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Sample records for oligopeptides

  1. Bacterial oligopeptide-binding proteins.

    Science.gov (United States)

    Monnet, V

    2003-10-01

    This review focuses on bacterial oligopeptide-binding proteins, which form part of the oligopeptide transport system belonging to the ATP-binding cassette family of transporters. Depending on the bacterial species, these binding proteins (OppA) capture peptides ranging in size from 2 to 18 amino acids from the environment and pass them on to the other components of the oligopeptide transport system for internalisation. Bacteria have developed several strategies to produce these binding proteins, which are periplasmic in Gram- bacteria and membrane-anchored in Gram+, with a higher stoichiometry (probably necessary for efficient transport) than the other components in the transport system. The expression of OppA-encoding genes is clearly modulated by external factors, especially nitrogen compounds, but the mechanisms of regulation are not always clear. The best-understood roles played by OppAs are internalisation of peptides for nutrition and recycling of muropeptides. It has, however, recently become clear that OppAs are also involved in sensing the external medium via specific or non-specific peptides.

  2. Synthesis of O-Phosphorylated Oligopeptides Using Phosphoramidite

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Reversible protein phosphorylation is of great importance in the regulation of many cellular processes. Structurally well-defined compounds are needed for the study of the roles of the phospho-proteins in biological processes. In this paper, O-phosphorylated oligopeptides were synthesized using bis-alkyloxy-N,N-dialkylphosphoramidite reacting with the oligopeptide followed by oxidation. Many hydroxyl groups in oligopeptides can be phosphorylated in one step.

  3. Human proton/oligopeptide transporter (POT) genes

    DEFF Research Database (Denmark)

    Botka, C. W.; Wittig, T. W.; Graul, R. C.

    2000-01-01

    The proton-dependent oligopeptide transporters (POT) gene family currently consists of approximately 70 cloned cDNAs derived from diverse organisms. In mammals, two genes encoding peptide transporters, PepT1 and PepT2 have been cloned in several species including humans, in addition to a rat...... the presence of several possible splice variants of hPHT1. A second closely related human EST-contig displayed high identity to a recently cloned mouse cDNA encoding cyclic adenosine monophosphate (cAMP)-inducible 1 protein (gi:4580995). This contig served to identify a PAC clone containing deduced exons...

  4. Human proton/oligopeptide transporter (POT) genes

    DEFF Research Database (Denmark)

    Botka, C. W.; Wittig, T. W.; Graul, R. C.

    2000-01-01

    The proton-dependent oligopeptide transporters (POT) gene family currently consists of approximately 70 cloned cDNAs derived from diverse organisms. In mammals, two genes encoding peptide transporters, PepT1 and PepT2 have been cloned in several species including humans, in addition to a rat...... histidine/peptide transporter (rPHT1). Because the Candida elegans genome contains five putative POT genes, we searched the available protein and nucleic acid databases for additional mammalian/human POT genes, using iterative BLAST runs and the human expressed sequence tags (EST) database. The apparent...... human orthologue of rPHT1 (expression largely confined to rat brain and retina) was represented by numerous ESTs originating from many tissues. Assembly of these ESTs resulted in a contiguous sequence covering approximately 95% of the suspected coding region. The contig sequences and analyses revealed...

  5. Three oligopeptide-binding proteins are involved in the oligopeptide transport of Streptococcus thermophilus.

    Science.gov (United States)

    Garault, Peggy; Le Bars, Dominique; Besset, Colette; Monnet, Veronique

    2002-01-04

    The functions necessary for bacterial growth strongly depend on the features of the bacteria and the components of the growth media. Our objective was to identify the functions essential to the optimum growth of Streptococcus thermophilus in milk. Using random insertional mutagenesis on a S. thermophilus strain chosen for its ability to grow rapidly in milk, we obtained several mutants incapable of rapid growth in milk. We isolated and characterized one of these mutants in which an amiA1 gene encoding an oligopeptide-binding protein (OBP) was interrupted. This gene was a part of an operon containing all the components of an ATP binding cassette transporter. Three highly homologous amiA genes encoding OBPs work with the same components of the ATP transport system. Their simultaneous inactivation led to a drastic diminution in the growth rate in milk and the absence of growth in chemically defined medium containing peptides as the nitrogen source. We constructed single and multiple negative mutants for AmiAs and cell wall proteinase (PrtS), the only proteinase capable of hydrolyzing casein oligopeptides outside the cell. Growth experiments in chemically defined medium containing peptides indicated that AmiA1, AmiA2, and AmiA3 exhibited overlapping substrate specificities, and that the whole system allows the transport of peptides containing from 3 to 23 residues.

  6. Oligopeptide M13 Phage Display in Pathogen Research

    Directory of Open Access Journals (Sweden)

    Michael Hust

    2013-10-01

    Full Text Available Phage display has become an established, widely used method for selection of peptides, antibodies or alternative scaffolds. The use of phage display for the selection of antigens from genomic or cDNA libraries of pathogens which is an alternative to the classical way of identifying immunogenic proteins is not well-known. In recent years several new applications for oligopeptide phage display in disease related fields have been developed which has led to the identification of various new antigens. These novel identified immunogenic proteins provide new insights into host pathogen interactions and can be used for the development of new diagnostic tests and vaccines. In this review we focus on the M13 oligopeptide phage display system for pathogen research but will also give examples for lambda phage display and for applications in other disease related fields. In addition, a detailed technical work flow for the identification of immunogenic oligopeptides using the pHORF system is given. The described identification of immunogenic proteins of pathogens using oligopeptide phage display can be linked to antibody phage display resulting in a vaccine pipeline.

  7. Oligopeptides as biomarkers of cyanobacterial subpopulations. Toward an understanding of their biological role.

    Science.gov (United States)

    Agha, Ramsy; Quesada, Antonio

    2014-06-23

    Cyanobacterial oligopeptides comprise a wide range of bioactive and/or toxic compounds. While current research is strongly focused on exploring new oligopeptide variants and their bioactive properties, the biological role of these compounds remains elusive. Oligopeptides production abilities show a remarkably patchy distribution among conspecific strains. This observation has prompted alternative approaches to unveil their adaptive value, based on the use of cellular oligopeptide compositions as biomarkers of intraspecific subpopulations or chemotypes in freshwater cyanobacteria. Studies addressing the diversity, distribution, and dynamics of chemotypes in natural systems have provided important insights into the structure and ecology of cyanobacterial populations and the adaptive value of oligopeptides. This review presents an overview of the fundamentals of this emerging approach and its most relevant findings, and discusses our current understanding of the role of oligopeptides in the ecology of cyanobacteria.

  8. Structure dependent spin selectivity in electron transport through oligopeptides

    Science.gov (United States)

    Kiran, Vankayala; Cohen, Sidney R.; Naaman, Ron

    2017-03-01

    The chiral-induced spin selectivity (CISS) effect entails spin-selective electron transmission through chiral molecules. In the present study, the spin filtering ability of chiral, helical oligopeptide monolayers of two different lengths is demonstrated using magnetic conductive probe atomic force microscopy. Spin-specific nanoscale electron transport studies elucidate that the spin polarization is higher for 14-mer oligopeptides than that of the 10-mer. We also show that the spin filtering ability can be tuned by changing the tip-loading force applied on the molecules. The spin selectivity decreases with increasing applied force, an effect attributed to the increased ratio of radius to pitch of the helix upon compression and increased tilt angles between the molecular axis and the surface normal. The method applied here provides new insights into the parameters controlling the CISS effect.

  9. Mechanistic study for immobilization of cysteine-labeled oligopeptides on UV-activated surfaces.

    Science.gov (United States)

    Ong, Lian Hao; Ding, Xiaokang; Yang, Kun-Lin

    2014-10-01

    In this study, we report immobilization of cysteine-labeled oligopeptides on UV activated surfaces decorated with N,N-dimethyl-n-octadecyl-3-aminopropyltrimethoxysilyl chloride (DMOAP). Our result shows that cysteine group, regardless of its position in the oligopeptide, is essential for successful immobilization of oligopeptide on the UV-activated surface. A possible reaction mechanism is nucleophilic addition of thiolates to surface aldehyde groups generated during UV activation. By using this technique, we are able to incorporate anchoring points into oligopeptides through cysteine residues. Furthermore, immobilized oligopeptides on the UV-activated surface is very stable even under harsh washing conditions. Finally, we show that an HPQ-containing oligopeptide can be immobilized on the UV-activated surface, but the final surface density and its ability to bind streptavidin are affected by the position of cysteine and HPQ. An oligopeptide with a cysteine at the N-terminus and a HPQ motif at the C-terminus gives the highest binding signal in the streptavidin-binding assay. This result is potentially useful for the development of functional oligopeptide microarrays for detecting target protein molecules.

  10. Mining gut microbiome oligopeptides by functional metaproteome display

    Science.gov (United States)

    Zantow, Jonas; Just, Sarah; Lagkouvardos, Ilias; Kisling, Sigrid; Dübel, Stefan; Lepage, Patricia; Clavel, Thomas; Hust, Michael

    2016-01-01

    Pathogen infections, autoimmune diseases, and chronic inflammatory disorders are associated with systemic antibody responses from the host immune system. Disease-specific antibodies can be important serum biomarkers, but the identification of antigens associated with specific immune reactions is challenging, in particular if complex communities of microorganisms are involved in the disease progression. Despite promising new diagnostic opportunities, the discovery of these serological markers becomes more difficult with increasing complexity of microbial communities. In the present work, we used a metagenomic M13 phage display approach to select immunogenic oligopeptides from the gut microbiome of transgenic mice suffering from chronic ileitis. We constructed three individual metaproteome phage display libraries with a library size of approximately 107 clones each. Using serum antibodies, we selected and validated three oligopeptides that induced specific antibody responses in the mouse model. This proof-of-concept study provides the first successful application of functional metaproteome display for the study of protein-protein interactions and the discovery of potential disease biomarkers. PMID:27703179

  11. A novel anti-inflammatory oligopeptide produced by Entamoeba histolytica.

    Science.gov (United States)

    Kretschmer, R R; Rico, G; Giménez, J A

    2001-02-01

    The monocyte locomotion inhibitory factor (MLIF), a heat-stable oligopeptide found in the supernatant fluid of Entamoeba histolytica axenic cultures was isolated by ultra-filtration, gel-sieve chromatography and high powered liquid chromatography (HPLC), and its primary structure (Met-Gln-Cys-Asn-Ser) established by Edman sequencing and mass-spectrometry (MS). A synthetic peptide had the same selective anti-inflammatory features as the native material in comparable concentrations: in vitro inhibition of the locomotion in human peripheral blood monocytes, and of the respiratory burst in the same cells and in human neutrophil polymorphonuclear leucocytes; and in vivo depression of delayed hypersensitivity skin reactions to dinitrochlorobenzene in guinea pigs. This oligopeptide is apparently synthesized by the ameba as suggested by [(35)S]-Cys and Met incorporation, probably as part of a larger molecule, from which it is cleaved by proteolysis. The full sequence was not found in the 431 available E. histolytica protein sequences. The factor may contribute to the unexpected paucity of the late inflammatory reaction found in advanced invasive amebiasis and, perhaps in consequence, to the regeneration without scarring (restitutio ad integrum) of the affected organs that is observed following successful treatment of this disease

  12. Structural Design of Oligopeptides for Intestinal Transport Model.

    Science.gov (United States)

    Hong, Seong-Min; Tanaka, Mitsuru; Koyanagi, Riho; Shen, Weilin; Matsui, Toshiro

    2016-03-16

    Glycyl-sarcosine (Gly-Sar) is a well-known model substrate for the intestinal uptake of dipeptides through peptide transporter 1 (PepT1). However, there are no other model peptides larger than tripeptides to evaluate their intestinal transport ability. In this study, we designed new oligopeptides based on the Gly-Sar structure in terms of protease resistance. Gly-Sar-Sar was found to be an appropriate transport model for tripeptides because it does not degrade during the transport across the rat intestinal membrane, while Gly-Gly-Sar was degraded to Gly-Sar during the 60 min transport. Caco-2 cell transport experiments revealed that the designed oligopeptides based on Gly-Sar-Sar showed a significantly (p transport ability by factors of 1/10-, 1/25-, and 1/40-fold for Gly-Sar-Sar, Gly-Sar-Sar-Sar, and Gly-Sar-Sar-Sar-Sar, respectively, compared to Gly-Sar (apparent permeability coefficient: 38.6 ± 11.4 cm/s). Cell experiments also showed that the designed tripeptide and Gly-Sar were transported across Caco-2 cell via PepT1, whereas the tetra- and pentapeptides were transported through the paracellular tight-junction pathway.

  13. Characterization of oligopeptide patterns in large protein sets

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    Persson Bengt

    2007-10-01

    Full Text Available Abstract Background Recent sequencing projects and the growth of sequence data banks enable oligopeptide patterns to be characterized on a genome or kingdom level. Several studies have focused on kingdom or habitat classifications based on the abundance of short peptide patterns. There have also been efforts at local structural prediction based on short sequence motifs. Oligopeptide patterns undoubtedly carry valuable information content. Therefore, it is important to characterize these informational peptide patterns to shed light on possible new applications and the pitfalls implicit in neglecting bias in peptide patterns. Results We have studied four classes of pentapeptide patterns (designated POP, NEP, ORP and URP in the kingdoms archaea, bacteria and eukaryotes. POP are highly abundant patterns statistically not expected to exist; NEP are patterns that do not exist but are statistically expected to; ORP are patterns unique to a kingdom; and URP are patterns excluded from a kingdom. We used two data sources: the de facto standard of protein knowledge Swiss-Prot, and a set of 386 completely sequenced genomes. For each class of peptides we looked at the 100 most extreme and found both known and unknown sequence features. Most of the known sequence motifs can be explained on the basis of the protein families from which they originate. Conclusion We find an inherent bias of certain oligopeptide patterns in naturally occurring proteins that cannot be explained solely on the basis of residue distribution in single proteins, kingdoms or databases. We see three predominant categories of patterns: (i patterns widespread in a kingdom such as those originating from respiratory chain-associated proteins and translation machinery; (ii proteins with structurally and/or functionally favored patterns, which have not yet been ascribed this role; (iii multicopy species-specific retrotransposons, only found in the genome set. These categories will affect the

  14. Discovery of Anti-Hypertensive Oligopeptides from Adlay Based on In Silico Proteolysis and Virtual Screening

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    Liansheng Qiao

    2016-12-01

    Full Text Available Adlay (Coix larchryma-jobi L. was the commonly used Traditional Chinese Medicine (TCM with high content of seed storage protein. The hydrolyzed bioactive oligopeptides of adlay have been proven to be anti-hypertensive effective components. However, the structures and anti-hypertensive mechanism of bioactive oligopeptides from adlay were not clear. To discover the definite anti-hypertensive oligopeptides from adlay, in silico proteolysis and virtual screening were implemented to obtain potential oligopeptides, which were further identified by biochemistry assay and molecular dynamics simulation. In this paper, ten sequences of adlay prolamins were collected and in silico hydrolyzed to construct the oligopeptide library with 134 oligopeptides. This library was reverse screened by anti-hypertensive pharmacophore database, which was constructed by our research team and contained ten anti-hypertensive targets. Angiotensin-I converting enzyme (ACE was identified as the main potential target for the anti-hypertensive activity of adlay oligopeptides. Three crystal structures of ACE were utilized for docking studies and 19 oligopeptides were finally identified with potential ACE inhibitory activity. According to mapping features and evaluation indexes of pharmacophore and docking, three oligopeptides were selected for biochemistry assay. An oligopeptide sequence, NPATY (IC50 = 61.88 ± 2.77 µM, was identified as the ACE inhibitor by reverse-phase high performance liquid chromatography (RP-HPLC assay. Molecular dynamics simulation of NPATY was further utilized to analyze interactive bonds and key residues. ALA354 was identified as a key residue of ACE inhibitors. Hydrophobic effect of VAL518 and electrostatic effects of HIS383, HIS387, HIS513 and Zn2+ were also regarded as playing a key role in inhibiting ACE activities. This study provides a research strategy to explore the pharmacological mechanism of Traditional Chinese Medicine (TCM proteins based on

  15. Quantitative serine protease assays based on formation of copper(II)-oligopeptide complexes.

    Science.gov (United States)

    Ding, Xiaokang; Yang, Kun-Lin

    2015-01-07

    A quantitative protease assay based on the formation of a copper-oligopeptide complex is developed. In this assay, when a tripeptide GGH fragment is cleaved from an oligopeptide chain by serine proteases, the tripeptide quickly forms a pink GGH/Cu(2+) complex whose concentration can be determined quantitatively by using UV-Vis spectroscopy. Therefore, activities of serine proteases can be determined from the formation rate of the GGH/Cu(2+) complex. This principle can be used to detect the presence of serine protease in a real-time manner, or measure proteolytic activities of serine protease cleaving different oligopeptide substrates. For example, by using this assay, we demonstrate that trypsin, a model serine protease, is able to cleave two oligopeptides GGGGKGGH () and GGGGRGGH (). However, the specificity constant (kcat/Km) for is higher than that of (6.4 × 10(3) mM(-1) min(-1)vs. 1.3 × 10(3) mM(-1) min(-1)). This result shows that trypsin is more specific toward arginine (R) than lysine (K) in the oligopeptide sequence.

  16. Optimization of Enzymatic Production of Oligopeptides from Apricot Almonds Meal with Neutrase and N120P

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    Chunyan Wang

    2010-12-01

    Full Text Available Neutrase 0.8L and N120P proteases were used for oligopeptide production from apricot almonds meal, and response surface design was carried out to optimize the effect of hydrolysis conditions on hydrolysis degree (DH and oligopeptide yield rate. Four independent variables were used to optimize the hydrolysis process: hydrolysis temperature (X1, enzyme-to substrate ratio (E/S (X2, substrate concentration (X3 and reaction time (X4. Statistical analysis indicated that the four variables, quadratic terms of X1, X3, and X4, and the interaction terms with X1 had a significant (p

  17. Chiral amplification of oligopeptides in two-dimensional crystalline self-assemblies on water

    DEFF Research Database (Denmark)

    Zepik, H.; Shavit, E.; Tang, M.;

    2002-01-01

    from chiral nonracemic mixtures. The crystalline structures on the water surface were determined by grazing incidence x-ray diffraction and the diastereomeric composition of the oligopeptides by matrix-assisted laser desorption time-of-flight mass spectrometry with enantio-labeling. These results...

  18. On the binding mechanism of the peptide receptor of the oligopeptide transport system of Lactococcus lactis

    NARCIS (Netherlands)

    Lanfermeijer, Frank C.; Detmers, Frank J.M.; Konings, Wil N.; Poolman, Bert

    2000-01-01

    Lactococcus lactis degrades exogenous proteins such as β-casein to peptides of 4–30 amino acids, and uses these as nitrogen sources. The binding protein or receptor (OppALl) of the oligopeptide transport system (Opp) of L.lactis has the unique capacity to bind peptides from five up to at least 20

  19. Salt-bridge Swapping in the EXXERFXYY Motif of Proton Coupled Oligopeptide Transporters

    DEFF Research Database (Denmark)

    Aduri, Nanda G; Prabhala, Bala K; Ernst, Heidi A

    2015-01-01

    Proton-coupled oligopeptide transporters (POTs) couple the inwards transport of di- or tripeptides with an inwards-directed transport of protons. Evidence from several studies of different POTs have pointed towards involvement of a highly conserved sequence motif, E1XXE2RFXYY (from here on referr...

  20. Peptide Selectivity of the Proton-Coupled Oligopeptide Transporter from Neisseria meningitidis

    DEFF Research Database (Denmark)

    Sharma, Neha; Aduri, Nanda G; Iqbal, Anna

    2016-01-01

    Peptide transport in living organisms is facilitated by either primary transport, hydrolysis of ATP, or secondary transport, cotransport of protons. In this study, we focused on investigating the ligand specificity of the Neisseria meningitidis proton-coupled oligopeptide transporter (NmPOT). It ...

  1. Organophosphorus Compound DEPBT as a Coupling Reagent for Oligopeptides and Peptoids Synthesis:Studies on Its Mechanism

    Institute of Scientific and Technical Information of China (English)

    HuiLIU; YunHuaYE; 等

    2002-01-01

    Some oligopeptides and peptoids were synthesized by applying the organophosphorus compound DEPBT as a coupling reagent. D-Biotin-OOBt was obtained unexpectedly. A proposed reaction mechanism for DEPBT-mediated coupling was proved.

  2. Pharmacokinetic assessment of an oligopeptide-based enteral formula in abdominal surgery patients.

    Science.gov (United States)

    Ziegler, F; Nitenberg, G; Coudray-Lucas, C; Lasser, P; Giboudeau, J; Cynober, L

    1998-01-01

    The specific effect of the molecular form of the nitrogen supply (oligopeptides and whole proteins) on amino acid kinetics during enteral feeding after surgery has not been assessed previously. In a prospective, randomized study, patients having undergone esophagectomy or gastrectomy for cancer received jejunal infusions of oligopeptide-based or whole-protein-based complete formulas (OPD and WPD, respectively) during two 9-h periods on 2 consecutive days in a crossover design. The OPD and WPD had identical energy compositions and amino acid profiles. Amino acid peripheral bioavailability (measurements of area under the curve of arterial blood concentrations), amino acid arteriovenous differences, and insulin and glucagon responses were measured. Amino acid peripheral bioavailability was higher (leucine: 54%, P bioavailability than the corresponding WPD. These results could be useful for a better definition of clinical indications of semi-elemental diets.

  3. Inclusion of Cu nano-cluster 1D arrays inside a C3-symmetric artificial oligopeptide via co-assembly

    Science.gov (United States)

    Gong, Ruiying; Li, Fei; Yang, Chunpeng; Wan, Xiaobo

    2015-12-01

    A peptide sequence N3-GVGV-OMe (G: glycine; V: valine) was attached to a benzene 1,3,5-tricarboxamide (BTA) derivative via ``click chemistry'' to afford a C3-symmetric artificial oligopeptide. The key feature of this oligopeptide is that the binding sites (triazole groups formed by click reaction) are located at the center, while the three oligopeptide arms with a strong tendency to assemble are located around it, which provides inner space to accommodate nanoparticles via self-assembly. The inclusion of Cu nanoclusters and the formation of one-dimensional (1D) arrays inside the nanofibers of the C3-symmetric artificial oligopeptide assembly were observed, which is quite different from the commonly observed nanoparticle growth on the surface of the pre-assembled oligopeptide nanofibers via the coordination sites located outside. Our finding provides an instructive concept for the design of other stable organic-inorganic hybrid 1D arrays with the inorganic nanoparticles inside.A peptide sequence N3-GVGV-OMe (G: glycine; V: valine) was attached to a benzene 1,3,5-tricarboxamide (BTA) derivative via ``click chemistry'' to afford a C3-symmetric artificial oligopeptide. The key feature of this oligopeptide is that the binding sites (triazole groups formed by click reaction) are located at the center, while the three oligopeptide arms with a strong tendency to assemble are located around it, which provides inner space to accommodate nanoparticles via self-assembly. The inclusion of Cu nanoclusters and the formation of one-dimensional (1D) arrays inside the nanofibers of the C3-symmetric artificial oligopeptide assembly were observed, which is quite different from the commonly observed nanoparticle growth on the surface of the pre-assembled oligopeptide nanofibers via the coordination sites located outside. Our finding provides an instructive concept for the design of other stable organic-inorganic hybrid 1D arrays with the inorganic nanoparticles inside. Electronic

  4. Lowry法测定寡肽的研究%Research of Lowry determination of oligopeptides

    Institute of Scientific and Technical Information of China (English)

    付玉梅; 许锦珍; 廖群; 郭勇

    2011-01-01

    Objective: To select suitable oligopeptide assay method. Methods: Determination of oligopeptide for the marine peptide powder and soybean peptide powder,compare the differences between the four kinds of methods ,Kjeldahl method;biuret method; Coomassie Brilliant Blue G250 method and Lowry method. Results : The measured results in the four kinds of methods have significant differences. Conclusion : Lowry method measured results are accurate , reliable, and the method of high sensitivity, easy to operate, the relative other three kinds of method is more suitable for assay of oligopeptides.%目的:探讨适合寡肽含量测定的方法.方法:通过测定海洋肽粉和大豆肽粉中寡肽含量,比较凯氏定氮法、双缩脲法、考马斯亮蓝(G250法和Lowry法4种方法之间的差异.结果:4种方法测得的结果具有明显差异.结论:Lowry法测得结果准确、可靠,灵敏度较高,易操作,较另外3种方法更适合于寡肽的含量测定.

  5. Purification and characterization of an anticoagulant oligopeptide from Whitmania pigra Whitman

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    Xiaobei Zheng

    2015-01-01

    Full Text Available Background: Dried Whitmania pigra is used for the treatment of cardiovascular and cerebrovascular diseases in traditional Chinese medicine and hot water and alcohol extracts also have anticogulant activity. However, a lower molecular weight and more stable anticogulant is needed. Objective: The objective of the following study is to purify and characterize of an anticoagulant oligopeptide from Hirudo (Whitmania pigra Whitman. Materials and Methods: Gel filtration on Sephadex G 50, ion exchange on diethylaminoethyl cellulose, and semi prepared high performance liquid chromatography were used to purify Hirudo. Automated coagulation analyzer was used for evaluating anticoagulant activity. Molecular weight was measured by Matrix assisted laser desorption ionization time of flight mass spectrometry. Amino acid sequence of the oligopeptide was measured by amino acid sequence analyzer. Results: A new anticoagulant, named whitide, isolated from Hirudo was purified, with a molecular weight 1997.1 Da. Amino acid sequence of the oligopeptide was identified as Gly-Pro-ALa-Gly-Hyp-Val-Gly-Ala-Hyp-Gly-Gly-Hyp-Gly-Val-Arg-Gly-Leu-Hyp-Gly-Asp-Arg-Gly. The results revealed that its amino acid sequence had strong homology to various types of collagen. Conclusion: Whitide might be an orally anticoagulant for its hot and trypsin stable.

  6. H(+)/peptide transporter (PEPT2) is expressed in human epidermal keratinocytes and is involved in skin oligopeptide transport.

    Science.gov (United States)

    Kudo, Michiko; Katayoshi, Takeshi; Kobayashi-Nakamura, Kumiko; Akagawa, Mitsugu; Tsuji-Naito, Kentaro

    2016-07-08

    Peptide transporter 2 (PEPT2) is a member of the proton-coupled oligopeptide transporter family, which mediates the cellular uptake of oligopeptides and peptide-like drugs. Although PEPT2 is expressed in many tissues, its expression in epidermal keratinocytes remains unclear. We investigated PEPT2 expression profile and functional activity in keratinocytes. We confirmed PEPT2 mRNA expression in three keratinocyte lines (normal human epidermal keratinocytes (NHEKs), immortalized keratinocytes, and malignant keratinocytes) by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. In contrast to PEPT1, PEPT2 expression in the three keratinocytes was similar or higher than that in HepG2 cells, used as PEPT2-positive cells. Immunolocalization analysis using human skin showed epidermal PEPT2 localization. We studied keratinocyte transport function by measuring the oligopeptide content using liquid chromatography/tandem mass spectrometry. Glycylsarcosine uptake in NHEKs was pH-dependent, suggesting that keratinocytes could absorb small peptides in the presence of an inward H(+) gradient. We also performed a skin-permeability test of several oligopeptides using skin substitute, suggesting that di- and tripeptides pass actively through the epidermis. In conclusion, PEPT2 is expressed in keratinocytes and involved in skin oligopeptide uptake. Copyright © 2016 DHC corporation. Published by Elsevier Inc. All rights reserved.

  7. Limited Stability of Microcystins in Oligopeptide Compositions of Microcystis aeruginosa (Cyanobacteria: Implications in the Definition of Chemotypes

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    Ramsy Agha

    2013-06-01

    Full Text Available The occurrence of diverse oligopeptides in cyanobacteria, including the cyanotoxins microcystins, has been recently used to classify individual clones into sub-specific oligopeptide chemotypes, whose composition and dynamics modulate microcystin concentrations in cyanobacterial blooms. Cyanobacterial chemotyping allows the study of the ecology of chemotypical subpopulations, which have been shown to possess dissimilar ecological traits. However, the stability of chemotypes under changing abiotic conditions is usually assumed and has not been assessed in detail. We monitored oligopeptide patterns of three strains of Microcystis aeruginosa under different nutrient and light conditions. MALDI-TOF MS revealed alterations in the microcystins signatures under N and P poor conditions and high light intensities (150 and 400 μmol photons m−2s−1. Variations in the general oligopeptide composition were caused by a gradual disappearance of microcystins with low relative intensity signals from the fingerprint. The extent of such variations seems to be closely related to physiological stress caused by treatments. Under identical clonal compositions, alterations in the oligopeptide fingerprint may be misinterpreted as apparent shifts in chemotype succession. We discuss the nature of such variations, as well as the consequent implications in the use of cyanobacterial chemotyping in studies at the subpopulation level and propose new guidance for the definition of chemotypes as a consistent subpopulation marker.

  8. Selective effect of zinc on uphill transport of oligopeptides into kidney brush border membrane vesicles.

    Science.gov (United States)

    Daniel, H; Adibi, S A

    1995-08-01

    Based on the involvement of zinc in hydrolysis of peptides, we hypothesized that Zn2+ may also play a role in peptide transport. To investigate this hypothesis, kidney brush border membrane vesicles (BBMV) were incubated for 30 min with different concentrations of ZnSO4 before use in uptake studies. This incubation increased by twofold the overshoot uptake of 3H-Gly-L-Gln, D-Leu-125I-Tyr and 3H-cephalexin (all high-affinity substrates for the oligopeptide/H+ symporter) without affecting passive and/or facilitated diffusion of these substrates. Zinc had no effect on the uptake of either glutamine or glucose by kidney BBMV. Among a group of metal ions (cobalt, iron, copper, cadmium, and manganese), only manganese and copper substantially stimulated the activity of the oligopeptide/H+ symporter. DTPA (a complexing agent) inhibited dipeptide uptake, which was reversed by the addition of zinc to the BBMV. Zinc treatment of BBMV reduced the EC50 value of inhibition of 3H-Gly-L-Gln uptake by unlabeled Gly-L-Gln by twofold (90 +/- 8 vs. 45 +/- 4 microM). Similarly, zinc treatment of BBMV reduced the EC50 value for inhibition of D-Leu-125I-Tyr uptake by bestatin from 80 +/- 4 to 40 +/- 3 mM. In conclusion, the data show that zinc has a selective effect on transport of nutrients into kidney BBMV. It stimulates uphill transport of oligopeptides by a modification of their affinity for the binding site of the membrane transporter.

  9. Focal accumulation of an apolipoprotein B-based synthetic oligopeptide in the healing rabbit arterial wall

    Energy Technology Data Exchange (ETDEWEB)

    Shih, I.L.; Lees, R.S.; Chang, M.Y.; Lees, A.M. (Harvard Univ., Boston, MA (USA))

    1990-02-01

    The functions of surface-accessible domains of apolipoprotein (apo) B, the protein moiety of low density lipoprotein (LDL), are unknown, aside from the LDL receptor-binding domain, which lies toward the carboxyl-terminal end of apoB. Since LDL accumulation in arterial lesions does not depend on recognition of LDLs by a cell-surface receptor, we synthesized an oligopeptide with the sequence of the trypsin-accessible domain of apoB that lies closest to the amino-terminal end of the protein and compared its biological activity to that of another synthetic oligopeptide with the sequence of the heparin- and apoB/apoE receptor-binding domains of apoE. (Tyrosine was added at the amino-terminal end of each peptide to facilitate radiolabeling.) The 18-amino acid apoB-based peptide included residues 1000-1016 of apoB, for which no function has been previously described. In radioautographs, the 125I-labeled peptide accumulated focally at the healing edges of regenerating endothelial islands in the balloon-catheter deendothelialized rabbit aorta. In contrast, the 21-residue apoE-based peptide, which included residues 129-148 of apoE, accumulated diffusely and uniformly throughout the deendothelialized areas of the aorta. The data show that focal binding of the apoB-based peptide can delineate arterial lesions and suggest that this arterial wall-binding domain of apoB mediates accumulation of LDLs in arterial lesions.

  10. The Oligopeptide Transporters: A Small Gene Family with a Diverse Group of Substrates and Functions?

    Institute of Scientific and Technical Information of China (English)

    Mark Lubkowitz

    2011-01-01

    T Genes in the Oligopeptide Transport family encode integral membrane proteins that are believed to translocate their substrates from either the extracellular environment or an organelle into the cytosol. Phylogenetic analyses of plant transporters have revealed two distant clades: the Yellow Stripe-Like (YSL) proteins and the so-called Oligopeptide Transporters (OPTs), for which the family was named. Three categories of substrates have been identified for this family: small peptides, secondary amino acids bound to metals, and glutathione. Notably, the YSL transporters are involved in metal homeostasis through the translocation of metal-chelates, indicating a level of conservation both in biological function as well as substrates. In contrast, the functions of OPT proteins seem to be less defined and, in this review, I will examine the supporting and contradictory evidence for the proposed roles of OPTs in such diverse functions as long-distance sulfur distribution, nitrogen mobilization, metal homeostasis, and heavy metal sequestration through the transport of glutathione, metal-chelates, and peptides.

  11. Effects of phenylalanine and threonine oligopeptides on milk protein synthesis in cultured bovine mammary epithelial cells.

    Science.gov (United States)

    Zhou, M M; Wu, Y M; Liu, H Y; Liu, J X

    2015-04-01

    This study was conducted to investigate the effects of phenylalanine (Phe) and threonine (Thr) oligopeptides on αs1 casein gene expression and milk protein synthesis in bovine mammary epithelial cells. Primary mammary epithelial cells were obtained from Holstein dairy cows and incubated in Dulbecco's modified Eagle's medium-F12 medium (DMEM/F12) containing lactogenic hormones (prolactin and glucocorticoids). Free Phe (117 μg/ml) was substituted partly with peptide-bound Phe (phenylalanylphenylalanine, phenylalanyl threonine, threonyl-phenylalanyl-phenylalanine) in the experimental media. After incubation with experimental medium, cells were collected for gene expression analysis and medium was collected for milk protein or amino acid determination. The results showed that peptide-bound Phe at 10% (11.7 μg/ml) significantly enhanced αs1 casein gene expression and milk protein synthesis as compared with equivalent amount of free Phe. When 10% Phe was replaced by phenylalanylphenylalanine, the disappearance of most essential amino acids increased significantly, and gene expression of peptide transporter 2 and some amino acid transporters was significantly enhanced. These results indicate that the Phe and Thr oligopeptides are important for milk protein synthesis, and peptide-bound amino acids could be utilised more efficiently in milk protein synthesis than the equivalent amount of free amino acids.

  12. Oligopeptide complex for targeted non-viral gene delivery to adipocytes

    Science.gov (United States)

    Won, Young-Wook; Adhikary, Partho Protim; Lim, Kwang Suk; Kim, Hyung Jin; Kim, Jang Kyoung; Kim, Yong-Hee

    2014-12-01

    Commercial anti-obesity drugs acting in the gastrointestinal tract or the central nervous system have been shown to have limited efficacy and severe side effects. Anti-obesity drug development is thus focusing on targeting adipocytes that store excess fat. Here, we show that an adipocyte-targeting fusion-oligopeptide gene carrier consisting of an adipocyte-targeting sequence and 9-arginine (ATS-9R) selectively transfects mature adipocytes by binding to prohibitin. Injection of ATS-9R into obese mice confirmed specific binding of ATS-9R to fat vasculature, internalization and gene expression in adipocytes. We also constructed a short-hairpin RNA (shRNA) for silencing fatty-acid-binding protein 4 (shFABP4), a key lipid chaperone in fatty-acid uptake and lipid storage in adipocytes. Treatment of obese mice with ATS-9R/shFABP4 led to metabolic recovery and body-weight reduction (>20%). The ATS-9R/shFABP4 oligopeptide complex could prove to be a safe therapeutic approach to regress and treat obesity as well as obesity-induced metabolic syndromes.

  13. A RGD-Containing Oligopeptide (K)16GRGDSPC: A Novel Vector for Integrin-Mediated Targeted Gene Delivery

    Institute of Scientific and Technical Information of China (English)

    PAN Haitao; ZHENG Qixin; GUO Xiaodong; LIU Yong; LI Changwen; SONG Yulin

    2006-01-01

    A 23 amino acid, bifunctional integrin-targeted synthetic oligopeptide was evaluated for ex vivo gene delivery to rabbit bone marrow stromal cells (BMSCs). Synthesis of the peptide (K)16GRGDSPC was performed on a solid-phase batch peptide synthesizer. BMSCs were transfected with plasmid DNA coding for luciferase by (K)16GRGDSPC and the transfection efficiency was assayed. The influences of chloroquine and polyethyleneimine on the transfection efficiency were also examined. The target specificity of (K)16GRGDSPC to mediate exogenous gene into BMSCs was analyzed using cell attachment test and gene delivery inhibition test. The results showed that the transfection efficiency of the oligopeptide vector was lower than that of Lipofectamine. But in the presence of endosomal buffer chloroquine or endosomal disrupting agent polyethyleneimine, the transfection efficiency of the vector was greatly enhanced. In addition, RGD-containing peptides inhibited BMSCs' attachment to the 96-well plates pretreated with fibronectin or vitronectin and significantly decreased the transfection efficiency of the oligopeptide vector. These studies demonstrated that oligopeptide (K)16GRGDSPC was an ideal novel targeted non-viral gene delivery vector, which was easy to be synthesized, high efficient and low cytotoxicity. The vector could effectively deliver exogenous gene into rat BMSCs.

  14. Critical role of a conserved transmembrane lysine in substrate recognition by the proton-coupled oligopeptide transporter YjdL

    DEFF Research Database (Denmark)

    Jensen, Johanne M; Aduri, Nanda Gowtham; Prabhala, Bala K

    2014-01-01

    Proton-coupled oligopeptide transporters (POTs) utilize an electrochemical proton gradient to accumulate peptides in the cytoplasm. Changing the highly conserved active-site Lys117 in the Escherichia coli POT YjdL to glutamine resulted in loss of ligand affinity as well as inability to distinguis...

  15. H{sup +}/peptide transporter (PEPT2) is expressed in human epidermal keratinocytes and is involved in skin oligopeptide transport

    Energy Technology Data Exchange (ETDEWEB)

    Kudo, Michiko; Katayoshi, Takeshi; Kobayashi-Nakamura, Kumiko [DHC Corporation Laboratories, Division 2, 2-42 Hamada, Mihama-ku, Chiba 261-0025 (Japan); Akagawa, Mitsugu [Department of Biological Chemistry, Division of Applied Life Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 599-8531 (Japan); Tsuji-Naito, Kentaro, E-mail: knaito@dhc.co.jp [DHC Corporation Laboratories, Division 2, 2-42 Hamada, Mihama-ku, Chiba 261-0025 (Japan)

    2016-07-08

    Peptide transporter 2 (PEPT2) is a member of the proton-coupled oligopeptide transporter family, which mediates the cellular uptake of oligopeptides and peptide-like drugs. Although PEPT2 is expressed in many tissues, its expression in epidermal keratinocytes remains unclear. We investigated PEPT2 expression profile and functional activity in keratinocytes. We confirmed PEPT2 mRNA expression in three keratinocyte lines (normal human epidermal keratinocytes (NHEKs), immortalized keratinocytes, and malignant keratinocytes) by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. In contrast to PEPT1, PEPT2 expression in the three keratinocytes was similar or higher than that in HepG2 cells, used as PEPT2-positive cells. Immunolocalization analysis using human skin showed epidermal PEPT2 localization. We studied keratinocyte transport function by measuring the oligopeptide content using liquid chromatography/tandem mass spectrometry. Glycylsarcosine uptake in NHEKs was pH-dependent, suggesting that keratinocytes could absorb small peptides in the presence of an inward H{sup +} gradient. We also performed a skin-permeability test of several oligopeptides using skin substitute, suggesting that di- and tripeptides pass actively through the epidermis. In conclusion, PEPT2 is expressed in keratinocytes and involved in skin oligopeptide uptake. -- Highlights: •PEPT2 is expressed in keratinocytes, which are more common than other skin cells. •Immunolocalization analysis using human skin revealed epidermal PEPT2 localization. •Keratinocytes could absorb small peptides in the presence of an inward H{sup +} gradient. •Di- and tripeptide pass actively through the epidermis.

  16. Chemical and enzymatic catalytic routes to polyesters and oligopeptides biobased materials

    Science.gov (United States)

    Zhu, Jianhui

    My Ph.D research focuses on the synthesis and property studies of different biobased materials, including polyesters, polyurethanes and oligopeptides. The first study describes the synthesis, crystal structure and physico-mechanical properties of a bio-based polyester prepared from 2,5-furandicarboxylic acid (FDCA) and 1,4-butanediol. Melt-polycondensation experiments were conducted by a two-stage polymerization using titanium tetraisopropoxide (Ti[OiPr] 4) as catalyst. Polymerization conditions (catalyst concentration, reaction time and 2nd stage reaction temperature) were varied to optimize poly(butylene furan dicarboxylate), PBF, molecular weight. A series of PBFs with different Mw were characterized by Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA), Dynamic Mechanical Thermal Analysis (DMTA), X-Ray diffraction and tensile testing. Influence of molecular weight and melting/crystallization enthalpy on PBF material tensile properties was explored. Cold-drawing tensile tests at room temperature for PBF with Mw 16K to 27K showed a brittle-to-ductile transition. When Mw reaches 38K, the Young's Modulus of PBF remains above 900 MPa, and the elongation at break increases to above 1000%. The mechanical properties, thermal properties and crystal structures of PBF were similar to petroleum derived poly(butylenes terephthalate), PBT. Fiber diagrams of uniaxially stretched PBF films were collected, indexed, and the unit cell was determined as triclinic (a=4.78(3) A, b=6.03(5) A, c=12.3(1) A, alpha=110.1(2)°, beta=121.1(3)°, gamma=100.6(2)°). A crystal structure was derived from this data and final atomic coordinates are reported. We concluded that there is a close similarity of the PBF structure to PBT alpha- and beta-forms. In the second study, a biobased long chain polyester polyol (PC14-OH) was synthesized from o-hydroxytetradecanoic acid (o-HOC14) and 1,4-butanediol. The first section about polyester polyurethanes describes the synthesis

  17. Generation of free radicals from lipid hydroperoxides by Ni2+ in the presence of oligopeptides.

    Science.gov (United States)

    Shi, X; Dalal, N S; Kasprzak, K S

    1992-11-15

    The generation of free radicals from lipid hydroperoxides by Ni2+ in the presence of several oligopeptides was investigated by electron spin resonance (ESR) utilizing 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap. Incubation of Ni2+ with cumene hydroperoxide or t-butyl hydroperoxide did not generate any detectable free radical. In the presence of glycylglycylhistidine (GlyGlyHis), however, Ni2+ generated cumene peroxyl (ROO.) radical from cumene hydroperoxide, with the free radical generation reaching its saturation level within about 3 min. The reaction was first order with respect to both cumene hydroperoxide and Ni2+. Similar results were obtained using t-butyl hydroperoxide, but the yield of t-butyl peroxyl radical generation was about 7-fold lower. Other histidine-containing oligopeptides such as beta-alanyl-L-histidine (carnosine), gamma-aminobutyryl-L-histidine (homocarnosine), and beta-alanyl-3-methyl-L-histidine (anserine) caused the generation of both cumene alkyl (R.) and cumene alkoxyl (RO.) radicals in the reaction of Ni2+ with cumene hydroperoxide. Similar results were obtained using t-butyl hydroperoxide. Glutathione also caused generation of R. and RO. radicals in the reaction of Ni2+ with cumene hydroperoxide but the yield was approximately 25-fold greater than that produced by the histidine-containing peptides, except GlyGlyHis. The ratio of DMPO/R. and DMPO/RO. produced with glutathione and cumene hydroperoxide was approximately 3:1. Essentially the same results were obtained using t-butyl hydroperoxide except that the ratio of DMPO/R. to DMPO/RO. was approximately 1:1. The free radical generation from cumene hydroperoxide reached its saturation level almost instantaneously while in the case of t-butyl hydroperoxide, the saturation level was reached in about 3 min. In the presence of oxidized glutathione, the Ni2+/cumene hydroperoxide system caused DMPO/.OH generation from DMPO without forming free hydroxyl radical. Since glutathione

  18. Intracellular polyamine pools, oligopeptide-binding protein A expression, and resistance to aminoglycosides in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Maria BR Acosta

    2005-11-01

    Full Text Available The role of intracellular free polyamine (putrescine and spermidine pools in multiple resistance to aminoglycoside antibiotics was investigated among in vitro selected kanamycin-resistant Escherichia coli J53 mutants expressing diminished oligopeptide-binding protein (OppA levels and/or defective ornithine decarboxylase (ODC activity. The results suggest that diminished OppA content, but not defective ODC activity expression, increased the relative concentration of free spermidine as compared to the wild type strain. Moreover, by adding exogenous polyamines or polyamine synthesis inhibitors to cultures with different mutant strains, a direct relationship between the intracellular OppA levels and resistance to kanamycin was revealed. Collectively these results further suggest a complex relation among OppA expression, aminoglycoside resistance and polyamine metabolism.

  19. Self-assembling monolayers of helical oligopeptides with applications in molecular electronics

    CERN Document Server

    Strong, A E

    1997-01-01

    prepared. Transformation of the two (Trt)Cys residues of the resin-bound peptide to the intramolecular disulphide by iodine was achieved in acetonitrile but not in DMF. CD suggested that the conformation of this peptide was a mixture of helix and random coil. Films of the peptide-disulphide and the peptide-dithiol adsorbed from protic solvents were characterised as multilayers by ellipsometry. However CV and ellipsometry showed that a monolayer was successfully prepared from acetonitrile. Future targets for improving and extending this method to form monolayers of linked disulphides are presented. The aim of this project was to develop a generic method of preparing a 'molecular architecture' containing functional groups on a surface at predetermined relative positions several nm apart. This would be of great utility in molecular electronics, chemical sensors and other fields. It was proposed that such an architecture could be prepared on gold using linked, helical oligopeptides that contained the components o...

  20. FRET study in oligopeptide-linked donor–acceptor system in PVA matrix

    Science.gov (United States)

    Shah, Sunil; Mandecki, Wlodek; Li, Ji; Gryczynski, Zygmunt; Borejdo, Julian; Gryczynski, Ignacy; Fudala, Rafal

    2016-12-01

    An oligopeptide: Lys-Gly-Pro-Arg-Ser-Leu-Ser-Gly-Lys-NH2, cleaved specifically by a matrix metalloproteinase 9 (MMP-9) at the Ser-Leu bond, was labeled on the ε-NH2 groups of lysine with donor (5, 6 TAMRA) and acceptor (HiLyte647) dye. The donor control was a peptide labeled with 5, 6 TAMRA only on the C-terminal lysine, and the acceptor control was free HiLyte647. Following three products were studied by dissolving in 10% (w/w) poly(vinyl alcohol) and dried on glass slides forming 200 micron films. Absorption spectra of the films show full additivity of donor and acceptor absorptions. A strong Fluorescence Resonance Energy Transfer (FRET) with an efficiency of about 85% was observed in the fluorescence emission and excitation spectra. The lifetime of the donor was shorter and heterogeneous compared with the donor control.

  1. Characterization and partial purification of an oligopeptide elicitor receptor from parsley (Petroselinum crispum).

    Science.gov (United States)

    Nennstiel, D; Scheel, D; Nürnberger, T

    1998-07-24

    Parsley cells recognize the fungal phytopathogen Phytophthora sojae through a plasma membrane receptor. A 13 amino acid oligopeptide fragment (Pep-13) of a 42 kDa fungal cell wall glycoprotein was shown to bind to the receptor and stimulate a complex defense response in cultured parsley cells. The Pep-13 binding site solubilized from parsley microsomal membranes by non-ionic detergents exhibited the same ligand affinity and ligand specificity as the membrane-bound receptor. Chemical crosslinking and photoaffinity labeling assays with [125I]Pep-13 revealed that a monomeric 100 kDa integral plasma membrane protein is sufficient for ligand binding and may thus constitute the ligand binding domain of the receptor. Ligand affinity chromatography of solubilized microsomal membrane protein on immobilized Pep-13 yielded a 5000-fold enrichment of specific receptor activity.

  2. Hg-coordination studies of oligopeptides containing cysteine, histidine and tyrosine by $^{199m}$Hg-TDPAC

    CERN Document Server

    Ctortecka, B; Mallion, S; Butz, T; Hoffmann, R

    1999-01-01

    In order to study the interaction of histidine- and tyrosine- containing peptide chains with Hg(II), the nuclear quadrupole interaction (NQI) of /sup 199m/Hg in the Hg complexes of the oligopeptides alanyl-alanyl-histidyl-alanyl-alanine-amid (AAHAA-NH /sub 2/) and alanyl-alanyl-tyrosyl-alanyl-alanine-amid (AAYAA-NH/sub 2/) was determined by time differential perturbed angular correlation and is compared with previous data on alanyl-alanyl-cysteyl-alanyl- alanyl (AACAA-OH). The /sup 199m/Hg-NQIs depend on the oligopeptide to Hg(II) stoichiometry and indicate that two-fold and four-fold coordinations occur for the bound Hg(II). (12 refs).

  3. The Research Development of Soybean Oligopeptide%大豆低聚肽的研究进展

    Institute of Scientific and Technical Information of China (English)

    吕育新; 梁金钟

    2013-01-01

    Soybean oligopeptide is a kind of small peptide mixture, which is gotten from soybean protein by separation and purification. This article summerized the methods of preparation, separation and purification for soybean oligopeptide, the physicochemical properties, functional characters and the application of it.%  大豆低聚肽是大豆蛋白经过分离和纯化等处理后得到的小肽混合物。本文概述了大豆低聚肽的制备和分离纯化方法、大豆低聚肽的理化特性和功能特性及大豆低聚肽的应用。

  4. [Interconnection between architecture of protein globule and disposition of conformational conservative oligopeptides in proteins from one protein family].

    Science.gov (United States)

    Batianovskiĭ, A V; Filatov, I V; Namiot, V A; Esipova, N G; Volotovskiĭ, I D

    2012-01-01

    It was shown that selective interactions between helical segments of macromolecules can realize in globular proteins in the segments characterized by the same periodicities of charge distribution i.e. between conformationally conservative oligopeptides. It was found that in the macromolecules of alpha-helical proteins conformationally conservative oligopeptides are disposed at a distance being characteristic of direct interactions. For representatives of many structural families of alpha-type proteins specific disposition of conformationally conservative segments is observed. This disposition is inherent to a particular structural family. Disposition of conformationally conservative segments is not related to homology of the amino acid sequence but reflects peculiarities of native 3D-architectures of protein globules.

  5. Spasmogenic activity of chemotactic N-formylated oligopeptides: identity of structure--function relationships for chemotactic and spasmogenic activities.

    OpenAIRE

    Marasco, W. A.; Fantone, J. C.; Ward, P. A.

    1982-01-01

    The chemotactic N-formylated oligopeptides are potent spasmogenic agents for guinea pig ileum. Structure-activity studies with various N-formylated peptides suggest the presence of a specific receptor that resembles in specificity the formyl peptide receptor on leukocytes. A competitive antagonist of the formyl peptide receptor on leukocytes also inhibits formyl peptide-induced ileum contraction, whereas the antihistamine diphenhydramine is without effect. The contractile response caused by t...

  6. Redox activity and multiple copper(I) coordination of 2His-2Cys oligopeptide.

    Science.gov (United States)

    Choi, DongWon; Alshahrani, Aisha A; Vytla, Yashodharani; Deeconda, Manogna; Serna, Victor J; Saenz, Robert F; Angel, Laurence A

    2015-02-01

    Copper binding motifs with their molecular mechanisms of selective copper(I) recognition are essential molecules for acquiring copper ions, trafficking copper to specific locations and controlling the potentially damaging redox activities of copper in biochemical processes. The redox activity and multiple Cu(I) binding of an analog methanobactin peptide-2 (amb2) with the sequence acetyl-His1-Cys2-Tyr3-Pro4-His5-Cys6 was investigated using ion mobility-mass spectrometry (IM-MS) and UV-Vis spectrophotometry analyses. The Cu(II) titration of amb2 showed oxidation of amb2 via the formation of intra- and intermolecular Cys-Cys disulfide bridges and the multiple Cu(I) coordination by unoxidized amb2 or the partially oxidized dimer and trimer of amb2. The principal product of these reactions was [amb2 + 3Cu(I)](+) which probably coordinates the three Cu(I) ions via two bridging thiolate groups of Cys2 and Cys6 and the δN6 of the imidazole groups of His6, as determined by geometry optimized structures at the B3LYP/LanL2DZ level of theory. The products observed by IM-MS showed direct correlation to spectral changes associated with disulfide bond formation in the UV-Vis spectrophotometric study. The results show that IM-MS analysis is a powerful technique for unambiguously determining the major ion species produced during the redox and metal binding chemistry of oligopeptides.

  7. Design of amphiphilic oligopeptides as models for fine tuning peptide assembly with plasmid DNA.

    Science.gov (United States)

    Goparaju, Geetha N; Gupta, Pardeep K

    2014-08-01

    We discuss the design of novel amphiphilic oligopeptides with hydrophobic and cationic amino acids to serve as models to understand peptide-DNA assembly. Biophysical and thermodynamic characterization of interaction of these amphiphilic peptides with plasmid DNA is presented. Peptides with at least +4 charges favor stable complex formation. Surface potential is dependent on the type of hydrophobic amino acid for a certain charge. Thermodynamically it is a spontaneous interaction between most of the peptides and plasmid DNA. Lys(7) and Tyr peptides with +4/+5 charges indicate cooperative binding with pDNA without saturation of interaction while Val(2)-Gly-Lys(4), Val-Gly-Lys(5), and Phe-Gly-Lys(5) lead to saturation of interaction indicating condensed pDNA within the range of N/Ps studied. We show that the biophysical properties of DNA-peptide complexes could be modulated by design and the peptides presented here could be used as building blocks for creating DNA-peptide complexes for various biomedical applications, mainly nucleic acid delivery.

  8. Protective Effects and Mechanism of Meretrix meretrix Oligopeptides against Nonalcoholic Fatty Liver Disease

    Science.gov (United States)

    Huang, Fangfang; Zhao, Shasha; Yu, Fangmiao; Yang, Zuisu; Ding, Guofang

    2017-01-01

    Meretrix meretrix oligopeptides (MMO) derived from shellfish have important medicinal properties. We previously obtained MMO from alcalase by hydrolysis processes. Here we examine the protective effects of MMO against nonalcoholic fatty liver disease (NAFLD) and explored the underlying mechanism. Human Chang liver cells were used in our experiments after exposure to palmitic acid at a final concentration of 15 μg/mL for 48 h to induce an overload of fatty acid as NAFLD model cells. Treatment with MMO for 24 h increased the viability of the NAFLD model cells by inhibiting apoptosis. MMO alleviated oxidative stress in the NAFLD model cells by preserving reactive oxygen species activity and increasing malondialdehyde and superoxide dismutase activity. MMO improved mitochondrial dysfunction by decreasing the mitochondrial membrane potential and increasing the activities of Na+/K+-ATPase and Ca2+/Mg2+-ATPase. In addition, MMO inhibited the activation of cell death-related pathways, based on reduced p-JNK, Bax expression, tumor necrosis factor-α, caspase-9, and caspase-3 activity in the NAFLD model cells, and Bcl-2 expression was enhanced in the NAFLD model cells compared with the control group. These findings indicate that MMO have antioxidant and anti-apoptotic effects on NAFLD model cells and may thus exert protective effects against NAFLD. PMID:28216552

  9. New proline-rich oligopeptides from the venom of African adders: Insights into the hypotensive effect of the venoms.

    Science.gov (United States)

    Kodama, Roberto T; Cajado-Carvalho, Daniela; Kuniyoshi, Alexandre K; Kitano, Eduardo S; Tashima, Alexandre K; Barna, Barbara F; Takakura, Ana Carolina; Serrano, Solange M T; Dias-Da-Silva, Wilmar; Tambourgi, Denise V; Portaro, Fernanda V

    2015-06-01

    The snakes from the Bitis genus are some of the most medically important venomous snakes in sub Saharan Africa, however little is known about the composition and effects of these snake venom peptides. Considering that the victims with Bitis genus snakes have exacerbate hypotension and cardiovascular disorders, we investigated here the presence of angiotensin-converting enzyme modulators on four different species of venoms. The peptide fractions from Bitis gabonica gabonica, Bitis nasicornis, Bitis gabonica rhinoceros and Bitis arietans which showed inhibitory activity on angiotensin-converting enzyme were subjected to mass spectrometry analysis. Eight proline-rich peptides were synthetized and their potencies were evaluated in vitro and in vivo. The MS analysis resulted in over 150 sequences, out of which 32 are new proline-rich oligopeptides, and eight were selected for syntheses. For some peptides, inhibition assays showed inhibitory potentials of cleavage of angiotensin I ten times greater when compared to bradykinin. In vivo tests showed that all peptides decreased mean arterial pressure, followed by tachycardia in 6 out of 8 of the tests. We describe here some new and already known proline-rich peptides, also known as bradykinin-potentiating peptides. Four synthetic peptides indicated a preferential inhibition of angiotensin-converting enzyme C-domain. In vivo studies show that the proline-rich oligopeptides are hypotensive molecules. Although proline-rich oligopeptides are known molecules, we present here 32 new sequences that are inhibitors of the angiotensin-converting enzyme and consistent with the symptoms of the victims of Bitis spp, who display severe hypotension. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Artificial neural network models for prediction of intestinal permeability of oligopeptides

    Directory of Open Access Journals (Sweden)

    Kim Min-Kook

    2007-07-01

    Full Text Available Abstract Background Oral delivery is a highly desirable property for candidate drugs under development. Computational modeling could provide a quick and inexpensive way to assess the intestinal permeability of a molecule. Although there have been several studies aimed at predicting the intestinal absorption of chemical compounds, there have been no attempts to predict intestinal permeability on the basis of peptide sequence information. To develop models for predicting the intestinal permeability of peptides, we adopted an artificial neural network as a machine-learning algorithm. The positive control data consisted of intestinal barrier-permeable peptides obtained by the peroral phage display technique, and the negative control data were prepared from random sequences. Results The capacity of our models to make appropriate predictions was validated by statistical indicators including sensitivity, specificity, enrichment curve, and the area under the receiver operating characteristic (ROC curve (the ROC score. The training and test set statistics indicated that our models were of strikingly good quality and could discriminate between permeable and random sequences with a high level of confidence. Conclusion We developed artificial neural network models to predict the intestinal permeabilities of oligopeptides on the basis of peptide sequence information. Both binary and VHSE (principal components score Vectors of Hydrophobic, Steric and Electronic properties descriptors produced statistically significant training models; the models with simple neural network architectures showed slightly greater predictive power than those with complex ones. We anticipate that our models will be applicable to the selection of intestinal barrier-permeable peptides for generating peptide drugs or peptidomimetics.

  11. Engineered bacterial hydrophobic oligopeptide repeats in a synthetic yeast prion, [REP-PSI+

    Directory of Open Access Journals (Sweden)

    Fátima eGasset-Rosa

    2015-04-01

    Full Text Available The yeast translation termination factor Sup35p, by aggregating as the [PSI+] prion, enables ribosomes to read-through stop codons, thus expanding the diversity of the Saccharomyces cerevisiae proteome. Yeast prions are functional amyloids that replicate by templating their conformation on native protein molecules, then assembling as large aggregates and fibers. Prions propagate epigenetically from mother to daughter cells by fragmentation of such assemblies. In the N-terminal prion-forming domain, Sup35p has glutamine/asparagine-rich oligopeptide repeats (OPRs, which enable propagation through chaperone-elicited shearing. We have engineered chimeras by replacing the polar OPRs in Sup35p by up to five repeats of a hydrophobic amyloidogenic sequence from the synthetic bacterial prionoid RepA-WH1. The resulting hybrid, [REP-PSI+], i was functional in a stop codon read-through assay in S. cerevisiae; ii generates weak phenotypic variants upon both its expression or transformation into [psi-] cells; iii these variants correlated with high molecular weight aggregates resistant to SDS during electrophoresis; and iv according to fluorescence microscopy, the fusion of the prion domains from the engineered chimeras to the reporter protein mCherry generated perivacuolar aggregate foci in yeast cells. All these are signatures of bona fide yeast prions. As assessed through biophysical approaches, the chimeras assembled as oligomers rather than as the fibers characteristic of [PSI+]. These results suggest that it is the balance between polar and hydrophobic residues in OPRs what determines prion conformational dynamics. In addition, our findings illustrate the feasibility of enabling new propagation traits in yeast prions by engineering OPRs with heterologous amyloidogenic sequence repeats.

  12. An Extracellular Oligopeptide Permease May Be a Potential Virulence Factor of Vibrio harveyi

    Institute of Scientific and Technical Information of China (English)

    HE Qingfang; CHEN Jixiang; LI Caifeng

    2011-01-01

    An oligopeptide permease A (OppA) was purified from the extracellular product of Vibrio harveyi SF-1.The molecular weight of the purified protein was estimated to be 58kDa on SDS-PAGE.The purified protein showed phospholipase C activity at the optimal values of temperature 50℃ and pH 8.0.The enzymatic activity decreased when the temperature increased to 40℃.The N-terminal sequence of the purified protein was determined as ADVPAGTKLA,which is similar to that of OppA.The OppA precursor gene was cloned from the genome of V.harveyi SF-1.The gene consisted of 1665 base pairs and encoded a 554 amino acid polypeptide,which showed a high similarity to those of the OppAs of V.harveyi and other Vibrio species.The gene was subcloned into pET-28a (+) and expressed in Escherichia coli.The expressed recombinant protein was purified by Ni-NTA metal affinity chromatography.The expressed recombinant protein showed a 58 kDa band on SDS-PAGE and exhibited phospholipase C activity with the optima of temperature 50℃ and pH 8.0.The purified protein was toxic to the flounder gill cells.An OppA mutant of V.harveyi SF-1 was constructed by homologous recombination.The mutant strain was less virulent when it was intraperitoneally inoculated to zebra fish,with the LD50 of 5.46x 105 CFU fish-1,compared to 3.11×104 CFU fish-1 of the wild-type strain,which indicated that the OppA might play an important role in the pathogenicity of V.harveyi.

  13. A Novel Vasoactive Proline-Rich Oligopeptide from the Skin Secretion of the Frog Brachycephalus ephippium.

    Directory of Open Access Journals (Sweden)

    Daniel Dias Rufino Arcanjo

    Full Text Available Proline-rich oligopeptides (PROs are a large family which comprises the bradykinin-potentiating peptides (BPPs. They inhibit the activity of the angiotensin I-converting enzyme (ACE and have a typical pyroglutamyl (Pyr/proline-rich structure at the N- and C-terminus, respectively. Furthermore, PROs decrease blood pressure in animals. In the present study, the isolation and biological characterization of a novel vasoactive BPP isolated from the skin secretion of the frog Brachycephalus ephippium is described. This new PRO, termed BPP-Brachy, has the primary structure WPPPKVSP and the amidated form termed BPP-BrachyNH2 inhibits efficiently ACE in rat serum. In silico molecular modeling and docking studies suggest that BPP-BrachyNH2 is capable of forming a hydrogen bond network as well as multiple van der Waals interactions with the rat ACE, which blocks the access of the substrate to the C-domain active site. Moreover, in rat thoracic aorta BPP-BrachyNH2 induces potent endothelium-dependent vasodilatation with similar magnitude as captopril. In DAF-FM DA-loaded aortic cross sections examined by confocal microscopy, BPP-BrachyNH2 was found to increase the release of nitric oxide (NO. Moreover, BPP-BrachyNH2 was devoid of toxicity in endothelial and smooth muscle cell cultures. In conclusion, the peptide BPP-BrachyNH2 has a novel sequence being the first BPP isolated from the skin secretion of the Brachycephalidae family. This opens for exploring amphibians as a source of new biomolecules. The BPP-BrachyNH2 is devoid of cytotoxicity and elicits endothelium-dependent vasodilatation mediated by NO. These findings open for the possibility of potential application of these peptides in the treatment of endothelial dysfunction and cardiovascular diseases.

  14. Hypocholesterolemic effect of sericin-derived oligopeptides in high-cholesterol fed rats.

    Science.gov (United States)

    Lapphanichayakool, Phakhamon; Sutheerawattananonda, Manote; Limpeanchob, Nanteetip

    2017-01-01

    The beneficial effect of cholesterol-lowering proteins and/or peptides derived from various dietary sources is continuously reported. A non-dietary protein from silk cocoon, sericin, has also demonstrated cholesterol-lowering activity. A sericin hydrolysate prepared by enzymatic hydrolysis was also expected to posses this effect. The present study was aimed at investigating the cholesterol-lowering effect of sericin peptides, so called "sericin-derived oligopeptides" (SDO) both in vivo and in vitro. The results showed that SDO at all three doses tested (10 mg kg(-1) day(-1), 50 mg kg(-1) day(-1), and 200 mg kg(-1) day(-1)) suppressed serum total and non-HDL cholesterol levels in rats fed a high-cholesterol diet. Triglyceride and HDL-cholesterol levels were not significantly changed among all groups. The fecal contents of bile acids and cholesterol did not differ among high-cholesterol fed rats. SDO dose-dependently reduced cholesterol solubility in lipid micelles, and inhibited cholesterol uptake in monolayer Caco-2 cells. SDO also effectively bound to all three types of bile salts including taurocholate, deoxytaurocholate, and glycodeoxycholate. Direct interaction with bile acids of SDO may disrupt micellar cholesterol solubility, and subsequently reduce the absorption of dietary cholesterol in intestines. Taking all data together, SDO or sericin peptides exhibit a beneficial effect on blood cholesterol levels and could be potentially used as a health-promoting dietary supplement or nutraceutical product.

  15. Effects of soya oligosaccharides and soya oligopeptides on lipid metabolism in hyperlipidaemic rats.

    Science.gov (United States)

    Xie, Shali; Zhu, Jundong; Zhang, Yanqi; Shi, Kai; Shi, Yuangang; Ma, Xiao

    2012-08-01

    In the present study, we aimed to examine the effects of soya oligosaccharides (SOS) and soya oligopeptides (SOP) on blood lipid levels, release of vasoactive substances, antioxidant activity and faecal bile acid (FBA) excretion in rats fed a high-fat diet (HFD). Male Sprague-Dawley rats were evenly divided into five groups according to diets as follows: regular diet (control), HFD, HFD enriched with 2 % of SOS (SOS), HFD enriched with 3 % of SOP (SOP) and HFD enriched with 2 % SOS and 3 % SOP (SOSP). The results showed that SOS and SOP significantly reduced plasma total cholesterol, LDL-cholesterol and TAG, whereas HDL-cholesterol concentration was significantly increased. Furthermore, SOS and SOP reduced plasma apoB, apoE and the apoB:apoAI ratio, whereas apoAI was significantly increased. Moreover, SOS and SOP also reduced plasma thromboxane A₂ (TXA₂) and the TXA₂:prostacyclin (PGI₂) ratio, whereas plasma PGI₂ and nitric oxide were significantly increased. In addition, SOS and SOP significantly reduced serum and liver malondialdehyde concentrations and increased FBA excretion. However, we did not observe obvious influences of SOS and SOP on superoxide dismutase activities in the liver of HFD-fed rats. The combination of 2 % SOS and 3 % SOP showed a more marked effect than SOS or SOP alone in improving the lipid profile, release of vasoactive substances and increasing FBA excretion (P < 0.05). In summary, SOS and SOP might help prevent atherosclerosis through improving abnormal blood lipid levels, regulating vasoactive substances and protecting against oxidative stress.

  16. Anti-Fatigue Effects of Small Molecule Oligopeptides Isolated from Panax ginseng C. A. Meyer in Mice.

    Science.gov (United States)

    Bao, Lei; Cai, Xiaxia; Wang, Junbo; Zhang, Yuan; Sun, Bin; Li, Yong

    2016-12-13

    Panax ginseng C. A. Meyer (ginseng) is an edible and medicinal Chinese herb, which is often used in Asian countries for physical fitness. Ginseng is reported to have a wide range of biological activity and pharmaceutical properties. There were more studies on ginsenosides and polysaccharides, but fewer studies on ginseng oligopeptides (GOP), which are small molecule oligopeptides isolated from ginseng. The present study was designed to evaluate the anti-fatigue effects of GOP in mice and explore the possible underlying mechanism. Mice were randomly divided into four experimental sets for the detection of different indicators. Each set of mice were then divided into four groups. The control group was administered distilled water, and three GOP intervention groups were administered 125, 250, and 500 mg/kg of body weight, respectively, of GOP by gavage each day. After 30 days of GOP treatment, it was observed that GOP could significantly increase the forced swimming time, enhance lactate dehydrogenase (LDH) activity and hepatic glycogen levels, and retard the accumulation of serum urea nitrogen (SUN) and blood lactic acid (BLA) in mice. GOP also markedly ameliorated fatigue-induced alterations of inoxidative stress biomarkers and antioxidant enzymes. Notably, GOP increased the mRNA expression of mitochondrial biogenesis factors and mitochondrial DNA content in skeletal muscles of mice. These results suggest that GOP possess anti-fatigue effects, which may be attributed to the inhibition of oxidative stress and the improvement of mitochondrial function in skeletal muscles. GOP could be a novel natural agent for relieving exercise fatigue.

  17. A genetic algorithm encoded with the structural information of amino acids and dipeptides for efficient conformational searches of oligopeptides.

    Science.gov (United States)

    Ru, Xiao; Song, Ce; Lin, Zijing

    2016-05-15

    The genetic algorithm (GA) is an intelligent approach for finding minima in a highly dimensional parametric space. However, the success of GA searches for low energy conformations of biomolecules is rather limited so far. Herein an improved GA scheme is proposed for the conformational search of oligopeptides. A systematic analysis of the backbone dihedral angles of conformations of amino acids (AAs) and dipeptides is performed. The structural information is used to design a new encoding scheme to improve the efficiency of GA search. Local geometry optimizations based on the energy calculations by the density functional theory are employed to safeguard the quality and reliability of the GA structures. The GA scheme is applied to the conformational searches of Lys, Arg, Met-Gly, Lys-Gly, and Phe-Gly-Gly representative of AAs, dipeptides, and tripeptides with complicated side chains. Comparison with the best literature results shows that the new GA method is both highly efficient and reliable by providing the most complete set of the low energy conformations. Moreover, the computational cost of the GA method increases only moderately with the complexity of the molecule. The GA scheme is valuable for the study of the conformations and properties of oligopeptides. © 2016 Wiley Periodicals, Inc.

  18. Fluorescent monitoring of copper-occupancy in His-ended catalytic oligo-peptides.

    Science.gov (United States)

    Inokuchi, Reina; Kawano, Tomonori

    2016-01-01

    Controlled generation of reactive oxygen species (ROS) is widely beneficial to various medical, environmental, and agricultural studies. As inspired by the functional motifs in natural proteins, our group has been engaged in development of catalytically active oligo-peptides as minimum-sized metalloenzymes for generation of superoxide anion, an active member of ROS. In such candidate molecules, catalytically active metal-binding minimal motif was determined to be X-X-H, where X can be most amino acids followed by His. Based on above knowledge, we have designed a series of minimal copper-binding peptides designated as G n H series peptides, which are composed of oligo-glycyl chains ended with C-terminal His residue such as GGGGGH sequence (G5H). In order to further study the role of copper binding to the peptidic catalysts sharing the X-X-H motif such as G5H-conjugated peptides, we should be able to score the occupancy of the peptide population by copper ion in the reaction mixture. Here, model peptides with Cu-binding affinity which show intrinsic fluorescence due to tyrosyl residue (Y) in the UV region (excitation at ca. 230 and 280 nm, and emission at ca. 320 nm) were synthesized to score the effect of copper occupancy. Synthesized peptides include GFP-derived fluorophore sequence, TFSYGVQ (designated as Gfp), and Gfp sequence fused to C-terminal G5H (Gfp-G5H). In addition, two Y-containing tri-peptides derived from natural GFP fluorophores, namely, TYG and SYG were fused to the G5H (TYG-G5H and SYG-G5H). Conjugation of metal-binding G5H sequence to GFP-fluorophore peptide enhanced the action of Cu(2+) on quenching of intrinsic fluorescence due to Y residue. Two other Y-containing peptides, TYG-G5H and SYG-G5H, also showed intrinsic fluorescence which is sensitive to addition of Cu(2+). There was linear relationship between the loading of Cu(2+) and the quenching of fluorescence in these peptide, suggesting that Cu(2+)-dependent quenching of Y

  19. Transport of β-Casein-derived Peptides by the Oligopeptide Transport System Is a Crucial Step in the Proteolytic Pathway of Lactococcus lactis

    NARCIS (Netherlands)

    Kunji, E.R S; Hagting, A; de Vries, C.J.; Juillard, V.; Haandrikman, A.J; Poolman, B.; Konings, W.N

    1995-01-01

    In the proteolytic pathway of Lactococcus lactis, milk proteins (caseins) are hydrolyzed extracellularly to oligopeptides by the proteinase (PrtP). The fate of these peptides, i.e. extracellular hydrolysis followed by amino acid uptake or transport followed by intracellular hydrolysis, has been addr

  20. Effect of condensation agents and minerals for oligopeptide formation under mild and hydrothermal conditions in related to chemical evolution of proteins

    Science.gov (United States)

    Kawamura, Kunio; Takeya, Hitoshi; Kushibe, Takao

    2009-07-01

    The role of condensation agents and minerals for oligopeptide formation was inspected to see whether minerals possess catalytic activity under mild and hydrothermal conditions. Under mild conditions, oligopeptide formation from negatively charged amino acids (Asp and Glu) using different minerals and the elongation of alanine oligopeptides ((Ala) 2-(Ala) 5) were attempted using apatite minerals. Oligo(Asp) up to 10 amino acid units from Asp were observed in the presence of 1-ethyl-3-(3-dimethylaminopropyl carbodiimide (EDC). Notable influence of minerals was not detected for the oligo(Asp) formation. Oligo(Asp) was gradually degraded by the further incubation in the presence of EDC in both the absence and presence of minerals. The formation of oligo(Glu) was less efficient in the presence of carbonyldiimidazole. The elongation from (Ala) 3, (Ala) 4, and (Ala) 5 and the formation of diketopiperazine from (Ala) 2 proceeded immediately in the presence of EDC in the meantime of the sample preparations. In addition, it was unexpected that the disappearance of the products and the reformation of the reactants occurred by the further incubation for 24 h; for instance, (Ala) 5 decreased but (Ala) 4 increased with increasing the reaction time in the reaction of (Ala) 4 with EDC. These facts suggest that the activation of the reactant amino acids or peptides immediately occurs. Under the simulated hydrothermal conditions, EDC did not enhance the formation of oligopeptides from Asp, Glu or Ala nor the spontaneous formation of (Ala) 5 from (Ala) 4.

  1. Separation of basic oligopeptides by ion-pairing reversed-phase chromatography

    Science.gov (United States)

    Xie, Wenchun

    The present thesis consist of five chapters. Chapter I introduces background information on the ion-pairing reversed-phase chromatography and liquid chromatography in the critical condition. Chapter II decribes our study on the isocratic separation of oligolysine (dp = 2 to 8) using a fixed content of acetonitrile (ACN) (23%) and different concentrations of HFBA in the mobile phase (0.6-30.6 mM) on a Waters XBridge Shield RP18® column. We found that the retention time of oligolysine increases as the dp increases, because of an increased number of HFBA bound to the peptides. Furthermore, when [HFBA] increased, the retention time increased at different rates. The greater the dp, the faster the rate. Based on a closed pairing model that presumes an equilibrium between an unpaired state and the paired state with a fixed number of HFBA molecules, an equation was derived for the retention factor of oligolysine. In Chapter III, we compare retention behaviors of oligolysine (dp = 2 to 8) and oligoarginine (dp = 2 to 8) when they are separated on the Waters XBridge Shield RP18® using fixed a ACN content (23%) and difference concentrations of HFBA (0.4-30.6 mM) in the mobile phase. The retention time of oligoarginine also increased at different rates as [HFBA] increased. The greater the dp, the faster the rate. The retention time of oligolysine is shorter than that of oligarginine having the dame dp. We applied Eq.1 to analyze the plot of ln k as a function of [HFBA] for each oligopeptide component to obtain the values for n, Kip,m, and βKd,ip. For oligolysine, n increases linearly as dp increase and oligoarginine exhibits an accelerated increase in n as dp rises. The plot of ln βKd,ip against dp followed a linear relationship for both peptides. In Chapter IV, we study the effect of mobile phase composition on the retention of oligolysine (dp = 2 to 8) on the Waters XBridge Shield RP18 ®. The ACN content was changed from 20% to 33% and the HFBA concentration from 0.7 to

  2. Spasmogenic activity of chemotactic N-formylated oligopeptides: identity of structure--function relationships for chemotactic and spasmogenic activities.

    Science.gov (United States)

    Marasco, W A; Fantone, J C; Ward, P A

    1982-12-01

    The chemotactic N-formylated oligopeptides are potent spasmogenic agents for guinea pig ileum. Structure-activity studies with various N-formylated peptides suggest the presence of a specific receptor that resembles in specificity the formyl peptide receptor on leukocytes. A competitive antagonist of the formyl peptide receptor on leukocytes also inhibits formyl peptide-induced ileum contraction, whereas the antihistamine diphenhydramine is without effect. The contractile response caused by the synthetic N-formylated peptides differs from those induced by acetylcholine, histamine, and substance P. In particular, a latent period after treatment with the N-formyl peptides is seen before the onset of the response, and a sustained contractile response is not maintained. In addition, tachyphylaxis does occur, but complete recovery of activity is seen after a 20- to 30-min rest period. These observations suggest broad biological roles of prokaryotic signal peptides from bacteria as acute inflammatory mediators.

  3. Critical role of a conserved transmembrane lysine in substrate recognition by the proton-coupled oligopeptide transporter YjdL.

    Science.gov (United States)

    Jensen, Johanne M; Aduri, Nanda G; Prabhala, Bala K; Jahnsen, Rasmus; Franzyk, Henrik; Mirza, Osman

    2014-10-01

    Proton-coupled oligopeptide transporters (POTs) utilize an electrochemical proton gradient to accumulate peptides in the cytoplasm. Changing the highly conserved active-site Lys117 in the Escherichia coli POT YjdL to glutamine resulted in loss of ligand affinity as well as inability to distinguish between a dipeptide ligand and the corresponding dipeptide amide. The radically changed pH(Bulk) profiles of Lys117Gln and Lys117Arg mutants indicate an important role of Lys117 in facilitating protonation of the transporter; a notion that is supported by the close proximity of Lys117 to the conserved ExxERFxYY POT motif previously shown to be involved in proton translocation. These results point toward a novel dual role of Lys117 in direct or indirect interaction with both proton and peptide.

  4. The oligopeptide transport system is essential for the development of natural competence in Streptococcus thermophilus strain LMD-9.

    Science.gov (United States)

    Gardan, Rozenn; Besset, Colette; Guillot, Alain; Gitton, Christophe; Monnet, Véronique

    2009-07-01

    In gram-positive bacteria, oligopeptide transport systems, called Opp or Ami, play a role in nutrition but are also involved in the internalization of signaling peptides that take part in the functioning of quorum-sensing pathways. Our objective was to reveal functions that are controlled by Ami via quorum-sensing mechanisms in Streptococcus thermophilus, a nonpathogenic bacterium widely used in dairy technology in association with other bacteria. Using a label-free proteomic approach combining one-dimensional electrophoresis with liquid chromatography-tandem mass spectrometry analysis, we compared the proteome of the S. thermophilus LMD-9 to that of a mutant deleted for the subunits C, D, and E of the ami operon. Both strains were grown in a chemically defined medium (CDM) without peptides. We focused our attention on proteins that were no more detected in the ami deletion mutant. In addition to the three subunits of the Ami transporter, 17 proteins fulfilled this criterion and, among them, 7 were similar to proteins that have been identified as essential for transformation in S. pneumoniae. These results led us to find a condition of growth, the early exponential state in CDM, that allows natural transformation in S. thermophilus LMD-9 to turn on spontaneously. Cells were not competent in M17 rich medium. Furthermore, we demonstrated that the Ami transporter controls the triggering of the competence state through the control of the transcription of comX, itself controlling the transcription of late competence genes. We also showed that one of the two oligopeptide-binding proteins of strain LMD-9 plays the predominant role in the control of competence.

  5. Anti-Fatigue Effects of Small Molecule Oligopeptides Isolated from Panax ginseng C. A. Meyer in Mice

    Science.gov (United States)

    Bao, Lei; Cai, Xiaxia; Wang, Junbo; Zhang, Yuan; Sun, Bin; Li, Yong

    2016-01-01

    Panax ginseng C. A. Meyer (ginseng) is an edible and medicinal Chinese herb, which is often used in Asian countries for physical fitness. Ginseng is reported to have a wide range of biological activity and pharmaceutical properties. There were more studies on ginsenosides and polysaccharides, but fewer studies on ginseng oligopeptides (GOP), which are small molecule oligopeptides isolated from ginseng. The present study was designed to evaluate the anti-fatigue effects of GOP in mice and explore the possible underlying mechanism. Mice were randomly divided into four experimental sets for the detection of different indicators. Each set of mice were then divided into four groups. The control group was administered distilled water, and three GOP intervention groups were administered 125, 250, and 500 mg/kg of body weight, respectively, of GOP by gavage each day. After 30 days of GOP treatment, it was observed that GOP could significantly increase the forced swimming time, enhance lactate dehydrogenase (LDH) activity and hepatic glycogen levels, and retard the accumulation of serum urea nitrogen (SUN) and blood lactic acid (BLA) in mice. GOP also markedly ameliorated fatigue-induced alterations of inoxidative stress biomarkers and antioxidant enzymes. Notably, GOP increased the mRNA expression of mitochondrial biogenesis factors and mitochondrial DNA content in skeletal muscles of mice. These results suggest that GOP possess anti-fatigue effects, which may be attributed to the inhibition of oxidative stress and the improvement of mitochondrial function in skeletal muscles. GOP could be a novel natural agent for relieving exercise fatigue. PMID:27983571

  6. Anti-Fatigue Effects of Small Molecule Oligopeptides Isolated from Panax ginseng C. A. Meyer in Mice

    Directory of Open Access Journals (Sweden)

    Lei Bao

    2016-12-01

    Full Text Available Panax ginseng C. A. Meyer (ginseng is an edible and medicinal Chinese herb, which is often used in Asian countries for physical fitness. Ginseng is reported to have a wide range of biological activity and pharmaceutical properties. There were more studies on ginsenosides and polysaccharides, but fewer studies on ginseng oligopeptides (GOP, which are small molecule oligopeptides isolated from ginseng. The present study was designed to evaluate the anti-fatigue effects of GOP in mice and explore the possible underlying mechanism. Mice were randomly divided into four experimental sets for the detection of different indicators. Each set of mice were then divided into four groups. The control group was administered distilled water, and three GOP intervention groups were administered 125, 250, and 500 mg/kg of body weight, respectively, of GOP by gavage each day. After 30 days of GOP treatment, it was observed that GOP could significantly increase the forced swimming time, enhance lactate dehydrogenase (LDH activity and hepatic glycogen levels, and retard the accumulation of serum urea nitrogen (SUN and blood lactic acid (BLA in mice. GOP also markedly ameliorated fatigue-induced alterations of inoxidative stress biomarkers and antioxidant enzymes. Notably, GOP increased the mRNA expression of mitochondrial biogenesis factors and mitochondrial DNA content in skeletal muscles of mice. These results suggest that GOP possess anti-fatigue effects, which may be attributed to the inhibition of oxidative stress and the improvement of mitochondrial function in skeletal muscles. GOP could be a novel natural agent for relieving exercise fatigue.

  7. Multiple sites of the cleavage of 21- and 25-mer encephalytogenic oligopeptides corresponding to human myelin basic protein (MBP by specific anti-MBP antibodies from patients with systemic lupus erythematosus.

    Directory of Open Access Journals (Sweden)

    Anna M Timofeeva

    Full Text Available IgGs from patients with multiple sclerosis and systemic lupus erythematosus (SLE purified on MBP-Sepharose in contrast to canonical proteases hydrolyze effectively only myelin basic protein (MBP, but not many other tested proteins. Here we have shown for the first time that anti-MBP SLE IgGs hydrolyze nonspecific tri- and tetrapeptides with an extreme low efficiency and cannot effectively hydrolyze longer 20-mer nonspecific oligopeptides corresponding to antigenic determinants (AGDs of HIV-1 integrase. At the same time, anti-MBP SLE IgGs efficiently hydrolyze oligopeptides corresponding to AGDs of MBP. All sites of IgG-mediated proteolysis of 21-and 25-mer encephalytogenic oligopeptides corresponding to two known AGDs of MBP were found by a combination of reverse-phase chromatography, TLC, and MALDI spectrometry. Several clustered major, moderate, and minor sites of cleavage were revealed in the case of 21- and 25-mer oligopeptides. The active sites of anti-MBP abzymes are localised on their light chains, while heavy chains are responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of abzymes provide high affinity to MBP and specificity of this protein hydrolysis. The affinity of anti-MBP abzymes for intact MBP is approximately 1000-fold higher than for the oligopeptides. The data suggest that all oligopeptides interact mainly with the light chains of different monoclonal abzymes of total pool of IgGs, which possesses a lower affinity for substrates, and therefore, depending on the oligopeptide sequences, their hydrolysis may be less specific than globular protein and can occur in several sites.

  8. Influence of Free Amino Acids, Oligopeptides, and Polypeptides on the Formation of Pyrazines in Maillard Model Systems.

    Science.gov (United States)

    Scalone, Gustavo Luis Leonardo; Cucu, Tatiana; De Kimpe, Norbert; De Meulenaer, Bruno

    2015-06-10

    Pyrazines are specific Maillard reaction compounds known to contribute to the unique aroma of many products. Most studies concerning the generation of pyrazines in the Maillard reaction have focused on amino acids, while little information is available on the impact of peptides and proteins. The present study investigated the generation of pyrazines in model systems containing whey protein, hydrolyzed whey protein, amino acids, and glucose. The impact of thermal conditions, ratio of reagents, and water activity (a(w)) on pyrazine formation was measured by headspace solid-phase microextraction with gas chromatography/mass spectrometry (HS-SPME-GC/MS. The presence of oligopeptides from hydrolyzed whey protein contributed significantly to an increased amount of pyrazines, while in contrast free amino acids generated during protein hydrolysis contributed to a lesser extent. The generation of pyrazines was enhanced at low a(w) (0.33) and high temperatures (>120 °C). This study showed that the role of peptides in the generation of pyrazines in Maillard reaction systems has been dramatically underestimated.

  9. Fluorescent sensors for activity and regulation of the nitrate transceptor CHL1/NRT1.1 and oligopeptide transporters.

    Science.gov (United States)

    Ho, Cheng-Hsun; Frommer, Wolf B

    2014-03-12

    To monitor nitrate and peptide transport activity in vivo, we converted the dual-affinity nitrate transceptor CHL1/NRT1.1/NPF6.3 and four related oligopeptide transporters PTR1, 2, 4, and 5 into fluorescence activity sensors (NiTrac1, PepTrac). Substrate addition to yeast expressing transporter fusions with yellow fluorescent protein and mCerulean triggered substrate-dependent donor quenching or resonance energy transfer. Fluorescence changes were nitrate/peptide-specific, respectively. Like CHL1, NiTrac1 had biphasic kinetics. Mutation of T101A eliminated high-affinity transport and blocked the fluorescence response to low nitrate. NiTrac was used for characterizing side chains considered important for substrate interaction, proton coupling, and regulation. We observed a striking correlation between transport activity and sensor output. Coexpression of NiTrac with known calcineurin-like proteins (CBL1, 9; CIPK23) and candidates identified in an interactome screen (CBL1, KT2, WNKinase 8) blocked NiTrac1 responses, demonstrating the suitability for in vivo analysis of activity and regulation. The new technology is applicable in plant and medical research. DOI: http://dx.doi.org/10.7554/eLife.01917.001.

  10. CgOpt1, a putative oligopeptide transporter from Colletotrichum gloeosporioides that is involved in responses to auxin and pathogenicity

    Directory of Open Access Journals (Sweden)

    Maor Rudy

    2009-08-01

    Full Text Available Abstract Background The fungus Colletotrichum gloeosporioides f. sp. aeschynomene produces high levels of indole-3-acetic acid (IAA in axenic cultures and during plant infection. We generated a suppression subtractive hybridization library enriched for IAA-induced genes and identified a clone, which was highly expressed in IAA-containing medium. Results The corresponding gene showed similarity to oligopeptide transporters of the OPT family and was therefore named CgOPT1. Expression of CgOPT1 in mycelia was low, and was enhanced by external application of IAA. cgopt1-silenced mutants produced less spores, had reduced pigmentation, and were less pathogenic to plants than the wild-type strain. IAA enhanced spore formation and caused changes in colony morphology in the wild-type strain, but had no effect on spore formation or colony morphology of the cgopt1-silenced mutants. Conclusion Our results show that IAA induces developmental changes in C. gloeosporioides. These changes are blocked in cgopt1-silenced mutants, suggesting that this protein is involved in regulation of fungal response to IAA. CgOPT1 is also necessary for full virulence, but it is unclear whether this phenotype is related to auxin.

  11. Stability, metabolism and transport of D-Asp(OBzl)-Ala--a model prodrug with affinity for the oligopeptide transporter

    DEFF Research Database (Denmark)

    Steffansen, B; Lepist, E I; Taub, M E

    1999-01-01

    The model prodrug D-Asp(OBzl)-Ala has previously been shown to have affinity and to be transported by the oligopeptide transporter PepT1 expressed in Caco-2 cells. The main objective of the present study was to investigate the aqueous stability of D-Asp(OBzl)-Ala and its in vitro metabolism...... in different gastrointestinal media arising from rats and humans, as well as in human plasma. The second major aim of the study was to evaluate our previous study in Caco-2 cell culture, by determining the effective intestinal permeability (Peff) of D-Asp(OBzl)-Ala in situ using the single-pass rat perfusion...... model. The aqueous stability studies show water, general buffer, as well as specific acid and base catalysis of D-Asp(OBzl)-Ala. The degradation of the model prodrug was independent of ionic strength. The half-lives in rat jejunal fluid and homogenate were >3 h. In human gastric and intestinal fluids...

  12. P2X7 receptor activation impairs exogenous MHC class I oligopeptides presentation in antigen presenting cells.

    Directory of Open Access Journals (Sweden)

    Alberto Baroja-Mazo

    Full Text Available Major histocompatibility complex class I (MHC I on antigen presenting cells (APCs is a potent molecule to activate CD8(+ T cells and initiate immunity. P2X7 receptors (P2X7Rs are present on the plasma membrane of APCs to sense the extracellular danger signal adenosine-5'-triphosphate (ATP. P2X7R activates the inflammasome and the release of IL-1β in macrophages and other immune cells to initiate the inflammatory response. Here we show that P2X7R stimulation by ATP in APCs decreased the amount of MHC I at the plasma membrane. Specific antagonism or genetic ablation of P2X7R inhibited the effects of ATP on levels of cellular MHC I. Furthermore, P2X7R stimulation was able to inhibit activation of CD8(+ T cells via specific MHC I-oligopeptide complexes. Our study suggests that P2X7R activation on APCs is a novel inhibitor of adaptive CD8(+ T cell immunity.

  13. Calculation of Relative Binding Free Energy in the Water-Filled Active Site of Oligopeptide-Binding Protein A

    Directory of Open Access Journals (Sweden)

    Manuela Maurer

    2016-04-01

    Full Text Available The periplasmic oligopeptide binding protein A (OppA represents a well-known example of water-mediated protein-ligand interactions. Here, we perform free-energy calculations for three different ligands binding to OppA, using a thermodynamic integration approach. The tripeptide ligands share a high structural similarity (all have the sequence KXK, but their experimentally-determined binding free energies differ remarkably. Thermodynamic cycles were constructed for the ligands, and simulations conducted in the bound and (freely solvated unbound states. In the unbound state, it was observed that the difference in conformational freedom between alanine and glycine leads to a surprisingly slow convergence, despite their chemical similarity. This could be overcome by increasing the softness parameter during alchemical transformations. Discrepancies remained in the bound state however, when comparing independent simulations of the three ligands. These difficulties could be traced to a slow relaxation of the water network within the active site. Fluctuations in the number of water molecules residing in the binding cavity occur mostly on a timescale larger than the simulation time along the alchemical path. After extensive simulations, relative binding free energies that were converged to within thermal noise could be obtained, which agree well with available experimental data.

  14. Antioxidant Effect of a Marine Oligopeptide Preparation from Chum Salmon (Oncorhynchus keta by Enzymatic Hydrolysis in Radiation Injured Mice

    Directory of Open Access Journals (Sweden)

    Zhigang Liu

    2011-11-01

    Full Text Available Marine oligopeptide preparation (MOP obtained from Chum Salmon (Oncorhynchus keta by the method of enzymatic hydrolysis, has been found to possess a radioprotective property through stimulation of the radiation-induced immunosuppression. The current study aimed to further investigate the free radicals scavenging and antioxidant effects of MOP in radiation injured mice. Female ICR mice (6–8 weeks old were randomly divided into 5 groups, i.e., blank control, irradiation control and MOP (0.225, 0.450 and 1.350 g/kg body weight plus an irradiation-treated group. The result revealed that MOP significantly increased the white blood cell counts after irradiation, and lessened the radiation-induced oxidative damage. These effects may be caused by augmentation of the activities of antioxidant enzymes, such as SOD and GSH-Px, reduction of the lipid peroxidation (MDA level in liver, and protection against radiation-induced apoptosis. Therefore, we propose that MOP be used as an ideal antioxidant to alleviate radiation-induced oxidation damage in cancer patients.

  15. Classification of functional oligopeptides and preparation of enzymatic hydrolysis%功能性短肽的分类及其酶解制备方法

    Institute of Scientific and Technical Information of China (English)

    李昊剑; 杨良嵘; 魏雪团; 邢慧芳; 刘会洲

    2012-01-01

    In recent years,the attention focused on nutritional,functional, physiological activities and applied of the functional oligopeptides derived from proteins was increasing. It had a wide variety of sources, functional oligopeptides could be obtained by appropriate enzymatic hydrolysis, with lowering blood pressure, improving immunity, promoting digestion, antioxidant, neural regulation, anti- bacterial and other biological activity.This paper reviewed the sources and classification,discussed the characteristics of oligopeptides in plant protein source and animal protein source, analyzed enzymatic hydrolysis to prepare peptides by selecting the protease and optimization of enzymatic hydrolysis conditions, it might provide the basis for its depth research and development.%近年来,蛋白质中功能性短肽的营养、功能性、生理活性和其应用价值越来越引起人们的关注。功能性短肽来源广泛,通过合适的蛋白酶酶解得到的功能性短肽,具有诸如降血压、提高免疫力、促进消化、抗氧化、神经调节和抗茵等生物活性。本文综述了功能性短肽的来源和分类,讨论了植物蛋白源短肽和动物蛋白源短肽的特点,通过选择蛋白酶和优化酶解条件分析了酶解制备短肽的方法,为开发和深入研究功能性短肽产品提供理论依据。

  16. Probing the putative active site of YjdL: an unusual proton-coupled oligopeptide transporter from E. coli.

    Directory of Open Access Journals (Sweden)

    Johanne Mørch Jensen

    Full Text Available YjdL from E. coli is an unusual proton-coupled oligopeptide transporter (POT. Unlike prototypical POTs, dipeptides are preferred over tripeptides, in particular dipeptides with a positively charged C-terminal residue. To further understand this difference in peptide specificity, the sequences of YjdL and YdgR, a prototypical E. coli POT, were compared in light of the crystal structure of a POT from Shewanella oneidensis. Several residues found in the putative active site were mutated and the activities of the mutated variants were assessed in terms of substrate uptake assays, and changes in specificity in terms of uptake inhibition. Most strikingly, changing the YjdL specific Asp392 to the conserved Ser in YjdL obliterated the preference for a positively charged C-terminal residue. Based on this unique finding and previously published results indicating that the dipeptide N-terminus may interact with Glu388, a preliminary orientation model of a dipeptide in the YjdL cavity is presented. Single site mutations of particularly Ala281 and Trp278 support the presented orientation. A dipeptide bound in the cavity of YjdL appears to be oriented such that the N-terminal side chain protrudes into a sub pocket that opens towards the extracellular space. The C-terminal side chain faces in the opposite direction into a sub pocket that faces the cytoplasm. These data indicated a stabilizing effect on a bulky N-terminal residue by an Ala281Phe variant and on the dipeptide backbone by Trp278. In the presented orientation model, Tyr25 and Tyr58 both appear to be in proximity of the dipeptide backbone while Lys117 appears to be in proximity of the peptide C-terminus. Mutational studies of these conserved residues highlight their functional importance.

  17. Fabrication of Thermo-Responsive Molecular Layers from Self-Assembling Elastin-Like Oligopeptides Containing Cell-Binding Domain for Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Tomoyuki Koga

    2015-01-01

    Full Text Available Novel thermo-responsive elastin-like oligopeptides containing cell-binding epitope (Arg-Gly-Asp-Ser sequence; arginine-glycine-aspartic acid-serine (RGDS-elastin-like peptides (ELP and RGDS-deg-ELP; were newly prepared as building blocks of self-assembled molecular layer for artificial extra cellular matrix. A detailed analysis of the conformation of the oligo(ELPs in water and their self-assembling behavior onto hydrophobic surfaces were performed by using circular dichroism, Fourier transform infrared spectroscopy (FTIR, atomic force microscopy and water contact angle measurements. The experimental results revealed that both oligo(ELPs self-assembled onto hydrophobic surfaces and formed molecular layers based on their thermo-responsive conformational change from hydrous random coil to dehydrated β-turn structure. Effective cell adhesion and spreading behaviors were observed on these self-assembled oligo(ELP layers. In addition, attached cells were found to be recovered successfully as a cell-sheet by temperature-induced disassembly of oligo(ELP layer. This achievement provides an important insight to construct novel oligopeptide-based nano-surfaces for the design of smart artificial extra-cellular matrix.

  18. Sulfonate group-modified FePtCu nanoparticles as a selective probe for LDI-MS analysis of oligopeptides from a peptide mixture and human serum proteins.

    Science.gov (United States)

    Kawasaki, Hideya; Akira, Tarui; Watanabe, Takehiro; Nozaki, Kazuyoshi; Yonezawa, Tetsu; Arakawa, Ryuichi

    2009-11-01

    Bare FePtCu nanoparticles (NPs) are first prepared for laser desorption/ionization mass spectroscopy (LDI-MS) analysis as affinity probes to selectively trap oppositely charged analytes from a sample solution. Our present results demonstrate bare FePtCu NPs to be a potentially useful matrix for surface-assisted laser desorption/ionization mass spectroscopy (SALDI-MS), for the analysis of small proteins and peptides. The upper detectable mass range of peptides was approximately 5 kDa, and the detection limit for peptides approximately 5 fmol. Sulfonate group-modified FePtCu nanoparticles (FePtCu-SO(3)(-) NPs), with ionization being independent of the solution pH, can interact with a positively charged analyte, and the analyte-bound NPs can be separated from the reaction supernatant by centrifugation or an external magnetic field. An oligopeptide, Gly-Gly-Tyr-Arg (GGYR) from an oligopeptide mixture containing Asp-Asp-Asp-Asp (DDDD), Gly-Gly-Gly-Gly (GGGG) and GGYR, was detected using SALDI-MS with FePtCu-SO(3)(-) NPs employing electrostatic interaction. Furthermore, FePtCu-SO(3)(-) NPs can detect lysozyme (Lyz) in human serum through the electrostatic attraction between positively charged Lyz and FePtCu-SO(3)(-) NPs at pH 8, while detection of negatively charged albumin in human serum is not possible.

  19. A major Sm epitope anchored to sequential oligopeptide carriers is a suitable antigenic substrate to detect anti-Sm antibodies.

    Science.gov (United States)

    Petrovas, C J; Vlachoyiannopoulos, P G; Tzioufas, A G; Alexopoulos, C; Tsikaris, V; Sakarellos-Daitsiotis, M; Sakarellos, C; Moutsopoulos, H M

    1998-11-01

    A sensitive, highly reproducible, solid-phase enzyme immunoassay (ELISA), was developed in order to investigate whether the synthetic heptapeptide PPGMRPP-a major epitope of the Sm autoantigen-anchored in five copies to a sequential oligopeptide carrier (SOC), [(PPGMRPP)5-SOC5] is a suitable antigenic substrate to identify anti-Sm/antibodies. Sera with different autoantibody specificities [45 anti-Sm, 40 anti-U1RNP, 40 anti-Ro (SSA)/La(SSB) positive, 21 Antinuclear antibody positive, but negative for antibodies to extractable nuclear antigens (ANA + /ENA - ) and 75 normal human sera, ANA negative] and 75 sera from patients with rheumatoid arthritis (RA) were tested for anti-(PPGMRPP)5-(SOC)5 reactivity in order to evaluate the specificity and sensitivity of the method to detect anti-Sm antibodies. RNA immunoprecipitation assays for the detection of anti-Sm and anti-U1RNP antibodies and counter immunoelectrophoresis (CIE) for the detection of anti-Ro(SSA) and anti-La(SSB) antibodies were used as reference techniques. The sensitivity of the method was 98% and the specificity was 68% for the determination of anti-Sm antibodies, while for the determination of anti-Sm and/or anti-U1RNP reactivity (antibodies to snRNPs) the corresponding values were 82% and 86%, respectively. In a comparison of the above assay with an ELISA, using Sm/U1RNP purified complex as immobilized antigen it was shown that the sensitivity of the anti-Sm/U1RNP ELISA in detecting anti-snRNPs was 74%; in addition sera with anti-Sm antibodies gave higher binding in the anti-(PPGMRPP)5-(SOC)5 ELISA compared with anti-Sm/U1RNP ELISA. Intra- and inter-assay precision was measured on four sera with reactivities extending into a wide range of absorbance values showed that the intra-assay coefficient of variation (CV%) ranged from 2.7 to 6 and the inter-assay CV% ranged from 9 to 14.5. These results indicate that the PPGMRPP peptide anchored to a pentameric SOC as a carrier is a suitable antigen for

  20. Cheese peptidomics: a detailed study on the evolution of the oligopeptide fraction in Parmigiano-Reggiano cheese from curd to 24 months of aging.

    Science.gov (United States)

    Sforza, S; Cavatorta, V; Lambertini, F; Galaverna, G; Dossena, A; Marchelli, R

    2012-07-01

    In this work, we performed a detailed evaluation of the evolution of the oligopeptide fractions in samples of Parmigiano-Reggiano cheese from the curd up to 24 mo of aging. The samples were taken from wheels produced the same day, in the same factory, from the same milk, during the same caseification process, thus simplifying the natural variability of a whey-based starter fermentation. This unique and homogeneous sampling plan, never reported before in the literature, provided a detailed study of the peptides produced by enzymatic events during Parmigiano-Reggiano aging. Given the large dimensions of the 35-kg wheels of Parmigiano-Reggiano, samples were taken from both the internal and external parts of the cheese, to evidence eventual differences in the oligopeptide composition of the different parts. Fifty-seven peptides were considered, being among the most abundant during at least one of the periods of ripening considered, and their semiquantification indicated that the peptide fraction of Parmigiano-Reggiano cheese constantly evolves during the aging period. Five trends in its evolution were outlined, which could be clearly correlated to the enzymatic activities present in the cheese, making it possible to discriminate cheeses according to their aging time. Several known bioactive peptides were also found to be present in Parmigiano-Reggiano cheese samples, and for the first time, the age at which they are most abundant has been identified. Aged cheeses have been shown to be dominated by nonproteolytic aminoacyl derivatives, a new class of peptide-like molecules recently reported. Finally, the changing peptide pattern may be related to the changing enzymatic activities occurring inside the cheeses during the aging period, which, in turn, are also related to the microbiological composition.

  1. Protein hydrolysate-induced cholecystokinin secretion from enteroendocrine cells is indirectly mediated by the intestinal oligopeptide transporter PepT1.

    Science.gov (United States)

    Liou, Alice P; Chavez, Diana I; Espero, Elvis; Hao, Shuzhen; Wank, Stephen A; Raybould, Helen E

    2011-05-01

    Dietary protein is a major stimulant for cholecystokinin (CCK) secretion by the intestinal I cell, however, the mechanism by which protein is detected is unknown. Indirect functional evidence suggests that PepT1 may play a role in CCK-mediated changes in gastric motor function. However, it is unclear whether this oligopeptide transporter directly or indirectly activates the I cell. Using both the CCK-expressing enteroendocrine STC-1 cell and acutely isolated native I cells from CCK-enhanced green fluorescent protein (eGFP) mice, we aimed to determine whether PepT1 directly activates the enteroendocrine cell to elicit CCK secretion in response to oligopeptides. Both STC-1 cells and isolated CCK-eGFP cells expressed PepT1 transcripts. STC-1 cells were activated, as measured by ERK(1/2) phosphorylation, by both peptone and the PepT1 substrate Cefaclor; however, the PepT1 inhibitor 4-aminomethyl benzoic acid (AMBA) had no effect on STC-1 cell activity. The PepT1-transportable substrate glycyl-sarcosine dose-dependently decreased gastric motility in anesthetized rats but had no affect on activation of STC-1 cells or on CCK secretion by CCK-eGFP cells. CCK secretion was significantly increased in response to peptone but not to Cefaclor, cephalexin, or Phe-Ala in CCK-eGFP cells. Taken together, the data suggest that PepT1 does not directly mediate CCK secretion in response to PepT1 specific substrates. PepT1, instead, may have an indirect role in protein sensing in the intestine.

  2. Application of HACCP System in Processing of Composite Beverage of Collagen Oligopeptide and Fruit Juice%HACCP在胶原蛋白寡肽果味饮料加工中的应用

    Institute of Scientific and Technical Information of China (English)

    刘海; 张嘉强; 任鹏康; 赵云涛

    2011-01-01

    Hazard in processing of composite beverage of collagen oligopeptide and fruit juice was analyzed by HACCP theory and method, and the key control points were determined. Supervise procedures and correct measures were set up to monitor the critical control points, and then the table of HACCP plan had been established. The application of HACCP system effectively guaranteed the safety of beverage of collagen oligopeptide and fruit juice.%应用HACCP原理和方法对胶原蛋白寡肽果味饮料的生产过程中可能出现的危害进行分析,确定关键控制点(CCP).在关键控制点提出了监控、纠偏措施,并制定了HACCP计划表.HACCP体系的实施有效保证了胶原蛋白寡肽果味饮料的品质安全.

  3. 海参寡肽:免疫调节作用及机制研究%Sea cucumber oligopeptides: Immunomodulatory effects and its mechanism

    Institute of Scientific and Technical Information of China (English)

    何丽霞; 陈启贺; 刘睿; 任金威; 孙彬; 李林; 徐腾; 王军波; 张召锋

    2016-01-01

    To investigate the immunomodulatory effects of sea cucumber oligopeptides and their mechanisms,a total of 250 specific pathogen free (SPF) female BALB/c mice are randomly divided into 5 groups:the control group,the whey protein group (with 0.30 g/kg of whey protein) and the groups with 0.15,0.30 and 0.60 g/kg of sea cucumber oligopeptides.The experiment takes 30 days.To determine the cell-mediated immunity,the humoral immunity,the macrophage phagocytosis and the natural killer (NK) cell activity in mice,the splenic lymphocyte proliferation and the delayed-type hypersensitivity assays (for the cell-mediated immunity),the IgM antibody response of spleen to sheep red blood cells (SRBC) and the serum hemolysin level assays (for the humoral immunity),the carbon clearance assay and the phagocytic capacity of peritoneal cavity phagocytes assays (for the macrophage phagocytosis) and the NK cell activity assays are performed.Furthermore,the flow cytometry is used to determine the T lymphocyte subsets.The results showed that the sea cucumber oligopeptides significantly increase the cell-mediated immunity,the humoral immunity,the macrophage phagocytosis and the NK cell activity in mice(P<0.05),and the effects are better than those of the whey protein.From the flow cytometry,it is observed that the sea cucumber oligopeptides significantly enhance the percentages of CD3+ and CD4+(P<0.05).In conclusion,the sea cucumber oligopeptides could increase the T lymphocyte cell number and the Th cell proportion to enhance the functions of the cell-mediated immunity,the humoral immunity,the macrophage phagocytosis and the NK cell activity,and furthermore to improve the immune functions in mice.%为研究海参寡肽对小鼠的免疫调节作用及机制,试验选取250只SPF级雌性BALB/c小鼠,随机分为5组:空白对照组,乳清蛋白组(0.30 g/kg),0.15、0.30、0.60 g/kg海参寡肽组.连续灌胃30 d后,通过测定ConA诱导的小鼠淋巴细胞转化实验、迟发型变

  4. Cell surface-engineered yeast displaying a histidine oligopeptide (hexa-His) has enhanced adsorption of and tolerance to heavy metal ions.

    Science.gov (United States)

    Kuroda, K; Shibasaki, S; Ueda, M; Tanaka, A

    2001-12-01

    A histidine oligopeptide (hexa-His) with the ability to chelate divalent heavy metal ions was displayed on the yeast cell surface for the purpose of enhanced adsorption of heavy metal ions. We genetically fused a hexa-His-encoding gene with the gene encoding the C-terminal half of alpha-agglutinin that includes a glycosylphosphatidylinositol anchor attachment signal sequence and attached the hexa-His peptide on the cell wall of Saccharomyces cerevisiae. This surface-engineered yeast adsorbed three to eight times more copper ions than the parent strain and was more resistant to copper (4 mM) than the parent (below 1 mM at pH 7.8). It was possible to recover about a half of the copper ions adsorbed by whole cells with EDTA treatment without disintegrating the cells. Thus, we succeeded in constructing a novel yeast cell with both tolerance to toxic contaminants and enhanced adsorption of metal ions onto the cell surface.

  5. The Combined Repetitive Oligopeptides of Clostridium difficile Toxin A Counteract Premature Cleavage of the Glucosyl-Transferase Domain by Stabilizing Protein Conformation

    Directory of Open Access Journals (Sweden)

    Alexandra Olling

    2014-07-01

    Full Text Available Toxin A (TcdA and B (TcdB from Clostridium difficile enter host cells by receptor-mediated endocytosis. A prerequisite for proper toxin action is the intracellular release of the glucosyltransferase domain by an inherent cysteine protease, which is allosterically activated by inositol hexaphosphate (IP6. We found that in in vitro assays, the C-terminally-truncated TcdA1–1065 was more efficient at IP6-induced cleavage compared with full-length TcdA. We hypothesized that the C-terminally-located combined repetitive oligopeptides (CROPs interact with the N-terminal part of the toxin, thereby preventing autoproteolysis. Glutathione-S-transferase (GST pull-down assays and microscale thermophoresis confirmed binding between the CROPs and the glucosyltransferase (TcdA1–542 or intermediate (TcdA1102–1847 domain of TcdA, respectively. This interaction between the N- and C-terminus was not found for TcdB. Functional assays revealed that TcdB was more susceptible to inactivation by extracellular IP6-induced cleavage. In vitro autoprocessing and inactivation of TcdA, however, significantly increased, either by acidification of the surrounding milieu or following exchange of its CROP domain by the homologous CROP domain of TcdB. Thus, TcdA CROPs contribute to the stabilization and protection of toxin conformation in addition to function as the main receptor binding domain.

  6. Effects of fasting and refeeding on gene expression of slc15a1a, a gene encoding an oligopeptide transporter (PepT1), in the intestine of Mozambique tilapia.

    Science.gov (United States)

    Orozco, Zenith Gaye A; Soma, Satoshi; Kaneko, Toyoji; Watanabe, Soichi

    2017-01-01

    The tissue distribution of slc15a1a, a gene that encodes an oligopeptide transporter, PepT1, and its response to fasting and refeeding were investigated in the intestinal epithelium of Mozambique tilapia for a better understanding of its role on nutrient absorption. The slc15a1a was predominantly expressed in the absorptive epithelia of the anterior part of the intestine, suggesting that digested oligopeptides are primarily absorbed in the anterior intestine. The response of slc15a1a to fasting was evaluated at 1, 2, 4, 7 and 14days after the last feeding. Fasting revealed a biphasic effect, where short-term fasting significantly upregulated slc15a1a expression and long-term fasting resulted in downregulation. The expression level continued to decrease and fell below the pre-fasted level from day 4 to 14. Proximal (the hepatic loop, HL) and distal parts (the proximal major coil, PMC) of the anterior intestine showed different magnitudes of responses to fasting; slc15a1a expression in the PMC showed greater upregulation and downregulation than that in the HL. Refeeding significantly stimulated slc15a1a expression at day 3, although the expression did not exceed the pre-fasted level. Observed responses of slc15a1a to fasting and refeeding suggest that the expression level of this gene can serve as a sensitive indicator of the changes that may occur in altering nutritional conditions. These findings contribute to a better understanding of the role of PepT1 in nutrition and of the complex mechanisms underlying the absorption of oligopeptides and amino acids in the intestine, and may lead to development of possible means to manipulate the absorption processes for the improvement of growth and other metabolic and physiological conditions in fish.

  7. Glutathione Utilization by Candida albicans Requires a Functional Glutathione Degradation (DUG) Pathway and OPT7, an Unusual Member of the Oligopeptide Transporter Family

    Science.gov (United States)

    Desai, Prashant Ramesh; Thakur, Anil; Ganguli, Dwaipayan; Paul, Sanjoy; Morschhäuser, Joachim; Bachhawat, Anand K.

    2011-01-01

    Candida albicans lacks the ability to survive within its mammalian host in the absence of endogenous glutathione biosynthesis. To examine the ability of this yeast to utilize exogenous glutathione, we exploited the organic sulfur auxotrophy of C. albicans met15Δ strains. We observed that glutathione is utilized efficiently by the alternative pathway of glutathione degradation (DUG pathway). The major oligopeptide transporters OPT1–OPT5 of C. albicans that were most similar to the known yeast glutathione transporters were not found to contribute to glutathione transport to any significant extent. A genomic library approach to identify the glutathione transporter of C. albicans yielded OPT7 as the primary glutathione transporter. Biochemical studies on OPT7 using radiolabeled GSH uptake revealed a Km of 205 μm, indicating that it was a high affinity glutathione transporter. OPT7 is unusual in several aspects. It is the most remote member to known yeast glutathione transporters, lacks the two highly conserved cysteines in the family that are known to be crucial in trafficking, and also has the ability to take up tripeptides. The transporter was regulated by sulfur sources in the medium. OPT7 orthologues were prevalent among many pathogenic yeasts and fungi and formed a distinct cluster quite remote from the Saccharomyces cerevisiae HGT1 glutathione transporter cluster. In vivo experiments using a systemic model of candidiasis failed to detect expression of OPT7 in vivo, and strains disrupted either in the degradation (dug3Δ) or transport (opt7Δ) of glutathione failed to show a defect in virulence. PMID:21994941

  8. Ginseng (Panax ginseng Meyer) oligopeptides regulate innate and adaptive immune responses in mice via increased macrophage phagocytosis capacity, NK cell activity and Th cells secretion.

    Science.gov (United States)

    He, Li-Xia; Ren, Jin-Wei; Liu, Rui; Chen, Qi-He; Zhao, Jian; Wu, Xin; Zhang, Zhao-Feng; Wang, Jun-Bo; Pettinato, Giuseppe; Li, Yong

    2017-09-06

    Traditionally used as a restorative medicine, ginseng (Panax ginseng Meyer) has been the most widely used and acclaimed herb in Chinese communities for thousands of years. To investigate the immune-modulating activity of ginseng oligopeptides (GOP), 420 healthy female BALB/c mice were intragastrically administered distilled water (control), whey protein (0.15 g per kg body weight (BW)), and GOP 0.0375, 0.075, 0.15, 0.3 and 0.6 g per kg BW for 30 days. Blood samples from mice were collected from the ophthalmic venous plexus and then sacrificed by cervical dislocation. Seven assays were conducted to determine the immunomodulatory effects of GOP on innate and adaptive immune responses, followed by flow cytometry to investigate spleen T lymphocyte sub-populations, multiplex sandwich immunoassays to investigate serum cytokine and immunoglobulin levels, and ELISA to investigate intestinally secreted immunoglobulin to study the mechanism of GOP affecting the immune system. Our results showed that GOP was able to enhance innate and adaptive immune responses in mice by improving cell-mediated and humoral immunity, macrophage phagocytosis capacity and NK cell activity. Notably, the use of GOP revealed a better immune-modulating activity compared to whey protein. We conclude that the immune-modulating activity might be due to the increased macrophage phagocytosis capacity and NK cell activity, and the enhancement of T and Th cells, as well as IL-2, IL-6 and IL-12 secretion and IgA, IgG1 and IgG2b production. These results indicate that GOP could be considered a good candidate that may improve immune functions if used as a dietary supplement, with a dosage that ranges from 0.3 to 0.6 g per kg BW.

  9. A redox-sensitive, oligopeptide-guided, self-assembling, and efficiency-enhanced (ROSE) system for functional delivery of microRNA therapeutics for treatment of hepatocellular carcinoma.

    Science.gov (United States)

    Hu, Qida; Wang, Kai; Sun, Xu; Li, Yang; Fu, Qihan; Liang, Tingbo; Tang, Guping

    2016-10-01

    Lack of efficient adjuvant therapy contributes to a high incidence of recurrence and metastasis of hepatocellular carcinoma (HCC). A novel therapeutic is required for adjuvant treatment of HCC. We developed a polymer-based nanosystem (ROSE) for functional gene therapy by synthesizing a supramolecular complex self-assembled from polycations and functional adamantyl modules. The ROSE system condensing tumor suppressor microRNA-34a (miR-34a) therapeutics becomes ROSE/miR-34a nanoparticles that could facilitate gene transfection in HCC cells with satisfied stability and efficiency, possibly due to proton sponge effect by polycations, PEGlyation protection, and controlled release by breakdown of disulfide bonds. Meanwhile, modification with a targeting oligopeptide SP94 in ROSE/miR-34a enables approximately higher affinity for LM3 HCC cells than hepatocytes in vitro and greater HCC specificity in vivo. Furthermore, ROSE/miR-34a nanoparticles significantly inhibits HCC cell proliferation and in vivo tumor growth, representing a notable effect improvement over conventional gene delivery strategies. ROSE/miR-34a, featuring redox-responsiveness, oligopeptide-guided specificity, self-assembly, and enhanced transfection, is therefore a potential therapeutic agent in future adjuvant therapy for HCC treatment.

  10. Secondary structure of synthetic oligopeptides

    CERN Document Server

    Martinez-Insua, M

    2000-01-01

    The secondary structure of three hydrophobic peptides P2, PRMo and P4 was studied by a combination of Circular Dichroism (CD), Fourier Transform InfraRed (FTIR) and Photoinduced Electron Transfer (PET). These peptides were fluorescence labelled in the central part of the backbone and contained two modified glutamic acid residues (relative positions i, i+4): one conjugated with the fluorescence methoxynapththalene electron donor (DON) and the other with the piperidone electron acceptor (ACC). The three peptides were synthesised to study the length dependence of the switch between alpha-helix and the 3 sub 1 sub 0 -helix conformations, previously observed for peptide PRM1 (Hungerford et al., Angew. Chem., Int. Ed. Engl., 1996, 35, 326-329). The CD and FTIR data indicated that peptides P2, PRMo and P4 adopt alpha-helical conformation in organic media in the temperature range studied and no conformational switch was detected. Furthermore, a mathematical correlation was observed in the PET data, questioning the ag...

  11. Effect of a strong interfacial electric field on the orientation of the dipole moment of thiolated aib-oligopeptides tethered to mercury on either the N- or C-terminus.

    Science.gov (United States)

    Becucci, Lucia; Guryanov, Ivan; Maran, Flavio; Guidelli, Rolando

    2010-05-05

    Four oligopeptides consisting of a sequence of alpha-aminoisobutyric acid (Aib) residues, thiolated at either the N- or C-terminus by means of a -(CH(2))(2)-SH anchor, were self-assembled on mercury, which is a substrate known to impart a high fluidity to self-assembled monolayers (SAMs). The surface dipole potential of these peptide SAMs was estimated in 0.1 M KCl aqueous solution at a negatively charged electrode, where the interfacial electric field is directed toward the metal. To the best of our knowledge, this is the first estimate of the surface dipole potential of peptide SAMs in aqueous solution. The procedure adopted consisted in measuring the charge involved in the gradual expansion of a peptide-coated mercury drop and then combining the resulting information with an estimate of the charge density experienced by diffuse layer ions. The dipole moment of the tethered thiolated peptides was found to be directed toward the metal, independent of whether they were thiolated at the C- or N-terminus. This result was confirmed by the effect of these SAMs on the kinetics and thermodynamics of the Eu(III)/Eu(II) redox couple. The combined outcome of these studies indicates that a strong interfacial electric field orients the dipole moment of peptide SAMs tethered to mercury, even against their "natural" dipole moment.

  12. Study on the effect of collagen oligopeptid on synthesis of hyaluronic acid and collagen%一种胶原蛋白寡肽促进皮肤胶原蛋白与透明质酸合成的研究

    Institute of Scientific and Technical Information of China (English)

    宋芹; 陈封政; 颜军; 郭晓强; 姚倩; 黄露萍; 苟小军

    2013-01-01

    研究罗非鱼鱼鳞胶原蛋白寡肽对人皮肤成纤维细胞透明质酸分泌以及对小鼠真皮中胶原蛋白合成的影响.采用WST-8法测定了该胶原蛋白寡肽对人皮肤成纤维细胞增殖的影响,ELISA法检测了胶原蛋白寡肽对人皮肤成纤维细胞透明质酸分泌的影响;以D-半乳糖衰老模型小鼠为实验对象,考察胶原蛋白寡肽对小鼠真皮中胶原蛋白合成的影响.结果表明,胶原蛋白寡肽能促进人皮肤成纤维细胞增殖(p<0.01),能促进人皮肤成纤维细胞透明质酸和小鼠真皮中胶原蛋白的合成(p<0.05)说明该胶原蛋白寡肽能有效促进透明质酸和胶原蛋白的合成.%To study the effect of collagen oligopeptid on cell proliferation and synthesis of hyaluronic acid in cultured human skin fibroblast cell, and the effect of collagen oligopeptid on type I collagen secretion of mouse skin. Proliferation viability was detected by WST-8 assay.ELISA was used to detect the secretion of hyaluronic acid of cultured human skin fibroblast Collagen oligopeptid were given to mice and D-galactosewere injected into mice for six weeks.Then,determine the type I collagen secretion of mouse skin.The results of the experiment showed that the collagen oligopeptidcould stimulate the human skin fibroblast proliferation ( p < 0.01 ), and increase the hyaluronic acid and type I collagen secretion(p<0.05).Collagen oligopeptid from tilapia fish scale could stimulate human skin fibroblast proliferation,and meanwhile increase type I collagen and hyaluronic acid secretion.

  13. Mecanismos de absorção de aminoácidos e oligopeptídios. Controle e implicações na dietoterapia humana Mechanisms of amino acids and oligopeptides absorption in humans

    Directory of Open Access Journals (Sweden)

    Patrícia Baston FRENHANI

    1999-12-01

    bactérias para produção de energia e aminoácidos, respectivamente.The mechanisms involved in the absorption of amino acids and oligopeptides are reviewed regarding their implications in human feedings. Brush border and basolateral membranes are crossed by amino acids and di-tripeptides by passive (facilitated or simple diffusion or active (Na+ or H+ co-transporters pathways. Active Na+-dependent system accurs mainly at brush border and simple diffusion at basolateral, both membranes have the passive facilitated transport. Free-amino acids use either passive or active transport systems whereas di-tripeptides do mainly active (H+ co-transporter. Brush border have distinctive transport system for amino acids and di-tripeptides. The former occurs mainly by active Na+ dependently whereas the later is active H+-dependent with little affinity for tetra or higher peptides. Free amino acids are transported at different speed by saturable, competitive carriers with specificity for basic, acidic or neutral amino acids. Di and tripeptides have at least two carriers both electrogenic and H+-dependent. The basolateral membrane transport of amino acids is mostly by facilitated diffusion while for di-tripeptides it is an active anion exchange associated process. The main regulation of amino acids and di-tripeptide transport is the presence o substrate at the mucosal membrane with higher the substrate higher the absorption. Di and tripeptides are more efficiently absorbed than free amino acids which in turns are better absorbed than oligopeptides. So di-tripeptides result in better N-retention and is particularly useful in cases of lower intestinal absorption capacity. The non-absorbed peptides are digested and fermented by colonic bacteria resulting short-chain fatty acids, dicarboxylic acids, phenolic compounds and ammonia. Short-chain fatty acid provides energy for colonocytes and bacteria and the ammonia not fixed by bacteria returns to the liver for ureagenesis.

  14. Model prodrugs for the intestinal oligopeptide transporter

    DEFF Research Database (Denmark)

    Nielsen, C U; Andersen, R; Brodin, Birger

    2001-01-01

    (sigma*) may influence the acid, water or base catalyzed model drug release rates, when released from series of D-Glu-Ala and D-Asp-Ala pro-moieties. Release rates were investigated in both aqueous solutions with varying pH, ionic strength, and buffer concentrations as well as in in vitro biological...... as Taft (sigma*) values, has a significant influence on the release rate of the model drug.......The human intestinal di/tri-peptide carrier, hPepT1, has been suggested as a target for increasing intestinal transport of low permeability compounds by creating prodrugs designed for the transporter. Model ester prodrugs using the stabilized dipeptides D-Glu-Ala and D-Asp-Ala as pro...

  15. Tailoring the volatility and stability of oligopeptides

    Science.gov (United States)

    Schätti, J.; Sezer, U.; Pedalino, S.; Cotter, J. P.; Mayor, M.

    2017-01-01

    Amino acids are essential building blocks of life, and fluorinated derivatives have gained interest in chemistry and medicine. Modern mass spectrometry has enabled the study of oligo‐ and polypeptides as isolated entities in the gas phase, but predominantly as singly or even multiply charged species. While laser desorption of neutral peptides into adiabatically expanding supersonic noble gas jets is possible, UV–VIS spectroscopy, electric or magnetic deflectometry as well as quantum interferometry would profit from the possibility to prepare thermally slow molecular beams. This has typically been precluded by the fragility of the peptide bond and the fact that a peptide would rather ‘fry’, i.e. denature and fragment than ‘fly’. Here, we explore how tailored perfluoroalkyl functionalization can reduce the intermolecular binding and thus increase the volatility of peptides and compare it to previously explored methylation, acylation and amidation of peptides. We show that this strategy is essential and enables the formation of thermal beams of intact neutral tripeptides, whereas only fragments were observed for an extensively fluoroalkyl‐decorated nonapeptide. © 2017 The Authors. Journal of Mass Spectrometry Published by John Wiley & Sons Ltd. PMID:28608445

  16. THE PROTECTIVE EFFECTS OF SOY OLIGOPEPTIDE ON MUSCLE ATROPHY IN WEIGHTLESSNESS-SIMULATED RATS%大豆低聚肽对模拟失重大鼠肌萎缩的防护作用

    Institute of Scientific and Technical Information of China (English)

    李红毅; 白树民; 黄贱英; 龙玥姣; 韩晓龙; 朱德兵

    2012-01-01

    Objective To investigate the protective effects of soy oligopeptide (SOP)on muscle atrophy in weightlessness-simulated rats. Method Weightlessness was simulated by tail-suspended model in rats. SD rats were randomly divided into 5 groups : ground control group (GC), tail-suspended control group (TC) and 3 tail-suspended nutrition intervention groups (NS, SI, S2, S3) according to the different doses of SOP (0.03, 0.1, 0.3 g/ml). They were fed normal diets and gived water or SOP solution by intragastric administration. The biochemical indices of serum and soleus (SOL) were measured after 4 w. Results Compared with GC group, the muscle wet weight (MWW)' of SOL and the ratio of MWW to body weight in rats were significantly decreased (P<0.05), while insulinlike growth fatory (IGF-1), testosterone (TESTO), SOD, GSH-Px in serum decreased (P<0.05). The expression of atrogin-1 protein in SOL was decreased by 32% in TC group. Compared with TC group, MWW of SOL and the ratio of MWW to body weight were significantly increased (P<0.01) in NS group. The level of SOD and GSH-Px increased significantly (P<0.05)in S2, S3 group as well as GF-1 in serum in SI, S2 group, and the expression of atrogin-1 protein in SOL was decreased in all NS groups, but significantly in S3 group (P<0.05).Conclusion SOP may promote the synthesis and inhibit the breakdown of SOL protein to some extent to lessen SOL atrophy in tail-suspended rats. The possible mechanism was through stimulating the secretion of anabolic hormone and the antioxidate ability and inhibiting the expression of atrogin-1 protein.%目的 观察大豆低聚肽对模拟失重条件下大鼠肌萎缩的对抗效果.方法 尾部悬吊法建立模拟失重模型,SD雄性大鼠随机分为地面对照组(GC组)、尾吊对照组(TC组)及尾吊营养干预组,按大豆低聚肽低、中、高剂量0.03,0.1,0.3 g/ml,(S1、S2、S3),GC组和尾吊营养干预组经人工灌胃的方式分别给予对应剂量的纯净水

  17. Sequence analysis and molecular structural simulation on oligopeptide binding protein A of Hps%副猪嗜血杆菌寡肽结合蛋白(OPPA)的序列分析及分子结构模拟

    Institute of Scientific and Technical Information of China (English)

    周伦江; 车勇良; 江斌; 陈如敬; 王隆柏; 魏宏; 吴学敏; 庄向生

    2012-01-01

    通过研究副猪嗜血杆菌(Haemophiius parasuis,Hps)寡肽结合蛋白(Oligopeptide Binding Protein A,OppA)的生物学特性,为副猪嗜血杆菌亚单位疫苗的研制奠定基础。本试验设计了1对特异性引物,应用PCR方法来扩增副猪嗜血杆菌肽聚糖相关脂蛋白的全基因序列,并进行克隆和测序。并且采用生物信息学方法,对Hps的oppA基因核苷酸及其对应氨基酸序列进行比对,选取其中的血清学5型分离株,对其OppA蛋白的分子结构、理化性质及结构功能域、蛋白质二级结构等重要参数进行了预测和分析,并对0ppA蛋白的三级结构进行了同源建模。PCR扩增出1638bp的目的片段;测序结果显示出不同血清型Hps之间oppA基因核苷酸序列相似性有一定的差异,而所对应的氨基酸序列却差异更小;OPPA蛋白的理论等电点为6.45,偏弱酸性;OppA蛋白的二级结构以α螺旋、不规则卷曲和8片层为主要构件;Hps的0ppA蛋白的氨基酸序列与鼠疫耶尔森氏菌2223蛋白的同源性为57.20%,以2223蛋白结构为模板成功构建了Hps的OppA蛋白三维结构分子模型,该OppA蛋白三维结构中与2223蛋白相似,也有3个结构域,在疏水口袋中有1条5肽结构。Hps的OppA蛋白的成功模建为OppA蛋白功能的深入研究提供了参考依据。%In order to provide a theoretical basis for developing the subunit vaccine of Hps,the bionomics of oligopep- tide binding protein A(OppA) was lucubrated, The oppA complete gene was amplified by PCR with a pair of specific primers designed and synthesized,and was cloned and sequenced. The Hps OppA was analyzed and predicted by the tools of bioinformatics in the following aspects:the composition and alignment of nucleic acid sequences and amino acid sequences, molecular structure, physical and chemical characters, secondary and tertiary structure of protein and so on. The results revealed that

  18. Transport Mechanism of Oligopeptide Transporter PepT1 and Drug Absorption Mediated by PepT1%寡肽转运载体PepT1的转运机制及其介导的药物吸收

    Institute of Scientific and Technical Information of China (English)

    刘畅; 魏刚; 陆伟跃

    2013-01-01

    寡肽转运载体PepT1广泛分布于人和动物体内的多种器官和组织,其底物主要为蛋白水解后的二肽和三肽.本文综合文献,阐述PepT1的体内分布及其质子依赖型的转运机制,从空间结构的角度分析其对底物分子的选择性,介绍评价底物与PepT1二者间亲和活性的体内外模型及PepT1底物修饰策略在促进药物吸收方面的应用.%Oligopeptide transporter PepT1 is widely distributed in various organs and tissues of human and animals,which substrates are mainly proteolytic dipeptides and tripeptides.Based on a literature review,the in vivo distribution of PepT1 and its proton-dependent transport mechanism are introduced and the structure of the transporter and its selectivity on the substrate molecules are elucidated in this paper.In addition,several in vitro and in vivo models for evaluation of PepTl-mediated transportation as well as the applications of suvstrate modification strategies to drug absorption are also described.

  19. Spontaneous kanamycin-resistant Escherichia coli mutant with altered periplasmic oligopeptide permease protein (OppA and impermeability to aminoglycosides Mutante espontâneo de Escherichia coli resistente à canamicina com expressão de oligopeptídeo permease periplasmática (OppA alterada e impermeabilidade aos aminoglicosídeos

    Directory of Open Access Journals (Sweden)

    Mônica B. Rodriguez

    1999-04-01

    Full Text Available A spontaneous kanamycin-resistant Escherichia coli mutant, showing cross resistance to five other aminoglycosides and absence of the OppA protein was isolated. [3H]-dihydrostreptomycin uptake is reduced in this mutant, implying that the oligopeptide transport system is involved in accumulation of aminoglycosides, although apparently not related with aminoglycoside permeability alteration due to bacterial adaptation to osmotic changes.Um mutante espontâneo de Escherichia coli foi selecionado com canamicina e mostrou resistência cruzada a cinco outros aminoglicosídeos e ausência da proteína OppA. A incorporação de diidroestreptomicina tritiada mostrou-se reduzida nesse mutante, implicando que o sistema de transporte de oligopeptídeos está envolvido na acumulação de aminoglicosídeos, embora aparentemente não esteja relacionado com a alteração de permeabilidade aos aminoglicosídeos decorrente da adaptação bacteriana a mudanças osmóticas.

  20. Screening for the mimetic homologous oligopeptides of keratins 14 and 17 related with psoriasis from phage random peptide library%应用噬菌体肽库研究银屑病相关的角蛋白K14,K17模拟表位

    Institute of Scientific and Technical Information of China (English)

    张亮; 刘玉峰; 杨乔欣; 任君萍; 黎志东

    2001-01-01

    目的获得与银屑病相关的角蛋白K14和K17同源序列的模拟表位,评估含有此基序的短肽与银屑病发病的关系. 方法将1株抗角蛋白K14和K17同源序列的单克隆抗体(mAb)5G5经亲和层析纯化后进行生物素标记,对噬菌体递呈的随机6肽库进行3轮淘洗并进行ELISA检测. 挑取10个阳性克隆进行DNA测序,分析所获数据,并进行竞争阻断实验. 结果氨基酸序列分析表明模拟短肽的基序为VL(x)AG,角蛋白K14、K17的同源序列及链球菌M蛋白均含有此基序. 携有这些短肽的噬菌体可与mAb 5G5特异结合,并可阻断单抗与角蛋白反应. 结论含有此基序的短肽可以模拟mAb 5G5识别的与银屑病相关的角蛋白K14和K17同源序列的抗原表位,为银屑病特异性短肽的研究提供了一个新途径,同时也为肽疫苗用于银屑病的治疗提供了崭新的思路.%AIM To acquire the mimetic homologous oligopeptides of human epidermal keratins 14 and 17 related with psoriasis and evaluate the effect of the oligopeptides on the pathogenesis of psoriasis. METHODS The mAb 5G5 recognizing the common epitope of human epidermal keratin K14 and K17 was purified by HiTrap Protein G affinity column, and was biotinylated by the biotin ester. A 6-mer phage random peptide library was biopanned for 3 cycles, then positive clones were identified by ELISA and DNA were extracted for sequencing. RESULTS Amino acid sequence analysis showed that 10 positive clones selected randomly had the consensus Amino acid sequence (motif) VL(x)AG. The motif VL(x)AG could be detected in the homologous amino acid sequence of keratins 14 and 17, and streptococcal M protein contained the motif too. The phages of positive clones reacted with mAb 5G5 specifically and prevented the interaction between mAb 5G5 and keratins with dose-dependent effects. CONCLUSION The motif could mimic the common epitope on human epidermal keratins 14,17 and streptococcal M protein, perhaps the

  1. Functional expression of oligopeptide transporter PepT1 in the gastrointestinal tract and its regulatory factors%寡肽转运体PepT1在胃肠道中的功能表达及调控因素

    Institute of Scientific and Technical Information of China (English)

    陈红旗; 秦环龙

    2010-01-01

    Many peptide transporters have been identified in mammals, among which PepT1 has been widely studied. PepT1, a member of proton-coupled oligopeptide transporter (POT) superfamily, is a peptide transporter of low affinity and high capacity and is mainly expressed in the brush border membrane of intestinal epithelial cells. PepT1 plays an important role in the absorption of di/tri-peptide (the degradation products of protein in intestinal tract). Meanwhile, it mediates the transport of peptide-like drugs and the bacterial products. Therefore,the changes of the functional expression of PepT1 in the gastrointestinal tract may dramatically affect the internal and external environmental stability and drug absorption. This paper reviews the structural features and function,distribution, transport mechanisms, and regulatory factors of PepT1.%目前在哺乳动物体内已发现多种肽转运载体,其中研究最为广泛的是PepT1.PepT1属于典型的依质子的寡肽转运体(POT)超家族成员,是一种H+依赖的、低亲和力/高容量寡肽转运体,主要表达在小肠上皮细胞刷状缘膜上.肠道PepT1除对消化道中蛋白质降解产物二肽、三肽具有转运吸收功能外,肽类似药物和部分细菌产物均可经该载体转运吸收;因此,PepT1在胃肠道中的功能表达变化,对于维持内外环境稳定及药物吸收具有重要的病理生理学意义.本文总结了PepT1的结构特征和功能、分布、转运机制及调控因素,初步探讨了需要进一步深入研究的问题.

  2. FTIR Vibrational Circular Dichroism Of Oligopeptides Related To Polyproline

    Science.gov (United States)

    Dukor, R. K.; Keiderling, Timothy A.

    1989-12-01

    Vibrational Circular Dichroism (VCD) data can be routinely measured by FTIR. On our FTIR VCD instrument it is possible to obtain a spectrum where baseline correction is accomplished with solvent only. Such capability is important for measuring biological systems. Several polypeptides that have been assigned to be in 'random coil' conformation all give an amide I VCD pattern which has the same sign and bandshape as poly-L-proline II but a smaller magnitude. This is consistent with a previous proposal by Tiffany & Krimm that the 'random coil' conformation of charged polypeptides actually has a significant local ordering in the form of a left-handed extended helix. To investigate this problem further we have studied the effects of temperature and chain length on the VCD bandshape of this conformational type. The latter studies were done with (Pro)n, n=2-7. Our results indicate that even at the level of (Pro)4 the VCD spectrum has the same bandshape, sign and intensity as found in the 'random coil' poly-L-glutamic acid spectrum. Data on various 'random coil' systems will be compared to the model studies we have done.

  3. Particle-Based Microarrays of Oligonucleotides and Oligopeptides.

    Science.gov (United States)

    Nesterov-Mueller, Alexander; Maerkle, Frieder; Hahn, Lothar; Foertsch, Tobias; Schillo, Sebastian; Bykovskaya, Valentina; Sedlmayr, Martyna; Weber, Laura K; Ridder, Barbara; Soehindrijo, Miriam; Muenster, Bastian; Striffler, Jakob; Bischoff, F Ralf; Breitling, Frank; Loeffler, Felix F

    2014-10-28

    In this review, we describe different methods of microarray fabrication based on the use of micro-particles/-beads and point out future tendencies in the development of particle-based arrays. First, we consider oligonucleotide bead arrays, where each bead is a carrier of one specific sequence of oligonucleotides. This bead-based array approach, appearing in the late 1990s, enabled high-throughput oligonucleotide analysis and had a large impact on genome research. Furthermore, we consider particle-based peptide array fabrication using combinatorial chemistry. In this approach, particles can directly participate in both the synthesis and the transfer of synthesized combinatorial molecules to a substrate. Subsequently, we describe in more detail the synthesis of peptide arrays with amino acid polymer particles, which imbed the amino acids inside their polymer matrix. By heating these particles, the polymer matrix is transformed into a highly viscous gel, and thereby, imbedded monomers are allowed to participate in the coupling reaction. Finally, we focus on combinatorial laser fusing of particles for the synthesis of high-density peptide arrays. This method combines the advantages of particles and combinatorial lithographic approaches.

  4. Particle-Based Microarrays of Oligonucleotides and Oligopeptides

    Directory of Open Access Journals (Sweden)

    Alexander Nesterov-Mueller

    2014-10-01

    Full Text Available In this review, we describe different methods of microarray fabrication based on the use of micro-particles/-beads and point out future tendencies in the development of particle-based arrays. First, we consider oligonucleotide bead arrays, where each bead is a carrier of one specific sequence of oligonucleotides. This bead-based array approach, appearing in the late 1990s, enabled high-throughput oligonucleotide analysis and had a large impact on genome research. Furthermore, we consider particle-based peptide array fabrication using combinatorial chemistry. In this approach, particles can directly participate in both the synthesis and the transfer of synthesized combinatorial molecules to a substrate. Subsequently, we describe in more detail the synthesis of peptide arrays with amino acid polymer particles, which imbed the amino acids inside their polymer matrix. By heating these particles, the polymer matrix is transformed into a highly viscous gel, and thereby, imbedded monomers are allowed to participate in the coupling reaction. Finally, we focus on combinatorial laser fusing of particles for the synthesis of high-density peptide arrays. This method combines the advantages of particles and combinatorial lithographic approaches.

  5. ABC transporters : Lessons from a bacterial oligopeptide uptake system

    NARCIS (Netherlands)

    Doeven, Mark Koop

    2006-01-01

    ABC transporters are proteins that are involved in the uptake of nutrients and excretion of harmful substances in biological cells. Defects in human ABC transporters can result in severe diseases. Too high ABC transporter activity can lead to non-responsiveness of cancercells to chemotherapy, and

  6. β-Sheet-induced chirogenesis in polymerization of oligopeptides.

    Science.gov (United States)

    Wagner, Nathaniel; Rubinov, Boris; Ashkenasy, Gonen

    2011-10-24

    The origin of long homochiral biopolymers in living systems has recently been the focus of intense research. In one particular research line, scientists studied, experimentally and theoretically, chiral amplification obtained during peptide formation by polymerization of amino acid building blocks. It was suggested that processes leading to temporal or spatial separation, and thus, to the growth of homochiral polymers at the expense of their heterochiral counterparts, can explain the chirality observed in larger molecules. We introduce a simple model and stochastic simulation for the polymerization of amino acids and β-sheet formation, showing the crucial effects of the β sheets on the distributions of peptide lengths. When chiral affinities are included, racemic β sheets of alternating homochiral strands lead to the formation of chiral peptides, the isotacticity of which increases with length, consistent with previous experimental results in aqueous solutions. The tendency to form isotactic peptides is shown for both initially racemic and initially nonracemic systems, as well as for closed and open systems. We suggest that these or similar mechanisms may explain the origin of chiroselectivity in prebiotic environments.

  7. Structural basis for polyspecificity in the POT family of proton-coupled oligopeptide transporters

    DEFF Research Database (Denmark)

    Lyons, Joseph A.; Parker, Joanne L.; Solcan, Nicolae

    2014-01-01

    An enigma in the field of peptide transport is the structural basis for ligand promiscuity, as exemplified by PepT1, the mammalian plasma membrane peptide transporter. Here, we present crystal structures of di‐ and tripeptide‐bound complexes of a bacterial homologue of PepT1, which reveal at leas...

  8. Abiotic protein fragmentation by manganese oxide: implications for a mechanism to supply soil biota with oligopeptides

    Energy Technology Data Exchange (ETDEWEB)

    Reardon, Patrick N.; Chacon, Stephany S.; Walter, Eric D.; Bowden, Mark E.; Washton, Nancy M.; Kleber, Markus W.

    2016-03-14

    Proteins facilitate a wide range of chemical transformations important in soil as well as being a major reservoir of soil nitrogen themselves. The interactions and reactions of proteins with soils and minerals are of key importance to our understanding of their functional persistence in the environment. We combined NMR and EPR spectroscopies to distinguish the reaction of a model protein with a redox active mineral surface (Birnessite, MnO2) from its response to a redox neutral phyllosilicate (Kaolinite). Our data demonstrate that birnessite fragments the model protein while kaolinite has little impact on the protein structure. NMR and EPR spectroscopies are shown to be valuable tools to observe these reactions and capture the extent of protein transformation together with the extent of mineral response. These data suggest that mineral surfaces can have both promoting and retarding roles in terrestrial nitrogen cycling, with redox active minerals acting as accelerators by catalyzing the breakdown of proteins and proteinaceous materials while phyllosilicates are more likely to act as preservative media.

  9. Regulation of Inflammatory Responses in Shock-Related Syndromes by Synthetic Oligopeptides and Steroids

    NARCIS (Netherlands)

    M. van der Zee (Marten)

    2010-01-01

    textabstractInflammation is the body’s way of responding to disturbances in homeostasis. Depending on the triggering event and the site of inflammation, the inflammatory response has different physiological purposes and pathological consequences (Figure 1). Inducers of inflammation are either foreig

  10. B cell epitopes on infliximab identified by oligopeptide microarray with unprocessed patient sera

    OpenAIRE

    Homann, Arne; Röckendorf, Niels; Kromminga, Arno; Frey, Andreas; Jappe, Uta

    2015-01-01

    Background Autoimmune diseases like rheumatoid arthritis and inflammatory bowel disease are treated with TNF-alpha-blocking antibodies such as infliximab and adalimumab. A common side effect of therapeutic antibodies is the induction of anti-drug antibodies, which may reduce therapeutic efficacy. Methods In order to reveal immunogenic epitopes on infliximab which are responsible for the adverse effects, sera from patients treated with infliximab were screened by ELISA for anti-infliximab anti...

  11. Organophosphorus Compound DEPBT as a Coupling Reagent for Oligopeptides and Peptoids Synthesis: Studies on Its Mechanism

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Some oligopeptidcs and peptoids were synthesized by applying the organophosphorus compound DEPBT as a coupling rcagent. D-Biotin-OOBt was obtained unexpcctcdly. A proposed reaction mechanism for DEPBT-mediated coupling was proved.

  12. Is Cu involved in prion oligopeptide stability? Experiments and numerical simulations

    Science.gov (United States)

    Minicozzi, V.; Morante, S.

    The high-sociological impact of neurodegenerative diseases (like Alzheimer disease, Transmissible Spongiform Encephalopathies, Parkinson disease, etc.) has renewed the interest of researchers in the study of misfolding processes and in particular of the rôle played by metals in plaque formation as their unbalanced concentration can be regarded as a possible concurrent cause of protein aggregation. Metals are essential players in many of the fundamental activities of cells. Storing, metabolism, and trafficking of metals through the cellular membrane and within the cytoplasm are mediated by many proteins via well-tuned mechanisms because of the toxicity of free ions. In this review article, we summarize the results of the most recent experimental and numerical investigations aimed at understanding the possible rôle of Cu in stabilizing the Prion protein structure and in the formation of protein polymers.

  13. Supramolecularly-knitted Tethered Oligopeptide/Single-walled Carbon Nanotube Organogels

    Science.gov (United States)

    Zou, Jiong; He, Xun; Fan, Jingwei; Raymond, Jeffery E.

    2014-01-01

    A facile polymerization of an allyl-functional N-carboxyanhydride (NCA) monomer is utilized to construct an A-B-A type triblock structure containing β-sheet-rich oligomeric peptide segments tethered by a poly(ethylene oxide) chain, which are capable of dispersing and gelating single-walled carbon nanotubes (SWCNTs) noncovalently in organic solvents, resulting in significant enhancement of the mechanical properties of polypeptide-based organogels. PMID:24961389

  14. A synthetic amino acid residue containing a new oligopeptide-based photosensitive fluorescent organogel.

    Science.gov (United States)

    Maiti, Dibakar Kumar; Banerjee, Arindam

    2013-01-01

    A synthetic amino acid (with a stilbene residue in the main chain) containing a tripeptide-based organogelator has been discovered. This peptide-based synthetic molecule 1 self-assembles in various organic solvents to form an organogel. The gel has been thoroughly characterized by using various microscopic techniques including field-emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), X-ray diffraction (XRD), UV-visible and fluorescence spectroscopy, and rheology. Morphological investigations using FESEM and AFM show a nanofibrillar network structure. Interestingly, the organogel is photoresponsive and a gel-sol transition occurred by irradiating the gel with UV light of 365 nm for 2 h as shown by the UV and fluorescence study. This photoresponsive fluorescent gel holds promise for new peptide-based soft materials with interesting applications.

  15. A Novel Vasoactive Proline-Rich Oligopeptide from the Skin Secretion of the Frog Brachycephalus ephippium

    DEFF Research Database (Denmark)

    Arcanjo, Daniel Dias Rufino; Vasconcelos, Andreanne Gomes; Comerma-Steffensen, Simón Gabriel

    2015-01-01

    -domain active site. Moreover, in rat thoracic aorta BPP-BrachyNH2 induces potent endothelium-dependent vasodilatation with similar magnitude as captopril. In DAF-FM DA-loaded aortic cross sections examined by confocal microscopy, BPP-BrachyNH2 was found to increase the release of nitric oxide (NO). Moreover...

  16. Carrier-mediated transport of oligopeptides in the human fibrosarcoma cell line HT1080

    National Research Council Canada - National Science Library

    Nakanishi, T; Tamai, I; Sai, Y; Sasaki, T; Tsuji, A

    1997-01-01

    To explore the feasibility of targeting human tumor cells via their transport systems, dipeptide uptake was studied in the human fibrosarcoma cell line HT1080 and the human fibroblast cell line IMR-90...

  17. Microstructured, functional PVA hydrogels through bioconjugation with oligopeptides under physiological conditions.

    Science.gov (United States)

    Chong, Siow-Feng; Smith, Anton A A; Zelikin, Alexander N

    2013-03-25

    In this work, bioconjugation techniques are developed to achieve peptide functionalization of poly(vinyl alcohol), PVA, as both a polymer in solution and within microstructured physical hydrogels, in both cases under physiological conditions. PVA is unique in that it is one of very few polymers with excellent biocompatibility and safety and has FDA approval for clinical uses in humans. However, decades of development have documented only scant opportunities in bioconjugation with PVA. As such, materials derived thereof fail to answer the call for functional biomaterials for advanced cell culture and tissue engineering applications. To address these limitations, PVA is synthesized with terminal thiol groups and conjugated with thiolated peptides using PVA in solution. Further, microstructured, surface-adhered PVA physical hydrogels are assembled, the available conjugation sites within the hydrogels are quantified, and quantitative kinetic data are collected on peptide conjugation to the hydrogels. The success of bioconjugation in the gel phase is quantified through the use of a cell-adhesive peptide and visualization of cell adhesion on PVA hydrogels as cell culture substrates. Taken together, the presented data establish a novel paradigm in bioconjugation and functionalization of PVA physical hydrogels. Coupled with an excellent safety profile of PVA, these results deliver a superior biomaterial for diverse biomedical applications.

  18. Biophysical characterization of the proton-coupled oligopeptide transporter YjdL

    DEFF Research Database (Denmark)

    Jensen, Johanne Mørch; Simonsen, Fie C.; Mastali, Amir

    2012-01-01

    calorimetrical studies exhibit a K(d) of 14 µM for binding of Ala-Lys to WT-YjdL. Expectedly, no binding could be detected for the tripeptide Ala-Ala-Lys. Surprisingly however, binding could not be detected for Ala-Gln, although earlier studies indicated inhibitory potencies of Ala-Gln to be comparable to Ala...

  19. Stability and in vitro metabolism of dipeptide model prodrugs with affinity for the oligopeptide transporter

    DEFF Research Database (Denmark)

    Lepist, E I; Kusk, T; Larsen, D H

    2000-01-01

    -Glu(OBzl)-Ala and Asp(OBzl)-Sar in aqueous solution and in relevant biological media and to compare these results with those of our previous study of D-Asp(OBzl)-Ala. Furthermore, the resulting aqueous stability and in vitro metabolism data are related to our previous affinity data to evaluate if Glu-Sar, D......-Glu-Ala, and Asp-Sar have potential as pro-moieties in these kinds of prodrugs. The degradation rates follow first-order kinetics, show maximun stability at pH 4-5 with maximum half-lives for Asp(OBzl)-Sar, Glu(OBzl)-Sar, and D-Glu(OBzl)-Ala of 115 h, 30 days and 152 days, respectively. The stability was dependent...... on buffer concentration, temperature, pH, and ionic strength. In biological media such as 80% human plasma, human gastric juice and intestinal fluid, and 10% rat jejunal homogenate at 37 degrees C, the half-lives were greater than 1 h except for the hydrolysis of Glu(OBzl)-Sar in 10% rat jejunal homogenate...

  20. CD studies of interaction of a bZIP oligopeptide model with DNA

    Science.gov (United States)

    Votavová, Hana; Točík, Zdeněk; Šponar, Jaroslav

    1999-05-01

    A leucine zipper (bZIP) binding peptide BP1 was constructed based on the DNA binding sequence of the GCN4 protein, slightly modified to make it more similar to the sequence of other bZIP proteins (Jun) with related DNA binding specificity. Selfcomplementary DNA hexadecanucleotides containing modified ATF/CRE target sites were used to study peptide-DNA complex formation. Four oligonucleotides contained substitutions of two GC or AT pairs by IC pairs in the ATF/CRE target sequence. In two other oligonucleotides there was a substitution of A by I in two AT pairs (mismatch IT pairs were presumably formed in the duplex) and one oligonucleotide contained I instead of C in two base pairs (IG mismatch in the duplex). Conformation changes of BP1 that occur on complex formation were studied by circular dichroism spectroscopy. The binding of peptide BP1 to oligonucleotides is accompanied by an increase of the α-helix content, which depends strongly on the oligonucleotide sequence. The substitution of two GC pairs within the specific binding site has either none or only a small effect. However, the substitution of two AT pairs within the binding site by IC strongly decreases the specificity of binding to a level observed with an oligonucleotide containing the C/EBP binding site, differing from the ATF/CRE site at four positions (Votavová et al., J. Biomol. Struct. Dyn. 3 (1997) 587). Similar results were obtained also with an oligonucleotide containing I instead of C in two base pairs (IG mismatch in the duplex). Two oligonucleotides with two substitutions of A by I but with unchanged T in the AT pairs (IT mismatch) showed smaller decrease in the α-helix formation on peptide binding than oligonucleotides, in which the whole AT pair was replaced by IC. The effect of such a substitution depends on the position of the original AT pairs in the target sequence, but the presence of T appears to be essential for specific peptide binding.

  1. Enhanced Intracellular Uptake of CdTe Quantum Dots by Conjugation of Oligopeptides

    Directory of Open Access Journals (Sweden)

    Moon-Jeong Choi

    2013-01-01

    Full Text Available Arg-Gly-Asp-Ser (RGDS, a typical membrane-permeable carrier peptide, was conjugated with mercaptoisobutyric acid-immobilized CdTe quantum dot (CTNPs to enhance the intracellular uptake of quantum dots. Mean size of mercaptoisobutyric acid-immobilized quantum dots (37 nm as determined by dynamic light scattering was increased up to 54 nm after RGDS immobilization. It was found, from in vitro cell culture experiment, that fibroblast (NIH 3T3 cells were well proliferated in the presence of RGDS-conjugated quantum dots (RCTNPs, and the intracellular uptake of CTNPs and RCTNPs was studied by means of ICP and fluorescence microscopy. As a result, the RCTNPs specifically bound to the membrane of NIH 3T3 cells and almost saturated after 6 hours incubation. The amount of RCTNPs uptaken by the cells was higher than that of CTNPs, demonstrating the enhancing effect of RGDS peptide conjugation on the intracellular uptake of quantum dots (QDs.

  2. Nutritional value of protein hydrolysis products (oligopeptides and free amino acids) as a consequence of absorption and metabolism kinetics

    Science.gov (United States)

    Rerat, A.

    1995-01-01

    When pigs were submitted to duodenal infusion of solutions containing a large percentage of small peptides (PEP) or free amino acids with the same pattern (AAL) amino acids appear in the portal blood more rapidly and more uniformly after infusion of PEP then after infusion of AAL, with the notable exception of methionine for which the opposite was true. These differences were lowered when a carbohydrate (maltose dextrin) was present in the solution, but nevertheless remained significant for the first hour after the infusion. The long-term (8-hour) uptake of free amino acids into the liver and the peripheral tissues differed in profile according to the nature of the duodenal infusion. Peripheral uptake was appreciably less well balanced after infusion of free amino acids (deficiency of threonine and phenylalanine) than after infusion of small peptides (deficiency of methionine). Accordingly, in the rat, under conditions of discontinuous enteral nutrition the mixture of small peptides was of greater nutritive value than the mixture of free amino acids. It thus appears that the absorption kinetics which results in important variations in the temporal distribution of free amino acids in the tissues may be at the origin of transitory imbalances in tissue amino acid uptake, and as a result of a lower nutritive value.

  3. Metal ion interaction of an oligopeptide fragment representing the regulatory metal binding site of a CueR protein

    DEFF Research Database (Denmark)

    Jancsó, Attila; Szokolai, Hajnalka; Roszahegyi, Livia

    2013-01-01

    Metalloregulatory proteins of the MerR family are transcriptional activators that sense/control the concentration of various metal ions inside bacteria.1 The Cu+ efflux regulator CueR, similarly to other MerR proteins, possesses a short multiple Cys-containing metal binding loop close to the C......-terminus. CueR has a high selectivity for Cu+, Ag+ and Au+, but exhibits no transcriptional activity for the divalent ions Hg2+ and Zn2+.2 The two Cys- residues of the metal binding loop were shown to settle M+ ions into a linear coordination environment but other factors may also play a role in the recognition...

  4. Metal ion interaction of an oligopeptide fragment representing the regulatory metal binding site of a CueR protein

    DEFF Research Database (Denmark)

    Jancsó, Attila; Szokolai, Hajnalka; Roszahegyi, Livia;

    2013-01-01

    Metalloregulatory proteins of the MerR family are transcriptional activators that sense/control the concentration of various metal ions inside bacteria.1 The Cu+ efflux regulator CueR, similarly to other MerR proteins, possesses a short multiple Cys-containing metal binding loop close to the C......-terminus. CueR has a high selectivity for Cu+, Ag+ and Au+, but exhibits no transcriptional activity for the divalent ions Hg2+ and Zn2+.2 The two Cys- residues of the metal binding loop were shown to settle M+ ions into a linear coordination environment but other factors may also play a role in the recognition...... of cognate metal ions.2 Nevertheless, it is an interesting question whether the same sequence, when removed from the protein, shows a flexibility to adopt different coordination environments and may efficiently bind metal ions having preferences for larger coordination numbers....

  5. Nutritional value of protein hydrolysis products (oligopeptides and free amino acids) as a consequence of absorption and metabolism kinetics

    Science.gov (United States)

    Rerat, A.

    1995-01-01

    When pigs were submitted to duodenal infusion of solutions containing a large percentage of small peptides (PEP) or free amino acids with the same pattern (AAL) amino acids appear in the portal blood more rapidly and more uniformly after infusion of PEP then after infusion of AAL, with the notable exception of methionine for which the opposite was true. These differences were lowered when a carbohydrate (maltose dextrin) was present in the solution, but nevertheless remained significant for the first hour after the infusion. The long-term (8-hour) uptake of free amino acids into the liver and the peripheral tissues differed in profile according to the nature of the duodenal infusion. Peripheral uptake was appreciably less well balanced after infusion of free amino acids (deficiency of threonine and phenylalanine) than after infusion of small peptides (deficiency of methionine). Accordingly, in the rat, under conditions of discontinuous enteral nutrition the mixture of small peptides was of greater nutritive value than the mixture of free amino acids. It thus appears that the absorption kinetics which results in important variations in the temporal distribution of free amino acids in the tissues may be at the origin of transitory imbalances in tissue amino acid uptake, and as a result of a lower nutritive value.

  6. Meat proteins had different effects on oligopeptide transporter PEPT1 in the small intestine of young rats.

    Science.gov (United States)

    Li, Mengjie; Li, Chunbao; Song, Shangxin; Zhao, Fan; Xu, Xinglian; Zhou, Guanghong

    2016-12-01

    The peptide transporter 1 (PEPT1) in the apical membrane of enterocytes is the central mechanism for regulating the absorption of di- and tripeptides. Dietary proteins may affect PEPT1 abundance and peptide absorption. The present study aimed to characterize changes in PEPT1 mRNA and PEPT1 protein levels in the duodenum and jejunum of young rats after 7-day diet intervention with casein (reference), soy, beef, pork, chicken and fish proteins and further evaluate the impact on the epithelial absorption capacity. RT-PCR and western blot analyses showed that: (1) PEPT1 protein level in duodenum was higher (p  0.05). The soy protein group had lower crypt depth and higher V/C ratio in the jejunum (p absorption capacity as reflected by gut morphology in small intestine.

  7. An anti-inflammatory oligopeptide produced by Entamoeba histolytica down-regulates the expression of pro-inflammatory chemokines.

    Science.gov (United States)

    Utrera-Barillas, Dolores; Velazquez, Juan R; Enciso, Antonio; Cruz, Samira Muñoz; Rico, Guadalupe; Curiel-Quesada, Everardo; Teran, Luis M; Kretschmer, Roberto R

    2003-10-01

    Axenically grown Entamoeba histolytica produces a pentapeptide (Met-Gln-Cys-Asn-Ser) with anti-inflammatory properties that, among others, inhibits the in vitro and in vivo locomotion of human monocytes, sparing polymorphonuclear leucocytes from this effect [hence the name originally given. Monocyte Locomotion Inhibitory Factor (MLIF)]. A synthetic construct of this peptide displays the same effects as the native material. We now added MLIF to resting and PMA-stimulated cells of a human monocyte cell line and measured the effect upon mRNA and protein expression of pro-inflammatory chemokines (RANTES, IP-10, MIP-1alpha, MIP-1beta, MCP-1, IL-8, I-309 and lymphotactin) and the shared CC receptor repertoire. The constitutive expression of these chemokines and the CC receptors was unaffected, whereas induced expression of MIP-1alpha, MIP-1beta, and I-309, and that of the CCR1 receptor--all involved in monocyte chemotaxis--was significantly inhibited by MLIF. This suggests that the inhibition of monocyte functions by MLIF may not only be exerted directly on these cells, but also--and perhaps foremost--through a conglomerate down-regulation of endogenous pro-inflammatory chemokines.

  8. P2X7 Receptor Activation Impairs Exogenous MHC Class I Oligopeptides Presentation in Antigen Presenting Cells

    OpenAIRE

    Alberto Baroja-Mazo; Maria Barberà-Cremades; Pablo Pelegrín

    2013-01-01

    Major histocompatibility complex class I (MHC I) on antigen presenting cells (APCs) is a potent molecule to activate CD8(+) T cells and initiate immunity. P2X7 receptors (P2X7Rs) are present on the plasma membrane of APCs to sense the extracellular danger signal adenosine-5'-triphosphate (ATP). P2X7R activates the inflammasome and the release of IL-1β in macrophages and other immune cells to initiate the inflammatory response. Here we show that P2X7R stimulation by ATP in APCs decreased the a...

  9. Epidermal growth factor inhibits glycyl sarcosine transport and hPepT1 expression in a human intestinal cell line

    DEFF Research Database (Denmark)

    Nielsen, Carsten Uhd; Amstrup, Jan; Steffansen, Bente;

    2001-01-01

    Intestinal oligopeptide transporter, growth factor, immunocytochemistry, laser scanning confocal microscopy......Intestinal oligopeptide transporter, growth factor, immunocytochemistry, laser scanning confocal microscopy...

  10. Quinine-Based Zwitterionic Chiral Stationary Phase as a Complementary Tool for Peptide Analysis: Mobile Phase Effects on Enantio- and Stereoselectivity of Underivatized Oligopeptides.

    Science.gov (United States)

    Ianni, Federica; Sardella, Roccaldo; Carotti, Andrea; Natalini, Benedetto; Lindner, Wolfgang; Lämmerhofer, Michael

    2016-01-01

    Peptide stereoisomer analysis is of importance for quality control of therapeutic peptides, the analysis of stereochemical integrity of bioactive peptides in food, and the elucidation of the stereochemistry of peptides from a natural chiral pool which often contains one or more D-amino acid residues. In this work, a series of model peptide stereoisomers (enantiomers and diastereomers) were analyzed on a zwitterionic ion-exchanger chiral stationary phase (Chiralpak ZWIX(+) 5 µm), in order to investigate the retention and separation performance for such compounds on this chiral stationary phase and elucidate its utility for this purpose. The goal of the study focused on 1) investigations of the effects of the sample matrix used to dissolve the peptide samples; 2) optimization of the mobile phase (enabling deriving information on factors of relevance for retention and separation); and 3) derivation of structure-selectivity relationships. It turned out that small di- and tripeptides can be well resolved under optimized conditions, typically with resolutions larger than 1.5. The optimized mobile phase often consisted of methanol-tetrahydrofuran-water (49:49:2; v/v/v) with 25 mM formic acid and 12.5 mM diethylamine. This work proposes some guidance on which mobile phases can be most efficiently used for peptide stereoisomer separations on Chiralpak ZWIX. Chirality 28:5-16, 2016. © 2015 Wiley Periodicals, Inc.

  11. 大豆低聚肽的功能性质及其应用%Functional properties and applications of soybean oligopeptide

    Institute of Scientific and Technical Information of China (English)

    易维学

    2001-01-01

    近年来,国内外对肽类物质的营养.及其功能性质进行了大量研究。结果表明,在动物体内低聚肽比蛋白质更易消化,吸收速度也快于氨基酸。此外,低聚肽无论是酪蛋白肽还是大豆肽,都表现出不同的生理活性,如具有ACEI(血管紧张素转化酶抑制剂)活性,即降血压作用、抗氧化作用、促进发酵作用及脂肪代谢作用等。目前,国内对大豆低聚肽的单一功能研究较多,但有系统的研究较

  12. TcOPT3, a member of oligopeptide transporters from the hyperaccumulator Thlaspi caerulescens, is a novel Fe/Zn/Cd/Cu transporter.

    Science.gov (United States)

    Hu, Yi Ting; Ming, Feng; Chen, Wei Wei; Yan, Jing Ying; Xu, Zheng Yu; Li, Gui Xin; Xu, Chun Yan; Yang, Jian Li; Zheng, Shao Jian

    2012-01-01

    Thlaspi caerulescens is a natural selected heavy metal hyperaccumulator that can not only tolerate but also accumulate extremely high levels of heavy metals in the shoots. Thus, to identify the transportors involved in metal long-distance transportation is very important for understanding the mechanism of heavy metal accumulation in this hyperaccumulator. We cloned and characterized a novel gene TcOPT3 of OPT family from T. caerulescens. TcOPT3 was pronouncedly expressed in aerial parts, including stem and leaf. Moreover, in situ hybridization analyses showed that TcOPT3 expressed in the plant vascular systems, especially in the pericycle cells that may be involved in the long-distance transportation. The expression of TcOPT3 was highly induced by iron (Fe) and zinc (Zn) deficiency, especially in the stem and leaf. Sub-cellular localization showed that TcOPT3 was a plasma membrane-localized protein. Furthermore, heterogonous expression of TcOPT3 by mutant yeast (Saccharomyces cerevisiae) complementation experiments demonstrated that TcOPT3 could transport Fe(2+) and Zn(2+). Moreover, expression of TcOPT3 in yeast increased metal (Fe, Zn, Cu and Cd) accumulation and resulted in an increased sensitivity to cadmium (Cd) and copper (Cu). Our data demonstrated that TcOPT3 might encode an Fe/Zn/Cd/Cu influx transporter with broad-substrate. This is the first report showing that TcOPT3 may be involved in metal long-distance transportation and contribute to the heavy metal hyperaccumulation.

  13. Dipeptide model prodrugs for the intestinal oligopeptide transporter. Affinity for and transport via hPepT1 in the human intestinal Caco-2 cell line

    DEFF Research Database (Denmark)

    Nielsen, C U; Andersen, R; Brodin, Birger

    2001-01-01

    The human intestinal di/tri-peptide carrier, hPepT1, has been suggested as a drug delivery target via increasing the intestinal transport of low permeability compounds by designing peptidomimetic prodrugs. Model ester prodrugs using the stabilized dipeptides D-Glu-Ala and D-Asp-Ala as pro......-moieties for benzyl alcohol have been shown to maintain affinity for hPepT1. The primary aim of the present study was to investigate if modifications of the benzyl alcohol model drug influence the corresponding D-Glu-Ala and D-Asp-Ala model prodrugs' affinity for hPepT1 in Caco-2 cells. A second aim...... was to investigate the transepithelial transport and hydrolysis parameters for D-Asp(BnO)-Ala and D-Glu(BnO)-Ala across Caco-2 cell monolayers. In the present study, all investigated D-Asp-Ala and D-Glu-Ala model prodrugs retained various degrees of affinity for hPepT1 in Caco-2 cells. These affinities are used...

  14. 酶法制备竹笋短肽的工艺优化%Optimization of Preparing Bamboo Shoot Oligopeptides by Enzymatic Hydrolysis

    Institute of Scientific and Technical Information of China (English)

    林倩; 王强; 刘红芝; 王春艳

    2011-01-01

    为提高竹笋短肽得率(TCA-NSI)及水解度(DH),本文对Protamex和Papain同步复合酶解竹笋蛋白制备竹笋短肽的方法进行了系统研究.在单因素试验基础上,进行了四因素五水平的二次正交旋转组合试验,建立了TCA-NSI及DH与各种影响因素的回归模型.结合实际生产,确定了Protamex和Papain酶解竹笋蛋白的最适条件为:复合酶比例1∶2、pH值7.0、温度58℃、酶用量9 000U/g、酶解时间179min、底物浓度2%.在此条件下,体系TCA-NSI为70.69±0.57%,水解度为25.07±0.71%.

  15. 复合蛋白酶水解大豆粕生产寡肽的技术研究%Technique to produce soya oligopeptides by using Protamax

    Institute of Scientific and Technical Information of China (English)

    陶沙; 王卫国

    2007-01-01

    文章介绍了大豆粕在动物生产中的一些作用,研究了利用复合蛋白酶水解豆粕生产大豆寡肽的最佳工艺,确定了最佳的酶添加量,原料最适底物浓度,水解时间,水解温度以及pH值,并对酶法脱苦做了研究.

  16. Several hPepT1-transported drugs are substrates of the Escherichia coli proton-coupled oligopeptide transporter YdgR

    DEFF Research Database (Denmark)

    Prabhala, Bala K; Aduri, Nanda G; Iqbal, Mazhar

    2017-01-01

    transported by hPepT1. The transport of these drugs was evaluated using the prototypical POT YdgR from E. coli. The transport studies were pursued through combining cell-based assays with liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. These investigations revealed that YdgR from E. coli...... is able to transport five (sulpiride, bestatin, valacyclovir, ampicillin and oseltamivir) drugs. Furthermore, cells not overexpressing YdgR were also able to transport these drugs in a POT-like manner. Orthologues of YdgR are found in several species in the gut microbiome; hence, our findings could have...

  17. New insights into the substrate specificities of proton-coupled oligopeptide transporters from E. coli by a pH sensitive assay

    DEFF Research Database (Denmark)

    Prabhala, Bala Krishna; Aduri, Nanda Gowtham; Jensen, Johanne Mørch

    2014-01-01

    of a label free transport assay using the hydrophilic pH sensitive dye pyranine and POT overexpressing E. coli cells. The results confirm and extend the functional knowledge on E. coli POTs. In contrast to previous assumptions, alanine and trialanine appears to be substrates of YjdL, albeit poor compared...

  18. TcOPT3, a Member of Oligopeptide Transporters from the Hyperaccumulator Thlaspi caerulescens, Is a Novel Fe/Zn/Cd/Cu Transporter

    Science.gov (United States)

    Hu, Yi Ting; Ming, Feng; Chen, Wei Wei; Yan, Jing Ying; Xu, Zheng Yu; Li, Gui Xin; Xu, Chun Yan; Yang, Jian Li; Zheng, Shao Jian

    2012-01-01

    Background Thlaspi caerulescens is a natural selected heavy metal hyperaccumulator that can not only tolerate but also accumulate extremely high levels of heavy metals in the shoots. Thus, to identify the transportors involved in metal long-distance transportation is very important for understanding the mechanism of heavy metal accumulation in this hyperaccumulator. Methodology/Principal Findings We cloned and characterized a novel gene TcOPT3 of OPT family from T. caerulescens. TcOPT3 was pronouncedly expressed in aerial parts, including stem and leaf. Moreover, in situ hybridization analyses showed that TcOPT3 expressed in the plant vascular systems, especially in the pericycle cells that may be involved in the long-distance transportation. The expression of TcOPT3 was highly induced by iron (Fe) and zinc (Zn) deficiency, especially in the stem and leaf. Sub-cellular localization showed that TcOPT3 was a plasma membrane-localized protein. Furthermore, heterogonous expression of TcOPT3 by mutant yeast (Saccharomyces cerevisiae) complementation experiments demonstrated that TcOPT3 could transport Fe2+ and Zn2+. Moreover, expression of TcOPT3 in yeast increased metal (Fe, Zn, Cu and Cd) accumulation and resulted in an increased sensitivity to cadmium (Cd) and copper (Cu). Conclusions Our data demonstrated that TcOPT3 might encode an Fe/Zn/Cd/Cu influx transporter with broad-substrate. This is the first report showing that TcOPT3 may be involved in metal long-distance transportation and contribute to the heavy metal hyperaccumulation. PMID:22761683

  19. TcOPT3, a member of oligopeptide transporters from the hyperaccumulator Thlaspi caerulescens, is a novel Fe/Zn/Cd/Cu transporter.

    Directory of Open Access Journals (Sweden)

    Yi Ting Hu

    Full Text Available BACKGROUND: Thlaspi caerulescens is a natural selected heavy metal hyperaccumulator that can not only tolerate but also accumulate extremely high levels of heavy metals in the shoots. Thus, to identify the transportors involved in metal long-distance transportation is very important for understanding the mechanism of heavy metal accumulation in this hyperaccumulator. METHODOLOGY/PRINCIPAL FINDINGS: We cloned and characterized a novel gene TcOPT3 of OPT family from T. caerulescens. TcOPT3 was pronouncedly expressed in aerial parts, including stem and leaf. Moreover, in situ hybridization analyses showed that TcOPT3 expressed in the plant vascular systems, especially in the pericycle cells that may be involved in the long-distance transportation. The expression of TcOPT3 was highly induced by iron (Fe and zinc (Zn deficiency, especially in the stem and leaf. Sub-cellular localization showed that TcOPT3 was a plasma membrane-localized protein. Furthermore, heterogonous expression of TcOPT3 by mutant yeast (Saccharomyces cerevisiae complementation experiments demonstrated that TcOPT3 could transport Fe(2+ and Zn(2+. Moreover, expression of TcOPT3 in yeast increased metal (Fe, Zn, Cu and Cd accumulation and resulted in an increased sensitivity to cadmium (Cd and copper (Cu. CONCLUSIONS: Our data demonstrated that TcOPT3 might encode an Fe/Zn/Cd/Cu influx transporter with broad-substrate. This is the first report showing that TcOPT3 may be involved in metal long-distance transportation and contribute to the heavy metal hyperaccumulation.

  20. Progress On Intestinal Oligopeptide Transporter PepT1 of Fishes%鱼类肠道小肽转运载体PepT1的研究进展

    Institute of Scientific and Technical Information of China (English)

    叶万里; 李英文

    2009-01-01

    从生理特性,cDNA序列和蛋白质分子结构,组织分布与表达这三个方面叙述了欧洲鳗鲡(Anguilla anguilla),罗非鱼(Oreochromis mossambicus),rockfish(Sebastes caurinus),银鱼(Chionodraco hamatus),斑马鱼(Danio rerio),亚洲泥鳅(Misgurnus anguillicaudatus),大西洋鳕鱼(Gadus morhua)和欧洲黑鲈(Dicentrarchus labrax)中有关肠道Ⅰ型肽转运载体(PepT1)方面的研究进展情况.

  1. Towards understanding the tandem mass spectra of protonated oligopeptides. 2: The proline effect in collision-induced dissociation of protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp).

    Science.gov (United States)

    Bleiholder, Christian; Suhai, Sándor; Harrison, Alex G; Paizs, Béla

    2011-06-01

    The product ion spectra of proline-containing peptides are commonly dominated by y(n) ions generated by cleavage at the N-terminal side of proline residues. This proline effect is investigated in the current work by collision-induced dissociation (CID) of protonated Ala-Ala-Xxx-Pro-Ala (Xxx includes Ala, Ser, Leu, Val, Phe, and Trp) in an electrospray/quadrupole/time-of-flight (QqTOF) mass spectrometer and by quantum chemical calculations on protonated Ala-Ala-Ala-Pro-Ala. The CID spectra of all investigated peptides show a dominant y(2) ion (Pro-Ala sequence). Our computational results show that the proline effect mainly arises from the particularly low threshold energy for the amide bond cleavage N-terminal to the proline residue, and from the high proton affinity of the proline-containing C-terminal fragment produced by this cleavage. These theoretical results are qualitatively supported by the experimentally observed y(2)/b(3) abundance ratios for protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp). In the post-cleavage phase of fragmentation the N-terminal oxazolone fragment with the Ala-Ala-Xxx sequence and Pro-Ala compete for the ionizing proton for these peptides. As the proton affinity of the oxazolone fragment increases, the y(2)/b(3) abundance ratio decreases.

  2. [Peptidyltransferase center of ribosomes. II. Effect of temperature on the activity of model peptide donors and a study of oligopeptide formation in a matrix-free system with ribosomes].

    Science.gov (United States)

    Kotusov, V V; Nikolaeva, L V; Kukhanova, M K; Kraevskiĭ, A A; Gottikh, B P

    1978-01-01

    The reaction of 3'(2')-O-(N-formylmethionyl)-adenosine 5'-phosphate with Phe-tRNA or CACCA-Phe catalysed with E. coli MRE-600 ribosomes and stimulated with cytidine 5'-phosphate was investigated. It was shown that the reaction with Phe-tRNA was stimulated within 2--3 times when the temperature has been raised from 0 to 40 degrees. On the contrary when the peptide acceptor was CACCA-Phe the yield of peptides synthesis dropped 5 times and more under the same conditions. Similar temperature influence was observed in the reaction with 50S subunits. The inhibition of peptide bond formation with pA and CpA at 0 degrees was achieved up to 80--90% but it was very low at 40 degrees. The synthesis of tri- and tetrapeptides was observed when the reaction of 3'(2')-O-(N-formylmethionyl)-adenosine 5'-phosphate was carried out either with Phe-tRNA or with CACCA-Phe.

  3. The deep processing of corn gluten meal and the application of high F ratio Oligopeptide%玉米蛋白粉的深加工与高F值寡肽的利用

    Institute of Scientific and Technical Information of China (English)

    郭冬生; 彭小兰

    2009-01-01

    玉米蛋白粉(CGM)主要成分是玉米醇溶蛋白(Zein),玉米醇溶蛋白中支链氨基酸(BCAA)相对含量很高,因此,可以从CGM中分离提取玉米黄素、玉米醇溶蛋白和高F值生物活性寡肽(F 值=BCAA/AAA,A从为芳香族氨基酸),高F值寡肽因其特有的生理功能而在生物医药和功能性食品等领域具有广阔的诱人应用前景.本文对玉米蛋白粉的加工开发利用做了系列综述,并展望了高F值生物活性寡肽的应用前景和潜在研究价值.

  4. Towards Understanding the Tandem Mass Spectra of Protonated Oligopeptides. 2: The Proline Effect in Collision-Induced Dissociation of Protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp)

    Science.gov (United States)

    Bleiholder, Christian; Suhai, Sándor; Harrison, Alex G.; Paizs, Béla

    2011-06-01

    The product ion spectra of proline-containing peptides are commonly dominated by y n ions generated by cleavage at the N-terminal side of proline residues. This proline effect is investigated in the current work by collision-induced dissociation (CID) of protonated Ala-Ala-Xxx-Pro-Ala (Xxx includes Ala, Ser, Leu, Val, Phe, and Trp) in an electrospray/quadrupole/time-of-flight (QqTOF) mass spectrometer and by quantum chemical calculations on protonated Ala-Ala-Ala-Pro-Ala. The CID spectra of all investigated peptides show a dominant y 2 ion (Pro-Ala sequence). Our computational results show that the proline effect mainly arises from the particularly low threshold energy for the amide bond cleavage N-terminal to the proline residue, and from the high proton affinity of the proline-containing C-terminal fragment produced by this cleavage. These theoretical results are qualitatively supported by the experimentally observed y 2 / b 3 abundance ratios for protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp). In the post-cleavage phase of fragmentation the N-terminal oxazolone fragment with the Ala-Ala-Xxx sequence and Pro-Ala compete for the ionizing proton for these peptides. As the proton affinity of the oxazolone fragment increases, the y 2 / b 3 abundance ratio decreases.

  5. Pharmacokinetics and safety of DTS-108, a human oligopeptide bound to SN-38 with an esterase-sensitive cross-linker in patients with advanced malignancies: a Phase I study

    Directory of Open Access Journals (Sweden)

    Coriat R

    2016-11-01

    Full Text Available Romain Coriat,1 Sandrine J Faivre,2 Olivier Mir,3 Chantal Dreyer,2 Stanislas Ropert,3 Mohammed Bouattour,2 Robert Desjardins,4 François Goldwasser,3 Eric Raymond5 1Gastroenterology and Digestive Oncology Unit, Cochin Teaching Hospital, Université Paris Descartes Sorbonne Paris Cité, Paris, 2Department of Medical Oncology, Beaujon Teaching Hospital, Université Paris Diderot, Paris 7, Clichy, 3Department of Medical Oncology, Cochin Teaching Hospital, Université Paris Descartes Sorbonne Paris Cité, Paris, France; 4Drais Pharmaceuticals, Bridgewater, NJ, USA; 5Groupe Hospitalier Paris Saint-Joseph, Paris, France Background: DTS-108 is a hydrosoluble prodrug, where the SN-38 moiety is covalently linked to a 20-amino acid vector peptide by a specific esterase-sensitive cross-linker, releasing 7-ethyl-10-hydroxycampthotecin (SN-38 by esterase bond cleavage. Methods: The pharmacokinetics of DTS-108, adverse events graded according to NCI-CTCv3.1, dose-limiting toxicities at cycle 1, the maximum tolerated dose (MTD, and the recommended Phase II dose (RP2D of intravenous DTS-108 (1–2 hours every 2 weeks were evaluated in a first-in-human Phase I study in patients with advanced/metastatic carcinomas, according to an accelerated dose escalation design. SN-38 and SN-38 glucuronide (SN-38G levels were evaluated with fluorescence high-performance liquid chromatography (HPLC test, then liquid chromatography–tandem mass spectrometry (LC/MS/MS methods. Results: Forty-two patients received DTS-108 across 14 dosing cohorts (range 3–416 mg/m2. At 416 mg/m2, three out of six patients had grade 4 neutropenia thereby defining the MTD and the RP2D at 313 mg/m2. Fluorescence HPLC was inaccurate to quantify DTS-108 and its metabolites (SN-38 and SN-38G. New processes and analytical LC/MS/MS methods for testing SN-38 were implemented. At a dose of 313 mg/m2, mean DTS-108, SN-38, and SN-38G area under the plasma concentration–time curve to infinity (coefficients of variation % were 439,293 (24%, 1,992 (34%, and 4,538 (46% h·ng/mL. Stable disease (according to Response Evaluation Criteria in Solid Tumors was observed in nine patients. Conclusion: Assessing SN-38 concentration using fluorescence HPLC is questionable since this method failed to monitor dose escalation of DTS-108, a new topoisomerase I inhibitor, due to ex vivo degradation. LC/MS/MS methods were consistent in evaluating SN-38 exposures allowing drug monitoring. The maximum tolerated dose of DTS-108 was 416 mg/m2. The RP2D for intravenous DTS-108 was 313 mg/m2 every 2 weeks in patients with advanced/metastatic solid tumors. Keywords: Phase I, topoisomerase I inhibitor, irinotecan, fluorescence HPLC

  6. A genome-wide analysis of nonribosomal peptide synthetase gene clusters and their peptides in a Planktothrix rubescens strain

    Directory of Open Access Journals (Sweden)

    Nederbragt Alexander J

    2009-08-01

    Full Text Available Abstract Background Cyanobacteria often produce several different oligopeptides, with unknown biological functions, by nonribosomal peptide synthetases (NRPS. Although some cyanobacterial NRPS gene cluster types are well described, the entire NRPS genomic content within a single cyanobacterial strain has never been investigated. Here we have combined a genome-wide analysis using massive parallel pyrosequencing ("454" and mass spectrometry screening of oligopeptides produced in the strain Planktothrix rubescens NIVA CYA 98 in order to identify all putative gene clusters for oligopeptides. Results Thirteen types of oligopeptides were uncovered by mass spectrometry (MS analyses. Microcystin, cyanopeptolin and aeruginosin synthetases, highly similar to already characterized NRPS, were present in the genome. Two novel NRPS gene clusters were associated with production of anabaenopeptins and microginins, respectively. Sequence-depth of the genome and real-time PCR data revealed three copies of the microginin gene cluster. Since NRPS gene cluster candidates for microviridin and oscillatorin synthesis could not be found, putative (gene encoded precursor peptide sequences to microviridin and oscillatorin were found in the genes mdnA and oscA, respectively. The genes flanking the microviridin and oscillatorin precursor genes encode putative modifying enzymes of the precursor oligopeptides. We therefore propose ribosomal pathways involving modifications and cyclisation for microviridin and oscillatorin. The microviridin, anabaenopeptin and cyanopeptolin gene clusters are situated in close proximity to each other, constituting an oligopeptide island. Conclusion Altogether seven nonribosomal peptide synthetase (NRPS gene clusters and two gene clusters putatively encoding ribosomal oligopeptide biosynthetic pathways were revealed. Our results demonstrate that whole genome shotgun sequencing combined with MS-directed determination of oligopeptides successfully

  7. NCBI nr-aa BLAST: CBRC-LAFR-01-0167 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-LAFR-01-0167 ref|ZP_01754031.1| oligopeptide ABC transporter permease protein [Roseo...bacter sp. SK209-2-6] gb|EBA17512.1| oligopeptide ABC transporter permease protein [Roseobacter sp. SK209-2-6] ZP_01754031.1 2.2 34% ...

  8. NCBI nr-aa BLAST: CBRC-LAFR-01-0849 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-LAFR-01-0849 ref|ZP_01754031.1| oligopeptide ABC transporter permease protein [Roseo...bacter sp. SK209-2-6] gb|EBA17512.1| oligopeptide ABC transporter permease protein [Roseobacter sp. SK209-2-6] ZP_01754031.1 0.091 35% ...

  9. NCBI nr-aa BLAST: CBRC-LAFR-01-1577 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-LAFR-01-1577 ref|ZP_01754031.1| oligopeptide ABC transporter permease protein [Roseo...bacter sp. SK209-2-6] gb|EBA17512.1| oligopeptide ABC transporter permease protein [Roseobacter sp. SK209-2-6] ZP_01754031.1 0.50 31% ...

  10. AcEST: DK945272 [AcEST

    Lifescience Database Archive (English)

    Full Text Available tr_hit_id A7GFH0 Definition tr|A7GFH0|A7GFH0_CLOBL Oligopeptide transporter, OPT family OS=Clostridium botulinum (strain Langela...GFH0|A7GFH0_CLOBL Oligopeptide transporter, OPT family OS=Clostridium botulinum (strain Langeland / NCTC 102

  11. N-Linked Glycosyl Auxiliary-Mediated Native Chemical Ligation on Aspartic Acid: Application towards N-Glycopeptide Synthesis.

    Science.gov (United States)

    Chai, Hua; Le Mai Hoang, Kim; Vu, Minh Duy; Pasunooti, Kalyan; Liu, Chuan-Fa; Liu, Xue-Wei

    2016-08-22

    A practical approach towards N-glycopeptide synthesis using an auxiliary-mediated dual native chemical ligation (NCL) has been developed. The first NCL connects an N-linked glycosyl auxiliary to the thioester side chain of an N-terminal aspartate oligopeptide. This intermediate undergoes a second NCL with a C-terminal thioester oligopeptide. Mild cleavage provides the desired N-glycopeptide.

  12. 两种质粒编码的Borrelia burgdorfei寡肽结合蛋白(OppA)的表达和纯化%Overexpression and purification of two oligopeptide binding proteins OppA4 and OppA5 encoded by plasmid genes of Borrelia burgdorfei

    Institute of Scientific and Technical Information of China (English)

    王行国; Linden H.Hu

    2005-01-01

    为了弄清Borrelia burgdorferi中OppA4和OppA5蛋白的生物学功能,oppAIV和oppAV基因分别被克隆并在大肠杆菌中表达.大肠杆菌BL21 CodonPlus (DE3)-RIL被用来克服由A+T丰富引起的密码偏差.十二烷基-β-D-麦芽糖苷和甘油分别用于提高脂蛋白的分离效果和维护蛋白的稳定性.经过金属离子亲和层析,OppA4和OppA5纯蛋白产量可达到1 mg/L细胞以上.

  13. 应用噬菌体肽库技术获得与猪鼻支原体蛋白P37结合的多肽序列%Identification of Oligopeptides Binding to Mycoplasma Hyorhinis P37 Using a Phage Display Library

    Institute of Scientific and Technical Information of China (English)

    杨华; 冯勤; 徐慧玉; 寿成超

    2011-01-01

    Phage display random heptapeptide library was screened with recombinant P37 in this study. The positive phage clones were identified by ELISA and were sequenced, and the amino acid sequences of the polypeptides displayed on phage were deduced. After GST-polypeptides fusion protein was constructed and expressed, its binding to P37 was determined by GST-pull down and Western blot. After 4 rounds of bio-panning, the enriched positive phage clones were identified by ELISA. Eighteen positive phage clones were sequenced and the peptide sequences were as follows, ACAPKPPWLC (12/18), RPLSIDPWSPHL (3/18), RPLSNDPWSPHL (1/18), QNMMSPIEGVRI (1/ 18) and WAPEKDYMQLMK (1/18). The results from GST-pull down and Western blot showed that peptide RPLSIDPWSPHL could interact with P37. The study will be helpful for identifying the protein reacting with P37.%本研究以猪鼻支原体P37蛋白作为筛选分子,对噬菌体展示随机肽库进行亲和淘选.利用ELISA法鉴定亲和力高的阳性噬菌体克隆,对其进行DNA测序和分析,据此推导随机多肽的氨基酸序列,然后构建、表达GST-多肽重组蛋白,经GST-pull down进一步鉴定该多肽与P37的结合力.经过4轮筛选和单克隆检测,获得18个有较强特异反应的阳性克隆,18个克隆包括了5种不同的小肽序列,它们分别为ACAPKPPWLC(12/18)、RPLSIDP-WSPHL(3/18)、RPLSNDPWSPHL(1/18)、QNMMSPIEGVRI (1/18)和WAPEKDYMQLMK(1/18).用ACAPKPPWLC、RPLSIDPWSPHL与GST重组的融合蛋白进行GST-pull down实验表明,RPLSIDPWSPHL多肽能与P37相互作用.本研究为进一步筛选与P37相互作用的蛋白提供了实验依据和结构基础.

  14. The role of terminal amino group and histidine at the fourth position in the metal ion binding of oligopeptides revisited: Copper(II) and nickel(II) complexes of glycyl-glycyl-glycyl-histamine and its N-Boc protected derivative.

    Science.gov (United States)

    Jancsó, Attila; Selmeczi, Katalin; Gizzi, Patrick; Nagy, Nóra V; Gajda, Tamás; Henry, Bernard

    2011-01-01

    Copper(II) and nickel(II) binding properties of two pseudo tetrapeptides, N-Boc-Gly-Gly-Gly-Histamine (BGGGHa) and Gly-Gly-Gly-Histamine (GGGHa) have been investigated by pH-potentiometric titrations, UV-visible-, EPR-, NMR- and ESI-HRMS (electrospray ionization high resolution MS) spectroscopies, in order to compare the role of N-terminal amino group and imidazole moiety at the fourth position in the complex formation processes. Substantially higher stabilities were determined for the ML complexes of GGGHa, compared to those of BGGGHa, supporting the coordination of the terminal amino group and the histamine imidazole of the non-protected ligand. A dimeric Cu(2)H(-2)L(2) species, formed through the deprotonation of peptide groups of the ligands, was found in the GGGHa-copper(II) system. Deprotonation and coordination of further amide nitrogens led to CuH(-2)L and, above pH~10, CuH(-3)L. Experimental data supports a {NH(2), 2 × N(amide),N(im)} macrochelate structure in CuH(-2)L whereas a {NH(2), 3 × N(amide)} coordination environment in CuH(-3)L. The first two amide deprotonation processes were found to be strongly cooperative with nickel(II) and spectroscopic studies proved the transformation of the octahedral parent complexes to square planar, yellow, diamagnetic species, NiH(-2)L and above pH~9, NiH(-3)L. In the basic pH-range deprotonation and coordination of the amide groups also took place in the BGGGHa containing systems, leading to complexes with a {3 × N(amide),N(im)} donor set, and in parallel the re-dissolving of precipitate. Above pH~11, a further proton release from the pyrrolic NH group of the imidazole ring of BGGGHa occurred providing an additional proof for the different binding modes of the two ligands. Copyright © 2010 Elsevier Inc. All rights reserved.

  15. Download this PDF file

    African Journals Online (AJOL)

    user

    2013-04-10

    Apr 10, 2013 ... role in regulating blood pressure. ACE acts as an exopeptidase ... ACE-inhibitory peptide was purified to determine the functional properties and .... Then, the resulting oligopeptides and free amino acids are absorbed into the ...

  16. Sequence Classification: 397684 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB TMH TMB TMB TMB Non-TMB >gi|15608423|ref|NP_215799.1| PROBABLE OLIGOPEPTIDE-TRANSPORT... INTEGRAL MEMBRANE PROTEIN ABC TRANSPORTER OPPB || http://www.ncbi.nlm.nih.gov/protein/15608423 ...

  17. Sequence Classification: 387964 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB TMH TMB TMB TMB Non-TMB >gi|31792475|ref|NP_854968.1| PROBABLE OLIGOPEPTIDE-TRANSPORT... INTEGRAL MEMBRANE PROTEIN ABC TRANSPORTER OPPB || http://www.ncbi.nlm.nih.gov/protein/31792475 ...

  18. Development of a functional food or drug against unloading-mediated muscle atrophy

    Science.gov (United States)

    Nikawa, Takeshi; Nakao, Reiko; Kagawa, Sachiko; Yamada, Chiharu; Abe, Manami; Tamura, Seiko; Kohno, Shohei; Sukeno, Akiko; Hirasaka, Katsuya; Okumura, Yuushi; Ishidoh, Kazumi

    The ubiquitin-proteasome pathway is a primary regulator of muscle protein turnover, providing a mechanism for selective degradation of regulatory and structural proteins. This pathway is constitutively active in muscle fibers and mediates both intracellular signaling events and normal muscle protein turnover. However, conditions of decreased muscle use, so called unloading, remarkably stimulate activity of this pathway, resulting in loss of muscle protein. In fact, we previously reported that expression of several ubiquitin ligase genes, such as MuRF-1, Cbl-b, and Siah-1A, which are rate-limiting enzymes of the ubiquitin-proteasome proteolytic pathway, are significantly up-regulated in rat skeletal muscle during spaceflight. Moreover, we found that Cbl-b-mediated ubiquitination and degradation of IRS-1, an important intermediates of IGF-1 signal transduction, contributes to muscle atrophy during unloading. Therefore, we hypothesized that inhibition of Cbl-b-mediated ubiquitination and degradation of IRS-1 leads to prevention of muscle atrophy during unloading. In this study, we aimed to evaluate oligopeptide as an inhibitor against ubiquitination of IRS-1 by Cbl-b. We synthesized various oligopeptides that may competitively inhibit the binding of Cbl-b to IRS-1 on the basis of their structures and screened inhibitory effects of these synthesized oligopeptides on Cbl-b-mediated ubiquitination of IRS-1 using in vitro ubiquitination systems. We found that two synthetic oligopeptides with specific amino acid sequences effectively inhibited interaction with Cbl-b and IRS-1, resulting in decreased ubiquitination and degradation of IRS-1 (Patent pending). In contrast, we also found inhibitory activity against Cbl-b-mediated ubiquitination of IRS-1 in soy protein-derived oligopeptides, whereas their inhibitory effects were weaker than those of synthetic oligopeptides. Our results suggest that specific oligopeptides may be available as a functional food against the muscle

  19. Identification of Novel Immunogenic Proteins of Neisseria gonorrhoeae by Phage Display.

    Directory of Open Access Journals (Sweden)

    Daniel O Connor

    Full Text Available Neisseria gonorrhoeae is one of the most prevalent sexually transmitted diseases worldwide with more than 100 million new infections per year. A lack of intense research over the last decades and increasing resistances to the recommended antibiotics call for a better understanding of gonococcal infection, fast diagnostics and therapeutic measures against N. gonorrhoeae. Therefore, the aim of this work was to identify novel immunogenic proteins as a first step to advance those unresolved problems. For the identification of immunogenic proteins, pHORF oligopeptide phage display libraries of the entire N. gonorrhoeae genome were constructed. Several immunogenic oligopeptides were identified using polyclonal rabbit antibodies against N. gonorrhoeae. Corresponding full-length proteins of the identified oligopeptides were expressed and their immunogenic character was verified by ELISA. The immunogenic character of six proteins was identified for the first time. Additional 13 proteins were verified as immunogenic proteins in N. gonorrhoeae.

  20. Identification of Novel Immunogenic Proteins of Neisseria gonorrhoeae by Phage Display.

    Science.gov (United States)

    Connor, Daniel O; Zantow, Jonas; Hust, Michael; Bier, Frank F; von Nickisch-Rosenegk, Markus

    2016-01-01

    Neisseria gonorrhoeae is one of the most prevalent sexually transmitted diseases worldwide with more than 100 million new infections per year. A lack of intense research over the last decades and increasing resistances to the recommended antibiotics call for a better understanding of gonococcal infection, fast diagnostics and therapeutic measures against N. gonorrhoeae. Therefore, the aim of this work was to identify novel immunogenic proteins as a first step to advance those unresolved problems. For the identification of immunogenic proteins, pHORF oligopeptide phage display libraries of the entire N. gonorrhoeae genome were constructed. Several immunogenic oligopeptides were identified using polyclonal rabbit antibodies against N. gonorrhoeae. Corresponding full-length proteins of the identified oligopeptides were expressed and their immunogenic character was verified by ELISA. The immunogenic character of six proteins was identified for the first time. Additional 13 proteins were verified as immunogenic proteins in N. gonorrhoeae.

  1. β-PEPTIDES CYCLOBUTANIQUES

    OpenAIRE

    Fernandez, Carlos

    2008-01-01

    The synthesis of β-amino acids, structural analogues of?-Amino acids, is an issue essential in the development of oligopeptides. A lot of work has been conducted on the behavior of β-peptide (sequence of β-amino acids) as well as peptides mixed (mixed β-and β- amino acids). As a result, the conformational preference of β-amino acids will induce the appearance of a three-dimensional structure of the oligopeptide ordered. Thus, several types of helices, sheets and elbows were observed in β-olig...

  2. miR-1279, miR-548j, miR-548m, and miR-548d-5p Binding Sites in CDSs of Paralogous and Orthologous PTPN12, MSH6, and ZEB1 Genes

    OpenAIRE

    Ivashchenko, Anatoliy T.; Issabekova, Assel S.; Berillo, Olga A.

    2013-01-01

    Only PTPN12, MSH6, and ZEB1 have significant miR-1279 binding sites among paralogous genes of human tyrosine phosphatase family, DNA mismatch repair family, and zinc finger family, respectively. All miRNA binding sites are located within CDSs of studied mRNAs. Nucleotide sequences of hsa-miR-1279 binding sites with mRNAs of human PTPN12, MSH6, and ZEB1 genes encode TKEQYE, EGSSDE, and GEKPYE oligopeptides, respectively. The conservation of miRNA binding sites encoding oligopeptides has been r...

  3. Improving the stability of alpha-conotoxin AuIB through N-to-C cyclization

    DEFF Research Database (Denmark)

    Armishaw, Christopher J; Jensen, Anders A; Balle, Lena D;

    2011-01-01

    Modification of alpha-conotoxin frameworks through cyclization via an oligopeptide linker has previously been shown as an effective strategy for improving in vivo stability. We have extended this strategy by investigating cyclic analogs of alpha-conotoxin AuIB, a selective alpha3beta4 nicotinic...... acetylcholine receptor antagonist, to examine a range of oligopeptide linker lengths on the oxidative formation of disulfide bonds, activity at nicotinic acetylcholine receptors, and stability to degradation by chymotrypsin. Upon non-directed random oxidation, the ribbon isomer formed preferentially...

  4. Front line defenders of the ecological niche! Screening the structural diversity of peptaibiotics from saprotrophic and fungicolous Trichoderma/Hypocrea species

    DEFF Research Database (Denmark)

    Röhrich, Christian René; Jaklitsch, Walter Michael; Voglmayr, Hermann

    2014-01-01

    Approximately 950 individual sequences of nonribosomally biosynthesised peptides are produced by the genus Trichoderma/Hypocreathat belong to a perpetually growing class of mostly linear antibiotic oligopeptides, which are rich in the non-proteinogenic α-aminoisobutyric acid (Aib). Thus, they are......Approximately 950 individual sequences of nonribosomally biosynthesised peptides are produced by the genus Trichoderma/Hypocreathat belong to a perpetually growing class of mostly linear antibiotic oligopeptides, which are rich in the non-proteinogenic α-aminoisobutyric acid (Aib). Thus...

  5. Escherichia coli Peptide Binding Protein OppA Has a Preference for Positively Charged Peptides

    NARCIS (Netherlands)

    Klepsch, M. M.; Kovermann, M.; Löw, C.; Balbach, J.; Permentier, H. P.; Fusetti, F.; de Gier, J. W.; Gier, Jan-Willem de; Slotboom, D. J.; Berntsson, R. P. -A.

    2011-01-01

    The Escherichia coli peptide binding protein OppA is an essential component of the oligopeptide transporter Opp. Based on studies on its orthologue from Salmonella typhimurium, it has been proposed that OppA binds peptides between two and five amino acids long, with no apparent sequence selectivity.

  6. Construction and Analysis of N-phosphoryl Peptide Libraries

    Institute of Scientific and Technical Information of China (English)

    Shu Xia CAO; Jian Chen ZHANG; Ming Yu NIU; Kui LU; Xin Cheng LIAO; Yu Fen ZHAO

    2004-01-01

    N-Phosphoryl peptide libraries were constructed by transformation from homo-oligopeptide libraries, which was synthesized by self-assembly of amino acids with the assistance of phosphorus oxychloride. Electrospray ionization mass spectrometry (ESI-MS) was used to monitor the reaction.

  7. Structure and Mode of Peptide Binding of Pheromone Receptor PrgZ

    NARCIS (Netherlands)

    Berntsson, Ronnie P. -A.; Schuurman-Wolters, Gea K.; Dunny, Gary; Slotboom, Dirk-Jan; Poolman, Bert

    2012-01-01

    Wepresent the crystal structure of the pheromone receptor protein PrgZ from Enterococcus faecalis in complex with the heptapeptide cCF10 (LVTLVFV), which is used in signaling between conjugative recipient and donor cells. Comparison of PrgZ with homologous oligopeptide-binding proteins (AppA and Opp

  8. Chemistry, structural insight and applications of β-sheet forming lipopeptides

    NARCIS (Netherlands)

    Cavalli, Silvia

    2007-01-01

    The research described in this thesis aimed to synthesize and investigate the physical properties of a new series of amphiphilic lipopeptides, ALPs. These molecules were composed of an amphiphilic oligopeptide domain, (Leu-Glu)n , interlinked by a succinyl moiety to the phospholipid

  9. Design, synthesis, and biological evaluation of CXCR4 ligands.

    Science.gov (United States)

    Mona, Christine E; Besserer-Offroy, Élie; Cabana, Jérôme; Leduc, Richard; Lavigne, Pierre; Heveker, Nikolaus; Marsault, Éric; Escher, Emanuel

    2016-11-02

    A combination of the CXCR4 inverse agonist T140 with N-terminal CXCL12 oligopeptides has produced the first nanomolar synthetic CXCR4 agonists. In these agonists, the inverse agonistic portion provides affinity whereas the N-terminal CXCL12 sequence induces receptor activation. Several CXCR4 crystal structures exist with either CVX15, an inverse agonist closely related to T140 and IT1t, a small molecule; we therefore attempted to produce another CXCL12 oligopeptide combination with IT1t. For this purpose, a primary amino group was introduced by total synthesis into one of the methyl groups of IT1t, serving as an anchoring point for the oligopeptide graft. The introduction of the oligopeptides on this analog however yielded antagonists, one compound displaying high affinity. On the other hand, the amino-substituted analogue itself proved to be an inverse agonist with a binding affinity of 2.6 nM compared to 11.5 nM for IT1t. This IT1t-like analog is hitherto one of the most potent non-peptidic CXCR4 inverse agonists reported.

  10. Improved Therapeutic Efficacy in Bone and Joint Disorders by Targeted Drug Delivery to Bone.

    Science.gov (United States)

    Takahashi, Tatsuo

    2016-01-01

     Site-specific drug delivery to bone is considered achievable using acidic amino acid (L-Asp or L-Glu) homopeptides known as acidic oligopeptides. We found that fluorescence-labeled acidic oligopeptides containing six or more residues bound strongly to hydroxyapatite, which is a major component of bone, and were selectively delivered to and retained in bone after systemic administration. We explored the applicability of this result for drug delivery by conjugation of estradiol and levofloxacin with an L-Asp hexapeptide. We also similarly tagged enzymes (tissue-nonspecific alkaline phosphatase, β-glucuronidase, and N-acetylgalactosamine-6-sulfate sulfatase) and decoy receptors (endogenous secretory receptor for advanced glycation end products and etanercept) to assess whether these would improve therapeutic efficacy. The L-Asp hexapeptide-tagged drugs, including enzymes and decoy receptors, were efficiently delivered to bone in comparison with the untagged drugs. An in vivo experiment confirmed the efficacy of L-Asp hexapeptide-tagged drugs on bone and joint disorders, although there was some loss of bioactivity of estradiol and levofloxacin in vitro, suggesting that the acidic hexapeptide was partly removed by hydrolysis in the body after delivery to bone. It was expected that the ester linkage to the hexapeptide would be susceptible to hydrolysis in situ, releasing the drug from the acidic oligopeptide. These results support the usefulness of acidic oligopeptides as bone-targeting carriers for therapeutic agents. We present some pharmacokinetic and pharmacological properties of the L-Asp hexapeptide-tagged drugs.

  11. Synthesis and comparison of antibody recognition of conjugates containing herpes simplex virus type 1 glycoprotein D epitope VII

    NARCIS (Netherlands)

    Mezo, G; de Oliveira, E; Krikorian, D; Feijlbrief, M; Jakab, A; Tsikaris, [No Value; Sakarellos, C; Welling-Wester, S; Andreu, D; Hudecz, F

    2003-01-01

    Synthetic oligopeptides comprising linear or continuous topographic B-cell epitope sequences of proteins might be considered as specific and small size antigens. It has been demonstrated that the strength and specificity of antibody binding could be altered by conjugation to macromolecules or by

  12. miR-1279, miR-548j, miR-548m, and miR-548d-5p binding sites in CDSs of paralogous and orthologous PTPN12, MSH6, and ZEB1 Genes.

    Science.gov (United States)

    Ivashchenko, Anatoliy T; Issabekova, Assel S; Berillo, Olga A

    2013-01-01

    Only PTPN12, MSH6, and ZEB1 have significant miR-1279 binding sites among paralogous genes of human tyrosine phosphatase family, DNA mismatch repair family, and zinc finger family, respectively. All miRNA binding sites are located within CDSs of studied mRNAs. Nucleotide sequences of hsa-miR-1279 binding sites with mRNAs of human PTPN12, MSH6, and ZEB1 genes encode TKEQYE, EGSSDE, and GEKPYE oligopeptides, respectively. The conservation of miRNA binding sites encoding oligopeptides has been revealed. MRNAs of many paralogs of zinc finger gene family have from 1 to 12 binding sites coding the same GEKPYE hexapeptide. MRNAs of PTPN12, MSH6, and ZEB1 orthologous genes from different animal species have binding sites for hsa-miR-1279 which consist of homologous oligonucleotides encoding similar human oligopeptides TKEQYE, EGSSDE, and GEKPYE. MiR-548j, miR-548m, and miR-548d-5p have homologous binding sites in the mRNA of PTPN12 orthologous genes which encode PRTRSC, TEATDI, and STASAT oligopeptides, respectively. All regions of miRNA are important for binding with the mRNA.

  13. miR-1279, miR-548j, miR-548m, and miR-548d-5p Binding Sites in CDSs of Paralogous and Orthologous PTPN12, MSH6, and ZEB1 Genes

    Directory of Open Access Journals (Sweden)

    Anatoliy T. Ivashchenko

    2013-01-01

    Full Text Available Only PTPN12, MSH6, and ZEB1 have significant miR-1279 binding sites among paralogous genes of human tyrosine phosphatase family, DNA mismatch repair family, and zinc finger family, respectively. All miRNA binding sites are located within CDSs of studied mRNAs. Nucleotide sequences of hsa-miR-1279 binding sites with mRNAs of human PTPN12, MSH6, and ZEB1 genes encode TKEQYE, EGSSDE, and GEKPYE oligopeptides, respectively. The conservation of miRNA binding sites encoding oligopeptides has been revealed. MRNAs of many paralogs of zinc finger gene family have from 1 to 12 binding sites coding the same GEKPYE hexapeptide. MRNAs of PTPN12, MSH6, and ZEB1 orthologous genes from different animal species have binding sites for hsa-miR-1279 which consist of homologous oligonucleotides encoding similar human oligopeptides TKEQYE, EGSSDE, and GEKPYE. MiR-548j, miR-548m, and miR-548d-5p have homologous binding sites in the mRNA of PTPN12 orthologous genes which encode PRTRSC, TEATDI, and STASAT oligopeptides, respectively. All regions of miRNA are important for binding with the mRNA.

  14. Materials taking a lesson from nature.

    Science.gov (United States)

    Tian, Liangfei; Croisier, Emmanuel; Frauenrath, Holger

    2013-01-01

    Structural biomaterials with their often extraordinary properties and versatile functions are typically constructed from very limited sets of building blocks and types of supramolecular interactions. In this review we discuss how, inspired by nature's design principles for protein-based materials, oligopeptide-modified polymers can be used as a versatile toolbox to program nanostructure and hierarchical structure formation in synthetic materials.

  15. Peptides as catalysts in the RNA world

    DEFF Research Database (Denmark)

    Wieczorek, Rafal; Dörr, Mark; Luisi, Pier Luigi

    , Stano P, Luisi PL. (2009) Ser-His catalyses the formation of peptides and PNAs. FEBS Lett. 583(1):153-6. Li Y, Zhao Y, Hatfield S, Wan R, Zhu Q, Li X, McMills M, Ma Y, Li J, Brown KL, He C, Liu F, Chen X. (2000) Dipeptide seryl-histidine and related oligopeptides cleave DNA, protein, and a carboxyl...

  16. An analysis of the secondary structure of spider spidroins I and II belonging to different species

    NARCIS (Netherlands)

    Ragulina, LE; Makeev, VY; Esipova, NG; Tumanyan, VG; Vlasov, PK; Bogush, VG; Debabov, VG

    2004-01-01

    We have analyzed the secondary structure of spidroin proteins of I and II types, related to spiders of different species. We used standard methods of secondary structure prediction NNPREDICT and JPRED and also analyzed the occurrences of oligopeptides with a preferred secondary structure with the

  17. Structure-function studies of BPP-BrachyNH2 and synthetic analogues thereof with Angiotensin I-Converting Enzyme

    DEFF Research Database (Denmark)

    Arcanjo, Daniel Dias Rufino; Vasconcelos, Andreanne Gomes; Nascimento, Lucas A

    2017-01-01

    The vasoactive proline-rich oligopeptide termed BPP-BrachyNH2 (H-WPPPKVSP-NH2) induces in vitro inhibitory activity of angiotensin I-converting enzyme (ACE) in rat blood serum. In the present study, the removal of N-terminal tryptophan or C-terminal proline from BPP-BrachyNH2 was investigated in ...

  18. Chemistry, structural insight and applications of β-sheet forming lipopeptides

    NARCIS (Netherlands)

    Cavalli, Silvia

    2007-01-01

    The research described in this thesis aimed to synthesize and investigate the physical properties of a new series of amphiphilic lipopeptides, ALPs. These molecules were composed of an amphiphilic oligopeptide domain, (Leu-Glu)n , interlinked by a succinyl moiety to the phospholipid 1,2-Dioleoyl-sn-

  19. 21 CFR 184.1553 - Peptones.

    Science.gov (United States)

    2010-04-01

    ... CONSUMPTION (CONTINUED) DIRECT FOOD SUBSTANCES AFFIRMED AS GENERALLY RECOGNIZED AS SAFE Listing of Specific..., oligopeptides, and amino acids that are produced by partial hydrolysis of casein, animal tissue, soy protein isolate, gelatin, defatted fatty tissue, egg albumin, or lactalbumin (whey protein). Peptones are...

  20. Escherichia coli Peptide Binding Protein OppA Has a Preference for Positively Charged Peptides

    NARCIS (Netherlands)

    Klepsch, M. M.; Kovermann, M.; Löw, C.; Balbach, J.; Permentier, H. P.; Fusetti, F.; de Gier, J. W.; Gier, Jan-Willem de; Slotboom, D. J.; Berntsson, R. P. -A.

    2011-01-01

    The Escherichia coli peptide binding protein OppA is an essential component of the oligopeptide transporter Opp. Based on studies on its orthologue from Salmonella typhimurium, it has been proposed that OppA binds peptides between two and five amino acids long, with no apparent sequence selectivity.

  1. An analysis of the secondary structure of spider spidroins I and II belonging to different species

    NARCIS (Netherlands)

    Ragulina, LE; Makeev, VY; Esipova, NG; Tumanyan, VG; Vlasov, PK; Bogush, VG; Debabov, VG

    2004-01-01

    We have analyzed the secondary structure of spidroin proteins of I and II types, related to spiders of different species. We used standard methods of secondary structure prediction NNPREDICT and JPRED and also analyzed the occurrences of oligopeptides with a preferred secondary structure with the he

  2. NCBI nr-aa BLAST: CBRC-OSAT-10-0002 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OSAT-10-0002 ref|NP_623489.1| ABC-type dipeptide/oligopeptide/nickel transport systems...peptide/nickel transport systems, permease components [Thermoanaerobacter tengcongensis MB4] NP_623489.1 1.9 30% ...

  3. Synthesis and comparison of antibody recognition of conjugates containing herpes simplex virus type 1 glycoprotein D epitope VII

    NARCIS (Netherlands)

    Mezo, G; de Oliveira, E; Krikorian, D; Feijlbrief, M; Jakab, A; Tsikaris, [No Value; Sakarellos, C; Welling-Wester, S; Andreu, D; Hudecz, F

    2003-01-01

    Synthetic oligopeptides comprising linear or continuous topographic B-cell epitope sequences of proteins might be considered as specific and small size antigens. It has been demonstrated that the strength and specificity of antibody binding could be altered by conjugation to macromolecules or by mod

  4. Chemo-enzymatic peptide synthesis : bioprocess engineering aspects

    NARCIS (Netherlands)

    Vossenberg, P.

    2012-01-01

      Peptides, in particular oligopeptides, play an important role in the fields of health care, nutrition and cosmetics. Chemical synthesis is currently the most mature technique for the synthesis of peptides that range in length from 5 to 80 amino acids. Chemical synthesis is, however, expected

  5. Chemo-enzymatic peptide synthesis : bioprocess engineering aspects

    NARCIS (Netherlands)

    Vossenberg, P.

    2012-01-01

      Peptides, in particular oligopeptides, play an important role in the fields of health care, nutrition and cosmetics. Chemical synthesis is currently the most mature technique for the synthesis of peptides that range in length from 5 to 80 amino acids. Chemical synthesis is, however, expected

  6. The Red Queen race between parasitic chytrids and their host, Planktothrix: a test using a time series reconstructed from sediment DNA.

    Directory of Open Access Journals (Sweden)

    Marcia Kyle

    Full Text Available Parasitic chytrid fungi (phylum Chytridiomycota are known to infect specific phytoplankton, including the filamentous cyanobacterium Planktothrix. Subspecies, or chemotypes of Planktothrix can be identified by the presence of characteristic oligopeptides. Some of these oligopeptides can be associated with important health concerns due to their potential for toxin production. However, the relationship between chytrid parasite and Planktothrix host is not clearly understood and more research is needed. To test the parasite-host relationship over time, we used a sediment core extracted from a Norwegian lake known to contain both multiple Planktothrix chemotype hosts and their parasitic chytrid. Sediment DNA of chytrids and Planktothrix was amplified and a 35-year coexistence was found. It is important to understand how these two antagonistic species can coexistence in a lake. Reconstruction of the time series showed that between 1979-1990 at least 2 strains of Planktothrix were present and parasitic pressure exerted by chytrids was low. After this period one chemotype became dominant and yet showed continued low susceptibility to chytrid parasitism. Either environmental conditions or intrinsic characteristics of Planktothrix could have been responsible for this continued dominance. One possible explanation could be found in the shift of Planktothrix to the metalimnion, an environment that typically consists of low light and decreased temperatures. Planktothrix are capable of growth under these conditions while the chytrid parasites are constrained. Another potential explanation could be due to the differences between cellular oligopeptide variations found between Planktothrix chemotypes. These oligopeptides can function as defense systems against chytrids. Our findings suggest that chytrid driven diversity was not maintained over time, but that the combination of environmental constraints and multiple oligopeptide production to combat chytrids

  7. The Red Queen race between parasitic chytrids and their host, Planktothrix: a test using a time series reconstructed from sediment DNA.

    Science.gov (United States)

    Kyle, Marcia; Haande, Sigrid; Ostermaier, Veronika; Rohrlack, Thomas

    2015-01-01

    Parasitic chytrid fungi (phylum Chytridiomycota) are known to infect specific phytoplankton, including the filamentous cyanobacterium Planktothrix. Subspecies, or chemotypes of Planktothrix can be identified by the presence of characteristic oligopeptides. Some of these oligopeptides can be associated with important health concerns due to their potential for toxin production. However, the relationship between chytrid parasite and Planktothrix host is not clearly understood and more research is needed. To test the parasite-host relationship over time, we used a sediment core extracted from a Norwegian lake known to contain both multiple Planktothrix chemotype hosts and their parasitic chytrid. Sediment DNA of chytrids and Planktothrix was amplified and a 35-year coexistence was found. It is important to understand how these two antagonistic species can coexistence in a lake. Reconstruction of the time series showed that between 1979-1990 at least 2 strains of Planktothrix were present and parasitic pressure exerted by chytrids was low. After this period one chemotype became dominant and yet showed continued low susceptibility to chytrid parasitism. Either environmental conditions or intrinsic characteristics of Planktothrix could have been responsible for this continued dominance. One possible explanation could be found in the shift of Planktothrix to the metalimnion, an environment that typically consists of low light and decreased temperatures. Planktothrix are capable of growth under these conditions while the chytrid parasites are constrained. Another potential explanation could be due to the differences between cellular oligopeptide variations found between Planktothrix chemotypes. These oligopeptides can function as defense systems against chytrids. Our findings suggest that chytrid driven diversity was not maintained over time, but that the combination of environmental constraints and multiple oligopeptide production to combat chytrids could have allowed one

  8. Thermal Stability, Sorption Properties and Morphology of Films of Dipeptide and Tripeptide Based on L-Glycine

    Directory of Open Access Journals (Sweden)

    Marat A. Ziganshin

    2015-12-01

    Full Text Available The effect of the number of amino acid residues in L-glycyl-L-glycine and L-glycyl-L-glycyl-L-glycine on thermal stability of powders, the sorption properties and surface morphology of thin films has been found. Dipeptide forms the film coated with disk-shaped nano-objects on the hydrophilic substrate, while tripeptide self-organizes to the film coated with nano-crystals on the hydrophobic substrate. Replacement of substrates (hydrophilic↔hydrophobic leads to the formation of smooth films of studied oligopeptides. Powders of oligopeptides do not form stable clathrates with water and organic compounds at room temperature. But their thin films are capable to bind organic or water vapors with high thermodynamic activity. Surprising difference in sorption selectivity of dipeptide and tripeptide has been observed. L-G

  9. In vitro and in vivo antimicrobial activity of a synthetic peptide derived from the C-terminal region of human chemokine CCL13 against Pseudomonas aeruginosa.

    Science.gov (United States)

    Cossio-Ayala, Mayte; Domínguez-López, Mariana; Mendez-Enriquez, Erika; Portillo-Téllez, María Del Carmen; García-Hernández, Enrique

    2017-08-01

    Chemokines are important mediators of immunological responses during inflammation and under steady-state conditions. In addition to regulating cell migration, some chemotactic cytokines have direct effects on bacteria. Here, we characterized the antibacterial ability of the synthetic oligopeptide CCL1357-75, which corresponds to the carboxyl-terminal region of the human chemokine CCL13. In vitro measurements indicated that CCL1357-75 disrupts the cell membrane of Pseudomonas aeruginosa through a mechanism coupled to an unordered-helicoidal conformational transition. In a murine pneumonic model, CCL1357-75 improved mouse survival and bacterial clearance and decreased neutrophil recruitment, proinflammatory cytokines and lung pathology compared with that observed in untreated infected animals. Overall, our study supports the ability of chemokines and/or chemokine-derived oligopeptides to act as direct defense agents against pathogenic bacteria and suggests their potential use as alternative antibiotics. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. [Evaluation of the endogenous intoxication syndrome in food toxic infections].

    Science.gov (United States)

    Marzhokhova, M Iu; Zhelikhazheva, Zh M

    2009-01-01

    To study the endogenous intoxication syndrome in patients with food toxic infections is essential in revealing the pathogenetic mechanisms underlying this disease. For this, the authors measured the level of low and average molecular weight, as well as their protein component--oligopeptides in plasma, red blood cells, and urine in the course of the disease. There were increased levels of the study parameters, which depended on the stage and degree of a pathological process. The determination of the level of low and average molecular weight and oligopeptides in plasma, red blood cells, and urine may serve as a marker of the intoxication syndrome; the level of the study parameters may be used as additional criteria for the severity of the process, the prediction of disease development and comorbidity, and as a criterion for recovery completeness.

  11. Fluorometric polyethyleneglycol-peptide hybrid substrates for quantitative assay of protein disulfide isomerase

    DEFF Research Database (Denmark)

    Christiansen, Camilla; St Hilaire, Phaedria M; Winther, Jakob R.

    2004-01-01

    In eukaryotic cells the enzyme protein disulfide isomerase (PDI) is responsible for the formation and reshuffling of disulfide bonds in secretory proteins. The reaction carried out by PDI involves interaction with a highly complex mixture of polypeptide molecules that are in the process of folding....... This means that PDI activity is typically measured in the context of a globular protein folding pathway. The absence of small, well-defined substrates for the quantitation of both oxidation and reduction reactions constitutes an inherent problem in the analysis of PDI activity. We describe a new type...... of substrate for PDI where two cysteine-containing oligopeptides are connected by an onameric ethylene glycol linker. We term such hybrid compounds PEGtides. The oligopeptides are each marked with a fluorescent aminobenzoic acid and a quenching nitrotyrosine group, respectively. The reversible formation...

  12. [Chromatographic analysis of low molecular weight fraction of cerebrospinal fluid in children with acute neuroinfections].

    Science.gov (United States)

    Alekseeva, L A; Shatik, S V; Sorokina, M N; Karasev, V V

    2002-05-01

    Low molecular-weight (oligopeptide) fraction of the cerebrospinal fluid was analyzed by high-performance reversed phase liquid chromatography in 30 children with bacterial and viral neuroinfections. The incidence and height of chromathoraphic peaks in bacterial meningitis depended on the disease etiology, stage, and severity. Qualitative and quantitative composition of low molecular-weight fraction of the liquor varied in patients with viral neuroinfections, depending on the severity of the cerebral parenchyma involvement. Differences in chromatographic profiles in complicated and uneventful course of neuroinfections indicate a possible damaging, protective, or regulatory effect of the liquor peptides. These data focus the attention on the role of oligopeptides in the genesis of neuroinfectious process, significance of search for peptide markers, their further isolation, identification, and development of test systems available for clinical application.

  13. Intestinal absorption of two dipeptides in Hartnup disease.

    Science.gov (United States)

    Asatoor, A M; Cheng, B; Edwards, K D; Lant, A F; Matthews, D M; Milne, M D; Navab, F; Richards, A J

    1970-05-01

    The results of oral tolerance tests of two dipeptides and of their constitutent amino acids are compared in normal subjects and in a case of Hartnup disease. In the control subjects the rate of absorption of phenylalanine from phenylalanyl-phenylalanine and of tryptophan from glycyl-tryptophan was slower than after the equivalent amount of the free amino acids. Absorption of the two essential amino acids (tryptophan and phenylalanine) in the patient was almost zero after administration in the free form, but was much greater after the dipeptide. Results of experiments on absorption and hydrolysis of the two peptides in the rat small intestine are also reported. It is suggested that whereas normal subjects absorb essential amino acids by a dual mechanism of mucosal uptake of free amino acids and oligopeptides, nutrition in Hartnup disease is largely dependent on uptake of oligopeptides containing the amino acids affected by the intestinal transport defect of the disease.

  14. Intestinal absorption of two dipeptides in Hartnup disease 1

    Science.gov (United States)

    Asatoor, A. M.; Cheng, B.; Edwards, K. D. G.; Lant, A. F.; Matthews, D. M.; Milne, M. D.; Navab, F.; Richards, A. J.

    1970-01-01

    The results of oral tolerance tests of two dipeptides and of their constitutent amino acids are compared in normal subjects and in a case of Hartnup disease. In the control subjects the rate of absorption of phenylalanine from phenylalanyl-phenylalanine and of tryptophan from glycyl-tryptophan was slower than after the equivalent amount of the free amino acids. Absorption of the two essential amino acids (tryptophan and phenylalanine) in the patient was almost zero after administration in the free form, but was much greater after the dipeptide. Results of experiments on absorption and hydrolysis of the two peptides in the rat small intestine are also reported. It is suggested that whereas normal subjects absorb essential amino acids by a dual mechanism of mucosal uptake of free amino acids and oligopeptides, nutrition in Hartnup disease is largely dependent on uptake of oligopeptides containing the amino acids affected by the intestinal transport defect of the disease. PMID:5428040

  15. Peptide-based protein capture agents with high affinity, selectivity, and stability as antibody replacements in biodetection assays

    Science.gov (United States)

    Coppock, Matthew B.; Farrow, Blake; Warner, Candice; Finch, Amethist S.; Lai, Bert; Sarkes, Deborah A.; Heath, James R.; Stratis-Cullum, Dimitra

    2014-05-01

    Current biodetection assays that employ monoclonal antibodies as primary capture agents exhibit limited fieldability, shelf life, and performance due to batch-to-batch production variability and restricted thermal stability. In order to improve upon the detection of biological threats in fieldable assays and systems for the Army, we are investigating protein catalyzed capture (PCC) agents as drop-in replacements for the existing antibody technology through iterative in situ click chemistry. The PCC agent oligopeptides are developed against known protein epitopes and can be mass produced using robotic methods. In this work, a PCC agent under development will be discussed. The performance, including affinity, selectivity, and stability of the capture agent technology, is analyzed by immunoprecipitation, western blotting, and ELISA experiments. The oligopeptide demonstrates superb selectivity coupled with high affinity through multi-ligand design, and improved thermal, chemical, and biochemical stability due to non-natural amino acid PCC agent design.

  16. Transcriptional responses in Lactococcus lactis subsp. cremoris to the changes in oxygen and redox potential during milk acidification

    DEFF Research Database (Denmark)

    Larsen, Nadja; Werner, Birgit Brøsted; Jespersen, Lene

    2016-01-01

    genes were detected during aerobic reduction phase. Upregulated genes were implicated in lactose utilization, glycogen biosynthesis, amino sugar metabolism, oxidation-reduction, pyrimidine biosynthesis and DNA integration processes. Genes of purine nucleotide biosynthesis and genes encoding amino acid......, multidrug resistance and ion ABC transporters were mostly downregulated, while oligopeptide transporter genes were reduced during oxygen depletion and induced at minimum Eh7. Significance and Impact of the Study: Understanding of gene responses in starter cultures to the changes of oxidation-reduction state...

  17. Chemical evolution of life-like system under hydrothermal environments: prebiotic formation, degradation, and functions regarding protein-like molecules and RNA

    Science.gov (United States)

    Kawamura, Kunio

    The accumulation of biopolymers without enzymes is an essential step for the chemical evolu-tion towards a primitive life-like system. Previously, we discussed the relationship between the RNA world hypothesis and the hydrothermal origin of life hypothesis on the basis of the em-pirical data of RNA behaviors under the hydrothermal environments examined using real-time monitoring technique for hydrothermal reactions within the millisecond to second time scale. On the other hand, we have also examined the stabilities and behaviors of amino acids, pep-tides, and proteins under the hydrothermal environments. These observations have shown the possibility that oligopeptides could have been accumulated under near submarine hydrother-mal vent environments on primitive Earth within the relatively short time scale. However, the formation of oligopeptides under the simulated hydrothermal conditions is not so effective in the absence of catalysts and condensation agents. Thus, the investigation of the roles of min-eral catalysis and condensation reagents are very important since these materials could have enhanced efficiently the formation of peptides and stabilize primitive protein-like molecules. Recently, we investigated the roles of condensation reagents for the elongation of oligopeptides in the presence of minerals. In addition, we have designed a mineral-mediated hydrothermal flow reactor system (MHFR), which enables monitoring hydrothermal reactions in the presence of solid particles. By using MHFR, we attempted to examine naturally occurring minerals, such as apatite and quartz, for the elongation of oligopeptides at temperatures over 200 o C within 10 -30 sec. According to these data, the chemical evolution of protein-like molecules on primitive Earth will be discussed.

  18. Physically Functional Materials

    OpenAIRE

    Berg, Rolf Henrik; Hvilsted, Søren; Ramanujam, P.S.

    2002-01-01

    The invention relates to novel monodisperse or polydisperse compounds, in general named DNO (diamino acid Nalpha-substituted oligopeptides), preferably low molecular weight polypeptides, e.g., based on ornithine, lysine, diaminobutyric acid, diaminopropionic acid, aminoethylglycine or other amino acids or peptides having azobenzenes or other physicially functional groups, e.g., photoresponsive groups, as side chains. These compounds may be synthesized using solid phase peptide synthesis techn...

  19. The Azobenzene Optical Storage Puzzle - Demands on the Polymer Scaffold?

    DEFF Research Database (Denmark)

    Hvilsted, Søren; Ramanujam, PS

    2001-01-01

    The basic mechanism of optical information storage utilizing the azobenzene photoaddressable moiety will briefly be introduced. A synthetically flexible polyester matrix covalently integrating cyanoazobenzene in regularly spaced side chains is particularly well suited for holographic storage...... of the nature of the main chain on polyester morphology and on the permanency of the induced anisotropy are discussed. Arguments for the design and methods of preparation of other very different polymer scaffolds supporting the cyanoazobenzene are elucidated. Whereas oligopeptides invariably form amorphous...

  20. The azobenzene optical storage puzzle - Demands on the polymer scaffold?

    DEFF Research Database (Denmark)

    Hvilsted, Søren; Ramanujam, P.S.

    2001-01-01

    The basic mechanism of optical information storage utilizing the azobenzene photoaddressable moiety will briefly be introduced. A synthetically flexible polyester matrix covalently integrating cyanoazobenzene in regularly spaced side chains is particularly well suited for holographic storage...... of the nature of the main chain on polyester morphology and on the permanency of the induced anisotropy are discussed. Arguments for the design and methods of preparation of other very different polymer scaffolds supporting the cyanoazobenzene are elucidated. Whereas oligopeptides invariably form amorphous...

  1. Self-assembled peptide nanoparticles as tumor microenvironment activatable probes for tumor targeting and imaging.

    Science.gov (United States)

    Zhao, Ying; Ji, Tianjiao; Wang, Hai; Li, Suping; Zhao, Yuliang; Nie, Guangjun

    2014-03-10

    Design of specific and sensitive imaging probes for targeting tumor microenvironment holds great promise to achieve precise detection and rapid responsiveness to neoplastic tissues. Dysregulated pH, one of the most remarkable hallmarks of tumor microenvironment, can be considered as a good specific trigger for the design of broad-spectrum and local-environment responsive imaging probes. However, the current existing design strategies for pH-responsive systems are insufficient to meet the needs for a rapid and tumor-specific diagnosis. Here we reported a novel biomimetic nanostructure based on oligopeptide self-assembly that can quickly switch into dissociated stage with active fluorescence property from self-assembled stage with quenched fluorescence activity when encountering a subtle pH-change in tumor microenvironment (pH 6.8 vs. 7.4). This oligopeptide-assembly is examined as tumor microenvironment activatable probes for both intratumoral and intravenous in vivo tumor imaging. Through the distinct fluorescent intensities, it is validated that the acidic tumor microenvironment can activate stronger fluorescence signals. The tailor-made self-assembled oligopeptide nanomaterials have the potential for efficient and specific in situ diagnosis of various solid tumors with a weakly acidic microenvironment, which is expected to be of crucial importance for clinical tumor diagnostics. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Crosslinking of DNA repair and replication proteins to DNA in cells treated with 6-thioguanine and UVA.

    Science.gov (United States)

    Gueranger, Quentin; Kia, Azadeh; Frith, David; Karran, Peter

    2011-07-01

    The DNA of patients taking immunosuppressive and anti-inflammatory thiopurines contains 6-thioguanine (6-TG) and their skin is hypersensitive to ultraviolet A (UVA) radiation. DNA 6-TG absorbs UVA and generates reactive oxygen species that damage DNA and proteins. Here, we show that the DNA damage includes covalent DNA-protein crosslinks. An oligonucleotide containing a single 6-TG is photochemically crosslinked to cysteine-containing oligopeptides by low doses of UVA. Crosslinking is significantly more efficient if guanine sulphonate (G(SO3))--an oxidized 6-TG and a previously identified UVA photoproduct--replaces 6-TG, suggesting that G(SO3) is an important reaction intermediate. Crosslinking occurs via oligopeptide sulphydryl and free amino groups. The oligonucleotide-oligopeptide adducts are heat stable but are partially reversed by reducing treatments. UVA irradiation of human cells containing DNA 6-TG induces extensive heat- and reducing agent-resistant covalent DNA-protein crosslinks and diminishes the recovery of some DNA repair and replication proteins from nuclear extracts. DNA-protein crosslinked material has an altered buoyant density and can be purified by banding in cesium chloride (CsCl) gradients. PCNA, the MSH2 mismatch repair protein and the XPA nucleotide excision repair (NER) factor are among the proteins detectable in the DNA-crosslinked material. These findings suggest that the 6-TG/UVA combination might compromise DNA repair by sequestering essential proteins.

  3. Biofunctionalization of titanium surfaces for osseintegration process improvement

    Energy Technology Data Exchange (ETDEWEB)

    Sevilla, P; Godoy, M; Salvagni, E; Rodriguez, D; Gil, F J, E-mail: pablo.sevilla@upc.edu

    2010-11-01

    This study aims to improve the osseointegration of titanium implants through surface immobilization of peptides that induce a beneficial biological response. This was carried out biofunctionalizating titanium surfaces by silanization and subsequent covalent binding of a peptide with a sequence that promotes cell adhesion. Objective: The development of a new technique of immobilization of oligopeptides on the surface of titanium by using 3-chloropropyltrietoxisilane (CPTES) as bonding agent between the surface of titanium and the peptide. Materials and methods: A physicochemical characterization of the surfaces through the techniques of XPS, ToF-SIMS and contact angle was performed. Also cell adhesion studies have been conducted to evaluate in vitro biological response. Results: Through the process of silanization the titanium surface is completely covered with CPTES, which allows the subsequent accession of oligopeptides. The cell adhesion results show a higher cell adhesion and cell extension on biofunctionalized samples. Conclusions: We developed a system of covalent binding of oligopeptides on titanium surfaces that can modify the biological response of the attached cells.

  4. A novel receptor-targeted gene delivery system for cancer gene therapy

    Institute of Scientific and Technical Information of China (English)

    田培坤; 任圣俊; 任常春; 滕青山; 曲淑敏; 姚明; 顾健人

    1999-01-01

    Some growth factor receptors, such as insulin like growth factor Ⅰ and Ⅱ receptor (IGF Ⅰ R, IGF Ⅱ R) and epidermal growth factor receptor (EGF R), have been proved to be over-expressed in a variety of human cancers derived from different tissue origins. Based on this molecular alteration, a polypeptide conjugate gene delivery system was designed and synthesized. It contains three essential moieties: a ligand oligopeptide (LOP) for receptor recognition, a polycationic polypeptide (PCP) such as protamine (PA) or poly-L-lysine (PL) as a backbone for DNA binding and an endosome-releasing oligopeptide (EROP) such as influenza baenagglutinin oligopeptide (HA20) for endosomolysis. These components are covalently conjugated as LOP-PCP-HA20 or in the form of a mixture of LOP-PCP and HA20-PCP. A 14 amino acid E5 was designed and synthesized as LOP for IGF Ⅰ R and IGF Ⅱ R, and a 16 amino acid GE7 as LOP for EGF R. Both E5 and GE7 systems could form stable complex with the plasmid DNA as E5-PCP/DNA/PCP-HA20 a

  5. Biofunctionalization of titanium surfaces for osseintegration process improvement

    Science.gov (United States)

    Sevilla, P.; Godoy, M.; Salvagni, E.; Rodríguez, D.; Gil, F. J.

    2010-11-01

    This study aims to improve the osseointegration of titanium implants through surface immobilization of peptides that induce a beneficial biological response. This was carried out biofunctionalizating titanium surfaces by silanization and subsequent covalent binding of a peptide with a sequence that promotes cell adhesion. Objective: The development of a new technique of immobilization of oligopeptides on the surface of titanium by using 3-chloropropyltrietoxisilane (CPTES) as bonding agent between the surface of titanium and the peptide. Materials and methods: A physicochemical characterization of the surfaces through the techniques of XPS, ToF-SIMS and contact angle was performed. Also cell adhesion studies have been conducted to evaluate in vitro biological response. Results: Through the process of silanization the titanium surface is completely covered with CPTES, which allows the subsequent accession of oligopeptides. The cell adhesion results show a higher cell adhesion and cell extension on biofunctionalized samples. Conclusions: We developed a system of covalent binding of oligopeptides on titanium surfaces that can modify the biological response of the attached cells.

  6. Morphologic, Phylogenetic and Chemical Characterization of a Brackish Colonial Picocyanobacterium (Coelosphaeriaceae with Bioactive Properties

    Directory of Open Access Journals (Sweden)

    Kerstin Häggqvist

    2016-04-01

    Full Text Available Despite their cosmopolitan distribution, knowledge on cyanobacteria in the family Coelosphaeriaceae is limited. In this study, a single species culture of a coelosphaeran cyanobacterium isolated from a brackish rock pool in the Baltic Sea was established. The strain was characterized by morphological features, partial 16S rRNA sequence and nonribosomal oligopeptide profile. The bioactivity of fractionated extracts against several serine proteases, as well as protein-serine/threonine phosphatases was studied. Phylogenetic analyses of the strain suggested a close relationship with Snowella litoralis, but its morphology resembled Woronichinia compacta. The controversial morphologic and phylogenetic results demonstrated remaining uncertainties regarding species division in this cyanobacteria family. Chemical analyses of the strain indicated production of nonribosomal oligopeptides. In fractionated extracts, masses and ion fragmentation spectra of seven possible anabaenopeptins were identified. Additionally, fragmentation spectra of cyanopeptolin-like peptides were collected in several of the fractions. The nonribosomal oligopeptide profile adds another potential identification criterion in future inter- and intraspecies comparisons of coelosphaeran cyanobacteria. The fractionated extracts showed significant activity against carboxypeptidase A and trypsin. Inhibition of these important metabolic enzymes might have impacts at the ecosystem level in aquatic habitats with high cyanobacteria densities.

  7. Pig kidney legumain: an asparaginyl endopeptidase with restricted specificity.

    Science.gov (United States)

    Dando, P M; Fortunato, M; Smith, L; Knight, C G; McKendrick, J E; Barrett, A J

    1999-01-01

    Legumain was recently discovered as a lysosomal endopeptidase in mammals [Chen, Dando, Rawlings, Brown, Young, Stevens, Hewitt, Watts and Barrett (1997) J. Biol. Chem. 272, 8090-8098], having been known previously only from plants and invertebrates. It has been shown to play a key role in processing of the C fragment of tetanus toxin for presentation by the MHC class-II system [Manoury, Hewitt, Morrice, Dando, Barrett and Watts (1998) Nature (London) 396, 695-699]. We examine here the specificity of the enzyme from pig kidney by use of protein, oligopeptide and synthetic arylamide substrates, all determinations being made at pH 5.8. In proteins, only about one in ten of the asparaginyl bonds were hydrolysed, and these were mostly predicted to be located at turns on the protein surface. Bonds that were not cleaved in tetanus toxin were cleaved when presented in oligopeptides, sometimes faster than an equivalent oligopeptide based on a bond that was cleaved in the protein. Legumain cleaved the bait region of rat alpha1-macroglobulin and was 'trapped' by the macroglobulin, as most other endopeptidases are, but did not interact with human alpha2-macroglobulin, which contains no asparagine residue in its bait region. Glycosylation of asparagine totally prevented hydrolysis by legumain. Specificity for arylamide substrates was evaluated with reference to benzyloxycarbonyl-Ala-Ala-Asn-aminomethylcoumarin, and the preference for the P3-position amino acid was Ala>Tyr(tertiary butyl)>Val>Pro>Phe=Tyr>Leu=Gly. There was no hydrolysis of substrate analogues containing mono- or di-N-methylasparagines, l-2-amino-3-ureidopropionic acid or citrulline in the P1 position. We conclude that mammalian legumain appears to be totally restricted to the hydrolysis of asparaginyl bonds in substrates of all kinds. There seem to be no strong preferences for particular amino acids in other subsites, and yet there are still unidentified factors that prevent hydrolysis of many asparaginyl bonds

  8. Molecular simulation of the reversible mechanical unfolding of proteins.

    Science.gov (United States)

    Rathore, Nitin; Yan, Qiliang; de Pablo, Juan J

    2004-03-22

    In this work we have combined a Wang-Landau sampling scheme [F. Wang and D. Landau, Phys. Rev. Lett. 86, 2050 (2001)] with an expanded ensemble formalism to yield a simple and powerful method for computing potentials of mean force. The new method is implemented to investigate the mechanical deformation of proteins. Comparisons are made with analytical results for simple model systems such as harmonic springs and Rouse chains. The method is then illustrated on a model 15-residue alanine molecule in an implicit solvent. Results for mechanical unfolding of this oligopeptide are compared to those of steered molecular dynamics calculations.

  9. Integration of Biomolecules with Metal-Organic Frameworks.

    Science.gov (United States)

    Zhuang, Jia; Young, Allison P; Tsung, Chia-Kuang

    2017-08-01

    Owing to the progressive development of metal-organic-frameworks (MOFs) synthetic processes and considerable potential applications in last decade, integrating biomolecules into MOFs has recently gain considerable attention. Biomolecules, including lipids, oligopeptides, nucleic acids, and proteins have been readily incorporated into MOF systems via versatile formulation methods. The formed biomolecule-MOF hybrid structures have shown promising prospects in various fields, such as antitumor treatment, gene delivery, biomolecular sensing, and nanomotor device. By optimizing biomolecule integration methods while overcoming existing challenges, biomolecule-integrated MOF platforms are very promising to generate more practical applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Total synthesis of feglymycin based on a linear/convergent hybrid approach using micro-flow amide bond formation

    Science.gov (United States)

    Fuse, Shinichiro; Mifune, Yuto; Nakamura, Hiroyuki; Tanaka, Hiroshi

    2016-11-01

    Feglymycin is a naturally occurring, anti-HIV and antimicrobial 13-mer peptide that includes highly racemizable 3,5-dihydroxyphenylglycines (Dpgs). Here we describe the total synthesis of feglymycin based on a linear/convergent hybrid approach. Our originally developed micro-flow amide bond formation enabled highly racemizable peptide chain elongation based on a linear approach that was previously considered impossible. Our developed approach will enable the practical preparation of biologically active oligopeptides that contain highly racemizable amino acids, which are attractive drug candidates.

  11. Derivation and Testing of Explicit Equations of Motion for Polymers Described by Internal Coordinates

    Science.gov (United States)

    Mazur, A. K.; Dorofeev, V. E.; Abagyan, R. A.

    1991-02-01

    General Lagrange's equations of motion for a system of polymeric molecules are obtained in an explicit form. They can be used for simulating molecular dynamics of large molecules. The molecular conformations are described by internal coordinates, i.e., bond lengths, valence angles, and torsion angles. The equations derived permit any internal degrees of freedom to be frozen. The method is applied to an oligopeptide in an α-helical conformation. Three models of the molecule with different degrees of fixation are compared. It is shown that the method permits one to increase significantly the time step in molecular dynamics calculations.

  12. Non-protein amino acids in peptide design

    Indian Academy of Sciences (India)

    S Aravinda; N Shamala; Rituparna S Roy; P Balaram

    2003-10-01

    An overview of the use of non-protein amino acids in the design of conformationally well-defined peptides, based on work from the author’s laboratory, is discussed. The crystal structures of several designed oligopeptides illustrate the use -aminoisobutyric acid (Aib) in the construction of helices, D-amino acids in the design of helix termination segments and DPro-Xxx segments for nucleating of -hairpin structures. - and -amino acid residues have been used to expand the range of designed polypeptide structures.

  13. Measles virus-substance P receptor interactions. Possible novel mechanism of viral fusion.

    OpenAIRE

    Harrowe, G; Mitsuhashi, M; Payan, D G

    1990-01-01

    Measles virus (MV) encodes the fusion protein (F) that mediates cell fusion and intercellular spread of the virus, and is homologous to the carboxy terminus of the neuropeptide substance P (SP). In addition, the oligopeptide Z-D-Phe-L-Phe-Gly, also homologous to F and SP, inhibits MV fusion with target cells. These observations raise the question of whether MV uses the SP receptor (SPR) during a specific phase of its infectious cycle. In this report, we examine the structural and functional c...

  14. Solution structure of a syndecan-4 cytoplasmic domain and its interaction with phosphatidylinositol 4,5-bisphosphate

    DEFF Research Database (Denmark)

    Lee, D; Oh, E S; Woods, A

    1998-01-01

    Syndecan-4, a transmembrane heparan sulfate proteoglycan, is a coreceptor with integrins in cell adhesion. It has been suggested to form a ternary signaling complex with protein kinase Calpha and phosphatidylinositol 4,5-bisphosphate (PIP2). Syndecans each have a unique, central, and variable (V......) region in their cytoplasmic domains, and that of syndecan-4 is critical to its interaction with protein kinase C and PIP2. Two oligopeptides corresponding to the variable region (4V) and whole domain (4L) of syndecan-4 cytoplasmic domain were synthesized for nuclear magnetic resonance (NMR) studies. Data...

  15. Remote Control of the Planar Chirality in Peptide-Bound Metallomacrocycles and Dynamic-to-Static Planar Chirality Control Triggered by Solvent-Induced 3(10)-to-α-Helix Transitions.

    Science.gov (United States)

    Mamiya, Fumihiko; Ousaka, Naoki; Yashima, Eiji

    2015-11-23

    The dynamic planar chirality in a peptide-bound Ni(II)-salphen-based macrocycle can be remotely controlled. First, a right-handed (P)-3(10)-helix is induced in the dynamic helical oligopeptides by a chiral amino acid residue far from the macrocyclic framework. The induced planar chirality remains dynamic in chloroform and acetonitrile, but is almost completely locked in fluoroalcohols as a result of the solvent-induced transition of the peptide chains from a 3(10)-helix to a wider α-helix, which freezes the rotation of the pendant peptide units around the macrocycle.

  16. UNIQUE ORAL DRUG DELIVERY SYSTEM

    Institute of Scientific and Technical Information of China (English)

    Raphael M. Ottenbrite; ZHAO Ruifeng; Sam Milstein

    1995-01-01

    An oral drug delivery system using proteinoid microspheres is discussed with respect to its unique dependence on pH. It has been found that certain drugs such as insulin and heparin can be encapsulated in proteinoid spheres at stomach pH's (1-3). These spheres also dissemble at intestinal pH's (6-7) releasing the drug for absorption. Using this technique low molecular weight heparin and human growth hormone have been orally delivered successfully to several animal species. Future work has been proposed to study the interaction and binding of the specific drugs with synthesized oligopeptides.

  17. Biased versus unbiased randomness in homo-polymers and copolymers of amino acids in the prebiotic world.

    Science.gov (United States)

    Mosqueira, Fernando G; Negron, Alicia; Ramos, Sergio; Polanco, Carlos

    2012-01-01

    The polymerization of amino acids under anhydrous prebiotic conditions was first studied several decades ago. Here we use a stochastic model stressing the relevant role of the polarity of amino acids in the formation of oligopeptides in a prebiotic milieu. Our goal is to outline the predominance of co-polypeptides over homo-polypeptides, resulting not only from the randomness, but also from polarity properties of amino acids. Our results conclude that there was a higher probability of the formation of co-polypeptides than of homo-polymers. Besides, we may hypothesize that the former would have a more ample spectrum of possible chemical functions than homo-polypeptides.

  18. A robust method of determination of high concentrations of peptides and proteins

    DEFF Research Database (Denmark)

    Levashov, Pavel A; Sutherland, Duncan S; Besenbacher, Flemming

    2009-01-01

    In this paper, we pioneer application of a unique method of protein determination by coloring peptide bonds for analysis of a variety of biomolecules with different grades of purity (e.g., oligopeptides, membrane, and glycol proteins). We demonstrated that the calibration curve for all studied...... molecules is universal and linear within 0.1 to 1.2mg protein content range. The assay thus can be used to analyze peptides without preliminary dilutions and calibration in up to 1g/ml solutions of peptides, which is crucial for many biotechnological processes, such as development of coatings, scaffolds...

  19. Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth

    DEFF Research Database (Denmark)

    Jakociune, Dziuginta; Herrero-Fresno, Ana; Jelsbak, Lotte

    2016-01-01

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common cause of egg borne salmonellosis in many parts of the world. This study analyzed gene expression of this bacterium during growth in whole egg, and whether highly expressed genes were essential for the growth. High quality......, di/oligopeptide transport system, biotin synthesis, ferrous iron transport system, and type III secretion system. Significant downregulation of 15 genes related to formate hydrogenlyase (FHL) and trehalose metabolism was observed. The results suggested that S. Enteritidis is starved for amino...

  20. Insight into the packing pattern of β2 fibrils: a model study of glutamic acid rich oligomers with 13C isotopic edited vibrational spectroscopy.

    Science.gov (United States)

    Chi, Heng; Welch, William R W; Kubelka, Jan; Keiderling, Timothy A

    2013-11-11

    Polyglutamic acid at low pH forms aggregates and self-assembles into a spiral, fibril-like superstructure formed as a β2-type sheet conformation that has a more compact intersheet packing than commonly found. This is stabilized by three-centered bifurcated hydrogen bonding of the amide carbonyl involving the protonated glutamic acid side chain. We report vibrational spectroscopic results and analyses for oligopeptides rich in glutamic acid enhanced with (13)C isotope labeling in a study modeling low pH poly-Glu self-assembly. Our results indicate bifurcated H-bonding and β2 aggregation can be attained in these model decamers, confirming they have the same conformations as poly-Glu. We also prepared conventional β1-sheet aggregates by rapid precipitation from the residual peptides in the higher pH supernatant. By comparing the isotope-enhanced IR and VCD spectra with theoretical predictions, we deduced that the oligo-Glu β2 structure is based on stacked, twisted, antiparallel β-sheets. The best fit to theoretical predictions was obtained for the strands being out of register, sequentially stepped by one residue, in a ladder-like fashion. The alternate β1 conformer for this oligopeptide was similarly shown to be antiparallel but was less ordered and apparently had a different registry in its aggregate structure.

  1. Identification of immunogenic proteins and generation of antibodies against Salmonella Typhimurium using phage display

    Directory of Open Access Journals (Sweden)

    Meyer Torsten

    2012-06-01

    Full Text Available Abstract Background Solely in Europoe, Salmonella Typhimurium causes more than 100,000 infections per year. Improved detection of livestock colonised with S. Typhimurium is necessary to prevent foodborne diseases. Currently, commercially available ELISA assays are based on a mixture of O-antigens (LPS or total cell lysate of Salmonella and are hampered by cross-reaction. The identification of novel immunogenic proteins would be useful to develop ELISA based diagnostic assays with a higher specificity. Results A phage display library of the entire Salmonella Typhimurium genome was constructed and 47 immunogenic oligopeptides were identified using a pool of convalescent sera from pigs infected with Salmonella Typhimurium. The corresponding complete genes of seven of the identified oligopeptids were cloned. Five of them were produced in E. coli. The immunogenic character of these antigens was validated with sera from pigs infeced with S. Tyhimurium and control sera from non-infected animals. Finally, human antibody fragments (scFv against these five antigens were selected using antibody phage display and characterised. Conclusion In this work, we identified novel immunogenic proteins of Salmonella Typhimurium and generated antibody fragments against these antigens completely based on phage display. Five immunogenic proteins were validated using a panel of positive and negative sera for prospective applications in diagnostics of Salmonela Typhimurium.

  2. Effective representation of amide III, II, I, and A modes on local vibrational modes: Analysis of ab initio quantum calculation results

    Science.gov (United States)

    Hahn, Seungsoo

    2016-10-01

    The Hamiltonian matrix for the first excited vibrational states of a protein can be effectively represented by local vibrational modes constituting amide III, II, I, and A modes to simulate various vibrational spectra. Methods for obtaining the Hamiltonian matrix from ab initio quantum calculation results are discussed, where the methods consist of three steps: selection of local vibrational mode coordinates, calculation of a reduced Hessian matrix, and extraction of the Hamiltonian matrix from the Hessian matrix. We introduce several methods for each step. The methods were assessed based on the density functional theory calculation results of 24 oligopeptides with four different peptide lengths and six different secondary structures. The completeness of a Hamiltonian matrix represented in the reduced local mode space is improved by adopting a specific atom group for each amide mode and reducing the effect of ignored local modes. The calculation results are also compared to previous models using C=O stretching vibration and transition dipole couplings. We found that local electric transition dipole moments of the amide modes are mainly bound on the local peptide planes. Their direction and magnitude are well conserved except amide A modes, which show large variation. Contrary to amide I modes, the vibrational coupling constants of amide III, II, and A modes obtained by analysis of a dipeptide are not transferable to oligopeptides with the same secondary conformation because coupling constants are affected by the surrounding atomic environment.

  3. ASSESSMENT OF THE DEGREE OF INTOXICATION AND THE FORECAST LEVEL OF THE INTEGRAL INDEX OF INTOXICATION UNDER CERTAIN INFECTIOUS DISEASES

    Directory of Open Access Journals (Sweden)

    M. Y. Marzhokhova

    2016-01-01

    Full Text Available The Purpose of this research was to study indicators syndrome of endogenous intoxication in patients with erysipelas and herpes infection that can be used to predict relapse in these diseases.Based on the goal were examined in 54 patients with primary, erythematous form of erysipelas of different localization and 42 patients with the localized form of herpes infection in the dynamics of these diseases.Determined the level of substances of low and medium molecular weight (HV and SMM, and oligopeptides, as well as the Integral index of intoxication by the method of M. I. Malakhov (1994 and Lowry (1991. To assess the value of the Integral index of intoxication in the prediction of relapses were calculated odds ratio.Revealed the dependence of studied parameters of disease and completeness of recovery.Level measurement HV and SMM and oligopeptides allows us to calculate the Integral index of intoxication, which can be used as a prognostic criterion of recurrence as erysipelas, and herpes infection.

  4. Binding of leachable components of polymethyl methacrylate (PMMA) and peptide on modified SPR chip

    Science.gov (United States)

    Szaloki, M.; Vitalyos, G.; Harfalvi, J.; Hegedus, Cs

    2013-12-01

    Many types of polymers are often used in dentistry, which may cause allergic reaction, mainly methyl methacrylate allergy due to the leachable, degradable components of polymerized dental products. The aim of this study was to investigate the interaction between the leachable components of PMMA and peptides by Fourier-transform Surface Plasmon Resonance (FT SPR). In our previous work binding of oligopeptides (Ph.D.-7 and Ph.D.-12 Peptide Library Kit) was investigated to PMMA surface by phage display technique. It was found that oligopeptides bounded specifically to PMMA surface. The most common amino acids were leucine and proline inside the amino acids sequences of DNA of phages. The binding of haptens, as formaldehyde and methacrylic acid, to frequent amino acids was to investigate on the modified gold SPR chip. Self assembled monolayer (SAM) modified the surface of gold chip and ensured the specific binding between the haptens and amino acids. It was found that amino acids bounded to modified SPR gold and the haptens bounded to amino acids by creating multilayer on the chip surface. By the application of phage display and SPR modern bioanalytical methods the interaction between allergens and peptides can be investigated.

  5. Characterization of a New Cell Envelope Proteinase PrtP from Lactobacillus rhamnosus CGMCC11055.

    Science.gov (United States)

    Guo, Tingting; Ouyang, Xudong; Xin, Yongping; Wang, Yue; Zhang, Susu; Kong, Jian

    2016-09-21

    Cell envelope proteinases (CEPs) play essential roles in lactic acid bacteria growth in milk and health-promoting properties of fermented dairy products. The genome of Lactobacillus rhamnosus CGMCC11055 possesses two putative CEP genes prtP and prtR2, and the PrtP displays the distinctive domain organization from PrtR2 reported. The PrtP was purified and biochemically characterized. The results showed that the optimal activity occurred at 44 °C, pH 6.5. p-Amidinophenylmethylsulfonyl fluoride obviously inhibited enzymatic activity, suggesting PrtP was a member of serine proteinases. Under the optimal conditions, β-casein was a favorite substrate over αS1- and κ-casein, and 35 oligopeptides were identified in the β-casein hydrolysate, including the phosphoserine peptide and bioactive isoleucine-proline-proline. By analysis of the amino acid sequences of those oligopeptides, proline was the preferred residue at the breakdown site. Therefore, we speculated that PrtP was a new type of CEPs from Lb. rhamnosus.

  6. A novel gene delivery system targeting cells expressing VEGF receptors

    Institute of Scientific and Technical Information of China (English)

    LIJUNMIN; JINGCHULUO; 等

    1999-01-01

    Two ligand oligopeptides GV1 and GV2 were designed according to the putative binding region of VEGF to its receptors.GV1,GV2 and endosome releasing oligopeptide HA20 were conjugated with poly-L-lysine or protamine and the resulting conjugates could interact with DNA in a noncovalent bond to form a complex.Using pSV2-β-galactosidase as a reporter gene,it has been demonstrated that exogenous gene was transferred into bovine aortic arch-derived endothelial cells (ABAE) and human malignant melanoma cell lines (A375) in vitro.In vivo experiments,exogenous gene was transferred into tumor vascular endothelial cells and tumor cells of subcutaneously transplanted human colon cancer LOVO,human malignant melanoma A375 and human hepatoma graft in nude mice.This system could also target gene to intrahepatically transplanted human hepatoma injected via portal vein in nude mice.These results are correlated with the relevant receptors(flt-1,flk-1/KDR) expression on the targeted cells and tissues.

  7. Collagen peptide and vitamin C additively attenuate age-related skin atrophy in Sod1-deficient mice.

    Science.gov (United States)

    Shibuya, Shuichi; Ozawa, Yusuke; Toda, Toshihiko; Watanabe, Kenji; Tometsuka, Chisa; Ogura, Takayuki; Koyama, Yoh-ichi; Shimizu, Takahiko

    2014-01-01

    Age-related skin thinning is correlated with a decrease in the content of collagen in the skin. Accumulating evidence suggests that collagen peptide (CP) and vitamin C (VC) transcriptionally upregulate type I collagen in vivo. However, the additive effects of CP and VC on age-related skin changes remain unclear. We herein demonstrate that CP and a VC derivative additively corrected age-related skin thinning via reduced oxidative damage in superoxide dismutase 1 (Sod1)-deficient mice. Co-treatment with these compounds significantly normalized the altered gene expression of Col1a1, Has2, and Ci1, a proton-coupled oligopeptide transporter, in Sod1(-/-) skin. The in vitro analyses further revealed that collagen oligopeptide, a digestive product of ingested CP, significantly promoted the bioactivity of the VC derivative with respect to the migration and proliferation of Sod1(-/-) fibroblasts. These findings suggest that combined treatment with CP and VC is effective in cases of age-related skin pathology.

  8. Enhanced Absorption and Growth Inhibition with Amino Acid Monoester Prodrugs of Floxuridine by Targeting hPEPT1 Transporters

    Directory of Open Access Journals (Sweden)

    Gordon L. Amidon

    2008-06-01

    Full Text Available A series of amino acid monoester prodrugs of floxuridine was synthesized and evaluated for the improvement of oral bioavailability and the feasibility of target drug delivery via oligopeptide transporters. All floxuridine 5′-amino acid monoester prodrugs exhibited PEPT1 affinity, with inhibition coefficients of Gly-Sar uptake (IC50 ranging from 0.7 – 2.3 mM in Caco-2 and 2.0 – 4.8 mM in AsPC-1 cells, while that of floxuridine was 7.3 mM and 6.3 mM, respectively. Caco-2 membrane permeabilities of floxuridine prodrugs (1.01 – 5.31 x 10-6 cm/sec and floxuridine (0.48 x 10-6 cm/sec were much higher than that of 5-FU (0.038 x 10-6 cm/sec. MDCK cells stably transfected with the human oligopeptide transporter PEPT1 (MDCK/hPEPT1 exhibited enhanced cell growth inhibition in the presence of the prodrugs. This prodrug strategy offers great potential, not only for increased drug absorption but also for improved tumor selectivity and drug efficacy.

  9. Comparative proteomic analysis of experimental evolution of the Bacillus cereus-Ketogulonicigenium vulgare co-culture.

    Directory of Open Access Journals (Sweden)

    Qian Ma

    Full Text Available The microbial co-culture system composing of Ketogulonicigenium vulgare and Bacillus cereus was widely adopted in industry for the production of 2-keto-gulonic acid (2-KGA, the precursor of vitamin C. We found serial subcultivation of the co-culture could enhance the yield of 2-KGA by 16% in comparison to that of the ancestral co-culture. To elucidate the evolutionary dynamics and interaction mechanisms of the two microbes, we performed iTRAQ-based quantitative proteomic analyses of the pure cultures of K. vulgare, B. cereus and their co-culture during serial subcultivation. Hierarchy cluster analyses of the proteomic data showed that the expression level of a number of crucial proteins associated with sorbose conversion and oligopeptide transport was significantly enhanced by the experimental evolution. In particular, the expression level of sorbose/sorbosone dehydrogenase was enhanced in the evolved K. vulgare, while the expression level of InhA and the transport efficiency of oligopeptides were increased in the evolved B. cereus. The decreased sporulating protein expression and increased peptide transporter expression observed in evolved B. cereus, together with the increased amino acids synthesis in evolved K. vulgare suggested that serial subcultivation result in enhanced synergistic cooperation between K. vulgare and B. cereus, enabling an increased production of 2-KGA.

  10. Common Genetic Denominators for Ca++-Based Skeleton in Metazoa: Role of Osteoclast-Stimulating Factor and of Carbonic Anhydrase in a Calcareous Sponge

    Science.gov (United States)

    Müller, Werner E. G.; Wang, Xiaohong; Grebenjuk, Vlad A.; Korzhev, Michael; Wiens, Matthias; Schloßmacher, Ute; Schröder, Heinz C.

    2012-01-01

    Calcium-based matrices serve predominantly as inorganic, hard skeletal systems in Metazoa from calcareous sponges [phylum Porifera; class Calcarea] to proto- and deuterostomian multicellular animals. The calcareous sponges form their skeletal elements, the spicules, from amorphous calcium carbonate (ACC). Treatment of spicules from Sycon raphanus with sodium hypochlorite (NaOCl) results in the disintegration of the ACC in those skeletal elements. Until now a distinct protein/enzyme involved in ACC metabolism could not been identified in those animals. We applied the technique of phage display combinatorial libraries to identify oligopeptides that bind to NaOCl-treated spicules: those oligopeptides allowed us to detect proteins that bind to those spicules. Two molecules have been identified, the (putative) enzyme carbonic anhydrase and the (putative) osteoclast-stimulating factor (OSTF), that are involved in the catabolism of ACC. The complete cDNAs were isolated and the recombinant proteins were prepared to raise antibodies. In turn, immunofluorescence staining of tissue slices and qPCR analyses have been performed. The data show that sponges, cultivated under standard condition (10 mM CaCl2) show low levels of transcripts/proteins for carbonic anhydrase or OSTF, compared to those animals that had been cultivated under Ca2+-depletion condition (1 mM CaCl2). Our data identify with the carbonic anhydrase and the OSTF the first two molecules which remain conserved in cells, potentially involved in Ca-based skeletal dissolution, from sponges (sclerocytes) to human (osteoclast). PMID:22506035

  11. Common genetic denominators for Ca++-based skeleton in Metazoa: role of osteoclast-stimulating factor and of carbonic anhydrase in a calcareous sponge.

    Science.gov (United States)

    Müller, Werner E G; Wang, Xiaohong; Grebenjuk, Vlad A; Korzhev, Michael; Wiens, Matthias; Schlossmacher, Ute; Schröder, Heinz C

    2012-01-01

    Calcium-based matrices serve predominantly as inorganic, hard skeletal systems in Metazoa from calcareous sponges [phylum Porifera; class Calcarea] to proto- and deuterostomian multicellular animals. The calcareous sponges form their skeletal elements, the spicules, from amorphous calcium carbonate (ACC). Treatment of spicules from Sycon raphanus with sodium hypochlorite (NaOCl) results in the disintegration of the ACC in those skeletal elements. Until now a distinct protein/enzyme involved in ACC metabolism could not been identified in those animals. We applied the technique of phage display combinatorial libraries to identify oligopeptides that bind to NaOCl-treated spicules: those oligopeptides allowed us to detect proteins that bind to those spicules. Two molecules have been identified, the (putative) enzyme carbonic anhydrase and the (putative) osteoclast-stimulating factor (OSTF), that are involved in the catabolism of ACC. The complete cDNAs were isolated and the recombinant proteins were prepared to raise antibodies. In turn, immunofluorescence staining of tissue slices and qPCR analyses have been performed. The data show that sponges, cultivated under standard condition (10 mM CaCl(2)) show low levels of transcripts/proteins for carbonic anhydrase or OSTF, compared to those animals that had been cultivated under Ca(2+)-depletion condition (1 mM CaCl(2)). Our data identify with the carbonic anhydrase and the OSTF the first two molecules which remain conserved in cells, potentially involved in Ca-based skeletal dissolution, from sponges (sclerocytes) to human (osteoclast).

  12. In Vitro Bioactivity Study of RGD-Coated Titanium Alloy Prothesis for Revision Total Hip Arthroplasty

    Directory of Open Access Journals (Sweden)

    Zhentao Man

    2016-01-01

    Full Text Available Total hip arthroplasty (THA is a common procedure for the treatment of end-stage hip joint disease, and the demand for revision THA will double by 2026. Ti6Al4V (Titanium, 6% Aluminum, and 4% Vanadium is a kind of alloy commonly used to make hip prothesis. To promote the osseointegration between the prothesis and host bone is very important for the revision THA. The peptide Arg-Gly-Asp (RGD could increase cell attachment and has been used in the vascular tissue engineering. In this study, we combined the RGD with Ti6Al4V alloy using the covalent cross-linking method to fabricate the functional Ti6Al4V alloy (FTA. The distribution of RGD oligopeptide on the FTA was even and homogeneous. The FTA scaffolds could promote mouse osteoblasts adhesion and spreading. Furthermore, the result of RT-qPCR indicated that the FTA scaffolds were more beneficial to osteogenesis, which may be due to the improvement of osteoblast adhesion by the RGD oligopeptide coated on FTA. Overall, the FTA scaffolds developed herein pave the road for designing and building more efficient prothesis for osseointegration between the host bone and prothesis in revision THA.

  13. Intracellular Peptides as Natural Regulators of Cell Signaling*S⃞

    Science.gov (United States)

    Cunha, Fernanda M.; Berti, Denise A.; Ferreira, Zulma S.; Klitzke, Clécio F.; Markus, Regina P.; Ferro, Emer S.

    2008-01-01

    Protein degradation by the ubiquitin proteasome system releases large amounts of oligopeptides within cells. To investigate possible functions for these intracellularly generated oligopeptides, we fused them to a cationic transactivator peptide sequence using reversible disulfide bonds, introduced them into cells, and analyzed their effect on G protein-coupled receptor (GPCR) signal transduction. A mixture containing four of these peptides (20–80 μm) significantly inhibited the increase in the extracellular acidification response triggered by angiotensin II (ang II) in CHO-S cells transfected with the ang II type 1 receptor (AT1R-CHO-S). Subsequently, either alone or in a mixture, these peptides increased luciferase gene transcription in AT1R CHO-S cells stimulated with ang II and in HEK293 cells treated with isoproterenol. These peptides without transactivator failed to affect GPCR cellular responses. All four functional peptides were shown in vitro to competitively inhibit the degradation of a synthetic substrate by thimet oligopeptidase. Overexpression of thimet oligopeptidase in both CHO-S and HEK293 cells was sufficient to reduce luciferase activation triggered by a specific GPCR agonist. Moreover, using individual peptides as baits in affinity columns, several proteins involved in GPCR signaling were identified, including α-adaptin A and dynamin 1. These results suggest that before their complete degradation, intracellular peptides similar to those generated by proteasomes can actively affect cell signaling, probably representing additional bioactive molecules within cells. PMID:18617518

  14. Intracellular peptides as natural regulators of cell signaling.

    Science.gov (United States)

    Cunha, Fernanda M; Berti, Denise A; Ferreira, Zulma S; Klitzke, Clécio F; Markus, Regina P; Ferro, Emer S

    2008-09-05

    Protein degradation by the ubiquitin proteasome system releases large amounts of oligopeptides within cells. To investigate possible functions for these intracellularly generated oligopeptides, we fused them to a cationic transactivator peptide sequence using reversible disulfide bonds, introduced them into cells, and analyzed their effect on G protein-coupled receptor (GPCR) signal transduction. A mixture containing four of these peptides (20-80 microm) significantly inhibited the increase in the extracellular acidification response triggered by angiotensin II (ang II) in CHO-S cells transfected with the ang II type 1 receptor (AT1R-CHO-S). Subsequently, either alone or in a mixture, these peptides increased luciferase gene transcription in AT1R CHO-S cells stimulated with ang II and in HEK293 cells treated with isoproterenol. These peptides without transactivator failed to affect GPCR cellular responses. All four functional peptides were shown in vitro to competitively inhibit the degradation of a synthetic substrate by thimet oligopeptidase. Overexpression of thimet oligopeptidase in both CHO-S and HEK293 cells was sufficient to reduce luciferase activation triggered by a specific GPCR agonist. Moreover, using individual peptides as baits in affinity columns, several proteins involved in GPCR signaling were identified, including alpha-adaptin A and dynamin 1. These results suggest that before their complete degradation, intracellular peptides similar to those generated by proteasomes can actively affect cell signaling, probably representing additional bioactive molecules within cells.

  15. Conformational distributions of denatured and unstructured proteins are similar to those of 20 Multiplication-Sign 20 blocked dipeptides

    Energy Technology Data Exchange (ETDEWEB)

    Oh, Kwang-Im [Korea University, Department of Chemistry (Korea, Republic of); Jung, Young-Sang; Hwang, Geum-Sook, E-mail: gshwang@kbsi.re.kr [Korea Basic Science Institute (Korea, Republic of); Cho, Minhaeng, E-mail: mcho@korea.ac.kr [Korea University, Department of Chemistry (Korea, Republic of)

    2012-05-15

    Understanding intrinsic conformational preferences of amino-acids in unfolded proteins is important for elucidating the underlying principles of their stability and re-folding on biological timescales. Here, to investigate the neighbor interaction effects on the conformational propensities of amino-acids, we carried out {sup 1}H NMR experiments for a comprehensive set of blocked dipeptides and measured the scalar coupling constants between alpha protons and amide protons as well as their chemical shifts. Detailed inspection of these NMR properties shows that, irrespective of amino-acid side-chain properties, the distributions of the measured coupling constants and chemical shifts of the dipeptides are comparatively narrow, indicating small variances of their conformation distributions. They are further compared with those of blocked amino-acids (Ac-X-NHMe), oligopeptides (Ac-GGXGG-NH{sub 2}), and native (lysozyme), denatured (lysozyme and outer membrane protein X from Escherichia coli), unstructured (Domain 2 of the protein 5A of Hepatitis C virus), and intrinsically disordered (hNlg3cyt: intracellular domain of human NL3) proteins. These comparative investigations suggest that the conformational preferences and local solvation environments of the blocked dipeptides are quite similar to not only those of other short oligopeptides but also those of denatured and natively unfolded proteins.

  16. Common genetic denominators for Ca++-based skeleton in Metazoa: role of osteoclast-stimulating factor and of carbonic anhydrase in a calcareous sponge.

    Directory of Open Access Journals (Sweden)

    Werner E G Müller

    Full Text Available Calcium-based matrices serve predominantly as inorganic, hard skeletal systems in Metazoa from calcareous sponges [phylum Porifera; class Calcarea] to proto- and deuterostomian multicellular animals. The calcareous sponges form their skeletal elements, the spicules, from amorphous calcium carbonate (ACC. Treatment of spicules from Sycon raphanus with sodium hypochlorite (NaOCl results in the disintegration of the ACC in those skeletal elements. Until now a distinct protein/enzyme involved in ACC metabolism could not been identified in those animals. We applied the technique of phage display combinatorial libraries to identify oligopeptides that bind to NaOCl-treated spicules: those oligopeptides allowed us to detect proteins that bind to those spicules. Two molecules have been identified, the (putative enzyme carbonic anhydrase and the (putative osteoclast-stimulating factor (OSTF, that are involved in the catabolism of ACC. The complete cDNAs were isolated and the recombinant proteins were prepared to raise antibodies. In turn, immunofluorescence staining of tissue slices and qPCR analyses have been performed. The data show that sponges, cultivated under standard condition (10 mM CaCl(2 show low levels of transcripts/proteins for carbonic anhydrase or OSTF, compared to those animals that had been cultivated under Ca(2+-depletion condition (1 mM CaCl(2. Our data identify with the carbonic anhydrase and the OSTF the first two molecules which remain conserved in cells, potentially involved in Ca-based skeletal dissolution, from sponges (sclerocytes to human (osteoclast.

  17. An Evaluation of the Critical Parameters for Abiotic Peptide Synthesis in Submarine Hydrothermal Systems

    Science.gov (United States)

    Cleaves, H. J.; Aubrey, A. D.; Bada, J. L.

    2009-04-01

    It has been proposed that oligopeptides may be formed in submarine hydrothermal systems (SHSs). Oligopeptides have been synthesized previously under simulated SHS conditions which are likely geochemically implausible. We have herein investigated the oligomerization of glycine under SHS-like conditions with respect to the limitations imposed by starting amino acid concentration, heating time, and temperature. When 10-1 M glycine solutions were heated at 250°C for diketopiperazine (DKP) were detectable. At 200°C, less oligomerization was noted. Peptides beyond glycylglycine (gly2) and DKP were not detected below 150°C. At 10-2 M initial glycine concentration and below, only gly2, DKP, and gly3 were detected, and then only above 200°C at < 20 min reaction time. Gly3 was undetectable at longer reaction times. The major parameters limiting peptide synthesis in SHSs appear to be concentration, time, and temperature. Given the expected low concentrations of amino acids, the long residence times and range of temperatures in SHSs, it is unlikely that SHS environments were robust sources of even simple peptides. Possible unexplored solutions to the problems presented here are also discussed.

  18. Exploring conformational space using a mean field technique with MOLS sampling

    Indian Academy of Sciences (India)

    P Arun Prasad; V Kanagasabai; J Arunachalam; N Gautham

    2007-08-01

    The computational identification of all the low energy structures of a peptide given only its sequence is not an easy task even for small peptides, due to the multiple-minima problem and combinatorial explosion. We have developed an algorithm, called the MOLS technique, that addresses this problem, and have applied it to a number of different aspects of the study of peptide and protein structure. Conformational studies of oligopeptides, including loop sequences in proteins have been carried out using this technique. In general the calculations identified all the folds determined by previous studies, and in addition picked up other energetically favorable structures. The method was also used to map the energy surface of the peptides. In another application, we have combined the MOLS technique, using it to generate a library of low energy structures of an oligopeptide, with a genetic algorithm to predict protein structures. The method has also been applied to explore the conformational space of loops in protein structures. Further, it has been applied to the problem of docking a ligand in its receptor site, with encouraging results.

  19. Function of fibrinogen gamma-chain dodecapeptide-conjugated latex beads under flow.

    Science.gov (United States)

    Takeoka, Shinji; Okamura, Yosuke; Teramura, Yuji; Watanabe, Naohide; Suzuki, Hidenori; Tsuchida, Eishun; Handa, Makoto; Ikeda, Yasuo

    2003-12-19

    In order to perform a fundamental study of platelet substitutes, novel particles that bound to activated platelets were prepared using two oligopeptides conjugated to latex beads. The oligopeptides were CHHLGGAKQAGDV (H12), which is a fibrinogen gamma-chain carboxy-terminal sequence (gamma 400-411), and CGGRGDF (RGD), which contains a fibrinogen alpha-chain sequence (alpha 95-98 RGDF). Both peptides contained an additional amino-terminal cysteine to enable conjugation. Human serum albumin was adsorbed onto the surface of latex beads (average diameter 1microm) and pyridyldisulfide groups were chemically introduced into the adsorbed protein. H12 or RGD peptides were then chemically linked to the modified surface protein via disulfide linkages. H12- or RGD-conjugated latex beads prepared in this way enhanced the in vitro thrombus formation of activated platelets on collagen-immobilized plates under flowing thrombocytopenic-imitation blood. Based on the result of flow cytometric analyses of agglutination, PAC-1 binding, antiP-selectin antibody binding, and annexin V binding, the H12-conjugated latex beads showed minimal interaction with non-activated platelets. These results indicate the excellent potential of H12-conjugated particles as a candidate for a platelet substitute.

  20. 鳖甲水提物W-A-80在模拟胃肠液中消化情况分析%Digestion Analysis of Water Extract W-A-80 from Trionycis Carapax in Simulated Gastrointestinal Fluid

    Institute of Scientific and Technical Information of China (English)

    韩秋俊; 徐士勋; 王鹏龙; 王艳慧; 程亚涛; 周燊; 褚福浩; 宋霁翔; 李国梁

    2013-01-01

    目的:研究鳖甲水提物W-A-80在模拟胃、肠环境中消化情况,探讨其体内转化形式.方法:采用水提醇沉法制备鳖甲水提物W-A-80,模拟胃肠液环境,于37℃消化.以寡肽产率为指标,通过双缩脲反应-酶联免疫检测仪法考察胃蛋白酶和胰蛋白酶于不同酶底比、酶解时间的寡肽(截留相对分子质量<3 500)产率变化;平衡透析法测定模拟胃肠液消化后所得寡肽与体外小鼠肝组织的蛋白结合率.结果:确定酶解条件为人工胃液酶底比1∶10,于37℃酶解3h,不同质量浓度寡肽与肝组织的蛋白结合率分别为31.63%,19.93%,9.65%;人工肠液酶底比1∶100,于37℃酶解11h,不同质量浓度寡肽与肝组织的蛋白结合率分别为48.75%,47.43%,22.72%;均明显高于空白对照的7.00%,5.31%,4.60%.结论:鳖甲水提物W-A-80经模拟胃肠液消化有大量寡肽产生,且活性显著强于原液,推测寡肽可能是鳖甲抗肝纤维化的物质基础.%Objective:To explore in vivo transformation form of aqueous extract W-A-80 from Trionycis Carapax through investigating its digestion in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF).Method:Aqueous extract W-A-80 from Trionycis Carapax was prepared by water extraction and alcohol precipitation method,it was digested at 37 ℃ in SGF and SIF.With yield of oligopeotides as index,effect of different enzyme-substrate ratios and reaction time of pepsin and trypsin on yield change of oligopeptides (interception relative molecular mass < 3 500) was investigated by enzyme-linked immunosorbent detector-biuret reaction; Equilibrium dialysis was adopted to determined protein binding rate of prepared oligopeptides with liver tissue of mice in vitro.Result:Optimum enzymatic hydrolysis conditions were determined that digested 3 h with pepsin enzyme substrate ratio of 1∶10 at 37 ℃ in SGF and 11 h with trypsin enzyme substrate ratio of 1∶100 at 37 ℃ in SIF

  1. Chemotaxis: the proper physiological response to evaluate phylogeny of signal molecules.

    Science.gov (United States)

    Köhidai, L

    1999-01-01

    In this review we summarize our results gained on the investigations focused to characterize ligand and signaling mechanisms required for the chemotaxis in the unicellular model Tetrahymena. Our data show that short chain signal molecules (amino acids, oligopeptides) are distinguished upon their physicochemical characteristics - lipophilicity, residual volumes and statistical distribution of side-chain distances (e.g. in proline containing dipeptides), while the vertebrate hormones have also specific attractant or repellent effects in the model (FSH vs. TSH). Hormonal imprinting developed by pretreatments has also special, signal molecule dependent effect (histamine vs. serotonin). It is shown that "chemotactic selection" of cells, by the new probe developed by us is a suitable tool to provide subpopulations possessing enhanced chemotactic receptor-effector mechanisms with respect to the selector signal molecules (IL-8, TNF-alpha).

  2. Microbial decomposition of keratin in nature—a new hypothesis of industrial relevance

    DEFF Research Database (Denmark)

    Lange, Lene; Huang, Yuhong; Kamp Busk, Peter

    2016-01-01

    Discovery of keratin-degrading enzymes from fungi and bacteria has primarily focused on finding one protease with efficient keratinase activity. Recently, an investigation was conducted of all keratinases secreted from a fungus known to grow on keratinaceous materials, such as feather, horn......, and hooves. The study demonstrated that a minimum of three keratinases is needed to break down keratin, an endo-acting, an exo-acting, and an oligopeptide-acting keratinase. Further, several studies have documented that disruption of sulfur bridges of the keratin structure acts synergistically...... with the keratinases to loosen the molecular structure, thus giving the enzymes access to their substrate, the protein structure. With such complexity, it is relevant to compare microbial keratin decomposition with the microbial decomposition of well-studied polymers such as cellulose and chitin. Interestingly...

  3. The geranyl-modified tryptophan residue is crucial for ComXRO-E-2 pheromone biological activity.

    Science.gov (United States)

    Tsuji, Fumitada; Kobayashi, Ko; Okada, Masahiro; Yamaguchi, Hisao; Ojika, Makoto; Sakagami, Youji

    2011-07-01

    The ComX pheromone is an isoprenoidal oligopeptide containing a modified tryptophan residue, which stimulates natural genetic competence in gram-positive bacteria, Bacillus. We have reported the structure of the ComX(RO-E-2) pheromone, which is produced by the RO-E-2 strain of Bacillus subtilis. ComX(RO-E-2) analogs with substituted amino acids and isoprenoid modified tryptophan residues (e.g., prenyl, geranyl, and farnesyl), were synthesized and examined for biological activity. These results indicate that Phe-Trp(∗)(Ger)-NH(2) is the minimum pharmacophore of the ComX(RO-E-2) pheromone. Furthermore, the length of the isoprenoid moiety (i.e., modification style), and the presence of double bonds, are crucial for biological activity. The modification style of the ComX pheromone is more important than the peptide sequence with respect to biological activity.

  4. Micro-X-ray fluorescence as a general high-throughput screening method for catalyst discovery and small molecule recognition.

    Science.gov (United States)

    Miller, Thomasin C; Mann, Grace; Havrilla, George J; Wells, Cyndi A; Warner, Benjamin P; Baker, R Tom

    2003-01-01

    A powerful high-throughput screening technique is described for the rapid screening of bead-based libraries for catalyst discovery and molecular recognition. Micro-X-ray fluorescence (MXRF) screens materials for elemental composition with mesoscale analysis. This method is nondestructive and requires minimal sample preparation and no special tags for analysis, and the screening time is dependent on the desired sensitivity. The speed, sensitivity, and simplicity of MXRF as a high-throughput screening technique were applied to screen bead-based libraries of oligopeptides for phosphate hydrolysis catalysts and molecular recognition of selective receptors for the degradation products and analogues of chemical warfare agents. This paper demonstrates the analytical or HTS capability of MXRF for combinatorial screening. It is meant only to show the capabilities of MXRF and is not meant as an exhaustive study of the catalyst and molecular recognition systems presented.

  5. Tetrapeptide-coumarin conjugate 3D networks based on hydrogen-bonded charge transfer complexes: gel formation and dye release.

    Science.gov (United States)

    Guo, Zongxia; Gong, Ruiying; Jiang, Yi; Wan, Xiaobo

    2015-08-14

    Oligopeptide-based derivatives are important synthons for bio-based functional materials. In this article, a Gly-(L-Val)-Gly-(L-Val)-coumarin (GVGV-Cou) conjugate was synthesized, which forms 3D networks in ethanol. The gel nanostructures were characterized by UV-vis spectroscopy, FT-IR spectroscopy, X-ray diffraction (XRD), SEM and TEM. It is suggested that the formation of charge transfer (CT) complexes between the coumarin moieties is the main driving force for the gel formation. The capability of the gel to encapsulate and release dyes was explored. Both Congo Red (CR) and Methylene Blue (MB) can be trapped in the CT gel matrix and released over time. The present gel might be used as a functional soft material for guest encapsulation and release.

  6. Secondary structure of fluorescence labelled synthetic peptides

    CERN Document Server

    Martin, A S

    2000-01-01

    A series of eight synthetic oligopeptides has been prepared and their secondary structures investigated using various techniques. The project represents a continuation of an investigation into thermally induced changes in secondary structure. Following the previously reported results, the change in structure was initially thought to represent a change from an alpha-helix at low temperature to 3 sub 1 sub 0 -helix at high temperature. However, the results reported herein suggest the peptides retain an alpha-helical configuration at all temperatures studied, but that this helix can adopt at least two related forms. The difference in the structures relates to the nature of the H-bonds which may or may not involve an additional interaction from water molecules or side-chains. The peptides were encouraged to adopt a helical configuration by the inclusion of alpha- aminoisobutyric acid (Aib) residues. Also, modified forms of glutamic acid were included in the sequences. These had pendant donor (4-methoxy naphthalen...

  7. In silico analysis of amino acid biosynthesis and proteolysis in Lactobacillus delbrueckii subsp. bulgaricus 2038 and the implications for bovine milk fermentation.

    Science.gov (United States)

    Zheng, Huajun; Liu, Enuo; Hao, Pei; Konno, Tomonobu; Oda, Munehiro; Ji, Zai-Si

    2012-08-01

    The amino acid biosynthesis pathway and proteolytic system of Lactobacillus delbrueckii subsp. bulgaricus 2038 (L. bulgaricus 2038), a mainstay of large-scale yogurt production, were modeled based on its genomic sequence. L. bulgaricus 2038 retains more potential for amino acid synthesis and a more powerful proteolytic system than other L. bulgaricus strains, but favors amino acid uptake over de novo synthesis. Free amino acids and peptides in bovine milk provide the main nitrogen sources; whey is more important than casein for L. bulgaricus during fermentation. Free amino acids are imported by amino acid permeases and by ABC-type transport systems whereas exogenous oligopeptides are imported by ABC-type proteins only. Histidine is neither synthesized nor imported singly, which might explain why L. bulgaricus cannot grow in synthetic media.

  8. Abiotic origin of biopolymers

    Science.gov (United States)

    Oro, J.; Stephen-Sherwood, E.

    1976-01-01

    A variety of methods have been investigated in different laboratories for the polymerization of amino acids and nucleotides under abiotic conditions. They include (1) thermal polymerization; (2) direct polymerization of certain amino acid nitriles, amides, or esters; (3) polymerization using polyphosphate esters; (4) polymerization under aqueous or drying conditions at moderate temperatures using a variety of simple catalysts or condensing agents like cyanamide, dicyandiamide, or imidazole; and (5) polymerization under similar mild conditions but employing activated monomers or abiotically synthesized high-energy compounds such as adenosine 5'-triphosphate (ATP). The role and significance of these methods for the synthesis of oligopeptides and oligonucleotides under possible primitive-earth conditions is evaluated. It is concluded that the more recent approach involving chemical processes similar to those used by contemporary living organisms appears to offer a reasonable solution to the prebiotic synthesis of these biopolymers.

  9. Probing the putative active site of YjdL

    DEFF Research Database (Denmark)

    Jensen, Johanne Mørch; Ismat, Fouzia; Szakonyi, Gerda;

    2012-01-01

    YjdL from E. coli is an unusual proton-coupled oligopeptide transporter (POT). Unlike prototypical POTs, dipeptides are preferred over tripeptides, in particular dipeptides with a positively charged C-terminal residue. To further understand this difference in peptide specificity, the sequences...... of YjdL and YdgR, a prototypical E. coli POT, were compared in light of the crystal structure of a POT from Shewanella oneidensis. Several residues found in the putative active site were mutated and the activities of the mutated variants were assessed in terms of substrate uptake assays, and changes...... pocket that opens towards the extracellular space. The C-terminal side chain faces in the opposite direction into a sub pocket that faces the cytoplasm. These data indicated a stabilizing effect on a bulky N-terminal residue by an Ala281Phe variant and on the dipeptide backbone by Trp278...

  10. Evidence for dual receptor-binding sites in Clostridium difficile toxin A.

    Science.gov (United States)

    Lambert, Gregory S; Baldwin, Michael R

    2016-12-01

    TcdA (308 kDa) and TcdB (270 kDa) disrupt the integrity of the intestinal epithelial barrier and provide an environment favorable for Clostridium difficile colonization. Recent evidence suggests that entry of TcdA into cells is mediated by at least two domains. Here, we report the characterization of a second receptor-binding domain (RBD2) for TcdA. While both the isolated combined repetitive oligopeptides (CROPs) and RBD2 fragments are rapidly internalized into cells under physiologic conditions, only the CROPs domain appreciably accumulates at the cell surface. Once internalized, CROPs and RBD2 are trafficked to late endosomal compartments. An internal deletion of RBD2 from TcdA holotoxin ablated toxicity in HT29 cells. These data are consistent with the recently proposed dual receptor model of cellular entry. © 2016 Federation of European Biochemical Societies.

  11. Antioxidant and cryoprotective effects of Amur sturgeon skin gelatin hydrolysate in unwashed fish mince.

    Science.gov (United States)

    Nikoo, Mehdi; Benjakul, Soottawat; Xu, Xueming

    2015-08-15

    Antioxidant and cryoprotective effects of Amur sturgeon skin gelatin hydrolysates prepared using different commercial proteases in unwashed fish mince were investigated. Gelatin hydrolysates prepared using either Alcalase or Flavourzyme, were effective in preventing lipid oxidation as evidenced by the lower thiobarbituric acid-reactive substances formation. Gelatin hydrolysates were able to retard protein oxidation as indicated by the retarded protein carbonyl formation and lower loss in sulfhydryl content. In the presence of gelatin hydrolysates, unwashed mince had higher transition temperature of myosin and higher enthalpy of myosin and actin as determined by differential scanning calorimetry. Based on low field proton nuclear magnetic resonance analysis, gelatin hydrolysates prevented the displacement of water molecules between the different compartments, thus stabilizing the water associated with myofibrils in unwashed mince induced by repeated freeze-thawing. Oligopeptides in gelatin hydrolysates more likely contributed to the cryoprotective effect. Thus, gelatin hydrolysate could act as both antioxidant and cryoprotectant in unwashed fish mince.

  12. Interaction of L-alanyl-L-valine and L-valyl-L-alanine with organic vapors: thermal stability of clathrates, sorption capacity and the change in the morphology of dipeptide films.

    Science.gov (United States)

    Ziganshin, Marat A; Gubina, Nadezhda S; Gerasimov, Alexander V; Gorbatchuk, Valery V; Ziganshina, Sufia A; Chuklanov, Anton P; Bukharaev, Anastas A

    2015-08-21

    The strong effect of the amino acid sequence in L-alanyl-L-valine and L-valyl-L-alanine on their sorption properties toward organic compounds and water, and the thermal stability of the inclusion compounds of these dipeptides have been found. Generally, L-valyl-L-alanine has a greater sorption capacity for the studied compounds, but the thermal stability of the L-alanyl-L-valine clathrates is higher. Unusual selectivity of L-valyl-L-alanine for vapors of few chloroalkanes was observed. The correlation between the change in the surface morphology of thin film of dipeptides and stoichiometry of their clathrates with organic compounds was found. This discovery may be used to predict the influence of vapors on the morphology of films of short-chain oligopeptides.

  13. Oxyma-based phosphates for racemization-free peptide segment couplings.

    Science.gov (United States)

    Mitachi, Katsuhiko; Kurosu, Yuki E; Hazlett, Brandon T; Kurosu, Michio

    2016-03-01

    Glyceroacetonide-Oxyma [(2,2-dimethyl-1,3-dioxolan-4-yl)methyl 2-cyano-2-(hydroxyimino)acetate (1)] displayed remarkable physico-chemical properties as an additive for peptide-forming reactions. Although racemization-free amide-forming reactions have been established for N-urethane-protected α-amino acids with EDCI, 1, and NaHCO3 in water or DMF-water media, amide-forming reactions of N-acyl-protected α-amino acids and segment couplings of oligopeptides still require further development. Diethylphosphoryl-glyceroacetonide-oxyma (DPGOx 3) exhibits relative stability in aprotic solvents and is an effective coupling reagent for N-acyl-protected α-amino acids and oligo peptide segments. The conditions reported here is also effective in lactam-forming reactions. Unlike most of the reported coupling reagents, simple aqueous work-up procedures can remove the reagents and by-products generated in the reactions.

  14. 6-Membered ring intermediates in polymerization of N-carboxyanhydride-L-a-arginine in H2O

    Institute of Scientific and Technical Information of China (English)

    XIN Liang; REN Jie; XIANG JunFeng; YAN Qin; XIE Yi; WANG KongJiang; LAI PingAn; BAI YaDuo

    2009-01-01

    In polymerization of N-carboxyanhydride-L-α-arginine (L-Arg-NCA) in H2O,nucleophilic reaction of guanidine group with the carbonyl group of L-Arg-NCA leads to quick intramolecular rearrangement,yielding a 6-membered ring intermediate 1-amidino-3-amino-2-piperidone,which is either elongated by another L-Arg-NCA yielding arginyl-1-amidino-3-amino-2-piperidone or hydrolyzed to L-α-arginine.The oligoarginines are formed mainly through hydrolysis of arginyl-1-amidino-3-amino-2-piperidones.This is a unique pathway in polymerization of L-Arg-NCA with regard to the usual pathway of elongations by reaction of N-carboxyanhydride-L-a-amino acid with L-a-amino acid or oligopeptides.

  15. Sonodelivery Facilitates Sustained Luciferase Expression from an Episomal Vector in Skeletal Muscle

    Directory of Open Access Journals (Sweden)

    Manoel Figueiredo Neto

    2015-07-01

    Full Text Available Successful gene delivery to skeletal muscle is a desirable goal, not only for treating muscle diseases, but also for immunization, treatment of metabolic disorders, and/or delivering gene expression that can treat systemic conditions, such as bone metastatic cancer, for example. Although naked DNA uptake into skeletal muscle is possible, it is largely inefficient in the absence of additional chemical or physical delivery methods. We describe a system for delivery of non-viral or plasmid DNA to skeletal muscle using ultrasound-assisted sonoporation of a nanoplex combining plasmid DNA and a branched polymer based on poly(cyclooctene-graft-oligopeptide. The materials and methods described herein promise to advance the field of sonodelivery and of gene delivery to muscle for therapeutic applications since a simple system is presented that enables long-term gene expression in vivo with the promise of a minimal inflammatory gene expression profile.

  16. High yield recombinant production of a self-assembling polycationic peptide for silica biomineralization.

    Science.gov (United States)

    Zerfaß, Christian; Braukmann, Sandra; Nietzsche, Sandor; Hobe, Stephan; Paulsen, Harald

    2015-04-01

    We report the recombinant bacterial expression and purification at high yields of a polycationic oligopeptide, P5S3. The sequence of P5S3 was inspired by a diatom silaffin, a silica precipitating peptide. Like its native model, P5S3 exhibits silica biomineralizing activity, but furthermore has unusual self-assembling properties. P5S3 is efficiently expressed in Escherichia coli as fusion with ketosteroid isomerase (KSI), which causes deposition in inclusion bodies. After breaking the fusion by cyanogen bromide reaction, P5S3 was purified by cation exchange chromatography, taking advantage of the exceptionally high content of basic amino acids. The numerous cationic charges do not prevent, but may even promote counterion-independent self-assembly which in turn leads to silica precipitation. Enzymatic phosphorylation, a common modification in native silica biomineralizing peptides, can be used to modify the precipitation activity.

  17. Inhibition of ALK enzymatic activity in T-cell lymphoma cells induces apoptosis and suppresses proliferation and STAT3 phosphorylation independently of Jak3

    DEFF Research Database (Denmark)

    Marzec, Michal; Kasprzycka, Monika; Ptasznik, Andrzej;

    2005-01-01

    Aberrant expression of the ALK tyrosine kinase as a chimeric protein with nucleophosmin (NPM) and other partners plays a key role in malignant cell transformation of T-lymphocytes and other cells. Here we report that two small-molecule, structurally related, quinazoline-type compounds, WHI-131...... and WHI-154, directly inhibit enzymatic activity of NPM/ALK as demonstrated by in vitro kinase assays using a synthetic tyrosine-rich oligopeptide and the kinase itself as the substrates. The inhibition of NPM/ALK activity resulted in malignant T cells in suppression of their growth, induction...... of apoptosis and inhibition of tyrosine phosphorylation of STAT3, the key effector of the NPM/ALK-induced oncogenesis. We also show that the STAT3 tyrosine phosphorylation is mediated in the malignant T cells by NPM/ALK independently of Jak3 kinase as evidenced by the presence of STAT3 phosphorylation...

  18. Glycopeptide dendrimers as Pseudomonas aeruginosa biofilm inhibitors.

    Science.gov (United States)

    Reymond, Jean-Louis; Bergmann, Myriam; Darbre, Tamis

    2013-06-01

    Synthetic glycopeptide dendrimers composed of a branched oligopeptide tree structure appended with glycosidic groups at its multiple N-termini were investigated for binding to the Pseudomonas aeruginosa lectins LecB and LecA. These lectins are partly responsible for the formation of antibiotic resistant biofilms in the human pathogenic bacterium P. aeruginosa, which causes lethal airway infections in immune-compromised and cystic fibrosis patients. Glycopeptide dendrimers with high affinity to the lectins were identified by screening of combinatorial libraries. Several of these dendrimers, in particular the LecB specific glycopeptide dendrimers FD2 and D-FD2 and the LecA specific glycopeptide dendrimers GalAG2 and GalBG2, also efficiently block P. aeruginosa biofilm formation and induce biofilm dispersal in vitro. Structure-activity relationship and structural studies are reviewed, in particular the observation that multivalency is essential to the anti-biofilm effect in these dendrimers.

  19. Wet chemical synthesis of soluble gold nanogaps

    DEFF Research Database (Denmark)

    Jain, Titoo; Tang, Qingxin; Bjørnholm, Thomas

    2014-01-01

    A central challenge in molecular electronics is to create electrode pairs separated by only a few nanometers that can accommodate a single molecule of interest to be optically or electrically characterized while residing in the gap. Current techniques for nanogap fabrication are largely based....... In addition, we introduce a novel fabrication approach in which we can grow AuNR nanogap electrodes in situ on prepatterned substrates, thus circumventing post-processing steps that potentially damage the nanogap environment. Due to the inherent optical properties of AuNRs, electromagnetic field enhancement...... in the nanogaps lets us spectroscopically characterize the molecules via surface-enhanced Raman scattering. We discuss the incorporation of oligopeptides functionalized with acetylene units having uniquely identifiable vibrational modes. This acetylene moiety allows chemical reactions to be performed in the gaps...

  20. 6-Membered ring intermediates in polymerization of N-carboxyanhydride-L-α-arginine in H2O

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    In polymerization of N-carboxyanhydride-L-α-arginine(L-Arg-NCA) in H2O,nucleophilic reaction of guanidine group with the carbonyl group of L-Arg-NCA leads to quick intramolecular rearrangement,yielding a 6-membered ring intermediate 1-amidino-3-amino-2-piperidone,which is either elongated by another L-Arg-NCA yielding arginyl-1-amidino-3-amino-2-piperidone or hydrolyzed to L-α-arginine.The oligoarginines are formed mainly through hydrolysis of arginyl-1-amidino-3-amino-2-piperidones.This is a unique pathway in polymerization of L-Arg-NCA with regard to the usual pathway of elongations by reaction of N-carboxyanhydride-L-α-amino acid with L-α-amino acid or oligopeptides.

  1. Synthesis of (N-[[sup 11]C]methyl)Y-29794, a competitive inhibitor of prolyl endopeptidase. [2-(8-dimethylaminoctylthio)-6-isopropyl-3-pyridyl-2-thienyl ketone

    Energy Technology Data Exchange (ETDEWEB)

    Charalambous, A.; Mangner, T.J.; Kilbourn, M.R. (Michigan Univ., Ann Arbor, MI (United States). Div. of Nuclear Medicine)

    1994-06-01

    Prolyl endopeptidase (PEP:[E.C.3.4.21.26]) is a widely distributed serine peptidase that cleaves a variety of oligopeptides in the brain and peripheral tissues. Y-29794 2-(8-dimethylaminooctylthio)-(6-isopropyl-3-pyridyl-2-thienyl ketone) is a potent competitive reversible inhibitor of this enzyme. In order to study the biodistribution of PEP in vivo we have synthesized (N[[sup 11]C]methyl)Y-29794, by [[sup 11]C]alkylation of the N-desmethyl precursor. The radiotracer was purified by silica gel Sep-Pak and was obtained in 10-17% yields (EOB: synthesis times shorter than 45 min) with >98% radiochemical purities and specific activities >550 Ci/mmol (EOS). (Author).

  2. Quorum Sensing Activity of Enterobacter asburiae Isolated from Lettuce Leaves

    Directory of Open Access Journals (Sweden)

    Kok-Gan Chan

    2013-10-01

    Full Text Available Bacterial communication or quorum sensing (QS is achieved via sensing of QS signaling molecules consisting of oligopeptides in Gram-positive bacteria and N-acyl homoserine lactones (AHL in most Gram-negative bacteria. In this study, Enterobacteriaceae isolates from Batavia lettuce were screened for AHL production. Enterobacter asburiae, identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS was found to produce short chain AHLs. High resolution triple quadrupole liquid chromatography mass spectrometry (LC/MS analysis of the E. asburiae spent supernatant confirmed the production of N-butanoyl homoserine lactone (C4-HSL and N–hexanoyl homoserine lactone (C6-HSL. To the best of our knowledge, this is the first report of AHL production by E. asburiae.

  3. A new tool in peptide engineering: a photoswitchable stilbene-type beta-hairpin mimetic.

    Science.gov (United States)

    Erdélyi, Máté; Karlén, Anders; Gogoll, Adolf

    2005-12-23

    Peptide secondary structure mimetics are important tools in medicinal chemistry, as they provide analogues of endogenous peptides with new physicochemical and pharmacological properties. The development, synthesis, photochemical investigation, and conformational analysis of a stilbene-type beta-hairpin mimetic capable of light-triggered conformational changes have been achieved. In addition to standard spectroscopic techniques (nuclear Overhauser effects, amide temperature coefficients, circular dichroism spectroscopy), the applicability of self-diffusion measurements (longitudinal eddy current delay pulsed-field gradient spin echo (LED-PGSE) NMR technique) in conformational studies of oligopeptides is demonstrated. The title compound shows photoisomerization of the stilbene chromophore, resulting in a change in solution conformation between an unfolded structure and a folded beta-hairpin.

  4. Studies on intestinal absorption of amino acids and a dipeptide in a case of Hartnup disease.

    Science.gov (United States)

    Navab, F; Asatoor, A M

    1970-05-01

    A severely affected case of Hartnup disease is reported, where the patient responded rapidly to nicotinamide. This supports the view that all the clinical features, except reduced stature from general nutritional defect, are secondary to tryptophan and nicotinamide deficiency rather than to an unknown toxic factor. Severe malabsorption of both tryptophan and phenylalanine was demonstrated. The dipeptide carnosine was absorbed normally whereas when the two constituent amino acids, beta-alanine and L-histidine, were ingested, absorption of the former was normal but that of the latter was grossly defective. The suggestion is advanced that in cases of Hartnup disease protein nutrition is maintained by intestinal uptake of amino acids as oligopeptides rather than as free amino acids. By contrast, both modes of absorption are probably important in normal subjects. Radiology of the small intestine is abnormal in Hartnup disease when a large amount of protein is admixed with the barium meal.

  5. Intestinal absorption of amino acids and peptides in Hartnup disorder.

    Science.gov (United States)

    Leonard, J V; Marrs, T C; Addison, J M; Burston, D; Clegg, K M; Lloyd, J K; Matthews, D M; Seakins, J W

    1976-04-01

    Absorption of free and peptide-bound amino acids was investigated in a girl with Hartnup disorder aged 26 months. Plasma levels of amino acids were followed after oral administration of (1) an amino acid mixture simulating casein and (2) an equivalent dose of a partial enzymic hydrolysate of casein containing oligopeptides in addition to free amino acids. The results suggested that many neutral amino acids were poorly absorbed when given in the free form, but much more readily absorbed when given as peptides. Unexpectedly, the results also suggested that glutamic acid was poorly absorbed when given in the free form. The results obtained with threonine could not be interpreted. There was an increased renal clearance of many neutral amino acids, including glycine, but clearance of proline was not increased. Most amino acids with an increased renal clearance also appeared to be poorly absorbed when given by mouth in the free form.

  6. Octaarginine-modified chitosan as a nonviral gene delivery vector: properties and in vitro transfection efficiency

    Science.gov (United States)

    Zhao, Xiaoli; Li, Zhaoyang; Liu, Wenguang; Lam, Wingmoon; Sun, Peng; Kao, Richard Y. T.; Luk, Keith D. K.; Lu, William W.

    2011-02-01

    Protein transduction domains (PTD) have been identified to have the capacity to facilitate molecular cargo to translocate through cell membrane. This study aims to utilize the cell membrane penetrating ability of octaarginine oligopeptide, a simplified prototype of the PTD, to enhance the transfection efficiency of chitosan. Octaarginine-modified chitosan (R8-CS) was synthesized as a gene transfer carrier by carbodiimide chemistry. The structure and composition of R8-CSs were characterized using FTIR and 1H NMR. Agarose gel electrophoresis assay showed that R8-CS could efficiently condense the DNA. The particle size of R8-CS/DNA complexes were determined to be around 100-200 nm. The nanoparticle complexes exhibited a spherical and compact morphology. R8-CS demonstrated higher transfection activity and lower cytotoxicity as compared to the unmodified chitosan and also showed good serum resistance.

  7. The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors.

    Science.gov (United States)

    Janek, Katharina; Niewienda, Agathe; Wöstemeyer, Johannes; Voigt, Jürgen

    2016-11-15

    Particular peptides generated from the vicilin-class(7S) globulin of the cocoa beans by acid-induced proteolysis during cocoa fermentation are essential precursors of the cocoa-specific aroma notes. As revealed by in vitro studies, the formation of the cocoa-specific aroma precursors depends on the particular cleavage specificity of the cocoa aspartic protease, which cannot be substituted by pepsin. Therefore, we have investigated the effects of aspartic protease inhibitors on both enzymes and comparatively studied their cleavage specificities using different protein substrates and MALDI-TOF mass spectrometric analyses of the generated oligopeptides. Three classes of cleavage sites have been identified and characterized: (I) sequences exclusively cleaved by the cocoa enzyme, (II) sequences cleaved by both pepsin and the cocoa enzyme, and (III) those cleaved exclusively by pepsin. In contrast to most aspartic proteases from other origins, basic amino acid residues, particularly lysine, were found to be abundant in the specific cleavage sites of the cocoa enzyme.

  8. Dataset of cocoa aspartic protease cleavage sites

    Directory of Open Access Journals (Sweden)

    Katharina Janek

    2016-09-01

    Full Text Available The data provide information in support of the research article, “The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors” (Janek et al., 2016 [1]. Three different protein substrates were partially digested with the aspartic protease isolated from cocoa beans and commercial pepsin, respectively. The obtained peptide fragments were analyzed by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS/MS and identified using the MASCOT server. The N- and C-terminal ends of the peptide fragments were used to identify the corresponding in-vitro cleavage sites by comparison with the amino acid sequences of the substrate proteins. The same procedure was applied to identify the cleavage sites used by the cocoa aspartic protease during cocoa fermentation starting from the published amino acid sequences of oligopeptides isolated from fermented cocoa beans.

  9. Desalted duck egg white peptides promote calcium uptake by counteracting the adverse effects of phytic acid.

    Science.gov (United States)

    Hou, Tao; Liu, Weiwei; Shi, Wen; Ma, Zhili; He, Hui

    2017-03-15

    The structure of the desalted duck egg white peptides-calcium chelate was characterized by fluorescence spectroscopy, fourier transform infrared spectroscopy, and dynamic light scattering. Characterization results showed structural folding and aggregation of amino acids or oligopeptides during the chelation process. Desalted duck egg white peptides enhanced the calcium uptake in the presence of oxalate, phosphate and zinc ions in Caco-2 monolayers. Animal model indicated that desalted duck egg white peptides effectively enhanced the mineral absorption and counteracted the deleterious effects of phytic acid. These findings suggested that desalted duck egg white peptides might promote calcium uptake in three pathways: 1) desalted duck egg white peptides bind with calcium to form soluble chelate and avoid precipitate; 2) the chelate is absorbed as small peptides by enterocyte; and 3) desalted duck egg white peptides regulate the proliferation and differentiation of enterocytes through the interaction with transient receptor potential vanilloid 6 calcium channel.

  10. Insertional mutagenesis of an industrial strain of Streptococcus thermophilus.

    Science.gov (United States)

    Labarre, C; Schirawski, J; van der Zwet, A; Fitzgerald, G F; van Sinderen, D

    2001-06-12

    Random mutagenesis of an industrial strain of Streptococcus thermophilus was achieved through an adapted version of a two-plasmid system. The mutagenesis strategy is based on random integration of derivatives of the non-replicative (Rep(-)) plasmid pORI19 by means of homologous recombination following a temperature shift that eliminates replication of the temperature-sensitive (Rep(ts)) helper plasmid pVE6007. In this way mutants were generated which were affected in bacteriophage sensitivity or sucrose metabolism. Homologues were identified of a protein related to folate metabolism from a bacteriophage-resistant mutant and of two subunits of an oligopeptide transport system from a mutant deficient in sucrose utilisation.

  11. BioR PREPARATION EFFECTS ON PRODUCTIVE INDEXES OF SUINA YOUTH

    Directory of Open Access Journals (Sweden)

    V. MACARI

    2013-12-01

    Full Text Available The problem of using biologicalliactive substancesin domestic animalfeedingis one of main problems of the world researchess. Our investigations studied the influence of BioR preparation on suina youth performances. BioR preparation is a biologically active one obtained using original tehnologies of oriented syhthesis, successive extraction, division and purification of bioactive priciples from cyanophit alga biomass Spirulina platensis. This preparation contains biologic active substances as aminoacids and oligopeptides, intermediary products of the glucide and lipid metabolisms, macro and microelements. The results of the investigations proved that the administration of BioR preparation had a positive influence on productive indices of suina namely: the body weight increased by 0.5-10.4%, daily body weight increased by 2.8-16.0%, but the specific consumption decreased by 18.3-23.0%.

  12. Tritium labeling of amino acids and peptides with liquid and solid tritium

    Energy Technology Data Exchange (ETDEWEB)

    Souers, P.C.; Coronado, P.R.; Peng, C.T.; Hua, R.L.

    1988-01-01

    Amino acids and peptides were labeled with liquid and solid tritium at 21/degree/K and 9/degree/K. At these low temperatures radiation degradation is minimal, and tritium incorporation increases with tritium concentration and exposure time. Ring saturation in L-phenylalanine does not occur. Peptide linkage in oligopeptides is stable toward tritium. Deiodination in 3-iodotyrosine and 3,5-diiodotyrosine occurs readily and proceeds in steps by losing one iodine atom at a time. Nickel and noble metal supported catalysts when used as supports for dispersion of the substrate promote tritium labeling at 21 K. Our study shows that both liquid and solid tritiums are potentially useful agents for labeling peptides and proteins.

  13. Tritium labeling of amino acids and peptides with liquid and solid tritium

    Energy Technology Data Exchange (ETDEWEB)

    Peng, C.T.; Hua, R.L.; Souers, P.C.; Coronado, P.R.

    1988-01-01

    Amino acids and peptides were labeled with liquid and solid tritium at 21 K and 9 K. At these low temperatures radiation degradation is minimal, and tritium incorporation increases with tritium concentration and exposure time. Ring saturation in L-phenyl-alanine does not occur. Peptide linkage in oligopeptides is stable toward tritium. Deiodination in 3-iodotyrosine and 3,5-diiodotyrosine occurs readily and proceeds in steps by losing one iodine atom at a time. Nickel and noble metal supported catalysts when used as supports for dispersion of the substrate promote tritium labeling at 21 K. Our study shows that both liquid and solid tritium are potentially useful agents for labeling peptides and proteins. 11 refs., 1 fig., 3 tabs.

  14. Production of Bioactive Peptides from Soybean Meal by Solid State Fermentation with Lactic Acid Bacteria and Protease

    Directory of Open Access Journals (Sweden)

    Naifu Wang

    2014-10-01

    Full Text Available In this study, soybean meal was first solid state fermented with different strains of Lactic Acid Bacteria (LAB. Among the strains used, Lactobacillus plantarum Lp6 was selected for further studies because of its highest Degree of Hydrolysis (DH of protein (2.49±0.08% in soybean meal after 72 h fermentation. Soybean meal fermented with L. plantarum Lp6 can also improve its DPPH radical scavenging and Angiotensin Converting Enzyme (ACE inhibitory activities. The addition of protease into soybean meal during the fermentation resulted in lowered IC50 of DPPH radical scavenging and ACE inhibitory activities, indicating more bioactive peptides were produced during fermentation. Molecular weight distribution analysis revealed the Extracts from Fermented Soybean Meal (EFSM was mainly composed of oligopeptides. These results indicated that soybean meal fermented with L. plantarum Lp6 and protease could be an easy and cheap method to produce functional food.

  15. Current prodrug strategies for improving oral absorption of nucleoside analogues

    Directory of Open Access Journals (Sweden)

    Youxi Zhang

    2014-04-01

    Full Text Available Nucleoside analogues are first line chemotherapy in various severe diseases: AIDS (acquired immunodeficiency disease syndrome, cytomegalovirus infections, cancer, etc. However, many nucleoside analogues exhibit poor oral bioavailability because of their high polarity and low intestinal permeability. In order to get around this drawback, prodrugs have been utilized to improve lipophilicity by chemical modification of the parent drug. Alternatively, prodrugs targeting transporters present in the intestine have been applied to promote the transport of the nucleoside analogues. Valacyclovir and valganciclovir are two classic valine ester prodrugs transported by oligopeptide transporter 1. The ideal prodrug achieves delivery of a parent drug by attaching a non-toxic moiety that is stable during transport, but is readily degraded to the parent drug once at the target. This article presents advances of prodrug approaches for enhancing oral absorption of nucleoside analogues.

  16. Inhibition of activity of the protease from bovine leukemia virus.

    Science.gov (United States)

    Ménard, A; Leonard, R; Llido, S; Geoffre, S; Picard, P; Berteau, F; Precigoux, G; Hospital, M; Guillemain, B

    1994-06-13

    In view of the close similarity between bovine leukemia virus (BLV) and human T-cell leukemia virus type I (HTLV-I) we investigated the possibility of developing specific inhibitors of the proteases of these retroviruses using the purified enzyme from BLV. We tested the ability of this protease to specifically cleave various short oligopeptide substrates containing cleavage sites of BLV and HTLV-I proteases, as well as a recombinant BLV Gag precursor. The best substrate, a synthetic decapeptide bearing the natural cleavage site between the matrix and the capsid proteins of BLV Gag precursor polyprotein, was used to develop an inhibition assay. We determined the relative inhibitory effect of synthetic Gag precursor-like peptides in which the cleavable site was replaced by a non-hydrolyzable moiety. The encouraging inhibitory effect of these compounds indicates that potent non-peptidic inhibitors for retroviral proteases are not unattainable.

  17. Re-Evaluation of Binding Properties of Recombinant Lymphocyte Receptors NKR-P1A and CD69 to Chemically Synthesized Glycans and Peptides

    Directory of Open Access Journals (Sweden)

    Daniel Rozbeský

    2014-01-01

    Full Text Available The binding of monosaccharides and short peptides to lymphocyte receptors (human CD69 and rat NKR-P1A was first reported in 1994 and then in a number of subsequent publications. Based on this observation, numerous potentially high-affinity saccharide ligands have been synthesized over the last two decades in order to utilize their potential in antitumor therapy. Due to significant inconsistencies in their reported binding properties, we decided to re-examine the interaction between multiple ligands and CD69 or NKR-P1A. Using NMR titration and isothermal titration calorimetry we were unable to detect the binding of the tested ligands such as N-acetyl-d-hexosamines and oligopeptides to both receptors, which contradicts the previous observations published in more than twenty papers over the last fifteen years.

  18. Liquid Crystalline Materials for Biological Applications.

    Science.gov (United States)

    Lowe, Aaron M; Abbott, Nicholas L

    2012-03-13

    Liquid crystals have a long history of use as materials that respond to external stimuli (e.g., electrical and optical fields). More recently, a series of investigations have reported the design of liquid crystalline materials that undergo ordering transitions in response to a range of biological interactions, including interactions involving proteins, nucleic acids, viruses, bacteria and mammalian cells. A central challenge underlying the design of liquid crystalline materials for such applications is the tailoring of the interface of the materials so as to couple targeted biological interactions to ordering transitions. This review describes recent progress toward design of interfaces of liquid crystalline materials that are suitable for biological applications. Approaches addressed in this review include the use of lipid assemblies, polymeric membranes containing oligopeptides, cationic surfactant-DNA complexes, peptide-amphiphiles, interfacial protein assemblies and multi-layer polymeric films.

  19. A secreted peptide growth factor, phytosulfokine, acting as a stimulatory factor of carrot somatic embryo formation.

    Science.gov (United States)

    Hanai, H; Matsuno, T; Yamamoto, M; Matsubayashi, Y; Kobayashi, T; Kamada, H; Sakagami, Y

    2000-01-01

    Somatic embryogenesis of the carrot (Daucus carota L.) depends on a set of factors, some of which accumulate in culture medium (conditioned medium, CM). When embryogenic cell clusters were transferred to an embryo-inducing medium, addition of CM derived from somatic embryo culture markedly stimulated somatic embryo formation. The active principles were purified using a simple bioassay system and identified to be phytosulfokines (PSKs), sulfated oligopeptide growth factors originally isolated from a CM derived from asparagus (Asparagus officinalis L.) mesophyll culture. Quantification studies using a competition ELISA system employing an anti-PSK-alpha polyclonal antibody showed that PSK production might be related to growth of cells, rather than development of somatic embryos. Thus the stimulatory effect of PSK on somatic embryo formation might be due to promotion of cell proliferation.

  20. Time-resolved fluorescence study of electron transfer in a model peptide system

    Science.gov (United States)

    Donald, Fiona; Hungerford, Graham; Moore, Barry D.; Birch, David J. S.

    1994-08-01

    At present there is a great deal of interest in the study of the transference of energy in biological systems. For example, electron transfer is of major importance in many synthetic and biological processes and in nature is mediated by proteins. Information regarding this process is therefore useful in leading to a greater understanding of phenomena such as photosynthesis and respiration. Previous work on protein systems has shown the electron transfer process to be complex to analyze because of the presence of competing pathways. This has led to the use of model systems to simplify the kinetics. We have synthesized novel model systems using peptides containing both a fluorescent methoxy- naphthalene donor and a dicyanoethylene group as a potential electron acceptor and observed fluorescence quenching for both dipeptide and oligopeptide systems. Biexponential fluorescence decay behavior was observed for all donor acceptor systems, with an increase in the amount of the shorter fluorescence decay component on increasing temperature.

  1. Structural diversity of marine cyclic peptides and their molecular mechanisms for anticancer, antibacterial, antifungal, and other clinical applications.

    Science.gov (United States)

    Lee, Yeji; Phat, Chanvorleak; Hong, Soon-Cheol

    2017-09-01

    Many cyclic peptides and analogues derived from marine sources are known to possess biological properties, including anticancer, antitumor, antibacterial, antifungal, antiparasitic, anti-inflammation, anti-proliferative, anti-hypertensive, cytotoxic, and antibiotic properties. These compounds demonstrate different activities and modes of action according to their structure such as cyclic oligopeptide, cyclic lipopeptide, cyclic glycopeptide and cyclic depsipeptide. The recent advances in application of the above-mentioned cyclic peptides were reported in dolastatins, soblidotin, didemnin B, aplidine, salinosporamide A, kahalalide F and bryostatin 1 and they are currently in clinical trials. These cyclic peptides are possible novel drugs discovered and developed from marine origin. Literature data concerning the potential properties of marine cyclic peptides were reviewed here, and the structural diversity and biological activities of marine cyclic peptides are discussed in relation to the molecular mechanisms of these marine cyclic peptides. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. A functional EXXEK motif is essential for proton coupling and active glucosinolate transport by NPF2.11

    DEFF Research Database (Denmark)

    Jørgensen, Morten Egevang; Olsen, Carl Erik; Geiger, Dietmar

    2015-01-01

    The proton-dependent oligopeptide transporter (POT/PTR) family shares a highly conserved E1X1X2E2RFXYY (E1X1X2E2R) motif across all kingdoms of life. This motif is suggested to have a role in proton coupling and active transport in bacterial homologs. For the plant POT/PTR family, also known......K motif variant in a plant NPF transporter. Using liquid chromatography-mass spectrometry (LC-MS)-based uptake assays and two-electrode voltage clamp (TEVC) electrophysiology, we demonstrate an essential role for the E1X1X2E2K motif for accumulation of substrate by NPF2.11. Our data suggest...

  3. Development of new peptide synthetic method of enzyme using the extraction reactivity; Chushutsu hanno wo mochiita shiki pepuchido koso goseiho no kaihatsu

    Energy Technology Data Exchange (ETDEWEB)

    Hirata, Makoto [Oita University, Oita (Japan)

    1999-03-05

    Recently, taste and bioactivation of large number of oligopeptide become clear, and the development of the efficient synthetic method becomes the urgency. In the production process by conventional enzyme reaction which combined the crystallization, because the solubility of the product to the water which is reaction solvent is low, the yield remained at about 60%, and the problem of reaction inhibition of the product by the crystal had also been indicated. In the enzyme synthesis of the aspartame in which he is the representative oligopeptide, it aimed at the establishment of the new synthesis method which can improve yield and reaction rate, while the segregation enzyme was continuously utilized. In this synthetic method, supply of organic solvent which dissolved the substrate, extraction of the substrate from organic solvent to water phase, synthesis reaction by the segregation enzyme in water phase, extraction of the aspartame which is a product from water phase to organic solvent progress, and they continuously progress by one complete mixing reactor. The process which controlled these speeds and yields was quantitatively analyzed, and material balance style considering substrate, enzyme and mass transfer of the product and enzyme reaction speed was deduced. The optimum operating condition for improving yield and productivity of the purpose product using this solution was examined, and optimum supply concentration and agitation speed of aspartic acid which was a substrate were started, and the optimum operating condition which realizes the improvement in high yield and productivity over 90% of the aspartame was clarified. Like this, it is that this research adopts features of liquid Citrus nobilis two-phase partition for the enzyme synthesis of the aspartame, and it is considered that there is a value, because it is the creative research which verified that the productivity can be greatly improved by the utilization of the chemical-engineering technique, and

  4. Active subsite properties, subsite residues and targeting to lysosomes or midgut lumen of cathepsins L from the beetle Tenebrio molitor.

    Science.gov (United States)

    Damasceno, Ticiane F; Dias, Renata O; de Oliveira, Juliana R; Salinas, Roberto K; Juliano, Maria A; Ferreira, Clelia; Terra, Walter R

    2017-08-25

    Cathepsins L are the major digestive peptidases in the beetle Tenebrio molitor. Two digestive cathepsins L (TmCAL2 and TmCAL3) from it had their 3D structures solved. The aim of this paper was to study in details TmCAL3 specificity and properties and relate them to its 3D structure. Recombinant TmCAL3 was assayed with 64 oligopeptides with different amino acid replacements in positions P2, P1, P1' and P2'. Results showed that TmCAL3 S2 specificity differs from the human enzyme and that its specificities also explain why on autoactivation two propeptide residues remain in the enzyme. Data on free energy of binding and of activation showed that S1 and S2' are mainly involved in substrate binding, S1' acts in substrate binding and catalysis, whereas S2 is implied mainly in catalysis. Enzyme subsite residues were identified by docking with the same oligopeptide used for kinetics. The subsite hydrophobicities were calculated from the efficiency of hydrolysis of different amino acid replacements in the peptide and from docking data. The results were closer for S1 and S2' than for S1' and S2, indicating that the residue subsites that were more involved in transition state binding are different from those binding the substrate seen in docking. Besides TmCAL1-3, there are nine other cathepsins L, most of them more expressed at midgut. They are supposed to be directed to lysosomes by a Drosophila-like Lerp receptor and/or motifs in their prodomains. The mannose 6-phosphate lysosomal sorting machinery is absent from T. molitor transcriptome. Cathepsin L direction to midgut contents seems to depend on overexpression. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Nanolayered Features of Collagen-like Peptides

    Science.gov (United States)

    Valluzzi, Regina; Bini, Elisabetta; Haas, Terry; Cebe, Peggy; Kaplan, David L.

    2003-01-01

    We have been investigating collagen-like model oligopeptides as molecular bases for complex ordered biomimetic materials. The collagen-like molecules incorporate aspects of native collagen sequence and secondary structure. Designed modifications to native primary and secondary structure have been incorporated to control the nanostructure and microstructure of the collagen-like materials produced. We find that the collagen-like molecules form a number of lyotropic rod liquid crystalline phases, which because of their strong temperature dependence in the liquid state can also be viewed as solvent intercalated thermotropic liquid crystals. The liquid crystalline phases formed by the molecules can be captured in the solid state by drying off solvent, resulting in solid nanopatterned (chemically and physically) thermally stable (to greater than 100 C) materials. Designed sequences which stabilize smectic phases have allowed a variety of nanoscale multilayered biopolymeric materials to be developed. Preliminary investigations suggest that chemical patterns running perpendicular to the smectic layer plane can be functionalized and used to localize a variety of organic, inorganic, and organometallic moieties in very simple multilayered nanocomposites. The phase behavior of collagen-like oligopeptide materials is described, emphasizing the correlation between mesophase, molecular orientation, and chemical patterning at the microscale and nanoscale. In many cases, the textures observed for smectic and hexatic phase collagens are remarkably similar to the complex (and not fully understood) helicoids observed in biological collagen-based tissues. Comparisons between biological morphologies and collagen model liquid crystalline (and solidified materials) textures may help us understand the molecular features which impart order and function to the extracellular matrix and to collagen-based mineralized tissues. Initial studies have utilized synthetic collagen-like peptides while

  6. Acceleration of vascular sprouting from fabricated perfusable vascular-like structures.

    Directory of Open Access Journals (Sweden)

    Tatsuya Osaki

    Full Text Available Fabrication of vascular networks is essential for engineering three-dimensional thick tissues and organs in the emerging fields of tissue engineering and regenerative medicine. In this study, we describe the fabrication of perfusable vascular-like structures by transferring endothelial cells using an electrochemical reaction as well as acceleration of subsequent endothelial sprouting by two stimuli: phorbol 12-myristate 13-acetate (PMA and fluidic shear stress. The electrochemical transfer of cells was achieved using an oligopeptide that formed a dense molecular layer on a gold surface and was then electrochemically desorbed from the surface. Human umbilical vein endothelial cells (HUVECs, adhered to gold-coated needles (ϕ600 μm via the oligopeptide, were transferred to collagen gel along with electrochemical desorption of the molecular layer, resulting in the formation of endothelial cell-lined vascular-like structures. In the following culture, the endothelial cells migrated into the collagen gel and formed branched luminal structures. However, this branching process was strikingly slow (>14 d and the cell layers on the internal surfaces became disrupted in some regions. To address these issues, we examined the effects of the protein kinase C (PKC activator, PMA, and shear stress generated by medium flow. Addition of PMA at an optimum concentration significantly accelerated migration, vascular network formation, and its stabilization. Exposure to shear stress reoriented the cells in the direction of the medium flow and further accelerated vascular network formation. Because of the synergistic effects, HUVECs began to sprout as early as 3 d of perfusion culture and neighboring vascular-like structures were bridged within 5 d. Although further investigations of vascular functions need to be performed, this approach may be an effective strategy for rapid fabrication of perfusable microvascular networks when engineering three-dimensional fully

  7. CodY Regulates the Activity of the Virulence Quorum Sensor PlcR by Controlling the Import of the Signaling Peptide PapR in Bacillus thuringiensis

    Science.gov (United States)

    Slamti, Leyla; Lemy, Christelle; Henry, Céline; Guillot, Alain; Huillet, Eugénie; Lereclus, Didier

    2016-01-01

    In Gram-positive bacteria, cell–cell communication mainly relies on cytoplasmic sensors of the RNPP family. Activity of these regulators depends on their binding to secreted signaling peptides that are imported into the cell. These quorum sensing regulators control important biological functions in bacteria of the Bacillus cereus group, such as virulence and necrotrophism. The RNPP quorum sensor PlcR, in complex with its cognate signaling peptide PapR, is the main regulator of virulence in B. cereus and Bacillus thuringiensis (Bt). Recent reports have shown that the global stationary phase regulator CodY, involved in adaptation to nutritional limitation, is required for the expression of virulence genes belonging to the PlcR regulon. However, the mechanism underlying this regulation was not described. Using genetics and proteomics approaches, we showed that CodY regulates the expression of the virulence genes through the import of PapR. We report that CodY positively controls the production of the proteins that compose the oligopeptide permease OppABCDF, and of several other Opp-like proteins. It was previously shown that the pore components of this oligopeptide permease, OppBCDF, were required for the import of PapR. However, the role of OppA, the substrate-binding protein (SBP), was not investigated. Here, we demonstrated that OppA is not the only SBP involved in the recognition of PapR, and that several other OppA-like proteins can allow the import of this peptide. Altogether, these data complete our model of quorum sensing during the lifecycle of Bt and indicate that RNPPs integrate environmental conditions, as well as cell density, to coordinate the behavior of the bacteria throughout growth. PMID:26779156

  8. The Pharmacogenetic Footprint of ACE Inhibition: A Population-Based Metabolomics Study.

    Directory of Open Access Journals (Sweden)

    Elisabeth Altmaier

    Full Text Available Angiotensin-I-converting enzyme (ACE inhibitors are an important class of antihypertensives whose action on the human organism is still not fully understood. Although it is known that ACE especially cleaves COOH-terminal dipeptides from active polypeptides, the whole range of substrates and products is still unknown. When analyzing the action of ACE inhibitors, effects of genetic variation on metabolism need to be considered since genetic variance in the ACE gene locus was found to be associated with ACE-concentration in blood as well as with changes in the metabolic profiles of a general population. To investigate the interactions between genetic variance at the ACE-locus and the influence of ACE-therapy on the metabolic status we analyzed 517 metabolites in 1,361 participants from the KORA F4 study. We replicated our results in 1,964 individuals from TwinsUK. We observed differences in the concentration of five dipeptides and three ratios of di- and oligopeptides between ACE inhibitor users and non-users that were genotype dependent. Such changes in the concentration affected major homozygotes, and to a lesser extent heterozygotes, while minor homozygotes showed no or only small changes in the metabolite status. Two of these resulting dipeptides, namely aspartylphenylalanine and phenylalanylserine, showed significant associations with blood pressure which qualifies them-and perhaps also the other dipeptides-as readouts of ACE-activity. Since so far ACE activity measurement is substrate specific due to the usage of only one oligopeptide, taking several dipeptides as potential products of ACE into account may provide a broader picture of the ACE activity.

  9. Construction of a specific biomineralization template of enamel%一种釉质特异性生物矿化模板的构建

    Institute of Scientific and Technical Information of China (English)

    许小会; 宁天云; 杨聪翀; 朱丽芳; 徐晓华; 祝小鹏; 李全利; 刘来奎

    2012-01-01

    目的 构建一种自组装类釉原蛋白两亲性寡肽,作为釉质仿生矿化的有机模板.方法 依据釉原蛋白分子在釉质形成中自组装成“纳米球”超分子结构的机制,参照目前“自组装多肽类生物材料”的设计特点,通过对釉原蛋白分子功能性残基进行改性,构建“类釉原蛋白两亲性寡肽”生物矿化模板,采用固相合成法合成并纯化,经质谱仪、高效液相色谱仪等鉴定.在寡肽溶液中加入1 mol/L的CaCl2溶液观察其能否自组装;将组装后的寡肽涂于透射电镜的铜网上,用2.5%的戊二醛固定1h后先置于常温或40℃下10 mmol/L的CaCl2溶液中1h,去离子水漂洗后再置于5 mmol/L的Na2HPO4溶液中1h,5个循环后取出自然干燥,通过扫描电镜( SEM),透射电镜(TEM)及选区电子衍射(SAEDS)观察表征.结果 类釉原蛋白两亲性寡肽序列为C18H35O- Thr - Lys - Arg - Glu - Glu - Val - Asp,高效液相色谱仪(HPLC)示多肽纯度为98.29%,质谱仪(MS)示肽的分子质量为1 142.41 u.通过Ca2+可使其自组装为纳米纤维超微结构的凝胶态.通过交替矿化发现组装后的类釉原蛋白两亲性寡肽能摄取溶液中的钙磷离子,在寡肽纳米纤维表面成核矿化,形成磷灰石晶体.结论 成功构建了一种类釉原蛋白两亲性寡肽,可以作为釉质仿生的特异性生物矿化模板,用于诱导釉质再矿化研究.%Objective Construct a self-assembly amelogenin-inspired oligopeptide amphiphile as organic matrix template for simulating mineralization of enamel. Methods Inspired by amelogenin that self-assembles into nanospherical supermolecular structures in enamels and according to the design features of "self-assembly polypeptide biological materials" ,a novel biomineralization template of " amelogenin-like oligopeptide amphiphile" was designed be modify the funtions of amelogenin. The oligopeptide amphiphile was synthesized by standard solid-phase peptide synthesis, purified and

  10. Abiogenic synthesis on terrestrial orbit

    Science.gov (United States)

    Simakov, Michael B.; Kuzicheva, Evgenia; Gontareva, Natalia

    Meteorites probably played a central role in the evolution of life. Due to the structure, they tend to adsorb organic compounds and catalyze a variety of organic reactions critical to scenarios of life’s origins. We have shown experimentally that extraterrestrial minerals can catalyze the formation of peptides and nucleotides. The present study was performed onboard different Russian space stations (BION, COSMOS, and MIR) with various duration, altitude, and radiation conditions. Irradiation of solid samples, free or admixed with certain minerals, was the major task of future space flight experiments, planned for performing onboard Russian space satellite Bion-M. The «simulated space ice conditions» experiments have shown the synthesis of simple biochemical compounds in the form of amino acid’s precursors and pyrimidine bases (uracil, cyrosine and thymine) of the nucleic acids. Our investigation dealt with further reaction of nucleic acid components to nucleotides - main components of RNA and DNA, and single aminoacids to oligopeptides. We investigated two types of reactions: (1) abiogenic synthesis of nucleotides from mixtures of nucleoside + inorganic phosphate; (2) abiogenic synthesis of dipeptides from mixtures of simple amino acids. The reaction mixture in the form of a solid film contains (1) nucleoside and dihydrogen phosphate; (2) two different amino acids. Seven different nucleosides (thymidine, cytidine, uracil, adenosine or deoxyadenosine, guanosine or deoxyguanosine) and four mixtures of aromatic (tyrosine or triptophan) and aliphatic (glycine or alanine) amino acids were investigated. Mixtures were irradiated as solid films with different sources of energy: (1) VUV-light of 145 nm; (2) high energy protons (2-6 MeV); and (3) were installed on the surface of biosputnik in outstanding container when they were exposed to the action of all spectra of the open space energy sources during the entire time of flight. We have shown experimentally that the

  11. Structural parameterization and functional prediction of antigenic polypeptome sequences with biological activity through quantitative sequence-activity models (QSAM) by molecular electronegativity edge-distance vector (VMED)

    Institute of Scientific and Technical Information of China (English)

    LI; ZhiLiang; WU; ShiRong; CHEN; ZeCong; YE; Nancy; YANG; ShengXi; LIAO; ChunYang; ZHANG; MengJun; YANG; Li; MEI; Hu; YANG; Yan; ZHAO; Na; ZHOU; Yuan; ZHOU; Ping; XIONG; Qing; XU; Hong; LIU; ShuShen; LING; ZiHua; CHEN; Gang; LI; GenRong

    2007-01-01

    Only from the primary structures of peptides, a new set of descriptors called the molecular electronegativity edge-distance vector (VMED) was proposed and applied to describing and characterizing the molecular structures of oligopeptides and polypeptides, based on the electronegativity of each atom or electronic charge index (ECI) of atomic clusters and the bonding distance between atom-pairs. Here, the molecular structures of antigenic polypeptides were well expressed in order to propose the automated technique for the computerized identification of helper T lymphocyte (Th) epitopes. Furthermore, a modified MED vector was proposed from the primary structures of polypeptides, based on the ECI and the relative bonding distance of the fundamental skeleton groups. The side-chains of each amino acid were here treated as a pseudo-atom. The developed VMED was easy to calculate and able to work. Some quantitative model was established for 28 immunogenic or antigenic polypeptides (AGPP) with 14 (1―14) Ad and 14 other restricted activities assigned as "1"(+) and "0"(-), respectively. The latter comprised 6 Ab(15-20), 3 Ak(21-23), 2 Ek(24-26), 2 H-2k(27 and 28) restricted sequences. Good results were obtained with 90% correct classification (only 2 wrong ones for 20 training samples) and 100% correct prediction (none wrong for 8 testing samples); while contrastively 100% correct classification (none wrong for 20 training samples) and 88% correct classification (1 wrong for 8 testing samples). Both stochastic samplings and cross validations were performed to demonstrate good performance. The described method may also be suitable for estimation and prediction of classes I and II for major histocompatibility antigen (MHC) epitope of human. It will be useful in immune identification and recognition of proteins and genes and in the design and development of subunit vaccines. Several quantitative structure activity relationship (QSAR) models were developed for various

  12. Effects of harmful cyanobacteria on the freshwater pathogenic free-living amoeba Acanthamoeba castellanii.

    Science.gov (United States)

    Urrutia-Cordero, Pablo; Agha, Ramsy; Cirés, Samuel; Lezcano, María Ángeles; Sánchez-Contreras, María; Waara, Karl-Otto; Utkilen, Hans; Quesada, Antonio

    2013-04-15

    Grazing is a major regulating factor in cyanobacterial population dynamics and, subsequently, considerable effort has been spent on investigating the effects of cyanotoxins on major metazoan grazers. However, protozoan grazers such as free-living amoebae can also feed efficiently on cyanobacteria, while simultaneously posing a major threat for public health as parasites of humans and potential reservoirs of opportunistic pathogens. In this study, we conducted several experiments in which the freshwater amoeba Acanthamoeba castellanii was exposed to pure microcystin-LR (MC-LR) and six cyanobacterial strains, three MC-producing strains (MC-LR, MC-RR, MC-YR, MC-WR, [Dha7] MC-RR) and three strains containing other oligopeptides such as anabaenopeptins and cyanopeptolins. Although the exposure to high concentrations of pure MC-LR yielded no effects on amoeba, all MC-producing strains inflicted high mortality rates on amoeba populations, suggesting that toxic effects must be mediated through the ingestion of toxic cells. Interestingly, an anabaenopeptin-producing strain caused the greatest inhibition of amoeba growth, indicating that toxic bioactive compounds other than MCs are of great importance for amoebae grazers. Confocal scanning microscopy revealed different alterations in amoeba cytoskeleton integrity and as such, the observed declines in amoeba densities could have indeed been caused via a cascade of cellular events primarily triggered by oligopeptides with protein-phosphatase inhibition capabilities such as MCs or anabaenopeptins. Moreover, inducible-defense mechanisms such as the egestion of toxic, MC-producing cyanobacterial cells and the increase of resting stages (encystation) in amoebae co-cultivated with all cyanobacterial strains were observed in our experiments. Consequently, cyanobacterial strains showed different susceptibilities to amoeba grazing which were possibly influenced by the potentiality of their toxic secondary metabolites. Hence, this

  13. Guiding plant virus particles to integrin-displaying cells

    Science.gov (United States)

    Hovlid, Marisa L.; Steinmetz, Nicole F.; Laufer, Burkhardt; Lau, Jolene L.; Kuzelka, Jane; Wang, Qian; Hyypiä, Timo; Nemerow, Glen R.; Kessler, Horst; Manchester, Marianne; Finn, M. G.

    2012-05-01

    Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity for several different cancer cell lines that express RGD-binding integrin receptors.Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity

  14. Spin-Dependent Transport through Chiral Molecules Studied by Spin-Dependent Electrochemistry

    Science.gov (United States)

    2016-01-01

    Conspectus Molecular spintronics (spin + electronics), which aims to exploit both the spin degree of freedom and the electron charge in molecular devices, has recently received massive attention. Our recent experiments on molecular spintronics employ chiral molecules which have the unexpected property of acting as spin filters, by way of an effect we call “chiral-induced spin selectivity” (CISS). In this Account, we discuss new types of spin-dependent electrochemistry measurements and their use to probe the spin-dependent charge transport properties of nonmagnetic chiral conductive polymers and biomolecules, such as oligopeptides, L/D cysteine, cytochrome c, bacteriorhodopsin (bR), and oligopeptide-CdSe nanoparticles (NPs) hybrid structures. Spin-dependent electrochemical measurements were carried out by employing ferromagnetic electrodes modified with chiral molecules used as the working electrode. Redox probes were used either in solution or when directly attached to the ferromagnetic electrodes. During the electrochemical measurements, the ferromagnetic electrode was magnetized either with its magnetic moment pointing “UP” or “DOWN” using a permanent magnet (H = 0.5 T), placed underneath the chemically modified ferromagnetic electrodes. The spin polarization of the current was found to be in the range of 5–30%, even in the case of small chiral molecules. Chiral films of the l- and d-cysteine tethered with a redox-active dye, toludin blue O, show spin polarizarion that depends on the chirality. Because the nickel electrodes are susceptible to corrosion, we explored the effect of coating them with a thin gold overlayer. The effect of the gold layer on the spin polarization of the electrons ejected from the electrode was investigated. In addition, the role of the structure of the protein on the spin selective transport was also studied as a function of bias voltage and the effect of protein denaturation was revealed. In addition to

  15. Cathepsin S-cleavable, multi-block HPMA copolymers for improved SPECT/CT imaging of pancreatic cancer.

    Science.gov (United States)

    Fan, Wei; Shi, Wen; Zhang, Wenting; Jia, Yinnong; Zhou, Zhengyuan; Brusnahan, Susan K; Garrison, Jered C

    2016-10-01

    This work continues our efforts to improve the diagnostic and radiotherapeutic effectiveness of nanomedicine platforms by developing approaches to reduce the non-target accumulation of these agents. Herein, we developed multi-block HPMA copolymers with backbones that are susceptible to cleavage by cathepsin S, a protease that is abundantly expressed in tissues of the mononuclear phagocyte system (MPS). Specifically, a bis-thiol terminated HPMA telechelic copolymer containing 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. Three maleimide modified linkers with different sequences, including cathepsin S degradable oligopeptide, scramble oligopeptide and oligo ethylene glycol, were subsequently synthesized and used for the extension of the HPMA copolymers by thiol-maleimide click chemistry. All multi-block HPMA copolymers could be labeled by (177)Lu with high labeling efficiency and exhibited high serum stability. In vitro cleavage studies demonstrated highly selective and efficient cathepsin S mediated cleavage of the cathepsin S-susceptible multi-block HPMA copolymer. A modified multi-block HPMA copolymer series capable of Förster Resonance Energy Transfer (FRET) was utilized to investigate the rate of cleavage of the multi-block HPMA copolymers in monocyte-derived macrophages. Confocal imaging and flow cytometry studies revealed substantially higher rates of cleavage for the multi-block HPMA copolymers containing the cathepsin S-susceptible linker. The efficacy of the cathepsin S-cleavable multi-block HPMA copolymer was further examined using an in vivo model of pancreatic ductal adenocarcinoma. Based on the biodistribution and SPECT/CT studies, the copolymer extended with the cathepsin S susceptible linker exhibited significantly faster clearance and lower non-target retention without compromising tumor targeting. Overall, these results indicate that

  16. Comparison of the influence of humidity and D-mannitol on the organization of tetraethylene glycol-terminated self-assembled monolayers and immobilized antimicrobial peptides.

    Science.gov (United States)

    Goel, Mohit; Marsh, E Neil G; Chen, Zhan; Abbott, Nicholas L

    2014-06-24

    We report the use of polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS) to characterize the effects of relative humidity (RH) and d-mannitol on the conformations of tetraethylene glycol (EG4)-terminated self-assembled monolayers (SAMs) and immobilized antimicrobial peptides (Cecropin P1 and a hybrid of Cecropin A (1-8) and Melittin (1-18)). These results are used to assess the extent to which d-mannitol can substitute for water in promoting conformational states of the SAMs and oligopeptides similar to those induced by hydration. Our measurements reveal a red shift of the COC asymmetric stretching vibration of the EG4-terminated SAMs with increasing humidity, consistent with a transition from a mixed all-trans/helical (7/2 helix) conformation at 0% RH to a predominantly helical conformation at 90% RH. Significantly, under dry conditions, a thin (2 nm in thickness) overlayer of d-mannitol generated the COC spectroscopic signature of the EG4-terminated SAM measured at high humidity. Comparisons of the effects of humidity and d-mannitol on the secondary structure of the two oligopeptides also revealed both to cause the amide I peak positions, which were measured in dry air (and without d-mannitol) to correspond to α-helical conformations, to undergo red-shifts. The magnitudes of the red-shifts, however, were more pronounced for dry d-mannitol than for high RH, with Cecropin P1 and the hybrid peptide exhibiting amide I peak positions under d-mannitol consistent with bulk aqueous solution secondary structures (random and β-sheet, respectively). These results are discussed in the context of prior reports of the tendency of d-mannitol to form glassy states in the absence of water. Overall, the results presented in this paper support the hypothesis that d-mannitol can substitute, in at least some ways, for the influence of water on the conformational states of biologically relevant molecules at interfaces. The results provide guidance for the

  17. 米渣蛋白肽的新型酶解工艺及产品结构表征%Characterization of rice residue protein peptide made by a new enzymolysis process

    Institute of Scientific and Technical Information of China (English)

    董瑞晨; 王深旗; 熊华; 黎文建; 赵士强; 张忠

    2013-01-01

    米渣经过前处理去除糖类、脂质后得到米渣蛋白,再经酶解、脱色等处理后喷雾干燥得到米蛋白肽粉 以溶解性蛋白含量为指标,采用正交实验法对酶解工艺进行优化,得到最佳条件为:胰蛋白酶与复合蛋白酶配比为3∶2、酶解固液比1∶9、温度50 ℃、pH8.0、时间3.0h,蛋白肽产品溶解性蛋白、短肽含量分别从酶解前的4.35%、0.97%提高至23.49%、16.35% 采用红外光谱、粒径分布和扫描电镜对产品米渣蛋白肤分子进行了表征,表明此工艺制得的蛋白肽纯度较高.%The rice protein peptide powder was obtained by the pretreatment of rice dreg for removing saccharides and lipids,and then by the enzymolysis and decolorization to the rice residue protein followed by spray drying. Using the contents of dissolubility protein and oligopeptide as indicators,an orthogonal experimental method was adopted to optimize the enzymolysis technology. The optimum conditions were attained:the ratio of trypsin and protamex 3:2,the proportion of enzymolysis solid to liquid 1:9,temperature 50℃,.pH8.0 and time 3.0h. The contents of dissolubility protein and oligopeptide were 23.49% and 16.35% respectively,compared with 4.35% and 0.97% which weren't treated with enzyme. And the product of rice residue protein peptide was characterized including infrared spectrometry,size distribution and scanning electron microscope,which proved the rice residue protein peptide made by method purity was high.

  18. Identification and characterisation of a novel acylpeptide hydrolase from Sulfolobus solfataricus: structural and functional insights.

    Directory of Open Access Journals (Sweden)

    Marta Gogliettino

    Full Text Available A novel acylpeptide hydrolase, named APEH-3(Ss, was isolated from the hypertermophilic archaeon Sulfolobus solfataricus. APEH is a member of the prolyl oligopeptidase family which catalyzes the removal of acetylated amino acid residues from the N terminus of oligopeptides. The purified enzyme shows a homotrimeric structure, unique among the associate partners of the APEH cluster and, in contrast to the archaeal APEHs which show both exo/endo peptidase activities, it appears to be a "true" aminopeptidase as exemplified by its mammalian counterparts, with which it shares a similar substrate specificity. Furthermore, a comparative study on the regulation of apeh gene expression, revealed a significant but divergent alteration in the expression pattern of apeh-3(Ss and apeh(Ss (the gene encoding the previously identified APEH(Ss from S. solfataricus, which is induced in response to various stressful growth conditions. Hence, both APEH enzymes can be defined as stress-regulated proteins which play a complementary role in enabling the survival of S. solfataricus cells under different conditions. These results provide new structural and functional insights into S. solfataricus APEH, offering a possible explanation for the multiplicity of this enzyme in Archaea.

  19. A photolabile hydrogel for guided three-dimensional cell growth and migration

    Science.gov (United States)

    Luo, Ying; Shoichet, Molly S.

    2004-04-01

    Tissue engineering aims to replace, repair or regenerate tissue/organ function, by delivering signalling molecules and cells on a three-dimensional (3D) biomaterials scaffold that supports cell infiltration and tissue organization. To control cell behaviour and ultimately induce structural and functional tissue formation on surfaces, planar substrates have been patterned with adhesion signals that mimic the spatial cues to guide cell attachment and function. The objective of this study is to create biochemical channels in 3D hydrogel matrices for guided axonal growth. An agarose hydrogel modified with a cysteine compound containing a sulphydryl protecting group provides a photolabile substrate that can be patterned with biochemical cues. In this transparent hydrogel we immobilized the adhesive fibronectin peptide fragment, glycine-arginine-glycine-aspartic acid-serine (GRGDS), in selected volumes of the matrix using a focused laser. We verified in vitro the guidance effects of GRGDS oligopeptide-modified channels on the 3D cell migration and neurite outgrowth. This method for immobilizing biomolecules in 3D matrices can generally be applied to any optically clear hydrogel, offering a solution to construct scaffolds with programmed spatial features for tissue engineering applications.

  20. From coordination chemistry to biological chemistry of aluminium.

    Science.gov (United States)

    Kiss, Tamas

    2013-11-01

    The paper gives a review on the importance of distribution of Al in biological fluids, primarily in the lights of the works of the author in Al chemistry. It starts with studies of interactions of Al(III) with small biomolecules, such as aliphatic and aromatic hydroxycarboxylic acids, and inorganic and organic phosphates. A significant part of this review deals with the problems of description of the biospeciation of Al(III) in serum, where besides the thermodynamic conditions the role of time is also considered in the case of this sluggish metal ion. The Al(III) complexes of the other large group of biomolecules, proteins and their building blocks (oligo)peptides and amino acids are also discussed, where the role of the type of the side chain donors and the extent of preorganisation are considered in the efficiency of metal ion binding. The application of low molecular mass chelator molecules in restoring the dysfunctioning metal ion (including Al(III)) homeostasis in the treatment of Alzheimer's disease is also discussed in the paper.

  1. Gene Expression Profiling of Clostridium botulinum under Heat Shock Stress

    Directory of Open Access Journals (Sweden)

    Wan-dong Liang

    2013-01-01

    Full Text Available During growth, C. botulinum is always exposed to different environmental changes, such as temperature increase, nutrient deprivation, and pH change; however, its corresponding global transcriptional profile is uncharacterized. This study is the first description of the genome-wide gene expression profile of C. botulinum in response to heat shock stress. Under heat stress (temperature shift from 37°C to 45°C over a period of 15 min, 176 C. botulinum ATCC 3502 genes were differentially expressed. The response included overexpression of heat shock protein genes (dnaK operon, groESL, hsp20, and htpG and downregulation of aminoacyl-tRNA synthetase genes (valS, queA, tyrR, and gatAB and ribosomal and cell division protein genes (ftsZ and ftsH. In parallel, several transcriptional regulators (marR, merR, and ompR families were induced, suggesting their involvement in reshuffling of the gene expression profile. In addition, many ABC transporters (oligopeptide transport system, energy production and conversion related genes (glpA and hupL, cell wall and membrane biogenesis related genes (fabZ, fabF, and fabG, flagella-associated genes (flhA, flhM, flhJ, flhS, and motAB, and hypothetical genes also showed changed expression patterns, indicating that they may play important roles in survival under high temperatures.

  2. On the temperature dependence of amide I frequencies of peptides in solution.

    Science.gov (United States)

    Amunson, Krista E; Kubelka, Jan

    2007-08-23

    The temperature dependence of the amide I vibrational frequencies of peptides in solution was investigated. In D2O, the amide I' bands of both an alpha-helical oligopeptide, the random-coil poly(L-lysine), and the simplest amide, N-methyl acetamide (NMA), exhibit linear frequency shifts of approximately 0.07 cm(-1)/degrees C with increasing temperature. Similar amide I frequency shifts are also observed for NMA in both polar (acetonitrile and DMSO) and nonpolar (1,4-dioxane) organic solvents, thus ruling out hydrogen-bonding strength as the cause of these effects. The experimental NMA amide I frequencies in the organic solvents can be accurately described by a simple theory based on the Onsager reaction field with temperature-dependent solvent dielectric properties and a solute molecular cavity. DFT-level calculations (BPW91/cc-pVDZ) for NMA with an Onsager reaction field confirm the significant contribution of the molecular cavity to the predicted amide I frequencies. Comparison of the computations to experimental data shows that the frequency-dependent response of the reaction field, taken into account by the index of refraction, is crucial for describing the amide I frequencies in polar solvents. The poor predictions of the model for the NMA amide I band in D2O might be due, in part, to the unknown temperature dependence of the refractive index of D2O in the mid-IR range, which was approximated by the available values in the visible region.

  3. Recent Advances in Chemoenzymatic Peptide Syntheses

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    Kenjiro Yazawa

    2014-09-01

    Full Text Available Chemoenzymatic peptide synthesis is the hydrolase-catalyzed stereoselective formation of peptide bonds. It is a clean and mild procedure, unlike conventional chemical synthesis, which involves complicated and laborious protection-deprotection procedures and harsh reaction conditions. The chemoenzymatic approach has been utilized for several decades because determining the optimal conditions for conventional synthesis is often time-consuming. The synthesis of poly- and oligopeptides comprising various amino acids longer than a dipeptide continues to pose a challenge owing to the lack of knowledge about enzymatic mechanisms and owing to difficulty in optimizing the pH, temperature, and other reaction conditions. These drawbacks limit the applications of the chemoenzymatic approach. Recently, a variety of enzymes and substrates produced using recombinant techniques, substrate mimetics, and optimal reaction conditions (e.g., frozen aqueous media and ionic liquids have broadened the scope of chemoenzymatic peptide syntheses. In this review, we highlight the recent advances in the chemoenzymatic syntheses of various peptides and their use in developing new materials and biomedical applications.

  4. Peptide chain elongation: A possible role of montmorillonite in prebiotic synthesis of protein precursors

    Science.gov (United States)

    Bujdák, Juraj; Faybíková, Katarína; Eder, Artur; Yongyai, Yongyos; Rode, Bernd M.

    1995-10-01

    Several studies have proven the ability of montmorillonite to catalyse amino acid condensation under assumed prebiotic conditions, simulating wetting-drying cycles. In this work, the oligomerization of short peptides gly2, gly3, gly4 and ala2 on Ca-and Cu-montmorillonite in drying-wetting cycles at 80 °C was studied. The catalytic effect of montmorillonite was found to be much higher than in the case of glycine oligomerization. From gly2 after 3 weeks, 10% oligomers (up to gly6, with gly3 as main products) are formed. Gly3 and gly4 give higher oligomers even after 1 cycle. Ala2 produces both ala3 and ala4, whereas ala does not produce any oligomers under these conditions. Heteroologomerization was observed: ala-gly-gly is formed from ala and gly2. Much higher yields are obtained using Ca-montmorillonite, because copper (II) oxidizes organic molecules. The influence of the reaction mechanism on the preferential oligomerization of oligopeptides is discussed.

  5. Transcriptional profiling of hexaploid wheat (Triticum aestivum L.) roots identifies novel, dehydration-responsive genes.

    Science.gov (United States)

    Mohammadi, Mohsen; Kav, Nat N V; Deyholos, Michael K

    2007-05-01

    We used a long-oligonucleotide microarray to identify transcripts that increased or decreased in abundance in roots of dehydration-tolerant hexaploid bread wheat, in response to withholding of water. We observed that the major classes of dehydration-responsive genes (e.g. osmoprotectants, compatible solutes, proteases, glycosyltransferases/hydrolases, signal transducers components, ion transporters) were generally similar to those observed previously in other species and osmotic stresses. More specifically, we highlighted increases in transcript expression for specific genes including those putatively related to the synthesis of asparagine, trehalose, oligopeptide transporters, metal-binding proteins, the gamma-aminobutyric acid (GABA) shunt and transcription factors. Conversely, we noted a decrease in transcript abundance for diverse classes of glutathione and sulphur-related enzymes, specific amino acids, as well as MATE-efflux carrier proteins. From these data, we identified a novel, dehydration-induced putative AP2/ERF transcription factor, which we predict to function as a transcriptional repressor. We also identified a dehydration-induced 'little protein' (LitP; predicted mass: 8 kDa) that is highly conserved across spermatophytes. Using qRT-PCR, we compared the expression patterns of selected genes between two related wheat genotypes that differed in their susceptibility to dehydration, and confirmed that these novel genes were highly inducible by water limitation in both genotypes, although the magnitude of induction differed.

  6. Isolation and Identification of Dipeptidyl Peptidase IV-Inhibitory Peptides from Trypsin/Chymotrypsin-Treated Goat Milk Casein Hydrolysates by 2D-TLC and LC-MS/MS.

    Science.gov (United States)

    Zhang, Ying; Chen, Ran; Ma, Huiqin; Chen, Shangwu

    2015-10-14

    New dipeptidyl peptidase IV (DPP-IV)-inhibitory peptides from trypsin/chymotrypsin-treated goat milk casein hydrolysates were isolated and identified by two-dimensional silica thin-layer chromatography (2D-TLC) combined to nano LC-MS/MS. 2D-TLC with chloroform/methanol/25% ammonia (2:2:1) and n-butanol/acetic acid/water (4:1:1) as the first- and second-dimension eluents, respectively, in analytical and semipreparative scales, was set up and verified by reversed-phase high-performance liquid chromatography (RP-HPLC) to be feasible and efficient to separate the hydrolysates. Five new DPP-IV-inhibitory peptides, four relatively large oligopeptides (MHQPPQPL, SPTVMFPPQSVL, VMFPPQSVL, and INNQFLPYPY), and AWPQYL were identified, and INNQFLPYPY showed a notable IC50 value of 40.08 μM as an uncompetitive inhibitor. Interactive effects on DPP-IV inhibition were also observed among separated fractions and pure synthetic peptide mixtures with concentration-dependent activity. The study gives new insights into goat casein hydrolysates with identified DPP-IV-inhibitory peptides efficiently isolated by 2D-TLC, which provides a simple and cost-efficient separation process and is compatible with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification.

  7. Purification and crystallization of the ABC-type transport substrate-binding protein OppA from Thermoanaerobacter tengcongensis

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Jinlan; Li, Xiaolu [State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Tsinghua University, Beijing 100005, People' s Republic of China (China); Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, People' s Republic of China (China); Feng, Yue; Zhang, Bo [Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, People' s Republic of China (China); Miao, Shiying [State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Tsinghua University, Beijing 100005, People' s Republic of China (China); Wang, Linfang, E-mail: lfwangz@yahoo.com [State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Tsinghua University, Beijing 100005, People' s Republic of China (China); Wang, Na, E-mail: nawang@tsinghua.edu.cn [Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, People' s Republic of China (China)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer We truncated the signal peptide of OppA{sub TTE0054} to make it express in Escherichia coli as a soluble protein. Black-Right-Pointing-Pointer Crystals of OppA{sub TTE0054} were grown by sitting-drop vapor diffusion method. Black-Right-Pointing-Pointer The crystal of OppA{sub TTE0054} diffracted to 2.25 A. -- Abstract: Di- and oligopeptide- binding protein OppAs play important roles in solute and nutrient uptake, sporulation, biofilm formation, cell wall muropeptides recycling, peptide-dependent quorum-sensing responses, adherence to host cells, and a variety of other biological processes. Soluble OppA from Thermoanaerobacter tengcongensis was expressed in Escherichia coli. The protein was found to be >95% pure with SDS-PAGE after a series of purification steps and the purity was further verified by mass spectrometry. The protein was crystallized using the sitting-drop vapour-diffusion method with PEG 400 as the precipitant. Crystal diffraction extended to 2.25 A. The crystal belonged to space group C222{sub 1}, with unit-cell parameters of a = 69.395, b = 199.572, c = 131.673 A, and {alpha} = {beta} = {gamma} = 90 Degree-Sign .

  8. Comparing serum responses to acute feedings of an extensively hydrolyzed whey protein concentrate versus a native whey protein concentrate in rats: a metabolomics approach.

    Science.gov (United States)

    Roberts, Michael D; Cruthirds, Clayton L; Lockwood, Christopher M; Pappan, Kirk; Childs, Thomas E; Company, Joseph M; Brown, Jacob D; Toedebusch, Ryan G; Booth, Frank W

    2014-02-01

    We examined how gavage feeding extensively hydrolyzed whey protein (WPH) versus a native whey protein concentrate (WPC) transiently affected serum biochemical profiles in rodents. Male Wistar rats (250-300 g) were 8 h fasted and subsequently fed isonitrogenous amounts of WPH or WPC, or remained unfed (control). Animals were sacrificed 15 min, 30 min, and 60 min post-gavage for serum extraction, and serum was analyzed using untargeted global metabolic profiling via gas chromatography/mass spectrometry (MS) and liquid chromatography/MS/MS platforms. We detected 333 serum metabolites amongst the experimental and control groups. Both WPH and WPC generally increased amino acids (1.2-2.8-fold), branched-chain amino acids (1.2-1.7-fold), and serum di- and oligo-peptides (1.1-2.7-fold) over the 60 min time course compared with control (q protein sources led to a dramatic increase in free fatty acids compared with control (up to 6-fold increases, q protein sources.

  9. Exploiting quorum sensing interfering strategies in gram-negative bacteria for the enhancement of environmental applications

    Directory of Open Access Journals (Sweden)

    Weiwei eZhang

    2016-01-01

    Full Text Available Quorum sensing (QS is a widespread intercellular form of communication to coordinate physiological processes and cooperative activities of bacteria at the population level, and it depends on the production, secretion, and detection of small diffusible autoinducers, such as acyl-homoserine lactones (AHLs, auto-inducing oligo-peptides (AIPs and autoinducer 2. In this review, the function of QS autoinducers of gram-negative bacteria in different aspects of wastewater treatment systems is examined. Based on research primarily performed over the past ten years, QS involvement in the formation of biofilm and aerobic granules and changes of the microbial community and degradation/transformation pathways is discussed. In particular, the QS pathway in the role of bacterial infections and disease prevention in aquaculture is addressed. Interference of QS autoinducer-regulated pathways is considered potential treatment for a variety of environmentally related problems. This review is expected to serve as a stepping stone for further study and development strategies based on the mediation of QS-regulated pathways to enhance applications in both wastewater treatment systems and aquaculture.

  10. Goadsporin, a chemical substance which promotes secondary metabolism and morphogenesis in streptomycetes. I. Purification and characterization.

    Science.gov (United States)

    Onaka, H; Tabata, H; Igarashi, Y; Sato, Y; Furumai, T

    2001-12-01

    Streptomycetes, which belong to the Gram-positive bacteria, produce secondary metabolites and sporulate. The timing of starting the secondary metabolite production and the sporulation depends on environmental conditions such as nitrogen and carbon sources. In order to obtain a tool for understanding the regulation mechanism, we carried out screening for chemical substances that induce secondary metabolism and sporulation in streptomycetes and found an active substance from the culture broth of Streptomyces sp. TP-A0584. This substance designated goadsporin promoted the formation of red pigment and sporulation at a concentration of 1 microM in Streptomyces lividans TK23 which does not produce the pigment under normal growth conditions. Goadsporin is an oligopeptide consisting of 19 amino acids with the molecular formula C72H97N19020S2. Sporulation and/or secondary metabolite production was induced in 36 streptomycetes strains among 42 strains tested. These results suggest that goadsporin acts on a common regulation pathway for sporulation and secondary metabolism in streptomycetes and can be a powerful tool to analyze the regulation mechanism.

  11. Effect of chestnut extract and chestnut fiber on viability of potential probiotic Lactobacillus strains under gastrointestinal tract conditions.

    Science.gov (United States)

    Blaiotta, Giuseppe; La Gatta, Barbara; Di Capua, Marika; Di Luccia, Aldo; Coppola, Raffaele; Aponte, Maria

    2013-12-01

    The main challenge to probiotics, during their passage through the gastrointestinal tract, are the acidic gastric secretions of the stomach, and the bile salts released into the duodenum. The survival of the strains, in this phase, is strongly influenced by the food used for their delivery. This work is part of a project studying the development of novel food processes, based on the use of chestnuts from cultivar "Castagna di Montella". In detail, the effect of indigestible chestnut fiber and of chestnut extract on the viability of selected lactic acid bacteria strains was evaluated. Among 28 cultures, twelve strains were selected, on the basis of tolerance to low pH values and bile salts, and submitted to exposition to simulated gastric or bile juice in presence of chestnut extract with or without immobilization in chestnut fiber. The presence of chestnut extract proved to play a significant role on the gastric tolerance improvement of lactobacilli. The recorded protective effect could not be simply related to the starch or reducing sugars content. RP-HPLC demonstrated that in the chestnut flour, there are one or more hydrophobic peptides or oligopeptides, which specifically offer a marked resistance to simulated gastric juice, albeit present at low concentration. These beneficial effects proved to be dependent by the cultivar used to produce the flour.

  12. Green route for the utilization of chrome shavings (chromium-containing solid waste) in tanning industry.

    Science.gov (United States)

    Rao, Jonnalagadda Raghava; Thanikaivelan, Palanisamy; Sreeram, Kalarical Janardhanan; Nair, Balachandran Unni

    2002-03-15

    Chromium-containing wastes from various industrial sectors are under critical review. Leather processing is one such industrial activity that generates chromium-bearing wastes in different forms. One of them is chrome shavings, and this contributes to an extent of 10% of the quantum of raw skins/hides processed, amounting to 0.8 million ton globally. In this study, the high protein content of chrome shavings has been utilized for reduction of chromium(VI) in the preparation of chrome tanning agent. This approach has been exploited for the development of two products: one with chrome shavings alone as reducing agent and the other with equal proportion of chrome shavings and molasses. The developed products exhibit more masking due to the formation of intermediate organic oligopeptides. This has been corroborated through the spectral, hydrolysis, and species-wise distribution studies. The formation of these organic masking agents helps in chrome tanning by shifting the precipitation point of chromium to relatively higher pH levels. Hence, the developed products find use as chrome tanning agents for leather processing, thus providing a means for better utilization of chrome shaving wastes.

  13. Identification, cloning and environmental factors modulation of a αβ defensin from the Lessepsian invasive mussel Brachidontes pharaonis (Bivalvia: Mytilidae

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    MG Parisi

    2015-10-01

    Full Text Available Immunological effectors of invasive species playing a role in addressing new colonization are still poorly studied. In the present study the cDNA sequence of the defensin from a Lessepsian invasive species, the Red Sea mussel Brachidontes pharaonis, was cloned using RACE method. Defensins are a class of widely known antimicrobial peptides (AMPs, oligopeptides with a broad spectrum of targeted organisms ranging from viruses to parasites. Analysis of BpDef sequence (262 bp revealed the presence of an ORF coding for 81 amino acids. The full-length amino acid sequence showed the highest similarity to antimicrobial peptides MGD1 and MGD2 sequence from Mytilus galloprovincialis. Phylogenetic analysis suggested that BpDef belongs to the αβ defensin AMPs with a typical domain structurally characterized by a α helix and two β sheets. BpDef mRNA is located in circulating hemocytes with small intra-cytoplasmic granules and with large granules. The transcription of defensin gene was modulated by the stress from temperatures and oxygenation condition. Temperatures of 20 °C did not stimulate a BpDef transcription over a short time. At 30 °C the kinetics of BpDef gene transcription showed up regulation after one day, while it was down regulated after six days, both under normoxia and hypoxia conditions.

  14. Untargeted metabolomics approach for unraveling robust biomarkers of nutritional status in fasted gilthead sea bream (Sparus aurata

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    Ruben Gil-Solsona

    2017-01-01

    Full Text Available A metabolomic study has been performed to identify sensitive and robust biomarkers of malnutrition in farmed fish, using gilthead sea bream (Sparus aurata as a model. The metabolomic fingerprinting of serum from fasted fish was assessed by means of ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. More than 15,000 different m/z ions were detected and Partial Least Squares–Discriminant analysis allowed a clear differentiation between the two experimental groups (fed and 10-day fasted fish with more than 90% of total variance explained by the two first components. The most significant metabolites (up to 45 were elucidated on the basis of their tandem mass spectra with a broad representation of amino acids, oligopeptides, urea cycle metabolites, L-carnitine-related metabolites, glutathione-related metabolites, fatty acids, lysophosphatidic acids, phosphatidylcholines as well as biotin- and noradrenaline-related metabolites. This untargeted approach highlighted important adaptive responses in energy and oxidative metabolism, contributing to identify robust and nutritionally-regulated biomarkers of health and metabolic condition that will serve to assess the welfare status of farmed fish.

  15. Identification of hepta-histidine as a candidate drug for Huntington’s disease by in silico-in vitro- in vivo-integrated screens of chemical libraries

    Science.gov (United States)

    Imamura, Tomomi; Fujita, Kyota; Tagawa, Kazuhiko; Ikura, Teikichi; Chen, Xigui; Homma, Hidenori; Tamura, Takuya; Mao, Ying; Taniguchi, Juliana Bosso; Motoki, Kazumi; Nakabayashi, Makoto; Ito, Nobutoshi; Yamada, Kazunori; Tomii, Kentaro; Okano, Hideyuki; Kaye, Julia; Finkbeiner, Steven; Okazawa, Hitoshi

    2016-09-01

    We identified drug seeds for treating Huntington’s disease (HD) by combining in vitro single molecule fluorescence spectroscopy, in silico molecular docking simulations, and in vivo fly and mouse HD models to screen for inhibitors of abnormal interactions between mutant Htt and physiological Ku70, an essential DNA damage repair protein in neurons whose function is known to be impaired by mutant Htt. From 19,468 and 3,010,321 chemicals in actual and virtual libraries, fifty-six chemicals were selected from combined in vitro-in silico screens; six of these were further confirmed to have an in vivo effect on lifespan in a fly HD model, and two chemicals exerted an in vivo effect on the lifespan, body weight and motor function in a mouse HD model. Two oligopeptides, hepta-histidine (7H) and Angiotensin III, rescued the morphological abnormalities of primary neurons differentiated from iPS cells of human HD patients. For these selected drug seeds, we proposed a possible common structure. Unexpectedly, the selected chemicals enhanced rather than inhibited Htt aggregation, as indicated by dynamic light scattering analysis. Taken together, these integrated screens revealed a new pathway for the molecular targeted therapy of HD.

  16. Oyster (Crassostrea gigas Hydrolysates Produced on a Plant Scale Have Antitumor Activity and Immunostimulating Effects in BALB/c Mice

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    Yu-Zhong Zhang

    2010-02-01

    Full Text Available Oyster extracts have been reported to have many bioactive peptides. But the function of oyster peptides produced by proteolysis is still unknown. In this study, the oligopeptide-enriched hydrolysates from oyster (Crassostrea gigas were produced using the protease from Bacillus sp. SM98011 at laboratory level, and scaled up to pilot (100 L and plant (1,000 L levels with the same conditions. And the antitumor activity and immunostimulating effects of the oyster hydrolysates in BALB/c mice were investigated. The growth of transplantable sarcoma-S180 was obviously inhibited in a dose-dependent manner in BALB/c mice given the oyster hydrolysates. Mice receiving 0.25, 0.5 and 1 mg/g of body weight by oral gavage had 6.8%, 30.6% and 48% less tumor growth, respectively. Concurrently, the weight coefficients of the thymus and the spleen, the activity of natural killer (NK cells, the spleen proliferation of lymphocytes and the phagocytic rate of macrophages in S180-bearing mice significantly increased after administration of the oyster hydrolysates. These results demonstrated that oyster hydrolysates produced strong immunostimulating effects in mice, which might result in its antitumor activity. The antitumor and immunostimulating effects of oyster hydrolysates prepared in this study reveal its potential for tumor therapy and as a dietary supplement with immunostimulatory activity.

  17. Comparative transcriptome analysis between csrA-disruption Clostridium acetobutylicum and its parent strain.

    Science.gov (United States)

    Tan, Yang; Liu, Zi-Yong; Liu, Zhen; Zheng, Hua-Jun; Li, Fu-Li

    2015-05-01

    The genome of Clostridium acetobutylicum contains the gene encoding CsrA, a carbon storage regulator. We investigated the function of CsrA in C. acetobutylicum by insertionally inactivating the encoding gene, CA_C2209 using the ClosTron. Disruption of csrA obviously decreases the growth of the organism and reduces the yield of acetone, butanol and ethanol (ABEs). Like the csrA in Escherichia coli, RNA-seq and β-galactosidase analysis revealed that csrA in C. acetobutylicum was closely involved in regulating multiple pathways including flagella assembly, oligopeptide transporting, iron uptake, and central carbon metabolism. It has also been newly demonstrated that csrA in C. acetobutylicum is related to the regulation of pathways involved in the phosphotransferase transporting systems, synthesis of riboflavin, and stage III sporulation. This research represented the first investigation of global regulation by CsrA in the strain belonging to Gram-positive bacteria through transcriptome analysis and provided the important theoretical evidence for improving solvent production by transcriptor engineering in C. acetobutylicum.

  18. Gene expression profiling of soft and firm Atlantic salmon fillet.

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    Thomas Larsson

    Full Text Available Texture of salmon fillets is an important quality trait for consumer acceptance as well as for the suitability for processing. In the present work we measured fillet firmness in a population of farmed Atlantic salmon with known pedigree and investigated the relationship between this trait and gene expression. Transcriptomic analyses performed with a 21 K oligonucleotide microarray revealed strong correlations between firmness and a large number of genes. Highly similar expression profiles were observed in several functional groups. Positive regression was found between firmness and genes encoding proteasome components (41 genes and mitochondrial proteins (129 genes, proteins involved in stress responses (12 genes, and lipid metabolism (30 genes. Coefficients of determination (R(2 were in the range of 0.64-0.74. A weaker though highly significant negative regression was seen in sugar metabolism (26 genes, R(2 = 0.66 and myofiber proteins (42 genes, R(2 = 0.54. Among individual genes that showed a strong association with firmness, there were extracellular matrix proteins (negative correlation, immune genes, and intracellular proteases (positive correlation. Several genes can be regarded as candidate markers of flesh quality (coiled-coil transcriptional coactivator b, AMP deaminase 3, and oligopeptide transporter 15 though their functional roles are unclear. To conclude, fillet firmness of Atlantic salmon depends largely on metabolic properties of the skeletal muscle; where aerobic metabolism using lipids as fuel, and the rapid removal of damaged proteins, appear to play a major role.

  19. The tenacious recognition of yeast telomere sequence by Cdc13 is fully exerted by a single OB-fold domain.

    Science.gov (United States)

    Lewis, Karen A; Pfaff, Danielle A; Earley, Jennifer N; Altschuler, Sarah E; Wuttke, Deborah S

    2014-01-01

    Cdc13, the telomere end-binding protein from Saccharomyces cerevisiae, is a multidomain protein that specifically binds telomeric single-stranded DNA (ssDNA) with exquisitely high affinity to coordinate telomere maintenance. Recent structural and genetic data have led to the proposal that Cdc13 is the paralog of RPA70 within a telomere-specific RPA complex. Our understanding of Cdc13 structure and biochemistry has been largely restricted to studies of individual domains, precluding analysis of how each domain influences the activity of the others. To better facilitate a comparison to RPA70, we evaluated the ssDNA binding of full-length S. cerevisiae Cdc13 to its minimal substrate, Tel11. We found that, unlike RPA70 and the other known telomere end-binding proteins, the core Cdc13 ssDNA-binding activity is wholly contained within a single tight-binding oligosaccharide/oligonucleotide/oligopeptide binding (OB)-fold. Because two OB-folds are implicated in dimerization, we also evaluated the relationship between dimerization and ssDNA-binding activity and found that the two activities are independent. We also find that Cdc13 binding exhibits positive cooperativity that is independent of dimerization. This study reveals that, while Cdc13 and RPA70 share similar domain topologies, the corresponding domains have evolved different and specialized functions.

  20. Analysis and update of the human solute carrier (SLC gene superfamily

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    He Lei

    2009-01-01

    Full Text Available Abstract The solute-carrier gene (SLC superfamily encodes membrane-bound transporters. The SLC superfamily comprises 55 gene families having at least 362 putatively functional protein-coding genes. The gene products include passive transporters, symporters and antiporters, located in all cellular and organelle membranes, except, perhaps, the nuclear membrane. Transport substrates include amino acids and oligopeptides, glucose and other sugars, inorganic cations and anions (H+, HCO3-, Cl-, Na+, K+, Ca2+, Mg2+, PO43-, HPO42-, H2PO4-, SO42-, C2O42-, OH-,CO32-, bile salts, carboxylate and other organic anions, acetyl coenzyme A, essential metals, biogenic amines, neurotransmitters, vitamins, fatty acids and lipids, nucleosides, ammonium, choline, thyroid hormone and urea. Contrary to gene nomenclature commonly assigned on the basis of evolutionary divergence http://www.genenames.org/, the SLC gene superfamily has been named based largely on transporter function by proteins having multiple transmembrane domains. Whereas all the transporters exist for endogenous substrates, it is likely that drugs, non-essential metals and many other environmental toxicants are able to 'hitch-hike' on one or another of these transporters, thereby enabling these moieties to enter (or leave the cell. Understanding and characterising the functions of these transporters is relevant to medicine, genetics, developmental biology, pharmacology and cancer chemotherapy.

  1. Peptide synthesis in early earth hydrothermal systems

    Science.gov (United States)

    Lemke, K.H.; Rosenbauer, R.J.; Bird, D.K.

    2009-01-01

    We report here results from experiments and thermodynamic calculations that demonstrate a rapid, temperature-enhanced synthesis of oligopeptides from the condensation of aqueous glycine. Experiments were conducted in custom-made hydrothermal reactors, and organic compounds were characterized with ultraviolet-visible procedures. A comparison of peptide yields at 260??C with those obtained at more moderate temperatures (160??C) gives evidence of a significant (13 kJ ?? mol-1) exergonic shift. In contrast to previous hydrothermal studies, we demonstrate that peptide synthesis is favored in hydrothermal fluids and that rates of peptide hydrolysis are controlled by the stability of the parent amino acid, with a critical dependence on reactor surface composition. From our study, we predict that rapid recycling of product peptides from cool into near-supercritical fluids in mid-ocean ridge hydrothermal systems will enhance peptide chain elongation. It is anticipated that the abundant hydrothermal systems on early Earth could have provided a substantial source of biomolecules required for the origin of life. Astrobiology 9, 141-146. ?? 2009 Mary Ann Liebert, Inc. 2009.

  2. Obligate Biotrophy Features Unraveled by the Genomic Analysis of the Rust Fungi, Melampsora larici-populina and Puccinia graminis f. sp. tritici

    Energy Technology Data Exchange (ETDEWEB)

    Duplessis, Sebastien; Cuomo, Christina A.; Lin, Yao-Cheng; Aerts, Andrea; Tisserant, Emilie; Veneault-Fourrey, Claire; Joly, David L.; Hacquard, Stephane; Amselem, Joelle; Cantarel, Brandi; Chiu, Readman; Couthinho, Pedro; Feau, Nicolas; Field, Matthew; Frey, Pascal; Gelhaye, Eric; Goldberg, Jonathan; Grabherr, Manfred; Kodira, Chinnappa; Kohler, Annegret; Kues, Ursula; Lindquist, Erika; Lucas, Susan; Mago, Rohit; Mauceli, Evan; Morin, Emmanuelle; Murat, Claude; Pangilinan, Jasmyn L.; Park, Robert; Pearson, Matthew; Quesneville, Hadi; Rouhier, Nicolas; Sakthikumar, Sharadha; Salamov, Asaf A.; Schmutz, Jeremy; Selles, Benjamin; Shapiro, Harris; Tangay, Philippe; Tuskan, Gerald A.; Peer, Yves Van de; Henrissat, Bernard; Rouze, Pierre; Ellis, Jeffrey G.; Dodds, Peter N.; Schein, Jacqueline E.; Zhong, Shaobin; Hamelin, Richard C.; Grigoriev, Igor V.; Szabo, Les J.; Martin1, Francis

    2011-04-27

    Rust fungi are some of the most devastating pathogens of crop plants. They are obligate biotrophs, which extract nutrients only from living plant tissues and cannot grow apart from their hosts. Their lifestyle has slowed the dissection of molecular mechanisms underlying host invasion and avoidance or suppression of plant innate immunity. We sequenced the 101 mega base pair genome of Melampsora larici-populina, the causal agent of poplar leaf rust, and the 89 mega base pair genome of Puccinia graminis f. sp. tritici, the causal agent of wheat and barley stem rust. We then compared the 16,841 predicted proteins of M. larici-populina to the 18,241 predicted proteins of P. graminis f. sp tritici. Genomic features related to their obligate biotrophic life-style include expanded lineage-specific gene families, a large repertoire of effector-like small secreted proteins (SSPs), impaired nitrogen and sulfur assimilation pathways, and expanded families of amino-acid, oligopeptide and hexose membrane transporters. The dramatic upregulation of transcripts coding for SSPs, secreted hydrolytic enzymes, and transporters in planta suggests that they play a role in host infection and nutrient acquisition. Some of these genomic hallmarks are mirrored in the genomes of other microbial eukaryotes that have independently evolved to infect plants, indicating convergent adaptation to a biotrophic existence inside plant cells

  3. Ampicillin-Ester Bonded Branched Polymers: Characterization, Cyto-, Genotoxicity and Controlled Drug-Release Behaviour

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    Ewa Oledzka

    2014-06-01

    Full Text Available The development and characterization of novel macromolecular conjugates of ampicillin using branched biodegradable polymers has been described in this study. The conjugates have been prepared coupling the β-lactam antibiotic with branched polymer matrices based on the natural oligopeptide core. The cyto- and genotoxicity of the synthesized polymers were evaluated with a bacterial luminescence test, two protozoan assays and Salmonella typhimurium TA1535. The presence of a newly formed covalent bond between the drug and the polymer matrices was confirmed by 1H-NMR and FTIR studies. A drug content (15.6 and 10.2 mole % in the macromolecular conjugates has been determined. The obtained macromolecular products have been subjected to further in vitro release studies. The total percentage of ampicillin released after 21 days of incubation was nearly 60% and 14% and this resulted from the different physicochemical properties of the polymeric matrices. This is the first report on the application of branched biodegradable polymeric matrices for the covalent conjugation of ampicillin. The obtained results showed that the synthesized macromolecular drug-conjugates might slowly release the active drug molecule and improve the pharmacokinetics of ampicillin.

  4. Molecular Recognition of PTS-1 Cargo Proteins by Pex5p: Implications for Protein Mistargeting in Primary Hyperoxaluria

    Directory of Open Access Journals (Sweden)

    Noel Mesa-Torres

    2015-02-01

    Full Text Available Peroxisomal biogenesis and function critically depends on the import of cytosolic proteins carrying a PTS1 sequence into this organelle upon interaction with the peroxin Pex5p. Recent structural studies have provided important insights into the molecular recognition of cargo proteins by Pex5p. Peroxisomal import is a key feature in the pathogenesis of primary hyperoxaluria type 1 (PH1, where alanine:glyoxylate aminotransferase (AGT undergoes mitochondrial mistargeting in about a third of patients. Here, we study the molecular recognition of PTS1 cargo proteins by Pex5p using oligopeptides and AGT variants bearing different natural PTS1 sequences, and employing an array of biophysical, computational and cell biology techniques. Changes in affinity for Pex5p (spanning over 3–4 orders of magnitude reflect different thermodynamic signatures, but overall bury similar amounts of molecular surface. Structure/energetic analyses provide information on the contribution of ancillary regions and the conformational changes induced in Pex5p and the PTS1 cargo upon complex formation. Pex5p stability in vitro is enhanced upon cargo binding according to their binding affinities. Moreover, we provide evidence that the rational modulation of the AGT: Pex5p binding affinity might be useful tools to investigate mistargeting and misfolding in PH1 by pulling the folding equilibria towards the native and peroxisomal import competent state.

  5. Red Queen Processes Drive Positive Selection on Major Histocompatibility Complex (MHC Genes.

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    Maciej Jan Ejsmond

    2015-11-01

    Full Text Available Major Histocompatibility Complex (MHC genes code for proteins involved in the incitation of the adaptive immune response in vertebrates, which is achieved through binding oligopeptides (antigens of pathogenic origin. Across vertebrate species, substitutions of amino acids at sites responsible for the specificity of antigen binding (ABS are positively selected. This is attributed to pathogen-driven balancing selection, which is also thought to maintain the high polymorphism of MHC genes, and to cause the sharing of allelic lineages between species. However, the nature of this selection remains controversial. We used individual-based computer simulations to investigate the roles of two phenomena capable of maintaining MHC polymorphism: heterozygote advantage and host-pathogen arms race (Red Queen process. Our simulations revealed that levels of MHC polymorphism were high and driven mostly by the Red Queen process at a high pathogen mutation rate, but were low and driven mostly by heterozygote advantage when the pathogen mutation rate was low. We found that novel mutations at ABSs are strongly favored by the Red Queen process, but not by heterozygote advantage, regardless of the pathogen mutation rate. However, while the strong advantage of novel alleles increased the allele turnover rate, under a high pathogen mutation rate, allelic lineages persisted for a comparable length of time under Red Queen and under heterozygote advantage. Thus, when pathogens evolve quickly, the Red Queen is capable of explaining both positive selection and long coalescence times, but the tension between the novel allele advantage and persistence of alleles deserves further investigation.

  6. One-pot synthesis of dextran decorated reduced graphene oxide nanoparticles for targeted photo-chemotherapy.

    Science.gov (United States)

    Hu, Yanfang; He, Liang; Ding, Jianxun; Sun, Diankui; Chen, Li; Chen, Xuesi

    2016-06-25

    Graphene-based nanocarriers show great potential in photo-chemotherapy, however, to prepare desired reduced graphene oxide (rGO) nanoparticles in a facile way is still a challenge. Herein, a novel strategy has been presented to prepare rGO nanoparticle using dextran (Dex) as a reducing agent. In this strategy, Dex was directly conjugated on rGO by hydrogen bond and then self-assemble to form rGO/Dex nanoparticles. After decorated by dextran, rGO-based nanoparticles not only show excellent biocompatibility but also can load anticancer drug for photo-chemotherapy. The data of fourier transform infrared (FT-IR) analysis, Raman spectrum analysis, thermos-gravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS), the transmission electron microscope (TEM) image and dynamic light scattering (DLS) measurements powerfully proved that the stable rGO-based nanoparticles with desired nanosize have been successfully prepared. To verify the photo-chemotherapy, anticancer drug, doxorubicin (DOX), has been loaded on rGO/Dex nanoparticles (rGO/DOX/Dex). And RGD, a kind of oligopeptide which can improve the intracellular uptake by αvβ3 recognition, also has been introduced (rGO/DOX/RDex). Compared with single chemotherapy, rGO/DOX/Dex and rGO/DOX/RDex combining the local specific chemotherapy and external near-infrared (NIR) photo-thermal therapy show higher therapeutic efficacy, endowing the desired rGO-based nanoparticle with great potential for cancer treatments.

  7. Champacyclin, a New Cyclic Octapeptide from Streptomyces Strain C42 Isolated from the Baltic Sea

    Directory of Open Access Journals (Sweden)

    Alexander Pesic

    2013-12-01

    Full Text Available New isolates of Streptomyces champavatii were isolated from marine sediments of the Gotland Deep (Baltic Sea, from the Urania Basin (Eastern Mediterranean, and from the Kiel Bight (Baltic Sea. The isolates produced several oligopeptidic secondary metabolites, including the new octapeptide champacyclin (1a present in all three strains. Herein, we report on the isolation, structure elucidation and determination of the absolute stereochemistry of this isoleucine/leucine (Ile/Leu = Xle rich cyclic octapeptide champacyclin (1a. As 2D nuclear magnetic resonance (NMR spectroscopy could not fully resolve the structure of (1a, additional information on sequence and configuration of stereocenters were obtained by a combination of multi stage mass spectrometry (MSn studies, amino acid analysis, partial hydrolysis and subsequent enantiomer analytics with gas chromatography positive chmical ionization/electron impact mass spectrometry (GC-PCI/EI-MS supported by comparison to reference dipeptides. Proof of the head-to-tail cyclization of (1a was accomplished by solid phase peptide synthesis (SPPS compared to an alternatively side chain cyclized derivative (2. Champacyclin (1a is likely synthesized by a non-ribosomal peptide synthetase (NRPS, because of its high content of (d-amino acids. The compound (1a showed antimicrobial activity against the phytopathogen Erwinia amylovora causing the fire blight disease of certain plants.

  8. Synergism and Mutualism in Non-Enzymatic RNA Polymerization

    Directory of Open Access Journals (Sweden)

    Hussein Kaddour

    2014-11-01

    Full Text Available The link between non-enzymatic RNA polymerization and RNA self-replication is a key step towards the “RNA world” and still far from being solved, despite extensive research. Clay minerals, lipids and, more recently, peptides were found to catalyze the non-enzymatic synthesis of RNA oligomers. Herein, a review of the main models for the formation of the first RNA polymers is presented in such a way as to emphasize the cooperation between life’s building blocks in their emergence and evolution. A logical outcome of the previous results is a combination of these models, in which RNA polymerization might have been catalyzed cooperatively by clays, lipids and peptides in one multi-component prebiotic soup. The resulting RNAs and oligopeptides might have mutualistically evolved towards functional RNAs and catalytic peptides, preceding the first RNA replication, thus supporting an RNA-peptide world. The investigation of such a system is a formidable challenge, given its complexity deriving from a tremendously large number of reactants and innumerable products. A rudimentary experimental design is outlined, which could be used in an initial attempt to study a quaternary component system.

  9. Synergism and Mutualism in Non-Enzymatic RNA Polymerization

    Science.gov (United States)

    Kaddour, Hussein; Sahai, Nita

    2014-01-01

    The link between non-enzymatic RNA polymerization and RNA self-replication is a key step towards the “RNA world” and still far from being solved, despite extensive research. Clay minerals, lipids and, more recently, peptides were found to catalyze the non-enzymatic synthesis of RNA oligomers. Herein, a review of the main models for the formation of the first RNA polymers is presented in such a way as to emphasize the cooperation between life’s building blocks in their emergence and evolution. A logical outcome of the previous results is a combination of these models, in which RNA polymerization might have been catalyzed cooperatively by clays, lipids and peptides in one multi-component prebiotic soup. The resulting RNAs and oligopeptides might have mutualistically evolved towards functional RNAs and catalytic peptides, preceding the first RNA replication, thus supporting an RNA-peptide world. The investigation of such a system is a formidable challenge, given its complexity deriving from a tremendously large number of reactants and innumerable products. A rudimentary experimental design is outlined, which could be used in an initial attempt to study a quaternary component system. PMID:25370531

  10. Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth.

    Science.gov (United States)

    Jakočiūnė, Džiuginta; Herrero-Fresno, Ana; Jelsbak, Lotte; Olsen, John Elmerdahl

    2016-05-01

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common cause of egg borne salmonellosis in many parts of the world. This study analyzed gene expression of this bacterium during growth in whole egg, and whether highly expressed genes were essential for the growth. High quality RNA was extracted from S. Enteritidis using a modified RNA-extraction protocol. Global gene expression during growth in whole egg was compared to growth in LB-medium using DNA array method. Twenty-six genes were significantly upregulated during growth in egg; these belonged to amino acid biosynthesis, di/oligopeptide transport system, biotin synthesis, ferrous iron transport system, and type III secretion system. Significant downregulation of 15 genes related to formate hydrogenlyase (FHL) and trehalose metabolism was observed. The results suggested that S. Enteritidis is starved for amino-acids, biotin and iron when growing in egg. However, site specific mutation of amino acid biosynthesis genes asnA (17.3 fold upregulated), asnB (18.6 fold upregulated), asnA/asnB and, serA (12.0 fold upregulated) and gdhA (3.7 fold upregulated), did not result in growth attenuation, suggesting that biosynthesis using the enzymes encoded from these genes may represent the first choice for S. Enteritidis when growing in egg, but when absent, the bacterium could use alternative ways to obtain the amino acids.

  11. Prebiotic significance of extraterrestrial ice photochemistry: detection of hydantoin in organic residues.

    Science.gov (United States)

    de Marcellus, Pierre; Bertrand, Marylène; Nuevo, Michel; Westall, Frances; Le Sergeant d'Hendecourt, Louis

    2011-11-01

    The delivery of extraterrestrial organic materials to primitive Earth from meteorites or micrometeorites has long been postulated to be one of the origins of the prebiotic molecules involved in the subsequent apparition of life. Here, we report on experiments in which vacuum UV photo-irradiation of interstellar/circumstellar ice analogues containing H(2)O, CH(3)OH, and NH(3) led to the production of several molecules of prebiotic interest. These were recovered at room temperature in the semi-refractory, water-soluble residues after evaporation of the ice. In particular, we detected small quantities of hydantoin (2,4-imidazolidinedione), a species suspected to play an important role in the formation of poly- and oligopeptides. In addition, hydantoin is known to form under extraterrestrial, abiotic conditions, since it has been detected, along with various other derivatives, in the soluble part of organic matter of primitive carbonaceous meteorites. This result, together with other related experiments reported recently, points to the potential importance of the photochemistry of interstellar "dirty" ices in the formation of organics in Solar System materials. Such molecules could then have been delivered to the surface of primitive Earth, as well as other telluric (exo-) planets, to help trigger first prebiotic reactions with the capacity to lead to some form of primitive biomolecular activity.

  12. Mucoadhesion vs mucus permeability of thiolated chitosan polymers and their resulting nanoparticles using a quartz crystal microbalance with dissipation (QCM-D).

    Science.gov (United States)

    Oh, Sejin; Borrós, Salvador

    2016-11-01

    The aim of this present study was to evaluate the combination properties between mucoadhesion/mucus permeability of thiolated chitosans (TC) and their resulting nanoparticles using a quartz crystal microbalance with dissipation (QCM-D). The QCM-D experiments were conducted at pH 4 or 6.8 to assess the interaction between thiolated polymers, with low (TCL), medium (TCM) and high (TCH) contents of free thiol groups, and native porcine gastric mucin (NPGM). TCL was chosen for further carriers as it showed higher permeability into the NPGM layer compared to TCM and TCH. In this study, we describe a formulation of a novel carrier comprised by positively charged TCL, negatively charged DNA and degradable oligopeptide-modified poly(β-amino ester)s (PBAEs), which were employed in order to approach for tuning particle size and surface charge of complexes. TCL/PBAE complexes with or without DNA were characterized using dynamic light scattering. Mechanism of adsorption or permeation of the TCL/PBAE/DNA complexes into the NPGM barrier was investigated with QCM-D, which is a highly sensitive technique for studying nanomechanical (viscoelastic) changes of the substrates. This work might provide that the QCM-D technique would be a promising method to monitor the dynamic behaviour between complexes and NPGM.

  13. Using mass spectrometry for identification of ABC transporters from Xanthomonas citri and mutants expressed in different growth conditions

    Energy Technology Data Exchange (ETDEWEB)

    Faria, J.N.; Balan, A. [Laboratorio Nacional de Biociencias - LNBIO, Campinas, SP (Brazil); Paes Leme, A.F. [Laboratorio Nacional de Luz Sincrotron (LNLS), Campinas, SP (Brazil)

    2012-07-01

    Full text: Xanthomonas citri is a phytopathogenic bacterium that infects citrus plants causing significant losses for the economy. In our group, we have focused on the identification and characterization of ABC transport proteins of this bacterium, in order to determinate their function for growth in vitro and in vivo, during infection. ABC transporters represent one of the largest families of proteins, which transport since small molecules as ions up to oligopeptides and sugars. In prokaryotic cells many works have reported the ABC transport function in pathogenesis, resistance, biofilm formation, infectivity and DNA repair, but until our knowledge, there is no data related to these transporters and X. citri. So, In order to determinate which transporters are expressed in X. citri, we started a proteomic analysis based on mono and bi-dimensional gels associated to mass spectrometry analyses. After growing X. citri and two different mutants deleted for ssuA and nitA genes in LB and minimum media, cellular extracts were obtained and used for preparation of mono and bi-dimensional gels. Seven bands covering the expected mass of ABC transporter components (20 kDa to 50 kDa) in SDS-PAGE were cut off the gel, treated with trypsin and submitted to the MS for protein identification. The results of 2D gels were good enough and will serve as a standard for development of similar experiments in large scale. (author)

  14. Chemical-to-mechanical energy conversion in biomacromolecular machines: a plasmon and optimum control theory for directional work. 1. General considerations.

    Science.gov (United States)

    Starikov, Evgeni B; Panas, Itai; Nordén, Bengt

    2008-07-17

    To rationalize coherence and mechanochemical aspects of proteins acting as molecular machines, a plasmon concept for dealing with protein nonequilibrium dynamics is introduced and tested with respect to thermodynamic consistency. A stochastic optimum-control theory for protein conformational diffusion is developed and the corresponding stochastic Newton's second law derived for optimum-controlled conformational diffusion in proteins. The plasmon concept is shown to be consistent with this theory, in that optical plasmons can pump entropy out of (or into) the protein, decreasing (or increasing) its conformational diffusion and, at the same time, help decrease intra- and intermolecular friction, as well as (potentially) break the symmetry of the latter. Instead, acoustic plasmons may break the spatial symmetry of a protein's "potential of mean force", thus converting it into an effective Brownian ratchet potential by applying quasistatic deformational corrections to the former. These concepts seem to be of rather general applicability and might also be useful when studying, for example, intercalation of cationic dyes into DNA duplexes, positively charged oligopeptide transduction through cell membranes, or even DNA translocation through nanopores.

  15. The cell's nucleolus: an emerging target for chemotherapeutic intervention.

    Science.gov (United States)

    Pickard, Amanda J; Bierbach, Ulrich

    2013-09-01

    The transient nucleolus plays a central role in the up-regulated synthesis of ribosomal RNA (rRNA) to sustain ribosome biogenesis, a hallmark of aberrant cell growth. This function, in conjunction with its unique pathohistological features in malignant cells and its ability to mediate apoptosis, renders this sub-nuclear structure a potential target for chemotherapeutic agents. In this Minireview, structurally and functionally diverse small molecules are discussed that have been reported to either interact with the nucleolus directly or perturb its function indirectly by acting on its dynamic components. These molecules include all major classes of nucleic-acid-targeted agents, antimetabolites, kinase inhibitors, anti-inflammatory drugs, natural product antibiotics, oligopeptides, as well as nanoparticles. Together, these molecules are invaluable probes of structure and function of the nucleolus. They also provide a unique opportunity to develop novel strategies for more selective and therefore better-tolerated chemotherapeutic intervention. In this regard, inhibition of RNA polymerase-I-mediated rRNA synthesis appears to be a promising mechanism for killing cancer cells. The recent development of molecules targeted at G-quadruplex-forming rRNA gene sequences, which are currently undergoing clinical trials, seems to attest to the success of this approach.

  16. Photoinduced electron transfer through peptide-based self-assembled monolayers chemisorbed on gold electrodes: directing the flow-in and flow-out of electrons through peptide helices.

    Science.gov (United States)

    Venanzi, Mariano; Gatto, Emanuela; Caruso, Mario; Porchetta, Alessandro; Formaggio, Fernando; Toniolo, Claudio

    2014-08-21

    Photoinduced electron transfer (PET) experiments have been carried out on peptide self-assembled monolayers (SAM) chemisorbed on a gold substrate. The oligopeptide building block was exclusively formed by C(α)-tetrasubstituted α-aminoisobutyric residues to attain a helical conformation despite the shortness of the peptide chain. Furthermore, it was functionalized at the C-terminus by a pyrene choromophore to enhance the UV photon capture cross-section of the compound and by a lipoic group at the N-terminus for linking to gold substrates. Electron transfer across the peptide SAM has been studied by photocurrent generation experiments in an electrochemical cell employing a gold substrate modified by chemisorption of a peptide SAM as a working electrode and by steady-state and time-resolved fluorescence experiments in solution and on a gold-coated glass. The results show that the electronic flow through the peptide bridge is strongly asymmetric; i.e., PET from the C-terminus to gold is highly favored with respect to PET in the opposite direction. This effect arises from the polarity of the Au-S linkage (Au(δ+)-S(δ-), junction effect) and from the electrostatic field generated by the peptide helix.

  17. Oyster (Crassostrea gigas) hydrolysates produced on a plant scale have antitumor activity and immunostimulating effects in BALB/c mice.

    Science.gov (United States)

    Wang, Yu-Kai; He, Hai-Lun; Wang, Guo-Fan; Wu, Hao; Zhou, Bai-Cheng; Chen, Xiu-Lan; Zhang, Yu-Zhong

    2010-02-02

    Oyster extracts have been reported to have many bioactive peptides. But the function of oyster peptides produced by proteolysis is still unknown. In this study, the oligopeptide-enriched hydrolysates from oyster (Crassostrea gigas) were produced using the protease from Bacillus sp. SM98011 at laboratory level, and scaled up to pilot (100 L) and plant (1,000 L) levels with the same conditions. And the antitumor activity and immunostimulating effects of the oyster hydrolysates in BALB/c mice were investigated. The growth of transplantable sarcoma-S180 was obviously inhibited in a dose-dependent manner in BALB/c mice given the oyster hydrolysates. Mice receiving 0.25, 0.5 and 1 mg/g of body weight by oral gavage had 6.8%, 30.6% and 48% less tumor growth, respectively. Concurrently, the weight coefficients of the thymus and the spleen, the activity of natural killer (NK) cells, the spleen proliferation of lymphocytes and the phagocytic rate of macrophages in S180-bearing mice significantly increased after administration of the oyster hydrolysates. These results demonstrated that oyster hydrolysates produced strong immunostimulating effects in mice, which might result in its antitumor activity. The antitumor and immunostimulating effects of oyster hydrolysates prepared in this study reveal its potential for tumor therapy and as a dietary supplement with immunostimulatory activity.

  18. Molecular interaction studies of hemostasis: fibrinogen ligand-human platelet receptor interactions

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Imshik; Marchant, Roger E

    2003-10-15

    The interactions between fibrinogen ligands and platelet receptor {alpha}{sub IIb}{beta}{sub 3} were studied under physiological conditions by atomic force microscopy (AFM). Two linear peptide sequences in fibrinogen, RGD and HHLGGAKQAGDV, play central roles in the regulation of hemostasis and thrombosis by facilitating adhesion and aggregation of platelets. In order to measure the interactions (i.e., debonding force), oligopeptides, GSSSGaaa, where aaa is -RGDSPA or -HHLGGAKQAGDV, were synthesized and grafted on to the surface of AFM probe tips. The interaction forces between a peptide-modified AFM probe tip and platelet surface were determined from pN to nN levels using AFM force measurements. Our results show that the zero kinetic off-rate, K{sub off}(0), for RGDSPA is significantly smaller than that for HHLGGAKQAGDV, under the consideration of flexible receptor surfaces. From our analysis, the K{sub off}(0), the single molecular binding energy E{sub b}, and the transition state x{sub b}, were extracted from the data, and estimated to be 1.53 s{sup -1}, -2.64x10{sup -20} J and 1.03 A for the RGD-{alpha}{sub IIb}{beta}{sub 3} system, and 47.58 s{sup -1}, 2.67x10{sup -20}, 1.09 A for the HHLGGAKQAGDV-{alpha}{sub IIb}{beta}{sub 3} system, respectively.

  19. A dipeptide transporter from the arbuscular mycorrhizal fungus Rhizophagus irregularis is upregulated in the intraradical phase

    Science.gov (United States)

    Belmondo, Simone; Fiorilli, Valentina; Pérez-Tienda, Jacob; Ferrol, Nuria; Marmeisse, Roland; Lanfranco, Luisa

    2014-01-01

    Arbuscular mycorrhizal fungi (AMF), which form an ancient and widespread mutualistic symbiosis with plants, are a crucial but still enigmatic component of the plant micro biome. Nutrient exchange has probably been at the heart of the success of this plant-fungus interaction since the earliest days of plants on land. To characterize genes from the fungal partner involved in nutrient exchange, and presumably important for the functioning of the AM symbiosis, genome-wide transcriptomic data obtained from the AMF Rhizophagus irregularis were exploited. A gene sequence, showing amino acid sequence and transmembrane domains profile similar to members of the PTR2 family of fungal oligopeptide transporters, was identified and called RiPTR2. The functional properties of RiPTR2 were investigated by means of heterologous expression in Saccharomyces cerevisiae mutants defective in either one or both of its di/tripeptide transporter genes PTR2 and DAL5. These assays showed that RiPTR2 can transport dipeptides such as Ala-Leu, Ala-Tyr or Tyr-Ala. From the gene expression analyses it seems that RiPTR2 responds to different environmental clues when the fungus grows inside the root and in the extraradical phase. PMID:25232358

  20. Enhancement of umami taste of hydrolyzed protein from wheat gluten by β-cyclodextrin.

    Science.gov (United States)

    Wang, Lihua; Xu, Baocai; Li, Linlin; Zhang, Mengke; Feng, Tao; Wang, Jinpeng; Jin, Zhengyu

    2016-10-01

    Wheat gluten was hydrolyzed by Flavourzyme and Neutrase at pH 7.0 and 50 °C for 8 h with β-cyclodextrin (β-CD) employed in the reaction. The hydrolysates were enzyme deactivated, cooled and centrifuged at 1500 × g for 15 min. Sensory and chemical characterization of wheat gluten hydrolysates WGH-1 (reaction conducted without β-CD), WGH-2 (reaction conducted with β-CD) and WGH-3 (β-CD added to WGH-1) was performed. WGH-2 revealed enhanced umami taste and higher hydrolyzing degree, total free amino acid amount, protein yield and umami taste amino acid (Glu + Asp) amount. High-performance liquid chromatography showed that the proportion of molecular weight 180-500 Da in WGH-2 was 11.5% higher than that in WGH-1. Further research indicated that β-CD had multiple effects on the hydrolysis. It could not only increase the solubility of wheat gluten but also form inclusion complexes with resultants. This can both promote the hydrolysis and protect oligopeptides from degradation. β-CD was found to have the ability to increase the umami taste of enzyme-hydrolyzed vegetable protein from wheat gluten. The reasons analyzed were that β-CD could take part in the hydrolysis process by improving the solubility of wheat gluten and form inclusion complexes with resultants. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  1. Spreading of a prion domain from cell-to-cell by vesicular transport in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Carmen I Nussbaum-Krammer

    2013-03-01

    Full Text Available Prion proteins can adopt self-propagating alternative conformations that account for the infectious nature of transmissible spongiform encephalopathies (TSEs and the epigenetic inheritance of certain traits in yeast. Recent evidence suggests a similar propagation of misfolded proteins in the spreading of pathology of neurodegenerative diseases including Alzheimer's or Parkinson's disease. Currently there is only a limited number of animal model systems available to study the mechanisms that underlie the cell-to-cell transmission of aggregation-prone proteins. Here, we have established a new metazoan model in Caenorhabditis elegans expressing the prion domain NM of the cytosolic yeast prion protein Sup35, in which aggregation and toxicity are dependent upon the length of oligopeptide repeats in the glutamine/asparagine (Q/N-rich N-terminus. NM forms multiple classes of highly toxic aggregate species and co-localizes to autophagy-related vesicles that transport the prion domain from the site of expression to adjacent tissues. This is associated with a profound cell autonomous and cell non-autonomous disruption of mitochondrial integrity, embryonic and larval arrest, developmental delay, widespread tissue defects, and loss of organismal proteostasis. Our results reveal that the Sup35 prion domain exhibits prion-like properties when expressed in the multicellular organism C. elegans and adapts to different requirements for propagation that involve the autophagy-lysosome pathway to transmit cytosolic aggregation-prone proteins between tissues.

  2. Chemical exploitation of native plant oils II. Subproject 4: Biodegradable tensides from domestic oils. Final report; Chemische Nutzung heimischer Pflanzenoele II. Teilvorhaben 4: Biologisch abbaubare Tenside aus heimischen Oelen. Abschlussbericht

    Energy Technology Data Exchange (ETDEWEB)

    Schneider, M.P.

    2002-07-01

    There are many domestic plants that yield large amounts of valuable raw materials such as fats and oils, proteins and carbohydrates. By linking lipophilic (fatty acids) with hydrophilic components like glycerine, amino acids, oligopeptides (protein hydrolysates), or mono- di- or oligosaccharides (starch hydrolysates), a wide range of combination products with surfactant properties is obtained. Target molecules are partial glycerides (mono and diglycerides), fatty acid modified amino acids, protein hydrolysates (fatty acid and protein condensates), and sugar esters. This project focused on the following subjects: 1. Sugar esters - enzymatic acylation of mono- and disaccharides and other sugar derivates. 2. Glycosides - enzymatic glycodisation of monosaccharides. 3. Fatty acid modified amino acids - enzymatic acylation of amino acids and protein hydrolysates. 4. Gemini tensides on the basis of amino acids. (orig.) [German] Zahlreiche Oelpflanzen und Getreidearten liefern preiswert und in grossen Mengen eine Vielzahl wertvoller Rohmaterialien wie Fette und Oele, Proteine und Kohlenhydrate. Durch Verknuepfung deren lipophiler Bestandteile (Fettsaeuren) mit hydrophilen Komponenten wie Glycerin, Aminosaeuren, Oligopeptiden (Proteinhydrolysaten) oder Mono, Di- oder Oligosacchariden (Staerkehydrolysaten) laesst sich eine breite Palette von Kombinationsprodukten mit oberflaechenaktiven Eigenschaften herstellen. Offensichtliche Zielmolekuele sind dabei Partialglyceride (Mono- und Diglyceride), fettsaeuremodifizierte Aminosaeuren, Proteinhydrolysate (Fettsaeure- Eiweiss-Kondensate) und Zuckerester. Ausgehend von unseren vielfaeltigen Vorarbeiten haben wir uns im Rahmen dieses Vorhabens schwerpunktmaessig mit den folgenden Themenkreisen beschaeftigt: 1. Zuckerester - enzymatische Acylierung von Mono- und Disacchariden sowie anderer Zuckerderivate. 2. Glycoside- enzymatische Glycosidierung von Monosacchariden. 3. Fettsaeuremodifizierte Aminosaeuren - enzymatische Acylierung von

  3. A novel physiological property of snake bradykinin-potentiating peptides-reversion of MK-801 inhibition of nicotinic acetylcholine receptors.

    Science.gov (United States)

    Nery, Arthur A; Trujillo, Cleber A; Lameu, Claudiana; Konno, Katsuhiro; Oliveira, Vitor; Camargo, Antonio C M; Ulrich, Henning; Hayashi, Mirian A F

    2008-10-01

    The first naturally occurring angiotensin-converting enzyme (ACE) inhibitors described are pyroglutamyl proline-rich oligopeptides, found in the venom of the viper Bothrops jararaca, and named as bradykinin-potentiating peptides (BPPs). Biochemical and pharmacological properties of these peptides were essential for the development of Captopril, the first active site-directed inhibitor of ACE, currently used for the treatment of human hypertension. However, a number of data have suggested that the pharmacological activity of BPPs could not only be explained by their inhibitory action on enzymatic activity of somatic ACE. In fact, we showed recently that the strong and long-lasting anti-hypertensive effect of BPP-10c [pheochromocytoma cell line, which following induction to neuronal differentiation expresses most of the nicotinic receptor subtypes. BPP-10c did not induce receptor-mediated ion flux, nor potentiated carbamoylcholine-provoked receptor activity as determined by whole-cell recording. This peptide, however, alleviated MK-801-induced inhibition of nicotinic acetylcholine receptor activity. Although more data are needed for understanding the mechanism of the BPP-10c effect on nicotinic receptor activity and its relationship with the anti-hypertensive activity, this work reveals possible therapeutic applications for BPP-10c in establishing normal acetylcholine receptor activity.

  4. Fecal Excretion of Orally Administered Collagen-Like Peptides in Rats: Contribution of the Triple-Helical Conformation to Their Stability.

    Science.gov (United States)

    Koide, Takaki; Yamamoto, Naoyuki; Taira, Kazuma B; Yasui, Hiroyuki

    2016-01-01

    Orally ingested peptides are generally digested in the gastrointestinal (GI) tract and absorbed in the form of oligopeptides. We previously reported that intravenously administered collagen-like triple-helical peptides circulated in the bloodstream and were excreted in their intact forms in urine nearly quantitatively. In the present study, we investigated the fates of orally administered collagen-like peptides in rats. (Pro-Hyp-Gly)10 (Hyp: 4-hydroxyproline), which formed a stable triple-helical structure, was stable in the GI tract, and 72.3±13.0% of the peptide was excreted in the feces. Its recovery ratio was similar to that of all-D-(Pro-Pro-Gly)10 (75.1±15.7%), the indigestible control. In contrast, (Pro-Hyp-Gly)5 and (Pro-Pro-Gly)10, the random coil conformations of which were dominant at body temperature, were not detected in fecal samples, indicating that they were digested by proteases. The high stability of the triple-helical conformation in mammalian bodies suggests the potential use of collagen-like peptides as novel scaffolds of peptide drugs.

  5. Purification and partial characterization of a tripeptidase from Pediococcus pentosaceus K9.2.

    Science.gov (United States)

    Simitsopoulou, M; Vafopoulou, A; Choli-Papadopoulou, T; Alichanidis, E

    1997-12-01

    A tripeptidase was purified from the cytoplasm of Pediococcus pentosaceus K9.2 by anion-exchange chromatography, gel filtration chromatography, and high-performance liquid chromatography. The molecular mass of the enzyme was estimated by gel filtration at 100,000 Da. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified peptidase showed one protein band of 45,000 Da. Optimal enzyme activity was obtained at pH 7.0 and at 50 degrees C. The peptidase hydrolyzed all tripeptides tested. Cleavage was not observed with dipeptides, oligopeptides, or amino acid-p-nitroanilide derivatives. Strong inhibition of activity was caused by EDTA, 1,10-phenanthroline, dithiothreitol, and beta-mercaptoethanol, whereas phenylmethylsulfonyl fluoride and sulfur-reactive reagents had no effect on peptidase activity. Mg2+, Mn2+, and Ca2+ stimulated the hydrolyzing activity of the enzyme. The 20 N-terminal amino acids of the tripeptidase from P. pentosaceus had 84% identity with those from the corresponding N-terminal region of the tripeptidase from Lactococcus lactis subsp. cremoris Wg2.

  6. A WW domain-containing yes-associated protein (YAP) is a novel transcriptional co-activator.

    Science.gov (United States)

    Yagi, R; Chen, L F; Shigesada, K; Murakami, Y; Ito, Y

    1999-05-04

    A protein module called the WW domain recognizes and binds to a short oligopeptide called the PY motif, PPxY, to mediate protein-protein interactions. The PY motif is present in the transcription activation domains of a wide range of transcription factors including c-Jun, AP-2, NF-E2, C/EBPalpha and PEBP2/CBF, suggesting that it plays an important role in transcriptional activation. We show here that mutation of the PY motif in the subregion of the activation domain of the DNA-binding subunit of PEBP2, PEBP2alpha, abolishes its transactivation function. Using yeast two-hybrid screening, we demonstrate that Yes-associated protein (YAP) binds to the PY motif of PEBP2alpha through its WW domain. The C-terminal region of YAP fused to the DNA-binding domain of GAL4 showed transactivation as strong as that of GAL4-VP16. Exogenously expressed YAP conferred transcription-stimulating activity on the PY motif fused to the GAL4 DNA-binding domain as well as to native PEBP2alpha. The osteocalcin promoter was stimulated by exogenous PEBP2alphaA and a dominant negative form of YAP strongly inhibited this activity, suggesting YAP involvement in this promoter activity in vivo. These results indicate that the PY motif is a novel transcription activation domain that functions by recruiting YAP as a strong transcription activator to target genes.

  7. [Comparative analysis of internal repeating segments in proteins of species from the three kingdoms of life].

    Science.gov (United States)

    Chen, Hao; Zhu, Sheng; Chen, Liang-Biao

    2005-03-01

    In 1970's, Ohno proposed that primordial proteins might evolve from periodic amplification of oligopeptides. Internal repeating segments in proteins may play important roles in functional evolution of proteins. In this study,a new method was designed to extract internal repeating segments from proteomes of 8 modern species belong to eukaryota, bacteria and archaea, respectively. The repeating patterns and the frequencies within proteomes of each kingdom were analyzed by matrix plot. Simple repeat segments were found in eukaryotic proteins with high frequencies,but were much lower in bacteria and none in archaea. Further analysis showed that, the biased usage of amino acids in the internal repeating segments was positively related to the frequencies of individual amino acids in the proteome of a given species. The correlation coefficient was up to 0.95 in prokaryota, with the eukaryota to be lower. The high frequency of simple repeat sequences in eukaryotic proteomes, as well as the disparate relationships of amino acid compositions between the internal repeating segments and their haboring eukaryotic proteomes imply that the fast evolution of simple repeat sequences could be one force that generates the high complexity of eukarytic proteomes.

  8. Integrating GHS into the Ghrelin System

    Directory of Open Access Journals (Sweden)

    Johannes D. Veldhuis

    2010-01-01

    Full Text Available Oligopeptide derivatives of metenkephalin were found to stimulate growth-hormone (GH release directly by pituitary somatotrope cells in vitro in 1977. Members of this class of peptides and nonpeptidyl mimetics are referred to as GH secretagogues (GHSs. A specific guanosine triphosphatate-binding protein-associated heptahelical transmembrane receptor for GHS was cloned in 1996. An endogenous ligand for the GHS receptor, acylghrelin, was identified in 1999. Expression of ghrelin and homonymous receptor occurs in the brain, pituitary gland, stomach, endothelium/vascular smooth muscle, pancreas, placenta, intestine, heart, bone, and other tissues. Principal actions of this peptidergic system include stimulation of GH release via combined hypothalamopituitary mechanisms, orexigenesis (appetitive enhancement, insulinostasis (inhibition of insulin secretion, cardiovascular effects (decreased mean arterial pressure and vasodilation, stimulation of gastric motility and acid secretion, adipogenesis with repression of fat oxidation, and antiapoptosis (antagonism of endothelial, neuronal, and cardiomyocyte death. The array of known and proposed interactions of ghrelin with key metabolic signals makes ghrelin and its receptor prime targets for drug development.

  9. Synthesis of albumin via a precursor protein in cell suspensions from rat liver.

    Science.gov (United States)

    Edwards, K; Schreiber, G; Dryburgh, H; Urban, J; Inglis, A S

    1976-03-16

    The mechanism of the biosynthesis of albumin was studied in cell suspensions from rat liver. The cells were prepared by continuous perfusion of the liver in situ with 0.05% collagenase and 0.10% hyaluronidase and incubated under conditions optimized for the incorporation of amino acids into protein. Seven minutes after starting the incubation L-[1-14C]leucine was added, followed after 25 min by a 15 or 30-min chase with an 830-fold excess of non-radioactive L-leucine. Total protein, an albumin-like protein, and albumin were isolated from samples withdrawn immediately of total protein was found to remain constant after addition of the non-radioactive L-leucine, whereas that of the albumin-like protein decreased and that of albumin increased with incubation time. The increase in albumin radioactivity accounted for the decrease in radioactivity of the albumin-like protein, suggesting that the latter is a precursor of albumin. The precursor protein differed from albumin by an oligopeptide extension at the N-terminal end.

  10. Hormones and endocrine disruptors in human seminal plasma.

    Science.gov (United States)

    Hampl, R; Kubatova, J; Heracek, J; Sobotka, V; Starka, L

    2013-07-01

    Seminal plasma represents a unique environment for maturation, nutrition, and protection of male germ cells from damaging agents. It contains an array of organic as well as inorganic chemicals, encompassing a number of biologically and immunologically active compounds, including hormones. Seminal plasma contains also various pollutants transferred from outer environment known as endocrine disruptors. They interfere with hormones at the receptor level, act as inhibitors of their biosynthesis, and affect hormone regulation.In this minireview, the main groups of hormones detected in seminal plasma are summarized. Seminal gonadal steroids were investigated mostly with aim to use them as biomarkers of impaired spermatogenesis (sperm count, motility, morphology). Concentrations of hormones in the seminal plasma often differ considerably from the blood plasma levels in dependence on their origin. In some instances (dihydrotestosterone, estradiol), their informative value is higher than determination in blood.Out of peptide hormones detected in seminal plasma, peptides of transforming growth factor beta family, especially antimullerian hormone, and oligopeptides related to thyrotropin releasing hormone have the high informative value, while assessment of seminal gonadotropins and prolactin does not bring advantage over determination in blood.Though there is a large body of information about the endocrine disruptors' impact on male reproduction, especially with their potential role in decline of male reproductive functions within the last decades, there are only scarce reports on their presence in seminal plasma. Herein, the main groups of endocrine disruptors found in seminal plasma are reviewed, and the use of their determination for investigation of fertility disorders is discussed.

  11. Metatranscriptome analysis reveals host-microbiome interactions in traps of carnivorous Genlisea species

    Directory of Open Access Journals (Sweden)

    Hieu X. Cao

    2015-07-01

    Full Text Available In the carnivorous plant genus Genlisea a unique lobster pot trapping mechanism supplements nutrition in nutrient-poor habitats. A wide spectrum of microbes frequently occurs in Genlisea’s leaf-derived traps without clear relevance for Genlisea carnivory. We sequenced the metatranscriptomes of subterrestrial traps versus the aerial chlorophyll-containing leaves of G. nigrocaulis and of G. hispidula. Ribosomal RNA assignment revealed soil-borne microbial diversity in Genlisea traps, with 92 genera of 19 phyla present in more than one sample. Microbes from 16 of these phyla including proteobacteria, green algae, amoebozoa, fungi, ciliates and metazoans, contributed additionally short-lived mRNA to the metatranscriptome. Furthermore, transcripts of 438 members of hydrolases (e.g. proteases, phosphatases, lipases, mainly resembling those of metazoans, ciliates and green algae, were found. Compared to aerial leaves, Genlisea traps displayed a transcriptional up-regulation of endogenous NADH oxidases generating reactive oxygen species as well as of acid phosphatases for prey digestion. A leaf-versus-trap transcriptome comparison reflects that carnivory provides inorganic P- and different forms of N-compounds (ammonium, nitrate, amino acid, oligopeptides and implies the need to protect trap cells against oxidative stress. The analysis elucidates a complex food web inside the Genlisea traps, and suggests ecological relationships between this plant genus and its entrapped microbiome.

  12. The M Protein of SARS-CoV: Basic Structural and Immunological Properties

    Institute of Scientific and Technical Information of China (English)

    Yongwu Hu; Jianping Shi; Xiangjun Tian; Feng Jiang; Xiaoqian Zhao; Jun Wang; Siqi Liu; Changqing Zeng; Jian Wang; Huanming Yang; Jie Wen; Lin Tang; Haijun Zhang; Xiaowei Zhang; Yan Li; Jing Wang; Yujun Han; Guoqing Li

    2003-01-01

    We studied structural and immunological properties of the SARS-CoV M (mem-brane) protein, based on comparative analyses of sequence features, phylogeneticinvestigation, and experimental results. The M protein is predicted to contain atriple-spanning transmembrane (TM) region, a single N-glycosylation site near itsN-terminus that is in the exterior of the virion, and a long C-terminal region inthe interior. The M protein harbors a higher substitution rate (0.6% correlated toits size) among viral open reading frames (ORFs) from published data. The foursubstitutions detected in the M protein, which cause non-synonymous changes,can be classified into three types. One of them results in changes of pI (isoelectricpoint) and charge, affecting antigenicity. The second changes hydrophobicity of theTM region, and the third one relates to hydrophilicity of the interior structure.Phylogenetic tree building based on the variations of the M protein appears tosupport the non-human origin of SARS-CoV. To investigate its immunogenicity,we synthesized eight oligopeptides covering 69.2% of the entire ORF and screenedthem by using ELISA (enzyme-linked immunosorbent assay) with sera from SARSpatients. The results confirmed our predictions on antigenic sites.

  13. Isolation and characterization of anti ROR1 single chain fragment variable antibodies using phage display technique.

    Science.gov (United States)

    Aghebati-Maleki, Leili; Younesi, Vahid; Jadidi-Niaragh, Farhad; Baradaran, Behzad; Majidi, Jafar; Yousefi, Mehdi

    2017-01-01

    Receptor tyrosine kinase-like orphan receptor (ROR1) belongs to one of the families of receptor tyrosine kinases (RTKs). RTKs are involved in the various physiologic cellular functions including proliferation, migration, survival, signaling and differentiation. Several RTKs are deregulated in various cancers implying the targeting potential of these molecules in cancer therapy. ROR1 has recently been shown to be expressed in various types of cancer cells but not in normal adult cells. Hence a molecular inhibitor of extracellular domain of ROR1 that inhibits ROR1-cell surface interaction is of great therapeutic importance. In an attempt to develop molecular inhibitors of ROR1, we screened single chain variable fragment (scFv) phage display libraries, Tomlinson I + J, against one specific synthetic oligopeptide from extracellular domain of ROR1 and selected scFvs were characterized using various immunological techniques. Several ROR1 specific scFvs were selected following five rounds of panning procedure. The scFvs showed specific binding to ROR1 using immunological techniques. Our results demonstrate successful isolation and characterization of specific ROR1 scFvs that may have great therapeutic potential in cancer immunotherapy.

  14. Microfluidics and nanoparticles based amperometric biosensor for the detection of cyanobacteria (Planktothrix agardhii NIVA-CYA 116) DNA.

    Science.gov (United States)

    Ölcer, Zehra; Esen, Elif; Ersoy, Aylin; Budak, Sinan; Sever Kaya, Dilek; Yağmur Gök, Mehmet; Barut, Serkan; Üstek, Duran; Uludag, Yildiz

    2015-08-15

    Some of the cyanobacteria produce protease inhibitor oligopeptides such as cyanopeptolins and cause drinking water contamination; hence, their detection has great importance to monitor the well-being of water sources that is used for human consumption. In the current study, a fast and sensitive nucleic acid biosensor assay has been described where cyanopeptolin coding region of one of the cyanobacteria (Planktothrix agardhii NIVA-CYA 116) genome has been used as target for monitoring of the fresh water resources. A biochip that has two sets of Au electrode arrays, each consist of shared reference/counter electrodes and 3 working electrodes has been used for the assay. The biochip has been integrated to a microfluidics system and all steps of the assay have been performed during the reagent flow to achieve fast and sensitive DNA detection. On-line hybridization of the target on to the capture probe immobilized surface resulted in a very short assay duration with respect to the conventional static assays. The binding of the avidin and enzyme modified Au nanoparticles to the biotinylated detection probe and the subsequent injection of the substrate enabled a real-time amperometric measurement with a detection limit of 6×10(-12) M target DNA (calibration curve r(2)=0.98). The developed assay enables fast and sensitive detection of cyanopeptolin producing cyanobacteria from freshwater samples and hence shows a promising technology for toxic microorganism detection from environmental samples.

  15. Heat-initiated prebiotic formation of peptides from glycine/aspartic acid and glycine/valine in aqueous environment and clay suspension

    Science.gov (United States)

    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.

  16. [Chronic inflammatory bowel diseases and nutrition].

    Science.gov (United States)

    Meier, R

    1996-01-01

    The etiology of inflammatory bowel disease is still unknown. Several potential mechanisms are discussed. The etiological and therapeutic importance of nutrition is controversial. Though changes in dietary habits and incidence of inflammatory bowel disease during the last century were in parallel, no specific nutritional factor has been isolated. No dietary prophylaxis of inflammatory bowel disease is yet known; all dietary therapies in inflammatory bowel disease aim to improve nutritional support and to diminish inflammation by bowel rest. Children and adolescents gain in weight and height. Total parenteral nutrition will not substantially reduce disease activity and operation rates. Total parenteral nutrition can only be recommended in ulcerative colitis patients with severe disease in the initial phase and in Crohn's patients with severe malnutrition and intestinal complications. Enteral nutrition support is less effective in ulcerative colitis than in Crohn's disease. Reported remission rates on enteral nutrition are 25% for ulcerative colitis and up to 80% for Crohn. However, in active Crohn's disease enteral nutrition is less effective than standard therapy with methylprednisolone and sulfasalizine. It is generally believed that nutrition therapy in combination with drugs is the best treatment modality. There is no evidence to support the importance of any combination of the formula diets such as elemental, oligopeptide, or polymeric formulations. Administration of formula diets by nasogastric tubes all show similar remission rates. Whether newer diets supplemented with arginine, glutamine, omega-3-fatty acids or short chain fatty acids increase remission rates is not known. Further studies in this field are warranted.

  17. Dipole Moment and Binding Energy of Water in Proteins from Crystallographic Analysis.

    Science.gov (United States)

    Morozenko, A; Leontyev, I V; Stuchebrukhov, A A

    2014-10-14

    The energetics of water molecules in proteins is studied using the water placement software Dowser. We compared the water position predictions for 14 high-resolution crystal structures of oligopeptide-binding protein (OppA) containing a large number of resolved internal water molecules. From the analysis of the outputs of Dowser with variable parameters and comparison with experimental X-ray data, we derived an estimate of the average dipole moment of water molecules located in the internal cavities of the protein and their binding energies. The water parameters thus obtained from the experimental data are then analyzed within the framework of charge-scaling theory developed recently by this group; the parameters are shown to be in good agreement with the predictions that the theory makes for the dipole moment in a protein environment. The water dipole in the protein environment is found to be much different from that in the bulk and in such models as SPC or TIPnP. The role of charge scaling due to electronic polarizability of the protein is discussed.

  18. Identification of hepta-histidine as a candidate drug for Huntington’s disease by in silico-in vitro- in vivo-integrated screens of chemical libraries

    Science.gov (United States)

    Imamura, Tomomi; Fujita, Kyota; Tagawa, Kazuhiko; Ikura, Teikichi; Chen, Xigui; Homma, Hidenori; Tamura, Takuya; Mao, Ying; Taniguchi, Juliana Bosso; Motoki, Kazumi; Nakabayashi, Makoto; Ito, Nobutoshi; Yamada, Kazunori; Tomii, Kentaro; Okano, Hideyuki; Kaye, Julia; Finkbeiner, Steven; Okazawa, Hitoshi

    2016-01-01

    We identified drug seeds for treating Huntington’s disease (HD) by combining in vitro single molecule fluorescence spectroscopy, in silico molecular docking simulations, and in vivo fly and mouse HD models to screen for inhibitors of abnormal interactions between mutant Htt and physiological Ku70, an essential DNA damage repair protein in neurons whose function is known to be impaired by mutant Htt. From 19,468 and 3,010,321 chemicals in actual and virtual libraries, fifty-six chemicals were selected from combined in vitro-in silico screens; six of these were further confirmed to have an in vivo effect on lifespan in a fly HD model, and two chemicals exerted an in vivo effect on the lifespan, body weight and motor function in a mouse HD model. Two oligopeptides, hepta-histidine (7H) and Angiotensin III, rescued the morphological abnormalities of primary neurons differentiated from iPS cells of human HD patients. For these selected drug seeds, we proposed a possible common structure. Unexpectedly, the selected chemicals enhanced rather than inhibited Htt aggregation, as indicated by dynamic light scattering analysis. Taken together, these integrated screens revealed a new pathway for the molecular targeted therapy of HD. PMID:27653664

  19. Structure-function studies of an engineered scaffold protein derived from stefin A. I: Development of the SQM variant.

    Science.gov (United States)

    Hoffmann, Toni; Stadler, Lukas Kurt Josef; Busby, Michael; Song, Qifeng; Buxton, Anthony T; Wagner, Simon D; Davis, Jason J; Ko Ferrigno, Paul

    2010-05-01

    Non-antibody scaffold proteins are used for a range of applications, especially the assessment of protein-protein interactions within human cells. The search for a versatile, robust and biologically neutral scaffold previously led us to design STM (stefin A triple mutant), a scaffold derived from the intracellular protease inhibitor stefin A. Here, we describe five new STM-based scaffold proteins that contain modifications designed to further improve the versatility of our scaffold. In a step-by-step approach, we introduced restriction sites in the STM open reading frame that generated new peptide insertion sites in loop 1, loop 2 and the N-terminus of the scaffold protein. A second restriction site in 'loop 2' allows substitution of the native loop 2 sequence with alternative oligopeptides. None of the amino acid changes interfered significantly with the folding of the STM variants as assessed by circular dichroism spectroscopy. Of the five scaffold variants tested, one (stefin A quadruple mutant, SQM) was chosen as a versatile, stable scaffold. The insertion of epitope tags at varying positions showed that inserts into loop 1, attempted here for the first time, were generally well tolerated. However, N-terminal insertions of epitope tags in SQM had a detrimental effect on protein expression.

  20. Oral collagen-derived dipeptides, prolyl-hydroxyproline and hydroxyprolyl-glycine, ameliorate skin barrier dysfunction and alter gene expression profiles in the skin.

    Science.gov (United States)

    Shimizu, Jun; Asami, Naoto; Kataoka, Aya; Sugihara, Fumihito; Inoue, Naoki; Kimira, Yoshifumi; Wada, Masahiro; Mano, Hiroshi

    2015-01-09

    Oral supplementation with collagen hydrolysate (CH) has been shown to improve the condition of the skin in humans and experimental animals. Several hydroxyproline-containing oligo-peptides were previously detected in human peripheral blood after the ingestion of CH, and the two dipeptides, prolyl-hydroxyproline (PO) and hydroxyprolyl-glycine (OG), have been proposed to have beneficial effects on human health. When HR-1 hairless mice were fed a HR-AD diet, which lacked magnesium and zinc, transepidermal water loss (TEWL) increased and water content of stratum corneum decreased. In the present study, we investigated the effects of dietary PO and OG on skin barrier dysfunction in HR-1 hairless mice. Mice were fed a HR-AD diet with or without PO (0.15%) and OG (0.15%) for 35 consecutive days. The administration of PO and OG significantly decreased TEWL, and significantly increased water content of stratum corneum. A DNA microarray analysis of the dorsal skin revealed differences in gene expression between the group administered PO and OG and the control group. We also identified muscle-related Gene Ontology as a result of analyzing the up-regulated genes. These results suggested that the administration of PO and OG improved skin barrier dysfunction and altered muscle-related gene expression.

  1. Molecular simulations of polycation-DNA binding exploring the effect of peptide chemistry and sequence in nuclear localization sequence based polycations.

    Science.gov (United States)

    Elder, Robert M; Jayaraman, Arthi

    2013-10-10

    Gene therapy relies on the delivery of DNA into cells, and polycations are one class of vectors enabling efficient DNA delivery. Nuclear localization sequences (NLS), cationic oligopeptides that target molecules for nuclear entry, can be incorporated into polycations to improve their gene delivery efficiency. We use simulations to study the effect of peptide chemistry and sequence on the DNA-binding behavior of NLS-grafted polycations by systematically mutating the residues in the grafts, which are based on the SV40 NLS (peptide sequence PKKKRKV). Replacing arginine (R) with lysine (K) reduces binding strength by eliminating arginine-DNA interactions, but placing R in a less hindered location (e.g., farther from the grafting point to the polycation backbone) has surprisingly little effect on polycation-DNA binding strength. Changing the positions of the hydrophobic proline (P) and valine (V) residues relative to the polycation backbone changes hydrophobic aggregation within the polycation and, consequently, changes the conformational entropy loss that occurs upon polycation-DNA binding. Since conformational entropy loss affects the free energy of binding, the positions of P and V in the grafts affect DNA binding affinity. The insight from this work guides synthesis of polycations with tailored DNA binding affinity and, in turn, efficient DNA delivery.

  2. The mbo operon is specific and essential for biosynthesis of mangotoxin in Pseudomonas syringae.

    Science.gov (United States)

    Carrión, Víctor J; Arrebola, Eva; Cazorla, Francisco M; Murillo, Jesús; de Vicente, Antonio

    2012-01-01

    Mangotoxin is an antimetabolite toxin produced by certain Pseudomonas syringae pv. syringae strains. This toxin is an oligopeptide that inhibits ornithine N-acetyl transferase, a key enzyme in the biosynthesis of ornithine and arginine. Previous studies have reported the involvement of the putative nonribosomal peptide synthetase MgoA in virulence and mangotoxin production. In this study, we analyse a new chromosomal region of P. syringae pv. syringae UMAF0158, which contains six coding sequences arranged as an operon (mbo operon). The mbo operon was detected in only mangotoxin-producing strains, and it was shown to be essential for the biosynthesis of this toxin. Mutants in each of the six ORFs of the mbo operon were partially or completely impaired in the production of the toxin. In addition, Pseudomonas spp. mangotoxin non-producer strains transformed with the mbo operon gained the ability to produce mangotoxin, indicating that this operon contains all the genetic information necessary for mangotoxin biosynthesis. The generation of a single transcript for the mbo operon was confirmed and supported by the allocation of a unique promoter and Rho-independent terminator. The phylogenetic analysis of the P. syringae strains harbouring the mbo operon revealed that these strains clustered together.

  3. Cold denaturation induces inversion of dipole and spin transfer in chiral peptide monolayers

    Science.gov (United States)

    Eckshtain-Levi, Meital; Capua, Eyal; Refaely-Abramson, Sivan; Sarkar, Soumyajit; Gavrilov, Yulian; Mathew, Shinto P.; Paltiel, Yossi; Levy, Yaakov; Kronik, Leeor; Naaman, Ron

    2016-02-01

    Chirality-induced spin selectivity is a recently-discovered effect, which results in spin selectivity for electrons transmitted through chiral peptide monolayers. Here, we use this spin selectivity to probe the organization of self-assembled α-helix peptide monolayers and examine the relation between structural and spin transfer phenomena. We show that the α-helix structure of oligopeptides based on alanine and aminoisobutyric acid is transformed to a more linear one upon cooling. This process is similar to the known cold denaturation in peptides, but here the self-assembled monolayer plays the role of the solvent. The structural change results in a flip in the direction of the electrical dipole moment of the adsorbed molecules. The dipole flip is accompanied by a concomitant change in the spin that is preferred in electron transfer through the molecules, observed via a new solid-state hybrid organic-inorganic device that is based on the Hall effect, but operates with no external magnetic field or magnetic material.

  4. Simulation and Numerical Modeling of the Self-assembly of an Optoelectronic Peptide

    Science.gov (United States)

    Mansbach, Rachael; Ferguson, Andrew

    We report molecular dynamics simulations of the self-assembly of synthetic π-conjugated oligopeptides into optoelectronic nanostructures. The electronic properties provide the basis for an array of organic electronic devices, such as light-emitting diodes, field-effect transistors, and solar cells. Control of the structure, stability, and kinetics of self-assembled organic electronics by tuning monomer chemistry and environmental conditions presents a powerful route to the fabrication of biocompatible ``designer materials.'' We have performed coarse-grained simulations of the self-assembly of several hundred peptides over microsecond time scales to probe the morphology and kinetics of aggregation with molecular-level detail. We have subsequently used this simulation data to parameterize a kinetic aggregation model based on Smoluchowski coagulation theory to enable prediction of aggregation dynamics on millisecond time scales. These numerical models are now being integrated into a multi-physics model of peptide aggregation in a microfluidic flow cell developed by our experimental collaborators to model the self-assembly of diverse peptide architectures under tailored flow-fields for the fabrication of biocompatible assemblies with defined morphology and optoelectronic function.

  5. Amino Acid and Peptide Utilization Profiles of the Fluoroacetate-Degrading Bacterium Synergistetes Strain MFA1 Under Varying Conditions.

    Science.gov (United States)

    Leong, Lex E X; Denman, Stuart E; Hugenholtz, Philip; McSweeney, Christopher S

    2016-02-01

    Synergistetes strain MFA1 is an asaccharolytic ruminal bacterium isolated based on its ability to degrade fluoroacetate, a plant toxin. The amino acid and peptide requirements of the bacterium were investigated under different culturing conditions. The growth of strain MFA1 and its fluoroacetate degradation rate were enhanced by peptide-rich protein hydrolysates (tryptone and yeast extract) compared to casamino acid, an amino acid-rich protein hydrolysate. Complete utilization and preference for arginine, asparagine, glutamate, glycine, and histidine as free amino acids from yeast extract were observed, while the utilization of serine, threonine, and lysine in free form and peptide-bound glutamate was stimulated during growth on fluoroacetate. A predominant peptide in yeast extract preferentially utilized by strain MFA1 was partially characterized by high-liquid performance chromatography-mass spectrometry as a hepta-glutamate oligopeptide. Similar utilization profiles of amino acids were observed between the co-culture of strain MFA1 with Methanobrevibacter smithii without fluoroacetate and pure strain MFA1 culture with fluoroacetate. This suggests that growth of strain MFA1 could be enhanced by a reduction of hydrogen partial pressure as a result of hydrogen removal by a methanogen or reduction of fluoroacetate.

  6. Anti-inflammatory defense mechanisms of Entamoeba histolytica.

    Science.gov (United States)

    Silva-García, Raúl; Rico-Rosillo, Guadalupe

    2011-02-01

    The monocyte locomotion inhibitory factor (MLIF), a heat-stable oligopeptide found in the supernatant fluid of Entamoeba histolytica axenic cultures, may contribute to the delayed inflammation observed in amoebic hepatic abscess. This factor was isolated by ultra-filtration and high powered liquid chromatography, obtaining a primary Met-Gln-Cys-Asn-Ser structure, identified afterwards as the carboxyl-terminal (…Cys-Asn-Ser) active site. The selective anti-inflammatory effects of the pentapeptide have been observed in both in vitro and in vivo models, using a synthetic pentapeptide to maintain the same anti-inflammatory conditions during the experimental assays. Anti-inflammatory effects observed include inhibition of human monocyte locomotion and the respiratory burst in monocytes and neutrophils, increasing expression of anti-inflammatory cytokines and inhibiting expression of the adhesion molecules VLA-4 and VCAM, among others. In this review, we will describe the effects of MLIF detected so far and how it might be used as a therapeutical agent against inflammatory diseases.

  7. Mass spectrometric detection, identification, and fragmentation of arseno-phytochelatins.

    Science.gov (United States)

    Schmied-Tobies, Maria I H; Arroyo-Abad, Uriel; Mattusch, Jürgen; Reemtsma, Thorsten

    2014-11-01

    Phytochelatins (PC) are cystein-rich oligopeptides in plants for coordination with toxic metals and metalloids via their thiol groups. The composition, structure, and mass spectrometric fragmentation of arseno-PC (As-PC) with PC of different degree of oligomerization (PC2-PC5) in solution were studied using liquid chromatography coupled in parallel to inductively coupled plasma mass spectrometry and electrospray ionization quadrupole time-of-flight mass spectrometry. As-PC were detected from As(PC2) to As(PC5) with an increasing number of isomers that differ in the position of thiol groups bound to As. Thermodynamic modeling supported the identification process in case of these isomers. Mass spectrometric fragmentation of the As-PC does not follow the established pattern of peptides but is governed by the formation of series of As-containing annular cations, which coordinate to As via S, N, or O. Structure proposals for 30 As-PC fragment ions in the range m/z 147.92 to m/z 1290.18 are elaborated. Many of these fragment ions are characteristic to several As-PC and may be suited for a screening for As-PC in plant extracts. The mass spectrometric data offer the perspective for a future more sensitive determination of As-PC by means of liquid chromatography tandem mass spectrometry with multiple reaction monitoring.

  8. First report of phytochelatins in a mushroom: induction of phytochelatins by metal exposure in Boletus edulis.

    Science.gov (United States)

    Collin-Hansen, Christian; Pedersen, Sindre A; Andersen, Rolf A; Steinnes, Eiliv

    2007-01-01

    Some species of macromycetes (mushrooms) consistently are found to contain high concentrations of toxic metals such as cadmium (Cd) and mercury (Hg), and consumption of wild-growing mushrooms is acknowledged as a significant source for Cd and Hg in humans. Yet little is known about the speciation of Cd and Hg in mushroom tissues. Here we present the first evidence of peptides of the phytochelatin family being responsible for binding a large fraction of Cd in caps of the macromycete Boletus edulis exposed to excess metals. Concentrations of Cd, Zn, Cu and Hg, as well as cytosolic Cd-binding capacity (CCBC), glutathione (GSH) and free proline (Pro) were quantified in fruiting bodies of B. edulis differentially exposed to a wide range of metals. Metal distribution among cytosolic compounds were investigated by size exclusion chromatography (SEC), followed by metal determinations with atomic absorption chromatography (AAS) and HR-ICP-MS. Cd-binding compounds in SEC elutates were investigated further by high performance liquid chromatography-mass spectrometry (HPLC-MS). CCBC was >90 times higher in the exposed group relative to the reference group (Mann-Whitney's P phytochelatins (PCs), a family of cystein-rich oligopeptides, was confirmed in Cd-containing SEC fractions by HPLC-MS. The appearance of more complex PCs was coupled to declining concentrations of GSH. To our knowledge this is the first report demonstrating the presence of PCs in a macromycete.

  9. Minor groove binder distamycin remodels chromatin but inhibits transcription.

    Directory of Open Access Journals (Sweden)

    Parijat Majumder

    Full Text Available The condensed structure of chromatin limits access of cellular machinery towards template DNA. This in turn represses essential processes like transcription, replication, repair and recombination. The repression is alleviated by a variety of energy dependent processes, collectively known as "chromatin remodeling". In a eukaryotic cell, a fine balance between condensed and de-condensed states of chromatin helps to maintain an optimum level of gene expression. DNA binding small molecules have the potential to perturb such equilibrium. We present herein the study of an oligopeptide antibiotic distamycin, which binds to the minor groove of B-DNA. Chromatin mobility assays and circular dichroism spectroscopy have been employed to study the effect of distamycin on chromatosomes, isolated from the liver of Sprague-Dawley rats. Our results show that distamycin is capable of remodeling both chromatosomes and reconstituted nucleosomes, and the remodeling takes place in an ATP-independent manner. Binding of distamycin to the linker and nucleosomal DNA culminates in eviction of the linker histone and the formation of a population of off-centered nucleosomes. This hints at a possible corkscrew type motion of the DNA with respect to the histone octamer. Our results indicate that distamycin in spite of remodeling chromatin, inhibits transcription from both DNA and chromatin templates. Therefore, the DNA that is made accessible due to remodeling is either structurally incompetent for transcription, or bound distamycin poses a roadblock for the transcription machinery to advance.

  10. One common structural feature of "words" in protein sequences and human texts.

    Science.gov (United States)

    Zemková, M; Trifonov, E N; Zahradník, D

    2014-01-01

    Frequently discussed analogy between genetic and human texts is explored by comparison of alternation of polar and non-polar amino-acid residues in proteins and alternation of consonants and vowels in human texts. In human languages, the usage of possible combinations of consonants and vowels is influenced by pronounceability of the combinations. Similarly, oligopeptide composition of proteins is influenced by requirements of protein folding and stability. One special type of structure often present in proteins is amphipathic α-helices in which polar and non-polar amino acids alternate with the period 3.5 residues, not unlike alternation of consonants and vowels. In this study, we evaluated the contribution made by amphipathic alternations to the protein sequence texts (20-24%). Their proportion is lower than respective values for alternating words in human texts (57-89%). The proteomes (full sets of proteins for selected organisms) were transformed into ranked sequences of n-grams (words of length n), including periodical amphipathic structures. Similarly, human texts were transformed into sequences of alternating consonants and vowels. Analysis of the vocabularies shows that in both types of texts (human languages and proteins) the alternating words are dominant or highly preferred, thus, strengthening the analogy between these two types of texts. The contribution of amphipathic words in the upper parts of the ranked lists for 10 analyzed proteomes varies between 58 and 74%. In human texts respective values range between 90 and 100%.

  11. A strategy for genetic modification of the spike-encoding segment of human reovirus T3D for reovirus targeting.

    Science.gov (United States)

    van den Wollenberg, D J M; van den Hengel, S K; Dautzenberg, I J C; Cramer, S J; Kranenburg, O; Hoeben, R C

    2008-12-01

    Human Orthoreovirus Type 3 Dearing is not pathogenic to humans and has been evaluated clinically as an oncolytic agent. Its transduction efficiency and the tumor cell selectivity may be enhanced by incorporating ligands for alternative receptors. However, the genetic modification of reoviruses has been difficult, and genetic targeting of reoviruses has not been reported so far. Here we describe a technique for generating genetically targeted reoviruses. The propagation of wild-type reoviruses on cells expressing a modified sigma 1-encoding segment embedded in a conventional RNA polymerase II transcript leads to substitution of the wild-type genome segment by the modified version. This technique was used for generating reoviruses that are genetically targeted to an artificial receptor expressed on U118MG cells. These cells lack the junction adhesion molecule-1 and therefore resist infection by wild-type reoviruses. The targeted reoviruses were engineered to carry the ligand for this receptor at the C terminus of the sigma 1 spike protein. This demonstrates that the C terminus of the sigma 1 protein is a suitable locale for the insertion of oligopeptide ligands and that targeting of reoviruses is feasible. The genetically targeted viruses can be propagated using the modified U118MG cells as helper cells. This technique may be applicable for the improvement of human reoviruses as oncolytic agents.

  12. Identification and characterization of MOR-CP, a cysteine protease induced by ozone and developmental senescence in maize (Zea mays L.) leaves.

    Science.gov (United States)

    Ahmad, Rafiq; Zuily-Fodil, Yasmine; Passaquet, Chantal; Bethenod, Olivier; Roche, Romain; Repellin, Anne

    2014-08-01

    Among the different classes of endoproteases, cysteine proteases are consistently associated with senescence, defense signaling pathways and cellular responses to abiotic stresses. The objectives of this work were to study the effects of various concentrations of ozone on gene expression and enzymatic activity for papain-like cysteine proteases (PLCPs), in the leaves of maize plants grown under field conditions. Leaves from ranks 12 and 10 (cob leaf) were harvested regularly over a long-term artificial ozone fumigation experiment (50 d). Tissues were tested for transcriptional and activity changes concerning cysteine proteases, using qRT-PCR for the newly identified ozone-responsive PLCP gene (Mor-CP) and synthetic oligopeptide Boc-Val-Leu-Lys-AMC as a PLCP-specific substrate, respectively. Results showed that developmental senescence induced a significant and progressive rise in CP activity, only in the older leaves 10 and had no effect on Mor-CP gene expression levels. On the other hand, ozone dramatically enhanced Mor-CP mRNA levels and global PLCP enzymatic activity in leaves 12 and 10, particularly toward the end of the treatment. Ozone impact was more pronounced in the older leaves 10. Together, these observations concurred to conclude that ozone stress enhances natural senescence processes, such as those related to proteolysis.

  13. Nanocarrier drugs in the treatment of brain tumors

    Institute of Scientific and Technical Information of China (English)

    Tereza Cerna; Marie Stiborova; Vojtech Adam; Rene Kizek; Tomas Eckschlager

    2016-01-01

    Nanoparticle-mediated targeted delivery of drugs might signiifcantly reduce the dosage and optimize their release properties, increase speciifcity and bioavailability, improve shelf life, and reduce toxicity. Some nanodrugs are able to overcome the blood-brain barrier that is an obstacle to treatment of brain tumors. Vessels in tumors have abnormal architecture and are highly permeable; moreover, tumors also have poor lymphatic drainage, allowing for accumulation of macromolecules greater than approximately 40 kDa within the tumor microenvironment. Nanoparticles exploit this feature, known as the enhanced permeability and retention effect, to target solid tumors. Active targeting, i.e. surface modiifcation of nanoparticles, is a way to decrease uptake in normal tissue and increase accumulation in a tumor, and it usually involves targeting surface membrane proteins that are upregulated in cancer cells. The targeting molecules are typically antibodies or their fragments; aptamers; oligopeptides or small molecules. There are currently several FDA-approved nanomedicines, but none approved for brain tumor therapy. This review, based both on the study of literature and on the authors own experimental work describes a comprehensive overview of preclinical and clinical research of nanodrugs in therapy of brain tumors.

  14. The byssus of the zebra mussel, Dreissena polymorpha. II: Structure and polymorphism of byssal polyphenolic protein families.

    Science.gov (United States)

    Rzepecki, L M; Waite, J H

    1993-10-01

    The byssus of the zebra mussel Dreissena polymorpha is the key element of its adhesive strategy. It consists of a bundle of threads tipped by adhesive plaques and attached to the mussel at the base of its byssal-synthesizing organ, the foot. Two polyphenolic protein precursors of the byssus have been purified from the foot. These precursors, Dpfp-1 and Dpfp-2 (Dreissena polymorpha foot protein), with apparent M(r) values of 76 and 26 K, respectively, contain 3,4-dihydroxyphenylalanine (DOPA) integrated into their primary sequence, but differ from previously characterized polyphenolic proteins from marine mussels. The related quagga mussel (Dreissena spp.?) has homologous proteins with significantly different compositions. The zebra mussel DOPA proteins are tandemly repetitive with unique oligopeptide motif sequences, contain tryptophan, and are O-glycosylated primarily on threonine residues. Galactosamine was the only carbohydrate detected after hydrolysis. Dpfp-1 constitutes a polymorphic family of polypeptides with, unusually, an acidic range of pI values between 5.3 and 6.5. The detection of carbohydrate in the thread and in the juncture between thread and plaque suggests that these two proteins are localized in those regions where they may function as lacquers or structural elements.

  15. Structure-Activity Relationship and Signaling of New Chimeric CXCR4 Agonists.

    Science.gov (United States)

    Mona, Christine E; Besserer-Offroy, Élie; Cabana, Jérôme; Lefrançois, Marilou; Boulais, Philip E; Lefebvre, Marie-Reine; Leduc, Richard; Lavigne, Pierre; Heveker, Nikolaus; Marsault, Éric; Escher, Emanuel

    2016-08-25

    The CXCR4 receptor binds with meaningful affinities only CXCL12 and synthetic antagonists/inverse agonists. We recently described high affinity synthetic agonists for this chemokine receptor, obtained by grafting the CXCL12 N-terminus onto the inverse agonist T140. While those chimeric molecules behave as agonists for CXCR4, their binding and activation mode are unknown. The present SAR of those CXCL12-oligopeptide grafts reveals the key determinants involved in CXCR4 activation. Position 3 (Val) controls affinity, whereas position 7 (Tyr) acts as an efficacy switch. Chimeric molecules bearing aromatic residues in position 3 possess high binding affinities for CXCR4 and are Gαi full agonists with robust chemotactic properties. Fine-tuning of electron-poor aromatic rings in position 7 enhances receptor activation. To rationalize these results, a homology model of a receptor-ligand complex was built using the published crystal structures of CXCR4. Molecular dynamics simulations reveal further details accounting for the observed SAR for this series.

  16. A novel nuclear DnaJ protein, DNAJC8, can suppress the formation of spinocerebellar ataxia 3 polyglutamine aggregation in a J-domain independent manner

    Energy Technology Data Exchange (ETDEWEB)

    Ito, Norie [Department of Pathology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556 (Japan); Department of Neurology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556 (Japan); Kamiguchi, Kenjiro; Nakanishi, Katsuya; Sokolovskya, Alice; Hirohashi, Yoshihiko; Tamura, Yasuaki; Murai, Aiko; Yamamoto, Eri; Kanaseki, Takayuki; Tsukahara, Tomohide; Kochin, Vitaly [Department of Pathology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556 (Japan); Chiba, Susumu [Department of Neurology, Clinical Brain Research Laboratory, Toyokura Memorial Hall, Sapporo Yamano-ue Hospital (Japan); Shimohama, Shun [Department of Neurology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556 (Japan); Sato, Noriyuki [Department of Pathology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556 (Japan); Torigoe, Toshihiko, E-mail: torigoe@sapmed.ac.jp [Department of Pathology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556 (Japan)

    2016-06-10

    Polyglutamine (polyQ) diseases comprise neurodegenerative disorders caused by expression of expanded polyQ-containing proteins. The cytotoxicity of the expanded polyQ-containing proteins is closely associated with aggregate formation. In this study, we report that a novel J-protein, DNAJ (HSP40) Homolog, Subfamily C, Member 8 (DNAJC8), suppresses the aggregation of polyQ-containing protein in a cellular model of spinocerebellar ataxia type 3 (SCA3), which is also known as Machado-Joseph disease. Overexpression of DNAJC8 in SH-SY5Y neuroblastoma cells significantly reduced the polyQ aggregation and apoptosis, and DNAJC8 was co-localized with the polyQ aggregation in the cell nucleus. Deletion mutants of DNAJC8 revealed that the C-terminal domain of DNAJC8 was essential for the suppression of polyQ aggregation, whereas the J-domain was dispensable. Furthermore, 22-mer oligopeptide derived from C-termilal domain could suppress the polyQ aggregation. These results indicate that DNAJC8 can suppress the polyQ aggregation via a distinct mechanism independent of HSP70-based chaperone machinery and have a unique protective role against the aggregation of expanded polyQ-containing proteins such as pathogenic ataxin-3 proteins.

  17. Efficient Absorption of X-Hydroxyproline (Hyp)-Gly after Oral Administration of a Novel Gelatin Hydrolysate Prepared Using Ginger Protease.

    Science.gov (United States)

    Taga, Yuki; Kusubata, Masashi; Ogawa-Goto, Kiyoko; Hattori, Shunji

    2016-04-13

    Recent studies have reported that oral intake of gelatin hydrolysate has various beneficial effects, such as reduction of joint pain and lowering of blood sugar levels. In this study, we produced a novel gelatin hydrolysate using a cysteine-type ginger protease having unique substrate specificity with preferential peptide cleavage with Pro at the P2 position. Substantial amounts of X-hydroxyproline (Hyp)-Gly-type tripeptides were generated up to 2.5% (w/w) concomitantly with Gly-Pro-Y-type tripeptides (5%; w/w) using ginger powder. The in vivo absorption of the ginger-degraded gelatin hydrolysate was estimated using mice. The plasma levels of collagen-derived oligopeptides, especially X-Hyp-Gly, were significantly high (e.g., 2.3-fold for Glu-Hyp-Gly, p < 0.05) compared with those of the control gelatin hydrolysate, which was prepared using gastrointestinal proteases and did not contain detectable X-Hyp-Gly. This study demonstrated that orally administered X-Hyp-Gly was effectively absorbed into the blood, probably due to the high protease resistance of this type of tripeptide.

  18. Proteinase K-catalyzed synthesis of linear and star oligo(L-phenylalanine) conjugates.

    Science.gov (United States)

    Ageitos, Jose M; Baker, Peter J; Sugahara, Michihiro; Numata, Keiji

    2013-10-14

    Chemoenzymatic synthesis of peptides is a green and clean chemical reaction that offers high yields without using organic synthesis and serves as an alternative to traditional peptide synthesis methods. This report describes the chemoenzymatic synthesis of oligo(L-phenylalanine) mediated by proteinase K from Tritirachium album, which is one of the most widely used proteases in molecular biological studies. The synthesized linear oligo-phenylalanine showed a unique self-assembly in aqueous solutions. To further functionalize linear oligo(L-phenylalanine) as a low-molecular-weight gelator, it was cosynthesized with tris(2-aminoethyl)amine to obtain star-oligo(L-phenylalanine), which was bioconjugated to demonstrate its self-assembly into fluorescent fibers. The self-assembled fibers of star-oligo(L-phenylalanine) formed fibrous networks with various branching ratios, which depended on the molecular weights and molecular aspect ratios of star-oligo(L-phenylalanine). This is the first study to demonstrate that proteinase K is a suitable enzyme for chemoenzymatic cosynthesis of oligopeptides and star-shaped heteropeptides.

  19. Identification and Relative Quantification of Bioactive Peptides Sequentially Released during Simulated Gastrointestinal Digestion of Commercial Kefir.

    Science.gov (United States)

    Liu, Yufang; Pischetsrieder, Monika

    2017-03-08

    Health-promoting effects of kefir may be partially caused by bioactive peptides. To evaluate their formation or degradation during gastrointestinal digestion, we monitored changes of the peptide profile in a model of (1) oral, (2) gastric, and (3) small intestinal digestion of kefir. Matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analyses revealed clearly different profiles between digests 2/3 and kefir/digest 1. Subsequent ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry identified 92 peptides in total (25, 25, 43, and 30, partly overlapping in kefir and digests 1, 2, and 3, respectively), including 16 peptides with ascribed bioactivity. Relative quantification in scheduled multiple reaction monitoring mode showed that many bioactive peptides were released by simulated digestion. Most prominently, the concentration of angiotensin-converting enzyme inhibitor β-casein203-209 increased approximately 10 000-fold after combined oral, gastric, and intestinal digestion. Thus, physiological digestive processes may promote bioactive peptide formation from proteins and oligopeptides in kefir. Furthermore, bioactive peptides present in certain compartments of the gastrointestinal tract may exert local physiological effects.

  20. Three complete turns of a 3(10)-helix at atomic resolution: the crystal structure of Z-(Aib)11-OtBu.

    Science.gov (United States)

    Gessmann, Renate; Brückner, Hans; Petratos, Kyriacos

    2003-01-01

    The crystal structure of the synthetic protected oligopeptide Z-(Aib)11-OtBu was determined by x-ray crystallography. The undecapeptide folds in a regular 3(10)-helix with nine consecutive 4 --> 1 hydrogen bonds. At present, this is the largest available structure of a homopeptide (including homopeptides consisting of standard amino acids) and also the longest observed regular 3(10)-helix at atomic resolution. Z-(Aib)11-OtBu crystallizes readily from hot ethanol-water mixture and is one of the crystals in which no solvent molecule is co-crystallized. In the crystal head-to-tail hydrogen bonded columns are formed in the [1 0 1] direction. Each helical column is surrounded by six others, whereby two are packed in parallel and four in antiparallel fashion. Helical columns are packed via apolar crystal contacts. The crystal structure of Z-(Aib)11-OtBu is compared with the crystal structures of Z-(Aib)10-OtBu and Z-(Aib)9-OtBu. The similarities and differences are analysed.

  1. Phytophthora parasitica elicitor-induced reactions in cells of Petroselinum crispum.

    Science.gov (United States)

    Fellbrich, G; Blume, B; Brunner, F; Hirt, H; Kroj, T; Ligterink, W; Romanski, A; Nürnberger, T

    2000-06-01

    Cultured parsley (Petroselinum crispum) cells respond to treatment with elicitors derived from different species of the genus Phytophthora with transcript accumulation of defense-associated genes and the production of furanocoumarin phytoalexins. Pep-25, an oligopeptide fragment of a Phytophthora sojae 42-kDa cell wall protein, and a cell wall elicitor preparation derived from Phytophthora parasitica (Pp-elicitor) stimulate accumulation of the same gene transcripts and formation of the same pattern of furanocoumarins. Treatment of cultured cells and protoplasts with proteinase-digested Pp-elicitor identified proteinaceous constituents as active eliciting compounds in parsley. Similar to Pep- 25, Pp-elicitor induced effluxes of K+ and Cl- and influxes of protons and Ca2+. Concomitantly, as monitored in aequorin-transgenic parsley cell lines both elicitors induced an immediate increase in the cytoplasmic Ca2+ concentration up to sustained levels of 175 nM (Pp-elicitor) or 300 nM (Pep-25), respectively. The signature of the Ca2+ response differed greatly between the two elicitors tested. Extracellular Ca2+ proved essential for activation of an oxidative burst, MAP kinase activity and phytoalexin production by either elicitor. While Pp-elicitor induced a qualitatively similar spectrum of defense responses as did Pep-25, elicitor-specific quantitative differences in response intensity and kinetics suggest activation of a conserved signaling cascade through separate ligand binding sites.

  2. Functional peptide nanocarriers for delivery of novel anti-RelA RNA interference agents as a topical treatment of atopic dermatitis.

    Science.gov (United States)

    Kanazawa, Takanori; Hamasaki, Tomohiro; Endo, Takahiro; Tamano, Kuniko; Sogabe, Kana; Seta, Yasuo; Ohgi, Tadaaki; Okada, Hiroaki

    2015-07-15

    Small interfering RNAs (siRNAs) are a potential treatment of atopic dermatitis (AD) because they can specifically silence the gene expression of AD-related factors. However, siRNA alone cannot exert a sufficiently strong therapeutic effect due to low delivery efficiency to the target tissues and cells; simply increasing the amount used is not possible due to the possibility of off-target effects. We previously reported a novel class of therapeutic RNA interference (RNAi) agents called nkRNA(®) and PnkRNA(®), which have been shown to be effective in several disease models, have greater resistance to nuclease degradation than canonical siRNAs, and do not induce any immunotoxicity. In the present study, we describe a non-invasive and effective transdermal RNAi therapeutic system for atopic dermatitis that uses the functional cell-penetrating stearoyl-oligopeptide OK-102 as a cytoplasm-responsive nanocarrier for nkRNA(®) and PnkRNA(®). The two RNAi agents were targeted against RelA, a subclass of NF-κB (nuclear factor kappa B), and, as part of OK-102 complexes, they strongly silenced RelA mRNA in macrophage cells and demonstrated a significant therapeutic effect in a mouse model of AD. It was shown that OK-102-complexed RNAi agents were an efficient therapeutic system for AD and caused no adverse reactions.

  3. Inhibition of Aeromonas sobria serine protease (ASP) by α2-macroglobulin.

    Science.gov (United States)

    Murakami, Yoji; Wada, Yoshihiro; Kobayashi, Hidetomo; Irie, Atsushi; Hasegawa, Makoto; Yamanaka, Hiroyasu; Okamoto, Keinosuke; Eto, Masatoshi; Imamura, Takahisa

    2012-10-01

    ASP is a serine protease secreted by Aeromonas sobria. ASP cleaves various plasma proteins, which is associated with onset of sepsis complications, such as shock and blood coagulation disorder. To investigate a host defense mechanism against this virulence factor, we examined the plasma for ASP inhibitor(s). Human plasma inhibited ASP activity for azocasein, which was almost completely abolished by treating plasma with methylamine, which inactivates α2-macroglobulin (α2-MG). The ASP-inhibitor complex in ASP-added plasma was not detected by immunoblotting using anti-ASP antibody; however, using gel filtration of the plasma ASP activity for an oligopeptide, the ASP substrate was eluted in the void fraction (Mw>200 000), suggesting ASP trapping by α2-MG. Indeed, human α2-MG inhibited ASP azocaseinolytic activity in a dose-dependent manner, rapidly forming a complex with the ASP. Fibrinogen degradation by ASP was completely inhibited in the presence of α2-MG. α1-Protease inhibitor, antithrombin, and α2-plasmin inhibitor neither inhibited ASP activity nor formed a complex with ASP. Surprisingly, ASP degraded these plasma serine protease inhibitors. Thus, α2-MG is the major ASP inhibitor in the human plasma and can limit ASP virulence activities in A. sobria infection sites. However, as shown by fluorescence correlation spectroscopy, slow ASP inhibition by α2-MG in plasma may indicate insufficient ASP control in vivo.

  4. Comparative study of (Asp)7-CHOL-modified liposome prepared using pre-insertion and post-insertion methods for bone targeting in vivo.

    Science.gov (United States)

    Zhang, Lijing; Cao, Hua; Zhang, Jiaxin; Yang, Chengli; Hu, Tingting; Li, Huili; Yang, Wu; He, Gu; Song, Xiangrong; Tong, Aiping; Guo, Gang; Li, Rui; Jiang, Yu; Liu, Jiyan; Cai, Lulu; Zheng, Yu

    2017-02-01

    Specific delivery of drugs to bone tissue is very challenging due to the architecture and structure of bone tissue. A seven-repeat sequence of aspartate, a representative bone-targeting oligopeptide, is preferentially used for targeted therapy for bone diseases. In this study, Asp7-cholesterol((Asp)7-CHOL) was synthesized and (Asp)7-CHOL-modified liposome loaded with doxorubicin (DOX) was successfully prepared using both pre-insertion (pre-L) and post-insertion (post-L) methods. The formulation was optimized according to particle size, zeta potential and the drug-loading efficiency of the liposome. In addition, the bone affinity of the (Asp)7-CHOL-modified liposome was evaluated using a hydroxyapatite (HA) absorption method. The results suggested that (Asp)7-CHOL-modified liposome show excellent HA absorption; pre-L showed slightly higher HA binding than post-L. However, post-L had a higher DOX entrapment efficiency than pre-L. In vivo imaging further demonstrated that pre-L showed a higher bone-targeting efficiency than post-L, which was consistent with in vitro results. In all, (Asp)7-CHOL-modified liposome showed excellent bone-targeting activity, suggesting their potential for use as a drug delivery system for bone disease-targeted therapies.

  5. THE EVALUATION OF PEPTIDE/HISTIDINE TRANSPORTER 1 (PHT1) FUNCTION: UPTAKE KINETICS UTILIZING A COS-7 STABLY TRANSFECTED CELL LINE.

    Science.gov (United States)

    Lindley, David J; Carl, Stephen M; Mowery, Stephanie A; Knipp, Gregory T

    2011-10-01

    There have been relatively few studies focused on the proton-dependent oligopeptide transporter (POT) superfamily member, Peptide/Histidine Transporter 1 (PHT1), with respect to its contribution to the ADME of peptides and peptide-based drugs. These studies were conducted to determine hPHT1-mediated, H(+)-dependent uptake kinetics of histidine, carnosine, Gly-Sar and valacyclovir in stably transfected hPHT1-COS-7 cells comparative to kinetics determined in an empty vector (Mock) stably transfected cell line. The results suggest that Gly-Sar appears to be a substrate for PHT1 based on efflux from the stably transfected hPHT1 COS-7 cells. Histidine and Gly-Sar concentration- and time-dependent studies suggest mixed-uptake kinetics. These studies suggest that stably transfected hPHT1-COS-7 cells exhibit different uptake kinetics than those observed in our previous studies and illustrate the requirement for experiments to delineate the physiological role of hPHT1.

  6. Simultaneous quantification of depolymerization and mineralization rates by a novel 15N tracing model

    Science.gov (United States)

    Andresen, Louise C.; Björsne, Anna-Karin; Bodé, Samuel; Klemedtsson, Leif; Boeckx, Pascal; Rütting, Tobias

    2016-09-01

    The depolymerization of soil organic matter, such as proteins and (oligo-)peptides, into monomers (e.g. amino acids) is currently considered to be the rate-limiting step for nitrogen (N) availability in terrestrial ecosystems. The mineralization of free amino acids (FAAs), liberated by the depolymerization of peptides, is an important fraction of the total mineralization of organic N. Hence, the accurate assessment of peptide depolymerization and FAA mineralization rates is important in order to gain a better process-based understanding of the soil N cycle. In this paper, we present an extended numerical 15N tracing model Ntrace, which incorporates the FAA pool and related N processes in order to provide a more robust and simultaneous quantification of depolymerization and gross mineralization rates of FAAs and soil organic N. We discuss analytical and numerical approaches for two forest soils, suggest improvements of the experimental work for future studies, and conclude that (i) when about half of all depolymerized peptide N is directly mineralized, FAA mineralization can be as important a rate-limiting step for total gross N mineralization as peptide depolymerization rate; (ii) gross FAA mineralization and FAA immobilization rates can be used to develop FAA use efficiency (NUEFAA), which can reveal microbial N or carbon (C) limitation.

  7. Transferable aspherical atom model refinement of protein and DNA structures against ultrahigh-resolution X-ray data.

    Science.gov (United States)

    Malinska, Maura; Dauter, Zbigniew

    2016-06-01

    In contrast to the independent-atom model (IAM), in which all atoms are assumed to be spherical and neutral, the transferable aspherical atom model (TAAM) takes into account the deformed valence charge density resulting from chemical bond formation and the presence of lone electron pairs. Both models can be used to refine small and large molecules, e.g. proteins and nucleic acids, against ultrahigh-resolution X-ray diffraction data. The University at Buffalo theoretical databank of aspherical pseudo-atoms has been used in the refinement of an oligopeptide, of Z-DNA hexamer and dodecamer duplexes, and of bovine trypsin. The application of the TAAM to these data improves the quality of the electron-density maps and the visibility of H atoms. It also lowers the conventional R factors and improves the atomic displacement parameters and the results of the Hirshfeld rigid-bond test. An additional advantage is that the transferred charge density allows the estimation of Coulombic interaction energy and electrostatic potential.

  8. Metalloproteinase activity secreted by fibrogenic cells in the processing of prolysyl oxidase. Potential role of procollagen C-proteinase.

    Science.gov (United States)

    Panchenko, M V; Stetler-Stevenson, W G; Trubetskoy, O V; Gacheru, S N; Kagan, H M

    1996-03-22

    Lysyl oxidase is secreted from fibrogenic cells as a 50-kDa proenzyme that is proteolytically processed to the mature enzyme in the extracellular space. To characterize the secreted proteinase activity, a truncated, recombinant form of lysyl oxidase was prepared as a proteinase substrate containing the sequence of the propeptide cleavage region. The processing proteinase activity secreted by cultured fibrogenic cells resists inhibitors of serine or aspartyl proteinases as well as tissue inhibitor of matrix metalloproteinases-2 (MMP-2) but is completely inhibited by metal ion chelators. Known metalloproteinases were tested for their activity toward this substrate. Carboxyl-terminal procollagen proteinase (C-proteinase), MMP-2, and conditioned fibrogenic cell culture medium cleave the lysyl oxidase substrate to the size of the mature enzyme. The NH2-terminal sequence generated by arterial smooth muscle conditioned medium and the C-proteinase but not by MMP-2, i.e. Asp-Asp-Pro-Tyr, was identical to that previously identified in mature lysyl oxidase isolated from connective tissue. The C-proteinase activity against the model substrate was inhibited by a synthetic oligopeptide mimic of the cleavage sequence (Ac-Met-Val-Gly-Asp-Asp-Pro-Tyr-Asn-amide), whereas this peptide also inhibited the generation of lysyl oxidase activity in the medium of fetal rat lung fibroblasts in culture. In toto, these results identify a secreted metalloproteinase activity participating in the activation of prolysyl oxidase, identify inhibitors of the processing activity, and implicate procollagen C-proteinase in this role.

  9. Quantitative proteomic analysis of CMS-related changes in Honglian CMS rice anther.

    Science.gov (United States)

    Sun, Qingping; Hu, Chaofeng; Hu, Jun; Li, Shaoqing; Zhu, Yingguo

    2009-10-01

    Honglian (HL) cytoplasmic male sterility (CMS) is one of the rice CMS types and has been widely used in hybrid rice production in China. The CMS line (Yuetai A, YTA) has a Yuetai B (maintainer line, YTB) nuclear genome, but has a rearranged mitochondrial (mt) genome consisting of Yuetai B. The fertility of hybrid (HL-6) was restored by restorer gene in nuclear genome of restorer line (9311). We used isotope-code affinity tag (ICAT) technology to perform the protein profiling of uninucleate stage rice anther and identify the CMS-HL related proteins. Two separate ICAT analyses were performed in this study: (1) anthers from YTA versus anthers from YTB, and (2) anthers from YTA versus anthers from HL-6. Based on the two analyses, a total of 97 unique proteins were identified and quantified in uninucleate stage rice anther under the error rate of less than 10%, of which eight proteins showed abundance changes of at least twofold between YTA and YTB. Triosephosphate isomerase, fructokinase II, DNA-binding protein GBP16 and ribosomal protein L3B were over-expressed in YTB, while oligopeptide transporter, floral organ regulator 1, kinase and S-adenosyl-L: -methionine synthetase were over-expressed in YTA. Reduction of the proteins associated with energy production and lesser ATP equivalents detected in CMS anther indicated that the low level of energy production played an important role in inducing CMS-HL.

  10. The Activation of Free Dipeptides Promoted by Strong Activating Agents in Water Does not Yield Diketopiperazines.

    Science.gov (United States)

    Beaufils, Damien; Jepaul, Sandra; Liu, Ziwei; Boiteau, Laurent; Pascal, Robert

    2016-03-01

    The activation of dipeptides was studied in the perspective of the abiotic formation of oligopeptides of significant length as a requirement for secondary structure formation. The formation of piperazin-2,5-diones (DKP), previously considered as a dead end when activating free dipeptides, was shown in this work to be efficiently suppressed when using strong activating agents (e.g., carbodiimides). This behaviour was explained by the fast formation of a 5(4H)-oxazolone intermediate at a rate that exceeds the time scale of the rotation of the peptide bond from the predominant trans-conformation into the cis-isomer required for DKP formation. No DKP was observed when using strong activating agents whereas phosphate mixed anhydrides or moderately activated esters were observed to predominantly yield DKP. The DKP side-reaction no longer constitutes a drawback for the C-terminus elongation of peptides. These results are considered as additional evidence that pathways involving strong activation are required to drive the emergence of living entities rather than close to equilibrium processes.

  11. A single-stage functionalization and exfoliation method for the production of graphene in water: stepwise construction of 2D-nanostructured composites with iron oxide nanoparticles.

    Science.gov (United States)

    Ihiawakrim, Dris; Ersen, Ovidiu; Melin, Frédéric; Hellwig, Petra; Janowska, Izabela; Begin, Dominique; Baaziz, Walid; Begin-Colin, Sylvie; Pham-Huu, Cuong; Baati, Rachid

    2013-10-07

    A practically simple top-down process for the exfoliation of graphene (GN) and few-layer graphene (FLG) from graphite is described. We have discovered that a biocompatible amphiphilic pyrene-based hexahistidine peptide is able to exfoliate, functionalize, and dissolve few layer graphene flakes in pure water under exceptionally mild, sustainable and virtually innocuous low intensity cavitation conditions. Large area functionalized graphene flakes with the hexahistidine oligopeptide (His₆-TagGN = His₆@GN) have been produced efficiently at room temperature and characterized by TEM, Raman, and UV spectroscopy. Conductivity experiments carried out on His₆-TagGN samples revealed superior electric performances as compared to reduced graphene oxide (rGO) and non-functionalized graphene, demonstrating the non-invasive features of our non-covalent functionalization process. We postulated a rational exfoliation mechanism based on the intercalation of the peptide amphiphile under cavitational chemistry. We also demonstrated the ability of His6-TagGN nanoassemblies to self-assemble spontaneously with inorganic iron oxide nanoparticles generating magnetic two-dimensional (2D) His₆-TagGN/Fe₃O₄ nanocomposites under mild and non-hydrothermal conditions. The set of original experiments described here open novel perspectives in the facile production of water dispersible high quality GN and FLG sheets that will improve and facilitate the interfacing, processing and manipulation of graphene for promising applications in catalysis, nanocomposite construction, integrated nanoelectronic devices and bionanotechnology.

  12. Temperature Effects on Donor-Acceptor Couplings in Peptides. A Combined Quantum Mechanics and Molecular Dynamics Study.

    Science.gov (United States)

    Wallrapp, Frank H; Voityuk, Alexander A; Guallar, Victor

    2010-10-12

    We report a quantum chemistry and molecular dynamics study on the temperature dependence of electronic coupling in two short model oligopeptides. Ten nanoseconds replica exchange molecular dynamics was performed on Trp-(Pro)3-Trp and Trp-(Pro)6-Trp peptides in the gas phase in combination with computation of the energy and electronic coupling for thermal hole transfer between Trp residues. The electron transfer parameters were estimated by using the semiempirical INDO/S method together with the charge fragment difference scheme. Conformational analysis of the derived trajectories revealed that the electronic coupling becomes temperature dependent when incorporating structural dynamics of the system. We demonstrate that Trp-(Pro)3-Trp, having only few degrees of freedom, results in relatively weak couplings at low and high temperature and a strong peak at 144 K, whereas the more flexible system Trp-(Pro)6-Trp shows monotonically decreased coupling. Only a few conformations with strong donor-acceptor couplings are shown to be crucial for the overall ET rates. Our results introduce the question whether the T dependence of ET coupling can also be found in large biological systems.

  13. The effect of fasting and refeeding on mRNA expression of PepT1 and gastrointestinal hormones regulating digestion and food intake in zebrafish (Danio rerio).

    Science.gov (United States)

    Koven, William; Schulte, Patricia

    2012-12-01

    In vertebrates, a significant part of ingested protein is absorbed as di- and tripeptides through a brush border membrane proton/oligopeptide transporter protein called PepT1. The aim of the present study was to determine the effect of short-term food deprivation and refeeding in adult zebrafish (Danio rerio) on gastrointestinal mRNA expression of PepT1 as well as on the satiety hormones cholecystokinin (CCK), gastrin-releasing peptide (GRP) and ghrelin (GHR) in order to elucidate a potential mechanism driving compensatory growth. Sixty adult zebrafish were stocked in a 40-L aquarium and fed daily a commercial flake diet to satiation for 10 days where the digestive tracts (DT) of sampled fish (n = 5) were dissected out. Samplings were repeated following 1, 2 and 5 days of food deprivation and after 1, 2 and 5 days of refeeding. The RNA was extracted from all sampled DTs and analyzed by quantitative real-time PCR for the mRNA expression of PepT1, rRNA 18S, CCK, GRP and GHR. PepT1 mRNA expression increased with successive refeedings reaching a level approximately 8 times higher than pre-fast levels. CCK, GRP and GHR mRNA levels also decreased during fasting, but increased only to pre-fasting levels with refeeding. Overall, the results suggest that PepT1 may be a contributing mechanism to compensatory growth that could influence CCK secretion and GRP and GHR activity.

  14. An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions.

    Science.gov (United States)

    Syedbasha, Mohameedyaseen; Linnik, Janina; Santer, Deanna; O'Shea, Daire; Barakat, Khaled; Joyce, Michael; Khanna, Nina; Tyrrell, D Lorne; Houghton, Michael; Egli, Adrian

    2016-01-01

    A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.

  15. Cyclopropane-derived peptidomimetics. Design, synthesis, and evaluation of novel enkephalin analogues.

    Science.gov (United States)

    Martin, S F; Dwyer, M P; Hartmann, B; Knight, K S

    2000-03-10

    It is known that peptide mimics containing trans-substituted cyclopropanes stabilize extended conformations of oligopeptides, and molecular modeling studies now suggest that the corresponding cis-cyclopropane dipeptide isosteres could stabilize a reverse turn. To begin to assess this possibility, a series of cis-substituted cyclopropanes were incorporated as replacements of the Gly(2)-Gly(3) and Phe(4)-Leu(5) dipeptide subunits in Leu-enkephalin (H(2)N-Tyr-Gly-Gly-Phe-Leu-OH), which is believed to bind to opiod receptors in a conformation containing a beta-turn. General methods for the synthesis of the cyclopropane-containing dipeptide isosteres -XaaPsi[COcpCO]Yaa- and -XaaPsi[NHcpNH]Yaa-were developed by a sequence that featured the enantioselective cyclization of allylic diazoacetates catalyzed by the chiral rhodium complexes Rh(2)[(5S)-MEPY](4) and Rh(2)[(5R)-MEPY](4). A useful modification of the Weinreb amidation procedure was applied to the opening of the intermediate lactones with dipeptides, and a novel method for the synthesis of substituted diaminocyclopropanes was also developed. The Leu-enkephalin analogues were tested in a panel of binding and functional assays, and although those derivatives containing cyclopropane replacements of the Gly(2)-Gly(3) exhibited low micromolar affinity for the mu-receptor, analogues containing such replacements for the Phe(4)-Leu(5) subunit did not bind with significant affinity to any of the opioid receptors. These results are discussed.

  16. EPOR-Based Purification and Analysis of Erythropoietin Mimetic Peptides from Human Urine by Cys-Specific Cleavage and LC/MS/MS

    Science.gov (United States)

    Vogel, Matthias; Thomas, Andreas; Schänzer, Wilhelm; Thevis, Mario

    2015-09-01

    The development of a new class of erythropoietin mimetic agents (EMA) for treating anemic conditions has been initiated with the discovery of oligopeptides capable of dimerizing the erythropoietin (EPO) receptor and thus stimulating erythropoiesis. The most promising amino acid sequences have been mounted on various different polymeric structures or carrier molecules to obtain highly active EPO-like drugs exhibiting beneficial and desirable pharmacokinetic profiles. Concomitant with creating new therapeutic options, erythropoietin mimetic peptide (EMP)-based drug candidates represent means to artificially enhance endurance performance and necessitate coverage by sports drug testing methods. Therefore, the aim of the present study was to develop a strategy for the comprehensive detection of EMPs in doping controls, which can be used complementary to existing protocols. Three model EMPs were used to provide proof-of-concept data. Following EPO receptor-facilitated purification of target analytes from human urine, the common presence of the cysteine-flanked core structure of EMPs was exploited to generate diagnostic peptides with the aid of a nonenzymatic cleavage procedure. Sensitive detection was accomplished by targeted-SIM/data-dependent MS2 analysis. Method characterization was conducted for the EMP-based drug peginesatide concerning specificity, linearity, precision, recovery, stability, ion suppression/enhancement, and limit of detection (LOD, 0.25 ng/mL). Additionally, first data for the identification of the erythropoietin mimetic peptides EMP1 and BB68 were generated, demonstrating the multi-analyte testing capability of the presented approach.

  17. Structure of the Mycobacterium tuberculosis proteasome and mechanism of inhibition by a peptidyl boronate

    Energy Technology Data Exchange (ETDEWEB)

    Hu,G.; Lin, G.; Wang, M.; Dick, L.; Xu, R.; Nathan, C.; Li, H.

    2006-01-01

    Mycobacterium tuberculosis (Mtb) has the remarkable ability to resist killing by human macrophages. The 750 kDa proteasome, not available in most eubacteria except Actinomycetes, appears to contribute to Mtb's resistance. The crystal structure of the Mtb proteasome at 3.0 Angstroms resolution reveals a substrate-binding pocket with composite features of the distinct {beta}1, {beta}2 and {beta}5 substrate binding sites of eukaryotic proteasomes, accounting for the broad specificity of the Mtb proteasome towards oligopeptides described in the companion article [Lin et al. (2006), Mol Microbiol doi:10.1111/j.1365-2958.2005.05035.x]. The substrate entrance at the end of the cylindrical proteasome appears open in the crystal structure due to partial disorder of the a-subunit N-terminal residues. However, cryo-electron microscopy of the core particle reveals a closed end, compatible with the density observed in negative-staining electron microscopy that depended on the presence of the N-terminal octapeptides of the a-subunits in the companion article, suggesting that the Mtb proteasome has a gated structure. We determine for the first time the proteasomal inhibition mechanism of the dipeptidyl boronate N-(4-morpholine)carbonyl-{beta}-(1-naphthyl)-l-alanine-l-leucine boronic acid (MLN-273), an analogue of the antimyeloma drug bortezomib. The structure improves prospects for designing Mtb-specific proteasomal inhibitors as a novel approach to chemotherapy of tuberculosis.

  18. The monocyte locomotion inhibitory factor an anti-inflammatory peptide; therapeutics originating from amebic abscess of the liver.

    Science.gov (United States)

    Velazquez, Juan R

    2011-01-01

    Entamoeba histolytica in culture produces a pentapeptide (MQCNS). This oligopeptide inhibits the in vitro and in vivo locomotion of human monocytes, hence its denomination Monocyte Locomotion Inhibitory Factor (MLIF). The original isolated peptide and its synthetic construct display similar effects, among others, being inhibition of the respiratory burst in monocytes and neutrophils, decrease of Dinitrochlorobenzene (DNCB) skin hypersensitivity in guinea pigs and gerbils, and delay of mononuclear leukocytes in human Rebuck skin windows with inhibition of vascular cell Very late antigen (VLA)-4 and Vascular adhesion molecules (VCAM) in endothelia and monocytes. The MLIF molecular mechanism of action is unknown, but data reveal its implication in Nuclear factor-kappa B (NF-κB) and Mitogenactivated protein kinase (MAPK) pathways. This could explain MLIF multiplicity of biological effects. On the other hand, the amebic peptide has been useful in treating experimental amebiasis of the liver. The amebic peptide is effective in reducing inflammation induced by carragenin and arthritis in a Collagen-induced arthritis (CIA) model. Microarray data from experimental arthritis revealed an MLIF gene expression profile that includes genes that are involved in apoptosis, cell adhesion, extracellular matrix, and inflammation / chemotaxis. MLIF could be involved in unsuspected biological factions because there is increasing data on the peptide effect on several cell activities. This review also presents uses of MLIF as described in patents.

  19. VEGF-A/NRP1 stimulates GIPC1 and Syx complex formation to promote RhoA activation and proliferation in skin cancer cells

    Directory of Open Access Journals (Sweden)

    Ayumi Yoshida

    2015-09-01

    Full Text Available Neuropilin-1 (NRP1 has been identified as a VEGF-A receptor. DJM-1, a human skin cancer cell line, expresses endogenous VEGF-A and NRP1. In the present study, the RNA interference of VEGF-A or NRP1 suppressed DJM-1 cell proliferation. Furthermore, the overexpression of the NRP1 wild type restored shNRP1-treated DJM-1 cell proliferation, whereas NRP1 cytoplasmic deletion mutants did not. A co-immunoprecipitation analysis revealed that VEGF-A induced interactions between NRP1 and GIPC1, a scaffold protein, and complex formation between GIPC1 and Syx, a RhoGEF. The knockdown of GIPC1 or Syx reduced active RhoA and DJM-1 cell proliferation without affecting the MAPK or Akt pathway. C3 exoenzyme or Y27632 inhibited the VEGF-A-induced proliferation of DJM-1 cells. Conversely, the overexpression of the constitutively active form of RhoA restored the proliferation of siVEGF-A-treated DJM-1 cells. Furthermore, the inhibition of VEGF-A/NRP1 signaling upregulated p27, a CDK inhibitor. A cell-penetrating oligopeptide that targeted GIPC1/Syx complex formation inhibited the VEGF-A-induced activation of RhoA and suppressed DJM-1 cell proliferation. In conclusion, this new signaling pathway of VEGF-A/NRP1 induced cancer cell proliferation by forming a GIPC1/Syx complex that activated RhoA to degrade the p27 protein.

  20. Variability or conservation of hepatitis C virus hypervariable region 1? Implications for immune responses

    Indian Academy of Sciences (India)

    Mario U Mondelli; Antonella Cerino; Annalisa Meola; Alfredo Nicosia

    2003-04-01

    The hypervariable region 1 (HVR1) of the E2 protein of hepatitis C virus (HCV) is highly heterogeneous in its primary sequence and is responsible for significant inter- and intra-individual variation of the infecting virus, which may represent an important pathogenetic mechanism leading to immune escape and persistent infection. A binding site for neutralizing antibodies (Ab) has also been allegedly identified in this region. Prospective studies of serological responses to synthetic oligopeptides derived from naturally-occurring HVR1 sequences showed promiscuous recognition of HVR1 variants in most patients via binding to C-terminal amino acid residues with conserved physicochemical properties. Monoclonal antibodies generated by immunization of mice with peptides derived from natural HVR1 sequences were shown to recognize several HVR1 variants in line with evidence gathered from studies using human sera. In addition, selected mAbs were able to bind HVR1 in the context of a complete soluble form of the E2 glycoprotein, indicating recognition of correctly folded sequences, and were shown to specifically capture circulating and recombinant HCV particles, suggesting that HVR1 is expressed on intact virus particles and therefore potentially able to interact with cellular receptor(s). These findings suggest that it is possible to induce a broadly reactive clonal immune response to multiple HCV variants and that this mechanism could be used in principle to induce protective immunity for a large repertoire of HCV variants.

  1. A single-stage functionalization and exfoliation method for the production of graphene in water: stepwise construction of 2D-nanostructured composites with iron oxide nanoparticles

    Science.gov (United States)

    Ihiawakrim, Dris; Ersen, Ovidiu; Melin, Frédéric; Hellwig, Petra; Janowska, Izabela; Begin, Dominique; Baaziz, Walid; Begin-Colin, Sylvie; Pham-Huu, Cuong; Baati, Rachid

    2013-09-01

    A practically simple top-down process for the exfoliation of graphene (GN) and few-layer graphene (FLG) from graphite is described. We have discovered that a biocompatible amphiphilic pyrene-based hexahistidine peptide is able to exfoliate, functionalize, and dissolve few layer graphene flakes in pure water under exceptionally mild, sustainable and virtually innocuous low intensity cavitation conditions. Large area functionalized graphene flakes with the hexahistidine oligopeptide (His6-TagGN = His6@GN) have been produced efficiently at room temperature and characterized by TEM, Raman, and UV spectroscopy. Conductivity experiments carried out on His6-TagGN samples revealed superior electric performances as compared to reduced graphene oxide (rGO) and non-functionalized graphene, demonstrating the non-invasive features of our non-covalent functionalization process. We postulated a rational exfoliation mechanism based on the intercalation of the peptide amphiphile under cavitational chemistry. We also demonstrated the ability of His6-TagGN nanoassemblies to self-assemble spontaneously with inorganic iron oxide nanoparticles generating magnetic two-dimensional (2D) His6-TagGN/Fe3O4 nanocomposites under mild and non-hydrothermal conditions. The set of original experiments described here open novel perspectives in the facile production of water dispersible high quality GN and FLG sheets that will improve and facilitate the interfacing, processing and manipulation of graphene for promising applications in catalysis, nanocomposite construction, integrated nanoelectronic devices and bionanotechnology.A practically simple top-down process for the exfoliation of graphene (GN) and few-layer graphene (FLG) from graphite is described. We have discovered that a biocompatible amphiphilic pyrene-based hexahistidine peptide is able to exfoliate, functionalize, and dissolve few layer graphene flakes in pure water under exceptionally mild, sustainable and virtually innocuous low

  2. Core-shell polymer nanoparticles for prevention of GSH drug detoxification and cisplatin delivery to breast cancer cells

    Science.gov (United States)

    Surnar, Bapurao; Sharma, Kavita; Jayakannan, Manickam

    2015-10-01

    Platinum drug delivery against the detoxification of cytoplasmic thiols is urgently required for achieving efficacy in breast cancer treatment that is over expressed by glutathione (GSH, thiol-oligopeptide). GSH-resistant polymer-cisplatin core-shell nanoparticles were custom designed based on biodegradable carboxylic functional polycaprolactone (PCL)-block-poly(ethylene glycol) diblock copolymers. The core of the nanoparticle was fixed as 100 carboxylic units and the shell part was varied using various molecular weight poly(ethylene glycol) monomethyl ethers (MW of PEGs = 100-5000 g mol-1) as initiator in the ring-opening polymerization. The complexation of cisplatin aquo species with the diblocks produced core-shell nanoparticles of 75 nm core with precise size control the particles up to 190 nm. The core-shell nanoparticles were found to be stable in saline solution and PBS and they exhibited enhanced stability with increase in the PEG shell thickness at the periphery. The hydrophobic PCL layer on the periphery of the cisplatin core behaved as a protecting layer against the cytoplasmic thiol residues (GSH and cysteine) and exhibited cisplatin and polymer drug core-shell nanoparticles showed similar cytotoxicity effects in the HeLa cells. In MCF-7 cells, the free cisplatin drug exhibited 50% cell death whereas complete cell death (100%) was accomplished by the polymer-cisplatin core-shell nanoparticles. Confocal microscopic images confirmed that the core-shell nanoparticles were taken up by the MCF-7 and HeLa cells and they were accumulated both at the cytoplasm as well at peri-nuclear environments. The present investigation lays a new foundation for the polymer-based core-shell nanoparticles approach for overcoming detoxification in platinum drugs for the treatment of GSH over-expressed breast cancer cells.Platinum drug delivery against the detoxification of cytoplasmic thiols is urgently required for achieving efficacy in breast cancer treatment that is over

  3. 抗糖尿病肽类药物研究进展%Research Advances of Peptides Counteracting Diabetes Mellitus

    Institute of Scientific and Technical Information of China (English)

    严国文; 李谦; 何书英; 朱杰; 吴梧桐

    2012-01-01

    Diabetes Mellitus ( DM) is a metabolic syndrome characterized by absolutely or comparatively insufficient insulin. DM is substantially hazardous to human health. The defects of traditional antihyperglycemic drugs have accelerated the development of peptide-based antidiabetic agents. Such agents frequently reported in published works are Clucagon-like peptide-1 (GLP-1) receptor agonists that include GLP-1 derivatives and GLP-1 analogs,amylin-based antidiabetic peptides (ABAPs) .animals or plants-derived antidiabetic peptides ( APDAPs) and antidiabetic oligopeptides ( ADOPs). This review summarizes the existing knowledge concerning advantages, drawbacks, developing thoughts and pharmaceutical effects of the four kinds of peptides, respectively.%糖尿病是以胰岛素相对或绝对不足为特征的代谢性疾病,严重危害人类健康,传统抗糖尿病药物各有缺点,从而催生了抗糖尿病肽类药物的面世.常见于文献报道的抗糖尿病肽有胰高血糖素样肽-1( Glucagonlike peptide-1,GLP-1)受体激动剂、基于胰淀素开发的抗糖尿病肽、动植物来源的抗糖尿病肽和抗糖尿病寡肽,其中前者又包括GLP-1衍生物和GLP-1类似物.该文对它们各自的优缺点,研发思路和药理效应作了综述.

  4. Mutations in Arabidopsis Yellow Stripe-Like1 and Yellow Stripe-Like3 Reveal Their Roles in Metal Ion Homeostasis and Loading of Metal Ions in Seeds1

    Science.gov (United States)

    Waters, Brian M.; Chu, Heng-Hsuan; DiDonato, Raymond J.; Roberts, Louis A.; Eisley, Robynn B.; Lahner, Brett; Salt, David E.; Walker, Elsbeth L.

    2006-01-01

    Here, we describe two members of the Arabidopsis (Arabidopsis thaliana) Yellow Stripe-Like (YSL) family, AtYSL1 and AtYSL3. The YSL1 and YSL3 proteins are members of the oligopeptide transporter family and are predicted to be integral membrane proteins. YSL1 and YSL3 are similar to the maize (Zea mays) YS1 phytosiderophore transporter (ZmYS1) and the AtYSL2 iron (Fe)-nicotianamine transporter, and are predicted to transport metal-nicotianamine complexes into cells. YSL1 and YSL3 mRNAs are expressed in both root and shoot tissues, and both are regulated in response to the Fe status of the plant. β-Glucuronidase reporter expression, driven by YSL1 and YSL3 promoters, reveals expression patterns of the genes in roots, leaves, and flowers. Expression was highest in senescing rosette leaves and cauline leaves. Whereas the single mutants ysl1 and ysl3 had no visible phenotypes, the ysl1ysl3 double mutant exhibited Fe deficiency symptoms, such as interveinal chlorosis. Leaf Fe concentrations are decreased in the double mutant, whereas manganese, zinc, and especially copper concentrations are elevated. In seeds of double-mutant plants, the concentrations of Fe, zinc, and copper are low. Mobilization of metals from leaves during senescence is impaired in the double mutant. In addition, the double mutant has reduced fertility due to defective anther and embryo development. The proposed physiological roles for YSL1 and YSL3 are in delivery of metal micronutrients to and from vascular tissues. PMID:16815956

  5. Genome-wide identification of genes essential for the survival of Streptococcus pneumoniae in human saliva.

    Directory of Open Access Journals (Sweden)

    Lilly M Verhagen

    Full Text Available Since Streptococcus pneumoniae transmits through droplet spread, this respiratory tract pathogen may be able to survive in saliva. Here, we show that saliva supports survival of clinically relevant S. pneumoniae strains for more than 24 h in a capsule-independent manner. Moreover, saliva induced growth of S. pneumoniae in growth-permissive conditions, suggesting that S. pneumoniae is well adapted for uptake of nutrients from this bodily fluid. By using Tn-seq, a method for genome-wide negative selection screening, we identified 147 genes potentially required for growth and survival of S. pneumoniae in saliva, among which genes predicted to be involved in cell envelope biosynthesis, cell transport, amino acid metabolism, and stress response predominated. The Tn-seq findings were validated by testing a panel of directed gene deletion mutants for their ability to survive in saliva under two testing conditions: at room temperature without CO2, representing transmission, and at 37 °C with CO2, representing in-host carriage. These validation experiments confirmed that the plsX gene and the amiACDEF and aroDEBC operons, involved in respectively fatty acid metabolism, oligopeptide transport, and biosynthesis of aromatic amino acids play an important role in the growth and survival of S. pneumoniae in saliva at 37 °C. In conclusion, this study shows that S. pneumoniae is well-adapted for growth and survival in human saliva and provides a genome-wide list of genes potentially involved in adaptation. This notion supports earlier evidence that S. pneumoniae can use human saliva as a vector for transmission.

  6. Microbial nitrogen dynamics in organic and mineral soil horizons along a latitudinal transect in western Siberia

    Science.gov (United States)

    Wild, Birgit; Schnecker, Jörg; Knoltsch, Anna; Takriti, Mounir; Mooshammer, Maria; Gentsch, Norman; Mikutta, Robert; Alves, Ricardo J. Eloy; Gittel, Antje; Lashchinskiy, Nikolay; Richter, Andreas

    2015-05-01

    Soil N availability is constrained by the breakdown of N-containing polymers such as proteins to oligopeptides and amino acids that can be taken up by plants and microorganisms. Excess N is released from microbial cells as ammonium (N mineralization), which in turn can serve as substrate for nitrification. According to stoichiometric theory, N mineralization and nitrification are expected to increase in relation to protein depolymerization with decreasing N limitation, and thus from higher to lower latitudes and from topsoils to subsoils. To test these hypotheses, we compared gross rates of protein depolymerization, N mineralization and nitrification (determined using 15N pool dilution assays) in organic topsoil, mineral topsoil, and mineral subsoil of seven ecosystems along a latitudinal transect in western Siberia, from tundra (67°N) to steppe (54°N). The investigated ecosystems differed strongly in N transformation rates, with highest protein depolymerization and N mineralization rates in middle and southern taiga. All N transformation rates decreased with soil depth following the decrease in organic matter content. Related to protein depolymerization, N mineralization and nitrification were significantly higher in mineral than in organic horizons, supporting a decrease in microbial N limitation with depth. In contrast, we did not find indications for a decrease in microbial N limitation from arctic to temperate ecosystems along the transect. Our findings thus challenge the perception of ubiquitous N limitation at high latitudes, but suggest a transition from N to C limitation of microorganisms with soil depth, even in high-latitude systems such as tundra and boreal forest.

  7. Theoretical modeling of peptide α-helical circular dichroism in aqueous solution.

    Science.gov (United States)

    Kaminský, Jakub; Kubelka, Jan; Bour, Petr

    2011-03-10

    Reliable modeling of protein and peptide circular dichroism (CD) spectra in the far UV presents a challenge for current theoretical approaches. In this study, the time-dependent density functional theory (TDDFT), configuration interaction with single excitation (CIS), and transition dipole coupling (TDC) were used to assess the most important factors contributing to the CD spectra of the α-helical secondary structure. The dependence on the peptide chain length and also the role of the flexibility and solvent environment were investigated with a model oligopeptide Ac-(Ala)(N)-NH-Me, (N = 1, ..., 18). Both the TDDFT and TDC-like methods suggest that the CD curve typical for the α-helix arises gradually, but its basic characteristic is discernible already for peptides with 4-5 amino acid residues. The calculated dependence was in a qualitative agreement with experimental spectra of short α-helices stabilized by the histidine-metal binding. The TDDFT computations of the CD were found to be unusually sensitive to the basis set and solvent model. Explicit hydration and temperature fluctuations of the peptide geometry, simulated with the aid of molecular dynamics (MD), significantly influenced the CD and absorption spectral shapes. An extensive averaging over MD configurations is thus required to obtain a converged spectral profile in cluster simulations. On the other hand, both the TDDFT and TDC models indicate only a minor influence of the alanine side chains. The CIS and TDC calculations also point toward a relatively small effect of the helix-helix interaction on the CD spectral profiles. For a model system of two helices, the CIS method predicted larger changes in the spectra than TDC. This suggests other than interactions between peptide chains, such as mutual polarization, can have a minor, but measurable, effect on the CD spectrum.

  8. Geometry optimizaion of aperiodic polymers by combining the elongation method with MOPAC program package

    Energy Technology Data Exchange (ETDEWEB)

    Mitani, Masaki; Aoki, Yuriko; Imamura, Akira [Hiroshima Univ. (Japan)

    1996-12-31

    Recently we have developed the elongation method, by which we can calculate the electronic structure of various types of periodic as well as aperiodic polymers with relatively much shorter computation time and much smaller memory size. The characteristic of the elongation method is to calculate the electronic structure of polymers by adding a monomer one by one as is the case for the polymerization reaction. The similar procedure can be applied to the geometry optimization of polymers in the frame of the elongation method, that is, the geometry optimization procedure is limited in a region in which the electronic structure changes against the elongation of the polymer. In this procedure, we can obtain the optimized geometry of the polymer efficiently with much smaller computation time. In the present study, we combined the elongation program with MOPAC program package which is used worldwide. The concrete geometry optimization procedure bases upon the Z-matrix procedure . When electron density on the relevant atoms does change its magnitude less than a threshold value, say 0.00001% against the elongation of the polymer, the corresponding geometrical parameters are thereafter fixed. For example, the length of the bond A-B is fixed when the electron densities on atoms A and B are frozen. The same procedure is applied to the bond angle and the dihedral angle. This optimization procedure were applied to polyethylene, polyacetylene, polyglycine and polyacetylene with positive and negative solitons. The calculated results are promising and this method is found to be reliable and applicable enough to be useful for the non-periodical polymers such as oligopeptides with various kinds of amino acid reridues. The obtained results will be given in the lecture in detail.

  9. Quorum Sensing Peptides Selectively Penetrate the Blood-Brain Barrier.

    Science.gov (United States)

    Wynendaele, Evelien; Verbeke, Frederick; Stalmans, Sofie; Gevaert, Bert; Janssens, Yorick; Van De Wiele, Christophe; Peremans, Kathelijne; Burvenich, Christian; De Spiegeleer, Bart

    2015-01-01

    Bacteria communicate with each other by the use of signaling molecules, a process called 'quorum sensing'. One group of quorum sensing molecules includes the oligopeptides, which are mainly produced by Gram-positive bacteria. Recently, these quorum sensing peptides were found to biologically influence mammalian cells, promoting i.a. metastasis of cancer cells. Moreover, it was found that bacteria can influence different central nervous system related disorders as well, e.g. anxiety, depression and autism. Research currently focuses on the role of bacterial metabolites in this bacteria-brain interaction, with the role of the quorum sensing peptides not yet known. Here, three chemically diverse quorum sensing peptides were investigated for their brain influx (multiple time regression technique) and efflux properties in an in vivo mouse model (ICR-CD-1) to determine blood-brain transfer properties: PhrCACET1 demonstrated comparatively a very high initial influx into the mouse brain (Kin = 20.87 μl/(g×min)), while brain penetrabilities of BIP-2 and PhrANTH2 were found to be low (Kin = 2.68 μl/(g×min)) and very low (Kin = 0.18 μl/(g×min)), respectively. All three quorum sensing peptides were metabolically stable in plasma (in vitro) during the experimental time frame and no significant brain efflux was observed. Initial tissue distribution data showed remarkably high liver accumulation of BIP-2 as well. Our results thus support the potential role of some quorum sensing peptides in different neurological disorders, thereby enlarging our knowledge about the microbiome-brain axis.

  10. Collagen metabolism of human osteoarthritic articular cartilage as modulated by bovine collagen hydrolysates.

    Directory of Open Access Journals (Sweden)

    Saskia Schadow

    Full Text Available Destruction of articular cartilage is a characteristic feature of osteoarthritis (OA. Collagen hydrolysates are mixtures of collagen peptides and have gained huge public attention as nutriceuticals used for prophylaxis of OA. Here, we evaluated for the first time whether different bovine collagen hydrolysate preparations indeed modulate the metabolism of collagen and proteoglycans from human OA cartilage explants and determined the chemical composition of oligopeptides representing collagen fragments. Using biophysical techniques, like MALDI-TOF-MS, AFM, and NMR, the molecular weight distribution and aggregation behavior of collagen hydrolysates from bovine origin (CH-Alpha®, Peptan™ B 5000, Peptan™ B 2000 were determined. To investigate the metabolism of human femoral OA cartilage, explants were obtained during knee replacement surgery. Collagen synthesis of explants as modulated by 0-10 mg/ml collagen hydrolysates was determined using a novel dual radiolabeling procedure. Proteoglycans, NO, PGE(2, MMP-1, -3, -13, TIMP-1, collagen type II, and cell viability were determined in explant cultures. Groups of data were analyzed using ANOVA and the Friedman test (n = 5-12. The significance was set to p≤0.05. We found that collagen hydrolysates obtained from different sources varied with respect to the width of molecular weight distribution, average molecular weight, and aggregation behavior. None of the collagen hydrolysates tested stimulated the biosynthesis of collagen. Peptan™ B 5000 elevated NO and PGE(2 levels significantly but had no effect on collagen or proteoglycan loss. All collagen hydrolysates tested proved not to be cytotoxic. Together, our data demonstrate for the first time that various collagen hydrolysates differ with respect to their chemical composition of collagen fragments as well as by their pharmacological efficacy on human chondrocytes. Our study underscores the importance that each collagen hydrolysate

  11. Comparative genome analysis of wheat blue dwarf phytoplasma, an obligate pathogen that causes wheat blue dwarf disease in China.

    Directory of Open Access Journals (Sweden)

    Wang Chen

    Full Text Available Wheat blue dwarf (WBD disease is an important disease that has caused heavy losses in wheat production in northwestern China. This disease is caused by WBD phytoplasma, which is transmitted by Psammotettix striatus. Until now, no genome information about WBD phytoplasma has been published, seriously restricting research on this obligate pathogen. In this paper, we report a new sequencing and assembling strategy for phytoplasma genome projects. This strategy involves differential centrifugation, pulsed-field gel electrophoresis, whole genome amplification, shotgun sequencing, de novo assembly, screening of contigs from phytoplasma and the connection of phytoplasma contigs. Using this scheme, the WBD phytoplasma draft genome was obtained. It was comprised of six contigs with a total size of 611,462 bp, covering ∼94% of the chromosome. Five-hundred-twenty-five protein-coding genes, two operons for rRNA genes and 32 tRNA genes were identified. Comparative genome analyses between WBD phytoplasma and other phytoplasmas were subsequently carried out. The results showed that extensive arrangements and inversions existed among the WBD, OY-M and AY-WB phytoplasma genomes. Most protein-coding genes in WBD phytoplasma were found to be homologous to genes from other phytoplasmas; only 22 WBD-specific genes were identified. KEGG pathway analysis indicated that WBD phytoplasma had strongly reduced metabolic capabilities. However, 46 transporters were identified, which were involved with dipeptides/oligopeptides, spermidine/putrescine, cobalt and Mn/Zn transport, and so on. A total of 37 secreted proteins were encoded in the WBD phytoplasma chromosome and plasmids. Of these, three secreted proteins were similar to the reported phytoplasma virulence factors TENGU, SAP11 and SAP54. In addition, WBD phytoplasma possessed several proteins that were predicted to play a role in its adaptation to diverse environments. These results will provide clues for research on

  12. Carbohydrate protease conjugates: Stabilized proteases for peptide synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Wartchow, C.A.; Wang, Peng; Bednarski, M.D.; Callstrom, M.R. [Ohio State Univ., Columbus, OH (United States)]|[Lawrence Berkeley Lab., CA (United States)

    1995-12-31

    The synthesis of oligopeptides using stable carbohydrate protease conjugates (CPCs) was examined in acetonitrile solvent systems. CPC[{alpha}-chymotrypsin] was used for the preparation of peptides containing histidine, phenylalanine, tryptophan in the P{sub 1} position in 60-93% yield. The CPC[{alpha}-chymotrypsin]-catalyzed synthesis of octamer Z-Gly-Gly-Phe-Gly-Gly-Phe-Gly-Gly-OEt from Z-Gly-Gly-Phe-Gly-Gly-Phe-OMe was achieved in 71% yield demonstrating that synthesis peptides containing both hydrophylic and hydrophobic amino acids. The P{sub 2} specificity of papain for aromatic residues was utilized for the 2 + 3 coupling of Z-Tyr-Gly-OMe to H{sub 2}N-Gly-Phe-Leu-OH to generate the leucine enkephalin derivative in 79% yield. Although papain is nonspecific for the hydrolysis of N-benzyloxycarbonyl amino acid methyl esters in aqueous solution, the rates of synthesis for these derivitives with nucleophile leucine tert-butyl ester differed by nearly 2 orders of magnitude. CPC[thermolysin] was used to prepare the aspartame precursor Z-Asp-Phe-OMe in 90% yield. The increased stability of CPCs prepared from periodate-modified poly(2-methacryl- amido-2-deoxy-D-glucose), poly(2-methacrylamido-2-deoxy-D-galactose), and poly(5-methacryl-amido-5-deoxy-D-ribose), carbohydrate materials designed to increase the aldehyde concentration in aqueous solution, suggests that the stability of CPCs is directly related to the aldehyde concentration of the carbohydrate material. Periodate oxidation of poly(2-methacrylamido-2-deoxy-D-glucose) followed by covalent attachment to {alpha}-chymotrypsin gave a CPC with catalytic activity in potassium phosphate buffer at 90{degrees}C for 2 h. 1 fig., 1 tab., 40 refs.

  13. Influence of Peptide Transporter 2 (PEPT2) on the Distribution of Cefadroxil in Mouse Brain: A Microdialysis Study.

    Science.gov (United States)

    Chen, Xiaomei; Keep, Richard F; Liang, Yan; Zhu, Hao-Jie; Hammarlund-Udenaes, Margareta; Hu, Yongjun; Smith, David E

    2017-02-10

    Peptide transporter 2 (PEPT2) is a high-affinity low-capacity transporter belonging to the proton-coupled oligopeptide transporter family. Although many aspects of PEPT2 structure-function are known, including its localization in choroid plexus and neurons, its regional activity in brain, especially extracellular fluid (ECF), is uncertain. In this study, the pharmacokinetics and regional brain distribution of cefadroxil, a ß-lactam antibiotic and PEPT2 substrate, were investigated in wildtype and Pept2 null mice using in vivo intracerebral microdialysis. Cefadroxil was infused intravenously over 4 hours at 0.15 mg/min/kg, and samples obtained from plasma, brain ECF, cerebrospinal fluid (CSF) and brain tissue. A permeability-surface area experiment was also performed in which 0.15 mg/min/kg cefadroxil was infused intravenously for 10 min, and samples obtained from plasma and brain tissues. Our results showed that PEPT2 ablation significantly increased the brain ECF and CSF levels of cefadroxil (2- to 2.5-fold). In contrast, there were no significant differences between wildtype and Pept2 null mice in the amount of cefadroxil in brain cells. The unbound volume of distribution of cefadroxil in brain was 60% lower in Pept2 null mice indicating an uptake function for PEPT2 in brain cells. Finally, PEPT2 did not affect the influx clearance of cefadroxil, thereby, ruling out differences between the two genotypes in drug entry across the blood-brain barriers. These findings demonstrate, for the first time, the impact of PEPT2 on brain ECF as well as the known role of PEPT2 in removing peptide-like drugs, such as cefadroxil, from the CSF to blood.

  14. New and cost effective cell-based assay for Dialyzed Leukocyte Extract (DLE)-induced Jurkat cells proliferation under azathioprine treatment.

    Science.gov (United States)

    Cardoso, F M; Tomkova, M; Petrovajova, D; Bubanova, M; Ragac, O; Hornakova, T

    2017-01-31

    The human Dialyzed Leukocyte Extract (DLE) is a heterogeneous mix of oligopeptides of assays exist: E-rosette test, induction of delayed type hypersensitivity in mice, leukocyte migration and IFN-γ secretion. The animal-origin materials and in vivo assays convey a considerable logistic, ethic and economic burden, meanwhile the available in vitro assays have been reported with limited reproducibility and sometimes contradictory results. Here we are reporting a new DLE biological activity cell-based assay. The A20 and Jurkat cell lines were treated with (+Aza) or without (-Aza) azathioprine, DLE (+DLE) or both (+Aza/+DLE). After 72h, the cell proliferation was analyzed by the MTT or BrdU incorporation assays. In +Aza/+DLE treated cells, we observed a significant higher proliferation, when compared with +Aza/-DLE. In the absence of Aza, cells did not present any proliferation difference between -DLE or +DLE treatments. Both assays, MTT and BrdU showed similar results, being the MTT test more cost effective and we select it for validation as DLE biological assay using Jurkat cells only. We tested three different lyophilized DLE batches and we found consistent results with acceptable assay reproducibility and linearity. The DLE capacity for rescuing Jurkat cell proliferation during +Aza treatment was consistent using different liquid and lyophilized DLE batches, presenting also consistent chromatographic profiles. Finally, DLE treatment in Jurkat cells did not result into significant IL-2 of IFN-γ secretion, and known lymphocyte proliferative drugs failed to rescue Jurkat cells viability in presence of +Aza, as +DLE treatment did in our MTT assay. In conclusion, our new cell-based MTT assay has excellent DLE biological activity consistency, robustness and is cost effective, presenting important advantages over previous DLE activity in vitro and in vivo assays.

  15. Molecular Characterization of Three Novel Phospholipase A2 Proteins from the Venom of Atheris chlorechis, Atheris nitschei and Atheris squamigera

    Directory of Open Access Journals (Sweden)

    He Wang

    2016-06-01

    Full Text Available Secretory phospholipase A2 (sPLA2 is known as a major component of snake venoms and displays higher-order catalytic hydrolysis functions as well as a wide range of pathological effects. Atheris is not a notoriously dangerous genus of snakes although there are some reports of fatal cases after envenomation due to the effects of coagulation disturbances and hemorrhaging. Molecular characterization of Atheris venom enzymes is incomplete and there are only a few reports in the literature. Here, we report, for the first time, the cloning and characterization of three novel cDNAs encoding phospholipase A2 precursors (one each from the venoms of the Western bush viper (Atheris chlorechis, the Great Lakes bush viper (Atheris nitschei and the Variable bush viper (Atheris squamigera, using a “shotgun cloning” strategy. Open-reading frames of respective cloned cDNAs contained putative 16 residue signal peptides and mature proteins composed of 121 to 123 amino acid residues. Alignment of mature protein sequences revealed high degrees of structural conservation and identity with Group II venom PLA2 proteins from other taxa within the Viperidae. Reverse-phase High Performance Liquid Chromatography (HPLC profiles of these three snake venoms were obtained separately and chromatographic fractions were assessed for phospholipase activity using an egg yolk suspension assay. The molecular masses of mature proteins were all identified as approximately 14 kDa. Mass spectrometric analyses of the fractionated oligopeptides arising from tryptic digestion of intact venom proteins, was performed for further structural characterization.

  16. Human transporters, PEPT1/2, facilitate melatonin transportation into mitochondria of cancer cells: An implication of the therapeutic potential.

    Science.gov (United States)

    Huo, Xiaokui; Wang, Chao; Yu, Zhenlong; Peng, Yulin; Wang, Shumei; Feng, Shengnan; Zhang, Shouji; Tian, Xiangge; Sun, Chengpeng; Liu, Kexin; Deng, Sa; Ma, Xiaochi

    2017-05-01

    Melatonin is present in virtually all organisms from bacteria to mammals, and it exhibits a broad spectrum of biological functions, including synchronization of circadian rhythms and oncostatic activity. Several functions of melatonin are mediated by its membrane receptors, but others are receptor-independent. For the latter, melatonin is required to penetrate membrane and enters intracellular compartments. However, the mechanism by which melatonin enters cells remains debatable. In this study, it was identified that melatonin and its sulfation metabolites were the substrates of oligopeptide transporter (PEPT) 1/2 and organic anion transporter (OAT) 3, respectively. The docking analysis showed that the binding of melatonin to PEPT1/2 was attributed to their low binding energy and suitable binding conformation in which melatonin was embedded in the active site of PEPT1/2 and fitted well with the cavity in three-dimensional space. PEPT1/2 transporters play a pivotal role in melatonin uptake in cells. Melatonin's membrane transportation via PEPT1/2 renders its oncostatic effect in malignant cells. For the first time, PEPT1/2 were identified to localize in the mitochondrial membrane of human cancer cell lines of PC3 and U118. PEPT1/2 facilitated the transportation of melatonin into mitochondria. Melatonin accumulation in mitochondria induced apoptosis of PC3 and U118 cells. Thus, PEPT1/2 can potentially be used as a cancer cell-targeted melatonin delivery system to improve the therapeutic effects of melatonin in cancer treatment. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Melatonin as a mitochondria-targeted antioxidant: one of evolution's best ideas.

    Science.gov (United States)

    Reiter, Russel J; Rosales-Corral, Sergio; Tan, Dun Xian; Jou, Mei Jie; Galano, Annia; Xu, Bing

    2017-09-01

    Melatonin is an ancient antioxidant. After its initial development in bacteria, it has been retained throughout evolution such that it may be or may have been present in every species that have existed. Even though it has been maintained throughout evolution during the diversification of species, melatonin's chemical structure has never changed; thus, the melatonin present in currently living humans is identical to that present in cyanobacteria that have existed on Earth for billions of years. Melatonin in the systemic circulation of mammals quickly disappears from the blood presumably due to its uptake by cells, particularly when they are under high oxidative stress conditions. The measurement of the subcellular distribution of melatonin has shown that the concentration of this indole in the mitochondria greatly exceeds that in the blood. Melatonin presumably enters mitochondria through oligopeptide transporters, PEPT1, and PEPT2. Thus, melatonin is specifically targeted to the mitochondria where it seems to function as an apex antioxidant. In addition to being taken up from the circulation, melatonin may be produced in the mitochondria as well. During evolution, mitochondria likely originated when melatonin-forming bacteria were engulfed as food by ancestral prokaryotes. Over time, engulfed bacteria evolved into mitochondria; this is known as the endosymbiotic theory of the origin of mitochondria. When they did so, the mitochondria retained the ability to synthesize melatonin. Thus, melatonin is not only taken up by mitochondria but these organelles, in addition to many other functions, also probably produce melatonin as well. Melatonin's high concentrations and multiple actions as an antioxidant provide potent antioxidant protection to these organelles which are exposed to abundant free radicals.

  18. Complete Sequences of Four Plasmids of Lactococcus lactis subsp. cremoris SK11 Reveal Extensive Adaptation to the Dairy Environment†

    Science.gov (United States)

    Siezen, Roland J.; Renckens, Bernadet; van Swam, Iris; Peters, Sander; van Kranenburg, Richard; Kleerebezem, Michiel; de Vos, Willem M.

    2005-01-01

    Lactococcus lactis strains are known to carry plasmids encoding industrially important traits. L. lactis subsp. cremoris SK11 is widely used by the dairy industry in cheese making. Its complete plasmid complement was sequenced and found to contain the plasmids pSK11A (10,372 bp), pSK11B (13,332 bp), pSK11L (47,165 bp), and pSK11P (75,814 bp). Six highly homologous repB-containing replicons were found, all belonging to the family of lactococcal theta-type replicons. Twenty-three complete insertion sequence elements segment the plasmids into numerous modules, many of which can be identified as functional units or containing functionally related genes. Plasmid-encoded functions previously known to reside on L. lactis SK11 plasmids were now mapped in detail, e.g., lactose utilization (lacR-lacABCDFEGX), the proteolytic system (prtM-prtP, pepO, pepF), and the oligopeptide permease system (oppDFBCA). Newly identified plasmid-encoded functions could facilitate the uptake of various cations, while the pabA and pabB genes could be essential for folate biosynthesis. A competitive advantage could be obtained by using the putative flavin adenine dinucleotide-dependent d-lactate dehydrogenase and oxalate:formate antiporter for enhanced ATP synthesis, while the activity of the predicted α-acetolactate decarboxylase may contribute to the formation of an additional electron sink. Various stress response proteins are plasmid encoded, which could enhance strain robustness. A substantial number of these “adaptation” genes have not been described before on L. lactis plasmids. Moreover, several genes were identified for the first time in L. lactis, possibly reflecting horizontal gene transfer. PMID:16332824

  19. Expression and characterization of common carp (Cyprinus carpio) matrix metalloproteinase-2 and its activity against type I collagen.

    Science.gov (United States)

    Wang, Ci; Zhan, Chun-Lan; Cai, Qiu-Feng; Du, Cui-Hong; Liu, Guang-Ming; Su, Wen-Jin; Cao, Min-Jie

    2014-05-10

    Matrix metalloproteinases (MMPs) play essential roles in the metabolism of animal collagen while few reports are available for MMPs in aquatic animals. In this study, we report the complete sequence of matrix metalloproteinase-2 (MMP-2) gene from common carp (Cyprinus carpio) skeletal muscle. The full-length cDNA of MMP-2 was 2792bp which contains an open reading frame of 1974bp, corresponding to a protein of 657 amino acid residues. Based on the structural feature of MMP-2, the gene of the catalytic domain containing 351 amino acid residues was cloned and expressed in Escherichia coli. SDS-PAGE showed that the truncated recombinant MMP-2 (trMMP-2) with molecular mass of approximately 38kDa was in the form of inclusion body. The trMMP-2 was further purified by immobilized metal ion affinity chromatography. After renaturation, similar to native MMP-2, the trMMP-2 exhibited high hydrolyzing activity toward gelatin as appeared on gelatin zymography and optimal activity was at pH 8.0 and 40°C. The activity of the trMMP-2 was completely suppressed by metalloproteinase inhibitors, including EDTA, EGTA and 1,10-phenanthroline while other proteinase inhibitors did not show any inhibitory effect. Divalent metal ion Ca(2+) was necessary for the gelatinolytic activity, suggesting it is a calcium-dependent metalloproteinase. Moreover, the trMMP-2 effectively hydrolyzed native type I collagen at 37°C and even at 4°C, implying its potential application value as a collagenase for preparation of biologically active oligopeptides.

  20. Disturbed intestinal nitrogen homeostasis in a mouse model of high-fat diet-induced obesity and glucose intolerance.

    Science.gov (United States)

    Do, Thi Thu Huong; Hindlet, Patrick; Waligora-Dupriet, Anne-Judith; Kapel, Nathalie; Neveux, Nathalie; Mignon, Virginie; Deloménie, Claudine; Farinotti, Robert; Fève, Bruno; Buyse, Marion

    2014-03-01

    The oligopeptide transporter peptide cotransporter-1 Slc15a1 (PEPT1) plays a major role in the regulation of nitrogen supply, since it is responsible for 70% of the dietary nitrogen absorption. Previous studies demonstrated that PEPT1 expression and function in jejunum are reduced in diabetes and obesity, suggesting a nitrogen malabsorption from the diet. Surprisingly, we reported here a decrease in gut nitrogen excretion in high-fat diet (HFD)-fed mice and further investigated the mechanisms that could explain this apparent contradiction. Upon HFD, mice exhibited an increased concentration of free amino acids (AAs) in the portal vein (60%) along with a selective increase in the expression of two AA transporters (Slc6a20a, Slc36a1), pointing to a specific and adaptive absorption of some AAs. A delayed transit time (+40%) and an increased intestinal permeability (+80%) also contribute to the increase in nitrogen absorption. Besides, HFD mice exhibited a 2.2-fold decrease in fecal DNA resulting from a reduction in nitrogen catabolism from cell desquamation and/or in the intestinal microbiota. Indeed, major quantitative (2.5-fold reduction) and qualitative alterations of intestinal microbiota were observed in feces of HFD mice. Collectively, our results strongly suggest that both increased AA transporters, intestinal permeability and transit time, and changes in gut microbiota are involved in the increased circulating AA levels. Modifications in nitrogen homeostasis provide a new insight in HFD-induced obesity and glucose intolerance; however, whether these modifications are beneficial or detrimental for the HFD-associated metabolic complications remains an open issue.

  1. Identification of residues critical for proton-coupled glutathione translocation in the yeast glutathione transporter, Hgt1p.

    Science.gov (United States)

    Zulkifli, Mohammad; Bachhawat, Anand Kumar

    2017-05-16

    The proton gradient acts as the driving force for the transport of many metabolites across fungal and plant plasma membranes. Identifying the mechanism of proton relay is critical for understanding the mechanism of transport mediated by these transporters. We investigated two strategies for identifying residues critical for proton-dependent substrate transport in the yeast glutathione transporter, Hgt1p, a member of the poorly understood oligopeptide transporter family of transporters. In the first strategy, we tried to identify the pH-independent mutants that could grow at higher pH when dependant on glutathione transport. Screening a library of 269 alanine mutants of the transmembrane domains (TMDs) along with a random mutagenesis strategy yielded two residues (E135K on the cusp of TMD2 and N710S on TMD12) that permitted growth on glutathione at pH 8.0. Further analysis revealed that these residues were not involved in proton symport even though they conferred better transport at a higher pH. The second strategy involved a knowledge-driven approach, targeting 31 potential residues based on charge, conservation and location. Mutation of these residues followed by functional and biochemical characterization revealed E177A, Y193A, D335A, Y374A, H445A and R554A as being defective in proton transport. Further analysis enabled possible roles of these residues to be assigned in proton relay. The implications of these findings in relation to Hgt1p and the suitability of these strategic approaches for identifying such residues are discussed. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  2. Study on the Production of Protein Feed with the Production of Soybean Peptide and the Optimization of Its Solid State Fermentation Technical Parameters%产大豆肽蛋白饲料的研制及其固态发酵工艺参数的优化研究

    Institute of Scientific and Technical Information of China (English)

    卢富山; 王平; 王潇

    2012-01-01

    就2种菌组合协同固态发酵生产功能大豆寡肽蛋白饲料工艺参数优化的工业化研究结果进行了总结。其模式为:2种菌组合→液态接种→发酵池封闭式发酵。筛选菌种:枯草芽胞杆菌和乳酸杆菌。经优化后发酵工艺参数:时间38h;pH值为7~8;初始培养温度(37±1)℃;芽胞杆菌菌液接种量2%,乳酸菌接种量为3%;料水比为2∶1;底物组成:豆粕90%、麸皮7%、玉米粉3%。%The industrialized research results of the optimization of the technical parameters of synergistic solid state fermentation of producing soybean oligopeptide protein feed with the two kinds of bacteria were summarized. The model was as follows: combination of two kinds of bacteria → liquid state inoculation→ closed fermentation in fermentation pool. The two kinds of bacteria were screened out: Bacillus subtilis and Lactobacillus. The optimized fermentation technical parameters were as follows: the fermentation time of 38 h, pH of 7~8, the initial temperature of (37±1) ℃, the inoculated volume of 2% Bacillus subtilis and 3% Lactobacillus, the feed-water ratio of 2∶1, the substrate composition of 90% soybean meal, 7% wheat bran and 3% maize meal.

  3. The Arabidopsis AtOPT3 protein functions in metal homeostasis and movement of iron to developing seeds.

    Science.gov (United States)

    Stacey, Minviluz G; Patel, Ami; McClain, William E; Mathieu, Melanie; Remley, Melissa; Rogers, Elizabeth E; Gassmann, Walter; Blevins, Dale G; Stacey, Gary

    2008-02-01

    The Arabidopsis thaliana AtOPT3 belongs to the oligopeptide transporter (OPT) family, a relatively poorly characterized family of peptide/modified peptide transporters found in archebacteria, bacteria, fungi, and plants. A null mutation in AtOPT3 resulted in embryo lethality, indicating an essential role for AtOPT3 in embryo development. In this article, we report on the isolation and phenotypic characterization of a second AtOPT3 mutant line, opt3-2, harboring a T-DNA insertion in the 5' untranslated region of AtOPT3. The T-DNA insertion in the AtOPT3 promoter resulted in reduced but sufficient AtOPT3 expression to allow embryo formation in opt3-2 homozygous seeds. Phenotypic analyses of opt3-2 plants revealed three interesting loss-of-function phenotypes associated with iron metabolism. First, reduced AtOPT3 expression in opt3-2 plants resulted in the constitutive expression of root iron deficiency responses regardless of exogenous iron supply. Second, deregulation of root iron uptake processes in opt3-2 roots resulted in the accumulation of very high levels of iron in opt3-2 tissues. Hyperaccumulation of iron in opt3-2 resulted in the formation of brown necrotic areas in opt3-2 leaves and was more pronounced during the seed-filling stage. Third, reduced AtOPT3 expression resulted in decreased accumulation of iron in opt3-2 seeds. The reduced accumulation of iron in opt3-2 seeds is especially noteworthy considering the excessively high levels of accumulated iron in other opt3-2 tissues. AtOPT3, therefore, plays a critical role in two important aspects of iron metabolism, namely, maintenance of whole-plant iron homeostasis and iron nutrition of developing seeds.

  4. ELECTROACTIVE AND NANOSTRUCTURED POLYMERS AS SCAFFOLD MATERIALS FOR NEURONAL AND CARDIAC TISSUE ENGINEERING

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Conducting polymer, polyaniline (PANI), has been studied as a novel electroactive and electrically conductive material for tissue engineering applications. The biocompatibility of the conductive polymer can be improved by (I) covalently grafting various adhesive peptides onto the surface of prefabricated conducting polymer flms or into the polymer structures during the synthesis, (ii) co-electrospinning or blending with natural proteins to form conducting nanofibers or films, and (iii) preparing conducting polymers using biopolymers, such as collagen, as templates. In this paper, we mainly describe and review the approaches of covalently attaching oligopeptides to PANI and electrospinning PANI-gelatin blend nanofibers. The employment of such modified conducting polymers as substrates for enhanced cell attachment, proliferation and differentiation has been investigated with neuronal PC-12 cells and H9c2 cardiac myoblasts. For the electrospun PANIgelatin fibers, depending on the concentrations of PANI, H9c2 cells initially displayed different morphologies on the fibrous substrates, but after one week all cultures reached confluence of similar densities and morphologies. Furthermore, we observed, that conductive PANI, when maintained in an aqueous physiologic environment, retained a significant level of electrical conductivity for at least 100 h, even though this conductivity was decreasing over time. Preliminary data show that the application of micro-current stimulates the differentiation of PC-12 cells. All the results demonstrate the potential for using PANI as an electroactive polymer in the culture of excitable cells and open the possibility of using this material as an electroactive scaffold for cardiac and/or neuronal tissue engineering applications that require biocompatibility of conductive polymers.

  5. Growth phase and pH influence peptide signaling for competence development in Streptococcus mutans.

    Science.gov (United States)

    Guo, Qiang; Ahn, Sang-Joon; Kaspar, Justin; Zhou, Xuedong; Burne, Robert A

    2014-01-01

    The development of competence by the dental caries pathogen Streptococcus mutans is mediated primarily through the alternative sigma factor ComX (SigX), which is under the control of multiple regulatory systems and activates the expression of genes involved in DNA uptake and recombination. Here we report that the induction of competence and competence gene expression by XIP (sigX-inducing peptide) and CSP (competence-stimulating peptide) is dependent on the growth phase and that environmental pH has a potent effect on the responses to XIP. A dramatic decline in comX and comS expression was observed in mid- and late-exponential-phase cells. XIP-mediated competence development and responses to XIP were optimal around a neutral pH, although mid-exponential-phase cells remained refractory to XIP treatment, and acidified late-exponential-phase cultures were resistant to killing by high concentrations of XIP. Changes in the expression of the genes for the oligopeptide permease (opp), which appears to be responsible for the internalization of XIP, could not entirely account for the behaviors observed. Interestingly, comS and comX expression was highly induced in response to endogenously overproduced XIP or ComS in mid-exponential-phase cells. In contrast to the effects of pH on XIP, competence induction and responses to CSP in complex medium were not affected by pH, although a decreased response to CSP in cells that had exited early-exponential phase was observed. Collectively, these results indicate that competence development may be highly sensitive to microenvironments within oral biofilms and that XIP and CSP signaling in biofilms could be spatially and temporally heterogeneous.

  6. A highly sensitive and simply operated protease sensor toward point-of-care testing.

    Science.gov (United States)

    Park, Seonhwa; Shin, Yu Mi; Seo, Jeongwook; Song, Ji-Joon; Yang, Haesik

    2016-04-21

    Protease sensors for point-of-care testing (POCT) require simple operation, a detection period of less than 20 minutes, and a detection limit of less than 1 ng mL(-1). However, it is difficult to meet these requirements with protease sensors that are based on proteolytic cleavage. This paper reports a highly reproducible protease sensor that allows the sensitive and simple electrochemical detection of the botulinum neurotoxin type E light chain (BoNT/E-LC), which is obtained using (i) low nonspecific adsorption, (ii) high signal-to-background ratio, and (iii) one-step solution treatment. The BoNT/E-LC detection is based on two-step proteolytic cleavage using BoNT/E-LC (endopeptidase) and l-leucine-aminopeptidase (LAP, exopeptidase). Indium-tin oxide (ITO) electrodes are modified partially with reduced graphene oxide (rGO) to increase their electrocatalytic activities. Avidin is then adsorbed on the electrodes to minimize the nonspecific adsorption of proteases. Low nonspecific adsorption allows a highly reproducible sensor response. Electrochemical-chemical (EC) redox cycling involving p-aminophenol (AP) and dithiothreitol (DTT) is performed to obtain a high signal-to-background ratio. After adding a C-terminally AP-labeled oligopeptide, DTT, and LAP simultaneously to a sample solution, no further treatment of the solution is necessary during detection. The detection limits of BoNT/E-LC in phosphate-buffered saline are 0.1 ng mL(-1) for an incubation period of 15 min and 5 fg mL(-1) for an incubation period of 4 h. The detection limit in commercial bottled water is 1 ng mL(-1) for an incubation period of 15 min. The developed sensor is selective to BoNT/E-LC among the four types of BoNTs tested. These results indicate that the protease sensor meets the requirements for POCT.

  7. Structural determinants of tobacco vein mottling virus protease substrate specificity.

    Science.gov (United States)

    Sun, Ping; Austin, Brian P; Tözsér, József; Waugh, David S

    2010-11-01

    Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMV protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac-RETVRFQSD) at 1.7-Å resolution. As observed in several crystal structures of TEV protease, the C-terminus (∼20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by ∼10-fold, an analogous truncation mutant of TVMV protease is significantly more active. Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1' position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters k(cat) and K(m) for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease.

  8. Generating new prions by targeted mutation or segment duplication.

    Science.gov (United States)

    Paul, Kacy R; Hendrich, Connor G; Waechter, Aubrey; Harman, Madison R; Ross, Eric D

    2015-07-14

    Yeasts contain various protein-based genetic elements, termed prions, that result from the structural conversion of proteins into self-propagating amyloid forms. Most yeast prion proteins contain glutamine/asparagine (Q/N)-rich prion domains that drive prion activity. Here, we explore two mechanisms by which new prion domains could evolve. First, it has been proposed that mutation and natural selection will tend to result in proteins with aggregation propensities just low enough to function under physiological conditions and thus that a small number of mutations are often sufficient to cause aggregation. We hypothesized that if the ability to form prion aggregates was a sufficiently generic feature of Q/N-rich domains, many nonprion Q/N-rich domains might similarly have aggregation propensities on the edge of prion formation. Indeed, we tested four yeast Q/N-rich domains that had no detectable aggregation activity; in each case, a small number of rationally designed mutations were sufficient to cause the proteins to aggregate and, for two of the domains, to create prion activity. Second, oligopeptide repeats are found in multiple prion proteins, and expansion of these repeats increases prion activity. However, it is unclear whether the effects of repeat expansion are unique to these specific sequences or are a generic result of adding additional aggregation-prone segments into a protein domain. We found that within nonprion Q/N-rich domains, repeating aggregation-prone segments in tandem was sufficient to create prion activity. Duplication of DNA elements is a common source of genetic variation and may provide a simple mechanism to rapidly evolve prion activity.

  9. An EGFR/HER2-Bispecific and enediyne-energized fusion protein shows high efficacy against esophageal cancer.

    Directory of Open Access Journals (Sweden)

    Xiao-Fang Guo

    Full Text Available Esophageal cancer is one of the most common cancers, and the 5-year survival rate is less than 10% due to lack of effective therapeutic agents. This study was to evaluate antitumor activity of Ec-LDP-Hr-AE, a recently developed bispecific enediyne-energized fusion protein targeting both epidermal growth factor receptor (EGFR and epidermal growth factor receptor 2 (HER2, on esophageal cancer. The fusion protein Ec-LDP-Hr-AE consists of two oligopeptide ligands and an enediyne antibiotic lidamycin (LDM for receptor binding and cell killing, respectively. The current study demonstrated that Ec-LDP-Hr had high affinity to bind to esophageal squamous cell carcinoma (ESCC cells, and enediyne-energized fusion protein Ec-LDP-Hr-AE showed potent cytotoxicity to ESCC cells with differential expression of EGFR and HER2. Ec-LDP-Hr-AE could cause significant G2-M arrest in EC9706 and KYSE150 cells, and it also induced apoptosis in ESCC cells in a dosage-dependent manner. Western blot assays showed that Ec-LDP-Hr-AE promoted caspase-3 and caspase-7 activities as well as PARP cleavage. Moreover, Ec-LDP-Hr-AE inhibited cell proliferation via decreasing phosphorylation of EGFR and HER2, and further exerted inhibition of the activation of their downstream signaling molecules. In vivo, at a tolerated dose, Ec-LDP-Hr-AE inhibited tumor growth by 88% when it was administered to nude mice bearing human ESCC cell KYSE150 xenografts. These results indicated that Ec-LDP-Hr-AE exhibited potent anti-caner efficacy on ESCC, suggesting it could be a promising candidate for targeted therapy of esophageal cancer.

  10. Isolation and characterization of anti c-met single chain fragment variable (scFv) antibodies.

    Science.gov (United States)

    Qamsari, Elmira Safaie; Sharifzadeh, Zahra; Bagheri, Salman; Riazi-Rad, Farhad; Younesi, Vahid; Abolhassani, Mohsen; Ghaderi, Sepideh Safaei; Baradaran, Behzad; Somi, Mohammad Hossein; Yousefi, Mehdi

    2017-12-01

    The receptor tyrosine kinase (RTK) Met is the cell surface receptor for hepatocyte growth factor (HGF) involved in invasive growth programs during embryogenesis and tumorgenesis. There is compelling evidence suggesting important roles for c-Met in colorectal cancer proliferation, migration, invasion, angiogenesis, and survival. Hence, a molecular inhibitor of an extracellular domain of c-Met receptor that blocks c-Met-cell surface interactions could be of great thera-peutic importance. In an attempt to develop molecular inhibitors of c-Met, single chain variable fragment (scFv) phage display libraries Tomlinson I + J against a specific synthetic oligopeptide from the extracellular domain of c-Met receptor were screened; selected scFv were then characterized using various immune techniques. Three c-Met specific scFv (ES1, ES2, and ES3) were selected following five rounds of panning procedures. The scFv showed specific binding to c-Met receptor, and significantly inhibited proliferation responses of a human colorectal carcinoma cell line (HCT-116). Moreover, anti- apoptotic effects of selected scFv antibodies on the HCT-116 cell line were also evaluated using Annexin V/PI assays. The results demonstrated rates of apoptotic cell death of 46.0, 25.5, and 37.8% among these cells were induced by use of ES1, ES2, and ES3, respectively. The results demonstrated ability to successfully isolate/char-acterize specific c-Met scFv that could ultimately have a great therapeutic potential in immuno-therapies against (colorectal) cancers.

  11. Significant mucosal sIgA production after a single oral or parenteral administration using in vivo CD40 targeting in the chicken.

    Science.gov (United States)

    Chou, Wen-Ko; Chen, Chang-Hsin; Vuong, Christine N; Abi-Ghanem, Daad; Waghela, Suryakant D; Mwangi, Waithaka; Bielke, Lisa R; Hargis, Billy M; Berghman, Luc R

    2016-10-01

    Many pathogens enter the host through mucosal surfaces and spread rapidly via the circulation. The most effective way to prevent disease is to establish mucosal and systemic immunity against the pathogen. However, current vaccination programs in poultry industry require repeated administrations of live-attenuated virus or large amounts (10 to 100μg) of antigen together with adjuvant to induce specific secretory IgA immune responses at the mucosal effector sites. In the present study, we show that a single administration of 0.4μg of oligopeptide complexed with an agonistic anti-chicken CD40 (chCD40) monoclonal antibody (Mab) effectively targets antigen-presenting cells of the bird's mucosa-associated lymphoid tissue in vivo, and induces peptide-specific secretory IgA (sIgA) in the trachea 7days post administration. Anti-chCD40 Mab-peptide complex was administered once to four-week old male Leghorns via various mucosal routes (orally, via cloacal drinking, or oculo-nasally) or via subcutaneous (s.c.) immunization. Immunization through any of the three mucosal induction routes induced significant peptide-specific mucosal sIgA responses 7 and 14days after immunization. Interestingly, s.c. injection of the complex also induced mucosal sIgA. Our data suggest in vivo targeting of CD40 as a potential adjuvant platform, particularly for the purpose of enhancing and speeding up mucosal vaccine responses in chickens, and potentially other food animals. This is the first study able to elicit specific sIgA immune responses in remote mucosal sites with a single administration of only 0.4μg of antigen.

  12. Two group A streptococcal peptide pheromones act through opposing Rgg regulators to control biofilm development.

    Science.gov (United States)

    Chang, Jennifer C; LaSarre, Breah; Jimenez, Juan C; Aggarwal, Chaitanya; Federle, Michael J

    2011-08-01

    Streptococcus pyogenes (Group A Streptococcus, GAS) is an important human commensal that occasionally causes localized infections and less frequently causes severe invasive disease with high mortality rates. How GAS regulates expression of factors used to colonize the host and avoid immune responses remains poorly understood. Intercellular communication is an important means by which bacteria coordinate gene expression to defend against host assaults and competing bacteria, yet no conserved cell-to-cell signaling system has been elucidated in GAS. Encoded within the GAS genome are four rgg-like genes, two of which (rgg2 and rgg3) have no previously described function. We tested the hypothesis that rgg2 or rgg3 rely on extracellular peptides to control target-gene regulation. We found that Rgg2 and Rgg3 together tightly regulate two linked genes encoding new peptide pheromones. Rgg2 activates transcription of and is required for full induction of the pheromone genes, while Rgg3 plays an antagonistic role and represses pheromone expression. The active pheromone signals, termed SHP2 and SHP3, are short and hydrophobic (DI[I/L]IIVGG), and, though highly similar in sequence, their ability to disrupt Rgg3-DNA complexes were observed to be different, indicating that specificity and differential activation of promoters are characteristics of the Rgg2/3 regulatory circuit. SHP-pheromone signaling requires an intact oligopeptide permease (opp) and a metalloprotease (eep), supporting the model that pro-peptides are secreted, processed to the mature form, and subsequently imported to the cytoplasm to interact directly with the Rgg receptors. At least one consequence of pheromone stimulation of the Rgg2/3 pathway is increased biogenesis of biofilms, which counteracts negative regulation of biofilms by RopB (Rgg1). These data provide the first demonstration that Rgg-dependent quorum sensing functions in GAS and substantiate the role that Rggs play as peptide receptors across the

  13. Electrophoretic Analysis on the Enzyme Hydrolysis of Chickpea Protein%鹰嘴豆分离蛋白酶解过程的电泳分析

    Institute of Scientific and Technical Information of China (English)

    2013-01-01

      对国产碱性蛋白酶降解鹰嘴豆分离蛋白的酶解过程及酶解产物的水解度和电泳结果进行了分析研究.结果表明,国产蛋白酶可有效降解鹰嘴豆分离蛋白为小分子蛋白肽;使用碱性蛋白酶、木瓜蛋白酶、菠萝蛋白酶顺序酶解鹰嘴豆分离蛋白3 h 时的水解度可达到35.42%以上,此时绝大多数鹰嘴豆分离蛋白被降解为小分子肽.实验结果为鹰嘴豆蛋白的开发利用与鹰嘴豆的精深加工提供了科学依据.%In this paper,we determined the degree of hydrolysis(DH) of the chickpea protein with three proteolytic enzymes of alcalase,papain and bromelain(domestic)at different times,and analysed the electrophoretic pattern of the hydrolytic products. The result shows that after treated with the proteases for three hours,the chickpea proteins were mostly hydrolysed into oligopeptides,and the DH value(35.42%)of chickpea protein hydrolyzing by the three proteases above-mentioned under each optimum conditions respectively in order was much higher than that of the DH of chickpea protein hydrolyzing by the three proteases simultaneously.

  14. Characterisation of the mgo operon in Pseudomonas syringae pv. syringae UMAF0158 that is required for mangotoxin production

    Directory of Open Access Journals (Sweden)

    Arrebola Eva

    2012-01-01

    Full Text Available Abstract Background Mangotoxin is an antimetabolite toxin that is produced by strains of Pseudomonas syringae pv. syringae; mangotoxin-producing strains are primarily isolated from mango tissues with symptoms of bacterial apical necrosis. The toxin is an oligopeptide that inhibits ornithine N-acetyl transferase (OAT, a key enzyme in the biosynthetic pathway of the essential amino acids ornithine and arginine. The involvement of a putative nonribosomal peptide synthetase gene (mgoA in mangotoxin production and virulence has been reported. Results In the present study, we performed a RT-PCR analysis, insertional inactivation mutagenesis, a promoter expression analysis and terminator localisation to study the gene cluster containing the mgoA gene. Additionally, we evaluated the importance of mgoC, mgoA and mgoD in mangotoxin production. A sequence analysis revealed an operon-like organisation. A promoter sequence was located upstream of the mgoB gene and was found to drive lacZ transcription. Two terminators were located downstream of the mgoD gene. RT-PCR experiments indicated that the four genes (mgoBCAD constitute a transcriptional unit. This operon is similar in genetic organisation to those in the three other P. syringae pathovars for which complete genomes are available (P. syringae pv. syringae B728a, P. syringae pv. tomato DC3000 and P. syringae pv. phaseolicola 1448A. Interestingly, none of these three reference strains is capable of producing mangotoxin. Additionally, extract complementation resulted in a recovery of mangotoxin production when the defective mutant was complemented with wild-type extracts. Conclusions The results of this study confirm that mgoB, mgoC, mgoA and mgoD function as a transcriptional unit and operon. While this operon is composed of four genes, only the last three are directly involved in mangotoxin production.

  15. Small RNA Transcriptome of the Oral Microbiome during Periodontitis Progression.

    Science.gov (United States)

    Duran-Pinedo, Ana E; Yost, Susan; Frias-Lopez, Jorge

    2015-10-01

    The oral microbiome is one of the most complex microbial communities in the human body, and due to circumstances not completely understood, the healthy microbial community becomes dysbiotic, giving rise to periodontitis, a polymicrobial inflammatory disease. We previously reported the results of community-wide gene expression changes in the oral microbiome during periodontitis progression and identified signatures associated with increasing severity of the disease. Small noncoding RNAs (sRNAs) are key players in posttranscriptional regulation, especially in fast-changing environments such as the oral cavity. Here, we expanded our analysis to the study of the sRNA metatranscriptome during periodontitis progression on the same samples for which mRNA expression changes were analyzed. We observed differential expression of 12,097 sRNAs, identifying a total of 20 Rfam sRNA families as being overrepresented in progression and 23 at baseline. Gene ontology activities regulated by the differentially expressed (DE) sRNAs included amino acid metabolism, ethanolamine catabolism, signal recognition particle-dependent cotranslational protein targeting to membrane, intron splicing, carbohydrate metabolism, control of plasmid copy number, and response to stress. In integrating patterns of expression of protein coding transcripts and sRNAs, we found that functional activities of genes that correlated positively with profiles of expression of DE sRNAs were involved in pathogenesis, proteolysis, ferrous iron transport, and oligopeptide transport. These findings represent the first integrated sequencing analysis of the community-wide sRNA transcriptome of the oral microbiome during periodontitis progression and show that sRNAs are key regulatory elements of the dysbiotic process leading to disease.

  16. MHC allele frequency distributions under parasite-driven selection: A simulation model

    Directory of Open Access Journals (Sweden)

    Radwan Jacek

    2010-10-01

    Full Text Available Abstract Background The extreme polymorphism that is observed in major histocompatibility complex (MHC genes, which code for proteins involved in recognition of non-self oligopeptides, is thought to result from a pressure exerted by parasites because parasite antigens are more likely to be recognized by MHC heterozygotes (heterozygote advantage and/or by rare MHC alleles (negative frequency-dependent selection. The Ewens-Watterson test (EW is often used to detect selection acting on MHC genes over the recent history of a population. EW is based on the expectation that allele frequencies under balancing selection should be more even than under neutrality. We used computer simulations to investigate whether this expectation holds for selection exerted by parasites on host MHC genes under conditions of heterozygote advantage and negative frequency-dependent selection acting either simultaneously or separately. Results In agreement with simple models of symmetrical overdominance, we found that heterozygote advantage acting alone in populations does, indeed, result in more even allele frequency distributions than expected under neutrality, and this is easily detectable by EW. However, under negative frequency-dependent selection, or under the joint action of negative frequency-dependent selection and heterozygote advantage, distributions of allele frequencies were less predictable: the majority of distributions were indistinguishable from neutral expectations, while the remaining runs resulted in either more even or more skewed distributions than under neutrality. Conclusions Our results indicate that, as long as negative frequency-dependent selection is an important force maintaining MHC variation, the EW test has limited utility in detecting selection acting on these genes.

  17. Characterization of poly-gamma-glutamate hydrolase encoded by a bacteriophage genome: possible role in phage infection of Bacillus subtilis encapsulated with poly-gamma-glutamate.

    Science.gov (United States)

    Kimura, Keitarou; Itoh, Yoshifumi

    2003-05-01

    Some Bacillus subtilis strains, including natto (fermented soybeans) starter strains, produce a capsular polypeptide of glutamate with a gamma-linkage, called poly-gamma-glutamate (gamma-PGA). We identified and purified a monomeric 25-kDa degradation enzyme for gamma-PGA (designated gamma-PGA hydrolase, PghP) from bacteriophage PhiNIT1 in B. subtilis host cells. The monomeric PghP internally hydrolyzed gamma-PGA to oligopeptides, which were then specifically converted to tri-, tetra-, and penta-gamma-glutamates. Monoiodoacetate and EDTA both inhibited the PghP activity, but Zn(2+) or Mn(2+) ions fully restored the enzyme activity inhibited by the chelator, suggesting that a cysteine residue(s) and these metal ions participate in the catalytic mechanism of the enzyme. The corresponding pghP gene was cloned and sequenced from the phage genome. The deduced PghP sequence (208 amino acids) with a calculated M(r) of 22,939 was not significantly similar to any known enzyme. Thus, PghP is a novel gamma-glutamyl hydrolase. Whereas phage PhiNIT1 proliferated in B. subtilis cells encapsulated with gamma-PGA, phage BS5 lacking PghP did not survive well on such cells. Moreover, all nine phages that contaminated natto during fermentation produced PghP, supporting the notion that PghP is important in the infection of natto starters that produce gamma-PGA. Analogous to polysaccharide capsules, gamma-PGA appears to serve as a physical barrier to phage absorption. Phages break down the gamma-PGA barrier via PghP so that phage progenies can easily establish infection in encapsulated cells.

  18. Role in proinflammatory response of YghJ, a secreted metalloprotease from neonatal septicemic Escherichia coli.

    Science.gov (United States)

    Tapader, Rima; Bose, Dipro; Basu, Pallabi; Mondal, Moumita; Mondal, Ayan; Chatterjee, Nabendu Sekhar; Dutta, Pujarini; Basu, Sulagna; Bhadra, Rupak K; Pal, Amit

    2016-11-01

    Neonatal sepsis is the invasion of microbial pathogens into blood stream and is associated with a systemic inflammatory response with production and release of a wide range of inflammatory mediators. The increased serum levels of cytokines were found to correlate with the severity and mortality in course of sepsis. There have been no reports on the role of microbial proteases in stimulation of proinflammatory response in neonatal sepsis. We have identified YghJ, a secreted metalloprotease from a neonatal septicemic Escherichia coli (NSEC) isolate. The protease was partially purified from culture supernatant by successive anion and gel filtration chromatography. MS/MS peptide sequencing of the protease showed homology with YghJ. YghJ was cloned, expressed and purified in pBAD TOPO expression vector. YghJ was found to be proteolytically active against Methoxysuccinyl Ala-Ala-Pro-Met-p-nitroanilide oligopeptide substrate, but not against casein and gelatin. YghJ showed optimal activity at pH 7-8 and at temperatures 37-40°C. YghJ showed clear changes in cellular morphologies of Int407, HT-29 and HEK293 cells. YghJ stimulated the secretion of cytokines IL-1α, IL-1β and TNF-α in murine macrophages (RAW 264.7) and IL-8 from human intestinal epithelial cells (HT-29). YghJ also down-regulated the production of anti-inflammatory cytokines such as IL-10. YghJ is present in both septicemic (78%) and fecal E. coli isolates (54%). However, expression and secretion of YghJ is significantly higher among the septicemic (89%) than the fecal isolates (33%). This is the first study to show the role of a microbial protease, YghJ in triggering proinflammatory response in NSEC.

  19. 血散薯的化学成分%Chemical Constituents from Stephania dielsiana

    Institute of Scientific and Technical Information of China (English)

    张毅; 张盛; 章海燕; 沈征武; 秦国伟

    2009-01-01

    目的:研究血散薯根的化学成分.方法:应用各种柱层析方法分离和纯化化合物.通过光谱方法及理化性质鉴定化合物结构.结果:从血散薯根中分离得到18个化合物.分别鉴定为sinoacufine(1),stephanine(2),ayuthianine(3),debyarosteplanine(4),cephamorphinanine(5),aknadinine(6),liriodenine(7),sinomenine(8),1-tetrahydropalmatine(9),(-)corydalmine(10).oxocrebanine(11),norcaneliltine(12),gomisin A(13),gomisin B(14),schisandrin C(15),6-O-benzoylgomisin(16).asterininB(17)和β-谷甾醇(18).结论:14个化合物3,5-8和10-18系首次从该植物中分得.%AIM: To study the chemical constituents from the roots of Stephania dielsiana. METHODS: Various column chromatographic techniques were used for isolation and purification. The structures were elucidated on the basis of spectral data and chemical evidences. RESULTS: Eighteen compounds were isolated and elucidated as twelve alkaloids: sinoacutine (1), stephanine (2), ayuthianine (3), dehydrostephanine (4), cephamorphinanine (5), aknadinine (6), liriodenine (7), sinomenine (8), 1-tetrahydropalmatine (9), (-)corydalmine (10), oxocrebanine (11), nor-canelilline (12), four lignans: gomisin A (13), gomisin B (14), schisandrin C (15), 6-O-benzoylgomisin (16), one oli-gopeptide asterinin B (17), and one sterol β-Sitosterol (18). CONCLUSION: Fourteen compounds 3, 5-8 and 10-18 were isolated from the plant for the first time.

  20. Pyrene-containing peptide-based fluorescent organogels: inclusion of graphene into the organogel.

    Science.gov (United States)

    Adhikari, Bimalendu; Nanda, Jayanta; Banerjee, Arindam

    2011-10-04

    The N-terminally pyrene-conjugated oligopeptide, Py-Phe-Phe-Ala-OMe, (Py=pyrene 1-butyryl acyl) forms transparent, stable, supramolecular fluorescent organogels in various organic solvents. One of these organogels was thoroughly studied using various techniques including transmission electron microscopy (TEM), field-emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), Fourier-transform infrared (FTIR) spectroscopy, photoluminescence (PL) spectroscopy, and rheology. Unfunctionalized and non-oxidized graphene was successfully incorporated into this fluorescent organogel in o-dichlorobenzene (ODCB) to form a stable hybrid organogel. Graphene is well dispersed into the gel medium by using non-covalent π-π stacking interactions with the pyrene-conjugated gelator peptide. In the presence of graphene, the minimum gelation concentration (mgc) of the hybrid organogel was lowered significantly. This suggests that there is a favorable interaction between the graphene and the gelator peptide within the hybrid organogel system. This hybrid organogel was characterized using TEM, AFM, FTIR, PL, and rheological studies. The TEM study of graphene-containing hybrid organogel revealed the presence of both graphene sheets and entangled gel nanofibers. The AFM study indicated the presence of 3 to 4 layers in exfoliated graphene in ODCB and the presence of both graphene nanosheets and the network of gel nanofibers in the hybrid gel system. The rheological investigation suggested that the flow of the hybrid organogel had become more resistant towards the applied angular frequency upon the incorporation of graphene into the organogel. The hybrid gel is about seven times more rigid than that of the native gel.

  1. Developmental expression, intracellular localization, and selenium content of the cysteine-rich protein associated with the mitochondrial capsules of mouse sperm.

    Science.gov (United States)

    Cataldo, L; Baig, K; Oko, R; Mastrangelo, M A; Kleene, K C

    1996-11-01

    The outer membranes of mitochondria of mammalian sperm are encased in a keratinous structure known as the mitochondrial capsule. The experiments in the present study were designed to resolve a controversy surrounding the intracellular localization, developmental expression, and selenium-content of a cysteine-rich 17-20 kD protein that has been reported to constitute the major structural protein in the mitochondrial capsule of mammals. An antibody to a synthetic oligopeptide based on the predicted sequence of mouse cysteinerich protein recognizes a 24 kD protein in epididymal sperm tails of mice. The 24 kD protein does not appear to be a selenoprotein because: (1) it is not labeled with 75Se-selenite in seminiferous tubule culture; (2) cleavage with cyanogen bromide and translation of T7 RNA polymerase transcripts in vitro indicate that the translation start site is located downstream of potential UGA selenocysteine codons in the mouse cysteine-rich mRNA; (3) the reading frame encoding the cysteine-rich protein in rat lacks inphase UGA selenocysteine codons. Light and electron microscopy immunocytochemistry detects the cysteine-rich protein first during step 11 of spermiogenesis in the mouse demonstrating that the cysteine-rich protein mRNA is under temporal translational control. Electron microscope immunocytochemistry reveals that the cysteine-rich protein is evenly distributed in the cytoplasm in spermatids in steps 11 through early step 16 in mouse, and that it is associated with the outer mitochondrial membranes of spermatids in late step 16 and epididymal spermatozoa.

  2. Clinical relevance of intestinal peptide uptake

    Institute of Scientific and Technical Information of China (English)

    Hugh; James; Freeman

    2015-01-01

    AIM: To determine available information on an independent peptide transporter 1(Pep T1) and its potential relevance to treatment, this evaluation was completed.METHODS: Fully published English language literature articles sourced through Pub Med related to protein digestion and absorption, specifically human peptide and amino acid transport, were accessed and reviewed.Papers from 1970 to the present, with particular emphasis on the past decade, were examined. In addition,abstracted information translated to English in Pub Med was also included. Finally, studies and reviews relevant to nutrient or drug uptake, particularly in human intestine were included for evaluation. This work represents a summary of all of these studies with particular reference to peptide transporter mediated assimilation of nutrients and pharmacologically active medications.RESULTS: Assimilation of dietary protein in humans involves gastric and pancreatic enzyme hydrolysis to luminal oligopeptides and free amino acids. During the ensuing intestinal phase, these hydrolytic products are transported into the epithelial cell and, eventually, the portal vein. A critical component of this process is the uptake of intact di-peptides and tri-peptides by an independent Pep T1. A number of "peptide-mimetic" pharmaceutical agents may also be transported through this carrier, important for uptake of different antibiotics, antiviral agents and angiotensin-converting enzyme inhibitors. In addition, specific peptide products of intestinal bacteria may also be transported by Pep T1, with initiation and persistence of an immune response including increased cytokine production and associated intestinal inflammatory changes. Interestingly, these inflammatory changes may also be attenuated with orallyadministered anti-inflammatory tripeptides administered as site-specific nanoparticles and taken up by this Pep T1 transport protein. CONCLUSION: Further evaluation of the role of this transporter in treatment of

  3. Colonic miRNA expression/secretion, regulated by intestinal epithelial PepT1, plays an important role in cell-to-cell communication during colitis.

    Directory of Open Access Journals (Sweden)

    Saravanan Ayyadurai

    Full Text Available PepT1 is a member of the proton-oligopeptide cotransporter family SLC15, which mediates the transport of di/tripeptides from intestinal lumen into epithelial cells. MicroRNAs (miRNAs, a small noncoding RNAs (21-23 nucleotides, post-transcriptionally regulate gene expression by binding to the 3'-untranslated regions (UTRs of their target mRNAs. Although the role of most miRNAs remains elusive, they have been implicated in vital cellular functions such as intestinal epithelial cells differentiation, proliferation, and apoptosis. In the present study, we investigated the effect of intestinal epithelial PepT1 expression on microRNA (miRNA expression/secretion in the colons of control mice and in mice with experimentally induced colonic inflammation (colitis. The colonic miRNA expression was deregulated in both colitis and control mice but the deregulation of miRNA expression/secretion was specific to colonic tissue and did not affect other tissues such as spleen and liver. Intestinal epithelial PepT1-dependent deregulation of colonic miRNA expression not only affects epithelial cells but also other cell types, such as intestinal macrophages. Importantly, we found the miRNA 23b which was known to be involved in inflammatory bowel disease was secreted and transported between cells to impose a gene-silencing effect on recipient intestinal macrophages. Based on our data, we may conclude that the expression of a specific protein, PepT1, in the intestine affects local miRNA expression/secretion in the colon on a tissue specific manner and may play an important role during the induction and progression of colitis. Colonic miRNA expression/secretion, regulated by intestinal epithelial PepT1, could play a crucial role in cell-to-cell communication during colitis.

  4. Electrochemical immobilization of Fluorescent labelled probe molecules on a FTO surface for affinity detection based on photo-excited current

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    Haruyama, Tetsuya; Wakabayashi, Ryo; Cho, Takeshi; Matsuyama, Sho-taro, E-mail: haruyama@life.kyutech.as.jp [Kyushu Institute of Technology, Department of Biological Functions and Engineering, Kitakyushu Science and Research Park, Hibikino, Kitakyushu, Fukuoka 808-0196 (Japan)

    2011-10-29

    Photo-excited current can be generated at a molecular interface between a photo-excited molecules and a semi-conductive material in appropriate condition. The system has been recognized for promoting photo-energy devices such as an organic dye sensitized solar-cell. The photo-current generated reactions are totally dependent on the interfacial energy reactions, which are in a highly fluctuated interfacial environment. The authors investigated the photo-excited current reaction to develop a smart affinity detection method. However, in order to perform both an affinity reaction and a photo-excited current reaction at a molecular interface, ordered fabrications of the functional (affinity, photo-excitation, etc.) molecules layer on a semi-conductive surface is required. In the present research, we would like to present the fabrication and functional performance of photo-excited current-based affinity assay device and its application for detection of endocrine disrupting chemicals. On the FTO surface, fluorescent pigment labelled affinity peptide was immobilized through the EC tag (electrochemical-tag) method. The modified FTO produced a current when it was irradiated with diode laser light. However, the photo current decreased drastically when estrogen (ES) coexisted in the reaction solution. In this case, immobilized affinity probe molecules formed a complex with ES and estrogen receptor (ER). The result strongly suggests that the photo-excited current transduction between probe molecule-labelled cyanine pigment and the FTO surface was partly inhibited by a complex that formed at the affinity oligo-peptide region in a probe molecule on the FTO electrode. The bound bulky complex may act as an impediment to perform smooth transduction of photo-excited current in the molecular interface. The present system is new type of photo-reaction-based analysis. This system can be used to perform simple high-sensitive homogeneous assays.

  5. Mechanisms of pH-gradient driven transport mediated by organic anion polypeptide transporters.

    Science.gov (United States)

    Leuthold, Simone; Hagenbuch, Bruno; Mohebbi, Nilufar; Wagner, Carsten A; Meier, Peter J; Stieger, Bruno

    2009-03-01

    Organic anion transporting polypeptides (humans OATPs, rodents Oatps) are expressed in most mammalian tissues and mediate cellular uptake of a wide variety of amphipathic organic compounds such as bile salts, steroid conjugates, oligopeptides, and a large list of drugs, probably by acting as anion exchangers. In the present study we aimed to investigate the role of the extracellular pH on the transport activity of nine human and four rat OATPs/Oatps. Furthermore, we aimed to test the concept that OATP/Oatp transport activity is accompanied by extrusion of bicarbonate. By using amphibian Xenopus laevis oocytes expressing OATPs/Oatps and mammalian cell lines stably transfected with OATPs/Oatps, we could demonstrate that in all OATPs/Oatps investigated, with the exception of OATP1C1, a low extracellular pH stimulated transport activity. This stimulation was accompanied by an increased substrate affinity as evidenced by lower apparent Michaelis-Menten constant values. OATP1C1 is lacking a highly conserved histidine in the third transmembrane domain, which was shown by site-directed mutagenesis to be critically involved in the pH dependency of OATPs/Oatps. Using online intracellular pH measurements in OATP/Oatp-transfected Chinese Hamster Ovary (CHO)-K1 cells, we could demonstrate the presence of a 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid-sensitive chloride/bicarbonate exchanger in CHO-K1 cells and that OATP/Oatp-mediated substrate transport is paralleled by bicarbonate efflux. We conclude that the pH dependency of OATPs/Oatps may lead to a stimulation of substrate transport in an acidic microenvironment and that the OATP/Oatp-mediated substrate transport into cells is generally compensated or accompanied by bicarbonate efflux.

  6. Large molecule protein feeding during the suckling period is required for the development of pancreatic digestive functions in rats.

    Science.gov (United States)

    Kinouchi, Toshi; Koyama, Satomi; Harada, Etsumori; Yajima, Takaji

    2012-12-15

    We examined if large molecule protein feeding during the suckling period is prerequisite for the proper development of pancreatic digestive functions. Most amino acids in breast milk exist as the constituent of large proteins and not as oligopeptides or free amino acids. Accumulating evidence indicates the nutritional importance of large protein feeding for suckling infants; however, evidence on the physiological significance remains small. We thus artificially reared rat pups on a standard rat formula with milk protein or a formula with milk protein hydrolysate from 7 to 21 days of age, and thereafter, fed a standard solid diet until 42 days of age. Pancreas weight and the stock of pancreatic digestive enzymes in the hydrolysate-fed rats were significantly lower than those in the protein-fed rats during and also after the suckling period. Plasma insulin, a stimulator of amylase synthesis, was also significantly low in the hydrolysate-fed rats compared with the protein-fed rats. At 28 days of age, we evaluated the pancreatic secretory ability in response to dietary protein and cholecystokinin (CCK) by means of pancreatic duct cannulation. Pancreatic secretion stimulated by dietary protein in the hydrolysate-fed rats was significantly weaker than that in the protein-fed rats. No significant difference was observed in the increasing rate of pancreatic enzyme secretion in response to CCK between the two groups. These results suggest that the presence of large proteins in breast milk is significant for the development of pancreatic digestive functions and the outcomes could remain even later on in life.

  7. A novel dipeptidyl aminopeptidase in rat brain membranes. Its isolation, purification, and characterization.

    Science.gov (United States)

    Hui, K S

    1988-05-15

    A new type of dipeptidyl aminopeptidase, which releases basic aminoacyl dipeptides from the NH2-terminal end of oligopeptides, was purified about 2100-fold with 6.8% recovery from rat brain membranes by column chromatography on Cellex D, Arg-Tyr-AH-Sepharose 4B, hydroxylapatite, and Sephadex G-75, after the membranes were solubilized with Nonidet P-40. Activity was assayed by high performance liquid chromatography (HPLC) using Arg0-Met5-enkephalin (Arg0-enk)* as substrate in the presence of bestatin, thiorphan, and captopril. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme is apparently homogeneous with a mass of 64,000 daltons. This thiol enzyme is optimally active at pH 7 and is selectively activated by Mn(II). It loses 94% of its activity after EDTA treatment and can be reactivated by Mn(II), Co(II), and Zn(II). It splits Arg0-enk into equimolar amounts of Arg-Tyr and Gly-Gly-Phe-Met with a Km of 100 microM, and Vmax of 3.8 mumol/mg of protein/min. Dipeptidyl aminopeptidase does not hydrolyze model substrates for dipeptidyl aminopeptidases I, II, III, and IV, aminoacyl beta-naphthylamides, actin, desmin, tubulin, glial fibrillary acidic protein, and cytoskeletal neurofilament proteins. The enzyme is insensitive to puromycin, but is inhibited by several neuropeptides. Angiotensin III is the most potent with a Ki of 0.3 microM. Substrate specificity, pH optimum, molecular weight, activators, and catalytic site demonstrate that this enzyme is distinct from dipeptidyl aminopeptidases previously described.

  8. Peptide Bond Synthesis by a Mechanism Involving an Enzymatic Reaction and a Subsequent Chemical Reaction.

    Science.gov (United States)

    Abe, Tomoko; Hashimoto, Yoshiteru; Zhuang, Ye; Ge, Yin; Kumano, Takuto; Kobayashi, Michihiko

    2016-01-22

    We recently reported that an amide bond is unexpectedly formed by an acyl-CoA synthetase (which catalyzes the formation of a carbon-sulfur bond) when a suitable acid and l-cysteine are used as substrates. DltA, which is homologous to the adenylation domain of nonribosomal peptide synthetase, belongs to the same superfamily of adenylate-forming enzymes, which includes many kinds of enzymes, including the acyl-CoA synthetases. Here, we demonstrate that DltA synthesizes not only N-(d-alanyl)-l-cysteine (a dipeptide) but also various oligopeptides. We propose that this enzyme catalyzes peptide synthesis by the following unprecedented mechanism: (i) the formation of S-acyl-l-cysteine as an intermediate via its "enzymatic activity" and (ii) subsequent "chemical" S → N acyl transfer in the intermediate, resulting in peptide formation. Step ii is identical to the corresponding reaction in native chemical ligation, a method of chemical peptide synthesis, whereas step i is not. To the best of our knowledge, our discovery of this peptide synthesis mechanism involving an enzymatic reaction and a subsequent chemical reaction is the first such one to be reported. This new process yields peptides without the use of a thioesterified fragment, which is required in native chemical ligation. Together with these findings, the same mechanism-dependent formation of N-acyl compounds by other members of the above-mentioned superfamily demonstrated that all members most likely form peptide/amide compounds by using this novel mechanism. Each member enzyme acts on a specific substrate; thus, not only the corresponding peptides but also new types of amide compounds can be formed.

  9. The Arabidopsis AtOPT3 Protein Functions in Metal Homeostasis and Movement of Iron to Developing Seeds1[W][OA

    Science.gov (United States)

    Stacey, Minviluz G.; Patel, Ami; McClain, William E.; Mathieu, Melanie; Remley, Melissa; Rogers, Elizabeth E.; Gassmann, Walter; Blevins, Dale G.; Stacey, Gary

    2008-01-01

    The Arabidopsis thaliana AtOPT3 belongs to the oligopeptide transporter (OPT) family, a relatively poorly characterized family of peptide/modified peptide transporters found in archebacteria, bacteria, fungi, and plants. A null mutation in AtOPT3 resulted in embryo lethality, indicating an essential role for AtOPT3 in embryo development. In this article, we report on the isolation and phenotypic characterization of a second AtOPT3 mutant line, opt3-2, harboring a T-DNA insertion in the 5′ untranslated region of AtOPT3. The T-DNA insertion in the AtOPT3 promoter resulted in reduced but sufficient AtOPT3 expression to allow embryo formation in opt3-2 homozygous seeds. Phenotypic analyses of opt3-2 plants revealed three interesting loss-of-function phenotypes associated with iron metabolism. First, reduced AtOPT3 expression in opt3-2 plants resulted in the constitutive expression of root iron deficiency responses regardless of exogenous iron supply. Second, deregulation of root iron uptake processes in opt3-2 roots resulted in the accumulation of very high levels of iron in opt3-2 tissues. Hyperaccumulation of iron in opt3-2 resulted in the formation of brown necrotic areas in opt3-2 leaves and was more pronounced during the seed-filling stage. Third, reduced AtOPT3 expression resulted in decreased accumulation of iron in opt3-2 seeds. The reduced accumulation of iron in opt3-2 seeds is especially noteworthy considering the excessively high levels of accumulated iron in other opt3-2 tissues. AtOPT3, therefore, plays a critical role in two important aspects of iron metabolism, namely, maintenance of whole-plant iron homeostasis and iron nutrition of developing seeds. PMID:18083798

  10. Characterization of the phytochelatin synthase of Schistosoma mansoni.

    Directory of Open Access Journals (Sweden)

    Debalina Ray

    2011-05-01

    Full Text Available Treatment for schistosomiasis, which is responsible for more than 280,000 deaths annually, depends exclusively on the use of praziquantel. Millions of people are treated annually with praziquantel and drug resistant parasites are likely to evolve. In order to identify novel drug targets the Schistosoma mansoni sequence databases were queried for proteins involved in glutathione metabolism. One potential target identified was phytochelatin synthase (PCS. Phytochelatins are oligopeptides synthesized enzymatically from glutathione by PCS that sequester toxic heavy metals in many organisms. However, humans do not have a PCS gene and do not synthesize phytochelatins. In this study we have characterized the PCS of S. mansoni (SmPCS. The conserved catalytic triad of cysteine-histidine-aspartate found in PCS proteins and cysteine proteases is also found in SmPCS, as are several cysteine residues thought to be involved in heavy metal binding and enzyme activation. The SmPCS open reading frame is considerably extended at both the N- and C-termini compared to PCS from other organisms. Multiple PCS transcripts are produced from the single encoded gene by alternative splicing, resulting in both mitochondrial and cytoplasmic protein variants. Expression of SmPCS in yeast increased cadmium tolerance from less than 50 µM to more than 1,000 µM. We confirmed the function of SmPCS by identifying PCs in yeast cell extracts using HPLC-mass spectrometry. SmPCS was found to be expressed in all mammalian stages of worm development investigated. Increases in SmPCS expression were seen in ex vivo worms cultured in the presence of iron, copper, cadmium, or zinc. Collectively, these results indicate that SmPCS plays an important role in schistosome response to heavy metals and that PCS is a potential drug target for schistosomiasis treatment. This is the first characterization of a PCS from a parasitic organism.

  11. Extracellular life cycle of ComS, the competence-stimulating peptide of Streptococcus thermophilus.

    Science.gov (United States)

    Gardan, Rozenn; Besset, Colette; Gitton, Christophe; Guillot, Alain; Fontaine, Laetitia; Hols, Pascal; Monnet, Véronique

    2013-04-01

    In streptococci, ComX is the alternative sigma factor controlling the transcription of the genes encoding the genetic transformation machinery. In Streptococcus thermophilus, comX transcription is controlled by a complex consisting of a transcriptional regulator of the Rgg family, ComR, and a signaling peptide, ComS, which controls ComR activity. Following its initial production, ComS is processed, secreted, and imported back into the cell by the Ami oligopeptide transporter. We characterized these steps and the partners interacting with ComS during its extracellular circuit in more detail. We identified the mature form of ComS and demonstrated the involvement of the membrane protease Eep in ComS processing. We found that ComS was secreted but probably not released into the extracellular medium. Natural competence was first discovered in a chemically defined medium without peptides. We show here that the presence of a high concentration of nutritional peptides in the medium prevents the triggering of competence. In milk, the ecological niche of S. thermophilus, competence was found to be functional, suggesting that the concentration of nutritional peptides was too low to interfere with ComR activation. The kinetics of expression of the comS, comR, and comX genes and of a late competence gene, dprA, in cultures inoculated at different initial densities revealed that the activation mechanism of ComR by ComS is more a timing device than a quorum-sensing mechanism sensu stricto. We concluded that the ComS extracellular circuit facilitates tight control over the triggering of competence in S. thermophilus.

  12. Proteins as "dopable" bio-electronic materials

    Science.gov (United States)

    Cahen, David

    2013-02-01

    Proteins are surprisingly good solid-state electronic conductors. This holds also for proteins without any known biological electron transfer function. How do they do it? To answer this question we measure solid-state electron transport (ETp) across proteins that are "dry" (only tightly bound water, to retain the conformation, still present). We compare results for the electron transfer (ET) protein, Azurin (Az), the proton-pumping membrane protein Bacteriorhodopsin (bR), and for Human and Bovine Serum Albumin (HSA and BSA). Clear differences between these proteins are seen, which preserve their structure in the solid state measurement configuration. Importantly for future bioelectronics, the results are sensitive to protein modification, e.g., removing or disconnecting the retinal in bR and removing or replacing the Cu redox centre in Az. These cofactors can thus be viewed as natural dopants for proteins. Insight in the ETp mechanism comes from temperature-dependent studies. Az shows 40-360K temperature-independent ETp across its 3.5 nm long axis, until its denaturation temperature, indicative of tunneling. Cu removal, replacement (by Zn) or deuteration changes this to thermally activated ETp. This suggests hopping and involvement of the amide backbone in the ETp. The latter, which rhymes with indications from ETp experiments on oligopeptide and simulations of ET in proteins, opens the way for modeling what otherwise is an awfully complex system. Below 200K all proteins and their variants show temperature-independent ETp. We can furthermore make a totally electrically inactive protein, HSA, into an efficient ETp medium by doping it with natural poly-ene. Putting our data in perspective by comparing them to all known protein ETp data in the literature, we conclude that, in general, proteins are well described as dopable molecular wires.

  13. Ribosome-Templated Azide-Alkyne Cycloadditions: Synthesis of Potent Macrolide Antibiotics by In Situ Click Chemistry.

    Science.gov (United States)

    Glassford, Ian; Teijaro, Christiana N; Daher, Samer S; Weil, Amy; Small, Meagan C; Redhu, Shiv K; Colussi, Dennis J; Jacobson, Marlene A; Childers, Wayne E; Buttaro, Bettina; Nicholson, Allen W; MacKerell, Alexander D; Cooperman, Barry S; Andrade, Rodrigo B

    2016-03-09

    Over half of all antibiotics target the bacterial ribosome-nature's complex, 2.5 MDa nanomachine responsible for decoding mRNA and synthesizing proteins. Macrolide antibiotics, exemplified by erythromycin, bind the 50S subunit with nM affinity and inhibit protein synthesis by blocking the passage of nascent oligopeptides. Solithromycin (1), a third-generation semisynthetic macrolide discovered by combinatorial copper-catalyzed click chemistry, was synthesized in situ by incubating either E. coli 70S ribosomes or 50S subunits with macrolide-functionalized azide 2 and 3-ethynylaniline (3) precursors. The ribosome-templated in situ click method was expanded from a binary reaction (i.e., one azide and one alkyne) to a six-component reaction (i.e., azide 2 and five alkynes) and ultimately to a 16-component reaction (i.e., azide 2 and 15 alkynes). The extent of triazole formation correlated with ribosome affinity for the anti (1,4)-regioisomers as revealed by measured Kd values. Computational analysis using the site-identification by ligand competitive saturation (SILCS) approach indicated that the relative affinity of the ligands was associated with the alteration of macrolactone+desosamine-ribosome interactions caused by the different alkynes. Protein synthesis inhibition experiments confirmed the mechanism of action. Evaluation of the minimal inhibitory concentrations (MIC) quantified the potency of the in situ click products and demonstrated the efficacy of this method in the triaging and prioritization of potent antibiotics that target the bacterial ribosome. Cell viability assays in human fibroblasts confirmed 2 and four analogues with therapeutic indices for bactericidal activity over in vitro mammalian cytotoxicity as essentially identical to solithromycin (1).

  14. The Effect of Ferula assa-foetida L and Carum copticum Hydroalcoholic Extract on the Expression Levels of Staphylococcus aureus Genes Involved in Quorum Sensing

    Science.gov (United States)

    Jomehpour, Najmeh; Eslami, Gilda; Khalili, Mohammad Bagher

    2016-01-01

    Background Quorum sensing is a microbial cell-to-cell communication process. Quorum sensing bacteria produce and release extracellular messenger molecules called autoinducers. Gram-positive and Gram-negative, homoserine lactones, and oligopeptides are autoinducers used to communicate and regulate gene expression. Objectives The goal of this study was to assess the impact of subinhibitory concentrations of Ferula assa-foetida l oleo-gum resin and Carum copticum fruit on the expression of tst and hld genes of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains. Methods This analytical study was performed using standard strains of MRSA (ATCC 33591) and MSSA (ATCC 29213). Suspensions of MRSA and MSSA bacteria were incubated at 37°C for 7 and 16 hours in the presence of ethanol extracts from F. assa-foetida and C. copticum. The expression of the hld and tst genes was then assessed using the real-time PCR protocol and SYBR Green Master Mix. The data analysis was carried out using the 2-ΔΔCT method. Results The hld gene expression (RNAIII) of MRSA after 7 and 16 hours of exposure to the sMIC of the F. assa-foetida extract showed a fold change of -1 and 0.08, respectively, in comparison with controls. After 7 and 16 hours of exposure to the sMIC of the C. copticum extract, the fold change was -0.23 and -0.27, respectively. After exposure to the sMIC of the C. copticum extract for 16 hours, the fold change in the expression of the tst (TSST-1) MSSA gene was 0.37 lower than that of the control sample. Conclusions The results indicate that sMICs of ethanol extracts from F. assa-foetida and C. copticum can be used to control the expression of virulence genes in pathogenic bacteria, such as MRSA and MSSA. PMID:27942357

  15. A novel pheromone quorum-sensing system controls the development of natural competence in Streptococcus thermophilus and Streptococcus salivarius.

    Science.gov (United States)

    Fontaine, Laetitia; Boutry, Céline; de Frahan, Marie Henry; Delplace, Brigitte; Fremaux, Christophe; Horvath, Philippe; Boyaval, Patrick; Hols, Pascal

    2010-03-01

    In streptococcal species, the key step of competence development is the transcriptional induction of comX, which encodes the alternative sigma factor sigma(X), which positively regulates genes necessary for DNA transformation. In Streptococcus species belonging to the mitis and mutans groups, induction of comX relies on the activation of a three-component system consisting of a secreted pheromone, a histidine kinase, and a response regulator. In Streptococcus thermophilus, a species belonging to the salivarius group, the oligopeptide transporter Ami is essential for comX expression under competence-inducing conditions. This suggests a different regulation pathway of competence based on the production and reimportation of a signal peptide. The objective of our work was to identify the main actors involved in the early steps of comX induction in S. thermophilus LMD-9. Using a transcriptomic approach, four highly induced early competence operons were identified. Among them, we found a Rgg-like regulator (Ster_0316) associated with a nonannotated gene encoding a 24-amino-acid hydrophobic peptide (Shp0316). Through genetic deletions, we showed that these two genes are essential for comX induction. Moreover, addition to the medium of synthetic peptides derived from the C-terminal part of Shp0316 restored comX induction and transformation of a Shp0316-deficient strain. These peptides also induced competence in S. thermophilus and Streptococcus salivarius strains that are poorly transformable or not transformable. Altogether, our results show that Ster_0316 and Shp0316, renamed ComRS, are the two members of a novel quorum-sensing system responsible for comX induction in species from the salivarius group, which differs from the classical phosphorelay three-component system identified previously in streptococci.

  16. Specificity of human rhinovirus 2A(pro) is determined by combined spatial properties of four cleavage site residues.

    Science.gov (United States)

    Neubauer, David; Aumayr, Martina; Gösler, Irene; Skern, Tim

    2013-07-01

    The 2A proteinase (2A(pro)) of human rhinoviruses cleaves the virally encoded polyprotein between the C terminus of VP1 and its own N terminus. Poor understanding of the 2A(pro) substrate specificity of this enzyme has hampered progress in developing inhibitors that may serve as antiviral agents. We show here that the 2A(pro) of human rhinovirus (HRV) 1A and 2 (rhinoviruses from genetic group A) cannot self-process at the HRV14 (a genetic group B rhinovirus) cleavage site. When the amino acids in the cleavage site of HRV2 2A(pro) (Ile-Ile-Thr-Thr-Ala*Gly-Pro-Ser-Asp) were singly or doubly replaced with the corresponding HRV14 residues (Asp-Ile-Lys-Ser-Tyr*Gly-Leu-Gly-Pro) at positions from P3 to P2', HRV1A and HRV2 2A(pro) cleavage took place at WT levels. However, when three or more positions of the HRV1A or 2 2A(pro) were substituted (e.g. at P2, P1 and P2'), cleavage in vitro was essentially eliminated. Introduction of the full HRV14 cleavage site into a full-length clone of the HRV1A and transfection of HeLa cells with a transcribed RNA did not give rise to viable virus. In contrast, revertant viruses bearing cysteine at the P1 position or proline at P2' were obtained when an RNA bearing the three inhibitory amino acids was transfected. Reversions in the enzyme affecting substrate specificity were not found in any of the in vivo experiments. Modelling of oligopeptide substrates onto the structure of HRV2 2A(pro) revealed no appreciable differences in residues of HRV2 and HRV14 in the respective substrate binding sites, suggesting that the overall shape of the substrate is important in determining binding efficiency.

  17. Do polyamines contribute to plant cell wall assembly by forming amide bonds with pectins?

    Science.gov (United States)

    Lenucci, Marcello; Piro, Gabriella; Miller, Janice G; Dalessandro, Giuseppe; Fry, Stephen C

    2005-11-01

    Two new reducing glycoconjugates [N-D-galacturonoyl-putrescinamide (GalA-Put) and N,N'-di-D-galacturonoyl-putrescinamide (GalA-Put-GalA)] and homogalacturonan-putrescine (GalAn-Put) conjugates were synthesised as model compounds representing possible amide (isopeptide) linkage points between a polyamine and either one or two pectic galacturonate residues. The amide bond(s) were stable to cold acid and alkali (2M TFA and 0.1M NaOH at 25 degrees C) but rapidly hydrolysed by these agents at 100 degrees C. The amide bond(s) were resistant to Driselase and to all proteinases tested, although Driselase digested GalAn-Put, releasing fragments such as GalA3-Put-GalA3. To trace the possible formation of GalA-polyamine amide bonds in vivo, we fed Arabidopsis and rose cell-cultures and chickpea internodes with [14C]Put. About 20% of the 14C taken up was released as 14CO2, indicating some catabolism. An additional approximately 73% of the 14C taken up (in Arabidopsis), or approximately 21% (in rose), became ethanol-insoluble, superficially suggestive of polysaccharide-Put covalent bonding. However, much of the ethanol-inextractable 14C was subsequently extractable by acidified phenol or by cold 1M TFA. The small proportion of radioactive material that stayed insoluble in both phenol and TFA was hydrolysable by Driselase or hot 6M HCl, yielding 14C-oligopeptides and/or amino acids (including Asp, Glu, Gly, Ala and Val); no free 14C-polyamines were released by hot HCl. We conclude that if pectin-polyamine amide bonds are present, they are a very minor component of the cell walls of cultured rose and Arabidopsis cells and chickpea internodes.

  18. Direct measurement of matrix metalloproteinase activity in 3D cellular microenvironments using a fluorogenic peptide substrate.

    Science.gov (United States)

    Leight, Jennifer L; Alge, Daniel L; Maier, Andrew J; Anseth, Kristi S

    2013-10-01

    Incorporation of degradable moieties into synthetic hydrogels has greatly increased the utility of these three-dimensional matrices for in vitro cell culture as well as tissue engineering applications. A common method for introducing degradability is the inclusion of oligopeptides sensitive to cleavage by matrix metalloproteinases (MMPs), enabling cell-mediated remodeling and migration within the material. While this strategy has been effective, characterization and measurement of cell-mediated degradation in these materials has remained challenging. There are 20+ MMP family members whose activity is regulated in space and time by a number of biochemical and biophysical cues. Thus, the typical approach of characterizing cleavage of degradable moieties in solution with recombinant enzymes does not easily translate to three-dimensional cell-mediated matrix remodeling. To address this challenge, we report here the synthesis of a cell-laden hydrogel matrix functionalized with a fluorogenic peptide substrate to provide real-time, quantitative monitoring of global MMP activity. Using this system, stimulation of MMP activity was observed with growth factor treatment in mammary epithelial cells and compared to classical zymography results. Further, the effect of biophysical cues on MMP activity of human mesenchymal stem cells was also investigated where more rigid hydrogels were observed to increase MMP activity. The regulation of MMP activity by these biochemical and biophysical cues highlights the need for in situ, real-time measurement of hydrogel degradation, and use of these functionalized hydrogels will aid in future rational design of degradable synthetic hydrogels for in vitro cell studies and tissue engineering applications. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Systemic lupus erythematosus: molecular cloning and analysis of 22 individual recombinant monoclonal kappa light chains specifically hydrolyzing human myelin basic protein.

    Science.gov (United States)

    Timofeeva, Anna M; Buneva, Valentina N; Nevinsky, Georgy A

    2015-10-01

    Antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis (MS) and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. Small pools of phage particles displaying light chains with different affinities for MBP were isolated by affinity chromatography on MBP-Sepharose, and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26-27 kDa). Seventy-two of 440 individual colonies were randomly chosen, expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography. Twenty-two of 72 MLChs have high affinity and efficiently hydrolyze only MBP (not other control proteins) demonstrating various pH optima in a 5.7-9.0 range and different substrate specificity in the hydrolysis of four different MBP oligopeptides. Four MLChs demonstrated serine protease-like and three thiol protease-like activities, while 11 MLChs were metalloproteases. The activity of three MLChs was inhibited by both phenylmethylsulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA), two other by EDTA and iodoacetamide, and one by PMSF, EDTA, and iodoacetamide. The ratio of relative activity in the presence of Ca(2+), Mg(2+), Mn(2+), Ni(2+), Zn(2+), Cu(2+), and Co(2+) was individual for each of 22 MLCh preparations. It is the first examples of human MLChs, which probably can possess two or even three different proteolytic activities. These observations suggest an extreme diversity of anti-MBP abzymes in SLE patients. The immune systems of individual SLE patients can generate a variety of anti-MBP abzymes, which can attack MBP of myelin-proteolipid sheath of axons and play an important role in MS and SLE pathogenesis.

  20. The role of metals in amyloid aggregation - Experiments and ab initio simulations

    Science.gov (United States)

    Minicozzi, V.; Morante, S.; Rossi, G. C.; Stellato, F.; Christian, N.; Jansen, K.

    With a combination of modern spectroscopic techniques and numerical first principle simulations it is possible to investigate the physico-chemical basis of the beta-amyloid aggregation phenomenon, which is suspected to be at the basis of the development of the Alzheimer disease. On the experimental side, in fact, X-ray absorption spectroscopy can be successfully used to determine the atomic structure around the metal binding site in samples where beta-amyloid peptides are complexed with either Cu2+ or Zn2+ ions. Exploiting spectroscopic information obtained on a selected set of fragments of the natural Abeta-peptide, the residues that along the sequence are coordinated to the metal are identified. Although copper data can be consistently interpreted assuming that oligopeptides encompassing the minimal 1-16 amino acidic sequence display a metal coordination mode which involves three Histidines (His6, His13, and His14), in complexes with zinc a four Histidines coordination mode is seen to be preferred. Lacking a fourth Histidine in the Abeta1-16 fragment, this geometrical arrangement hints to a Zn2+ promoted inter-peptide aggregation mode. On the theoretical side, first principle ab initio molecular dynamics simulations of the Car-Parrinello type, which have proved to be of invaluable help in understanding the microscopic mechanisms of chemical bonding both in solid-state physics and structural biophysics, have been employed in an effort to give a microscopic basis and find a phenomenological interpretation of the body of available experimental data on Abeta-peptides-metal complexes. Using medium size PC-clusters as well as larger parallel platforms, it is possible to deal with systems comprising 300-500 atoms and 1,000-2,000 electrons for simulation times as long as 2-3 ps. We present structural results that nicely compare with NMR and XAS data.

  1. Metallic nanocone array photonic substrate for high-uniformity surface deposition and optical detection of small molecules

    Science.gov (United States)

    Coppé, Jean-Philippe; Xu, Zhida; Chen, Yi; Logan Liu, G.

    2011-06-01

    Molecular probe arrays printed on solid surfaces such as DNA, peptide, and protein microarrays are widely used in chemical and biomedical applications especially genomic and proteomic studies (Pollack et al 1999 Nat. Genet. 23 41-6, Houseman et al 2002 Nat. Biotechnol. 20 270-4, Sauer et al 2005 Nat. Rev. Genet. 6 465-76) as well as surface imaging and spectroscopy (Mori et al 2008 Anal. Biochem. 375 223-31, Liu et al 2006 Nat. Nanotechnol. 1 47-52, Liu 2010 IEEE J. Sel. Top. Quantum Electron. 16 662-71). Unfortunately the printed molecular spots on solid surfaces often suffer low distribution uniformity due to the lingering 'coffee stain' (Deegan et al 1997 Nature 389 827-9) problem of molecular accumulations and blotches, especially around the edge of deposition spots caused by solvent evaporation and convection processes. Here we present, without any surface chemistry modification, a unique solid surface of high-aspect-ratio silver-coated silicon nanocone arrays that allows highly uniform molecular deposition and thus subsequent uniform optical imaging and spectroscopic molecular detection. Both fluorescent Rhodamine dye molecules and unlabeled oligopeptides are printed on the metallic nanocone photonic substrate surface as circular spot arrays. In comparison with the printed results on ordinary glass slides and silver-coated glass slides, not only high printing density but uniform molecular distribution in every deposited spot is achieved. The high-uniformity and repeatability of molecular depositions on the 'coffee stain'-free nanocone surface is confirmed by laser scanning fluorescence imaging and surface enhanced Raman imaging experiments. The physical mechanism for the uniform molecular deposition is attributed to the superhydrophobicity and localized pinned liquid-solid-air interface on the silver-coated silicon nanocone surface. The unique surface properties of the presented nanocone surface enabled high-density, high-uniformity probe spotting beneficial

  2. Gene activation by UV light, fungal elicitor or fungal infection in Petroselinum crispum is correlated with repression of cell cycle-related genes.

    Science.gov (United States)

    Logemann, E; Wu, S C; Schröder, J; Schmelzer, E; Somssich, I E; Hahlbrock, K

    1995-12-01

    The effects of UV light or fungal elicitors on plant cells have so far been studied mostly with respect to defense-related gene activation. Here, an inverse correlation of these stimulatory effects with the activities of several cell cycle-related genes is demonstrated. Concomitant with the induction of flavonoid biosynthetic enzymes in UV-irradiated cell suspension cultures of parsley (Petroselinum crispum), total histone synthesis declined to about half the initial rate. A subclass of the histone H3 gene family was selected to demonstrate the close correlation of its expression with cell division, both in intact plants and cultured cells. Using RNA-blot and run-on transcription assays, it was shown that one arbitrarily selected subclass of each of the histone H2A, H2B, H3 and H4 gene families and of the genes encoding a p34cdc2 protein kinase and a mitotic cyclin were transcriptionally repressed in UV-irradiated as well as fungal elicitor-treated parsley cells. The timing and extent of repression differed between the two stimuli; the response to light was more transient and smaller in magnitude. These differential responses to light and elicitor were inversely correlated with the induction of phenylalanine ammonia-lyase, a key enzyme of phenylpropanoid metabolism. Essentially the same result was obtained with a defined oligopeptide elicitor, indicating that the same signaling pathway is responsible for defense-related gene activation and cell cycle-related gene repression. A temporary (UV light) or long-lasting (fungal elicitor) cessation of cell culture growth is most likely due to an arrest of cell division which may be a prerequisite for full commitment of the cells to transcriptional activation of full commitment of the cells to transcriptional activation of pathways involved in UV protection or pathogen defense. This conclusion is corroborated by the observation that the histone H3 mRNA level greatly declined around fungal infection sites in young parsley

  3. Divergent evolution of the M3A family of metallopeptidases in plants.

    Science.gov (United States)

    Kmiec, Beata; Teixeira, Pedro F; Murcha, Monika W; Glaser, Elzbieta

    2016-07-01

    Plants, as stationary organisms, have developed mechanisms allowing them efficient resource reallocation and a response to changing environmental conditions. One of these mechanisms is proteome remodeling via a broad peptidase network present in various cellular compartments including mitochondria and chloroplasts. The genome of the model plant Arabidopsis thaliana encodes as many as 616 putative peptidase-coding genes organized in 55 peptidase families. In this study, we describe the M3A family of peptidases, which comprises four members: mitochondrial and chloroplastic oligopeptidase (OOP), cytosolic oligopeptidase (CyOP), mitochondrial octapeptidyl aminopeptidase 1 (Oct1) and plant-specific protein of M3 family (PSPM3) of unknown function. We have analyzed the evolutionary conservation of M3A peptidases across plant species and the functional specialization of the three distinct subfamilies. We found that the subfamily-containing OOP and CyOP-like peptidases, responsible for oligopeptide degradation in the endosymbiotic organelles (OOP) or in the cytosol (CyOP), are highly conserved in all kingdoms of life. The Oct1-like peptidase subfamily involved in pre-protein maturation in mitochondria is conserved in all eukaryotes, whereas the PSPM3-like protein subfamily is strictly conserved in higher plants only and is of unknown function. Specific characteristics within PSPM3 sequences, i.e. occurrence of a N-terminal transmembrane domain and amino acid changes in distal substrate-binding motif, distinguish PSPM3 proteins from other members of M3A family. We performed peptidase activity measurements to analyze the role of substrate-binding residues in the different Arabidopsis M3A paralogs. © 2016 Scandinavian Plant Physiology Society.

  4. Niche specialization of novel Thaumarchaeota to oxic and hypoxic acidic geothermal springs of Yellowstone National Park.

    Science.gov (United States)

    Beam, Jacob P; Jay, Zackary J; Kozubal, Mark A; Inskeep, William P

    2014-04-01

    Novel lineages of the phylum Thaumarchaeota are endemic to thermal habitats, and may exhibit physiological capabilities that are not yet observed in members of this phylum. The primary goals of this study were to conduct detailed phylogenetic and functional analyses of metagenome sequence assemblies of two different thaumarchaeal populations found in high-temperature (65-72 °C), acidic (pH~3) iron oxide and sulfur sediment environments of Yellowstone National Park (YNP). Metabolic reconstruction was coupled with detailed geochemical measurements of each geothermal habitat and reverse-transcriptase PCR to confirm the in situ activity of these populations. Phylogenetic analyses of ribosomal and housekeeping proteins place these archaea near the root of the thaumarchaeal branch. Metabolic reconstruction suggests that these populations are chemoorganotrophic and couple growth with the reduction of oxygen or nitrate in iron oxide habitats, or sulfur in hypoxic sulfur sediments. The iron oxide population has the potential for growth via the oxidation of sulfide to sulfate using a novel reverse sulfate reduction pathway. Possible carbon sources include aromatic compounds (for example, 4-hydroxyphenylacetate), complex carbohydrates (for example, starch), oligopeptides and amino acids. Both populations contain a type III ribulose bisphosphate carboxylase/oxygenase used for carbon dioxide fixation or adenosine monophosphate salvage. No evidence for the oxidation of ammonia was obtained from de novo sequence assemblies. Our results show that thermoacidophilic Thaumarchaeota from oxic iron mats and hypoxic sulfur sediments exhibit different respiratory machinery depending on the presence of oxygen versus sulfide, represent deeply rooted lineages within the phylum Thaumarchaeota and are endemic to numerous sites in YNP.

  5. Enzymatic activation of double-targeted 5'-O-L-valyl-decitabine prodrug by biphenyl hydrolase-like protein and its molecular design basis.

    Science.gov (United States)

    Tao, Wenhui; Zhao, Dongyang; Sun, Mengchi; Li, Meng; Zhang, Xiangyu; He, Zhonggui; Sun, Yinghua; Sun, Jin

    2017-04-01

    A primary focus of this research was to explore the activation process and mechanism of decitabine (5-aza-2'-deoxycytidine, DAC) prodrug. Recently, it has been reported that biphenyl hydrolase-like protein (BPHL) can play an important role in the activation of some amino acid nucleoside prodrugs with a general preference for hydrophobic amino acids and 5'-esters. Therefore, we put forward a bold hypothesis that this novel enzyme may be primarily responsible for the activation process of DAC prodrug as well. 5'-O-L-valyl-decitabine (L-val-DAC) was synthesized before and can be transported across biological membranes by the oligopeptide transporter (PEPT1), granting it much greater utility in vivo. In this report, L-val-DAC was found to be a good substrate of BPHL protein (K m 0.59 mM; k cat/K m 553.69 mM(-1) s(-1)). After intestinal absorption, L-val-DAC was rapidly and almost completely hydrolyzed to DAC and L-valine. The catalysis was mainly mediated by the BPHL hydrolase and resulted in the intestinal first-pass effect of L-val-DAC after oral administration in Sprague-Dawley rats with cannulated jugular and portal veins. The structural insights using computational molecular docking showed that BPHL had a unique binding mode for L-val-DAC. As a fundamental basis, the simulation was employed to explain the catalytic mechanism in molecular level. In conclusion, BPHL was at least one of the primary candidate enzymes for L-val-DAC prodrug activation. This promising double-targeted prodrug approach have more advantages than the traditional targeted designs due to its higher transport and more predictable activation, thereby leading to a favorable property for oral delivery.

  6. HNF4alpha dysfunction as a molecular rational for cyclosporine induced hypertension.

    Directory of Open Access Journals (Sweden)

    Monika Niehof

    Full Text Available Induction of tolerance against grafted organs is achieved by the immunosuppressive agent cyclosporine, a prominent member of the calcineurin inhibitors. Unfortunately, its lifetime use is associated with hypertension and nephrotoxicity. Several mechanism for cyclosporine induced hypertension have been proposed, i.e. activation of the sympathetic nervous system, endothelin-mediated systemic vasoconstriction, impaired vasodilatation secondary to reduction in prostaglandin and nitric oxide, altered cytosolic calcium translocation, and activation of the renin-angiotensin system (RAS. In this regard the molecular basis for undue RAS activation and an increased signaling of the vasoactive oligopeptide angiotensin II (AngII remain elusive. Notably, angiotensinogen (AGT is the precursor of AngII and transcriptional regulation of AGT is controlled by the hepatic nuclear factor HNF4alpha. To better understand the molecular events associated with cyclosporine induced hypertension, we investigated the effect of cyclosporine on HNF4alpha expression and activity and searched for novel HNF4alpha target genes among members of the RAS cascade. Using bioinformatic algorithm and EMSA bandshift assays we identified angiotensin II receptor type 1 (AGTR1, angiotensin I converting enzyme (ACE, and angiotensin I converting enzyme 2 (ACE2 as genes targeted by HNF4alpha. Notably, cyclosporine represses HNF4alpha gene and protein expression and its DNA-binding activity at consensus sequences to AGT, AGTR1, ACE, and ACE2. Consequently, the gene expression of AGT, AGTR1, and ACE2 was significantly reduced as evidenced by quantitative real-time RT-PCR. While RAS is composed of a sophisticated interplay between multiple factors we propose a decrease of ACE2 to enforce AngII signaling via AGTR1 to ultimately result in vasoconstriction and hypertension. Taken collectively we demonstrate cyclosporine to repress HNF4alpha activity through calcineurin inhibitor mediated inhibition

  7. Metallic nanocone array photonic substrate for high-uniformity surface deposition and optical detection of small molecules

    Energy Technology Data Exchange (ETDEWEB)

    Coppe, Jean-Philippe [Kinogea Inc., 2168 Shattuck Ave, Berkeley, CA 94704 (United States); Xu Zhida; Chen Yi; Logan Liu, G, E-mail: loganliu@illinois.edu [Micro and Nanotechnology Laboratory, Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, Urbana, IL (United States)

    2011-06-17

    Molecular probe arrays printed on solid surfaces such as DNA, peptide, and protein microarrays are widely used in chemical and biomedical applications especially genomic and proteomic studies (Pollack et al 1999 Nat. Genet. 23 41-6, Houseman et al 2002 Nat. Biotechnol. 20 270-4, Sauer et al 2005 Nat. Rev. Genet. 6 465-76) as well as surface imaging and spectroscopy (Mori et al 2008 Anal. Biochem. 375 223-31, Liu et al 2006 Nat. Nanotechnol. 1 47-52, Liu 2010 IEEE J. Sel. Top. Quantum Electron. 16 662-71). Unfortunately the printed molecular spots on solid surfaces often suffer low distribution uniformity due to the lingering 'coffee stain' (Deegan et al 1997 Nature 389 827-9) problem of molecular accumulations and blotches, especially around the edge of deposition spots caused by solvent evaporation and convection processes. Here we present, without any surface chemistry modification, a unique solid surface of high-aspect-ratio silver-coated silicon nanocone arrays that allows highly uniform molecular deposition and thus subsequent uniform optical imaging and spectroscopic molecular detection. Both fluorescent Rhodamine dye molecules and unlabeled oligopeptides are printed on the metallic nanocone photonic substrate surface as circular spot arrays. In comparison with the printed results on ordinary glass slides and silver-coated glass slides, not only high printing density but uniform molecular distribution in every deposited spot is achieved. The high-uniformity and repeatability of molecular depositions on the 'coffee stain'-free nanocone surface is confirmed by laser scanning fluorescence imaging and surface enhanced Raman imaging experiments. The physical mechanism for the uniform molecular deposition is attributed to the superhydrophobicity and localized pinned liquid-solid-air interface on the silver-coated silicon nanocone surface. The unique surface properties of the presented nanocone surface enabled high-density, high-uniformity probe

  8. Separation of polar mushroom toxins by mixed-mode hydrophilic and ionic interaction liquid chromatography-electrospray ionization-mass spectrometry.

    Science.gov (United States)

    Chung, Wai-cheung; Tso, Sau-ching; Sze, Sai-tim

    2007-02-01

    Reversed-phase liquid chromatography (RPLC) is commonly used to analyze nonvolatile contaminants and naturally occurring toxins in foods. However, polar compounds, such as hydrophilic polypeptides and quaternary ammonium salts, are often not satisfactorily separated by RPLC and present a challenge for analytical scientists. In this study, hydrophilic interaction liquid chromatography (HILIC), on an amide-based stationary phase in combination with electrospray ionization (ESI) tandem mass spectrometry (MS-MS), is successfully employed to simultaneously separate polar mushroom toxins, including amanitins and phallotoxins, which are cyclic oligopeptides and muscarine, a quaternary ammonium compound, in mushrooms. The sensitivity of different ionization modes is studied, and the positive ionization mode is found to provide a more sensitive and effective tool for the unambiguous identification of the concerned polar toxins because of their characteristic fragmentation patterns. The properties of the mobile phase are also found to have significant impacts on the separation. At a high acetonitrile (ACN) concentration, hydrophilic interaction dominates, and all analytes under study demonstrate a much higher affinity with the stationary phase. The addition of methanol (MeOH) as a modifier could further enhance the HILIC separation for amanitins, phallotoxins, and muscarine. Valley-to-valley separation is achieved upon the optimatizatiqn of the mobile phase (comprising of ACN, MeOH, and ammonium formate buffer at pH approximately 3.5) and the solvent gradient. HILIC coupled with ESI-MS-MS is demonstrated to be a novel technique for the simultaneous separation and confirmatory analysis of the concerned polar toxins by providing an environment of solubility and retention that could not be achieved through the use of RPLC.

  9. Selective inhibition of dipeptidyl peptidase 4 by targeting a substrate-specific secondary binding site.

    Science.gov (United States)

    Kühn-Wache, Kerstin; Bär, Joachim W; Hoffmann, Torsten; Wolf, Raik; Rahfeld, Jens-Ulrich; Demuth, Hans-Ulrich

    2011-03-01

    Dipeptidyl peptidase 4/CD26 (DP4) is a multifunctional serine protease liberating dipeptide from the N-terminus of (oligo)peptides which can modulate the activity of these peptides. The enzyme is involved in physiological processes such as blood glucose homeostasis and immune response. DP4 substrate specificity is characterized in detail using synthetic dipeptide derivatives. The specificity constant k(cat)/K(m) strongly depends on the amino acid in P₁-position for proline, alanine, glycine and serine with 5.0 x 10⁵ M⁻¹ s⁻¹, 1.8 x 10⁴ M⁻¹ s⁻¹, 3.6 x 10² M⁻¹ s⁻¹, 1.1 x 10² M⁻¹ s⁻¹, respectively. By contrast, kinetic investigation of larger peptide substrates yields a different pattern. The specific activity of DP4 for neuropeptide Y (NPY) cleavage comprising a proline in P₁-position is the same range as the k(cat)/K(m) values of NPY derivatives containing alanine or serine in P₁-position with 4 x 10⁵ M⁻¹ s⁻¹, 9.5 x 10⁵ M⁻¹ s⁻¹ and 2.1 x 10⁵ M⁻¹ s⁻¹, respectively. The proposed existence of an additional binding region outside the catalytic center is supported by measurements of peptide substrates with extended chain length. This 'secondary' binding site interaction depends on the amino acid sequence in P₄'-P₈'-position. Interactions with this binding site could be specifically blocked for substrates of the GRF/glucagon peptide family. By contrast, substrates not belonging to this peptide family and dipeptide derivative substrates that only bind to the catalytic center of DP4 were not inhibited. This more selective inhibition approach allows, for the first time, to distinguish between substrate families by substrate-discriminating inhibitors.

  10. A single-chain Fv reactive with the Goodpasture antigen.

    Science.gov (United States)

    Ross, C N; Turner, N; Savage, P; Cashman, S J; Spooner, R A; Pusey, C D

    1996-06-01

    Goodpasture's disease is defined by the presence of autoantibodies to the glomerular basement membrane and characterized clinically by rapidly progressive glomerulonephritis and pulmonary hemorrhage. P1, a murine monoclonal antibody to the Goodpasture antigen (the noncollagenous domain of the alpha 3 chain of type IV collagen, alpha 3(IV)NC1), has been a valuable reagent in investigating the pathogenesis of this disorder. The purpose of this study was to generate and characterize a recombinant form of P1 as a single-chain Fv (scFv). First strand cDNA was made from RNA extracted from the P1 hybridoma cell line, and DNA encoding the antibody light and heavy chain variable domains was amplified by polymerase chain reaction, using universal oligonucleotides. The purified products were ligated sequentially into an expression plasmid separated by a sequence encoding a 15 amino acid flexible oligopeptide linker. The resulting scFv was expressed in E. coli. Functional scFv, designated HBR-3, was obtained by denaturing and refolding the expressed product. HBR-3 was shown by ELISA, immunoblotting, and immunohistologic techniques, to have the same specificity for alpha 3(IV)NC1 as P1 and autoantibodies from patients with Goodpasture's disease. HBR-3 and P1 were shown to have similar affinity for their mutual ligand. On sections of normal human kidney, the scFv bound only to glomerular basement membrane and distal tubular basement membrane. It did not bind to the glomerular basement membrane of patients with Alport's syndrome, in whom the Goodpasture antigen is often not expressed in an antigenic form. We have, therefore, generated a scFv which reproduces the specific binding properties of the parent monoclonal antibody, P1. The potential of HBR-3 as a diagnostic reagent in Alport's syndrome has been demonstrated. The development of this recombinant molecule should permit new approaches to the investigation of Goodpasture's disease.

  11. Helix stability of oligoglycine, oligoalanine, and oligo-β-alanine dodecamers reflected by hydrogen-bond persistence.

    Science.gov (United States)

    Liu, Chengyu; Ponder, Jay W; Marshall, Garland R

    2014-11-01

    Helices are important structural/recognition elements in proteins and peptides. Stability and conformational differences between helices composed of α- and β-amino acids as scaffolds for mimicry of helix recognition has become a theme in medicinal chemistry. Furthermore, helices formed by β-amino acids are experimentally more stable than those formed by α-amino acids. This is paradoxical because the larger sizes of the hydrogen-bonding rings required by the extra methylene groups should lead to entropic destabilization. In this study, molecular dynamics simulations using the second-generation force field, AMOEBA (Ponder, J.W., et al., Current status of the AMOEBA polarizable force field. J Phys Chem B, 2010. 114(8): p. 2549-64.) explored the stability and hydrogen-bonding patterns of capped oligo-β-alanine, oligoalanine, and oligoglycine dodecamers in water. The MD simulations showed that oligo-β-alanine has strong acceptor+2 hydrogen bonds, but surprisingly did not contain a large content of 3(12) -helical structures, possibly due to the sparse distribution of the 3(12) -helical structure and other structures with acceptor+2 hydrogen bonds. On the other hand, despite its backbone flexibility, the β-alanine dodecamer had more stable and persistent <3.0 Å hydrogen bonds. Its structure was dominated more by multicentered hydrogen bonds than either oligoglycine or oligoalanine helices. The 3(1) (PII) helical structure, prevalent in oligoglycine and oligoalanine, does not appear to be stable in oligo-β-alanine indicating its competition with other structures (stacking structure as indicated by MD analyses). These differences are among the factors that shape helical structural preferences and the relative stabilities of these three oligopeptides. © 2014 Wiley Periodicals, Inc.

  12. Genome-Wide Identification of Genes Essential for the Survival of Streptococcus pneumoniae in Human Saliva

    Science.gov (United States)

    Verhagen, Lilly M.; de Jonge, Marien I.; Burghout, Peter; Schraa, Kiki; Spagnuolo, Lorenza; Mennens, Svenja; Eleveld, Marc J.; van der Gaast-de Jongh, Christa E.; Zomer, Aldert; Hermans, Peter W. M.; Bootsma, Hester J.

    2014-01-01

    Since Streptococcus pneumoniae transmits through droplet spread, this respiratory tract pathogen may be able to survive in saliva. Here, we show that saliva supports survival of clinically relevant S. pneumoniae strains for more than 24 h in a capsule-independent manner. Moreover, saliva induced growth of S. pneumoniae in growth-permissive conditions, suggesting that S. pneumoniae is well adapted for uptake of nutrients from this bodily fluid. By using Tn-seq, a method for genome-wide negative selection screening, we identified 147 genes potentially required for growth and survival of S. pneumoniae in saliva, among which genes predicted to be involved in cell envelope biosynthesis, cell transport, amino acid metabolism, and stress response predominated. The Tn-seq findings were validated by testing a panel of directed gene deletion mutants for their ability to survive in saliva under two testing conditions: at room temperature without CO2, representing transmission, and at 37°C with CO2, representing in-host carriage. These validation experiments confirmed that the plsX gene and the amiACDEF and aroDEBC operons, involved in respectively fatty acid metabolism, oligopeptide transport, and biosynthesis of aromatic amino acids play an important role in the growth and survival of S. pneumoniae in saliva at 37°C. In conclusion, this study shows that S. pneumoniae is well-adapted for growth and survival in human saliva and provides a genome-wide list of genes potentially involved in adaptation. This notion supports earlier evidence that S. pneumoniae can use human saliva as a vector for transmission. PMID:24586856

  13. Two group A streptococcal peptide pheromones act through opposing Rgg regulators to control biofilm development.

    Directory of Open Access Journals (Sweden)

    Jennifer C Chang

    2011-08-01

    Full Text Available Streptococcus pyogenes (Group A Streptococcus, GAS is an important human commensal that occasionally causes localized infections and less frequently causes severe invasive disease with high mortality rates. How GAS regulates expression of factors used to colonize the host and avoid immune responses remains poorly understood. Intercellular communication is an important means by which bacteria coordinate gene expression to defend against host assaults and competing bacteria, yet no conserved cell-to-cell signaling system has been elucidated in GAS. Encoded within the GAS genome are four rgg-like genes, two of which (rgg2 and rgg3 have no previously described function. We tested the hypothesis that rgg2 or rgg3 rely on extracellular peptides to control target-gene regulation. We found that Rgg2 and Rgg3 together tightly regulate two linked genes encoding new peptide pheromones. Rgg2 activates transcription of and is required for full induction of the pheromone genes, while Rgg3 plays an antagonistic role and represses pheromone expression. The active pheromone signals, termed SHP2 and SHP3, are short and hydrophobic (DI[I/L]IIVGG, and, though highly similar in sequence, their ability to disrupt Rgg3-DNA complexes were observed to be different, indicating that specificity and differential activation of promoters are characteristics of the Rgg2/3 regulatory circuit. SHP-pheromone signaling requires an intact oligopeptide permease (opp and a metalloprotease (eep, supporting the model that pro-peptides are secreted, processed to the mature form, and subsequently imported to the cytoplasm to interact directly with the Rgg receptors. At least one consequence of pheromone stimulation of the Rgg2/3 pathway is increased biogenesis of biofilms, which counteracts negative regulation of biofilms by RopB (Rgg1. These data provide the first demonstration that Rgg-dependent quorum sensing functions in GAS and substantiate the role that Rggs play as peptide

  14. In Silico Analysis of the Metabolic Potential and Niche Specialization of Candidate Phylum "Latescibacteria" (WS3.

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    Noha H Youssef

    Full Text Available The "Latescibacteria" (formerly WS3, member of the Fibrobacteres-Chlorobi-Bacteroidetes (FCB superphylum, represents a ubiquitous candidate phylum found in terrestrial, aquatic, and marine ecosystems. Recently, single-cell amplified genomes (SAGs representing the "Latescibacteria" were obtained from the anoxic monimolimnion layers of Sakinaw Lake (British Columbia, Canada, and anoxic sediments of a coastal lagoon (Etoliko lagoon, Western Greece. Here, we present a detailed in-silico analysis of the four SAGs to gain some insights on their metabolic potential and apparent ecological roles. Metabolic reconstruction suggests an anaerobic fermentative mode of metabolism, as well as the capability to degrade multiple polysaccharides and glycoproteins that represent integral components of green (Charophyta and Chlorophyta and brown (Phaeophycaea algae cell walls (pectin, alginate, ulvan, fucan, hydroxyproline-rich glycoproteins, storage molecules (starch and trehalose, and extracellular polymeric substances (EPSs. The analyzed SAGs also encode dedicated transporters for the uptake of produced sugars and amino acids/oligopeptides, as well as an extensive machinery for the catabolism of all transported sugars, including the production of a bacterial microcompartment (BMC to sequester propionaldehyde, a toxic intermediate produced during fucose and rhamnose metabolism. Finally, genes for the formation of gas vesicles, flagella, type IV pili, and oxidative stress response were found, features that could aid in cellular association with algal detritus. Collectively, these results indicate that the analyzed "Latescibacteria" mediate the turnover of multiple complex organic polymers of algal origin that reach deeper anoxic/microoxic habitats in lakes and lagoons. The implications of such process on our understanding of niche specialization in microbial communities mediating organic carbon turnover in stratified water bodies are discussed.

  15. Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro.

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    Alessandro Cinti

    Full Text Available Several biomedical applications, such as xenotransplantation, require multiple genes simultaneously expressed in eukaryotic cells. Advances in genetic engineering technologies have led to the development of efficient polycistronic vectors based on the use of the 2A self-processing oligopeptide. The aim of this work was to evaluate the protective effects of the simultaneous expression of a novel combination of anti-inflammatory human genes, ENTPD1, E5NT and HO-1, in eukaryotic cells. We produced an F2A system-based multicistronic construct to express three human proteins in NIH3T3 cells exposed to an inflammatory stimulus represented by tumor necrosis factor alpha (TNF-α, a pro-inflammatory cytokine which plays an important role during inflammation, cell proliferation, differentiation and apoptosis and in the inflammatory response during ischemia/reperfusion injury in several organ transplantation settings. The protective effects against TNF-α-induced cytotoxicity and cell death, mediated by HO-1, ENTPD1 and E5NT genes were better observed in cells expressing the combination of genes as compared to cells expressing each single gene and the effect was further improved by administrating enzymatic substrates of the human genes to the cells. Moreover, a gene expression analyses demonstrated that the expression of the three genes has a role in modulating key regulators of TNF-α signalling pathway, namely Nemo and Tnfaip3, that promoted pro-survival phenotype in TNF-α injured cells. These results could provide new insights in the research of protective mechanisms in transplantation settings.

  16. A new daunomycin-peptide conjugate: synthesis, characterization and the effect on the protein expression profile of HL-60 cells in vitro.

    Science.gov (United States)

    Orbán, Erika; Manea, Marilena; Marquadt, Andreas; Bánóczi, Zoltán; Csík, Gabriella; Fellinger, Erzsébet; Bosze, Szilvia; Hudecz, Ferenc

    2011-10-19

    Daunomycin (Dau) is a DNA-binding antineoplastic agent in the treatment of various types of cancer, such as osteosarcomas and acute myeloid leukemia. One approach to improve its selectivity and to decrease the side effects is the conjugation of Dau with oligopeptide carriers, which might alter the drug uptake and intracellular fate. Here, we report on the synthesis, characterization, and in vitro biological properties of a novel conjugate in which Dau is attached, via an oxime bond, to one of the cancer specific small peptides (LTVSPWY) selected from a random phage peptide library. The in vitro cytostatic effect and cellular uptake of Dau═Aoa-LTVSPWY-NH(2) conjugate were studied on various human cancer cell lines expressing different levels of ErbB2 receptor which could be targeted by the peptide. We found that the new daunomycin-peptide conjugate is highly cytostatic and could be taken up efficiently by the human cancer cells studied. However, the conjugate was less effective than the free drug itself. RP-HPLC data indicate that the conjugate is stable at least for 24 h in the pH 2.5-7.0 range of buffers, as well as in cell culture medium. The conjugate in the presence of rat liver lysosomal homogenate, as indicated by LC-MS analysis, could be degraded. The smallest, Dau-containing metabolite (Dau═Aoa-Leu-OH) identified and prepared expresses DNA-binding ability. In order to get insight on the potential mechanism of action, we compared the protein expression profile of HL-60 human leukemia cells after treatment with the free and peptide conjugated daunomycin. Proteomic analysis suggests that the expression of several proteins has been altered. This includes three proteins, whose expression was lower (tubulin β chain) or markedly higher (proliferating cell nuclear antigen and protein kinase C inhibitor protein 1) after administration of cells with Dau-conjugate vs free drug.

  17. Identification of salivary mucin MUC7 binding proteins from Streptococcus gordonii

    Directory of Open Access Journals (Sweden)

    Thornton David J

    2009-08-01

    Full Text Available Abstract Background The salivary mucin MUC7 (previously known as MG2 can adhere to various strains of streptococci that are primary colonizers and predominant microorganisms of the oral cavity. Although there is a growing interest in interaction between oral pathogens and salivary mucins, studies reporting the specific binding sites on the bacteria are rather limited. Identification and characterization of the specific interacting proteins on the bacterial cell surface, termed adhesins, are crucial to further understand host-pathogen interactions. Results We demonstrate here, using purified MUC7 to overlay blots of SDS-extracts of Streptococcus gordonii cell surface proteins, 4 MUC7-binding bands, with apparent molecular masses of 62, 78, 84 and 133 kDa from the Streptococcus gordonii strain, PK488. Putative adhesins were identified by in-gel digestion and subsequent nanoLC-tandem mass spectrometry analysis of resultant peptides. The 62 kDa and 84 kDa bands were identified as elongation factor (EF Tu and EF-G respectively. The 78 kDa band was a hppA gene product; the 74 kDa oligopeptide-binding lipoprotein. The 133 kDa band contained two proteins; alpha enolase and DNA-directed RNA polymerase, beta' subunit. Some of these proteins, for example alpha enolase are expected to be intracellular, however, flow cytometric analysis confirmed its location on the bacterial surface. Conclusion Our data demonstrated that S. gordonii expressed a number of putative MUC7 recognizing proteins and these contribute to MUC7 mucin binding of this streptococcal strain.

  18. The low-AGE content of low-fat vegan diets could benefit diabetics - though concurrent taurine supplementation may be needed to minimize endogenous AGE production.

    Science.gov (United States)

    McCarty, Mark F

    2005-01-01

    Increased endogenous generation of advanced glycation endproducts (AGEs) contributes importantly to the vascular complications of diabetes, in part owing to activation of the pro-inflammatory RAGE receptor. However, AGE-altered oligopeptides with RAGE-activating potential can also be absorbed from the diet, and indeed make a significant contribution to the plasma and tissue pool of AGEs; this contribution is especially prominent when compromised renal function impairs renal clearance of AGEs. Perhaps surprisingly, foods rich in both protein and fat, and cooked at high heat, tend to be the richest dietary sources of AGEs, whereas low-fat carbohydrate-rich foods tend to be relatively low in AGEs. Conceivably, this reflects the fact that the so-called "AGEs" in the diet are generated primarily, not by glycation reactions, but by interactions between oxidized lipids and protein; such reactions are known to give rise to certain prominent AGEs, such as epsilonN-carboxymethyl-lysine and methylglyoxal. Although roasted nuts and fried or broiled tofu are relatively high in AGEs, low-fat plant-derived foods, including boiled or baked beans, typically are low in AGEs. Thus, a low-AGE content may contribute to the many benefits conferred to diabetics by a genuinely low-fat vegan diet. Nonetheless, the plasma AGE content of healthy vegetarians has been reported to be higher than that of omnivores - suggesting that something about vegetarian diets may promote endogenous AGE production. Some researchers have proposed that the relatively high-fructose content of vegetarian diets may explain this phenomenon, but there so far is no clinical evidence that normal intakes of fructose have an important impact on AGE production. An alternative or additional possibility is that the relatively poor taurine status of vegetarians up-regulates the physiological role of myeloperoxidase-derived oxidants in the generation of AGEs - in which case, taurine supplementation might be expected to

  19. Core-shell polymer nanoparticles for prevention of GSH drug detoxification and cisplatin delivery to breast cancer cells.

    Science.gov (United States)

    Surnar, Bapurao; Sharma, Kavita; Jayakannan, Manickam

    2015-11-14

    Platinum drug delivery against the detoxification of cytoplasmic thiols is urgently required for achieving efficacy in breast cancer treatment that is over expressed by glutathione (GSH, thiol-oligopeptide). GSH-resistant polymer-cisplatin core-shell nanoparticles were custom designed based on biodegradable carboxylic functional polycaprolactone (PCL)-block-poly(ethylene glycol) diblock copolymers. The core of the nanoparticle was fixed as 100 carboxylic units and the shell part was varied using various molecular weight poly(ethylene glycol) monomethyl ethers (MW of PEGs = 100-5000 g mol(-1)) as initiator in the ring-opening polymerization. The complexation of cisplatin aquo species with the diblocks produced core-shell nanoparticles of 75 nm core with precise size control the particles up to 190 nm. The core-shell nanoparticles were found to be stable in saline solution and PBS and they exhibited enhanced stability with increase in the PEG shell thickness at the periphery. The hydrophobic PCL layer on the periphery of the cisplatin core behaved as a protecting layer against the cytoplasmic thiol residues (GSH and cysteine) and exhibited drug detoxification. In vitro drug-release studies revealed that the core-shell nanoparticles were ruptured upon exposure to lysosomal enzymes like esterase at the intracellular compartments. Cytotoxicity studies were performed both in normal wild-type mouse embryonic fibroblast cells (Wt-MEFs), and breast cancer (MCF-7) and cervical cancer (HeLa) cell lines. Free cisplatin and polymer drug core-shell nanoparticles showed similar cytotoxicity effects in the HeLa cells. In MCF-7 cells, the free cisplatin drug exhibited 50% cell death whereas complete cell death (100%) was accomplished by the polymer-cisplatin core-shell nanoparticles. Confocal microscopic images confirmed that the core-shell nanoparticles were taken up by the MCF-7 and HeLa cells and they were accumulated both at the cytoplasm as well at peri-nuclear environments

  20. Interferon-γ and proliferation responses to Salmonella enterica Serotype Typhi proteins in patients with S. Typhi Bacteremia in Dhaka, Bangladesh.

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    Alaullah Sheikh

    2011-06-01

    Full Text Available Salmonella enterica serotype Typhi is a human-restricted intracellular pathogen and the cause of typhoid fever. Cellular immune responses are required to control and clear Salmonella infection. Despite this, there are limited data on cellular immune responses in humans infected with wild type S. Typhi.For this work, we used an automated approach to purify a subset of S. Typhi proteins identified in previous antibody-based immuno-affinity screens and antigens known to be expressed in vivo, including StaF-putative fimbrial protein-STY0202, StbB-fimbrial chaperone-STY0372, CsgF-involved in curli production-STY1177, CsgD- putative regulatory protein-STY1179, OppA-periplasmic oligopeptide binding protein precursor-STY1304, PagC-outer membrane invasion protein-STY1878, and conserved hypothetical protein-STY2195; we also generated and analyzed a crude membrane preparation of S. Typhi (MP. In comparison to samples collected from uninfected Bangladeshi and North American participants, we detected significant interferon-γ responses in PBMCs stimulated with MP, StaF, StbB, CsgF, CsgD, OppA, STY2195, and PagC in patients bacteremic with S. Typhi in Bangladesh. The majority of interferon-γ expressing T cells were CD4 cells, although CD8 responses also occurred. We also assessed cellular proliferation responses in bacteremic patients, and confirmed increased responses in infected individuals to MP, StaF, STY2195, and PagC in convalescent compared to acute phase samples and compared to controls. StaF is a fimbrial protein homologous to E. coli YadK, and contains a Pfam motif thought to be involved in cellular adhesion. PagC is expressed in vivo under the control of the virulence-associated PhoP-regulon required for intra-macrophage survival of Salmonella. STY2195 is a conserved hypothetical protein of unknown function.This is the first analysis of cellular immune responses to purified S. Typhi antigens in patients with typhoid fever. These results indicate

  1. 新生隐球菌TUP1△株群体效应分子的筛选鉴定%Identification of quorum sensing molecular in Cryptococcus neoformans TUP1△ strain

    Institute of Scientific and Technical Information of China (English)

    朱定衡; 车付彬; 徐楠; 陈江汉

    2010-01-01

    Objective To clarify the quorum sensing and identify the quorum sensing molecular of Cryptococcus neoformans. Methods The growth of Cryptococcus neoformans TUP1 △ strain at different inoculum size was observed. The culture filtrate of high cell density TUP1△ strain was collected and plated on medium with low density TUP1△ cell, and the growth of low cell density TUP1△ was recorded. Finally, the active molecule in culture filtrate was purified and identified.Results The growth of Cryptococcus neoformans TUP1△ strain was density dependent and the colony could be normally formed only above a specific density. The filtrate from a high cell density TUP1△culture could promote the growth of low cell density TUP1△, and the active molecule in this culture filtrate was identified to be an oligopeptide composed of 11 amino acids pepride, named quorum sensing peptide 1(QSP1). QSP1 was artificially synthesized, which could also promote the colony forming of TUP1△ strain inoculated at low cell density. Conclusion Quorum sensing exists in Cryptococcus neoformans, and the quorum sensing molecular is QSP1.%目的 明确新生隐球菌群体感应现象,分离鉴定其群体效应分子.方法 观察以不同密度接种的新生隐球菌TUP1△株生长情况,取高密度生长的培养滤过液加入低密度接种TUP1△株的培养基中,观察滤过液促其生长作用,提纯并分析滤过液中的活性物质.结果 新生隐球菌TUP1△株具有密度依赖生长特性,只有高于特定密度接种才能形成正常菌落,从高密度生长菌落培养滤过液中分离出一种11肽,可促低密度TUP1△株生长,命名为quorum sensing peptide 1(QSP1).人工合成该活性物质QSP1,同样可使低密度接种的TUP1△菌株形成正常菌落.结论 新生隐球菌存在群体感应现象,其群体效应分子为QSP1.

  2. A sialoreceptor binding motif in the Mycoplasma synoviae adhesin VlhA.

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    Meghan May

    Full Text Available Mycoplasma synoviae depends on its adhesin VlhA to mediate cytadherence to sialylated host cell receptors. Allelic variants of VlhA arise through recombination between an assemblage of promoterless vlhA pseudogenes and a single transcription promoter site, creating lineages of M. synoviae that each express a different vlhA allele. The predicted full-length VlhA sequences adjacent to the promoter of nine lineages of M. synoviae varying in avidity of cytadherence were aligned with that of the reference strain MS53 and with a 60-a.a. hemagglutinating VlhA C-terminal fragment from a Tunisian lineage of strain WVU1853(T. Seven different sequence variants of an imperfectly conserved, single-copy, 12-a.a. candidate cytadherence motif were evident amid the flanking variable residues of the 11 total sequences examined. The motif was predicted to adopt a short hairpin structure in a low-complexity region near the C-terminus of VlhA. Biotinylated synthetic oligopeptides representing four selected variants of the 12-a.a. motif, with the whole synthesized 60-a.a. fragment as a positive control, differed (P<0.01 in the extent they bound to chicken erythrocyte membranes. All bound to a greater extent (P<0.01 than scrambled or irrelevant VlhA domain negative control peptides did. Experimentally introduced branched-chain amino acid (BCAA substitutions Val3Ile and Leu7Ile did not significantly alter binding, whereas fold-destabilizing substitutions Thr4Gly and Ala9Gly tended to reduce it (P<0.05. Binding was also reduced to background levels (P<0.01 when the peptides were exposed to desialylated membranes, or were pre-saturated with free sialic acid before exposure to untreated membranes. From this evidence we conclude that the motif P-X-(BCAA-X-F-X-(BCAA-X-A-K-X-G binds sialic acid and likely mediates VlhA-dependent M. synoviae attachment to host cells. This conserved mechanism retains the potential for fine-scale rheostasis in binding avidity, which could be a

  3. 多菌种固态发酵对豆粕、棉籽粕和花生粕组成的混合蛋白原料的影响%Effect of multi-strain solid state fermentation on mixed protein feed composed of soybean meal, cottonseed meal and peanut meal

    Institute of Scientific and Technical Information of China (English)

    李旺军; 方华; 夏佳吉; 王玲; 季春源

    2011-01-01

    The active bacteria number and nutrient substances were analyzed during mixed culture solid state fermentation by Bacillus subitilis and Lactobacillus bulgaricus ,? With soybean meal, cottonseed meal and peanut meal as mixed solid protein feed. The Results showed that the colony formation unit (cfu) of Bacillus subitilis and Lactobacillus bulgaricus were 3. 1×108 and 4. 45×108 in fermented products, respectively. The content of lactate was 1. 98% {Meanwhile, the large molecular weight of proteins was nearly degraded after fermentation. Compared to the unfermented protein feed,the content of crude protein increased by 7. 89%,and the contents of oligo-peptide increased by 129. 36%. The anti-nutritional factors (stachyose and raffi-nose) almost completely degraded, as well as producing a certain amount of reducing sugar.%以枯草芽孢杆菌和保加利亚乳杆菌为试验菌株,对豆粕、棉籽粕和花生粕组成的混合蛋白原料样品进行多菌种发酵处理,检测发酵过程中的活菌数和营养物质的变化.结果表明:发酵结束后混合蛋白原料样品中枯草芽孢杆菌活菌数为3.1×108 cfu/g,保加利亚乳杆菌活菌数为4.45×108 cfu/g,乳酸含量为1.98%,大分子蛋白几乎完全分解成小分子蛋白,粗蛋白质含量比发酵前提高了7.89%,寡肽含量比发酵前提高了129.36%,抗营养因子(水苏糖和棉籽糖)几乎完全降解,同时产生了一定量的还原糖.

  4. Genes involved in protein metabolism of the probiotic lactic acid bacterium Lactobacillus delbrueckii UFV H2b20.

    Science.gov (United States)

    Do Carmo, A P; da Silva, D F; De Oliveira, M N V; Borges, A C; De Carvalho, A F; De Moraes, C A

    2011-09-01

    A basic requirement for the prediction of the potential use of lactic acid bacteria (LAB) in the dairy industry is the identification of specific genes involved in flavour-forming pathways. The probiotic Lactobacillus delbrueckii UFV H2b20 was submitted to a genetic characterisation and phylogenetic analysis of genes involved in protein catabolism. Eight genes belonging to this system were identified, which possess a closely phylogenetic relationship to NCFM strains representative, as it was demonstrated for oppC and oppBII, encoding oligopeptide transport system components. PepC, PepN, and PepX might be essential for growth of LAB, probiotic or not, since the correspondent genes are always present, including in L. delbrueckii UFV H2b20 genome. For pepX gene, a probable link between carbohydrate catabolism and PepX expression may exists, where it is regulated by PepR1/CcpA-like, a common feature between Lactobacillus strains and also in L. delbrueckii UFV H2b20. The well conserved evolutionary history of the ilvE gene is evidence that the pathways leading to branched-chain amino acid degradation, such as isoleucine and valine, are similar among L. delbrueckii subsp. bulgaricus strains and L. delbrueckii UFV H2b20. Thus, the involvement of succinate in flavour formation can be attributed to IlvE activity. The presence of aminopeptidase G in L. delbrueckii UFV H2b20 genome, which is absent in several strains, might improve the proteolytic activity and effectiveness. The nucleotide sequence encoding PepG revealed that it is a cysteine endopeptidase, belonging to Peptidase C1 superfamily; sequence analysis showed 99% identity with L. delbrueckii subsp. bulgaricus ATCC 11842 pepG, whereas protein sequence analysis revealed 100% similarity with PepG from the same organism. The present study proposes a schematic model to explain how the proteolytic system of the probiotic L. delbrueckii UFV H2b20 works, based on the components identified so far.

  5. New Synthesis Route of Hydrogel through A Bioinspired Supramolecular Approach: Gelation, Binding Interaction, and in Vitro Dressing.

    Science.gov (United States)

    Cheng, Chieh; Tang, Meng-Che; Wu, Chung-Shu; Simon, Turibius; Ko, Fu-Hsiang

    2015-09-02

    Peptide-based supramolecular hydrogels have been comprehensively investigated in biomaterial applications because of their unique bioactivity, biofunctionality, and biocompatible features. However, the presence of organic building blocks in peptide-based hydrogels often results in low mechanical stability. To expand their practical use and range of applications, it is necessary to develop the tool kit available to prepare bioinspired, peptide-based supramolecular hydrogels with improved mechanical stability. In this paper, we present an innovative electrostatic and cross-linking approach in which naphthyl-Phe-Phe-Cys (NapFFC) oligopeptides are combined with gold nanoparticles (AuNPs) and calcium ions (Ca(2+)) to produce peptide-based supramolecular hydrogels. We further investigate the interactions among NapFFC, AuNPs and Ca(2+) by microscopy. The morphology of the nanofibrous network constructions and the binding forces exhibited from the hydrogel demonstrated that the combination of two mechanisms successfully enhanced the mechanical stability through the formation of a densely entangled fibrous network of peptide multimers that is attributed to the AuNP linkage and Ca(2+)-induced agglomeration. UV-vis spectrophotometry and fluorescence analysis were also used to demonstrate the enhanced stability of the hydrogel under various conditions such as thermal, solvent erosion, pH value and sonication. All results indicate that the presence of AuNPs and Ca(2+) can strengthen the prepared hydrogel by more than doubling the diameter of NapFFC nanofibers, enabling the formation of stronger frameworks and slowing the release of components. Further experiments confirmed that HeLa cells can grow on the bioinspired NapFFC-AuNP hydrogel and exhibit high cell viability and that these cells were killed on contact with a hydrogel containing a drug. Our peptide-based supramolecular hydrogels prepared from the observed electrostatic and cross-linking mechanisn exhibited a

  6. Effect of 5-aminolevulinic acid on erythropoiesis: A preclinical in vitro characterization for the treatment of congenital sideroblastic anemia

    Energy Technology Data Exchange (ETDEWEB)

    Fujiwara, Tohru [Department of Hematology and Rheumatology, Tohoku University Graduate School, Sendai (Japan); Department of Molecular Hematology/Oncology, Tohoku University Graduate School, Sendai (Japan); Okamoto, Koji; Niikuni, Ryoyu [Department of Hematology and Rheumatology, Tohoku University Graduate School, Sendai (Japan); Takahashi, Kiwamu [SBI Pharmaceuticals Co., Ltd., Tokyo (Japan); Okitsu, Yoko; Fukuhara, Noriko; Onishi, Yasushi [Department of Hematology and Rheumatology, Tohoku University Graduate School, Sendai (Japan); Ishizawa, Kenichi [Department of Hematology and Rheumatology, Tohoku University Graduate School, Sendai (Japan); Clinical Research, Innovation and Education Center, Tohoku University Hospital, Sendai (Japan); Ichinohasama, Ryo [Department of Hematopathology, Tohoku University Graduate School, Sendai (Japan); Nakamura, Yukio [Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki (Japan); Nakajima, Motowo; Tanaka, Tohru [SBI Pharmaceuticals Co., Ltd., Tokyo (Japan); Harigae, Hideo, E-mail: harigae@med.tohoku.ac.jp [Department of Hematology and Rheumatology, Tohoku University Graduate School, Sendai (Japan); Department of Molecular Hematology/Oncology, Tohoku University Graduate School, Sendai (Japan)

    2014-11-07

    Highlights: • Treatment with ALA induces erythroid differentiation of K562 cells. • Transportation of ALA into erythroid cells occurs predominantly via SLC36A1. • ALA restores defects in ALAS2 in human iPS cell-derived erythroblasts. • ALA may represent a novel therapeutic option for CSA caused by ALAS2 mutations. - Abstract: Congenital sideroblastic anemia (CSA) is a hereditary disorder characterized by microcytic anemia and bone marrow sideroblasts. The most common form of CSA is attributed to mutations in the X-linked gene 5-aminolevulinic acid synthase 2 (ALAS2). ALAS2 is a mitochondrial enzyme, which utilizes glycine and succinyl-CoA to form 5-aminolevulinic acid (ALA), a crucial precursor in heme synthesis. Therefore, ALA supplementation could be an effective therapeutic strategy to restore heme synthesis in CSA caused by ALAS2 defects. In a preclinical study, we examined the effects of ALA in human erythroid cells, including K562 cells and human induced pluripotent stem cell-derived erythroid progenitor (HiDEP) cells. ALA treatment resulted in significant dose-dependent accumulation of heme in the K562 cell line. Concomitantly, the treatment substantially induced erythroid differentiation as assessed using benzidine staining. Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed significant upregulation of heme-regulated genes, such as the globin genes [hemoglobin alpha (HBA) and hemoglobin gamma (HBG)] and the heme oxygenase 1 (HMOX1) gene, in K562 cells. Next, to investigate the mechanism by which ALA is transported into erythroid cells, quantitative RT-PCR analysis was performed on previously identified ALA transporters, including solute carrier family 15 (oligopeptide transporter), member (SLC15A) 1, SLC15A2, solute carrier family 36 (proton/amino acid symporter), member (SLC36A1), and solute carrier family 6 (neurotransmitter transporter), member 13 (SLC6A13). Our analysis revealed that SLC36A1 was abundantly

  7. Prion protein insertional mutations increase aggregation propensity but not fiber stability

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    True Heather L

    2008-03-01

    Full Text Available Abstract Background Mutations in the PRNP gene account for ~15% of all prion disease cases. Little is understood about the mechanism of how some of these mutations in PRNP cause the protein to aggregate into amyloid fibers or cause disease. We have taken advantage of a chimeric protein system to study the oligopeptide repeat domain (ORD expansions of the prion protein, PrP, and their effect on protein aggregation and amyloid fiber formation. We replaced the ORD of the yeast prion protein Sup35p with that from wild type and expanded ORDs of PrP and compared their biochemical properties in vitro. We previously determined that these chimeric proteins maintain the [PSI+] yeast prion phenotype in vivo. Interestingly, we noted that the repeat expanded chimeric prions seemed to be able to maintain a stronger strain of [PSI+] and convert from [psi-] to [PSI+] with a much higher frequency. In this study we have attempted to understand the biochemical properties of these chimeric proteins and to establish a system to study the properties of the ORD of PrP both in vivo and in vitro. Results Investigation of the chimeric proteins in vitro reveals that repeat-expansions increase aggregation propensity and that the kinetics of fiber formation depends on the number of repeats. The fiber formation reactions are promiscuous in that the chimeric protein containing 14 repeats can readily cross-seed fiber formation of proteins that have the wild type number of repeats. Morphologically, the amyloid fibers formed by repeat-expanded proteins associate with each other to form large clumps that were not as prevalent in fibers formed by proteins containing the wild type number of repeats. Despite the increased aggregation propensity and lateral association of the repeat expanded proteins, there was no corresponding increase in the stability of the fibers formed. Therefore, we predict that the differences in fibers formed with different repeat lengths may not be due to

  8. POSSIM: Parameterizing Complete Second-Order Polarizable Force Field for Proteins.

    Science.gov (United States)

    Li, Xinbi; Ponomarev, Sergei Y; Sigalovsky, Daniel L; Cvitkovic, John P; Kaminski, George A

    2014-11-11

    Previously, we reported development of a fast polarizable force field and software named POSSIM (POlarizable Simulations with Second order Interaction Model). The second-order approximation permits the speed up of the polarizable component of the calculations by ca. an order of magnitude. We have now expanded the POSSIM framework to include a complete polarizable force field for proteins. Most of the parameter fitting was done to high-level quantum mechanical data. Conformational geometries and energies for dipeptides have been reproduced within average errors of ca. 0.5 kcal/mol for energies of the conformers (for the electrostatically neutral residues) and 9.7° for key dihedral angles. We have also validated this force field by running Monte Carlo simulations of collagen-like proteins in water. The resulting geometries were within 0.94 Å root-mean-square deviation (RMSD) from the experimental data. We have performed additional validation by studying conformational properties of three oligopeptides relevant in the context of N-glycoprotein secondary structure. These systems have been previously studied with combined experimental and computational methods, and both POSSIM and benchmark OPLS-AA simulations that we carried out produced geometries within ca. 0.9 Å RMSD of the literature structures. Thus, the performance of POSSIM in reproducing the structures is comparable with that of the widely used OPLS-AA force field. Furthermore, our fitting of the force field parameters for peptides and proteins has been streamlined compared with the previous generation of the complete polarizable force field and relied more on transferability of parameters for nonbonded interactions (including the electrostatic component). The resulting deviations from the quantum mechanical data are similar to those achieved with the previous generation; thus, the technique is robust, and the parameters are transferable. At the same time, the number of parameters used in this work was

  9. Hidrolisados protéicos de arroz com baixo teor de finilalanina, obtidos pela ação da corolase PP e uso do carvão ativado Rice protein hydrolysates with low phenylalanine content, prepared by the action of corolase PP and the use of activated carbon

    Directory of Open Access Journals (Sweden)

    Carolina Schaper Bizzotto

    2006-04-01

    indicated that the higher oligopeptide content was obtained when corolase PP was used with an enzyme:substrate ratio of 1% and the initial protein concentration of 1,56 g 100 mL-1.

  10. Design of different strategies of multivalent DNA-based vaccination against rabies and canine distemper in mice and dogs

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    Touihri Leila

    2012-12-01

    Full Text Available Abstract Background During the vaccination campaigns, puppies younger than 3 months old are not targeted and remain unvaccinated for at least the first year of their lives. Almost half of the reported rabid dogs are 6 months or younger. Hence, we should recommend the vaccination against rabies of young puppies. Unfortunately, owing to the exposure of puppies to infections with either canine parvovirus (CPV or distemper virus (CDV after the intervention of the vaccinators, owners are reluctant to vaccinate puppies against rabies. Therefore, it is necessary to include the CPV and CDV valences in the vaccine against rabies. Multivalent DNA-based vaccination in dogs, including rabies and distemper valences, could help in raising vaccine coverage. Methods We have designed monovalent and multivalent DNA-based vaccine candidates for in vitro and in vivo assays. These plasmids encode to the rabies virus glycoprotein and/or the canine distemper virus hemagglutinin. The first strategy of multivalent DNA-based vaccination is by mixing plasmids encoding to a single antigen each. The second is by simply fusing the genes of the antigens together. The third is by adding the foot and mouth disease virus (FMDV 2A oligopeptide gene into the antigen genes. The last strategy is by the design and use of a bicistronic plasmid with an “Internal Ribosome Entry Site” (IRES domain. Results The monovalent construct against canine distemper was efficiently validated by inducing higher humoral immune responses compared to cell-culture-derived vaccine both in mice and dogs. All multivalent plasmids efficiently expressed both valences after in vitro transfection of BHK-21 cells. In BALB/c mice, the bicistronic IRES-dependant construct was the most efficient inducer of virus-neutralizing antibodies against both valences. It was able to induce better humoral immune responses compared to the administration of either cell-culture-derived vaccines or monovalent plasmids. The

  11. Matrix metalloproteinases-2/9-sensitive peptide-conjugated polymer micelles for site-specific release of drugs and enhancing tumor accumulation: preparation and in vitro and in vivo evaluation

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    Zhang X

    2016-04-01

    Full Text Available Xiaoyan Zhang, Xiaofei Wang, Weitong Zhong, Xiaoqing Ren, Xianyi Sha, Xiaoling FangKey Laboratory of Smart Drug Delivery, Ministry of Education and People’s Liberation Army of China, School of Pharmacy, Fudan University, Shanghai, People’s Republic of China Abstract: Since elevated expression of matrix metalloproteinase (MMP-2 and MMP-9 is commonly observed in several malignant tumors, MMPs have been widely reported as key factors in the design of drug delivery systems. Several strategies have been proposed to develop MMPs-responsive nanoparticles to deliver chemotherapeutics to malignant solid tumors. A stimuli-responsive drug delivery system, which could be cleaved by MMPs, was proposed in this study. By inserting an MMP-2/9 cleavable oligopeptide GPVGLIGK-NH2 (GK8 as spacer between α-tocopherol succinate (α-TOS and methoxy-polyethylene glycol molecular weight (MW 2000 Da activated by N-hydroxysuccinimide (mPEG2K-NHS, mPEG2K-GK8-α-TOS (TGK was synthesized as the primary ingredient for MMP-2/9-sensitive micelles composed of D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS and TGK (n:n =40:60, TGK micelles. mPEG2K-α-TOS (T2K was similarly synthesized as nonsensitive control. The TGK micelles showed better stability than nonsensitive micelles composed of TPGS and T2K (n:n =40:60, T2K micelles owing to the inserted peptide. Fluorescence resonance energy transfer results indicated that TGK micelles could be successfully cleaved by MMP-2/9. Effective drug release was demonstrated in the presence of collagenase type IV, a mixture of MMP-2 and MMP-9. Compared with nonsensitive micelles, docetaxel (DTX-loaded TGK micelles showed a fold higher cellular uptake in HT1080 cells. While the half-maximal inhibitory concentration (IC50 of TGK and T2K micelles were similar (P>0.05 in MCF-7 cells (MMP-2/9 underexpression, the IC50 values of the aforementioned micelles were 0.064±0.006 and 0.122±0.009 µg/mL, respectively, in HT1080 cells (MMP

  12. Enzymatic characterization of a novel Xaa-Pro aminopeptidase XpmA from Aspergillus oryzae expressed in Escherichia coli.

    Science.gov (United States)

    Matsushita-Morita, Mayumi; Tada, Sawaki; Suzuki, Satoshi; Hattori, Ryota; Kusumoto, Ken-Ichi

    2017-07-08

    Xaa-Pro aminopeptidases are peptidases responsible for the cleavage of any amino acid N-terminally adjacent to a proline residue. We identified a gene encoding a putative Xaa-Pro aminopeptidase in the genome of the filamentous fungus Aspergillus oryzae (genome database number: AO090701000720) and named this gene xpmA. We produced its enzyme in a C-terminally His6-tag-fused form in an Escherichia coli expression system and purified it. The purified recombinant XpmA (rXpmA) showed hydrolysis activity toward Xaa-Pro-oligopeptides, especially the two dipeptides Ala-Pro and Phe-Pro. The molecular weight of rXpmA was estimated to be 69 kDa by SDS-PAGE and 126 kDa by gel filtration, suggesting that it is a homodimer. The enzyme was activated by various divalent metal ions such as Mn(2+), Co(2+), and Mg(2+); in particular, the enzyme activity was increased 27.6-times relative to the no-addition control by 1 mM Mn(2+). Additionally, 10 mM EDTA suppressed its activity to 0.26-times of the control level. Therefore, rXpmA was a metalloprotease. Optimal hydrolytic activity of rXpmA was observed at 50°C and pH 8.5-9.0. The enzyme was stable up to 50°C and from pH 4.0 to 11.0. rXpmA showed substrate inhibition by Leu-Pro, Ser-Pro and Arg-Pro at concentrations over 4 mM, 10 mM, and 3 mM, respectively. NaCl increased the enzyme activity in the concentration range 0.5-3.0 M, suggesting that the enzyme is halophilic. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  13. Polyclonal antibodies against the TLA1 protein also recognize with high specificity the D2 reaction center protein of PSII in the green alga Chlamydomonas reinhardtii.

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    Mitra, Mautusi; Dewez, David; García-Cerdán, Jose Gines; Melis, Anastasios

    2012-04-01

    The Chlamydomonas reinhardtii DNA-insertional transformant truncated light-harvesting antenna 1 (tla1) mutant, helped identify the novel TLA1 gene (GenBank Accession # AF534570-71) as an important genetic determinant in the chlorophyll antenna size of photosynthesis. Down-regulation in the amount of the TLA1 23 kDa protein in the cell resulted in smaller chlorophyll antenna size for both photosystems (in Tetali et al. Planta 225:813-829, 2007). Specific polyclonal antibodies, raised against the recombinant TLA1 protein, showed a cross-reaction with the predicted 23 kDa TLA1 protein in C. reinhardtii protein extracts, but also showed a strong cross-reaction with a protein band migrating to 28.5 kDa. Questions of polymorphism, or posttranslational modification of the TLA1 protein were raised as a result of the unexpected 28.5 kDa cross-reaction. Work in this paper aimed to elucidate the nature of the unexpected 28.5 kDa cross-reaction, as this was deemed to be important in terms of the functional role of the TLA1 protein in the regulation of the chlorophyll antenna size of photosynthesis. Immuno-precipitation of the 28.5 kDa protein, followed by LC-mass spectrometry, showed amino acid sequences ascribed to the psbD/D2 reaction center protein of PSII. The common antigenic determinant between TLA1 and D2 was shown to be a stretch of nine conserved amino acids V-F-L(V)LP-GNAL in the C-terminus of the two proteins, constituting a high antigenicity "GNAL" domain. Antibodies raised against the TLA1 protein containing this domain recognized both the TLA1 and the D2 protein. Conversely, antibodies raised against the TLA1 protein minus the GNAL domain specifically recognized the 23 kDa TLA1 protein and failed to recognize the 28.5 kDa D2 protein. D2 antibodies raised against an oligopeptide containing this domain also cross-reacted with the TLA1 protein. It is concluded that the 28.5 kDa cross-reaction of C. reinhardtii protein extracts with antiTLA1 antibodies is due to

  14. Transcriptome sequencing of Mycosphaerella fijiensis during association with Musa acuminata reveals candidate pathogenicity genes.

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    Noar, Roslyn D; Daub, Margaret E

    2016-08-30

    Mycosphaerella fijiensis, causative agent of the black Sigatoka disease of banana, is considered the most economically damaging banana disease. Despite its importance, the genetics of pathogenicity are poorly understood. Previous studies have characterized polyketide pathways with possible roles in pathogenicity. To identify additional candidate pathogenicity genes, we compared the transcriptome of this fungus during the necrotrophic phase of infection with that during saprophytic growth in medium. Transcriptome analysis was conducted, and the functions of differentially expressed genes were predicted by identifying conserved domains, Gene Ontology (GO) annotation and GO enrichment analysis, Carbohydrate-Active EnZymes (CAZy) annotation, and identification of genes encoding effector-like proteins. The analysis showed that genes commonly involved in secondary metabolism have higher expression in infected leaf tissue, including genes encoding cytochrome P450s, short-chain dehydrogenases, and oxidoreductases in the 2-oxoglutarate and Fe(II)-dependent oxygenase superfamily. Other pathogenicity-related genes with higher expression in infected leaf tissue include genes encoding salicylate hydroxylase-like proteins, hydrophobic surface binding proteins, CFEM domain-containing proteins, and genes encoding secreted cysteine-rich proteins characteristic of effectors. More genes encoding amino acid transporters, oligopeptide transporters, peptidases, proteases, proteinases, sugar transporters, and proteins containing Domain of Unknown Function (DUF) 3328 had higher expression in infected leaf tissue, while more genes encoding inhibitors of peptidases and proteinases had higher expression in medium. Sixteen gene clusters with higher expression in leaf tissue were identified including clusters for the synthesis of a non-ribosomal peptide. A cluster encoding a novel fusicoccane was also identified. Two putative dispensable scaffolds were identified with a large proportion of

  15. Metatranscriptomics reveals the diversity of genes expressed by eukaryotes in forest soils.

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    Coralie Damon

    Full Text Available Eukaryotic organisms play essential roles in the biology and fertility of soils. For example the micro and mesofauna contribute to the fragmentation and homogenization of plant organic matter, while its hydrolysis is primarily performed by the fungi. To get a global picture of the activities carried out by soil eukaryotes we sequenced 2×10,000 cDNAs synthesized from polyadenylated mRNA directly extracted from soils sampled in beech (Fagus sylvatica and spruce (Picea abies forests. Taxonomic affiliation of both cDNAs and 18S rRNA sequences showed a dominance of sequences from fungi (up to 60% and metazoans while protists represented less than 12% of the 18S rRNA sequences. Sixty percent of cDNA sequences from beech forest soil and 52% from spruce forest soil had no homologs in the GenBank/EMBL/DDJB protein database. A Gene Ontology term was attributed to 39% and 31.5% of the spruce and beech soil sequences respectively. Altogether 2076 sequences were putative homologs to different enzyme classes participating to 129 KEGG pathways among which several were implicated in the utilisation of soil nutrients such as nitrogen (ammonium, amino acids, oligopeptides, sugars, phosphates and sulfate. Specific annotation of plant cell wall degrading enzymes identified enzymes active on major polymers (cellulose, hemicelluloses, pectin, lignin and glycoside hydrolases represented 0.5% (beech soil-0.8% (spruce soil of the cDNAs. Other sequences coding enzymes active on organic matter (extracellular proteases, lipases, a phytase, P450 monooxygenases were identified, thus underlining the biotechnological potential of eukaryotic metatranscriptomes. The phylogenetic affiliation of 12 full-length carbohydrate active enzymes showed that most of them were distantly related to sequences from known fungi. For example, a putative GH45 endocellulase was closely associated to molluscan sequences, while a GH7 cellobiohydrolase was closest to crustacean sequences, thus

  16. Differential gene expression during the moult cycle of Antarctic krill (Euphausia superba

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    Gaten Edward

    2010-10-01

    Full Text Available Abstract Background All crustaceans periodically moult to renew their exoskeleton. In krill this involves partial digestion and resorption of the old exoskeleton and synthesis of new cuticle. Molecular events that underlie the moult cycle are poorly understood in calcifying crustaceans and even less so in non-calcifying organisms such as krill. To address this we constructed an Antarctic krill cDNA microarray in order to generate gene expression profiles across the moult cycle and identify possible activation pathways. Results A total of 26 different cuticle genes were identified that showed differential gene expression across the moult cycle. Almost all cuticle genes were up regulated during premoult and down regulated during late intermoult. There were a number of transcripts with significant sequence homology to genes potentially involved in the synthesis, breakdown and resorption of chitin. During early premoult glutamine synthetase, a gene involved in generating an amino acid used in the synthesis of glucosamine, a constituent of chitin, was up regulated more than twofold. Mannosyltransferase 1, a member of the glycosyltransferase family of enzymes that includes chitin synthase was also up regulated during early premoult. Transcripts homologous to a β-N-acetylglucosaminidase (β-NAGase precursor were expressed at a higher level during late intermoult (prior to apolysis than during premoult. This observation coincided with the up regulation during late intermoult, of a coatomer subunit epsilon involved in the production of vesicles that maybe used to transport the β-NAGase precursors into the exuvial cleft. Trypsin, known to activate the β-NAGase precursor, was up regulated more than fourfold during premoult. The up regulation of a predicted oligopeptide transporter during premoult may allow the transport of chitin breakdown products across the newly synthesised epi- and exocuticle layers. Conclusion We have identified many genes

  17. [Features of immune proteasome expression in the development of rat central nervous system].

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    Orlova, A Sh; Liupina, Iu V; Abaturova, S B; Sharova, N P

    2014-01-01

    Formation of the central nervous system in ontogeny and function in adult mammals are controlled by universal ubiquitin-proteasome proteolytic system. The aim of this work was to study the dynamics of expression of immune proteasomes in comparison with the dynamics of ChLA and CLA proteasome and expression of the transcription factor Zif268 in the structures of the brain (cortex, hippocampus, and brainstem) in embryonic (E19, E21 days of embryonic development) and early postnatal (P1, P3, P4, P5, P7, P15 days of post-natal development) development in rats. ChLA and CLA in clarified homogenates of rat brain structures were determined by hydrolysis of fluorogenic commercial oligopeptides Suc-LLVY-AMC and Z-LLG-AMC, respectively. In the cortex and hippocampus of the brain was observed upregulation of immune subunits LMP7 during the active formation of biochemical mediatory structure and efferent neuronal projections at the period P7-P15. In the cerebral cortex during this period ChLA and CLA also are increased. In all structures of the brain the LMP2 immune subunits content was significantly increased at the period P7-P15. Contents of proteolytic constitutive subunit β1 in all structures decreased by P4 compare to P1 levels and was increased on P15 relative to the P1 levels. However, the level of expression of proteolytic constitutive subunit β5 increased in cortex, hippocampus and brainstem from E21 and reached maximum values on P3, P5 and P1, respectively with a sharp decrease to P7 in all studied structures. In all structures expression of LM P2 immune subunits and β1 constitutive subunits increased simultaneously with LMP7 immune subunits and sharply on P15. Also shown a positive correlation of increased expression regulator PA28 and constitutive β5 subunits in the hippocampus during the period P3-P5 and in the brainstem at the period P1-P5. The peculiarity of the studied brain regions during P7-P15 of rat early development is a correlation of expression of

  18. Proteome analysis of a recombinant Bacillus megaterium strain during heterologous production of a glucosyltransferase

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    Jahn Dieter

    2005-05-01

    polyhydroxybutyrate production. No expression increases were found with cytosolic chaperones such as GroEL and DnaK during dextransucrase production and secretion, whereas a strong up-regulation of the oligopeptide-binding protein OppA was observed in correlation with an increased secretion of dextransucrase into the culture medium.

  19. Fisiopatología de la diarrea aguda

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    Raúl L. Riverón Corteguera

    1999-06-01

    Full Text Available Es un artículo de revisión de los aspectos importantes de la fisiopatología de la diarrea. Se hace una descripción de la fisiopatología intestinal que incluye la anatomía del intestino y de su superficie de absorción y la estructura funcional de la mucosa intestinal. La fisiopatología normal de los líquidos intestinales. Mecanismo de absorción del agua y los electrólitos. Absorción de sodio por difusión electrogénica, unido al ion cloro, intercambio con el ion hidrógeno y unido a sustancias orgánicas como glucosa, aminoácidos y algunos oligopéptidos; secreción intestinal de agua y electrólitos. Control intracelular de la secreción. Mediadores y moduladores extracelulares del transporte intestinal. Factores que aumentan la absorción y reducen la secreción. Factores que estimulan la secreción y reducen la absorción Mecanismo fisiopatológico de la diarrea. Clasificación de la diarrea infecciosa aguda: acuosa secretoria y osmótica; diarrea con sangre invasiva y no invasiva.This a review of some important aspects of the physiopathology of diarrhea. A description is made of the intestinal physiopathology, including the anatomy of the intestine and of its surface of absorption, as well as the functional structure of the intestinal mucosa: the normal physiopathology of the intestinal fluids; the mechanism of absorption of water and electrolites; the absorption of sodium by electrogenic difussion, joined to the chloride ion; the interchange with the hydrogen ion and attached to organic substances, such as glucose, aminoacids and some oligopeptides; the intestinal secretion of water and electrolites; the intracellular control of secretion, the extracellular mediators and modulators of intestinal transport; the factors that increase absorption and reduce secretion; the factors that stimulate secretion and absorption; and the physiopathological mechanism of diarrhea. The classification of acute infectious diarrhea in aqueous

  20. Phenylalanine 664 of dipeptidyl peptidase (DPP) 7 and Phenylalanine 671 of DPP11 mediate preference for P2-position hydrophobic residues of a substrate.

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    Rouf, Shakh M A; Ohara-Nemoto, Yuko; Ono, Toshio; Shimoyama, Yu; Kimura, Shigenobu; Nemoto, Takayuki K

    2013-01-01

    Dipeptidyl peptidases (DPPs) are crucial for the energy metabolism in Porphyromonas gingivalis, a Gram-negative proteolytic and asaccharolytic anaerobic rod causing chronic periodontitis. Three DPPs, DPPIV specific for Pro, DPP7 for hydrophobic residues and DPP11 for Asp/Glu at the P1 position, are expressed in the bacterium. Like DPP7, DPP11 belongs to the S46 protease family, and they share 38.7% sequence identity. Although DPP11 is preferential for hydrophobic residues at the P2 position, it has been reported that DPP7 has no preference at the P2 position. In the present study, we defined the detailed P2 substrate preference of DPP7 and the amino acid residue responsible for the specificity. DPP7 most efficiently hydrolyzed Met-Leu-dipeptidyl-4-methylcoumaryl-7-amide (MCA) carrying hydrophobic residues at the P1 position with k cat/Km of 10.62 ± 2.51 μM(-1) s(-1), while it unexpectedly cleaved substrates with hydrophilic (Gln, Asn) or charged (Asp, Arg) residues. Examination with 21 dipeptidyl MCA demonstrated that DPP7-peptidase activity was dependent on hydrophobicity of the P2- as well as P1-position residue, thus it correlated best with the sum of the hydrophobicity index of P1- and P2-amino acid residues. Hydrophobicity of the P1 and P2 positions ensured efficient enzyme catalysis by increasing k cat and lowering Km values, respectively. Substitution of hydrophobic residues conserved in the S46 DPP7/DPP11 family to Ala revealed that Phe664 of DPP7 and Phe671 of DPP11 primarily afforded hydrophobic P2 preference. A modeling study suggested that Phe664 and Gly666 of DPP7 and Phe671 and Arg673 of DPP11 being associated with the P2- and P1-position residues, respectively, are located adjacent to the catalytic Ser648/Ser655. The present results expand the substrate repertoire of DPP7, which ensures efficient degradation of oligopeptides in asaccharolytic bacteria.