic protease. The method is similar to our earlier field-cycling experiment [Ivanov, D., and Redfield, A. R. (1998) Z. Naturforsch. A 53, 269-272] but uses a simple Hartmann-Hahn transfer from proton to (11)B before field cycle and direct (11)B observe after it. Pure quadrupole resonance is sensitive to the boron coordination geometry. For example, trigonal boron in neutral phenylboronic acid, which was used as a model compound, resonates at 1450 kHz, while the resonance of the tetrahedral phenylboronic acid anion appears at approximately 600 kHz. In the complex of the MeOSuc-Ala-Ala-Pro-boroVal inhibitor with the enzyme the quadrupole resonance signal was observed at 600-650 kHz, which indicates tetrahedral boron coordination in the active site. The quadrupole frequency of the MeOSuc-Ala-Ala-Pro-boroPhe enzyme-inhibitor complex, in which a boron-histidine bond is known to be formed, was found to be the same within experimental error as in the MeOSuc-Ala-Ala-Pro-boroVal enzyme-inhibitor adduct, suggesting that the boron coordination geometry in the enzyme-MeOSuc-Ala-Ala-Pro-boroPhe adduct is also close to tetrahedral. PMID:11814352

  3. Polyanionic Carboxyethyl Peptide Nucleic Acids (ce-PNAs): Synthesis and DNA Binding

    PubMed Central

    Kirillova, Yuliya; Boyarskaya, Nataliya; Dezhenkov, Andrey; Tankevich, Mariya; Prokhorov, Ivan; Varizhuk, Anna; Eremin, Sergei; Esipov, Dmitry; Smirnov, Igor; Pozmogova, Galina

    2015-01-01

    New polyanionic modifications of polyamide nucleic acid mimics were obtained. Thymine decamers were synthesized from respective chiral α- and γ-monomers, and their enantiomeric purity was assessed. Here, we present the decamer synthesis, purification and characterization by MALDI-TOF mass spectrometry and an investigation of the hybridization properties of the decamers. We show that the modified γ-S-carboxyethyl-T10 PNA forms a stable triplex with polyadenine DNA. PMID:26469337

  4. Promotion of alpha-cyano-4-hydroxycinnamic acid and peptide cocrystallization within levitated droplets with net charge.

    PubMed

    Bogan, Michael J; Bakhoum, Samuel F W; Agnes, George R

    2005-02-01

    Many reactions occur as a result of charge imbalance within or between reactive species in reaction vessels that have zero net charge. Here, chemical processes taking place within reaction vessels having net excess charge were studied. For mass spectroscopists, a familiar example of vessels that defy electroneutrality are the charged droplets produced by an electrospray ion source. Evidence is presented that control of the magnitude of the net charge contained in a reaction vessel, in this case a levitated droplet, can be used to promote nucleation and crystal growth of a mixture of an organic acid, alpha-cyano-4-hydroxycinnamic acid (CHCA), with one or more peptides. This phenomenon was first observed during our ongoing development of wall-less sample preparation (WaSP), electrodynamic charged droplet processing methodology capable of creating micrometer-sized sample spots for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) from subnanoliter volumes of sample material. Peptide ion signal-to-noise (S/N) ratios obtained by MALDI-TOF-MS from sample spots created from droplets that had high relative magnitude of net charge were consistently greater than those detected from sample spots created from droplets that had lower net charge. To study this unexpected phenomenon further, WaSP methodology was developed to process different mass-to-charge (m/z) droplets levitated in an electrodynamic balance (EDB), facilitating their deposition onto different positions of a target to create arrays of droplet residues ordered from highest to lowest m/z. This capability allowed simultaneous levitation with subsequent separation of a population of droplets created from a single starting solution, but the droplets had varied magnitudes of net charge. After the droplets were ejected from the EDB and collected on a glass slide or MALDI plate, the solids contained in the deposited droplets were characterized using microscopy and MALDI

  5. Salicylic acid signaling controls the maturation and localization of the arabidopsis defense protein ACCELERATED CELL DEATH6.

    PubMed

    Zhang, Zhongqin; Shrestha, Jay; Tateda, Chika; Greenberg, Jean T

    2014-08-01

    ACCELERATED CELL DEATH6 (ACD6) is a multipass membrane protein with an ankyrin domain that acts in a positive feedback loop with the defense signal salicylic acid (SA). This study implemented biochemical approaches to infer changes in ACD6 complexes and localization. In addition to forming endoplasmic reticulum (ER)- and plasma membrane (PM)-localized complexes, ACD6 forms soluble complexes, where it is bound to cytosolic HSP70, ubiquitinated, and degraded via the proteasome. Thus, ACD6 constitutively undergoes ER-associated degradation. During SA signaling, the soluble ACD6 pool decreases, whereas the PM pool increases. Similarly, ACD6-1, an activated version of ACD6 that induces SA, is present at low levels in the soluble fraction and high levels in the PM. However, ACD6 variants with amino acid substitutions in the ankyrin domain form aberrant, inactive complexes, are induced by a SA agonist, but show no PM localization. SA signaling also increases the PM pools of FLAGELLIN SENSING2 (FLS2) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1). FLS2 forms complexes ACD6; both FLS2 and BAK1 require ACD6 for maximal accumulation at the PM in response to SA signaling. A plausible scenario is that SA increases the efficiency of productive folding and/or complex formation in the ER, such that ACD6, together with FLS2 and BAK1, reaches the cell surface to more effectively promote immune responses. PMID:24923602

  6. Side-chain-to-tail cyclization of ribosomally derived peptides promoted by aryl and alkyl amino-functionalized unnatural amino acids.

    PubMed

    Frost, John R; Wu, Zhijie; Lam, Yick Chong; Owens, Andrew E; Fasan, Rudi

    2016-06-28

    A strategy for the production of side-chain-to-tail cyclic peptides from ribosomally derived polypeptide precursors is reported. Two genetically encodable unnatural amino acids, bearing either an aryl or alkyl amino group, were investigated for their efficiency toward promoting the formation of medium to large-sized peptide macrocycles via intein-mediated side-chain-to-C-terminus cyclization. While only partial cyclization was observed with precursor proteins containing para-amino-phenylalanine, efficient peptide macrocyclization could be achieved using O-2-aminoethyl-tyrosine as the reactive moiety. Conveniently, the latter was generated upon quantitative, post-translational reduction of the azido-containing counterpart, O-2-azidoethyl-tyrosine, directly in E. coli cells. This methodology could be successfully applied for the production of a 12 mer cyclic peptide with enhanced binding affinity for the model target protein streptavidin as compared to the acyclic counterpart (KD: 5.1 μM vs. 22.4 μM), thus demonstrating its utility toward the creation and investigation of novel, functional macrocyclic peptides. PMID:27064594

  7. Effect of Fluorescent Labels on Peptide and Amino Acid Sample Dimensionality in Two Dimensional nLC × μFFE Separations.

    PubMed

    Geiger, Matthew; Bowser, Michael T

    2016-02-16

    Multidimensional separations present a unique opportunity for generating the high peak capacities necessary for the analysis of complex biological mixtures. We have coupled nano liquid chromatography with micro free flow electrophoresis (nLC × μFFE) to produce high peak capacity separations of peptide and amino acid mixtures. Currently, μFFE largely relies on laser-induced fluorescence (LIF) detection. We have demonstrated that the choice of fluorescent label significantly affects the fractional coverage and peak capacity of nLC × μFFE separations of peptides and amino acids. Of the labeling reagents assessed, Chromeo P503 performed the best for nLC × μFFE separations of peptides. A nLC × μFFE analysis of a Chromeo P503-labeled BSA tryptic digest produced a 2D separation that made effective use of the available separation space (48%), generating a corrected peak capacity of 521 in a 5 min separation window (104 peaks/min). nLC × μFFE separations of NBD-F-labeled peptides produced similar fractional coverage and peak capacity, but this reagent was able to react with multiple reaction sites, producing an unnecessarily complex analyte mixture. NBD-F performed the best for nLC × μFFE separations of amino acids. NBD-F-labeled amino acids produced a 2D separation that covered 36% of the available separation space, generating a corrected peak capacity of 95 in a 75 s separation window (76 peaks/min). Chromeo P503 and Alexa Fluor 488-labeled amino acids were not effectively separated in the μFFE dimension, giving 2D separations with poor fractional coverage and peak capacity. PMID:26757484

  8. Transport of free and peptide-bound glycated amino acids: synthesis, transepithelial flux at Caco-2 cell monolayers, and interaction with apical membrane transport proteins.

    PubMed

    Hellwig, Michael; Geissler, Stefanie; Matthes, René; Peto, Anett; Silow, Christoph; Brandsch, Matthias; Henle, Thomas

    2011-05-16

    In glycation reactions, the side chains of protein-bound nucleophilic amino acids such as lysine and arginine are post-translationally modified to a variety of derivatives also known as Maillard reaction products (MRPs). Considerable amounts of MRPs are taken up in food. Here we have studied the interactions of free and dipeptide-bound MRPs with intestinal transport systems. Free and dipeptide-bound derivatives of N(6)-(1-fructosyl)lysine (FL), N(6)-(carboxymethyl)lysine (CML), N(6)-(1-carboxyethyl)lysine (CEL), formyline, argpyrimidine, and methylglyoxal-derived hydroimidazolone 1 (MG-H1) were synthesized. The inhibition of L-[(3)H]lysine and [(14) C]glycylsarcosine uptakes was measured in Caco-2 cells which express the H(+)/peptide transporter PEPT1 and lysine transport system(s). Glycated amino acids always displayed lower affinities than their unmodified analogues towards the L-[(3)H]lysine transporter(s). In contrast, all glycated dipeptides except Ala-FL were medium- to high-affinity inhibitors of [(14)C]Gly-Sar uptake. The transepithelial flux of the derivatives across Caco-2 cell monolayers was determined. Free amino acids and intact peptides derived from CML and CEL were translocated to very small extents. Application of peptide-bound MRPs, however, led to elevation (up to 80-fold) of the net flux and intracellular accumulation of glycated amino acids, which were hydrolyzed from the dipeptides inside the cells. We conclude 1) that free MRPs are not substrates for the intestinal lysine transporter(s), and 2) that dietary MRPs are absorbed into intestinal cells in the form of dipeptides, most likely by the peptide transporter PEPT1. After hydrolysis, hydrophobic glycated amino acids such as pyrraline, formyline, maltosine, and argpyrimidine undergo basolateral efflux, most likely by simple diffusion down their concentration gradients. PMID:21538757

  9. Dual Role of Hydrophobic Racemic Thioesters of α-Amino Acids in the Generation of Isotactic Peptides and Co-peptides in Water; Implications for the Origin of Homochirality

    NASA Astrophysics Data System (ADS)

    Illos, Roni A.; Clodic, Gilles; Bolbach, Gerard; Weissbuch, Isabelle; Lahav, Meir

    2010-02-01

    Thioesters of α-amino acids are considered as plausible monomers for the generation of the primeval peptides. DL-Leucine-thioethyl esters (LeuSEt), where the L-enantiomer was tagged with deuterium atoms, undergo polycondensation in water or in bicarbonate or imidazole buffer solutions to yield mainly heterochiral (atactic) peptides and diketopiperazine, as analyzed by MALDI-TOF and ESI mass-spectrometry. In variance, when polymerization of DL(d10) -Leu, first activated with N,N'-carbonyldiimidazole, then initiated with ethanethiol or with DL(d3) -LeuSEt yielded a library of peptides up to 30 detectable residues where those of homochiral sequence (isotactic) are the dominant diastereoisomers. At these conditions, racemic β-sheets are formed and operate as stereoselective templates in the process of chain-elongation. Isotopic L: L(d10)-Leu co-peptides were obtained in the polymerization of L(d10)-Leu with L-LeuSEt. By contrast, mixtures of oligo- D-Leu and oligo- L(d10)-Leu were obtained in the polymerization of mixtures of D-LeuSEt with activated L(d10)-Leu. Isotactic co-peptides containing Leu and Val residues were formed in the polymerization of mixtures of activated DL(d8)-Val with DL(d3) -LeuSEt in water, implying that the racemic β-sheets exert regio-enantio-selection but not chemo-selection. A reaction pathway is suggested, where LeuSEt operates both as initiator of the reaction as well as a multimer.

  10. Antimicrobial Peptides in 2014

    PubMed Central

    Wang, Guangshun; Mishra, Biswajit; Lau, Kyle; Lushnikova, Tamara; Golla, Radha; Wang, Xiuqing

    2015-01-01

    This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms. PMID:25806720

  11. Antimicrobial peptides in 2014.

    PubMed

    Wang, Guangshun; Mishra, Biswajit; Lau, Kyle; Lushnikova, Tamara; Golla, Radha; Wang, Xiuqing

    2015-01-01

    This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms. PMID:25806720

  12. Aerosolized Medications for Gene and Peptide Therapy.

    PubMed

    Laube, Beth L

    2015-06-01

    Inhalation therapy has matured to include drugs that: (1) deliver nucleic acids that either lead to the restoration of a gene construct or protein coding sequence in a population of cells or suppress or disrupt production of an abnormal gene product (gene therapy); (2) deliver peptides that target lung diseases such as asthma, sarcoidosis, pulmonary hypertension, and cystic fibrosis; and (3) deliver peptides to treat diseases outside the lung whose target is the systemic circulation (systemic drug delivery). These newer applications for aerosol therapy are the focus of this paper, and I discuss the status of each and the challenges that remain to their successful development. Drugs that are highlighted include: small interfering ribonucleic acid to treat lung cancer and Mycobacterium tuberculosis; vectors carrying the normal alpha-1 antitrypsin gene to treat alpha-1 antitrypsin deficiency; vectors carrying the normal cystic fibrosis transmembrane conductance regulator gene to treat cystic fibrosis; vasoactive intestinal peptide to treat asthma, pulmonary hypertension, and sarcoidosis; glutathione to treat cystic fibrosis; granulocyte-macrophage colony-stimulating factor to treat pulmonary alveolar proteinosis; calcitonin for postmenopausal osteoporosis; and insulin to treat diabetes. The success of these new aerosol applications will depend on many factors, such as: (1) developing gene therapy formulations that are safe for acute and chronic administrations to the lung, (2) improving the delivery of the genetic material beyond the airway mucus barrier and cell membrane and transferring the material to the cell cytoplasm or the cell nucleus, (3) developing aerosol devices that efficiently deliver genetic material and peptides to their lung targets over a short period of time, (4) developing devices that increase aerosol delivery to the lungs of infants, (5) optimizing the bioavailability of systemically delivered peptides, and (6) developing peptide formulations for

  13. Changes of Phosphatidylcholine and Fatty Acids in Germ Cells during Testicular Maturation in Three Developmental Male Morphotypes of Macrobrachium rosenbergii Revealed by Imaging Mass Spectrometry

    PubMed Central

    Siangcham, Tanapan; Chansela, Piyachat; Hayasaka, Takahiro; Masaki, Noritaka; Sroyraya, Morakot; Poljaroen, Jaruwan; Suwansa-ard, Saowaros; Engsusophon, Attakorn; Hanna, Peter J.; Sobhon, Prasert; Setou, Mitsutoshi

    2015-01-01

    Testis maturation, germ cell development and function of sperm, are related to lipid composition. Phosphatidylcholines (PCs) play a key role in the structure and function of testes. As well, increases of polyunsaturated fatty acids (PUFA) and highly unsaturated fatty acids (HUFA), especially arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are essential for male fertility. This study is the first report to show the composition and distribution of PCs and total fatty acids (FAs) in three groups of seminiferous tubules (STs) classified by cellular associations [i.e., A (STs with mostly early germ cells), B (STs with mostly spermatids), and C (STs with spermatozoa)], in three morphotypes of Macrobrachium rosenbergii, [i.e., small male (SM), orange claw male (OC), and blue claw male (BC)]. Thin layer chromatography exhibited levels of PCs reaching maxima in STs of group B. Imaging mass spectrometry showed remarkably high signals corresponding to PC (16:0/18:1), PC (18:0/18:2), PC (18:2/20:5), and PC (16:0/22:6) in STs of groups A and B. Moreover, most signals were detected in the early developing cells and the intertubular area, but not at the area containing spermatozoa. Finally, gas chromatography-mass spectrometry indicated that the major FAs present in the testes were composed of 14:0, 16:0, 17:0, 18:0, 16:1, 18:1, 18:2, 20:1, 20:2, 20:4, 20:5, and 22:6. The testes of OC contained the greatest amounts of these FAs while the testes of BC contained the least amounts of these FAs, and there was more EPA (20:5) in the testes of SM and OC than those in the BC. The increasing amounts of FAs in the SM and OC indicate that they are important for spermatogenesis and spermiogenesis. This knowledge will be useful in formulating diets containing PUFA and HUFA for prawn broodstocks in order to improve testis development, and lead to increased male fecundity. PMID:25781176

  14. High-affinity homologous peptide nucleic acid probes for targeting a quadruplex-forming sequence from a MYC promoter element.

    PubMed

    Roy, Subhadeep; Tanious, Farial A; Wilson, W David; Ly, Danith H; Armitage, Bruce A

    2007-09-18

    Guanine-rich DNA and RNA sequences are known to fold into secondary structures known as G-quadruplexes. Recent biochemical evidence along with the discovery of an increasing number of sequences in functionally important regions of the genome capable of forming G-quadruplexes strongly indicates important biological roles for these structures. Thus, molecular probes that can selectively target quadruplex-forming sequences (QFSs) are envisioned as tools to delineate biological functions of quadruplexes as well as potential therapeutic agents. Guanine-rich peptide nucleic acids have been previously shown to hybridize to homologous DNA or RNA sequences forming PNA-DNA (or RNA) quadruplexes. For this paper we studied the hybridization of an eight-mer G-rich PNA to a quadruplex-forming sequence derived from the promoter region of the MYC proto-oncogene. UV melting analysis, fluorescence assays, and surface plasmon resonance experiments reveal that this PNA binds to the MYC QFS in a 2:1 stoichiometry and with an average binding constant Ka = (2.0 +/- 0.2) x 10(8) M(-1) or Kd = 5.0 nM. In addition, experiments carried out with short DNA targets revealed a dependence of the affinity on the sequence of bases in the loop region of the DNA. A structural model for the hybrid quadruplex is proposed, and implications for gene targeting by G-rich PNAs are discussed. PMID:17718513

  15. Targeted disruption of the CCR5 gene in human hematopoietic stem cells stimulated by peptide nucleic acids

    PubMed Central

    Schleifman, Erica B.; Bindra, Ranjit; Leif, Jean; Campo, Jacob del; Rogers, Faye A.; Uchil, Pradeep; Kutsch, Olaf; Shultz, Leonard D.; Kumar, Priti; Greiner, Dale L.; Glazer, Peter M.

    2011-01-01

    SUMMARY Peptide nucleic acids (PNAs) bind duplex DNA in a sequence-specific manner, creating triplex structures that can provoke DNA repair and produce genome modification. CCR5 encodes a chemokine receptor required for HIV-1 entry into human cells and individuals carrying mutations in this gene are resistant to HIV-1 infection. Transfection of human cells with PNAs targeted to the CCR5 gene, plus donor DNAs designed to introduce stop codons mimicking the naturally occurring CCR5-delta32 mutation, produced 2.46% targeted gene modification. CCR5 modification was confirmed at the DNA, RNA and protein levels and was shown to confer resistance to infection with HIV-1. Targeting of CCR5 was achieved in human CD34+ hematopoietic stem cells (HSCs) with subsequent engraftment into mice and persistence of the gene modification more than four months post-transplantation. This work suggests a therapeutic strategy for CCR5 knockout in HSCs from HIV-1-infected individuals, rendering cells resistant to HIV-1 and preserving immune system function. PMID:21944757

  16. Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids.

    PubMed

    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

  17. Picoliter droplet-based digital peptide nucleic acid clamp PCR and dielectric sorting for low abundant K-ras mutations

    NASA Astrophysics Data System (ADS)

    Zhang, Huidan; Sperling, Ralph; Rotem, Assaf; Shan, Lianfeng; Heyman, John; Zhang, Yizhe; Weitz, David

    2012-02-01

    Colorectal cancer (CRC) remains the second leading cause of cancer-related mortality in the US, and the 5-year survival of metastatic CRC (mCRC) is less than 10%. Although monoclonal antibodies against epidermal growth factor receptor (EGFR) provide incremental improvements in survival, approximately 40% of mCRC patients with activating KRAS mutations won't benefit from this therapy. Peptide nucleic acid (PNA), a synthetic non-extendable oligonucleotides, can bind strongly to completely complementary wild-type KRAS by Watson-Crick base pairing and suppress its amplification during PCR, while any mutant allele will show unhindered amplification. The method is particularly suitable for the simultaneously detection of several adjoining mutant sites, just as mutations of codons 12 and 13 of KRAS gene where there are totally 12 possible mutation types. In this work, we describe the development and validation of this method, based on the droplet-based digital PCR. Using a microfluidic system, single target DNA molecule is compartmentalized in microdroplets together with PNA specific for wild-type KRAS, thermocycled and the fluorescence of each droplet was detected, followed by sorting and sequencing. It enables the precise determination of all possible mutant KRAS simultaneously, and the precise quantification of a single mutated KRAS in excess background unmutated KRAS.

  18. Fluorinated Peptide Nucleic Acids with Fluoroacetyl Side Chain Bearing 5-(F/CF3)-Uracil: Synthesis and Cell Uptake Studies.

    PubMed

    Ellipilli, Satheesh; Palvai, Sandeep; Ganesh, Krishna N

    2016-08-01

    Fluorine incorporation into organic molecules imparts favorable physicochemical properties such as lipophilicity, solubility and metabolic stability necessary for drug action. Toward such applications using peptide nucleic acids (PNA), we herein report the chemical synthesis of fluorinated PNA monomers and biophysical studies of derived PNA oligomers containing fluorine in in the acetyl side chain (-CHF-CO-) bearing nucleobase uracil (5-F/5-CF3-U). The crystal structures of fluorinated racemic PNA monomers reveal interesting base pairing of enantiomers and packing arrangements directed by the chiral F substituent. Reverse phase HPLC show higher hydrophobicity of fluorinated PNA oligomers, dependent on the number and site of the fluorine substitution: fluorine on carbon adjacent to the carbonyl group induces higher lipophilicity than fluorine on nucleobase or in the backbone. The PNA oligomers containing fluorinated bases form hybrids with cDNA/RNA with slightly lower stability compared to that of unmodified aeg PNA, perhaps due to electronic effects. The uptake of fluorinated homooligomeric PNAs by HeLa cells was as facile as that of nonfluorinated PNA. In conjunction with our previous work on PNAs fluorinated in backbone and at N-terminus, it is evident that the fluorinated PNAs have potential to emerge as a new class of PNA analogues for applications in functional inhibition of RNA. PMID:27391099

  19. Investigation on natural diets of larval marine animals using peptide nucleic acid-directed polymerase chain reaction clamping.

    PubMed

    Chow, Seinen; Suzuki, Sayaka; Matsunaga, Tadashi; Lavery, Shane; Jeffs, Andrew; Takeyama, Haruko

    2011-04-01

    The stomach contents of the larvae of marine animals are usually very small in quantity and amorphous, especially in invertebrates, making morphological methods of identification very difficult. Nucleotide sequence analysis using polymerase chain reaction (PCR) is a likely approach, but the large quantity of larval (host) DNA present may mask subtle signals from the prey genome. We have adopted peptide nucleic acid (PNA)-directed PCR clamping to selectively inhibit amplification of host DNA for this purpose. The Japanese spiny lobster (Panulirus japonicus) and eel (Anguilla japonica) were used as model host and prey organisms, respectively. A lobster-specific PNA oligomer (20 bases) was designed to anneal to the sequence at the junction of the 18 S rDNA gene and the internal transcribed spacer 1 (ITS1) of the lobster. PCR using eukaryote universal primers for amplifying the ITS1 region used in conjunction with the lobster-specific PNA on a mixed DNA template of lobster and eel demonstrated successful inhibition of lobster ITS1 amplification while allowing efficient amplification of eel ITS1. This method was then applied to wild-caught lobster larvae of P. japonicus and P. longipes bispinosus collected around Ryukyu Archipelago, Japan. ITS1 sequences of a wide variety of animals (Ctenophora, Cnidaria, Crustacea, Teleostei, Mollusca, and Chaetognatha) were detected. PMID:20535520

  20. Use of peptide nucleic acid probes for rapid detection and enumeration of viable bacteria in recreational waters and beach sand.

    PubMed

    Esiobu, Nwadiuto

    2006-01-01

    Environmental monitoring and public health risk assessments require methods that are rapid and quantitative with defined sensitivity and specificity thresholds. Although several molecular techniques have been developed to rapidly detect bacteria in complex matrices, the challenge to simultaneously detect and enumerate only viable cells remains a limiting factor to their routine application. This chapter describes the use of peroxidase-labeled peptide nucleic acid (PNA) probes to simultaneously detect and count live Staphylococcus aureus, a human pathogen in sea water and beach sand. Mixed bacteria from the environmental sample were immobilized on polyvinylidene difluoride membrane filters and allowed to form microcolonies during a 5-h incubation on Tryptic soy agar plates. PNA probes targeting species-specific regions of the 16S rRNA sequences of S. aureus were then used to hybridize the target bacteria in situ. Probes were detected by capturing chemiluminiscence on instant (e.g., Polaroid) films. Each viable cell (i.e., rRNA producing) is detected as a light spot from its microcolony on the film after scanning the image into a computer. This rapid in situ hybridization technique is simple and highly sensitive and could be developed into portable kits for monitoring pathogens and indicators in the environment. PMID:16957353

  1. Peptide nucleic acid (PNA) is capable of enhancing hammerhead ribozyme activity with long but not with short RNA substrates.

    PubMed Central

    Jankowsky, E; Strunk, G; Schwenzer, B

    1997-01-01

    Long RNA substrates are inefficiently cleaved by hammerhead ribozymes in trans. Oligonucleotide facilitators capable of affecting the ribozyme activity by interacting with the substrates at the termini of the ribozyme provide a possibility to improve ribozyme mediated cleavage of long RNA substrates. We have examined the effect of PNA as facilitator in vitro in order to test if even artificial compounds have facilitating potential. Effects of 12mer PNA- (peptide nucleic acid), RNA- and DNA-facilitators of identical sequence were measured with three substrates containing either 942, 452 or 39 nucleotides. The PNA facilitator enhances the ribozyme activity with both, the 942mer and the 452mer substrate to a slightly smaller extent than RNA and DNA facilitators. This effect was observed up to PNA facilitator:substrate ratios of 200:1. The enhancement becomes smaller as the PNA facilitator:substrate ratio exceeds 200:1. With the 39mer substrate, the PNA facilitator decreases the ribozyme activity by more than 100-fold, even at PNA facilitator:substrate ratios of 1:1. Although with long substrates the effect of the PNA facilitator is slightly smaller than the effect of identical RNA or DNA facilitators, PNA may be a more practical choice for potential applications in vivo because PNA is much more resistant to degradation by cellular enzymes. PMID:9207013

  2. Effects of Hypoxanthine Substitution in Peptide Nucleic Acids Targeting KRAS2 Oncogenic mRNA Molecules: Theory and Experiment

    PubMed Central

    Sanders, Jeffrey M.; Wampole, Matthew E.; Chen, Chang-Po; Sethi, Dalip; Singh, Amrita; Dupradeau, François-Yves; Wang, Fan; Gray, Brian D.; Thakur, Mathew L.; Wickstrom, Eric

    2013-01-01

    Genetic disorders can arise from single base substitutions in a single gene. A single base substitution for wild type guanine in the twelfth codon of KRAS2 mRNA occurs frequently to initiate lung, pancreatic, and colon cancer. We have observed single base mismatch specificity in radioimaging of mutant KRAS2 mRNA in tumors in mice by in vivo hybridization with radiolabeled peptide nucleic acid (PNA) dodecamers. We hypothesized that multi-mutant specificity could be achieved with a PNA dodecamer incorporating hypoxanthine, which can form Watson-Crick basepairs with adenine, cytosine, thymine, and uracil. Using molecular dynamics simulations and free energy calculations, we show that hypoxanthine substitutions in PNAs are tolerated in KRAS2 RNA-PNA duplexes where wild type guanine is replaced by mutant uracil or adenine in RNA. To validate our predictions, we synthesized PNA dodecamers with hypoxanthine, and then measured the thermal stability of RNA-PNA duplexes. Circular dichroism thermal melting results showed that hypoxanthine-containing PNAs are more stable in duplexes where hypoxanthine-adenine and hypoxanthine-uracil base pairs are formed than single mismatch duplexes or duplexes containing hypoxanthine-guanine opposition. PMID:23972113

  3. MUSI: an integrated system for identifying multiple specificity from very large peptide or nucleic acid data sets

    PubMed Central

    Kim, TaeHyung; Tyndel, Marc S.; Huang, Haiming; Sidhu, Sachdev S.; Bader, Gary D.; Gfeller, David; Kim, Philip M.

    2012-01-01

    Peptide recognition domains and transcription factors play crucial roles in cellular signaling. They bind linear stretches of amino acids or nucleotides, respectively, with high specificity. Experimental techniques that assess the binding specificity of these domains, such as microarrays or phage display, can retrieve thousands of distinct ligands, providing detailed insight into binding specificity. In particular, the advent of next-generation sequencing