Application of Statistical Thermodynamics To Predict the Adsorption Properties of Polypeptides in Reversed-Phase HPLC.

Science.gov (United States)

Tarasova, Irina A; Goloborodko, Anton A; Perlova, Tatyana Y; Pridatchenko, Marina L; Gorshkov, Alexander V; Evreinov, Victor V; Ivanov, Alexander R; Gorshkov, Mikhail V

2015-07-07

The theory of critical chromatography for biomacromolecules (BioLCCC) describes polypeptide retention in reversed-phase HPLC using the basic principles of statistical thermodynamics. However, whether this theory correctly depicts a variety of empirical observations and laws introduced for peptide chromatography over the last decades remains to be determined. In this study, by comparing theoretical results with experimental data, we demonstrate that the BioLCCC: (1) fits the empirical dependence of the polypeptide retention on the amino acid sequence length with R(2) > 0.99 and allows in silico determination of the linear regression coefficients of the log-length correction in the additive model for arbitrary sequences and lengths and (2) predicts the distribution coefficients of polypeptides with an accuracy from 0.98 to 0.99 R(2). The latter enables direct calculation of the retention factors for given solvent compositions and modeling of the migration dynamics of polypeptides separated under isocratic or gradient conditions. The obtained results demonstrate that the suggested theory correctly relates the main aspects of polypeptide separation in reversed-phase HPLC.

Measles virus-specified polypeptides in infected cells

International Nuclear Information System (INIS)

Vainionpaepae, R.

1979-01-01

The synthesis of wild-type measles virus-specified polypeptides in Vero cells in pulse-chase experiments, in cells with synchronized protein synthesis by high salt concentration, and in the presence of proteolytic enzyme inhibitors was analyzed by polyacrylamide slab-gel electrophoresis. Six major (L, G, 2, NP, 5 and M) structural polypeptides were identified in infected cells. The results of pulse-chase experiments suggested that most of the structural polypeptides were synthesized at their final length. Polypeptide M was found to be sensitive to trypsin. In TLCK-treated cells its molecular weight was about 1000-2000 daltons higher than in untreated cells. A minor virus-specific polypeptide with a molecular weight of about 23,000 was found as a very faint and diffuse band. In addition, three nonstructural polypeptides with molecular weights of 65,000, 38,000 and 18,000 were also detected. The experiments with proteolytic enzyme inhibitors and with synchronized protein synthesis suggested that the polypeptide with a molecular weight of 65,000 might be a precursor of the structural polypeptide 5. (author)

Temperature-dependent morphology of hybrid nanoflowers from elastin-like polypeptides

Energy Technology Data Exchange (ETDEWEB)

Ghosh, Koushik; Balog, Eva Rose M.; Sista, Prakash; Williams, Darrick J.; Martinez, Jennifer S., E-mail: jenm@lanl.gov, E-mail: rcrocha@lanl.gov; Rocha, Reginaldo C., E-mail: jenm@lanl.gov, E-mail: rcrocha@lanl.gov [Center for Integrated Nanotechnologies, Materials Physics and Applications Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 (United States); Kelly, Daniel [Chemistry Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 (United States)

2014-02-01

We report a method for creating hybrid organic-inorganic “nanoflowers” using calcium or copper ions as the inorganic component and a recombinantly expressed elastin-like polypeptide (ELP) as the organic component. Polypeptides provide binding sites for the dynamic coordination with metal ions, and then such noncovalent complexes become nucleation sites for primary crystals of metal phosphates. We have shown that the interaction between the stimuli-responsive ELP and Ca{sup 2+} or Cu{sup 2+}, in the presence of phosphate, leads to the growth of micrometer-sized particles featuring nanoscale patterns shaped like flower petals. The morphology of these flower-like composite structures is dependent upon the temperature of growth and has been characterized by scanning electron microscopy. The composition of nanoflowers has also been analyzed by energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and X-ray diffraction. The temperature-dependent morphologies of these hybrid nanostructures, which arise from the controllable phase transition of ELPs, hold potential for morphological control of biomaterials in emerging applications such as tissue engineering and biocatalysis.

Temperature-dependent morphology of hybrid nanoflowers from elastin-like polypeptides

Directory of Open Access Journals (Sweden)

Koushik Ghosh

2014-02-01

Full Text Available We report a method for creating hybrid organic-inorganic “nanoflowers” using calcium or copper ions as the inorganic component and a recombinantly expressed elastin-like polypeptide (ELP as the organic component. Polypeptides provide binding sites for the dynamic coordination with metal ions, and then such noncovalent complexes become nucleation sites for primary crystals of metal phosphates. We have shown that the interaction between the stimuli-responsive ELP and Ca2+ or Cu2+, in the presence of phosphate, leads to the growth of micrometer-sized particles featuring nanoscale patterns shaped like flower petals. The morphology of these flower-like composite structures is dependent upon the temperature of growth and has been characterized by scanning electron microscopy. The composition of nanoflowers has also been analyzed by energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and X-ray diffraction. The temperature-dependent morphologies of these hybrid nanostructures, which arise from the controllable phase transition of ELPs, hold potential for morphological control of biomaterials in emerging applications such as tissue engineering and biocatalysis.

Biosynthesis and characterization of a non-repetitive polypeptide derived from silk fibroin heavy chain

Energy Technology Data Exchange (ETDEWEB)

Yang, Gaoqiang; Wu, Mingyang; Yi, Honggen; Wang, Jiannan, E-mail: wangjn@suda.edu.cn

2016-02-01

Silk fibroin heavy chain is the major protein component of Bombyx mori silk fibroin and is composed of 12 repetitive and 11 non-repetitive regions, with the non-repetitive domain consisting of a hydrophilic polypeptide chain. In order to determine the biomedical function of the non-repetitive domain or potentially use it to modify hydrophobic biomaterials, high-purity isolation is necessary. Previously, we cloned and extended a gene motif (f(1)) encoding the non-repetitive domain. Here, this motif and its multimers are inserted into a glutathione S-transferase (GST)-tagged fusion-protein expression vector. Motif f(1) and multimers f(4) and f(8) were expressed in Escherichia coli BL21 cells following isopropyl β-D-1-thiogalactopyranoside induction, purified by GST-affinity chromatography, and single bands of purified fusion proteins GST-F(1), GST-F(4), and GST-F(8), were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Target polypeptides F(1), F(4), and F(8), were cleaved clearly from the GST-fusion tag following thrombin digestion. Mass spectrometry results indicate that the molecular weights associated with fusion proteins GST-F(1), GST-F(4), and GST-F(8) are 31.5, 43.8, and 59.0 kDa, respectively, and with the cleaved polypeptides F(1), F(4), and F(8) are 4.8, 16.8, and 32.8 kDa, respectively. The F(1), F(4), and F(8) polypeptide chains are negatively charged with isoelectric points (pI) of 3.3, 3.2, and 3.0, respectively. The molecular weight and pI values of the polypeptide chains are consistent with the predicted values and the amino acid compositions similar to predicted sequences. FTIR and CD results show the molecular conformation of F(1) was mainly random coil, and more stable α-helix structure formed in longer molecular chain. - Highlights: • A non-repetitive domain and its multimers of silk fibroin were expressed by E. coli. • The corresponding target polypeptides F(1), F(4) and F(8) were cleaved clearly. • Their

Common spectrum of polypeptides occurs in secretion granule membranes of different exocrine glands

International Nuclear Information System (INIS)

Cameron, R.S.; Cameron, P.L.; Castle, J.D.

1986-01-01

A highly purified membrane preparation from rat parotid secretion granules has been used as a comparative probe to examine the extent of compositional overlap in granule membranes of three other exocrine secretory tissues - pancreatic, lacrimal, and submandibular - from several standpoints. First, indirect immunofluorescent studies using a polyclonal polyspecific anti-parotid granule membrane antiserum has indicated a selective staining of granule membrane profiles in all acinar cells of all tissues. Second, highly purified granule membrane subfractions have been isolated from each exocrine tissue; comparative two-dimensional (isoelectric focusing; SDS) PAGE of radioiodinated granule membranes has identified 10-15 polypeptides of identical pI and apparent molecular mass. These species are likely to be integral membrane components since they are not extracted by either saponin-sodium sulfate or sodium carbonate (pH 11.5) treatments, and they do not have counterparts in the granule content. Finally, the identity among selected parotid and pancreatic radioiodinated granule membrane polypeptides has been documented using two-dimensional peptide mapping of chymotryptic and tryptic digests. These findings clearly indicate that exocrine secretory granules, irrespective of the nature of stored secretion, comprise a type of vesicular carrier with a common (and probably refined) membrane composition. Conceivably, the polypeptides identified carry out general functions related to exocrine secretion

Methods for using polypeptides having cellobiohydrolase activity

Science.gov (United States)

Morant, Marc D; Harris, Paul

2016-08-23

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polynucleotides encoding polypeptides having beta-glucosidase activity

Science.gov (United States)

Harris, Paul; Golightly, Elizabeth

2010-03-02

The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having catalase activity and polynucleotides encoding same

Energy Technology Data Exchange (ETDEWEB)

Liu, Ye; Duan, Junxin; Zhang, Yu; Tang, Lan

2017-05-02

Provided are isolated polypeptides having catalase activity and polynucleotides encoding the polypeptides. Also provided are nucleic acid constructs, vectors and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Ring-Opening Polymerization of N-Carboxyanhydrides for Preparation of Polypeptides and Polypeptide-Based Hybrid Materials with Various Molecular Architectures

KAUST Repository

Pahovnik, David

2015-09-01

Different synthetic approaches utilizing ring-opening polymerization of N-carboxyanhydrides for preparation of polypeptide and polypeptide-based hybrid materials with various molecular architectures are described. An overview of polymerization mechanisms using conventional (various amines) as well as some recently developed initiators (hexamethyldisilazane, N-heterocyclic persistent carbenes, etc.) is presented, and their benefits and drawbacks for preparation of polypeptides with well-defined chain lengths and chain-end functionality are discussed. Recent examples from literature are used to illustrate different possibilities for synthesis of pure polypeptide materials with different molecular architectures bearing various functional groups, which are introduced either by modification of amino acids, before they are transformed into corresponding Ncarboxyanhydrides, or by post-polymerization modifications using protective groups and/or orthogonal functional groups. Different approaches for preparation of polypeptide-based hybrid materials are discussed as well using examples from recent literature. Syntheses of simple block copolymers or copolymers with more complex molecular architectures (graft and star copolymers) as well as modifications of nanoparticles and other surfaces with polypeptides are described.

Stimuli-Triggered Sol-Gel Transitions of Polypeptides Derived from α-Amino Acid N-Carboxyanhydride (NCA) Polymerizations.

Science.gov (United States)

He, Xun; Fan, Jingwei; Wooley, Karen L

2016-02-18

The past decade has witnessed significantly increased interest in the development of smart polypeptide-based organo- and hydrogel systems with stimuli responsiveness, especially those that exhibit sol-gel phase-transition properties, with an anticipation of their utility in the construction of adaptive materials, sensor designs, and controlled release systems, among other applications. Such developments have been facilitated by dramatic progress in controlled polymerizations of α-amino acid N-carboxyanhydrides (NCAs), together with advanced orthogonal functionalization techniques, which have enabled economical and practical syntheses of well-defined polypeptides and peptide hybrid polymeric materials. One-dimensional stacking of polypeptides or peptide aggregations in the forms of certain ordered conformations, such as α helices and β sheets, in combination with further physical or chemical cross-linking, result in the construction of three-dimensional matrices of polypeptide gel systems. The macroscopic sol-gel transitions, resulting from the construction or deconstruction of gel networks and the conformational changes between secondary structures, can be triggered by external stimuli, including environmental factors, electromagnetic fields, and (bio)chemical species. Herein, the most recent advances in polypeptide gel systems are described, covering synthetic strategies, gelation mechanisms, and stimuli-triggered sol-gel transitions, with the aim of demonstrating the relationships between chemical compositions, supramolecular structures, and responsive properties of polypeptide-based organo- and hydrogels. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Elucidation of the biosynthesis of eicosapentaenoic acid in the microalga Porphyridium cruentum. II. Studies with radiolabeled precursors

International Nuclear Information System (INIS)

Khozin, I.; Adlerstein, D.; Bigongo, C.; Heimer, Y.M.; Cohen, Z.

1997-01-01

In the course of the study of the biosynthesis of the fatty acid eicosapentaenoic acid (EPA) in the microalga Porphyridium cruentum, cells were pulse-labeled with various radiolabeled fatty acid precursors. Our data show that the major end products of the biosynthesis are EPA-containing galactolipids of a eukaryotic and prokaryotic nature. The prokaryotic molecular species contain EPA and arachidonic acid at the sn-1 position and C16 fatty acids, mainly 16:0, at the sn-2 positions, whereas in the eukaryotic species both positions are occupied by EPA or arachidonic acid. However, we suggest that both the eukaryotic and prokaryotic molecular species are formed in two pathways, omega 6 and omega 3, which involve cytoplasmic and chloroplastic lipids. In the omega 6 pathway, cytoplasmic 18:2-phosphatidylcholine (PC) is converted to 20:4 omega 6-PC by a sequence that includes a delta 6 desaturase, an elongation step, and a delta 5 desaturase. In the minor omega 3 pathway, 18:2-PC is presumably desaturated to 18:3 omega 3, which is sequentially converted by the enzymatic sequence of the omega 6 pathway to 20:5 omega 3-PC. The products of both pathways are exported, as their diacylglycerol moieties, to the chloroplast to be galactosylated into their respective monogalactosyldiacylglycerol molecular species. The 20:4 omega 6 in both eukaryotic and prokaryotic monogalactosyldiacylglycerol can be further desaturated to EPA by a chloroplastic delta 17 (omega 3) desaturase

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

Science.gov (United States)

Morant, Marc D.; Harris, Paul

2015-10-13

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

Energy Technology Data Exchange (ETDEWEB)

Spodsberg, Nikolaj

2018-02-06

The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Investigation of Gelatin Polypeptides of Jellyfish (Rhopilema esculentum for Their Antioxidant Activity in vitro

Directory of Open Access Journals (Sweden)

Yong-Liang Zhuang

2010-01-01

Full Text Available Jellyfish gelatin was hydrolyzed by different proteases to obtain antioxidative polypeptides. The gelatin hydrolysate obtained by progressive hydrolysis using trypsin and Properase E exhibited the highest hydrolysis degree and antioxidant activity. Three series of gelatin polypeptides (SCP1, SCP2 and SCP3 were obtained by ultrafiltrating the gelatin hydrolysate through molecular mass cut-off membranes of 10, 6 and 2 kDa, respectively. Amino acid composition analysis showed that SCP3 had the highest total hydrophobic amino acid content. The in vitro antioxidant tests demonstrated that SCP2 had the strongest hydroxyl radical and hydrogen peroxide scavenging activities and metal chelating ability, while SCP3 showed the highest reducing power, antioxidant activity in linoleic acid emulsion system and superoxide anion radical scavenging activity. The results support the feasibility of jellyfish gelatin as a natural antioxidant polypeptide provider, and enzymatic hydrolysis and ultrafiltration could be potent future processing technologies to utilize the abundant jellyfish resource.

Ring-Opening Polymerization of N-Carboxyanhydrides for Preparation of Polypeptides and Polypeptide-Based Hybrid Materials with Various Molecular Architectures

KAUST Repository

Pahovnik, David; Hadjichristidis, Nikolaos

2015-01-01

Different synthetic approaches utilizing ring-opening polymerization of N-carboxyanhydrides for preparation of polypeptide and polypeptide-based hybrid materials with various molecular architectures are described. An overview of polymerization

Polypeptides having xylanase activity and polynucleotides encoding same

Energy Technology Data Exchange (ETDEWEB)

Spodsberg, Nikolaj; Shaghasi, Tarana

2017-06-20

The present invention relates to polypeptides having xylanase activity, catalytic domains, and carbohydrate binding domains, and polynucleotides encoding the polypeptides, catalytic domains, and carbohydrate binding domains. The present invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains, and carbohydrate binding domains.

Phase transitions in polypeptides: analysis of energy fluctuations

DEFF Research Database (Denmark)

Yakubovich, Alexander V.; Solov'yov, Ilia; Solov'yov, Andrey V.

2009-01-01

The helix random coil transition in alanine, valine, and leucine polypeptides consisting of 30 amino acids is studied in vacuo using the Langevin molecular dynamics approach. The influence of side chain radicals on internal energy and heat capacity of the polypeptides is discussed. The heat...... of simulation time. This study provides a comparison of methods for the description of structural transitions in polypeptides....

Polypeptides having beta-glucosidase activity and polynucleotides encoding same

Science.gov (United States)

Harris, Paul; Golightly, Elizabeth

2012-11-27

The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

Science.gov (United States)

Maiyuran, Suchindra; Kramer, Randall; Harris, Paul

2013-10-29

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Chirality-selected phase behaviour in ionic polypeptide complexes

Science.gov (United States)

Perry, Sarah L.; Leon, Lorraine; Hoffmann, Kyle Q.; Kade, Matthew J.; Priftis, Dimitrios; Black, Katie A.; Wong, Derek; Klein, Ryan A.; Pierce, Charles F.; Margossian, Khatcher O.; Whitmer, Jonathan K.; Qin, Jian; de Pablo, Juan J.; Tirrell, Matthew

2015-01-01

Polyelectrolyte complexes present new opportunities for self-assembled soft matter. Factors determining whether the phase of the complex is solid or liquid remain unclear. Ionic polypeptides enable examination of the effects of stereochemistry on complex formation. Here we demonstrate that chirality determines the state of polyelectrolyte complexes, formed from mixing dilute solutions of oppositely charged polypeptides, via a combination of electrostatic and hydrogen-bonding interactions. Fluid complexes occur when at least one of the polypeptides in the mixture is racemic, which disrupts backbone hydrogen-bonding networks. Pairs of purely chiral polypeptides, of any sense, form compact, fibrillar solids with a β-sheet structure. Analogous behaviour occurs in micelles formed from polypeptide block copolymers with polyethylene oxide, where assembly into aggregates with either solid or fluid cores, and eventually into ordered phases at high concentrations, is possible. Chirality is an exploitable tool for manipulating material properties in polyelectrolyte complexation. PMID:25586861

Vasoactive intestinal polypeptide and other preprovasoactive intestinal polypeptide-derived peptides in the female and male genital tract: localization, biosynthesis, and functional and clinical significance

DEFF Research Database (Denmark)

Ottesen, B; Fahrenkrug, J

1995-01-01

Vasoactive intestinal polypeptide, a neuropeptide with wide distribution in the central and peripheral nervous system, has a broad spectrum of biologic actions. The demonstration of vasoactive intestinal polypeptide containing nerve fibers within the female and male genital tract 17 years ago...... indicated a putative role for this peptide in the local nervous control of reproductive functions. The genes encoding the preprovasoactive intestinal polypeptide precursor molecule and the vasoactive intestinal polypeptide receptor have been identified. The gene expression has been studied by the use...... in the genital tracts (i.e., blood flow and nonvascular smooth muscle relaxation). In the ovary vasoactive intestinal polypeptide seems to play an important role as regulator and/or modulator of folliculogenesis and steroidogenesis. In the male genital tract vasoactive intestinal polypeptide seems to participate...

[New drug developments of snake venom polypeptides and progress].

Science.gov (United States)

Fu, Sihai; Feng, Mei; Xiong, Yan

2017-11-28

The value of snake venom polypeptides in clinical application has drawn extensive attention, and the development of snake polypeptides into new drugs with anti-tumor, anti-inflammatory, antithrombotic, analgesic or antihypertensive properties has become the recent research hotspot. With the rapid development of molecular biology and biotechnology, the mechanisms of snake venom polypeptides are also gradually clarified. Numerous studies have demonstrated that snake venom polypeptides exert their pharmacological effects by regulating ion channels, cell proliferation, apoptosis, intracellular signaling pathway, and expression of cytokine as well as binding to relevant active sites or receptors.

Caffeine-water-polypeptide interaction in aqueous solution

Science.gov (United States)

Ghabi, Habib; Dhahbi, Mahmoud

1999-04-01

The interaction of caffeine monomer with the synthetic polypeptides polyasparagine (pAg) and polyaspartic acid (pAsp) was studied by UV spectrophotometry. The results show that different types of interactions are possible depending on the nature of polypeptide. The form of the complex was discussed.

Polypeptides having beta-glucosidase activity and polynucleotides encoding the same

Science.gov (United States)

Brown, Kimberly; Harris, Paul

2013-12-17

The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Chimeric polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

Science.gov (United States)

Wogulis, Mark; Sweeney, Matthew; Heu, Tia

2017-06-14

The present invention relates to chimeric GH61 polypeptides having cellulolytic enhancing activity. The present invention also relates to polynucleotides encoding the chimeric GH61 polypeptides; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the chimeric GH61 polypeptides.

Tunable drug loading and release from polypeptide multilayer nanofilms

Science.gov (United States)

Jiang, Bingbing; Li, Bingyun

2009-01-01

Polypeptide multilayer nanofilms were prepared using electrostatic layer-by-layer self-assembly nanotechnology. Small charged drug molecules (eg, cefazolin, gentamicin, and methylene blue) were loaded in polypeptide multilayer nanofilms. Their loading and release were found to be pH-dependent and could also be controlled by changing the number of film layers and drug incubation time, and applying heat-treatment after film formation. Antibioticloaded polypeptide multilayer nanofilms showed controllable antibacterial properties against Staphylococcus aureus. The developed biodegradable polypeptide multilayer nanofilms are capable of loading both positively- and negatively-charged drug molecules and promise to serve as drug delivery systems on biomedical devices for preventing biomedical device-associated infection, which is a significant clinical complication for both civilian and military patients. PMID:19421369

Immunogenic compositions comprising human immunodeficiency virus (HIV) mosaic Nef proteins

Science.gov (United States)

Korber, Bette T [Los Alamos, NM; Perkins, Simon [Los Alamos, NM; Bhattacharya, Tanmoy [Los Alamos, NM; Fischer, William M [Los Alamos, NM; Theiler, James [Los Alamos, NM; Letvin, Norman [Boston, MA; Haynes, Barton F [Durham, NC; Hahn, Beatrice H [Birmingham, AL; Yusim, Karina [Los Alamos, NM; Kuiken, Carla [Los Alamos, NM

2012-02-21

The present invention relates to mosaic clade M HIV-1 Nef polypeptides and to compositions comprising same. The polypeptides of the invention are suitable for use in inducing an immune response to HIV-1 in a human.

Self-assembling chimeric polypeptide-doxorubicin conjugate nanoparticles that abolish tumours after a single injection

Science.gov (United States)

Andrew Mackay, J.; Chen, Mingnan; McDaniel, Jonathan R.; Liu, Wenge; Simnick, Andrew J.; Chilkoti, Ashutosh

2009-12-01

New strategies to self-assemble biocompatible materials into nanoscale, drug-loaded packages with improved therapeutic efficacy are needed for nanomedicine. To address this need, we developed artificial recombinant chimeric polypeptides (CPs) that spontaneously self-assemble into sub-100-nm-sized, near-monodisperse nanoparticles on conjugation of diverse hydrophobic molecules, including chemotherapeutics. These CPs consist of a biodegradable polypeptide that is attached to a short Cys-rich segment. Covalent modification of the Cys residues with a structurally diverse set of hydrophobic small molecules, including chemotherapeutics, leads to spontaneous formation of nanoparticles over a range of CP compositions and molecular weights. When used to deliver chemotherapeutics to a murine cancer model, CP nanoparticles have a fourfold higher maximum tolerated dose than free drug, and induce nearly complete tumour regression after a single dose. This simple strategy can promote co-assembly of drugs, imaging agents and targeting moieties into multifunctional nanomedicines.

Compositions and Methods for the Treatment of Pierce's Disease

Energy Technology Data Exchange (ETDEWEB)

Gupta, Goutam (Santa Fe, NM)

2008-10-07

Chimeric anti-microbial proteins, compositions, and methods for the therapeutic and prophylactic treatment of plant diseases caused by the bacterial pathogen Xylella fastidiosa are provided. The anti-microbial proteins of the invention generally comprise a surface recognition domain polypeptide, capable of binding to a bacterial membrane component, fused to a bacterial lysis domain polypeptide, capable of affecting lysis or rupture of the bacterial membrane, typically via a fused polypeptide linker. In particular, methods and compositions for the treatment or prevention of Pierce's disease of grapevines are provided. Methods for the generation of transgenic Vitus vinefera plants expressing xylem-secreted anti-microbial chimeras are also provided.

Star-Shaped Polypeptides: Synthesis and Opportunities for Delivery of Therapeutics.

Science.gov (United States)

Byrne, Mark; Murphy, Robert; Kapetanakis, Antonios; Ramsey, Joanne; Cryan, Sally-Ann; Heise, Andreas

2015-09-17

Significant advances in the synthesis of polypeptides by N-carboxyanhydride (NCA) polymerisation over the last decade have enabled the design of advanced polypeptide architectures such as star-shaped polypeptides. These materials combine the functionality offered by amino acids with the flexibility of creating stable nanoparticles with adjustable cargo space for therapeutic delivery. This review highlights recent advances in the synthesis of star polypeptides by NCA polymerisation followed by a critical review of the applications of this class of polymer in the delivery of therapeutic agents. This includes examples of traditional small-molecule drugs as well as the emerging class of biologics such as genetic therapeutics (gene delivery). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Polypeptide profiles of human oocytes and preimplantation embryos.

Science.gov (United States)

Capmany, G; Bolton, V N

1993-11-01

The polypeptides that direct fertilization and early development until activation of the embryonic genome occurs, at the 4-8 cell stage in the human, are exclusively maternal in origin, and are either synthesized during oogenesis or translated later from maternal mRNA. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and silver stain, we have visualized and compared the polypeptides present in different populations of human oocytes and cleavage stage embryos obtained after superovulation and insemination in vitro. Two polypeptide patterns were resolved, differing in the region of mol. wt 69 kDa. The distribution of these patterns showed no correlation with the ability of individual oocytes to achieve fertilization and develop normally to the 8-cell stage.

Proteolytic processing of poliovirus polypeptides: antibodies to polypeptide P3-7c inhibit cleavage at glutamine-glycine pairs

International Nuclear Information System (INIS)

Hanecak, R.; Semler, B.L.; Anderson, C.W.; Wimmer, E.

1982-01-01

Proteolytic processing of poliovirus polypeptides was examined by the addition of antibodies directed against the viral proteins P3-7c and P2-X to a cell-free translation extract prepared from infected HeLa cells. Antisera to P3-7c specifically inhibited in vitro processing at Gln-Gly pairs. Partial amino acid sequence analysis revealed a second Tyr-Gly pair that is utilized in protein processing. Neither Tyr-Gly cleavage is affected by antibody to P3-7C. Anti-P3-7c antibodies react not only with P3-7c but also with P3-6a and P3-2, two viral polypeptides NH 2 -coterminal with P3-7c. Preimmune and anti-P2-X antibodies had no effect on the processing of poliovirus proteins in vitro. The authors conclude that the activity responsible for processing poliovirus polypeptides at Gln-Gly pairs resides in the primary structure of P3-7c and not in P2-X

UV cross-linking of polypeptides associated with 3'-terminal exons

International Nuclear Information System (INIS)

Stolow, D.T.; Berget, S.M.

1990-01-01

Association of nuclear proteins with chimeric vertebrate precursor RNAs containing both polyadenylation signals and an intron was examined by UV cross-linking. One major difference in cross-linking pattern was observed between this chimeric precursor RNA and precursors containing only polyadenylation or splicing signals. The heterogeneous nuclear ribonucleoprotein (hnRNP) polypeptide C cross-linked strongly to sequences downstream of the A addition site in polyadenylation precursor RNA containing only the polyadenylation signal from the simian virus 40 (SV40) late transcription unit. In contrast, the hnRNP C polypeptide cross-linked to chimeric RNA containing the same SV40 late poly(A) cassette very poorly, at a level less than 5% of that observed with the precursor RNA containing just the poly(A) site. Observation that cross-linking of the hnRNP C polypeptide to elements within the SV40 late poly(A) site was altered by the presence of an upstream intron suggests differences in the way nuclear factors associate with poly(A) sites in the presence and absence of an upstream intron. Cross-linking of C polypeptide to chimeric RNA increased with RNAs mutated for splicing or polyadenylation consensus sequences and under reaction conditions (high magnesium) that inhibited polyadenylation. Furthermore, cross-linking of hnRNP C polypeptide to precursors containing just the SV40 late poly(A) site was eliminated in the presence of competing poly(U); polyadenylation, however, was unaffected. Correlation of loss of activity with high levels of hnRNP C polypeptide cross-linking raises questions about the specificity of the interaction between the hnRNP C polypeptide and polyadenylation precursor RNAs in vitro

Biochemical map of polypeptides specified by foot-and-mouth disease virus.

OpenAIRE

Grubman, M J; Robertson, B H; Morgan, D O; Moore, D M; Dowbenko, D

1984-01-01

Pulse-chase labeling of foot-and-mouth disease virus-infected bovine kidney cells revealed stable and unstable viral-specific polypeptides. To identify precursor-product relationships among these polypeptides, antisera against a number of structural and nonstructural viral-specific polypeptides were used. Cell-free translations programmed with foot-and-mouth disease virion RNA or foot-and-mouth disease virus-infected bovine kidney cell lysates, which were shown to contain almost identical pol...

Polypeptide based hydrogels

OpenAIRE

Hanay, Saltuk

2018-01-01

There is a need for biocompatible, biodegradable, 3-D printable and stable hydrogels especially in the areas of tissue engineering, drug delivery, bio-sensing technologies and antimicrobial coatings. The main aim of this Ph.D. work was to fabricate polypeptide based hydrogel which may find a potential application in those fields. Focusing on tyrosine or tryptophan-containing copolypeptides prepared by NCarboxyanhydride (NCA) polymerizations, three different crosslinking strategies have been t...

A pH- and temperature-responsive bioresorbable injectable hydrogel based on polypeptide block copolymers for the sustained delivery of proteins in vivo.

Science.gov (United States)

Turabee, Md Hasan; Thambi, Thavasyappan; Duong, Huu Thuy Trang; Jeong, Ji Hoon; Lee, Doo Sung

2018-02-27

Sustained delivery of protein therapeutics is limited owing to the fragile nature of proteins. Despite its great potential, delivery of proteins without any loss of bioactivity remains a challenge in the use of protein therapeutics in the clinic. To surmount this shortcoming, we report a pH- and temperature-responsive in situ-forming injectable hydrogel based on comb-type polypeptide block copolymers for the controlled delivery of proteins. Polypeptide block copolymers, composed of hydrophilic polyethylene glycol (PEG), temperature-responsive poly(γ-benzyl-l-glutamate) (PBLG), and pH-responsive oligo(sulfamethazine) (OSM), exhibit pH- and temperature-induced sol-to-gel transition behavior in aqueous solutions. Polypeptide block copolymers were synthesized by combining N-carboxyanhydride-based ring-opening polymerization and post-functionalization of the chain-end using N-hydroxy succinimide ester activated OSM. The physical properties of polypeptide-based hydrogels were tuned by varying the composition of temperature- and pH-responsive PBLG and OSM in block copolymers. Polypeptide block copolymers were non-toxic to human embryonic kidney cells at high concentrations (2000 μg mL -1 ). Subcutaneous administration of polypeptide block copolymer sols formed viscoelastic gel instantly at the back of Sprague-Dawley (SD) rats. The in vivo gels exhibited sustained degradation and were found to be bioresorbable in 6 weeks without any noticeable inflammation at the injection site. Anionic characteristics of hydrogels allow efficient loading of a cationic model protein, lysozyme, through electrostatic interaction. Lysozyme-loaded polypeptide block copolymer sols readily formed a viscoelastic gel in vivo and sustained lysozyme release for at least a week. Overall, the results demonstrate an elegant approach to control the release of certain charged proteins and open a myriad of therapeutic possibilities in protein therapeutics.

Simultaneous Polymerization and Polypeptide Particle Production via Reactive Spray-Drying.

Science.gov (United States)

Glavas, Lidija; Odelius, Karin; Albertsson, Ann-Christine

2016-09-12

A method for producing polypeptide particles via in situ polymerization of N-carboxyanhydrides during spray-drying has been developed. This method was enabled by the development of a fast and robust synthetic pathway to polypeptides using 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as an initiator for the ring-opening polymerization of N-carboxyanhydrides. The polymerizations finished within 5 s and proved to be very tolerant toward impurities such as amino acid salts and water. The formed particles were prepared by mixing the monomer, N-carboxyanhydride of l-glutamic acid benzyl ester (NCAGlu) and the initiator (DBU) during the atomization process in the spray-dryer and were spherical with a size of ∼1 μm. This method combines two steps; making it a straightforward process that facilitates the production of polypeptide particles. Hence, it furthers the use of spray-drying and polypeptide particles in the pharmaceutical industry.

Characterization of cell surface polypeptides of unfertilized, fertilized, and protease-treated zona-free mouse eggs

International Nuclear Information System (INIS)

Boldt, J.; Gunter, L.E.; Howe, A.M.

1989-01-01

The polypeptide composition of unfertilized, fertilized, and protease-treated zona-free mouse eggs was evaluated in this study. Zona-free eggs were radioiodinated by an Iodogen-catalyzed reaction. Light microscopic autoradiography of egg sections revealed that labeling was restricted to the cell surface. Labeled eggs were solubilized, and cell surface polypeptides were identified by one-dimensional SDS polyacrylamide gel electrophoresis and autoradiography. The unfertilized egg demonstrated 8-10 peptides that incorporated 125 I, with major bands observed at approximately 145-150, 94, and 23 kilodaltons (kD). Zona-free eggs fertilized in vitro and then radiolabeled demonstrated several new bands in comparison to unfertilized eggs, with a major band appearing at approximately 36 kD. Treatment of radiolabeled unfertilized eggs with either trypsin or chymotrypsin (1 mg/ml for 5-20 min) caused enzyme-specific modifications in labeled polypeptides. Trypsin (T) treatment resulted in time-dependant modification of the three major peptides at 145-150, 94, and 23 kD. Chymotrypsin (CT) treatment, in contrast, was associated with loss or modification of the 94 kD band, with no apparent effect on either the 145-150 or 23 kD band. Taken together with previous data indicating that T or CT egg treatment interferes with sperm-egg attachment and fusion, these results suggest a possible role for the 94 kD protein in sperm-egg interaction

The influence of the side-chain sequence on the structure-activity correlations of immunomodulatory branched polypeptides. Synthesis and conformational analysis of new model polypeptides.

Science.gov (United States)

Mezö, G; Hudecz, F; Kajtár, J; Szókán, G; Szekerke, M

1989-10-01

New branched polypeptides were synthesized for a detailed study of the influence of the side-chain structure on the conformation and biological properties. The first subset of polypeptides were prepared by coupling of tetrapeptides to poly[L-Lys]. These polymers contain either DL-Ala3-X [poly[Lys-(X-DL-Ala3)n

Induction of protein body formation in plant leaves by elastin-like polypeptide fusions

Directory of Open Access Journals (Sweden)

Joensuu Jussi J

2009-08-01

Full Text Available Abstract Background Elastin-like polypeptides are synthetic biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the simple non-chromatographic purification of recombinant proteins. In addition, elastin-like polypeptide fusions have been shown to enhance the accumulation of a range of different recombinant proteins in plants, thus addressing the major limitation of plant-based expression systems, which is a low production yield. This study's main objectives were to determine the general utility of elastin-like polypeptide protein fusions in various intracellular compartments and to elucidate elastin-like polypeptide's mechanism of action for increasing recombinant protein accumulation in the endoplasmic reticulum of plants. Results The effect of elastin-like polypeptide fusions on the accumulation of green fluorescent protein targeted to the cytoplasm, chloroplasts, apoplast, and endoplasmic reticulum was evaluated. The endoplasmic reticulum was the only intracellular compartment in which an elastin-like polypeptide tag was shown to significantly enhance recombinant protein accumulation. Interestingly, endoplasmic reticulum-targeted elastin-like polypeptide fusions induced the formation of a novel type of protein body, which may be responsible for elastin-like polypeptide's positive effect on recombinant protein accumulation by excluding the heterologous protein from normal physiological turnover. Although expressed in the leaves of plants, these novel protein bodies appeared similar in size and morphology to the prolamin-based protein bodies naturally found in plant seeds. The elastin-like polypeptide-induced protein bodies were highly mobile organelles, exhibiting various dynamic patterns of movement throughout the cells, which were dependent on intact actin microfilaments and a functional actomyosin motility system. Conclusion An endoplasmic reticulum-targeted elastin-like polypeptide fusion approach

Fabrication of polypeptide-based piezoelectric composite polymer film

International Nuclear Information System (INIS)

Farrar, Dawnielle; West, James E.; Busch-Vishniac, Ilene J.; Yu, Seungju M.

2008-01-01

A new class of molecular composite piezoelectric material was produced by simultaneous poling and curing of a homogeneous solution comprising poly(γ-benzyl α,L-glutamate) and methylmethacrylate via corona discharge methods. This film exhibited high piezoelectricity (d 33 = 23 pC N -1 ), and its mechanical characteristics (modulus = 450 MPa) were similar to those of low molecular weight poly(methylmethacrylate). As it is produced via solution-based fabrication processes, the composite film is conducive to miniaturization for small sensors with integrated electronics, and could also potentially be used in piezoelectric coating applications

Basal serum pancreatic polypeptide is dependent on age and gender in an adult population

DEFF Research Database (Denmark)

Brimnes Damholt, M; Rasmussen, B K; Hilsted, L

1997-01-01

This study is the first epidemiologically based study of basal levels of serum pancreatic polypeptide (s-PP). The basal level of serum PP has become a field of interest mainly due to the role of PP as an endocrine tumour marker, and as a marker of pancreatic neuroendocrine function after pancreas...... a monospecific radioimmunoassay. Fasting serum pancreatic polypeptide depended on age and gender. The results demonstrated that fasting pancreatic polypeptide levels increase exponentially with age. Fitted separately for each sex, basal serum pancreatic polypeptide was found to increase by approximately 3% per...... reports on the fasting levels of serum pancreatic polypeptide are most likely due to lack of adjustment for age and gender. Thus, variation due to age and gender should be considered in evaluating fasting levels of serum pancreatic polypeptide. Whether similar considerations are important when evaluating...

Side-chain-controlled self-assembly of polystyrene-polypeptide miktoarm star copolymers

KAUST Repository

Junnila, Susanna

2012-03-27

We show how the self-assembly of miktoarm star copolymers can be controlled by modifying the side chains of their polypeptide arms, using A 2B and A 2B 2 type polymer/polypeptide hybrids (macromolecular chimeras). Initially synthesized PS 2PBLL and PS 2PBLL 2 (PS, polystyrene; PBLL, poly(ε-tert-butyloxycarbonyl-l-lysine) ) miktoarms were first deprotected to PS 2PLLHCl and PS 2PLLHCl 2 miktoarms (PLLHCl, poly(l-lysine hydrochloride)) and then complexed ionically with sodium dodecyl sulfonate (DS) to give the supramolecular complexes PS 2PLL(DS) and PS 2(PLL(DS)) 2. The solid-state self-assemblies of these six miktoarm systems were studied by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and small- and wide-angle X-ray scattering (SAXS, WAXS). The side chains of the polypeptide arms were observed to have a large effect on the solubility, polypeptide conformation, and self-assembly of the miktoarms. Three main categories were observed: (i) lamellar self-assemblies at the block copolymer length scale with packed layers of α-helices in PS 2PBLL and PS 2PBLL 2; (ii) charge-clustered polypeptide micelles with less-defined conformations in a nonordered lattice within a PS matrix in PS 2PLLHCl and PS 2PLLHCl 2; (iii) lamellar polypeptide-surfactant self-assemblies with β-sheet conformation in PS 2PLL(DS) and PS 2(PLL(DS)) 2 which dominate over the formation of block copolymer scale structures. Differences between the 3- and 4-arm systems illustrate how packing frustration between the coil-like PS arms and rigid polypeptide conformations can be relieved by the right number of arms, leading to differences in the extent of order. © 2012 American Chemical Society.

Avian leukosis virus is a versatile eukaryotic platform for polypeptide display

International Nuclear Information System (INIS)

Khare, Pranay D.; Russell, Stephen J.; Federspiel, Mark J.

2003-01-01

Display technology refers to methods of generating libraries of modularly coded biomolecules and screening them for particular properties. Retroviruses are good candidates to be a eukaryotic viral platform for the display of polypeptides synthesized in eukaryotic cells. Here we demonstrate that avian leukosis virus (ALV) provides an ideal platform for display of nonviral polyaeptides expressed in a eukaryotic cell substrate. Different sizes of polypeptides were genetically fused to the extreme N-terminus of the ALV envelope glycoprotein in an ALV infectious clone containing an alkaline phosphatase reporter gene. The chimeric envelope glycoproteins were efficiently incorporated into virions and were stably displayed on the surface of the virions through multiple virus replication cycles. The foreign polypeptides did not interfere with the attachment and entry functions of the underlying ALV envelope glycoproteins. The displayed polypeptides were fully functional and could efficiently mediate attachment of the recombinant viruses to their respective cognate receptors. This study demonstrates that ALV is an ideal display platform for the generation and selection of libraries of polypeptides where there is a need for expression, folding, and posttranslational modification in the endoplasmic reticulum of eukaryotic cells

Methods and compositions for targeting macromolecules into the nucleus

Science.gov (United States)

Chook, Yuh Min

2013-06-25

The present invention includes compositions, methods and kits for directing an agent across the nuclear membrane of a cell. The present invention includes a Karyopherin beta2 translocation motif in a polypeptide having a slightly positively charged region or a slightly hydrophobic region and one or more R/K/H-X.sub.(2-5)-P-Y motifs. The polypeptide targets the agent into the cell nucleus.

Polypeptide synthesis in alphavirus-infected aedes albopictus cells during the establishment of persistent infection

International Nuclear Information System (INIS)

Richardson, M.A.; Boulton, R.W.; Raghow, R.S.; Dalgarno, L.

1980-01-01

Polypeptide synthesis was examined in mosquito cells during the establishment of a persistent infection with two alphaviruses, Ross River virus (RRV) and Semliki Forest virus (SFV), and in vertebrate cells cytopathically-infected with the same viruses. In Aedes albopictus cells, RRV reached peak titres at 34-48 hours p.i. At 12 hours 85 per cent of cells assayed as infected by infective centre assay; by 48 hours when persistence was established, virus production was reduced and <5 per cent of cells assayed as infected. There was not shutdown of host polypeptide synthesis during infection. Viral polypeptide synthesis was maximal between 10 and 24 hours p.i. The major viral polypeptides labelled were nucleocapsid protein and envelope protein(s).The precursor polypeptide p95 which was prominent in infected BHK cells was not detected in mosquito cells. Similar results were obtained on SFV infection. During the establishment of persistence there was a coordinate decline in the synthesis of RRV polypeptides, reaching undetectable levels by 72 hours p.i. Subculturing persistently-infected cells led to a small increase in viral polypeptide synthesis and virus titre. In contrast, during RRV growth in BHK cells host protein synthesis was severely inhibited and by 9-11 hours p.i. virus-specific polypeptide synthesis represented more than 90 per cent of total protein synthetic activity. (author)

Heat shock induced change in protein ubiquitination in Chlamydomonas

International Nuclear Information System (INIS)

Shimogawara, K.; Muto, S.

1989-01-01

Ubiquitin was purified from pea (Pisum sativum L.) and its antibody was produced. Western blot analysis showed that the antibody cross-reacted with ubiquitins from a green alga Chlamydomonas reinhardtii, a brown alga Laminaria angustata and a red alga Porphyridium cruentum but not with ubiquitin from a blue-green alga Synechococcus sp. In Chlamydomonas, the antibody also reacted with some ubiquitinated proteins including 28- and 31-kDa polypeptides. The isoelectric points of Chlamydomonas ubiquitin and the 28- and 31-kDa ubiquitinated proteins were 8.0, 8.9 and 10.3, respectively. The ubiquitinated proteins, including the 28- and 31-kDa polypeptides were detected after in vitro ATP-dependent ubiquitination of Chlamydomonas cell extract with l25 I-labeled bovine ubiquitin. Heat treatment of Chlamydomonas cells (>40°C) caused drastic increase of ubiquitinated proteins with high mol wt (>60kDa), and coordinated redistribution or decrease of other ubiquitinated proteins and free ubiquitin. Quantitative analysis revealed that the 28- and 31-kDa ubiquitinated proteins showed different responses against heat stress, i.e. the former being more sensitive than the latter. (author)

Quantitative assessments of the distinct contributions of polypeptide backbone amides versus sidechain groups to chain expansion via chemical denaturation

Science.gov (United States)

Holehouse, Alex S.; Garai, Kanchan; Lyle, Nicholas; Vitalis, Andreas; Pappu, Rohit V.

2015-01-01

In aqueous solutions with high concentrations of chemical denaturants such as urea and guanidinium chloride (GdmCl) proteins expand to populate heterogeneous conformational ensembles. These denaturing environments are thought to be good solvents for generic protein sequences because properties of conformational distributions align with those of canonical random coils. Previous studies showed that water is a poor solvent for polypeptide backbones and therefore backbones form collapsed globular structures in aqueous solvents. Here, we ask if polypeptide backbones can intrinsically undergo the requisite chain expansion in aqueous solutions with high concentrations of urea and GdmCl. We answer this question using a combination of molecular dynamics simulations and fluorescence correlation spectroscopy. We find that the degree of backbone expansion is minimal in aqueous solutions with high concentrations denaturants. Instead, polypeptide backbones sample conformations that are denaturant-specific mixtures of coils and globules, with a persistent preference for globules. Therefore, typical denaturing environments cannot be classified as good solvents for polypeptide backbones. How then do generic protein sequences expand in denaturing environments? To answer this question, we investigated the effects of sidechains using simulations of two archetypal sequences with amino acid compositions that are mixtures of charged, hydrophobic, and polar groups. We find that sidechains lower the effective concentration of backbone amides in water leading to an intrinsic expansion of polypeptide backbones in the absence of denaturants. Additional dilution of the effective concentration of backbone amides is achieved through preferential interactions with denaturants. These effects lead to conformational statistics in denaturing environments that are congruent with those of canonical random coils. Our results highlight the role of sidechain-mediated interactions as determinants of the

A de novo designed 11 kDa polypeptide: model for amyloidogenic intrinsically disordered proteins.

Science.gov (United States)

Topilina, Natalya I; Ermolenkov, Vladimir V; Sikirzhytski, Vitali; Higashiya, Seiichiro; Lednev, Igor K; Welch, John T

2010-07-01

A de novo polypeptide GH(6)[(GA)(3)GY(GA)(3)GE](8)GAH(6) (YE8) has a significant number of identical weakly interacting beta-strands with the turns and termini functionalized by charged amino acids to control polypeptide folding and aggregation. YE8 exists in a soluble, disordered form at neutral pH but is responsive to changes in pH and ionic strength. The evolution of YE8 secondary structure has been successfully quantified during all stages of polypeptide fibrillation by deep UV resonance Raman (DUVRR) spectroscopy combined with other morphological, structural, spectral, and tinctorial characterization. The YE8 folding kinetics at pH 3.5 are strongly dependent on polypeptide concentration with a lag phase that can be eliminated by seeding with a solution of folded fibrillar YE8. The lag phase of polypeptide folding is concentration dependent leading to the conclusion that beta-sheet folding of the 11-kDa amyloidogenic polypeptide is completely aggregation driven.

Radiolysis of polypeptide

International Nuclear Information System (INIS)

Ogura, Isao; Nakamura, Katsuichi; Tanaka, Hiroshi; Takahashi, Katsuhiro; Ozaki, Makoto

1981-01-01

Almost the same results were obtained from the additional dipeptide, Gly-DL-Ala and DL-Ala-DL-Phe, by the γ-irradiation as previous report. Tri and tetrapeptide consisted of the same amino acid signified good stability than the others. Every polypeptide composed from sulfur contained amino acid exhaled the smell of hydrogen sulfide by the irradiation. It seemed that the stability by the difference of position of amino group in amino acid increased in order α, β, γ ... amino acid and that by the existence of hydroxyl group became smaller. (author)

The Transcriptome of the Zoanthid Protopalythoa variabilis (Cnidaria, Anthozoa) Predicts a Basal Repertoire of Toxin-like and Venom-Auxiliary Polypeptides.

Science.gov (United States)

Huang, Chen; Morlighem, Jean-Étienne Rl; Zhou, Hefeng; Lima, Érica P; Gomes, Paula B; Cai, Jing; Lou, Inchio; Pérez, Carlos D; Lee, Simon Ming; Rádis-Baptista, Gandhi

2016-10-05

Protopalythoa is a zoanthid that, together with thousands of predominantly marine species, such as hydra, jellyfish, and sea anemones, composes the oldest eumetazoan phylum, i.e., the Cnidaria. Some of these species, such as sea wasps and sea anemones, are highly venomous organisms that can produce deadly toxins for preying, for defense or for territorial disputes. Despite the fact that hundreds of organic and polypeptide toxins have been characterized from sea anemones and jellyfish, practically nothing is known about the toxin repertoire in zoanthids. Here, based on a transcriptome analysis of the zoanthid Protopalythoa variabilis, numerous predicted polypeptides with canonical venom protein features are identified. These polypeptides comprise putative proteins from different toxin families: neurotoxic peptides, hemostatic and hemorrhagic toxins, membrane-active (pore-forming) proteins, protease inhibitors, mixed-function venom enzymes, and venom auxiliary proteins. The synthesis and functional analysis of two of these predicted toxin products, one related to the ShK/Aurelin family and the other to a recently discovered anthozoan toxin, displayed potent in vivo neurotoxicity that impaired swimming in larval zebrafish. Altogether, the complex array of venom-related transcripts that are identified in P. variabilis, some of which are first reported in Cnidaria, provides novel insight into the toxin distribution among species and might contribute to the understanding of composition and evolution of venom polypeptides in toxiferous animals. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The Research on the Impact of Maca Polypeptide on Sport Fatigue.

Science.gov (United States)

Miao, Hua

2015-01-01

In order to study the effect of maca polypeptide on sport fatigue, this paper selected 40 male mice, and they were randomly divided into group A, B, C and D. group A, B and C were fed food with different concentrations of maca polypeptide, and group D was control group. After two weeks of feeding, measured physiological indexes of mice, including blood glucose, urea nitrogen and creatinine. At last gived the experimental results, as well as the analysis. Experimental results show that maca polypeptide can improve the ability of anti-fatigue mice, and in a certain concentration range, the higher the concentration, the better the resistance to fatigue.

Tuning Ice Nucleation with Supercharged Polypeptides

NARCIS (Netherlands)

Yang, Huige; Ma, Chao; Li, Kaiyong; Liu, Kai; Loznik, Mark; Teeuwen, Rosalie; van Hest, Jan C. M.; Zhou, Xin; Herrmann, Andreas; Wang, Jianjun

2016-01-01

Supercharged unfolded polypeptides (SUPs) are exploited for controlling ice nucleation via tuning the nature of charge and charge density of SUPs. The results show that positively charged SUPs facilitate ice nucleation, while negatively charged ones suppress it. Moreover, the charge density of the

T-Stimulator effect on cotton protein composition and synthesis in salinization stress

International Nuclear Information System (INIS)

Ibragimova, E.A.; Nazirova, E.R.; Samarkhodjaeva, N.R.; Nalbandyan, A.A.; Babaev, T.A.

2004-01-01

Full text: T-stimulator was established to possess a wide spectrum of physiological effects, to enhance plant adaptation to thermal stress and to increase plant resistance to pathogens. Plant adaptation to unfavorable conditions manifests in changes in many links of metabolism, that of proteins included. We studied effect of cottonseed treatment with T-stimulator on composition and synthesis of plasma membrane proteins upon chloride salinization by means of the radioisotope method. Electrophoretic fractionation of cottonseed plasma membrane proteins showed absence of more than 40 polypeptides with molecular mass from 10 to more than 100 kDa in the cotton root membranes. Major fractions-polypeptides with molecular mass of 61, 53, 46, 25, 21, 20 and 18 kDa constitute about 50% of the total polypeptide composition. The salinization significantly affects the total membrane protein output, proportion of some polypeptides and their synthesis rate. Analysis of phoreogram radioautographs showed that 2-hour exposition of cotton roots to 35 S methionine suppresses synthesis of major polypeptides with molecular mass of 63, 61 and 53 kDa, that of low molecular polypeptides (46, 20, 18 kDa) increasing. Changes in the proportion of major polypeptides in cotton plasma membranes, reduction in rate of biosynthesis of high molecular fractions with the general suppression of label inclusion in the membrane fraction are the evidence for a disturbance in biosynthesis of some membrane proteins in cotton tissue cells upon salinization. The inhibiting effect of salinization on the protein-synthesizing system was observed in plants treated with T-stimulator, but the rate of synthesis in plasma membranes of the treated plants was found significantly higher. The activation of some plasma membrane proteins under T-stimulator effect suggests an association with the increase in adaptation of the treated plants to the disturbing effect of salinization

Ultrastructural and biochemical detection of biotin and biotinylated polypeptides in Schistosoma mansoni

Directory of Open Access Journals (Sweden)

Santos P.R.P.

1997-01-01

Full Text Available Biotinylation is proposed for the identification of surface proteins in Schistosoma mansoni using the streptavidin-HRP conjugate for the detection of labeled polypeptides. However, control samples also showed several endogenous biotinylated polypeptides. In an attempt to determine the possibility of nonspecific binding between the streptavidin-HRP conjugate and polypeptides from S. mansoni, the conjugate was blocked with biotinamidecaproate-N-hydroxysuccinimide ester (BcapNHS before biotin-streptavidin blotting. No bands were detected on the nitrocellulose sheet, demonstrating the specific recognition of biotin by the streptavidin present in the conjugate. Whole cercariae and cercarial bodies and tails showed several endogenous biotinylated polypeptides. The biotin concentration was 13 µg/190,000 cercariae. Adult worms presented less endogenous biotinylated polypeptides than cercariae. These results may be due to changes in the environment from aerobic to anaerobic conditions when cercarial bodies (schistosomula are transformed into adult worms and a decrease in CO2 production may occur. Cercariae, cercarial bodies and adult male worms were examined by transmission electron microscopy employing an avidin-colloidal gold conjugate for the detection of endogenous biotin. Gold particles were distributed mainly on the muscle fibers, but dispersed granules were observed in the tegument, mitochondria and cytosol. The discovery of endogenous biotin in S. mansoni should be investigated in order to clarify the function of this vitamin in the parasite

Comparison between the polypeptide profile of halophilic bacteria and salt tolerant plants.

Science.gov (United States)

Muñoz, G; González, C; Flores, P; Prado, B; Campos, V

1997-12-01

Changes in the polypeptide profile induced by salt stress in halotolerant and halophilic bacteria, isolated from the Atacama desert (northern Chile), were compared with those in the cotyledons of Prosopis chilensis (Leguminoseae) seedlings, a salt tolerant plant. SDS-PAGE analyses show the presence of four predominant polypeptides, with molecular weights around 78, 70, 60 and 44 kDa respectively, both in bacteria and in cotyledons from P. chilensis seedlings raised under salt stress conditions. Moreover, the 60 and 44 kDa polypeptides seem to be salt responsive, since their concentration increases with increasing NaCl in the growth medium. Our results suggest a common mechanism for salt tolerance in prokaryotes and in eukaryotes.

The mining of toxin-like polypeptides from EST database by single residue distribution analysis.

Science.gov (United States)

Kozlov, Sergey; Grishin, Eugene

2011-01-31

Novel high throughput sequencing technologies require permanent development of bioinformatics data processing methods. Among them, rapid and reliable identification of encoded proteins plays a pivotal role. To search for particular protein families, the amino acid sequence motifs suitable for selective screening of nucleotide sequence databases may be used. In this work, we suggest a novel method for simplified representation of protein amino acid sequences named Single Residue Distribution Analysis, which is applicable both for homology search and database screening. Using the procedure developed, a search for amino acid sequence motifs in sea anemone polypeptides was performed, and 14 different motifs with broad and low specificity were discriminated. The adequacy of motifs for mining toxin-like sequences was confirmed by their ability to identify 100% toxin-like anemone polypeptides in the reference polypeptide database. The employment of novel motifs for the search of polypeptide toxins in Anemonia viridis EST dataset allowed us to identify 89 putative toxin precursors. The translated and modified ESTs were scanned using a special algorithm. In addition to direct comparison with the motifs developed, the putative signal peptides were predicted and homology with known structures was examined. The suggested method may be used to retrieve structures of interest from the EST databases using simple amino acid sequence motifs as templates. The efficiency of the procedure for directed search of polypeptides is higher than that of most currently used methods. Analysis of 39939 ESTs of sea anemone Anemonia viridis resulted in identification of five protein precursors of earlier described toxins, discovery of 43 novel polypeptide toxins, and prediction of 39 putative polypeptide toxin sequences. In addition, two precursors of novel peptides presumably displaying neuronal function were disclosed.

Processes for the production of hydroxycinnamic acids using polypeptides having tyrosine ammonia lyase activity

DEFF Research Database (Denmark)

2016-01-01

The present invention generally relates to the field of biotechnology as it applies to the production of hydroxycinnamic acids using polypeptides having tyrosine ammonia lyase activity. More particularly, the present invention pertains to polypeptides having tyrosine ammonia lyase activity and high...... substrate specificity towards tyrosine, which makes them particularly suitable in the production of p-coumaric acid and other hydroxycinnamic acids. The present invention thus provides processes for the production of p-coumaric acid and other hydroxycinnamic acids employing these polypeptides as well...

Recuperation of uranyl ions from effluents by means of microbiological collectors

International Nuclear Information System (INIS)

Cecal, A.; Palamaru, I.; Humelnicu, D.; Goanta, M.; Rudic, V.; Salaru, V.V.; Gulea, A.

1997-01-01

This paper deals with the study of bioaccumulation of uranyl ions (UO 2 2+ ) from industrial effluents, using microbiological collectors: Nostoc linkia sp., Tolipotrix sp., Spirulina sp., Porphyridium cruentum and also the glucide extract of P. cruentum. The values of retaining degree of UO 2 2+ on the biomass, for several experimental conditions, were established between 14.22 and 91.99%

Biosynthesis of human sialophorins and analysis of the polypeptide core

International Nuclear Information System (INIS)

Remold-O'Donnell, E.; Kenney, D.; Rosen, F.S.

1987-01-01

Biosynthesis was examined of sialophorin (formerly called gpL115) which is altered in the inherited immunodeficiency Wiskott-Aldrich syndrome. Sialophorin is greater than 50% carbohydrate, primarily O-linked units of sialic acid, galactose, and galactosamine. Pulse-labeling with [ 35 S]methionine and chase incubation established that sialophorin is synthesized in CEM lymphoblastoid cells as an Mr 62,000 precursor which is converted within 45 min to mature glycosylated sialophorin, a long-lived molecule. Experiments with tunicamycin and endoglycosidase H demonstrated that sialophorin contains N-linked carbohydrate (approximately two units per molecule) and is therefore an N,O-glycoprotein. Pulse-labeling of tunicamycin-treated CEM cells together with immunoprecipitation provided the means to isolate the [ 35 S]-methionine-labeled polypeptide core of sialophorin and determine its molecular weight (58,000). This datum allowed us to express the previously established composition on a per molecule basis and determine that sialophorin molecules contain approximately 520 amino acid residues and greater than or equal to 100 O-linked carbohydrate units. A recent study showed that various blood cells express sialophorin and that there are two molecular forms: lymphocyte/monocyte sialophorin and platelet/neutrophil sialophorin. Biosynthesis of the two forms was compared by using sialophorin of CEM cells and sialophorin of MOLT-4 cells (another lymphoblastoid line) as models for lymphocyte/monocyte sialophorin and platelet/neutrophil sialophorin, respectively. The time course of biosynthesis and the content of N units were found to be identical for the two sialophorin species. [ 35 S]Methionine-labeled polypeptide cores of CEM sialophorin and MOLT sialophorin were isolated and compared by electrophoresis, isoelectrofocusing, and a newly developed peptide mapping technique

Fabrication of genetically engineered polypeptide@quantum dots hybrid nanogels for targeted imaging

Science.gov (United States)

Yang, Jie; Yao, Ming-Hao; Zhao, Dong-Hui; Zhang, Xiao-Shuai; Jin, Rui-Mei; Zhao, Yuan-Di; Liu, Bo

2017-08-01

Nanogels have been widely used as multifunctional drug delivery carriers because of high water content, biocompatibility, and high loading capability. We designed and biosynthesized two triblock artificial polypeptides PC10A and PC10ARGD as vehicles for encapsulating hydrophobic materials. These polypeptides can form nanogels by self-assembly when the concentration is below 2% ( w/ v). The physical properties of nanogels, including size, surface potential, and targeting domain, are able to be tuned. Hydrophobic materials from molecular size to nano-size can be loaded into the polypeptide nanogels to form hybrid nanogels. Hydrophobic quantum dots CdSe@ZnS below 10 nM were loaded into the polypeptide nanogels by ultrasonic treatment. Encapsulation endows hydrophobic QDs with good tunability of size, water solubility, stability, targeting, and biocompatibility. PC10ARGD nanogels and PC10ARGD@QDs hybrid nanogels showed excellent biocompatibility, which the cellular viabilities of HeLa and MCF-7 cells treated with 1% PC10ARGD nanogels and PC10ARGD@QDs hybrid nanogels contained 20 nM QDs were above 90 and 80%, respectively. PC10ARGD@QDs hybrid nanogels with an arginine-glycine-aspartic acid motif present efficient receptor-mediated endocytosis in α v β 3 overexpressing HeLa cells but not in the control MCF-7 cells as analyzed by confocal microscopy. These results demonstrate that such polypeptide nanogels as nanocarriers are expected to have great potential applications in biomedicine.

Effect of oxygen on morphogenesis and polypeptide expression by Mucor racemosus

International Nuclear Information System (INIS)

Phillips, G.J.; Borgia, P.T.

1985-01-01

The morphology of Mucor racemosus in cultures continuously sparged with nitrogen gas was investigated. When appropriate precautions were taken to prevent oxygen from entering the cultures, the morphology of the cells was uniformly yeastlike irrespective of the N 2 flow rate. When small amounts of oxygen entered the cultures the resulting microaerobic conditions evoked mycelial development. Polypeptides synthesized by aerobic mycelia, microaerobic mycelia, anaerobic yeasts, and yeasts grown in a CO 2 atmosphere were compared by two-dimensional gel electrophoresis. The results indicated that a large number of differences in polypeptide expression exist when microaerobic mycelia or anaerobic yeasts are compared with aerobic mycelia and that these alterations correlate with a change from an oxidative to a fermentative metabolic mode. The authors hypothesize that oxygen regulates the expression of polypeptides involved in both the metabolic mode and in morphogenesis

Structural variation and inhibitor binding in polypeptide deformylase from four different bacterial species.

Science.gov (United States)

Smith, Kathrine J; Petit, Chantal M; Aubart, Kelly; Smyth, Martin; McManus, Edward; Jones, Jo; Fosberry, Andrew; Lewis, Ceri; Lonetto, Michael; Christensen, Siegfried B

2003-02-01

Polypeptide deformylase (PDF) catalyzes the deformylation of polypeptide chains in bacteria. It is essential for bacterial cell viability and is a potential antibacterial drug target. Here, we report the crystal structures of polypeptide deformylase from four different species of bacteria: Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, and Escherichia coli. Comparison of these four structures reveals significant overall differences between the two Gram-negative species (E. coli and H. influenzae) and the two Gram-positive species (S. pneumoniae and S. aureus). Despite these differences and low overall sequence identity, the S1' pocket of PDF is well conserved among the four enzymes studied. We also describe the binding of nonpeptidic inhibitor molecules SB-485345, SB-543668, and SB-505684 to both S. pneumoniae and E. coli PDF. Comparison of these structures shows similar binding interactions with both Gram-negative and Gram-positive species. Understanding the similarities and subtle differences in active site structure between species will help to design broad-spectrum polypeptide deformylase inhibitor molecules.

The mining of toxin-like polypeptides from EST database by single residue distribution analysis

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Grishin Eugene

2011-01-01

Full Text Available Abstract Background Novel high throughput sequencing technologies require permanent development of bioinformatics data processing methods. Among them, rapid and reliable identification of encoded proteins plays a pivotal role. To search for particular protein families, the amino acid sequence motifs suitable for selective screening of nucleotide sequence databases may be used. In this work, we suggest a novel method for simplified representation of protein amino acid sequences named Single Residue Distribution Analysis, which is applicable both for homology search and database screening. Results Using the procedure developed, a search for amino acid sequence motifs in sea anemone polypeptides was performed, and 14 different motifs with broad and low specificity were discriminated. The adequacy of motifs for mining toxin-like sequences was confirmed by their ability to identify 100% toxin-like anemone polypeptides in the reference polypeptide database. The employment of novel motifs for the search of polypeptide toxins in Anemonia viridis EST dataset allowed us to identify 89 putative toxin precursors. The translated and modified ESTs were scanned using a special algorithm. In addition to direct comparison with the motifs developed, the putative signal peptides were predicted and homology with known structures was examined. Conclusions The suggested method may be used to retrieve structures of interest from the EST databases using simple amino acid sequence motifs as templates. The efficiency of the procedure for directed search of polypeptides is higher than that of most currently used methods. Analysis of 39939 ESTs of sea anemone Anemonia viridis resulted in identification of five protein precursors of earlier described toxins, discovery of 43 novel polypeptide toxins, and prediction of 39 putative polypeptide toxin sequences. In addition, two precursors of novel peptides presumably displaying neuronal function were disclosed.

Brachytherapy Using Elastin-Like Polypeptides with (131)I Inhibit Tumor Growth in Rabbits with VX2 Liver Tumor.

Science.gov (United States)

Liu, Xinpei; Shen, Yiming; Zhang, Xuqian; Lin, Rui; Jia, Qiang; Chang, Yixiang; Liu, Wenge; Liu, Wentian

2016-10-01

Brachytherapy is a targeted type of radiotherapy utilized in the treatment of cancers. Elastin-like polypeptides are a unique class of genetically engineered peptide polymers that have several attractive properties for brachytherapy. To explore the feasibility and application of brachytherapy for VX2 liver tumor using elastin-like polypeptides with (131)I so as to provide reliable experimental evidence for a new promising treatment of liver cancer. Elastin-like polypeptide as carrier was labeled with (131)I using the iodogen method. Ten eligible rabbits with VX2 liver tumor were randomly divided into the treatment group (n = 5) and control group (n = 5). The treatment group received brachytherapy using elastin-like polypeptide with (131)I, and in the control group, elastin-like polypeptide was injected into the VX2 liver tumor as a control. Periodic biochemical and imaging surveillances were required to assess treatment efficacy. The stability of elastin-like polypeptide with (131)I in vitro was maintained at over 96.8 % for 96 h. Biochemistry and imaging indicated brachytherapy using elastin-like polypeptide with (131)I for liver tumor can improve liver function and inhibit tumor growth (P Elastin-like polypeptide can be an ideal carrier of (131)I and have high labeling efficiency, radiochemical purity and stability. Brachytherapy using elastin-like polypeptide with (131)I for liver tumor is a useful therapy that possesses high antitumor efficacy advantages.

Moisture absorption and retention properties, and activity in alleviating skin photodamage of collagen polypeptide from marine fish skin.

Science.gov (United States)

Hou, Hu; Li, Bafang; Zhang, Zhaohui; Xue, Changhu; Yu, Guangli; Wang, Jingfeng; Bao, Yuming; Bu, Lin; Sun, Jiang; Peng, Zhe; Su, Shiwei

2012-12-01

Collagen polypeptides were prepared from cod skin. Moisture absorption and retention properties of collagen polypeptides were determined at different relative humidities. In addition, the protective effects of collagen polypeptide against UV-induced damage to mouse skin were evaluated. Collagen polypeptides had good moisture absorption and retention properties and could alleviate the damage induced by UV radiation. The action mechanisms of collagen polypeptide mainly involved enhancing immunity, reducing the loss of moisture and lipid, promoting anti-oxidative properties, inhibiting the increase of glycosaminoglycans, repairing the endogenous collagen and elastin protein fibres, and maintaining the ratio of type III to type I collagen. Copyright © 2012 Elsevier Ltd. All rights reserved.

Polycondensation of Asparagine-comprising Dipeptides in Aqueous Media-A Simulation of Polypeptide Formation in Primordial Earth Hydrosphere

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Munegumi, Toratane; Tanikawa, Naoya

2017-09-01

Asparagine and aspartic acid might have mutually transformed in the primordial hydrosphere of the earth, if ammonia and aspartic acid had existed in equilibrium. These amino acids seem to contribute to polypeptides, while the simple amino acids glycine and alanine easily form cyclic dipeptides and do not achieve long peptide chains. Asparagine-comprising dipeptides contribute some kinds of activation forms of dipeptides because these can polymerize faster than asparagine only. The new finding of polypeptide formation suggests a pathway of sequential polypeptides to evolve a diversity of polypeptides.

Polycondensation of Asparagine-comprising Dipeptides in Aqueous Media-A Simulation of Polypeptide Formation in Primordial Earth Hydrosphere.

Science.gov (United States)

Munegumi, Toratane; Tanikawa, Naoya

2017-09-01

Asparagine and aspartic acid might have mutually transformed in the primordial hydrosphere of the earth, if ammonia and aspartic acid had existed in equilibrium. These amino acids seem to contribute to polypeptides, while the simple amino acids glycine and alanine easily form cyclic dipeptides and do not achieve long peptide chains. Asparagine-comprising dipeptides contribute some kinds of activation forms of dipeptides because these can polymerize faster than asparagine only. The new finding of polypeptide formation suggests a pathway of sequential polypeptides to evolve a diversity of polypeptides.

Congenital deficiency of two polypeptide subunits of the iron-protein fragment of mitochondrial complex I.

Science.gov (United States)

Moreadith, R W; Cleeter, M W; Ragan, C I; Batshaw, M L; Lehninger, A L

1987-02-01

Recently, we described a patient with severe lactic acidosis due to congenital complex I (NADH-ubiquinone oxidoreductase) deficiency. We now report further enzymatic and immunological characterizations. Both NADH and ferricyanide titrations of complex I activity (measured as NADH-ferricyanide reductase) were distinctly altered in the mitochondria from the patient's tissues. In addition, antisera against complex I immunoprecipitated NADH-ferricyanide reductase from the control but not the patient's mitochondria. However, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of complex I polypeptides demonstrated that the majority of the 25 polypeptides comprising complex I were present in the affected mitochondria. A more detailed analysis using subunit selective antisera against the main polypeptides of the iron-protein fragments of complex I revealed a selective absence of the 75- and 13-kD polypeptides. These findings suggest that the underlying basis for this patient's disease was a congenital deficiency of at least two polypeptides comprising the iron-protein fragment of complex I, which resulted in the inability to correctly assemble a functional enzyme complex.

Generation of polypeptide-templated gold nanoparticles using ionizing radiation.

Science.gov (United States)

Walker, Candace Rae; Pushpavanam, Karthik; Nair, Divya Geetha; Potta, Thrimoorthy; Sutiyoso, Caesario; Kodibagkar, Vikram D; Sapareto, Stephen; Chang, John; Rege, Kaushal

2013-08-13

Ionizing radiation, including γ rays and X-rays, are high-energy electromagnetic radiation with diverse applications in nuclear energy, astrophysics, and medicine. In this work, we describe the use of ionizing radiation and cysteine-containing elastin-like polypeptides (C(n)ELPs, where n = 2 or 12 cysteines in the polypeptide sequence) for the generation of gold nanoparticles. In the presence of C(n)ELPs, ionizing radiation doses higher than 175 Gy resulted in the formation of maroon-colored gold nanoparticle dispersions, with maximal absorbance at 520 nm, from colorless metal salts. Visible color changes were not observed in any of the control systems, indicating that ionizing radiation, gold salt solution, and C(n)ELPs were all required for nanoparticle formation. The hydrodynamic diameters of nanoparticles, determined using dynamic light scattering, were in the range of 80-150 nm, while TEM imaging indicated the formation of gold cores 10-20 nm in diameter. Interestingly, C2ELPs formed 1-2 nm diameter gold nanoparticles in the absence of radiation. Our results describe a facile method of nanoparticle formation in which nanoparticle size can be tailored based on radiation dose and C(n)ELP type. Further improvements in these polypeptide-based systems can lead to colorimetric detection of ionizing radiation in a variety of applications.

Synthetic profiles of polypeptides of human oocytes and normal and abnormal preimplantation embryos.

Science.gov (United States)

Capmany, G; Bolton, V N

1999-09-01

There is considerable variation in the rate of development in vitro of individual preimplantation human embryos. The relationship between the rate of development and patterns of polypeptide synthesis in individual embryos was examined using SDS-PAGE and autoradiography. After incubation in [35S]methionine, 19 polypeptide bands were identified that change between fertilization and the morula stage. Although changes in two of the bands occurred in embryos that were developing normally and in ageing oocytes, and are thus independent of fertilization, the changes identified in the remaining 17 bands occurred only after fertilization. In embryos that were developing abnormally, as assessed by delayed cleavage, cleavage arrest or extensive fragmentation, the alteration in polypeptide synthetic profiles increased with increasing abnormality.

Characterization of an amidated form of pancreatic polypeptide from the daddy sculpin (Cottus scorpius).

Science.gov (United States)

Conlon, J M; Schmidt, W E; Gallwitz, B; Falkmer, S; Thim, L

1986-12-30

The primary structure of pancreatic polypeptide from the teleostean fish, Cottus scorpius (daddy sculpin) was established as: YPPQPESPGGNASPEDWAKYHAAVRHYVNLITRQRYNH2 The presence of a COOH-terminally alpha-amidated amino acid was established using an HPLC method of general applicability. Although the peptide shows strong homology towards anglerfish pancreatic polypeptide (86%), homology towards porcine peptide YY (PYY) (61%) and porcine neuropeptide Y (NPY) (61%) was greater than towards porcine pancreatic polypeptide (PP) (47%). This result supports suggestions that the gene duplication events which led to PP, NPY and PYY formation took place after the time of divergence of fish and mammals.

Molecular diversity and hypoglycemic polypeptide-P content of Momordica charantia in different accessions and different seasons.

Science.gov (United States)

Tian, Miao; Zeng, Xiang-Qing; Song, Huan-Lei; Hu, Shan-Xin; Wang, Fu-Jun; Zhao, Jian; Hu, Zhi-Bi

2015-04-01

Momordica charantia (MC) has been used for treating diabetes mellitus from ancient times in Asia, Africa and South America. There are many MC accessions in local markets. Polypeptide-P as a main hypoglycemic component in MC was first studied in this experiment to illustrate the different contents in MC of different accessions and different harvesting times. Nineteen MC accessions collected from different regions were clustered into three groups using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) molecular markers. Content of polypeptide-P in the tested MC accessions was detected by western blot (WB) method. The WB results revealed that polypeptide-P was detected in MC accessions harvested in June and July but not in September and October. Furthermore, Polypeptide-P content corresponded well with the MC accessions. Our results suggest that the MC accessions and the harvesting times or the weather during harvest play significant roles in high content of polypeptide-P. © 2014 Society of Chemical Industry.

Cotranslational structure acquisition of nascent polypeptides monitored by NMR spectroscopy.

Science.gov (United States)

Eichmann, Cédric; Preissler, Steffen; Riek, Roland; Deuerling, Elke

2010-05-18

The folding of proteins in living cells may start during their synthesis when the polypeptides emerge gradually at the ribosomal exit tunnel. However, our current understanding of cotranslational folding processes at the atomic level is limited. We employed NMR spectroscopy to monitor the conformation of the SH3 domain from alpha-spectrin at sequential stages of elongation via in vivo ribosome-arrested (15)N,(13)C-labeled nascent polypeptides. These nascent chains exposed either the entire SH3 domain or C-terminally truncated segments thereof, thus providing snapshots of the translation process. We show that nascent SH3 polypeptides remain unstructured during elongation but fold into a compact, native-like beta-sheet assembly when the entire sequence information is available. Moreover, the ribosome neither imposes major conformational constraints nor significantly interacts with exposed unfolded nascent SH3 domain moieties. Our data provide evidence for a domainwise folding of the SH3 domain on ribosomes without significant population of folding intermediates. The domain follows a thermodynamically favorable pathway in which sequential folding units are stabilized, thus avoiding kinetic traps during the process of cotranslational folding.

Nanostructured complexes of polyelectrolytes and charged polypeptides

Czech Academy of Sciences Publication Activity Database

Müller, M.; Ouyang, W.; Bohatá, Karolína; Kessler, B.

2010-01-01

Roč. 12, Sp. Iss. 9 (2010), B519-B528 ISSN 1438-1656. [Sino-German Symposium on Advanced Biomedical Nanostructures /1./. Jena, 26.10.2009-30.10.2009] Institutional research plan: CEZ:AV0Z40500505 Keywords : situ ATR-FTIR * alpha-helical polypeptides * multilayer films Subject RIV: CD - Macromolecular Chemistry Impact factor: 1.746, year: 2010

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

Science.gov (United States)

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

1985-01-16

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

Investigation of the pathophysiological mechanisms of migraine attacks induced by pituitary adenylate cyclase-activating polypeptide-38

DEFF Research Database (Denmark)

Amin, Faisal Mohammad; Hougaard, Anders; Schytz, Henrik W

2014-01-01

Pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) and vasoactive intestinal polypeptide are structurally and functionally closely related but show differences in migraine-inducing properties. Mechanisms responsible for the difference in migraine induction are unknown. Here, for the ...

The Generation of Dehydroalanine Residues in Protonated Polypeptides: Ion/Ion Reactions for Introducing Selective Cleavages

Science.gov (United States)

Peng, Zhou; Bu, Jiexun; McLuckey, Scott A.

2017-09-01

We examine a gas-phase approach for converting a subset of amino acid residues in polypeptide cations to dehydroalanine (Dha). Subsequent activation of the modified polypeptide ions gives rise to specific cleavage N-terminal to the Dha residue. This process allows for the incorporation of selective cleavages in the structural characterization of polypeptide ions. An ion/ion reaction within the mass spectrometer between a multiply protonated polypeptide and the sulfate radical anion introduces a radical site into the multiply protonated polypeptide reactant. Subsequent collisional activation of the polypeptide radical cation gives rise to radical side chain loss from one of several particular amino acid side chains (e.g., leucine, asparagine, lysine, glutamine, and glutamic acid) to yield a Dha residue. The Dha residues facilitate preferential backbone cleavages to produce signature c- and z-ions, demonstrated with cations derived from melittin, mechano growth factor (MGF), and ubiquitin. The efficiencies for radical side chain loss and for subsequent generation of specific c- and z-ions have been examined as functions of precursor ion charge state and activation conditions using cations of ubiquitin as a model for a small protein. It is noted that these efficiencies are not strongly dependent on ion trap collisional activation conditions but are sensitive to precursor ion charge state. Moderate to low charge states show the greatest overall yields for the specific Dha cleavages, whereas small molecule losses (e.g., water/ammonia) dominate at the lowest charge states and proton catalyzed amide bond cleavages that give rise to b- and y-ions tend to dominate at high charge states. [Figure not available: see fulltext.

Pituitary adenylate cyclase activating polypeptide reduces A-type K+ currents and caspase activity in cultured adult mouse olfactory neurons.

Science.gov (United States)

Han, P; Lucero, M T

2005-01-01

Pituitary adenylate cyclase activating polypeptide has been shown to reduce apoptosis in neonatal cerebellar and olfactory receptor neurons, however the underlying mechanisms have not been elucidated. In addition, the neuroprotective effects of pituitary adenylate cyclase activating polypeptide have not been examined in adult tissues. To study the effects of pituitary adenylate cyclase activating polypeptide on neurons in apoptosis, we measured caspase activation in adult olfactory receptor neurons in vitro. Interestingly, we found that the protective effects of pituitary adenylate cyclase activating polypeptide were related to the absence of a 4-aminopyridine (IC50=144 microM) sensitive rapidly inactivating potassium current often referred to as A-type current. In the presence of 40 nM pituitary adenylate cyclase activating polypeptide 38, both A-type current and activated caspases were significantly reduced. A-type current reduction by pituitary adenylate cyclase activating polypeptide was blocked by inhibiting the phospholipase C pathway, but not the adenylyl cyclase pathway. Our observation that 5 mM 4-aminopyridine mimicked the caspase inhibiting effects of pituitary adenylate cyclase activating polypeptide indicates that A-type current is involved in apoptosis. This work contributes to our growing understanding that potassium currents are involved with the activation of caspases to affect the balance between cell life and death.

Polysaccharide-producing microalgae

Energy Technology Data Exchange (ETDEWEB)

Braud, J.P.; Chaumont, D.; Gudin, C.; Thepenier, C.; Chassin, P.; Lemaire, C.

1982-11-01

The production of extracellular polysaccharides is studied with Nostoc sp (cyanophycus), Porphiridium cruentum, Rhodosorus marinus, Rhodella maculata (rhodophyci) and Chlamydomonas mexicana (chlorophycus). The polysaccharides produced are separated by centrifugation of the culture then precipitation with alcohol. Their chemical structure was studied by infrared spectrometry and acid hydrolysis. By their rheological properties and especially their insensitivity to temperatrure and pH variations the polysaccharides produced by Porphryridium cruentum and Rhodella maculata appear as suitable candidates for industrial applications.

Molecular cloning and protein structure of a human blood group Rh polypeptide

International Nuclear Information System (INIS)

Cherif-Zahar, B.; Bloy, C.; Le Van Kim, C.; Blanchard, D.; Bailly, P.; Hermand, P.; Salmon, C.; Cartron, J.P.; Colin, Y.

1990-01-01

cDNA clones encoding a human blood group Rh polypeptide were isolated from a human bone marrow cDNA library by using a polymerase chain reaction-amplified DNA fragment encoding the known common N-terminal region of the Rh proteins. The entire primary structure of the Rh polypeptide has been deduced from the nucleotide sequence of a 1384-base-pair-long cDNA clone. Translation of the open reading frame indicates that the Rh protein is composed of 417 amino acids, including the initiator methionine, which is removed in the mature protein, lacks a cleavable N-terminal sequence, and has no consensus site for potential N-glycosylation. The predicted molecular mass of the protein is 45,500, while that estimated for the Rh protein analyzed in NaDodSO 4 /polyacrylamide gels is in the range of 30,000-32,000. These findings suggest either that the hydrophobic Rh protein behaves abnormally on NaDodSO 4 gels or that the Rh mRNA may encode a precursor protein, which is further matured by a proteolytic cleavage of the C-terminal region of the polypeptide. Hydropathy analysis and secondary structure predictions suggest the presence of 13 membrane-spanning domains, indicating that the Rh polypeptide is highly hydrophobic and deeply buried within the phospholipid bilayer. These results suggest that the expression of the Rh gene(s) might be restricted to tissues or cell lines expressing erythroid characters

Phase transition in polypeptides: a step towards the understanding of protein folding

DEFF Research Database (Denmark)

Yakubovich, Alexander V.; Solov'yov, Ilia; Solov'yov, Andrey V.

2006-01-01

We present a formalism which turns out to be very successful in the description of the polypeptide folding. We consider this process as a first-order phase transition and develop a theory which is free of model parameters and is based solely on fundamental physical principles. It describes...... essential thermodynamical properties of the system such as heat capacity, the phase transition temperature and others from the analysis of the polypeptide potential energy surface calculated within ab initio density functional theory and parameterized by two dihedral angles. This problem is viewed...

Immunoassay of serum polypeptide hormones by using 125I-labelled anti(-immunoglobulin G) antibodies.

Science.gov (United States)

Beck, P; Nicholas, H

1975-03-01

1. A technique for indirectly labelling antibodies to polypeptide hormones, by combining them with radioactively labelled anti-(immunoglobulin G) is described. (a) 125I-labelled anti-(rabbit immunoglobulin G) and anti-(guinea-pig immunoglobulin G) antibodies with high specific radioactivity were prepared after purification of the antibodies on immunoadsorbents containing the respective antigens. (b) Rabbit immunoglobulin G antibodies to human growth hormone, porcine glucagon and guinea-pig immunoglobulin G antibodies to bovine insulin and bovine parathyroid hormone were combined with immunoadsorbents containing the respective polypeptide hormone antigen. (c) The immunoglobulin G antibodies to the polypeptide hormones were reacted with 125-I-labelled anti-(immunoglobulin G) antibodies directed against the appropriate species of immunoglobulin G,and the anti-hormone antibodies were combined with the hormone-containing immunoadsorbent. (d) 125I-labelled anti-(immunoglobulin G) antibodies and anti-hormone antibodies were simultaneously eluted from the hormone-containing immunoadsorbent by dilute HCl, pH 2.0. After elution the anti-(immunoglobulin G) antibodies and antihormone antibodies were allowed to recombine at pH 8.0 and 4 degrees C. 2. The resultant immunoglobulin G-anti-immunoglobulin G complex was used in immunoradiometric (labelled antibody) and two-site assays of the respective polypeptide hormone. 3. By using these immunoassays, concentrations down to 90pg of human growth hormone/ml, 100 pg of bovine insulin/ml, 80 pg of bovine parathyroid hormone/ml and 150 pg of glucagon/ml were readily detected. Assays of human plasma for growth hormone and insulin by these methods showed good agreement with results obtained by using a directly 125I-labelled anti-hormone antibody in an immunoradiometric assay of human growth hormone or by radioimmunoassay of human insulin. 4. The method described allows immunoradiometric or two-site assays to be performed starting with as

Sequence Directionality Dramatically Affects LCST Behavior of Elastin-Like Polypeptides.

Science.gov (United States)

Li, Nan K; Roberts, Stefan; Quiroz, Felipe Garcia; Chilkoti, Ashutosh; Yingling, Yaroslava G

2018-04-30

Elastin-like polypeptides (ELP) exhibit an inverse temperature transition or lower critical solution temperature (LCST) transition phase behavior in aqueous solutions. In this paper, the thermal responsive properties of the canonical ELP, poly(VPGVG), and its reverse sequence poly(VGPVG) were investigated by turbidity measurements of the cloud point behavior, circular dichroism (CD) measurements, and all-atom molecular dynamics (MD) simulations to gain a molecular understanding of mechanism that controls hysteretic phase behavior. It was shown experimentally that both poly(VPGVG) and poly(VGPVG) undergo a transition from soluble to insoluble in aqueous solution upon heating above the transition temperature ( T t ). However, poly(VPGVG) resolubilizes upon cooling below its T t , whereas the reverse sequence, poly(VGPVG), remains aggregated despite significant undercooling below the T t . The results from MD simulations indicated that a change in sequence order results in significant differences in the dynamics of the specific residues, especially valines, which lead to extensive changes in the conformations of VPGVG and VGPVG pentamers and, consequently, dissimilar propensities for secondary structure formation and overall structure of polypeptides. These changes affected the relative hydrophilicities of polypeptides above T t , where poly(VGPVG) is more hydrophilic than poly(VPGVG) with more extended conformation and larger surface area, which led to formation of strong interchain hydrogen bonds responsible for stabilization of the aggregated phase and the observed thermal hysteresis for poly(VGPVG).

Chronic changes in pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulation in rats.

Science.gov (United States)

Han, Xun; Ran, Ye; Su, Min; Liu, Yinglu; Tang, Wenjing; Dong, Zhao; Yu, Shengyuan

2017-01-01

Background Preclinical experimental studies revealed an acute alteration of pituitary adenylate cyclase-activating polypeptide in response to a single activation of the trigeminovascular system, which suggests a potential role of pituitary adenylate cyclase-activating polypeptide in the pathogenesis of migraine. However, changes in pituitary adenylate cyclase-activating polypeptide after repeated migraine-like attacks in chronic migraine are not clear. Therefore, the present study investigated chronic changes in pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulations in the rat. Methods A rat model of chronic migraine was established by repeated chemical dural stimulations using an inflammatory soup for a different numbers of days. The pituitary adenylate cyclase-activating polypeptide levels were quantified in plasma, the trigeminal ganglia, and the trigeminal nucleus caudalis using radioimmunoassay and Western blotting in trigeminal ganglia and trigeminal nucleus caudalis tissues. Western blot analysis and real-time polymerase chain reaction were used to measure the protein and mRNA expression of pituitary adenylate cyclase-activating polypeptide-related receptors (PAC1, VPAC1, and VPAC2) in the trigeminal ganglia and trigeminal nucleus caudalis to identify changes associated with repetitive applications of chemical dural stimulations. Results All rats exhibited significantly decreased periorbital nociceptive thresholds to repeated inflammatory soup stimulations. Radioimmunoassay and Western blot analysis demonstrated significantly decreased pituitary adenylate cyclase-activating polypeptide levels in plasma and trigeminal ganglia after repetitive chronic inflammatory soup stimulation. Protein and mRNA analyses of pituitary adenylate cyclase-activating polypeptide-related receptors demonstrated significantly increased PAC1 receptor protein and mRNA expression in the trigeminal ganglia, but not

Adhesive polypeptides of Staphylococcus aureus identified using a novel secretion library technique in Escherichia coli

Directory of Open Access Journals (Sweden)

Holm Liisa

2011-05-01

Full Text Available Abstract Background Bacterial adhesive proteins, called adhesins, are frequently the decisive factor in initiation of a bacterial infection. Characterization of such molecules is crucial for the understanding of bacterial pathogenesis, design of vaccines and development of antibacterial drugs. Because adhesins are frequently difficult to express, their characterization has often been hampered. Alternative expression methods developed for the analysis of adhesins, e.g. surface display techniques, suffer from various drawbacks and reports on high-level extracellular secretion of heterologous proteins in Gram-negative bacteria are scarce. These expression techniques are currently a field of active research. The purpose of the current study was to construct a convenient, new technique for identification of unknown bacterial adhesive polypeptides directly from the growth medium of the Escherichia coli host and to identify novel proteinaceous adhesins of the model organism Staphylococcus aureus. Results Randomly fragmented chromosomal DNA of S. aureus was cloned into a unique restriction site of our expression vector, which facilitates secretion of foreign FLAG-tagged polypeptides into the growth medium of E. coli ΔfliCΔfliD, to generate a library of 1663 clones expressing FLAG-tagged polypeptides. Sequence and bioinformatics analyses showed that in our example, the library covered approximately 32% of the S. aureus proteome. Polypeptides from the growth medium of the library clones were screened for binding to a selection of S. aureus target molecules and adhesive fragments of known staphylococcal adhesins (e.g coagulase and fibronectin-binding protein A as well as polypeptides of novel function (e.g. a universal stress protein and phosphoribosylamino-imidazole carboxylase ATPase subunit were detected. The results were further validated using purified His-tagged recombinant proteins of the corresponding fragments in enzyme-linked immunoassay and

Identification of UDPG-binding polypeptides and purified (1,3)-β-glucan synthase by photoaffinity labelling with 5-azido-UDPG

International Nuclear Information System (INIS)

Frost, D.J.; Wu, A.; Read, S.M.; Wasserman, B.P.; Drake, R.R.; Haley, B.E.

1989-01-01

The photoaffinity probe 5-azido-uridine 5'-β-[ 32 P]-diphosphate glucose was used to identify the major UDPG-binding polypeptide of red beet (1,3)-β-glucan synthase. Glucan synthase was purified from plasma membranes by sequential solubilization with CHAPS followed by product entrapment. Two major polypeptides at 72 and 54 kD were labelled by probe. Labelling of both was abolished with increasing levels of cold UDPG. However, labelling of the 54 kD polypeptide was dependent upon the presence of divalent cations. These data suggest that the 54 kD polypeptide is a substrate-binding and cation-regulated component of the glucan synthase complex

The interdomain flexible linker of the polypeptide GalNAc transferases dictates their long-range glycosylation preferences

DEFF Research Database (Denmark)

Rivas, Matilde De Las; Lira-Navarrete, Erandi; Daniel, Earnest James Paul

2017-01-01

The polypeptide GalNAc-transferases (GalNAc-Ts), that initiate mucin-type O-glycosylation, consist of a catalytic and a lectin domain connected by a flexible linker. In addition to recognizing polypeptide sequence, the GalNAc-Ts exhibit unique long-range N- A nd/or C-terminal prior glycosylation ...

High-yield recombinant expression and purification of marginally soluble, short elastin-like polypeptides.

Science.gov (United States)

Bahniuk, Markian S; Alshememry, Abdullah K; Unsworth, Larry D

2016-12-01

The protocol described here is designed as an extension of existing techniques for creating elastin-like polypeptides. It allows for the expression and purification of elastin-like polypeptide (ELP) constructs that are poorly expressed or have very low transition temperatures. DNA concatemerization has been modified to reduce issues caused by methylation sensitivity and inefficient cloning. Linearization of the modified expression vector has been altered to greatly increase cleavage efficiency. The purification regimen is based upon using denaturing metal affinity chromatography to fully solubilize and, if necessary, pre-concentrate the target peptide before purification by inverse temperature cycling (ITC). This protocol has been used to express multiple leucine-containing elastin-like polypeptides, with final yields of 250-660 mg per liter of cells, depending on the specific construct. This was considerably greater than previously reported yields for similar ELPs. Due to the relative hydrophobicity of the tested constructs, even compared with commonly employed ELPs, conventional methods would not have been able to be purify these peptides.

Induction of salivary polypeptides associated with parotid hypertrophy by gallotannins administered topically into the mouse mouth.

Science.gov (United States)

Gho, Francesca; Peña-Neira, Alvaro; López-Solís, Remigio O

2007-02-01

Isoproterenol-induced salivary polypeptides (IISP), a group of proline-rich proteins synthesized by mouse parotids, have been considered as markers for isoproterenol-induced parotid hypertrophy. Rodents fed diets containing high-tannin cereals (sorghum), also develop parotid hypertrophy. To test whether tannins are directly involved in provoking sialotrophic growth, we studied the effect of intraperitoneal and topical oral administrations of tannic acid (TA) on the induction of IISP polypeptides in endogamic mice (A/Snell). TA was characterized by HPLC chromatography and spectral analysis and shown to be composed solely of gallotannins, a complex family of glucose and gallic acid esters. IISP polypeptides were monitored in saliva by SDS-polyacrylamide gel electrophoresis during 36 h after ending TA stimulation. Single daily intraperitoneal administrations of TA for 3 consecutive days (0.033 mg/g bw/day), at variance of parallel administrations of isoproterenol (0.042 mg/g bw/day) failed to induce IISP polypeptides. However, repeated topical applications of TA into the mouse mouths (1.21 mg/g bw divided into three equal doses given at 4-h intervals within a single day) resulted in unequivocal induction of IISP polypeptides. That response was clearly intensified by increasing the stimulation frequency to eight equivalent doses given at 1.5-h intervals within a single day (corresponding to 3.23 mg/g bw) and even further by repeating this protocol for 3 days. Under these productive schemes of stimulations by TA, electrophoretic fractionation of parotid homogenates showed new polypeptide bands migrating in parallel to salivary IISP. These results suggest that topically administered gallotannins are effective inducers of trophic growth in mouse parotids.

Improvement of Learning and Memory Induced by Cordyceps Polypeptide Treatment and the Underlying Mechanism

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Guangxin Yuan

2018-01-01

Full Text Available Our previous research revealed that Cordyceps militaris can improve the learning and memory, and although the main active ingredient should be its polypeptide complexes, the underlying mechanism of its activity remains poorly understood. In this study, we explored the mechanisms by which Cordyceps militaris improves learning and memory in a mouse model. Mice were given scopolamine hydrobromide intraperitoneally to establish a mouse model of learning and memory impairment. The effects of Cordyceps polypeptide in this model were tested using the Morris water maze test; serum superoxide dismutase activity; serum malondialdehyde levels; activities of acetyl cholinesterase, Na+-k+-ATPase, and nitric oxide synthase; and gamma aminobutyric acid and glutamate contents in brain tissue. Moreover, differentially expressed genes and the related cellular signaling pathways were screened using an mRNA expression profile chip. The results showed that the genes Pik3r5, Il-1β, and Slc18a2 were involved in the effects of Cordyceps polypeptide on the nervous system of these mice. Our findings suggest that Cordyceps polypeptide may improve learning and memory in the scopolamine-induced mouse model of learning and memory impairment by scavenging oxygen free radicals, preventing oxidative damage, and protecting the nervous system.

Immune-tolerant elastin-like polypeptides (iTEPs) and their application as CTL vaccine carriers.

Science.gov (United States)

Cho, S; Dong, S; Parent, K N; Chen, M

2016-01-01

Cytotoxic T lymphocyte (CTL) vaccine carriers are known to enhance the efficacy of vaccines, but a search for more effective carriers is warranted. Elastin-like polypeptides (ELPs) have been examined for many medical applications but not as CTL vaccine carriers. We aimed to create immune tolerant ELPs using a new polypeptide engineering practice and create CTL vaccine carriers using the ELPs. Four sets of novel ELPs, termed immune-tolerant elastin-like polypeptide (iTEP) were generated according to the principles dictating humoral immunogenicity of polypeptides and phase transition property of ELPs. The iTEPs were non-immunogenic in mice. Their phase transition feature was confirmed through a turbidity assay. An iTEP nanoparticle (NP) was assembled from an amphiphilic iTEP copolymer plus a CTL peptide vaccine, SIINFEKL. The NP facilitated the presentation of the vaccine by dendritic cells (DCs) and enhanced vaccine-induced CTL responses. A new ELP design and development practice was established. The non-canonical motif and the immune tolerant nature of the iTEPs broaden our insights about ELPs. ELPs, for the first time, were successfully used as carriers for CTL vaccines. It is feasible to concurrently engineer both immune-tolerant and functional peptide materials. ELPs are a promising type of CTL vaccine carriers.

Kinetics of Internal-Loop Formation in Polypeptide Chains: A Simulation Study

Science.gov (United States)

Doucet, Dana; Roitberg, Adrian; Hagen, Stephen J.

2007-01-01

The speed of simple diffusional motions, such as the formation of loops in the polypeptide chain, places one physical limit on the speed of protein folding. Many experimental studies have explored the kinetics of formation of end-to-end loops in polypeptide chains; however, protein folding more often requires the formation of contacts between interior points on the chain. One expects that, for loops of fixed contour length, interior loops will form more slowly than end-to-end loops, owing to the additional excluded volume associated with the “tails”. We estimate the magnitude of this effect by generating ensembles of randomly coiled, freely jointed chains, and then using the theory of Szabo, Schulten, and Schulten to calculate the corresponding contact formation rates for these ensembles. Adding just a few residues, to convert an end-to-end loop to an internal loop, sharply decreases the contact rate. Surprisingly, the relative change in rate increases for a longer loop; sufficiently long tails, however, actually reverse the effect and accelerate loop formation slightly. Our results show that excluded volume effects in real, full-length polypeptides may cause the rates of loop formation during folding to depart significantly from the values derived from recent loop-formation experiments on short peptides. PMID:17208979

Role of Side-Chain Molecular Features in Tuning Lower Critical Solution Temperatures (LCSTs) of Oligoethylene Glycol Modified Polypeptides.

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Gharakhanian, Eric G; Deming, Timothy J

2016-07-07

A series of thermoresponsive polypeptides has been synthesized using a methodology that allowed facile adjustment of side-chain functional groups. The lower critical solution temperature (LCST) properties of these polymers in water were then evaluated relative to systematic molecular modifications in their side-chains. It was found that in addition to the number of ethylene glycol repeats in the side-chains, terminal and linker groups also have substantial and predictable effects on cloud point temperatures (Tcp). In particular, we found that the structure of these polypeptides allowed for inclusion of polar hydroxyl groups, which significantly increased their hydrophilicity and decreased the need to use long oligoethylene glycol repeats to obtain LCSTs. The thioether linkages in these polypeptides were found to provide an additional structural feature for reversible switching of both polypeptide conformation and thermoresponsive properties.

Synthesis and Characterization of Methyl Cellulose/Keratin Hydrolysate Composite Membranes

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Bernd M. Liebeck

2017-03-01

Full Text Available It is known that aqueous keratin hydrolysate solutions can be produced from feathers using superheated water as solvent. This method is optimized in this study by varying the time and temperature of the heat treatment in order to obtain a high solute content in the solution. With the dissolved polypeptides, films are produced using methyl cellulose as supporting material. Thereby, novel composite membranes are produced from bio-waste. It is expected that these materials exhibit both protein and polysaccharide properties. The influence of the embedded keratin hydrolysates on the methyl cellulose structure is investigated using Fourier transform infrared spectroscopy (FTIR and wide angle X-ray diffraction (WAXD. Adsorption peaks of both components are present in the spectra of the membranes, while the X-ray analysis shows that the polypeptides are incorporated into the semi-crystalline methyl cellulose structure. This behavior significantly influences the mechanical properties of the composite films as is shown by tensile tests. Since further processing steps, e.g., crosslinking, may involve a heat treatment, thermogravimetric analysis (TGA is applied to obtain information on the thermal stability of the composite materials.

CDNA encoding a polypeptide including a hevein sequence

Science.gov (United States)

Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

1995-03-21

A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

Strategies to Fabricate Polypeptide-Based Structures via Ring-Opening Polymerization of N-Carboxyanhydrides

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Carmen M. González-Henríquez

2017-10-01

Full Text Available In this review, we provide a general and clear overview about the different alternatives reported to fabricate a myriad of polypeptide architectures based on the ring-opening polymerization of N-carbonyanhydrides (ROP NCAs. First of all, the strategies for the preparation of NCA monomers directly from natural occurring or from modified amino acids are analyzed. The synthetic alternatives to prepare non-functionalized and functionalized NCAs are presented. Protection/deprotection protocols, as well as other functionalization chemistries are discussed in this section. Later on, the mechanisms involved in the ROP NCA polymerization, as well as the strategies developed to reduce the eventually occurring side reactions are presented. Finally, a general overview of the synthetic strategies described in the literature to fabricate different polypeptide architectures is provided. This part of the review is organized depending on the complexity of the macromolecular topology prepared. Therefore, linear homopolypeptides, random and block copolypeptides are described first. The next sections include cyclic and branched polymers such as star polypeptides, polymer brushes and highly branched structures including arborescent or dendrigraft structures.

Glucose-dependent Insulinotropic Polypeptide

DEFF Research Database (Denmark)

Christensen, Mikkel B; Calanna, Salvatore; Holst, Jens Juul

2014-01-01

CONTEXT: Patients with type 2 diabetes mellitus (T2DM) have clinically relevant disturbances in the effects of the hormone glucose-dependent insulinotropic polypeptide (GIP). OBJECTIVE: We aimed to evaluate the importance of the prevailing plasma glucose levels for the effect of GIP on responses......: During fasting glycemia (plasma glucose ∼8 mmol/L), GIP elicited significant increments in both insulin and glucagon levels, resulting in neutral effects on plasma glucose. During insulin-induced hypoglycemia (plasma glucose ∼3 mmol/L), GIP elicited a minor early-phase insulin response and increased...... glucagon levels during the initial 30 minutes, resulting in less glucose needed to be infused to maintain the clamp (29 ± 8 vs 49 ± 12 mg × kg(-1), P glucose ∼12 mmol/L), GIP augmented insulin secretion throughout the clamp, with slightly less glucagon...

Effects on DPPH inhibition of egg-white protein polypeptides treated by pulsed electric field technology.

Science.gov (United States)

Wang, Ke; Wang, Jia; Liu, Bolong; Lin, Songyi; Zhao, Ping; Liu, Jingbo; Jones, Gregory; Huang, Hsiang-Chi

2013-05-01

Egg-white protein polypeptides are potentially used as a functional ingredient in food products. In this study, the effects on DPPH inhibition of egg-white protein polypeptides ranging from 10 to 30 kDa treated by pulsed electric field (PEF) technology were investigated. 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) inhibition (%) was used to evaluate the antioxidant activity of polypeptides. In order to develop and optimize a pulsed electric field (PEF) mathematical model for improving the antioxidant activity, we have investigated three variables, including concentration (6, 8 and 10 mg mL(-1)), electric field intensity (10, 20 and 30 kV cm(-1)) and pulse frequency (2000, 2350 and 2700 Hz) and subsequently optimized them by response surface methodology (RSM). The concentration (8 mg mL(-1)), electric field intensity (10 kV cm(-1)) and pulse frequency (2000 Hz) were found to be the optimal conditions under which the DPPH inhibition increased 28.44%, compared to the sample without PEF treatment. Both near-infrared spectroscopy (NIR) and mid-infrared spectroscopy (MIR) were used to analyze the change of functional groups. The results showed that PEF technology could improve the antioxidant activity of antioxidant polypeptides from egg-white protein under the optimized conditions. © 2012 Society of Chemical Industry.

Polymer-Block-Polypeptides and Polymer-Conjugated Hybrid Materials as Stimuli-Responsive Nanocarriers for Biomedical Applications.

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John, Johnson V; Johnson, Renjith P; Heo, Min Seon; Moon, Byeong Kyu; Byeon, Seong Jin; Kim, Il

2015-01-01

Stimuli-responsive nanocarriers are a class of soft materials that includes natural polymers, synthetic polymers, and polypeptides. Recently, modern synthesis tools such as atom transfer radical polymerization, reversible addition-fragmentation chain transfer polymerization, nitroxide-mediated radical polymerization, ring-opening polymerization of α-amino acid N-carboxyanhydrides, and various "click" chemistry strategies were simultaneously employed for the design and synthesis of nanosized drug delivery vehicles. Importantly, the research focused on the improvement of the nanocarrier targetability and the site-specific, triggered release of therapeutics with high drug loading efficiency and minimal drug leakage during the delivery to specific targets. In this context, nanocarriers responsive to common stimuli such as pH, temperature, redox potential, light, etc. have been widely used for the controlled delivery of therapeutics to pathological sites. Currently, different synthesis and self-assembly strategies improved the drug loading efficacy and targeted delivery of therapeutic agents to the desired site. In particular, polypeptide-containing hybrid materials have been developed for the controlled delivery of therapeutic agents. Therefore, stimuli-sensitive synthetic polypeptide-based materials have been extensively investigated in recent years. This review focuses on recent advances in the development of polymer-block-polypeptides and polymer-conjugated hybrid materials that have been designed and evaluated for various stimuli-responsive drug and gene delivery applications.

Smart systems related to polypeptide sequences

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Lourdes Franco

2016-03-01

Full Text Available Increasing interest for the application of polypeptide-based smart systems in the biomedical field has developed due to the advantages given by the peptidic sequence. This is due to characteristics of these systems, which include: biocompatibility, potential control of degradation, capability to provide a rich repertoire of biologically specific interactions, feasibility to self-assemble, possibility to combine different functionalities, and capability to give an environmentally responsive behavior. Recently, applications concerning the development of these systems are receiving greater attention since a targeted and programmable release of drugs (e.g. anti-cancer agents can be achieved. Block copolymers are discussed due to their capability to render differently assembled architectures. Hybrid systems based on silica nanoparticles are also discussed. In both cases, the selected systems must be able to undergo fast changes in properties like solubility, shape, and dissociation or swelling capabilities. This review is structured in different chapters which explain the most recent advances on smart systems depending on the stimuli to which they are sensitive. Amphiphilic block copolymers based on polyanionic or polycationic peptides are, for example, typically employed for obtaining pH-responsive systems. Elastin-like polypeptides are usually used as thermoresponsive polymers, but performance can be increased by using techniques which utilize layer-by-layer electrostatic self-assembly. This approach offers a great potential to create multilayered systems, including nanocapsules, with different functionality. Recent strategies developed to get redox-, magnetic-, ultrasound-, enzyme-, light- and electric-responsive systems are extensively discussed. Finally, some indications concerning the possibilities of multi-responsive systems are discussed.

Folding and self-assembly of polypeptides: Dynamics and thermodynamics from molecular simulation

Science.gov (United States)

Fluitt, Aaron Michael

Empowered by their exquisite three-dimensional structures, or "folds," proteins carry out biological tasks with high specificity, efficiency, and fidelity. The fold that optimizes biological function represents a stable configuration of the constituent polypeptide molecule(s) under physiological conditions. Proteins and polypeptides are not static, however: battered by thermal motion, they explore a distribution of folds that is determined by the sequence of amino acids, the presence and identity of other molecules, and the thermodynamic conditions. In this dissertation, we apply molecular simulation techniques to the study of two polypeptides that have unusually diffuse distributions of folds under physiological conditions: polyglutamine (polyQ) and islet amyloid polypeptide (IAPP). Neither polyQ nor IAPP adopts a predominant fold in dilute aqueous solution, but at sufficient concentrations, both are prone to self-assemble into stable, periodic, and highly regular aggregate structures known as amyloid. The appearance of amyloid deposits of polyQ in the brain, and of IAPP in the pancreas, are associated with Huntington's disease and type 2 diabetes, respectively. A molecular view of the mechanism(s) by which polyQ and IAPP fold and self-assemble will enhance our understanding of disease pathogenesis, and it has the potential to accelerate the development of therapeutics that target early-stage aggregates. Using molecular simulations with spatial and temporal resolution on the atomic scale, we present analyses of the structural distributions of polyQ and IAPP under various conditions, both in and out of equilibrium. In particular, we examine amyloid fibers of polyQ, the IAPP dimer in solution, and single IAPP fragments at a lipid bilayer. We also benchmark the molecular models, or "force fields," available for such studies, and we introduce a novel simulation algorithm.

Bioleaching of UO22+ ions from poor uranium ores by means of cyanobacteria

International Nuclear Information System (INIS)

Cecal, A.; Humelnicu, D.; Popa, K.; Palamaru, I.; Nemtoi, G.; Rudic, V.; Gulea, A.

2000-01-01

Uranium (VI) leaching, as uranyl ions, from ores with a poor content in util minerals, using some algae as: Porphyridium cruentum (Smith and Soerly) Naegeli, Spirulina platensis CNM-CB-02 and Nostoc linkia (Roth) Born and Flah was studied. The basic composition of these ores allowed the self-maintenance and self-development of these microorganisms, which have facilitated then the leaching of the uranium (VI) as UO 2 2+ ions. The microbial leaching degree was comprised between 40-90%, depending on the used alga and experimental conditions. (author)

Light Scattering Characterization of Elastin-Like Polypeptide Trimer Micelles

Science.gov (United States)

Tsuper, Ilona; Terrano, Daniel; Maraschky, Adam; Holland, Nolan; Streletzky, Kiril

The elastin-like polypeptides (ELP) nanoparticles are composed of three-armed star polypeptides connected by a negatively charged foldon. Each of the three arms extending from the foldon domain includes 20 repeats of the (GVGVP) amino acid sequence. The ELP polymer chains are soluble at room temperature and become insoluble at the transition temperature (close to 50 ° C), forming micelles. The size and shape of the micelle are dependent on the temperature and the pH of the solution, and on the concentration of the phosphate buffered saline (PBS). The depolarized dynamic light scattering (DDLS) was employed to study the structure and dynamics of micelles at 62 ° C. The solution was maintained at an approximate pH level of 7.3 - 7.5, while varying PBS concentration. At low salt concentrations (60 mM) displayed an apparent elongation of the micelles evident by a significant VH signal, along with a surge in the apparent Rh. A model of micelle growth (and potential elongation) with increase in salt concentration is considered.

In vitro and in vivo phosphorylation of polypeptides in plasma membrane and tonoplast-enriched fractions from barley roots

International Nuclear Information System (INIS)

Garbarino, J.E.; Hurkman, W.J.; Tanaka, C.K.; DuPont, F.M.

1991-01-01

Phosphorylation of polypeptides in membrane fractions from barley (Hordeum vulgare L. cv CM 72) roots was compared in in vitro and in vivo assays to assess the potential role of protein kinases in modification of membrane transport. Membrane fractions enriched in endoplasmic reticulum, tonoplast, and plasma membrane were isolated using sucrose gradients and the membrane polypeptides separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis. When the membrane fractions were incubated with γ[p 32 P]ATP, phosphorylation occurred almost exclusively in the plasma membrane fraction. Phosphorylation of a band at 38 kilodaltons increased as the concentration of Mg 2+ was decreased from millimolar to micromolar levels. Phosphorylation of bands at 125, 86, 58, 46 and 28 kilodaltons required millimolar Mg 2+ concentrations and was greatly enhanced by Ca 2+ . When roots of intact plants were labeled with [ 32 P]orthophosphate, polypeptides at approximately 135, 166, 90, 46 to 53, 32, 28, and 19 kilodaltons were labeled in the plasma membrane fraction and polypeptides at approximately 73, 66, and 48 kilodaltons were labeled in the tonoplast fraction. Treatment of the roots of intact plants with 150 millimolar NaCl resulted in increased phosphorylation of some polypeptides while treatment with 100 mM NaCl had no effect

In vivo guided vascular regeneration with a non-porous elastin-like polypeptide hydrogel tubular scaffold.

Science.gov (United States)

Mahara, Atsushi; Kiick, Kristi L; Yamaoka, Tetsuji

2017-06-01

Herein, we demonstrate a new approach for small-caliber vascular reconstruction using a non-porous elastin-like polypeptide hydrogel tubular scaffold, based on the concept of guided vascular regeneration (GVR). The scaffolds are composed of elastin-like polypeptide, (Val-Pro-Gly-Ile-Gly) n , for compliance matching and antithrombogenicity and an Arg-Gly-Asp (RGD) motif for connective tissue regeneration. When the polypeptide was mixed with an aqueous solution of β-[Tris(hydroxymethyl)phosphino]propionic acid at 37°C, the polypeptide hydrogel was rapidly formed. The elastic modulus of the hydrogel was 4.4 kPa. The hydrogel tubular scaffold was formed in a mold and reinforced with poly(lactic acid) nanofibers. When tubular scaffolds with an inner diameter of 1 mm and length of 5 mm were implanted into rat abdominal aortae, connective tissue grew along the scaffold luminal surface from the flanking native tissues, resulting in new blood vessel tissue with a thickness of 200 μm in 1 month. In contrast, rats implanted with control scaffolds without the RGD motif died. These results indicate that the non-porous hydrogel tubular scaffold containing the RGD motif effectively induced rapid tissue regeneration and that GVR is a promising strategy for the regeneration of small-diameter blood vessels. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1746-1755, 2017. © 2017 Wiley Periodicals, Inc.

Membrane fractions active in poliovirus RNA replication contain VPg precursor polypeptides

International Nuclear Information System (INIS)

Takegami, T.; Semler, B.L.; Anderson, C.W.; Wimmer, E.

1983-01-01

The poliovirus specific polypeptide P3-9 is of special interest for studies of viral RNA replication because it contains a hydrophobic region and, separated by only seven amino acids from that region, the amino acid sequence of the genome-linked protein VPg. Membraneous complexes of poliovirus-infected HeLa cells that contain poliovirus RNA replicating proteins have been analyzed for the presence of P3-9 by immunoprecipitation. Incubation of a membrane fraction rich in P3-9 with proteinase leaves the C-terminal 69 amino acids of P3-9 intact, an observation suggesting that this portion is protected by its association with the cellular membrane. These studies have also revealed two hitherto undescribed viral polypeptides consisting of amino acid sequences of the P2 andf P3 regions of the polyprotein. Sequence analysis by stepwise Edman degradation show that these proteins are 3b/9 (M/sub r/77,000) and X/9 (M/sub r/50,000). 3b/9 and X/9 are membrane bound and are turned over rapidly and may be direct precursors to proteins P2-X and P3-9 of the RNA replication complex. P2-X, a polypeptide void of hydrophobic amino acid sequences but also found associated with membranes, is rapidly degraded when the membraneous complex is treated with trypsin. It is speculated that P2-X is associated with membranes by its affinity to the N-terminus of P3-9

Liver fatty acid binding protein is the mitosis-associated polypeptide target of a carcinogen in rat hepatocytes

International Nuclear Information System (INIS)

Bassuk, J.A.; Tsichlis, P.N.; Sorof, S.

1987-01-01

Hepatocytes in normal rat liver were found previously to contain a cytoplasmic 14,000-dalton polypeptide (p14) that is associated with mitosis and is the principal early covalent target of activated metabolites of the carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene). The level of immunohistochemically detected p14 was low when growth activity of hepatocytes was low, was markedly elevated during mitosis in normal and regenerating livers, but was very high throughout interphase during proliferation of hyperplastic and malignant hepatocytes induced in rat liver by a carcinogen (N-2-fluorenylacetamide or 3'-methyl-4-dimethylaminoazobenzene). The authors report here that p14 is the liver fatty acid binding protein. The nucleotide sequence of p14 cDNA clones, isolated by screening a rat liver cDNA library in bacteriophage λgt11 using p14 antiserum, was completely identical to part of the sequence reported for liver fatty acid binding protein. Furthermore, the two proteins shared the following properties: size of mRNA, amino acid composition, molecular size according to NaDodSO 4 gel electrophoresis, and electrophoretic mobilities in a Triton X-100/acetic acid/urea gel. The two polypeptides bound oleic acid similarly. Finally, identical elevations of cytoplasmic immunostain were detected specifically in mitotic hepatocytes with either antiserum. The collected findings are suggestive that liver fatty acid binding protein may carry ligands that promote hepatocyte division and may transport certain activated chemical carcinogens

Homoallylglycine residues are superior precursors to orthogonally modified thioether containing polypeptides.

Science.gov (United States)

Perlin, Pesach; Gharakhanian, Eric G; Deming, Timothy J

2018-06-12

Homoallylglycine N-carboxyanhydride, Hag NCA, monomers were synthesized and used to prepare polypeptides containing Hag segments with controllable lengths of up to 245 repeats. Poly(l-homoallylglycine), GHA, was found to adopt an α-helical conformation, which provided good solubility in organic solvents and allowed high yield functionalization of its alkene side-chains via radical promoted addition of thiols. The conformations of these derivatives were shown to be switchable between α-helical and disordered states in aqueous media using thioether alkylation or oxidation reactions. Incorporation of GHA segments into block copolymers with poly(l-methionine), M, segments provided a means to orthogonally modify thioether side-chains different ways in separate copolypeptide domains. This approach allows preparation of functional polypeptides containing discrete domains of oxidized and alkylated thioether containing residues, where chain conformation and functionality of each domain can be independently modified.

Salt- and pH-Triggered Helix-Coil Transition of Ionic Polypeptides under Physiology Conditions.

Science.gov (United States)

Yuan, Jingsong; Zhang, Yi; Sun, Yue; Cai, Zhicheng; Yang, Lijiang; Lu, Hua

2018-06-11

Controlling the helix-coil transition of polypeptides under physiological conditions is an attractive way toward smart functional materials. Here, we report the synthesis of a series of tertiary amine-functionalized ethylene glycol (EG x )-linked polypeptide electrolytes with their secondary structures tunable under physiological conditions. The resultant polymers, denoted as P(EG x DMA-Glu) ( x = 1, 2, and 3), show excellent aqueous solubility (>20 mg/mL) regardless of their charge states. Unlike poly-l-lysine that can form a helix only at pH above 10, P(EG x DMA-Glu) undergo a pH-dependent helix-coil switch with their transition points within the physiological range (pH ∼5.3-6.5). Meanwhile, P(EG x DMA-Glu) exhibit an unusual salt-induced helical conformation presumably owing to the unique properties of EG x linkers. Together, the current work highlights the importance of fine-tuning the linker chemistry in achieving conformation-switchable polypeptides and represents a facile approach toward stimuli-responsive biopolymers for advanced biological applications.

Zonadhesin D3-polypeptides vary among species but are similar in Equus species capable of interbreeding.

Science.gov (United States)

Tardif, Steve; Brady, Heidi A; Breazeale, Kelly R; Bi, Ming; Thompson, Leslie D; Bruemmer, Jason E; Bailey, Laura B; Hardy, Daniel M

2010-02-01

Zonadhesin is a rapidly evolving protein in the sperm acrosome that confers species specificity to sperm-zona pellucida adhesion. Though structural variation in zonadhesin likely contributes to its species-specific function, the protein has not previously been characterized in organisms capable of interbreeding. Here we compared properties of zonadhesin in several animals, including the horse (Equus caballus), donkey (E. asinus), and Grevy's zebra (E. grevyi) to determine if variation in zonadhesin correlates with ability of gametes to cross-fertilize. Zonadhesin localized to the apical acrosomes of spermatozoa from all three Equus species, similar to its localization in other animals. Likewise, in horse and donkey testis, zonadhesin was detected only in germ cells, first in the acrosomal granule of round spermatids and then in the developing acrosomes of elongating spermatids. Among non-Equus species, D3-domain polypeptides of mature, processed zonadhesin varied markedly in size and detergent solubility. However, zonadhesin D3-domain polypeptides in horse, donkey, and zebra spermatozoa exhibited identical electrophoretic mobility and detergent solubility. Equus zonadhesin D3-polypeptides (p110/p80 doublet) were most similar in size to porcine and bovine zonadhesin D3-polypeptides (p105). Sequence comparisons revealed that the horse zonadhesin precursor's domain content and arrangement are similar to those of zonadhesin from other large animals. Partial sequences of horse and donkey zonadhesin were much more similar to each other (>99% identity) than they were to orthologous sequences of human, pig, rabbit, and mouse zonadhesin (52%-72% identity). We conclude that conservation of zonadhesin D3-polypeptide properties correlates with ability of Equus species to interbreed.

An anti-cancer WxxxE-containing azurin polypeptide inhibits Rac1-dependent STAT3 and ERK/GSK-3β signaling in breast cancer cells.

Science.gov (United States)

Zhang, Zhe; Luo, Zhiyong; Min, Wenpu; Zhang, Lin; Wu, Yaqun; Hu, Xiaopeng

2017-06-27

In our previous study, we characterized a mycoplasmal small GTPase-like polypeptide of 240 amino acids that possesses an N-terminal WVLGE sequence. The N-terminal WVLGE sequence promotes activation of Rac1 and subsequent host cancer cell proliferation. To investigate the function of the WxxxE motif in the interaction with Rac1 and host tumor progression, we synthesized a 35-amino acid WVLGE-containing polypeptide derived from a cell-penetrating peptide derived from the azurin protein. We verified that the WVLGE-containing polypeptide targeted MCF-7 cells rather than MCF-10A cells. However, the WVLGE-containing polypeptide inhibited activation of Rac1 and induced cellular phenotypes that resulted from inhibition of Rac1. In addition, the WVLGE-containing polypeptide down-regulated phosphorylation of the STAT3 and ERK/GSK-3β signaling pathways, and this effect was abolished by either stimulation or inhibition of Rac1 activity. We also found that the WVLGE-containing polypeptide has a Rac1-dependent potential to suppress breast cancer growth in vitro and in vivo. We suggest that by acting as a Rac1 inhibitor, this novel polypeptide may be useful for the treatment of breast cancer.

cDNA encoding a polypeptide including a hevein sequence

Energy Technology Data Exchange (ETDEWEB)

Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

2000-07-04

A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

cDNA encoding a polypeptide including a hevein sequence

Energy Technology Data Exchange (ETDEWEB)

Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

1999-05-04

A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli. 12 figs.

cDNA encoding a polypeptide including a hevein sequence

Energy Technology Data Exchange (ETDEWEB)

Raikhel, Natasha V. (Okemos, MI); Broekaert, Willem F. (Dilbeek, BE); Chua, Nam-Hai (Scarsdale, NY); Kush, Anil (New York, NY)

1999-05-04

A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

cDNA encoding a polypeptide including a hevein sequence

Energy Technology Data Exchange (ETDEWEB)

Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

1995-03-21

A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1,018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli. 11 figures.

Functional polypeptides obtained by living ring opening polymerizations of N-carboxyanhydrides

NARCIS (Netherlands)

Habraken, G.J.M.

2011-01-01

N-Carboxyanhydride ring opening polymerization (NCA ROP) is a method to prepare polypeptides with a high degree of polymerization in large quantities. The living polymerization technique of NCA ROP gave the opportunity to synthesize many polymer architectures with well-defined blocks and copolymers

Side-chain-controlled self-assembly of polystyrene-polypeptide miktoarm star copolymers

KAUST Repository

Junnila, Susanna; Houbenov, Nikolay; Karatzas, A.; Hadjichristidis, Nikolaos; Hirao, Akira; Iatrou, Hermis; Ikkala, Olli T.

2012-01-01

polypeptide-surfactant self-assemblies with β-sheet conformation in PS 2PLL(DS) and PS 2(PLL(DS)) 2 which dominate over the formation of block copolymer scale structures. Differences between the 3- and 4-arm systems illustrate how packing frustration between

Study of local conformation and molecular movements of homo-polypeptides in aqueous solutions by using magnetic resonance and relaxation

International Nuclear Information System (INIS)

Perly, Bruno

1980-01-01

The objective of this research thesis is to study local conformations and mobilities of some typical homo-polypeptides by using techniques of magnetic resonance. By using these techniques, it is possible to make highly local observations of molecular elements which allows very efficient analysis of structural and dynamic properties of several biologically important compounds to be performed, and the study of their interactions. After a presentation of the general properties of the studied polypeptides, of magnetic resonance and of magnetic relaxation, the author presents some elements of macromolecular dynamics and movement models. Then, he reports the study of local conformations and structural transitions, applications of spin marking to the dynamic study of polypeptides, a dynamic study of the polypeptide skeleton under the form of statistic balls, the study of local movements of side chains by using nuclear relaxation, the study of the coupling of movements of main and side chains, and of the nuclear relaxation induced by a radical spin marker

Structure and biochemical composition of desmosomes and tonofilaments isolated from calf muzzle epidermis.

Science.gov (United States)

Drochmans, P; Freudenstein, C; Wanson, J C; Laurent, L; Keenan, T W; Stadler, J; Leloup, R; Franke, W W

1978-11-01

Complexes of plasma membrane segments with desmosomes and attached tonofilaments were separated from the stratum spinosum cells of calf muzzle by means of moderately alkaline buffers of low ionic strength and mechanical homogenization. These structures were further fractionated by the use of various treatments including sonication, sucrose gradient centrifugation, and extraction with buffers containing high concentrations of salt, urea, citric acid, or detergents. Subfractions enriched in desmosome-tonofilament-complexes and tonofilament fragments were studied in detail. The desmosome structures such as the midline, the trilaminar membrane profile, and the desmosomal plaque appeared well preserved and were notably resistant to the various treatments employed. Fractions containing desmosome-tonofilament complexes were invariably dominated by the nonmembranous proteins of the tonofilaments which appeared as five major polypeptide bands (apparent molecular weights: 48,000; 51,000; 58,000; 60,000; 68,000) present in molar ratios of approx. 2:1:1:2:2. Four of these polypeptide bands showed electrophoretic mobilities similar to those of prekeratin polypeptides from bovine hoof. However, the largest polypeptide (68,000 mol wt) migrated significantly less in polyacrylamide gels than the largest component of the hoof prekeratin (approximately 63,000 mol wt). In addition, a series of minor bands, including carbohydrate-containing proteins, were identified and concluded to represent constituents of the desmosomal membrane. The analysis of protein-bound carbohydrates (total 270 microgram/mg phospholipid in desmosome-enriched subfractions) showed the presence of relatively high amounts of glucosamine, mannose, galactose, and sialic acids. These data as well as the lipid composition (e.g., high ratio of cholesterol to phospholipids, relatively high contents of sphingomyelin and gangliosides, and fatty acid pattern) indicate that the desmosomal membrane is complex in protein and

Well-defined (co)polypeptides bearing pendant alkyne groups

KAUST Repository

Zhao, Wei

2016-03-18

A novel metal-free strategy, using hydrogen-bonding catalytic ring opening polymerization of acetylene-functionalized N-carboxy anhydrites of α-amino acids, was developed for the synthesis of well-defined polypeptides bearing pendant alkyne groups. This method provides an efficient way to synthesize novel alkyne-functionalized homopolypeptides (A) and copolypeptides, such as AB diblock (B: non-functionalized), ABA triblock and star-AB diblock, as well as linear and star random copolypeptides, precursors of a plethora complex macromolecular architectures by click chemistry.

Well-defined (co)polypeptides bearing pendant alkyne groups

KAUST Repository

Zhao, Wei; Gnanou, Yves; Hadjichristidis, Nikolaos

2016-01-01

A novel metal-free strategy, using hydrogen-bonding catalytic ring opening polymerization of acetylene-functionalized N-carboxy anhydrites of α-amino acids, was developed for the synthesis of well-defined polypeptides bearing pendant alkyne groups. This method provides an efficient way to synthesize novel alkyne-functionalized homopolypeptides (A) and copolypeptides, such as AB diblock (B: non-functionalized), ABA triblock and star-AB diblock, as well as linear and star random copolypeptides, precursors of a plethora complex macromolecular architectures by click chemistry.

Zonadhesin D3-Polypeptides Vary among Species but Are Similar in Equus Species Capable of Interbreeding1

Science.gov (United States)

Tardif, Steve; Brady, Heidi A.; Breazeale, Kelly R.; Bi, Ming; Thompson, Leslie D.; Bruemmer, Jason E.; Bailey, Laura B.; Hardy, Daniel M.

2009-01-01

Zonadhesin is a rapidly evolving protein in the sperm acrosome that confers species specificity to sperm-zona pellucida adhesion. Though structural variation in zonadhesin likely contributes to its species-specific function, the protein has not previously been characterized in organisms capable of interbreeding. Here we compared properties of zonadhesin in several animals, including the horse (Equus caballus), donkey (E. asinus), and Grevy's zebra (E. grevyi) to determine if variation in zonadhesin correlates with ability of gametes to cross-fertilize. Zonadhesin localized to the apical acrosomes of spermatozoa from all three Equus species, similar to its localization in other animals. Likewise, in horse and donkey testis, zonadhesin was detected only in germ cells, first in the acrosomal granule of round spermatids and then in the developing acrosomes of elongating spermatids. Among non-Equus species, D3-domain polypeptides of mature, processed zonadhesin varied markedly in size and detergent solubility. However, zonadhesin D3-domain polypeptides in horse, donkey, and zebra spermatozoa exhibited identical electrophoretic mobility and detergent solubility. Equus zonadhesin D3-polypeptides (p110/p80 doublet) were most similar in size to porcine and bovine zonadhesin D3-polypeptides (p105). Sequence comparisons revealed that the horse zonadhesin precursor's domain content and arrangement are similar to those of zonadhesin from other large animals. Partial sequences of horse and donkey zonadhesin were much more similar to each other (>99% identity) than they were to orthologous sequences of human, pig, rabbit, and mouse zonadhesin (52%–72% identity). We conclude that conservation of zonadhesin D3-polypeptide properties correlates with ability of Equus species to interbreed. PMID:19794156

Permanent Electric Dipole-Dipole Interactions in Lyotropic Polypeptide Liquid Crystals

OpenAIRE

MORI, Norio; Norio, MORI; Research Associate, Department of Industrial Chemistry

1981-01-01

The interaction energy between two adjacent α-helical molecules was calculated taking into account for permanent electric dipoles locating orl the helical core of a polymer mainchain in order to explain the cholesteric structure of lyotropic polypeptide liquid crystals. It was concluded that the dipole-dipole interactions were responsible for the formation of the cholesteric structure.

Elastin-like polypeptides: Therapeutic applications for an emerging class of nanomedicines.

Science.gov (United States)

Despanie, Jordan; Dhandhukia, Jugal P; Hamm-Alvarez, Sarah F; MacKay, J Andrew

2016-10-28

Elastin-like polypeptides (ELPs) constitute a genetically engineered class of 'protein polymers' derived from human tropoelastin. They exhibit a reversible phase separation whereby samples remain soluble below a transition temperature (T t ) but form amorphous coacervates above T t . Their phase behavior has many possible applications in purification, sensing, activation, and nanoassembly. As humanized polypeptides, they are non-immunogenic, substrates for proteolytic biodegradation, and can be decorated with pharmacologically active peptides, proteins, and small molecules. Recombinant synthesis additionally allows precise control over ELP architecture and molecular weight, resulting in protein polymers with uniform physicochemical properties suited to the design of multifunctional biologics. As such, ELPs have been employed for various uses including as anti-cancer agents, ocular drug delivery vehicles, and protein trafficking modulators. This review aims to offer the reader a catalogue of ELPs, their various applications, and potential for commercialization across a broad spectrum of fields. Copyright © 2015. Published by Elsevier B.V.

Characterization of mutants expressing thermostable D1 and D2 polypeptides of photosystem II in the cyanobacterium Synechococcus elongatus PCC 7942.

Science.gov (United States)

Haraguchi, Norihisa; Kaseda, Jun; Nakayama, Yasumune; Nagahama, Kazuhiro; Ogawa, Takahira; Matsuoka, Masayoshi

2018-06-08

Photosystem II complex embedded in thylakoid membrane performs oxygenic photosynthesis where the reaction center D1/D2 heterodimer accommodates all components of the electron transport chain. To express thermostable D1/D2 heterodimer in a cyanobacterium Synechococcus elongatus PCC 7942, we constructed a series of mutant strains whose psbA1 and psbD1 genes encoding, respectively, the most highly expressed D1 and D2 polypeptides were replaced with those of a thermophilic strain, Thermosynechococcus vulcanus. Because the C-terminal 16 amino acid sequences of D1 polypeptides should be processed prior to maturation but diverge from each other, we also constructed the psbA1ΔC-replaced strain expressing a thermostable D1 polypeptide devoid of the C-terminal extension. The psbA1/psbD1-replaced strain showed decreased growth rate and oxygen evolution rate, suggesting inefficient photosystem II. Immunoblot analyses for thermostable D1, D2 polypeptides revealed that the heterologous D1 protein was absent in thylakoid membrane from any mutant strains with psbA1, psbA1ΔC, and psbA1/psbD1-replacements, whereas the heterologous D2 protein was present in thylakoid membrane as well as purified photosystem II complex from the psbA1/psbD1-replaced strain. In the latter strain, the compensatory expression of psbA3 and psbD2 genes was elevated. These data suggest that heterologous D2 polypeptide could be combined with the host D1 polypeptide to form chimeric D1/D2 heterodimer, whereas heterologous D1 polypeptide even without the C-terminal extension was unable to make complex with the host D2 polypeptide. Since the heterologous D1 could not be detected even in the whole cells of psbA1/psbD1-replaced strain, the rapid degradation of unprocessed or unassembled heterologous D1 was implicated. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Double-hydrophobic elastin-like polypeptides with added functional motifs: Self-assembly and cytocompatibility.

Science.gov (United States)

Le, Duc H T; Tsutsui, Yoko; Sugawara-Narutaki, Ayae; Yukawa, Hiroshi; Baba, Yoshinobu; Ohtsuki, Chikara

2017-09-01

We have recently developed a novel double-hydrophobic elastin-like triblock polypeptide called GPG, designed after the uneven distribution of two different hydrophobic domains found in elastin, an extracellular matrix protein providing elasticity and resilience to tissues. Upon temperature trigger, GPG undergoes a sequential self-assembling process to form flexible beaded nanofibers with high homogeneity and excellent dispersibility in water. Given that GPG might be a potential elastin-mimetic material, we sought to explore the biological activities of this block polypeptide. Besides GPG, several functionalized derivatives were also constructed by fusing functional motifs such as KAAK or KAAKGRGDS at the C-terminal of GPG. Although the added motifs affected the kinetics of fiber formation and β-sheet contents, all three GPGs assembled into beaded nanofibers at the physiological temperature. The resulting GPG nanofibers preserved their beaded structures in cell culture medium; therefore, they were coated on polystyrene substrates to study their cytocompatibility toward mouse embryonic fibroblasts, NIH-3T3. Among the three polypeptides, GPG having the cell-binding motif GRGDS derived from fibronectin showed excellent cell adhesion and cell proliferation properties compared to other conventional materials, suggesting its promising applications as extracellular matrices for mammalian cells. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2475-2484, 2017. © 2017 Wiley Periodicals, Inc.

Architecture effects on multivalent interactions by polypeptide-based multivalent ligands

Science.gov (United States)

Liu, Shuang

protein materials, including structural as well as functional proteins. Therefore, polypeptide-based multivalent scaffolds are used to display ligands to assess the contribution of different architectural parameters to the multivalent binding events. In this work, a family of alanine-rich alpha-helical glycopolypeptides was designed and synthesized by a combination of protein engineering and chemical coupling, to display two types of saccharide ligands for two different multivalent binding systems. The valencies, chain length and spacing between adjacent ligands of these multivalent ligands were designed in order to study architecture effects on multivalent interactions. The polypeptides and their glycoconjugates were characterized via various methods, including SDS-PAGE, NMR, HPLC, amino acid analysis (AAA), MALDI, circular dichroism (CD) and GPC. In the first multivalent binding system, cholera toxin B pentamer (CT B5) was chosen to be the protein receptor due to its well-characterized structure, lack of significant steric interference of binding to multiple binding sites, and requirement of only simple monosaccharide as ligands. Galactopyranoside was incorporated into polypeptide scaffolds through amine-carboxylic acid coupling to the side chains of glutamic acid residues. The inhibition and binding to CT B5 of these glycopolypeptide ligands were evaluated by direct enzyme-linked assay (DELA). As a complement method, weak affinity chromatography (WAC) was also used to evaluate glycopolypeptides binding to a CT B5 immobilized column. The architecture effects on CT B 5 inhibition are discussed. In the second system, cell surface receptor L-selectin was targeted by polypeptide-based multivalent ligands containing disulfated galactopyranoside ligands, due to its important roles in various immunological activities. The effects of glycopolypeptide architectural variables L-selectin shedding were evaluated via ELISA-based assays. These polypeptide-based multivalent ligands

cDNA encoding a polypeptide including a hev ein sequence

Energy Technology Data Exchange (ETDEWEB)

Raikhel, Natasha V. (Okemos, MI); Broekaert, Willem F. (Dilbeek, BE); Chua, Nam-Hai (Scarsdale, NY); Kush, Anil (New York, NY)

2000-07-04

A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

Organic anion transporting polypeptide 1B transporters modulate hydroxyurea pharmacokinetics

OpenAIRE

Walker, Aisha L.; Lancaster, Cynthia S.; Finkelstein, David; Ware, Russell E.; Sparreboom, Alex

2013-01-01

Hydroxyurea is currently the only FDA-approved drug that ameliorates the pathophysiology of sickle cell anemia. Unfortunately, substantial interpatient variability in the pharmacokinetics (PK) of hydroxyurea may result in variation of the drug's efficacy. However, little is known about mechanisms that modulate hydroxyurea PK. Recent in vitro studies identifying hydroxyurea as a substrate for organic anion transporting polypeptide (OATP1B) transporters prompted the current investigation assess...

Functional Modification of Thioether Groups in Peptides, Polypeptides, and Proteins

OpenAIRE

Deming, TJ

2017-01-01

Recent developments in the modification of methionine and other thioether-containing residues in peptides, polypeptides, and proteins are reviewed. Properties and potential applications of the resulting functionalized products are also discussed. While much of this work is focused on natural Met residues, modifications at other side-chain residues have also emerged as new thioether-containing amino acids have been incorporated into peptidic materials. Functional modification of thioether-cont...

NMR study of the cooperative behavior of thermotropic model polypeptides

Czech Academy of Sciences Publication Activity Database

Kurková, Dana; Kříž, Jaroslav; Rodríguez-Cabello, J. C.; Arias, F. J.

2007-01-01

Roč. 56, č. 2 (2007), s. 186-194 ISSN 0959-8103 R&D Projects: GA AV ČR IAA400500604 Grant - others:Spanish Ministry of Science and Culture(ES) A002/02; MAT2000-1764-C02; MAT2001-1853-C02-01; MAT2003- Institutional research plan: CEZ:AV0Z40500505 Keywords : thermotropic polymers * cooperativity * synthetic polypeptides Subject RIV: CD - Macromolecular Chemistry Impact factor: 1.557, year: 2007

DNA-interactive properties of crotamine, a cell-penetrating polypeptide and a potential drug carrier.

Directory of Open Access Journals (Sweden)

Pei-Chun Chen

Full Text Available Crotamine, a 42-residue polypeptide derived from the venom of the South American rattlesnake Crotalus durissus terrificus, has been shown to be a cell-penetrating protein that targets chromosomes, carries plasmid DNA into cells, and shows specificity for actively proliferating cells. Given this potential role as a nucleic acid-delivery vector, we have studied in detail the binding of crotamine to single- and double-stranded DNAs of different lengths and base compositions over a range of ionic conditions. Agarose gel electrophoresis and ultraviolet spectrophotometry analysis indicate that complexes of crotamine with long-chain DNAs readily aggregate and precipitate at low ionic strength. This aggregation, which may be important for cellular uptake of DNA, becomes less likely with shorter chain length. 25-mer oligonucleotides do not show any evidence of such aggregation, permitting the determination of affinities and size via fluorescence quenching experiments. The polypeptide binds non-cooperatively to DNA, covering about 5 nucleotide residues when it binds to single (ss or (ds double stranded molecules. The affinities of the protein for ss- vs. ds-DNA are comparable, and inversely proportional to salt levels. Analysis of the dependence of affinity on [NaCl] indicates that there are a maximum of ∼3 ionic interactions between the protein and DNA, with some of the binding affinity attributable to non-ionic interactions. Inspection of the three-dimensional structure of the protein suggests that residues 31 to 35, Arg-Trp-Arg-Trp-Lys, could serve as a potential DNA-binding site. A hexapeptide containing this sequence displayed a lower DNA binding affinity and salt dependence as compared to the full-length protein, likely indicative of a more suitable 3D structure and the presence of accessory binding sites in the native crotamine. Taken together, the data presented here describing crotamine-DNA interactions may lend support to the design of more

Two-dimensional electrophoretic analysis of transformation-sensitive polypeptides during chemically, spontaneously, and oncogene-induced transformation of rat liver epithelial cells

DEFF Research Database (Denmark)

Wirth, P J; Luo, L D; Fujimoto, Y

1992-01-01

; AFB), spontaneously, and oncogene (v-Ha-ras, v-raf, and v-myc/v-raf)-induced transformation of RLE cells. Two-dimensional mapping of [35S]methionine-labeled whole cell lysate, cell-free in vitro translation products and [32P]orthophosphate-labeled polypeptides revealed subsets of polypeptides specific...... for each transformation modality. A search of the RLE protein database indicated the specific subcellular location for the majority of these transformation-sensitive proteins. Significant alterations in the expression of the extracellular matrix protein, fibronectin, as well as tropomyosin......- and intermediate filament-related polypeptides (vimentin, beta-tubulin, the cytokeratins, and actin) were observed among the various transformant cell lines. Immunoprecipitation and Western immunoblot analysis of tropomyosin expression in four individual AFB-, as well as four spontaneously induced, and each...

Formation of a [sup(99m)Tc]polypeptide hormone: characterization and chemical quality control by ampholyte displacement radiochromatography

International Nuclear Information System (INIS)

Sundrehagen, E.

1983-01-01

Sup(99m)Tc-complexes with the polypeptide hormone secretin in very low concentration were formed by the concentrated hydrochloric acid/vacuum evaporation/gentisic acid method. The sup(99m)Tc-secretin was characterized by a modified ampholyte radiochromatographic procedure, in addition to thin layer chromatography, gel chromatography and paper electrophoresis. High radiochemical purity and specific radioactivity were obtained. In vivo distribution studies were performed, and the conditions necessary for application of [sup(99m)Tc]polypeptides as scintigraphic agents are discussed. (author)

Dynamic enzyme docking to the ribosome coordinates N-terminal processing with polypeptide folding.

Science.gov (United States)

Sandikci, Arzu; Gloge, Felix; Martinez, Michael; Mayer, Matthias P; Wade, Rebecca; Bukau, Bernd; Kramer, Günter

2013-07-01

Newly synthesized polypeptides undergo various cotranslational maturation steps, including N-terminal enzymatic processing, chaperone-assisted folding and membrane targeting, but the spatial and temporal coordination of these steps is unclear. We show that Escherichia coli methionine aminopeptidase (MAP) associates with ribosomes through a charged loop that is crucial for nascent-chain processing and cell viability. MAP competes with peptide deformylase (PDF), the first enzyme to act on nascent chains, for binding sites at the ribosomal tunnel exit. PDF has extremely fast association and dissociation kinetics, which allows it to frequently sample ribosomes and ensure the processing of nascent chains after their emergence. Premature recruitment of the chaperone trigger factor, or polypeptide folding, negatively affect processing efficiency. Thus, the fast ribosome association kinetics of PDF and MAP are crucial for the temporal separation of nascent-chain processing from later maturation events, including chaperone recruitment and folding.

Induction of a M/sub r/ 21,000 polypeptide in an Arthrobacter Sp. by dye-sensitized photooxidation

International Nuclear Information System (INIS)

Franzi, J.J.

1985-01-01

Irradiation of aerobic cultures of an Arthrobacter species with near-UV light and oxygen induced synthesis of a cell surface protein, M/sub r/ 21,000 polypeptide. Visible light, oxygen and a sensitizing dye were also effective in induction. Far-UV light, bleomycin and nalidixic acid, all inducers of the recA protein in Escherichia coli, were ineffective inducers of this protein. Furthermore, X-irradiation and radical-generating oxidants failed to induce synthesis of the M/sub r/ 21,000 polypeptide. DNA binding dyes proved to be capable of inducing synthesis of this protein or inhibiting dye-mediated stimulation of synthesis of this protein. For example, dGdC-specific dyes (e.g. methylene blue, neutral red, acridine orange or ethidium bromide) were efficient inducers of the M/sub r/ 21,000 polypeptide. Also methylene blue and neutral red were more efficient inducers than were acridine orange or ethidium bromide, which could be explained by the greater dGdC specificity and, possibly by the greater photoreactivity of methylene blue and neutral red. dAdT-specific dyes such as methyl green or daunomycin effectively inhibited dye-mediated induction. Rose bengal is an anionic dye which does not bind to DNA but does mediate the photooxidation of deoxyguanosine residues in DNA. It is an efficient inducer of the M/sub r/ 21,000 polypeptide. Induction with this dye is nearly eliminated when novobiocin, an inhibitor of DNA gyrase (topoisomerase II) which mediates relaxation, is added in conjunction with rose bengal

Induction of a M/sub r/ 21,000 polypeptide in an Arthrobacter Sp. by dye-sensitized photooxidation

Energy Technology Data Exchange (ETDEWEB)

Franzi, J.J.

1985-01-01

Irradiation of aerobic cultures of an Arthrobacter species with near-UV light and oxygen induced synthesis of a cell surface protein, M/sub r/ 21,000 polypeptide. Visible light, oxygen and a sensitizing dye were also effective in induction. Far-UV light, bleomycin and nalidixic acid, all inducers of the recA protein in Escherichia coli, were ineffective inducers of this protein. Furthermore, X-irradiation and radical-generating oxidants failed to induce synthesis of the M/sub r/ 21,000 polypeptide. DNA binding dyes proved to be capable of inducing synthesis of this protein or inhibiting dye-mediated stimulation of synthesis of this protein. For example, dGdC-specific dyes (e.g. methylene blue, neutral red, acridine orange or ethidium bromide) were efficient inducers of the M/sub r/ 21,000 polypeptide. Also methylene blue and neutral red were more efficient inducers than were acridine orange or ethidium bromide, which could be explained by the greater dGdC specificity and, possibly by the greater photoreactivity of methylene blue and neutral red. dAdT-specific dyes such as methyl green or daunomycin effectively inhibited dye-mediated induction. Rose bengal is an anionic dye which does not bind to DNA but does mediate the photooxidation of deoxyguanosine residues in DNA. It is an efficient inducer of the M/sub r/ 21,000 polypeptide. Induction with this dye is nearly eliminated when novobiocin, an inhibitor of DNA gyrase (topoisomerase II) which mediates relaxation, is added in conjunction with rose bengal.

Double network physical gels from elastin-like polypeptide block copolymers: nanoscale control of thermoresponsive reinforcement

Science.gov (United States)

Glassman, Matthew; Olsen, Bradley

2014-03-01

Triblock copolymers with associative protein midblocks and thermoresponsive endblocks form shear thinning hydrogels with a low yield stress at low temperatures, but can be reinforced by a self-assembled network of the endblock aggregates. Here, we compare the use of bioengineered elastin-like polypeptides (ELPs) to synthetic poly(N-isopropylacrylamide) (PNIPAM) as endblocks to control the self-assembly of the reinforcing network. The temperature dependence of the mechanics of these hydrogels is a strong function of the domain size and morphology in the endblock network. Despite the architectural similarities, triblock ELP fusions and PNIPAM bioconjugates exhibit distinct reinforcement maxima at fixed block composition and polymer concentration, and these differences can be attributed to the nanostructural features of the two systems. Furthermore, in ELP fusions, the amino acid sequence can be readily modified to manipulate the solvation kinetics of the endblock domains. Finally, various endblocks have been combined to form triblock terpolymer hydrogels, demonstrating how the choice of thermoresponsive blocks can be used to tune the reinforcement of shear thinning hydrogels.

Biomimetic Synthesis of Gelatin Polypeptide-Assisted Noble-Metal Nanoparticles and Their Interaction Study

Science.gov (United States)

Liu, Ying; Liu, Xiaoheng; Wang, Xin

2011-12-01

Herein, the generation of gold, silver, and silver-gold (Ag-Au) bimetallic nanoparticles was carried out in collagen (gelatin) solution. It first showed that the major ingredient in gelatin polypeptide, glutamic acid, acted as reducing agent to biomimetically synthesize noble metal nanoparticles at 80°C. The size of nanoparticles can be controlled not only by the mass ratio of gelatin to gold ion but also by pH of gelatin solution. Interaction between noble-metal nanoparticles and polypeptide has been investigated by TEM, UV-visible, fluorescence spectroscopy, and HNMR. This study testified that the degradation of gelatin protein could not alter the morphology of nanoparticles, but it made nanoparticles aggregated clusters array (opposing three-dimensional α-helix folding structure) into isolated nanoparticles stabilized by gelatin residues. This is a promising merit of gelatin to apply in the synthesis of nanoparticles. Therefore, gelatin protein is an excellent template for biomimetic synthesis of noble metal/bimetallic nanoparticle growth to form nanometer-sized device.

Effect of Sequence Blockiness on the Morphologies of Surface-grafted Elastin-like Polypeptides

Science.gov (United States)

Albert, Julie; Sintavanon, Kornkanok; Mays, Robin; MacEwan, Sarah; Chilkoti, Ashutosh; Genzer, Jan

2014-03-01

The inter- and intra- molecular interactions among monomeric units of copolymers and polypeptides depend strongly on monomer sequence distribution and dictate the phase behavior of these species both in solution and on surfaces. To study the relationship between sequence and phase behavior, we have designed a series of elastin-like polypeptides (ELPs) with controlled monomer sequences that mimic copolymers with various co-monomer sequence distributions and attached them covalently to silicon substrates from buffer solutions at temperatures below and above the bulk ELPs' lower critical solution temperatures (LCSTs). The dependence of ELP grafting density on solution temperature was examined by ellipsometry and the resultant surface morphologies were examined in air and under water with atomic force microscopy. Depositions performed above the LCST resulted in higher grafting densities and greater surface roughness of ELPs relative to depositions carried out below the LCST. In addition, we are using gradient substrates to examine the effect of ELP grafting density on temperature responsiveness.

Serological Reactivity and Identification of Immunoglobulin E-Binding Polypeptides of Ganoderma applanatum Crude Spore Cytoplasmic Extract in Puerto Rican Subjects.

Science.gov (United States)

Vilá-Héreter, Frances; Rivera-Mariani, Félix E; Bolaños-Rosero, Benjamín

2017-01-01

The allergenic potential of Ganoderma applanatum basidiospores has been demonstrated previously in Puerto Rico. However, basidiomycete allergens are not available for inclusion in allergy diagnostic panels. Therefore, we sought to confirm allergic sensitization to G. applanatum crude spore cytoplasmic extract through reactivity in serological assays and detection of immunoglobulin E (IgE)-binding polypeptides. Via an indirect ELISA, serological reactivity was compared between groups of individuals with different allergic profiles. Group 1 (n = 51) consisted of individuals with sIgE to the allergens included in the diagnostic panels; group 2 (n = 14) comprised individuals with no sIgE to the allergens tested; and group 3 (n = 22) included individuals with no allergic history. To visualize IgE-binding polypeptides, group 1 sera were examined via Western blotting (WB). Polypeptide bands with the highest reactivity were analyzed by mass spectrometry (MS) for putative identification. The serological reactivity of group 1 was significantly higher than that of group 3 in an indirect ELISA (p = 0.03). Sixty-five percent of group 1 individuals showed reactivity to polypeptide bands in WB. Bands of 81 and 56 kDa had the highest reactivity proportions among the reactive sera, followed by a 45-kDa band. MS analysis of these 3 polypeptides suggests that they are basidiomycete-derived enzymes with aconitate hydratase, catalase, and enolase functions. G. applanatum spores have allergenic components recognized by Puerto Rican individuals, which could eventually be considered as markers in cases of fungal allergy and be included in diagnostic allergen panels in Puerto Rico and tropical regions. © 2017 S. Karger AG, Basel.

Accelerated evolution of the pituitary adenylate cyclase-activating polypeptide precursor gene during human origin

DEFF Research Database (Denmark)

Wang, Yin-Qiu; Qian, Ya-Ping; Yang, Su

2005-01-01

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide abundantly expressed in the central nervous system and involved in regulating neurogenesis and neuronal signal transduction. The amino acid sequence of PACAP is extremely conserved across vertebrate species, indicating a...

Membrane polypeptide in rabbit erythrocytes associated with the inhibition of L-lactate transport by a synthetic anhydride of lactic acid

International Nuclear Information System (INIS)

Donovan, J.A.; Jennings, M.L.

1985-01-01

The synthetic lactyl anhydride isobutylcarbonyl lactyl anhydride (iBCLA), a selective and potent inhibitor of L-(+)-lactate transport in rabbit erythrocytes, reduces the chemical labeling of a 40-50-kdalton polypeptide by tritiated 4,4'-diisothiocyanato-2,2'-dihydrostilbenedisulfonate ([ 3 H]H 2 DIDS). iBCLA does so in a dose-dependent manner at concentrations that strongly inhibit lactate-lactate exchange but not chloride-phosphate exchange. These labeling experiments and inhibition reversal studies using iBCLA, p-(chloro-mercuri)benzenesulfonic acid (pCMBS), and dithiothreitol (DDT) suggest that iBCLA does not act at sulfhydryl groups but at or near an amino group that is near a disulfide linkage in the polypeptide which catalyzes lactate transport. These experiments support the association between specific monocarboxylate transport and a 40-50-kdalton membrane-bound polypeptide of the rabbit erythrocyte

Pituitary adenylate cyclase-activating polypeptide stimulates renin secretion via activation of PAC1 receptors

DEFF Research Database (Denmark)

Hautmann, Matthias; Friis, Ulla G; Desch, Michael

2007-01-01

Besides of its functional role in the nervous system, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in the regulation of cardiovascular function. Therefore, PACAP is a potent vasodilator in several vascular beds, including the renal vasculature. Because...

Wall-associated kinase-like polypeptide mediates nutritional status perception and response

Science.gov (United States)

Yang, Zhenbiao; Karr, Stephen

2014-02-11

The disclosure relates to methods for modulating plant growth and organogenesis using dominant-negative receptor-like kinases. The disclosure further provides a method for increasing plant yield relative to corresponding wild type plants comprising modulating the expression in a plant of a nucleic acid encoding a Wall-Associated Kinase-like 14 polypeptide or a homolog thereof, and selecting for plants having increased yield or growth on a nutrient deficient substrate.

Sustained Release of Antibiotics from Injectable and Thermally Responsive Polypeptide Depots

OpenAIRE

Adams, Samuel B.; Shamji, Mohammed F.; Nettles, Dana L.; Hwang, Priscilla; Setton, Lori A.

2009-01-01

Biodegradable polymeric scaffolds are of interest for delivering antibiotics to local sites of infection in orthopaedic applications, such as bone and diarthrodial joints. The objective of this study was to develop a biodegradable scaffold with ease of drug loading in aqueous solution, while providing for drug depot delivery via syringe injection. Elastin-like polypeptides (ELPs) were used for this application, biopolymers of repeating pentapeptide sequences that were thermally triggered to u...

Prohormone convertase 1/3 is essential for processing of the glucose-dependent insulinotropic polypeptide precursor

DEFF Research Database (Denmark)

Ugleholdt, Randi; Poulsen, Marie-Louise H; Holst, Peter J

2006-01-01

The physiology of the incretin hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), and their role in type 2 diabetes currently attract great interest. Recently we reported an essential role for prohormone convertase (PC) 1/3 in the cleavage of intesti......The physiology of the incretin hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), and their role in type 2 diabetes currently attract great interest. Recently we reported an essential role for prohormone convertase (PC) 1/3 in the cleavage....../3 is essential and sufficient for the production of the intestinal incretin hormone GIP, whereas PC2, although capable of cleaving proGIP, does not participate in intestinal proGIP processing and is not found in intestinal GIP-expressing cells....

Elastin-like Polypeptide Linkers for Single-Molecule Force Spectroscopy.

Science.gov (United States)

Ott, Wolfgang; Jobst, Markus A; Bauer, Magnus S; Durner, Ellis; Milles, Lukas F; Nash, Michael A; Gaub, Hermann E

2017-06-27

Single-molecule force spectroscopy (SMFS) is by now well established as a standard technique in biophysics and mechanobiology. In recent years, the technique has benefitted greatly from new approaches to bioconjugation of proteins to surfaces. Indeed, optimized immobilization strategies for biomolecules and refined purification schemes are being steadily adapted and improved, which in turn has enhanced data quality. In many previously reported SMFS studies, poly(ethylene glycol) (PEG) was used to anchor molecules of interest to surfaces and/or cantilever tips. The limitation, however, is that PEG exhibits a well-known trans-trans-gauche to all-trans transition, which results in marked deviation from standard polymer elasticity models such as the worm-like chain, particularly at elevated forces. As a result, the assignment of unfolding events to protein domains based on their corresponding amino acid chain lengths is significantly obscured. Here, we provide a solution to this problem by implementing unstructured elastin-like polypeptides as linkers to replace PEG. We investigate the suitability of tailored elastin-like polypeptides linkers and perform direct comparisons to PEG, focusing on attributes that are critical for single-molecule force experiments such as linker length, monodispersity, and bioorthogonal conjugation tags. Our results demonstrate that by avoiding the ambiguous elastic response of mixed PEG/peptide systems and instead building the molecular mechanical systems with only a single bond type with uniform elastic properties, we improve data quality and facilitate data analysis and interpretation in force spectroscopy experiments. The use of all-peptide linkers allows alternative approaches for precisely defining elastic properties of proteins linked to surfaces.

N-terminal and C-terminal heparin-binding domain polypeptides derived from fibronectin reduce adhesion and invasion of liver cancer cells

International Nuclear Information System (INIS)

Tang, Nan-Hong; Chen, Yan-Lin; Wang, Xiao-Qian; Li, Xiu-Jin; Wu, Yong; Zou, Qi-Lian; Chen, Yuan-Zhong

2010-01-01

Fibronectin (FN) is known to be a large multifunction glycoprotein with binding sites for many substances, including N-terminal and C-terminal heparin-binding domains. We investigated the effects of highly purified rhFNHN29 and rhFNHC36 polypeptides originally cloned from the two heparin-binding domains on the adhesion and invasion of highly metastatic human hepatocellular carcinoma cells (MHCC97H) and analyzed the underlying mechanism involved. The MHCC97H cells that adhered to FN in the presence of various concentrations of rhFNHN29 and rhFNHC36 polypeptides were stained with crystal violet and measured, and the effects of rhFNHN29 and rhFNHC36 on the invasion of the MHCC97H cells were then detected using the Matrigel invasion assay as well as a lung-metastasis mouse model. The expression level of integrins and focal adhesion kinase (FAK) phosphotyrosyl protein was examined by Western blot, and the activity of matrix metalloproteinases (MMPs) and activator protein 1 (AP-1) was analyzed by gelatin zymography and the electrophoretic mobility band-shift assay (EMSA), respectively. Both of the polypeptides rhFNHN29 and rhFNHC36 inhibited adhesion and invasion of MHCC97H cells; however, rhFNHC36 exhibited inhibition at a lower dose than rhFNHN29. These inhibitory effects were mediated by integrin αvβ3 and reversed by a protein tyrosine phosphatase inhibitor. Polypeptides rhFNHN29 and rhFNHC36 abrogated the tyrosine phosphorylation of focal adhesion kinase (p-FAK) and activation of activator protein 1 (AP-1), resulting in the decrease of integrin αv, β3 and β1 expression as well as the reduction of MMP-9 activity. Polypeptides rhFNHN29 and rhFNHC36 could potentially be applicable to human liver cancer as anti-adhesive and anti-invasive agents

TISSUE POLYPEPTIDE-SPECIFIC ANTIGEN - A DISCRIMINATIVE PARAMETER BETWEEN PROSTATE-CANCER AND BENIGN PROSTATIC HYPERTROPHY

NARCIS (Netherlands)

MARRINK, J; OOSTEROM, R; BONFRER, HMG; SCHRODER, FH; MENSINK, HJA

1993-01-01

The serum concentration of the cell proliferation marker TPS (tissue polypeptide-specific antigen) was compared with the tumour marker PSA (prostate specific antigen). PSA was found elevated in 50% of the benign prostatic hypertrophy (BPH) patients, in 88% of the patients with active prostate cancer

Regulation of the subunit composition of plastidic glutamine synthetase of the wild-type and of the phytochrome-deficient aurea mutant of tomato by blue/UV-A- or by UV-B-light

International Nuclear Information System (INIS)

Migge, A.; Carrayol, E.; Hirel, B.; Lohmann, M.; Meya, G.; Becker, T.W.

1998-01-01

The photomorphogenetic aurea mutant of tomato severely deficient in spectrophotometrically active phytochromes was used to study the light-regulation of the single-copy nuclear gene encoding plastidic glutamine synthetase (GS-2; EC 6.1.3.2). The de-etiolation of dark-grown aurea mutant seedling cotyledons showed an obligatory dependency on blue light. A limited red light-responsiveness of etiolated aurea cotyledons is, however, retained as seen by the stimulation of both the GS-2 transcript and protein level in the cotyledons of aurea seedlings during growth in red light. The subunits of the octameric GS-2 enzyme were represented by polypeptides with similar electrophoretic mobilities (polypeptides a) in etiolated wild-type or aurea mutant cotyledons. GS-2 proteins with similar apparent molecular masses were also seen in the cotyledons of red light-grown aurea mutant seedlings. In contrast, GS-2 polypeptides with different apparent molecular masses (polypeptides a and b) were detected in the cotyledons of wild-type seedlings grown in red light. This difference indicates that the (post-translational) modification of tomato GS-2 subunit composition is mediated by the photoreceptor phytochrome. The illumination of etiolated wild-type or aurea cotyledons with UV-A- or UV-B-light light resulted in an increase in both the GS-2 transcript and protein level. Following illumination of etiolated wild-type seedlings with UV-A-light, the relative proportion of the GS-2 polypeptides a and b was similar than upon irradiation with blue light but different than after exposure to UV-B- or red light. This result suggests the involvement of a blue/ UV-A-light-specific photoreceptor in the regulation of tomato GS-2 subunit composition. (author)

Primary structure, gene organization and polypeptide expression of poliovirus RNA

Energy Technology Data Exchange (ETDEWEB)

Kitamura, N. (State Univ. of New York, Stony Brook); Semler, B.L.; Rothberg, P.G.

1981-06-18

The primary structure of the poliovirus genome has been determined. The RNA molecule is 7433 nucleotides long, polyadenylated at the 3' terminus, and covalently linked to a small protein (VPg) at the 5' terminus. An open reading frame of 2207 consecutive triplets spans over 89% of the nucleotide sequence and codes for the viral polyprotein NCVPOO. Twelve viral polypeptides have been mapped by amino acid sequence analysis and were found to be proteolytic cleavage products of the polyprotein, cleavages occurring predominantly at Gln-Gly pairs.

High-resolution polypeptide structure and dynamics in anisotropic environments: The gramicidin channel

Energy Technology Data Exchange (ETDEWEB)

Cross, T.A.; Lee, K.C.; Ketchem, R.R.; Hu, W.; Lazo, N.D.; Huo, S. [Florida State Univ., Tallahassee, FL (United States)

1994-12-01

To understand the details of macromolecular function, high-resolution structural and dynamic detail is essential. The polypeptide fold of the gramicidin channel has been effectively modeled for the past 20 years, yet the functional changes in conductance and channel lifetime associated with amino acid substitutions cannot be predicted. To accomplish this goal, high-resolution electrostatic modeling and the precise orientation of all dipoles are required. Furthermore, an enhanced knowledge of the complex molecular environment of this membrane-bound peptide is needed. An aqueous environment is relatively uniform and achiral. The membrane environment is very heterogenous and chiral. A knowledge of the interactions, specific and nonspecific, between peptide and lipid will aid in developing a better understanding of this environment. To accomplish this goal, it is necessary to study the peptide in an extended lipid bilayer, rather than in a vesicular or micellar form. These latter environments are likely to possess increased dynamics, increased water penetration, and distorted interactions between the polypeptide and membrane surface. To perform NMR studies on bilayer bound peptides, solid state NMR methods are required, and for specific site information, isotopic labels are incorporated using solid phase peptide synthesis.

Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin

International Nuclear Information System (INIS)

Hicks, G.R.; Rayle, D.L.; Jones, A.M.; Lomax, T.L.

1989-01-01

Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7- 3 H]IAA([ 3 H]N 3 IAA), in a manner similar to the accumulation of [ 3 H]IAA. The association of the [ 3 H]N 3 IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [ 3 H]N 3 IAA to plasma membrane vesicles prior to exposure to UV light and detected by subsequent NaDodSO 4 /PAGE and fluorography. When the reaction temperature was lowered to -196 degree C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors

Tuning calcium carbonate growth through physical confinement and templating with amyloid-like polypeptide aggregates

Science.gov (United States)

Colaco, Martin Francis

The creation of useful composite materials requires precise control of the interface between the components in order to tune the overall shape and material properties. Despite the current research into nanotechnology, our ability to create materials with nanoscale precision is nascent. However, nature has a paradigm for the creation of finely structured composites under mild conditions called biomineralization. Through control of protein template assembly, solution conditions, and physical confinement, organisms are able to create useful optical and structural materials, such as bones, teeth, and mollusk shells. The objective of this thesis is to elucidate the importance of these various controls in synthetic systems to further our ability to create nanostructured materials. We begin by examining the formation of self-assembled monolayers (SAMs) of organosilanes on silica oxides. The formation of functionalized surfaces can help control the mineralization of amorphous or crystalline calcium carbonate. Long-chained organosilanes organize on surfaces to form dense, solid-like films, with the terminal groups determining the hydrophobicity and stereochemistry of the film. Our work has shown that uniform hydrophobic and hydrophilic films can be formed by using cleaned silica over glass or mica and through a vapor phase reaction over a liquid one. Additionally, we showed that mixed SAMs with phase-separated domains could be created through the selection of organosilanes and reaction conditions. We have built on these functionalized surfaces through the use of microfabrication and a gas permeable polymer to create three-dimensionally confined microcrystallizers. Other researchers have shown that one-dimensional confinement with a multi-functional surface (patterned with a small nucleating ordered region in a disordered SAM) can stabilize the creation of an amorphous calcium carbonate film before a single, large, micropatterned crystal is grown. Our work has determined

The generalized model of polypeptide chain describing the helix-coil transition in biopolymers

International Nuclear Information System (INIS)

Mamasakhlisov, E.S.; Badasyan, A.V.; Tsarukyan, A.V.; Grigoryan, A.V.; Morozov, V.F.

2005-07-01

In this paper we summarize some results of our theoretical investigations of helix-coil transition both in single-strand (polypeptides) and two-strand (polynucleotides) macromolecules. The Hamiltonian of the Generalized Model of Polypeptide Chain (GMPC) is introduced to describe the system in which the conformations are correlated over some dimensional range Δ (it equals 3 for polypeptide, because one H-bond fixes three pairs of rotation, for double strand DNA it equals to one chain rigidity because of impossibility of loop formation on the scale less than Δ). The Hamiltonian does not contain any parameter designed especially for helix-coil transition and uses pure molecular microscopic parameters (the energy of hydrogen bond formation, reduced partition function of repeated unit, the number of repeated units fixed by one hydrogen bond, the energies of interaction between the repeated units and the solvent molecules). To calculate averages we evaluate the partition function using the transfer-matrix approach. The GMPC allowed to describe the influence of a number of factors, affecting the transition, basing on a unified microscopic approach. Thus we obtained, that solvents change transition temperature and interval in different ways, depending on type of solvent and on energy of solvent- macromolecule interaction; stacking on the background of H-bonding increases stability and decreases cooperativity of melting. For heterogeneous DNA we could analytically derive well known formulae for transition temperature and interval. In the framework of GMPC we calculate and show the difference of two order parameters of helix-coil transition - the helicity degree, and the average fraction of repeated units in helical conformation. Given article has the aim to review the results obtained during twenty years in the context of GMPC. (author)

Peptides and polypeptides as scaffolds for optoelectronics and biomaterials applications

Science.gov (United States)

Charati, Manoj B.

Peptides and polypeptides are emerging as a new class of biomaterials due to their unique structural, physiochemical, mechanical, and biological properties. The development of peptide and protein-based biomaterials is driven by the convergence of convenient techniques for peptide/protein engineering and its importance in applications as smart biomaterials. The thesis is divided in two parts; the first part highlights the importance of incorporation of non-natural amino acids into peptides and proteins. In particular, incorporation on p-bromophenylalanine in short alpha-helical peptide templates to control the association of chromophores is discussed. In the second part, design of a multi-component, biocompatible polypeptide with superior elasticity is discussed. Part 1. Novel peptide templates to control association of chromophores. Tailor made peptide and protein materials have many versatile applications, as both conformation and functional group position can be controlled. Such control may have intriguing applications in the development of hybrid materials for electroactive applications. A critical need in fabricating devices from organic semiconducting materials is to achieve control over the conformation and distance between two conjugated chains. Controlling chromophore spacing and orientation with required precision over nanometer length scale poses a greater challenge. Here we propose a peptide based template to control the alignment of the methylstilbene and Oxa-PPV chromophores with desired orientations and spacing. The hybrid peptides were characterized via CD, exciton coupled CD, 1H NMR and photoluminescence experiments. It is observed that slight change in the orientation of molecules has pronounced effect on the photo-physical behavior of the molecules. Characterization of the hybrid peptides via circular dichroism (CD) confirmed the helical character of the designed peptides and indicated that inclusion of non-natural amino acids has significant

Recombinant DNA specifying the human amyloid. beta. precursor protein (ABPP) encodes a 95-kDa polypeptide

Energy Technology Data Exchange (ETDEWEB)

Mita, S; Sadlock, J; Herbert, J; Schon, E A

1988-10-11

Although the ABPP gene give rise to multiple mRNAs, the primary translation product of this gene is unknown. The longest published cDNA sequences predict a 770-aa polypeptide of 87 kDa. However, in immunoblots, ABPP migrated as a single species of >92 kDa in rat brain, and in human, as a species of 95-100 kDa in non-membrane bound form, as multiple species of 110-135 kDa in membrane-associated form and as a 130-kDa species in fibroblasts. The sizes of these larger species relative to the MW of ABPP predicted from the cDNA sequences have been attributed to postranslational modification. However, the authors have isolated a cDNA (lambdaHAP2) from a human fetal muscle lambdagt11 cDNA library encoding an 843-aa polypeptide with a deduced MW of 94,642. This cDNA contains both exons encoding an 843-aa polypeptide with a deduced MW of 94.642. This cDNA contains both exons encoding the protease inhibitor domains. Primer extension analysis indicates that the 5' terminus of this cDNA is 14 nt from a transcriptional start site. This cDNA, encoding the longest ABPP described to date, may explain some of the observations on the sizes of tissue-derived ABPP.

Peptides, polypeptides and peptide-polymer hybrids as nucleic acid carriers.

Science.gov (United States)

Ahmed, Marya

2017-10-24

Cell penetrating peptides (CPPs), and protein transduction domains (PTDs) of viruses and other natural proteins serve as a template for the development of efficient peptide based gene delivery vectors. PTDs are sequences of acidic or basic amphipathic amino acids, with superior membrane trespassing efficacies. Gene delivery vectors derived from these natural, cationic and cationic amphipathic peptides, however, offer little flexibility in tailoring the physicochemical properties of single chain peptide based systems. Owing to significant advances in the field of peptide chemistry, synthetic mimics of natural peptides are often prepared and have been evaluated for their gene expression, as a function of amino acid functionalities, architecture and net cationic content of peptide chains. Moreover, chimeric single polypeptide chains are prepared by a combination of multiple small natural or synthetic peptides, which imparts distinct physiological properties to peptide based gene delivery therapeutics. In order to obtain multivalency and improve the gene delivery efficacies of low molecular weight cationic peptides, bioactive peptides are often incorporated into a polymeric architecture to obtain novel 'polymer-peptide hybrids' with improved gene delivery efficacies. Peptide modified polymers prepared by physical or chemical modifications exhibit enhanced endosomal escape, stimuli responsive degradation and targeting efficacies, as a function of physicochemical and biological activities of peptides attached onto a polymeric scaffold. The focus of this review is to provide comprehensive and step-wise progress in major natural and synthetic peptides, chimeric polypeptides, and peptide-polymer hybrids for nucleic acid delivery applications.

Biosensor compositions and methods of use

Science.gov (United States)

Bayley, Hagan P.; Howorka, Stefan G.; Movileanu, Liviu

2005-07-12

Provided are pore-subunit polypeptides covalently linked to one or more sensing moieties, and uses of these modified polypeptides to detect and/or measure analytes or physical characteristics within a given sample.

Tissue polypeptide antigen activity in cerebrospinal fluid

DEFF Research Database (Denmark)

Bach, F; Söletormos, Georg; Dombernowsky, P

1991-01-01

Tissue polypeptide antigen (TPpA) in the cerebrospinal fluid (CSF) was measured in 59 consecutive breast cancer patients with suspected central nervous system (CNS) metastases. Subsequently, we determined that 13 patients had parenchymal brain metastases, 10 had leptomeningeal carcinomatosis......, and 36 had no CNS involvement. The concentration of TPpA, which is a nonspecific marker for cell proliferation, was significantly higher in patients with CNS metastases than in those without it (P less than .0001; Mann-Whitney test). A tentative cutoff value for CNS metastases was set at 95 U/L TPp...... metastases, no correlation was found between TPpA activity in corresponding CSF and blood samples (correlation coefficient, Spearman's rho = .4; P greater than .1). In three patients treated for leptomeningeal carcinomatosis, the measurements of CSF TPpA showed correlation between the presence of tumor cells...

Natural polypeptide scaffolds: beta-sheets, beta-turns, and beta-hairpins.

Science.gov (United States)

Rotondi, Kenneth S; Gierasch, Lila M

2006-01-01

This paper provides an introduction to fundamental conformational states of polypeptides in the beta-region of phi,psi space, in which the backbone is extended near to its maximal length, and to more complex architectures in which extended segments are linked by turns and loops. There are several variants on these conformations, and they comprise versatile scaffolds for presentation of side chains and backbone amides for molecular recognition and designed catalysts. In addition, the geometry of these fundamental folds can be readily mimicked in peptidomimetics. Copyright 2005 Wiley Periodicals, Inc.

Functional Modification of Thioether Groups in Peptides, Polypeptides, and Proteins.

Science.gov (United States)

Deming, Timothy J

2017-03-15

Recent developments in the modification of methionine and other thioether-containing residues in peptides, polypeptides, and proteins are reviewed. Properties and potential applications of the resulting functionalized products are also discussed. While much of this work is focused on natural Met residues, modifications at other side-chain residues have also emerged as new thioether-containing amino acids have been incorporated into peptidic materials. Functional modification of thioether-containing amino acids has many advantages and is a complementary methodology to the widely utilized methods for modification at cysteine residues.

Accumulation of New Polypeptides in Ri T-DNA-Transformed Roots of Tomato (Lycopersicon esculentum) during the Development of Vesicular-Arbuscular Mycorrhizae.

Science.gov (United States)

Simoneau, P; Louisy-Louis, N; Plenchette, C; Strullu, D G

1994-06-01

Root-inducing transferred-DNA (Ri T-DNA)-transformed roots of tomato (Lycopersicon esculentum) were in vitro inoculated with surface-sterilized vesicular-arbuscular mycorrhizal leek root pieces. About 1 week after inoculation, the infection of the transformed root culture by the fungal endophyte was confirmed by photonic microscopy. Total proteins were extracted from the mycorrhizal roots and analyzed by two-dimensional polyacrylamide gel electrophoresis. Control gels were run with proteins extracted from noninoculated roots mixed with purified intraradical vesicles and extraradical hyphae. Comparison of the resulting patterns revealed the presence of two polypeptides with estimated apparent masses of 24 and 39 kDa that were detected only in infected roots. Polypeptides with similar migration parameters were not detected in roots challenged with spore extracts, suggesting that the accumulation of the polypeptides was directly linked to root colonization by the fungus rather than to induction by fungus-derived elicitors.

Vasoactive intestinal polypeptide (VIP) in the pig pancreas

DEFF Research Database (Denmark)

Poulsen, Steen Seier

1984-01-01

Vasoactive intestinal polypeptide (VIP) in the pig pancreas is localized to nerves, many of which travel along the pancreatic ducts. VIP stimulates pancreatic fluid and bicarbonate secretion like secretin. Electrical vagal stimulation in the pig causes an atropine-resistant profuse secretion...... of bicarbonate-rich pancreatic juice. In an isolated perfused preparation of the pig pancreas with intact vagal nerve supply, electrical vagal stimulation caused an atropine-resistant release of VIP, which accurately parallelled the exocrine secretion of juice and bicarbonate. Perfusion of the pancreas...... with a potent VIP-antiserum inhibited the effect of vagal stimulation on the exocrine secretion. It is concluded, that VIP is responsible for (at least part of) the neurally controlled fluid and bicarbonate secretion from the pig pancreas....

Elastin-like polypeptides: the power of design for smart cell encapsulation.

Science.gov (United States)

Bandiera, Antonella

2017-01-01

Cell encapsulation technology is still a challenging issue. Innovative methodologies such as additive manufacturing, and alternative bioprocesses, such as cell therapeutic delivery, where cell encapsulation is a key tool are rapidly gaining importance for their potential in regenerative medicine. Responsive materials such as elastin-based recombinant expression products have features that are particularly attractive for cell encapsulation. They can be designed and tailored to meet desired requirements. Thus, they represent promising candidates for the development of new concept-based materials that can be employed in this field. Areas covered: An overview of the design and employment of elastin-like polypeptides for cell encapsulation is given to outline the state of the art. Special attention is paid to the design of the macromolecule employed as well as to the method of matrix formation and the biological system involved. Expert opinion: As a result of recent progress in regenerative medicine there is a compelling need for materials that provide specific properties and demonstrate defined functional features. Rationally designed materials that may adapt according to applied external stimuli and that are responsive to biological systems, such as elastin-like polypeptides, belong to this class of smart material. A run through the components described to date represents a good starting point for further advancement in this area. Employment of these components in cell encapsulation application will promote its advance toward 'smart cell encapsulation technology'.

An Investigation of the Polypeptide, Poly - L - Glutamic Acid, Using Neutron Inelastic Scattering

International Nuclear Information System (INIS)

Whittemore, W.L.

1968-01-01

The polypeptides are synthetic polymers of amino acids with many similarities to natural proteins. In a large number of cases, one of the conformations for both the synthetic and natural proteins is the α - helix. The simplest of the synthetic polymers with no side chains is polyglycine and the simplest of the synthetic polymers with a small side chain (methyl group) is polyalanine. Dispersion curves have been computed by Gupta for both of these polymers. Polyglutamic acid is similar to polyalanine in that the composition of the basic residue and radius of helix is the same. Polyglutamic acid has a more complicated side chain which will contribute a number of additional natural frequencies that are expected to be essentially independent of conformation. On the other hand, the dispersion curves already derived for polyalanine in the α -helix form should be correct in many specific details for polyglutamic acid. An experimental study has been undertaken for polyglutamic acid at room temperature using the techniques of inelastic neutron scattering. In the first measurements, 'cold' neutrons from a reactor were used to investigate the energy level structure up to ≃ 3 kT for both conformations of the polymer. In addition, the scattering of monoenergetic high-energy neutrons ( > 0.15 eV) provided- by an electron Linac was used to study energy levels above 3 kT. These latter measurements permit comparisons to be made between the calculated and measured results for a much larger range of frequencies (and hence permit a check for a larger number of dispersion curves). This extension of the experimental results to higher frequencies has made it possible to check on the earlier assumption that only the lower frequencies are altered when the conformation is changed. This assumption underlies the evaluation of changes in internal energy with conformation from only the 'cold' neutron data, as is done with the present data. An experiment was performed to evaluate the

An Investigation of the Polypeptide, Poly - L - Glutamic Acid, Using Neutron Inelastic Scattering

Energy Technology Data Exchange (ETDEWEB)

Whittemore, W. L. [Gulf General Atomic Incorporated, San Diego, CA (United States)

1968-09-15

The polypeptides are synthetic polymers of amino acids with many similarities to natural proteins. In a large number of cases, one of the conformations for both the synthetic and natural proteins is the {alpha} - helix. The simplest of the synthetic polymers with no side chains is polyglycine and the simplest of the synthetic polymers with a small side chain (methyl group) is polyalanine. Dispersion curves have been computed by Gupta for both of these polymers. Polyglutamic acid is similar to polyalanine in that the composition of the basic residue and radius of helix is the same. Polyglutamic acid has a more complicated side chain which will contribute a number of additional natural frequencies that are expected to be essentially independent of conformation. On the other hand, the dispersion curves already derived for polyalanine in the {alpha} -helix form should be correct in many specific details for polyglutamic acid. An experimental study has been undertaken for polyglutamic acid at room temperature using the techniques of inelastic neutron scattering. In the first measurements, 'cold' neutrons from a reactor were used to investigate the energy level structure up to Asymptotically-Equal-To 3 kT for both conformations of the polymer. In addition, the scattering of monoenergetic high-energy neutrons ( > 0.15 eV) provided- by an electron Linac was used to study energy levels above 3 kT. These latter measurements permit comparisons to be made between the calculated and measured results for a much larger range of frequencies (and hence permit a check for a larger number of dispersion curves). This extension of the experimental results to higher frequencies has made it possible to check on the earlier assumption that only the lower frequencies are altered when the conformation is changed. This assumption underlies the evaluation of changes in internal energy with conformation from only the 'cold' neutron data, as is done with the present data. An experiment was

Injectable, Biomolecule-Responsive Polypeptide Hydrogels for Cell Encapsulation and Facile Cell Recovery through Triggered Degradation.

Science.gov (United States)

Xu, Qinghua; He, Chaoliang; Zhang, Zhen; Ren, Kaixuan; Chen, Xuesi

2016-11-16

Injectable hydrogels have been widely investigated in biomedical applications, and increasing demand has been proposed to achieve dynamic regulation of physiological properties of hydrogels. Herein, a new type of injectable and biomolecule-responsive hydrogel based on poly(l-glutamic acid) (PLG) grafted with disulfide bond-modified phloretic acid (denoted as PLG-g-CPA) was developed. The hydrogels formed in situ via enzymatic cross-linking under physiological conditions in the presence of horseradish peroxidase and hydrogen peroxide. The physiochemical properties of the hydrogels, including gelation time and the rheological property, were measured. Particularly, the triggered degradation of the hydrogel in response to a reductive biomolecule, glutathione (GSH), was investigated in detail. The mechanical strength and inner porous structure of the hydrogel were influenced by the addition of GSH. The polypeptide hydrogel was used as a three-dimensional (3D) platform for cell encapsulation, which could release the cells through triggered disruption of the hydrogel in response to the addition of GSH. The cells released from the hydrogel were found to maintain high viability. Moreover, after subcutaneous injection into rats, the PLG-g-CPA hydrogels with disulfide-containing cross-links exhibited a markedly faster degradation behavior in vivo compared to that of the PLG hydrogels without disulfide cross-links, implying an interesting accelerated degradation process of the disulfide-containing polypeptide hydrogels in the physiological environment in vivo. Overall, the injectable and biomolecule-responsive polypeptide hydrogels may serve as a potential platform for 3D cell culture and easy cell collection.

99mTc-HYNIC-derivatized ternary ligand complexes for 99mTc-labeled polypeptides with low in vivo protein binding

International Nuclear Information System (INIS)

Ono, Masahiro; Arano, Yasushi; Mukai, Takahiro; Fujioka, Yasushi; Ogawa, Kazuma; Uehara, Tomoya; Saga, Tsuneo; Konishi, Junji; Saji, Hideo

2001-01-01

6-Hydrazinopyridine-3-carboxylic acid (HYNIC) is a representative agent used to prepare technetium-99m ( 99m Tc)-labeled polypeptides with tricine as a coligand. However, 99m Tc-HYNIC-labeled polypeptides show delayed elimination rates of the radioactivity not only from the blood but also from nontarget tissues such as the liver and kidney. In this study, a preformed chelate of tetrafluorophenol (TFP) active ester of [ 99m Tc](HYNIC)(tricine)(benzoylpyridine: BP) ternary complex was synthesized to prepare 99m Tc-labeled polypeptides with higher stability against exchange reactions with proteins in plasma and lysosomes using the Fab fragment of a monoclonal antibody and galactosyl-neoglycoalbumin (NGA) as model polypeptides. When incubated in plasma, [ 99m Tc](HYNIC-Fab)(tricine)(BP) showed significant reduction of the radioactivity in high molecular weight fractions compared with [ 99m Tc](HYNIC-Fab)(tricine) 2. When injected into mice, [ 99m Tc](HYNIC-NGA)(tricine)(BP) was metabolized to [ 99m Tc](HYNIC-lysine)(tricine)(BP) in the liver with no radioactivity detected in protein-bound fractions in contrast to the observations with [ 99m Tc](HYNIC-NGA)(tricine) 2. In addition, [ 99m Tc](HYNIC-NGA)(tricine)(BP) showed significantly faster elimination rates of the radioactivity from the liver as compared with [ 99m Tc](HYNIC-NGA)(tricine) 2. Similar results were observed with 99m Tc-labeled Fab fragments where [ 99m Tc](HYNIC-Fab)(tricine)(BP) exhibited significantly faster elimination rates of the radioactivity not only from the blood but also from the kidney. These findings indicated that conjugation of [ 99m Tc](HYNIC)(tricine)(BP) ternary ligand complex to polypeptides accelerated elimination rates of the radioactivity from the blood and nontarget tissues due to low binding of the [ 99m Tc](HYNIC)(tricine)(BP) complex with proteins in the blood and in the lysosomes. Such characteristics would render the TFP active ester of [ 99m Tc](HYNIC)(tricine)(BP) complex

Gastric inhibitory polypeptide (GIP) dose-dependently stimulates glucagon secretion in healthy human subjects at euglycaemia

DEFF Research Database (Denmark)

Meier, J J; Gallwitz, B; Siepmann, N

2003-01-01

AIMS/HYPOTHESIS: In the isolated perfused pancreas, gastric inhibitory polypeptide (GIP) has been shown to enhance glucagon secretion at basal glucose concentrations, but in healthy humans no glucagonotropic effect of GIP has yet been reported. Therefore, we studied the effect of GIP on glucagon ...

Similarities in the induction of synthesis of a cell-surface polypeptide in Arthrobacter sp. by near-UV irradiation and photodynamic conditions

International Nuclear Information System (INIS)

Hoober, J.K.; Franzi, J.

1983-01-01

Irradiation of aerobic suspensions of Arthrobacter sp. with near-UV light (310-400 nm) induced synthesis of a 21 000 dalton, cell-surface polypeptide. Synthesis of this polypeptide also was induced by visible light in the presence of photodynamic dyes. Induction of the polypeptide in ear-UV light and with visible light plus dyes was inhibited by histidine. Hemin inhibited induction in near-UV light and in visible light with methylene blue, neutral red and acriflavin, which are cationic dyes, but failed to inhibit induction in visible light with rose bengal, an anionic dye. These results suggested that inhibition by hemin required electrostatically favored interaction between the anionic porphyrin and the sensitizer, and that the near-UV light effect was mediated by a cationic or neutral endogenous sensitizer. The similarities in the responses of the cells to near-UV irradiation and visible light plus dyes suggested that the mechanism of induction under the two conditions was the same. (author)

N-(2-hydroxy) propyl-3-trimethylammonium chitosan chloride: An immune-enhancing adjuvant for hepatitis E virus recombinant polypeptide vaccine in mice.

Science.gov (United States)

Tao, Wei; Zheng, Hai-Qun; Fu, Ting; He, Zhuo-Jing; Hong, Yan

2017-08-03

Adjuvants are essential for enhancing vaccine potency by improving the humoral and/or cell-mediated immune response to vaccine antigens. This study was performed to evaluate the immuno-enhancing characteristic of N-(2-hydroxy) propyl-3-trimethylammonium chitosan chloride (HTCC), the cationically modified chitosan, as an adjuvant for hepatitis E virus (HEV) recombinant polypeptide vaccine. Animal experiments showed that HTCC provides adjuvant activity when co-administered with HEV recombinant polypeptide vaccine by intramuscularly route. Vaccination using HTCC as an adjuvant was associated with increases of the serum HEV-specific IgG antibodies, splenocytes proliferation and the growths of CD4 + CD8 - T lymphocytes and IFN-γ-secreting T lymphocytes in peripheral blood. These findings suggested that HTCC had strong immuno-enhancing effect. Our findings are the first to demonstrate that HTCC is safe and effective in inducing a good antibody response and stimulating Th1-biased immune responses for HEV recombinant polypeptide vaccine.

In-situ crosslinkable and self-assembling elastin-like polypeptide block copolymers for cartilage tissue repair

Science.gov (United States)

Lim, Dong Woo

This work describes the development of genetically engineered elastin-like polypeptide (ELP) block copolymers as in-situ gelling scaffolds for cartilage tissue repair. The central hypothesis underlying this work is that ELP based biopolymers can be exploited as injectable biomaterials by rapid chemical crosslinking. To prove this, gene libraries encoding ELP having different molecular weights and amino acid sequences, and ELP block copolymers composed of various ELP blocks having diverse amino acid composition, length, and phase transition behavior were synthesized by recursive directional ligation, expressed in E. Coli and purified by inverse transition cycling. Mannich-type condensation of hydroxymethylphosphines (HMPs) with primary- and secondary-amines of amino acids was developed as a new crosslinking method of polypeptides. Chemically crosslinked ELP hydrogels were formed rapidly in an aqueous solution by reaction of ELPs containing periodic lysine residues with HMPs. The crosslinking density and mechanical property of the ELP hydrogels were controlled at the sequence level by varying the Lys density in ELPs composed of mono-block as well as by segregation of the Lys residues within specific blocks of tri-block architectures. Fibroblasts embedded in ELP hydrogels survived the crosslinking process and were viable after in vitro culture for at least 3 days. The DNA content of fibroblasts within the tri-block gels was significantly higher than that in the mono-block gels at day 3. These results suggest that the HMP crosslinked ELP block copolymer hydrogels show finely tuned mechanical properties and different microenvironments for cell viability as well as potential as in-situ crosslinkable biopolymers for tissue repair applications with load-bearing environments. As an alternative, rheological behavior of the ELP block copolymers and ELP-grafted hyaluronic acids (HAs) as artificial extracellular matrices (ECMs) showed that they were thermally aggregated into

The Beads of Translation: Using Beads to Translate mRNA into a Polypeptide Bracelet

Science.gov (United States)

Dunlap, Dacey; Patrick, Patricia

2012-01-01

During this activity, by making beaded bracelets that represent the steps of translation, students simulate the creation of an amino acid chain. They are given an mRNA sequence that they translate into a corresponding polypeptide chain (beads). This activity focuses on the events and sites of translation. The activity provides students with a…

Reaction mechanisms in the radiolysis of peptides, polypeptides and proteins

International Nuclear Information System (INIS)

Garrison, W.M.

1985-01-01

The purpose of this review is to bring together and to correlate the wide variety of experimental studies that provide information on the reaction products and reaction mechanisms involved in the radiolysis of peptides, polypeptides and proteins (including chromosomal proteins) in both aqueous and solid-state systems. The comparative radiation chemistry of these systems is developed in terms of specific reactions of the peptide main-chain and the aliphatic, aromatic-unsaturated and sulfur-containing side-chains. Information obtained with the various experimental techniques of product analysis, competition kinetics, spin-trapping, pulse radiolysis and ESR spectroscopy is included. 147 refs

Reaction mechanisms in the radiolysis of peptides, polypeptides and proteins

Energy Technology Data Exchange (ETDEWEB)

Garrison, W.M.

1985-01-01

The purpose of this review is to bring together and to correlate the wide variety of experimental studies that provide information on the reaction products and reaction mechanisms involved in the radiolysis of peptides, polypeptides and proteins (including chromosomal proteins) in both aqueous and solid-state systems. The comparative radiation chemistry of these systems is developed in terms of specific reactions of the peptide main-chain and the aliphatic, aromatic-unsaturated and sulfur-containing side-chains. Information obtained with the various experimental techniques of product analysis, competition kinetics, spin-trapping, pulse radiolysis and ESR spectroscopy is included. 147 refs.

Chemical modification as an approach for the identification of UDPG-binding polypeptides of UDPG-glucose: (1,3)-Beta-glucan synthase

International Nuclear Information System (INIS)

Mason, T.L.

1989-01-01

The lysine-reactive chemical modification reagents uridine diphosphate pyridoxal (UDP-pyridoxal) and formaldehyde (HCHO) were used to identify UDPG-binding polypeptides of UDP-glucose: (1,3)-β-D-glucan synthase (GS) from red beet storage tissue. Complete enzyme inactivation occurred after exposure to micromolar levels of UDP-pyridoxal and millimolar levels of HCHO. Divalent cations (Mg 2+ and Ca 2+ , particularly Ca 2+ ) were required by both for inactivation. Substrate (UDPG) and chelators (EDTA and EGTA) protected plasma membrane GS (PMGS) against UDP-pyridoxal and HCHO inhibition. UDPG protected CHAPS solubilized GS (CSGS) against UDP-pyridoxal inactivation, but not against HCHO. It was concluded that beet GS contains a lysine residue at the UDPG-binding site. When PMGS was directly labeled with UDP[ 3 H]-pyridoxal or [ 14 C]HCHO, random labeling occurred. Therefore, a multi-step labeling procedure was developed. Nonessential lysine residues were first blocked with HCHO while 5 mM UDPG protected the active site lysine. Background labeling was reduced 4-fold. Membranes were recovered by centrifugation and the active site lysine exposed to [ 14 C] HCHO. Major labeled polypeptides were at 200, 76, and 54 kD. Minor polypeptides were seen at 94, 82, 68, 60, and 20-25 kD. CSGS was labeled by a modified multi-step procedure. CSGS was blocked by reaction with UDP-pyridoxal in the presence of UDPG. CSGS was then recovered by product entrapment and labeled with [ 14 C]HCHO. Background labeling was reduced by 8-fold and potential UDPG-binding polypeptides narrowed to 68, 54, 25 and 22 kD

Pancreatic polypeptide controls energy homeostasis via Npy6r signaling in the suprachiasmatic nucleus in mice.

Science.gov (United States)

Yulyaningsih, Ernie; Loh, Kim; Lin, Shu; Lau, Jackie; Zhang, Lei; Shi, Yanchuan; Berning, Britt A; Enriquez, Ronaldo; Driessler, Frank; Macia, Laurence; Khor, Ee Cheng; Qi, Yue; Baldock, Paul; Sainsbury, Amanda; Herzog, Herbert

2014-01-07

Y-receptors control energy homeostasis, but the role of Npy6 receptors (Npy6r) is largely unknown. Young Npy6r-deficient (Npy6r(-/-)) mice have reduced body weight, lean mass, and adiposity, while older and high-fat-fed Npy6r(-/-) mice have low lean mass with increased adiposity. Npy6r(-/-) mice showed reduced hypothalamic growth hormone releasing hormone (Ghrh) expression and serum insulin-like growth factor-1 (IGF-1) levels relative to WT. This is likely due to impaired vasoactive intestinal peptide (VIP) signaling in the suprachiasmatic nucleus (SCN), where we found Npy6r coexpressed in VIP neurons. Peripheral administration of pancreatic polypeptide (PP) increased Fos expression in the SCN, increased energy expenditure, and reduced food intake in WT, but not Npy6r(-/-), mice. Moreover, intraperitoneal (i.p.) PP injection increased hypothalamic Ghrh mRNA expression and serum IGF-1 levels in WT, but not Npy6r(-/-), mice, an effect blocked by intracerebroventricular (i.c.v.) Vasoactive Intestinal Peptide (VPAC) receptors antagonism. Thus, PP-initiated signaling through Npy6r in VIP neurons regulates the growth hormone axis and body composition. Copyright © 2014 Elsevier Inc. All rights reserved.

Fibrillar dimer formation of islet amyloid polypeptides

Energy Technology Data Exchange (ETDEWEB)

Chiu, Chi-cheng [Univ. of Chicago, IL (United States); Argonne National Lab. (ANL), Argonne, IL (United States); de Pablo, Juan J. [Univ. of Chicago, IL (United States); Argonne National Lab. (ANL), Argonne, IL (United States)

2015-05-08

Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 – 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 – 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

Fibrillar dimer formation of islet amyloid polypeptides

Directory of Open Access Journals (Sweden)

Chi-cheng Chiu

2015-09-01

Full Text Available Amyloid deposits of human islet amyloid polypeptide (hIAPP, a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 – 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 – 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

Mathematical Modelling of Glucose-Dependent Insulinotropic Polypeptide and Glucagon-like Peptide-1 following Ingestion of Glucose

DEFF Research Database (Denmark)

Røge, Rikke M; Bagger, Jonatan I; Alskär, Oskar

2017-01-01

The incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), play an important role in glucose homeostasis by potentiating glucose-induced insulin secretion. Furthermore, GLP-1 has been reported to play a role in glucose homeostasis by inhibiting ...

The synthesis and characterization of polypeptide-adriamycin conjugates and its complexes with adriamycin. Part I

NARCIS (Netherlands)

Heeswijk, W.A.R.; Hoes, C.J.T.; Stoffer, T.; Eenink, M.J.D.; Potman, W.; Feijen, Jan

1985-01-01

Poly(α-l-glutamic acid) (PGA) was grafted with amino acid and oligopeptide spacers up to 5 amino acids with the use of N,N'-carbonyldiimidazole and 2,3-dihydro-1,2-benz-isothiazole-3-on-1, 1-dioxide (saccharin) as an additive, and these polypeptides were characterized. The antitumor antibiotic

Inhibition of the coated vesicle proton pump and labeling of a 17,000-dalton polypeptide by N,N'-dicyclohexylcarbodiimide

International Nuclear Information System (INIS)

Arai, H.; Berne, M.; Forgac, M.

1987-01-01

N,N'-Dicyclohexylcarbodiimide (DCCD) inhibits 100% of proton transport and 80-85% of (Mg2+)-ATPase activity in clathrin-coated vesicles. Half-maximum inhibition of proton transport is observed at 10 microM DCCD after 30 min. Although treatment of the coated vesicle (H+)-ATPase with DCCD has no effect on ATP hydrolysis in the detergent-solubilized state, sensitivity of proton transport and ATPase activity to DCCD is restored following reconstitution into phospholipid vesicles. In addition, treatment of the detergent-solubilized enzyme with DCCD followed by reconstitution gives a preparation that is blocked in both proton transport and ATP hydrolysis. These results suggest that although the coated vesicle (H+)-ATPase can react with DCCD in either a membrane-bound or detergent-solubilized state, inhibition of ATPase activity is only manifested when the pump is present in sealed membrane vesicles. To identify the subunit responsible for inhibition of the coated vesicle (H+)-ATPase by DCCD, we have labeled the partially purified enzyme with [ 14 C]DCCD. A single polypeptide of molecular weight 17,000 is labeled. The extremely hydrophobic nature of this polypeptide is indicated by its extraction with chloroform:methanol. The 17,000-dalton protein can be labeled to a maximum stoichiometry of 0.99 mol of DCCD/mol of protein with 100% inhibition of proton transport occurring at a stoichiometry of 0.15-0.20 mol of DCCD/mol of protein. Amino acid analysis of the chloroform:methanol extracted 17,000-dalton polypeptide reveals a high percentage of nonpolar amino acids. The similarity in properties of this protein and the DCCD-binding subunit of the coupling factor (H+)-ATPases suggests that the 17,000-dalton polypeptide may function as part of a proton channel in the coated vesicle proton pump

Morphological variation of stimuli-responsive polypeptide at air–water interface

International Nuclear Information System (INIS)

Shin, Sungchul; Ahn, Sungmin; Cheng, Jie; Chang, Hyejin; Jung, Dae-Hong; Hyun, Jinho

2016-01-01

Graphical abstract: - Highlights: • It is the first report on the interfacial properties of ELP monolayers formed at the air–water interface. • ELP monolayers could be prepared with high stability at the air–water interface. • The compressive behavior of thermo-sensitive ELP monolayers was imaged. • The SERS spectra showed a change in the ELP secondary structure at different preparation conditions. - Abstract: The morphological variation of stimuli-responsive polypeptide molecules at the air–water interface as a function of temperature and compression was described. The surface pressure–area (π–A) isotherms of an elastin-like polypeptide (ELP) monolayer were obtained under variable external conditions, and Langmuir–Blodgett (LB) monolayers were deposited onto a mica substrate for characterization. As the compression of the ELP monolayer increased, the surface pressure increased gradually, indicating that the ELP monolayer could be prepared with high stability at the air–water interface. The temperature in the subphase of the ELP monolayer was critical in the preparation of LB monolayers. The change in temperature induced a shift in the π–A isotherms as well as a change in ELP secondary structures. Surprisingly, the compression of the ELP monolayer influenced the ELP secondary structure due to the reduction in the phase transition temperature with decreasing temperature. The change in the ELP secondary structure formed at the air–water interface was investigated by surface-enhanced Raman scattering. Moreover, the morphology of the ELP monolayer was subsequently imaged using atomic force microscopy. The temperature responsive behavior resulted in changes in surface morphology from relatively flat structures to rugged labyrinth structures, which suggested conformational changes in the ELP monolayers.

Morphological variation of stimuli-responsive polypeptide at air–water interface

Energy Technology Data Exchange (ETDEWEB)

Shin, Sungchul; Ahn, Sungmin; Cheng, Jie [Department of Biosystems and Biomaterials Science and Engineering, Seoul National University, Seoul 151-921 (Korea, Republic of); Chang, Hyejin; Jung, Dae-Hong [Department of Chemical Education, Seoul National University, Seoul 151-741 (Korea, Republic of); Hyun, Jinho, E-mail: jhyun@snu.ac.kr [Department of Biosystems and Biomaterials Science and Engineering, Seoul National University, Seoul 151-921 (Korea, Republic of); Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921 (Korea, Republic of); Center for Food and Bioconvergence, Seoul National University, Seoul 151-921, Republic of Korea. (Korea, Republic of)

2016-12-01

Graphical abstract: - Highlights: • It is the first report on the interfacial properties of ELP monolayers formed at the air–water interface. • ELP monolayers could be prepared with high stability at the air–water interface. • The compressive behavior of thermo-sensitive ELP monolayers was imaged. • The SERS spectra showed a change in the ELP secondary structure at different preparation conditions. - Abstract: The morphological variation of stimuli-responsive polypeptide molecules at the air–water interface as a function of temperature and compression was described. The surface pressure–area (π–A) isotherms of an elastin-like polypeptide (ELP) monolayer were obtained under variable external conditions, and Langmuir–Blodgett (LB) monolayers were deposited onto a mica substrate for characterization. As the compression of the ELP monolayer increased, the surface pressure increased gradually, indicating that the ELP monolayer could be prepared with high stability at the air–water interface. The temperature in the subphase of the ELP monolayer was critical in the preparation of LB monolayers. The change in temperature induced a shift in the π–A isotherms as well as a change in ELP secondary structures. Surprisingly, the compression of the ELP monolayer influenced the ELP secondary structure due to the reduction in the phase transition temperature with decreasing temperature. The change in the ELP secondary structure formed at the air–water interface was investigated by surface-enhanced Raman scattering. Moreover, the morphology of the ELP monolayer was subsequently imaged using atomic force microscopy. The temperature responsive behavior resulted in changes in surface morphology from relatively flat structures to rugged labyrinth structures, which suggested conformational changes in the ELP monolayers.

Effects of vasoactive intestinal polypeptide on heart rate in relation to vagal cardioacceleration in conscious dogs

NARCIS (Netherlands)

Roossien, A; Brunstig, J.R; Nijmeijer, A; Zaagsma, Hans; Zijlstra, W.G

Objective: The vagal cardiac accelerator (VCA) system takes part in the nervous control of the heart rate. In the present study we tried to adduce evidence that vasoactive intestinal polypeptide (VLP) contributes to vagally induced cardioacceleration. Methods: The effect of VIP on heart rate and

Thermally Targeted Delivery of a c-Myc Inhibitory Peptide In Vivo Using Elastin-like Polypeptide

Science.gov (United States)

2009-10-01

cytoplasm. Also, in a subset of cells, Bac-ELP1⁎-H1 showed very bright nuclear staining exclusive of nucleoli (Fig. 5, lower right, arrows). 3.6. Time...localization was very bright relative to the amount of polypeptide in the cytoplasm, and it appeared to be nucleoplasmic and excluded from nucleoli . The

Hemoglobin variants as models for investigation of dissociation of intact polypeptide chains by ESI tandem mass spectrometry

International Nuclear Information System (INIS)

Light, K.J.; Loo, J.A.; Edmonds, C.G.; Smith, R.D.

1991-06-01

Electrospray ionization mass spectroscopy (ESI-MS) is rapidly becoming a practical biochemical tool for peptide and protein sequence analysis. The utility of ESI-MS is through use of Collisionally Activated Dissociation (ESI-CAD-MS). Human hemoglobin (Hb, ∼62 kDa) consists of four polypeptide chains and a prosthetic heme group. There are over 400 Hb variants, characterized by amino acid substitutions in either the alpha or beta polypeptide chains. We investigated ESI-CAD-MS as a tool for rapidly analyzing amino acid substitutions, using eight Hb beta chain variants. The approximate location of the modification can be deduced from comparison of the CAD mass spectra and observance of the mass shifts of the fragment ion containing the substitution. Fragmentation occurs preferentially at the amino terminus of proline residues. For most substitutions, differences in CAD mass spectra were not seen. 2 figs

Cancer Nano technology Using Elastin-Like Polypeptides

International Nuclear Information System (INIS)

Siti Najila Mohd Janib

2014-01-01

Despite progress in understanding cancer biology, this knowledge has not translated into comparable advances in the clinic. Two fundamental problems currently stalling the efficient treatment of cancer have been detecting cancer early enough for successful treatment and avoiding excessive toxicity to normal tissues. In view of this, cancer still remains one of the leading causes of mortality worldwide, affecting over 10 million new patients every year. Clearly the development of novel approaches for early detection and treatment of cancer is urgently needed to increase patient survival. Recently, nano technology-based systems have emerged as novel therapeutic modalities for cancer treatment. Tiny man made nanoparticles, much smaller than a virus, are being developed to package, transport, and deliver imaging and therapeutic agents. Co-inclusion of these agents, into nano carriers might be advantageous because they increase solubility of hydrophobic drugs, enhance permeability across physiological barriers, alter drug biodistribution, increase local bioavailability and reduce side effects. Initial findings have been promising and nanoparticles have been shown to deliver therapeutic agents to target cells and effect tumor growth. To this end our lab is investigating a class of biodegradable and biocompatible polymers known as elastin-like polypeptides (ELP). Elastin like polypeptide is a bio polymer derived from the structural motif found in mammalian elastin protein and has a sequence dependent transition temperature that can be used as nano carriers to treat diseases. ELPs are characterized by the pentameric repeat VPGXG, where X can be any amino acid. All functional ELPs undergo inverse phase transition whereby below its transition temperature, they exist in a solubilized form while above its transition temperature they undergo phase separation which leads to their aggregation in solution. This process is reversible. Phase transition can also be triggered by other

NMR and rotational angles in solution conformation of polypeptides

Science.gov (United States)

Bystrov, V. F.

1985-01-01

Professor San-Ichiro Mizushima and Professor Yonezo Morino's classical contributions provided unique means and firm basis for understanding of conformational states and internal rotation in polypeptide molecules. Now the NMR spectroscopy is the best choice to study molecular conformation, mechanism of action and structure-functional relationships of peptide and proteins in solution under conditions approaching those of their physiological environments. Crucial details of spatial structure and interactions of these molecules in solution are revealed by using proton-proton and carbon-proton vicinal coupling constants, proton nuclear Overhauser effect and spectral perturbation techniques. The results of NMR conformational analysis are presented for valinomycin "bracelet", gramicidin A double helices, honey-bee neurotoxin apamin, scorpion insectotoxins and snake neurotoxins of long and short types.

UDP-[14C]glucose-labelable polypeptides from pea: Possible components of glucan synthase I activity

International Nuclear Information System (INIS)

Ray, P.M.; Dhugga, K.S.; Gallaghar, S.R.

1989-01-01

A membrane-bound polypeptide doublet of about 40 kD can be rapidly labeled with UDP-[ 14 C]glucose under the assay conditions for glucan synthase I (GS-I). Label seems covalently bound, and chases when unlabeled UDPG is added; it might represent a covalent intermediate in polysaccharide synthesis. Labeling and GS-I activity show several common features: they co-sediment with Golgi membranes in sucrose gradients; they depend similarly on Mg 2+ or Mn 2+ (not Ca 2+ ); they decrease dramatically from stem apex to base, and are higher in epidermis than internal tissue; they show similar sensitivities to several inhibitors. But the doublet still labels after polysaccharide-synthesizing activity has been destroyed by Triton X-100. The doublet polypeptides might be glucosyl tranferases whose ability to transfer glucose units to a glucan chain is detergent-sensitive, but to accept glucose from UDPG is not; or they might be detergent-insensitive primary glucose acceptors, from which a distinct, detergent-sensitive transferase(s) move(s) these units to glucan chains

Microalgae respond differently to nitrogen availability during culturing

Indian Academy of Sciences (India)

Variations in the exogenous nitrogen level are known to significantly affect the physiological status and metabolism of microalgae. However, responses of red, green and yellow-green algae to nitrogen (N) availability have not been compared yet. Porphyridium cruentum, Scenedesmus incrassatulus and Trachydiscus ...

Design and synthesis of elastin-like polypeptides for an ideal nerve conduit in peripheral nerve regeneration

International Nuclear Information System (INIS)

Hsueh, Yu-Sheng; Savitha, S.; Sadhasivam, S.; Lin, Feng-Huei; Shieh, Ming-Jium

2014-01-01

The study involves design and synthesis of three different elastin like polypeptide (ELP) gene monomers namely ELP1, ELP2 and ELP3 that encode for ELP proteins. The formed ELPs were assessed as an ideal nerve conduit for peripheral nerve regeneration. ELP1 was constructed with a small elongated pentapeptide carrying VPGVG sequence to mimic the natural polypeptide ELP. The ELP2 was designed by the incorporation of 4-penta peptide chains to improve the biocompatibility and mechanical strength. Thus, the third position in unique VPGVG was replaced with alanine to VPAVG and in a similar way modified to VPGKG, VPGEG and VPGIG with the substitution of lysine, glutamic acid and isoleucine. In ELP3, fibronectin C5 domain endowed with REDV sequence was introduced to improve the cell attachment. The ELP1, ELP2 and ELP3 proteins expressed by Escherichia coli were purified by inverse transition cycling (ITC). The purified ELPs were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The Schwann cell (SC) morphology and cell adhesion were assessed by fabrication of ELP membrane cross-linked with glutaraledhyde. The Schwann cell proliferation was measured by WST-1 assay. Immunofluorostaining of Schwann cells was accomplished with SC specific phenotypic marker, S100. - Highlights: • Design and synthesis of three gene monomers of elastin like polypeptides (ELP1, 2 and 3) were reported. • Molecular weight of ITC purified ELP1, ELP2 and ELP3 was in the range of 37–38 kDa. • Schwann cell adhesion was found to be prominent in ELP3 and could be used as nerve conduit for peripheral nerve regeneration

Design and synthesis of elastin-like polypeptides for an ideal nerve conduit in peripheral nerve regeneration

Energy Technology Data Exchange (ETDEWEB)

Hsueh, Yu-Sheng [Institute of Biomedical Engineering, College of Engineering, National Taiwan University, Taipei 100, Taiwan (China); Institute of Biomedical Engineering, College of Medicine, National Taiwan University, Taipei 100, Taiwan (China); Savitha, S. [Institute of Biomedical Engineering, College of Engineering, National Taiwan University, Taipei 100, Taiwan (China); Department of Biotechnology, Sree Sastha Institute of Engineering and Technology, Chennai (India); Institute of Biomedical Engineering, College of Medicine, National Taiwan University, Taipei 100, Taiwan (China); Sadhasivam, S. [Division of Biomedical Engineering and Nanomedicine Research, National Health Research Institutes, Miaoli 350, Taiwan (China); Lin, Feng-Huei, E-mail: double@ntu.edu.tw [Institute of Biomedical Engineering, College of Engineering, National Taiwan University, Taipei 100, Taiwan (China); Division of Biomedical Engineering and Nanomedicine Research, National Health Research Institutes, Miaoli 350, Taiwan (China); Institute of Biomedical Engineering, College of Medicine, National Taiwan University, Taipei 100, Taiwan (China); Shieh, Ming-Jium [Institute of Biomedical Engineering, College of Engineering, National Taiwan University, Taipei 100, Taiwan (China); College of Medicine, National Taiwan University Hospital, Taipei 100, Taiwan (China); Institute of Biomedical Engineering, College of Medicine, National Taiwan University, Taipei 100, Taiwan (China)

2014-05-01

The study involves design and synthesis of three different elastin like polypeptide (ELP) gene monomers namely ELP1, ELP2 and ELP3 that encode for ELP proteins. The formed ELPs were assessed as an ideal nerve conduit for peripheral nerve regeneration. ELP1 was constructed with a small elongated pentapeptide carrying VPGVG sequence to mimic the natural polypeptide ELP. The ELP2 was designed by the incorporation of 4-penta peptide chains to improve the biocompatibility and mechanical strength. Thus, the third position in unique VPGVG was replaced with alanine to VPAVG and in a similar way modified to VPGKG, VPGEG and VPGIG with the substitution of lysine, glutamic acid and isoleucine. In ELP3, fibronectin C5 domain endowed with REDV sequence was introduced to improve the cell attachment. The ELP1, ELP2 and ELP3 proteins expressed by Escherichia coli were purified by inverse transition cycling (ITC). The purified ELPs were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The Schwann cell (SC) morphology and cell adhesion were assessed by fabrication of ELP membrane cross-linked with glutaraledhyde. The Schwann cell proliferation was measured by WST-1 assay. Immunofluorostaining of Schwann cells was accomplished with SC specific phenotypic marker, S100. - Highlights: • Design and synthesis of three gene monomers of elastin like polypeptides (ELP1, 2 and 3) were reported. • Molecular weight of ITC purified ELP1, ELP2 and ELP3 was in the range of 37–38 kDa. • Schwann cell adhesion was found to be prominent in ELP3 and could be used as nerve conduit for peripheral nerve regeneration.

Vasoactive intestinal polypeptide (VIP) in cirrhosis: arteriovenous extraction in different vascular beds

DEFF Research Database (Denmark)

Henriksen, Jens Henrik Sahl; Staun-Olsen, P; Fahrenkrug, J

1980-01-01

The concentration of vasoactive intestinal polypeptide (VIP) was determined in peripheral venous plasma from 136 patients with liver cirrhosis without gastrointestinal bleeding or coma and from 112 controls. In eight patients (cirrhosis, six; fibrosis, one; steatosis, one) arteriovenous extraction...... is significantly elevated in peripheral plasma from patients with cirrhosis, probably due to porto-systemic shunting and/or compromised hepatic elimination. Hepatic elimination is still likely to account for the inactivation of most of the VIP escaping from the neurosynapses throughout the body in patients...

Interactions of protein content and globulin subunit composition of soybean proteins in relation to tofu gel properties.

Science.gov (United States)

James, Andrew T; Yang, Aijun

2016-03-01

The content and globulin subunit composition of soybean proteins are known to affect tofu quality and food-grade soybeans usually have higher levels of proteins. We studied the tofu quality of soybeans with high (44.8%) or low (39.1%) protein content and with or without the 11S globulin polypeptide, 11SA4. Both protein content and 11SA4 significantly affected tofu gel properties. Soybeans containing more protein had smaller seeds which produced significantly firmer (0.663 vs.0.557 N, pseed size, tofu hardness and water holding capacity and led to significant changes to the profile of storage protein subunits, which may have contributed to the improvement in tofu gel properties. These results suggest that, in combination with higher protein content, certain protein subunits or their polypeptides can also be targeted in selecting soybeans to further improve soy food quality. Copyright © 2015 Elsevier Ltd. All rights reserved.

Conformational energy calculations on polypeptides and proteins: use of a statistical mechanical procedure for evaluating structure and properties.

Science.gov (United States)

Scheraga, H A; Paine, G H

1986-01-01

We are using a variety of theoretical and computational techniques to study protein structure, protein folding, and higher-order structures. Our earlier work involved treatments of liquid water and aqueous solutions of nonpolar and polar solutes, computations of the stabilities of the fundamental structures of proteins and their packing arrangements, conformations of small cyclic and open-chain peptides, structures of fibrous proteins (collagen), structures of homologous globular proteins, introduction of special procedures as constraints during energy minimization of globular proteins, and structures of enzyme-substrate complexes. Recently, we presented a new methodology for predicting polypeptide structure (described here); the method is based on the calculation of the probable and average conformation of a polypeptide chain by the application of equilibrium statistical mechanics in conjunction with an adaptive, importance sampling Monte Carlo algorithm. As a test, it was applied to Met-enkephalin.

Impaired pancreatic polypeptide response to a meal in type 1 diabetic patients

DEFF Research Database (Denmark)

Rasmussen, M H; Carstensen, H; List, S

1993-01-01

The pancreatic polypeptide (PP) response to a mixed meal was investigated in seven insulin-dependent diabetics without measurable signs of diabetic autonomic neuropathy, and in seven healthy subjects. Since acute changes in metabolic regulation might influence the meal-induced PP response...... is independent of short-term changes in metabolic control. Since the response was attenuated in the insulin-dependent diabetic patients, who had no otherwise measurable signs of neuropathy, the PP response to a meal could be a sensitive indicator of dysfunction of the reflex arc controlling PP secretion...

A fluorescence-based method for direct measurement of submicrosecond intramolecular contact formation in biopolymers: an exploratory study with polypeptides.

Science.gov (United States)

Hudgins, Robert R; Huang, Fang; Gramlich, Gabriela; Nau, Werner M

2002-01-30

A fluorescent amino acid derivative (Fmoc-DBO) has been synthesized, which contains 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) as a small, hydrophilic fluorophore with an extremely long fluorescence lifetime (325 ns in H2O and 505 ns in D2O under air). Polypeptides containing both the DBO residue and an efficient fluorescence quencher allow the measurement of rate constants for intramolecular end-to-end contact formation. Bimolecular quenching experiments indicated that Trp, Cys, Met, and Tyr are efficient quenchers of DBO (k(q) = 20, 5.1, 4.5, and 3.6 x 10(8) M(-1) x s(-1) in D2O), while the other amino acids are inefficient. The quenching by Trp, which was selected as an intrinsic quencher, is presumed to involve exciplex-induced deactivation. Flexible, structureless polypeptides, Trp-(Gly-Ser)n-DBO-NH2, were prepared by standard solid-phase synthesis, and the rates of contact formation were measured through the intramolecular fluorescence quenching of DBO by Trp with time-correlated single-photon counting, laser flash photolysis, and steady-state fluorometry. Rate constants of 4.1, 6.8, 4.9, 3.1, 2.0, and 1.1 x 10(7) s(-1) for n = 0, 1, 2, 4, 6, and 10 were obtained. Noteworthy was the relatively slow quenching for the shortest peptide (n = 0). The kinetic data are in agreement with recent transient absorption studies of triplet probes for related peptides, but the rate constants are significantly larger. In contrast to the flexible structureless Gly-Ser polypeptides, the polyproline Trp-Pro4-DBO-NH2 showed insignificant fluorescence quenching, suggesting that a high polypeptide flexibility and the possibility of probe-quencher contact is essential to induce quenching. Advantages of the new fluorescence-based method for measuring contact formation rates in biopolymers include high accuracy, fast time range (100 ps-1 micros), and the possibility to perform measurements in water under air.

The lectin domain of UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-T4 directs its glycopeptide specificities

DEFF Research Database (Denmark)

Hassan, H; Reis, C A; Bennett, E P

2000-01-01

The initiation step of mucin-type O-glycosylation is controlled by a large family of homologous UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Differences in kinetic properties, substrate specificities, and expression patterns of these isoenzymes provide for diff...

Binding Properties of Streptococcus gordonii SspA and SspB (Antigen I/II Family) Polypeptides Expressed on the Cell Surface of Lactococcus lactis MG1363

OpenAIRE

Holmes, Ann R.; Gilbert, Christophe; Wells, Jeremy M.; Jenkinson, Howard F.

1998-01-01

The oral bacterium Streptococcus gordonii expresses two cell wall-associated polypeptides, designated SspA (1,542 amino acid residues) and SspB (1,462 amino acid residues), that have 70% sequence identity. These polypeptides are members of the antigen I/II family of oral streptococcal adhesins and mediate the binding of streptococci to salivary glycoproteins, collagen, and other oral microorganisms such as Actinomyces naeslundii. To determine if SspA and SspB have differential binding propert...

Rapid measurement of 3J(H N-H alpha) and 3J(N-H beta) coupling constants in polypeptides.

Science.gov (United States)

Barnwal, Ravi Pratap; Rout, Ashok K; Chary, Kandala V R; Atreya, Hanudatta S

2007-12-01

We present two NMR experiments, (3,2)D HNHA and (3,2)D HNHB, for rapid and accurate measurement of 3J(H N-H alpha) and 3J(N-H beta) coupling constants in polypeptides based on the principle of G-matrix Fourier transform NMR spectroscopy and quantitative J-correlation. These experiments, which facilitate fast acquisition of three-dimensional data with high spectral/digital resolution and chemical shift dispersion, will provide renewed opportunities to utilize them for sequence specific resonance assignments, estimation/characterization of secondary structure with/without prior knowledge of resonance assignments, stereospecific assignment of prochiral groups and 3D structure determination, refinement and validation. Taken together, these experiments have a wide range of applications from structural genomics projects to studying structure and folding in polypeptides.

HPLC of the Polypeptides in a Hydrolyzate of Egg-White Lysozyme. An Experiment for the Undergraduate Biochemistry Laboratory.

Science.gov (United States)

Richardson, W. S., III; Burns, L.

1988-01-01

Describes a simple high-performance liquid chromatography experiment for undergraduate biochemistry laboratories. The experiment illustrates the separation of polypeptides by a step gradient elution using a single pump instrument with no gradient attachments. Discusses instrumentation, analysis, a sample preparation, and results. (CW)

A radioimmunoassay of gastric inhibitory polypeptide in human plasma

International Nuclear Information System (INIS)

Sarson, D.L.; Bryant, M.G.; Bloom, S.R.

1980-01-01

A sensitive radioimmunoassay for the measurement of human gastric inhibitory polypeptide (GIP), using pure porcine GIP, has been developed. Cross-reactivity of the antiserum with all available mammalian gut peptide preparations was negligible with the exception of glucagon when it was approximately 1%. Two major molecular forms of GIP were detectable in plasma and tissue extracts, one of large molecular size and the other corresponding to the elution coefficient of pure porcine standard. Concentrations of GIP in plasma from 50 normal subjects after overnight fasting were 9+-1.0(S.E.M.) pmol/1 rising to a peak of 34+-2.8 pmol/1 following the ingestion of a small mixed test meal. Ingestion of glucose or fat resulted in a similar rise of plasma GIP, whereas no change was observed after the ingestion of protein. (author)

Prevention of adsorption losses during radioimmunoassay of polypeptide hormones: effectiveness of albumins, gelatin, caseins, Tween 20 and plasma

International Nuclear Information System (INIS)

Livesey, J.H.; Donald, R.A.

1982-01-01

It is well known that polypeptide hormones adsorb to glass and plastic surfaces and that this adsorption may be reduced by adding a relatively large quantity of another protein. Consequently proteins (or sometimes detergents) are added almost universally to peptide hormone radioimmunassays to minimise loss of analyte by adsorption. This study was undertaken because there are few reports of the relative effectiveness of the proteins so used. The results suggest that moderate concentrations of the widely used albumins of Tween 20 do not always adequately prevent the adsorption of hormonal polypeptides to surfaces. Casein and alkali-treated casein appear to be more effective than the adsorption inhibitors in general use in radioimmunoassay for the range of hormones and adsorptive surfaces tested. They were also found to be very effective for preventing the adsorption of radio-labelled human luteinizing hormone, human growth hormone and Tyr-somatostatin. (Auth.)

Islet amyloid polypeptide in the control of food intake : An experimental study in the rat

OpenAIRE

Arnelo, Urban

1997-01-01

Control of food intake and satiety are physiologically complex processes, thatonly partly are understood. Several hormonal peptides have been proposed to mediatesatiety. Islet amyloid polypeptide (IAPP) is a recently discovered 37 amino acidpeptide, mainly produced by the pancreatic ß-cells. Initially, IAPP was shownto impair glucose tolerance at supra-physiological plasma concentrations and wasspeculated to be involved in the development of type-2 diabetes. More recent stud...

Glucose-dependent insulinotropic polypeptide may enhance fatty acid re-esterification in subcutaneous abdominal adipose tissue in lean humans

DEFF Research Database (Denmark)

Asmar, Meena; Simonsen, Lene; Madsbad, Sten

2010-01-01

Glucose-dependent insulinotropic polypeptide (GIP) has been implicated in lipid metabolism in animals. In humans, however, there is no clear evidence of GIP effecting lipid metabolism. The present experiments were performed in order to elucidate the effects of GIP on regional adipose tissue metab...

Mosaic protein and nucleic acid vaccines against hepatitis C virus

Science.gov (United States)

Yusim, Karina; Korber, Bette T. M.; Kuiken, Carla L.; Fischer, William M.

2013-06-11

The invention relates to immunogenic compositions useful as HCV vaccines. Provided are HCV mosaic polypeptide and nucleic acid compositions which provide higher levels of T-cell epitope coverage while minimizing the occurrence of unnatural and rare epitopes compared to natural HCV polypeptides and consensus HCV sequences.

Amphotericin B-conjugated polypeptide hydrogels as a novel innovative strategy for fungal infections

Science.gov (United States)

Shu, Chang; Li, Tengfei; Yang, Wen; Li, Duo; Ji, Shunli; Ding, Li

2018-03-01

The present work is focused on the design and development of novel amphotericin B (AmB)-conjugated biocompatible and biodegradable polypeptide hydrogels to improve the antifungal activity. Using three kinds of promoting self-assembly groups (2-naphthalene acetic acid (Nap), naproxen (Npx) and dexamethasone (Dex)) and polypeptide sequence (Phe-Phe-Asp-Lys-Tyr, FFDKY), we successfully synthesized the Nap-FFDK(AmB)Y gels, Npx-FFDK(AmB)Y gels and Dex-FFDK(AmB)Y gels. The AmB-conjugated hydrogelators are highly soluble in different aqueous solutions. The cryo-transmission electron microscopy and scanning electron microscopy micrographs of hydrogels afford nanofibres with a width of 20-50 nm. Powder X-ray diffraction analyses demonstrate that the crystalline structures of the AmB and Dex are changed into amorphous structures after the formation of hydrogels. Circular dichroism spectra of the solution of blank carriers and the corresponding drug deliveries further help elucidate the molecular arrangement in gel phase, indicating the existence of turn features. The in vitro drug releases suggest that the AmB-conjugated hydrogels are suitable as drug-controlled release vehicles for hydrophobic drugs. The antifungal effect of AmB-conjugated hydrogels significantly exhibits the antifungal activity against Candida albicans. The results of the present study indicated that the AmB-conjugated hydrogels are suitable carriers for poorly water soluble drugs and for enhancement of therapeutic efficacy of antifungal drugs.

Tissue polypeptide-specific antigen (TPS) determinations before and during intermittent maximal androgen blockade in patients with metastatic prostatic carcinoma

NARCIS (Netherlands)

Kil, P. J. M.; Goldschmidt, H. M. J.; Wieggers, B. J. A.; Kariakine, O. B.; Studer, U. E.; Whelan, P.; Hetherington, J.; de Reijke, Th M.; Hoekstra, J. W.; Collette, L.

2003-01-01

To evaluate the prognostic significance of serially measured tissue polypeptide-specific antigen (TPS) levels in patients with metastatic prostatic carcinoma treated with intermittent maximal androgen blockade (MAB). To determine its value with respect to predicting response to treatment and time to

Target organs for avian pancreatic polypeptide

International Nuclear Information System (INIS)

Kimmel, J.R.; Pollock, H.G.

1981-01-01

The problem of the physiological function of pancreatic polypeptide (PP) has been approached by attempting to identify target organs. Avian PP (aPP) labeled with 125I at either the C-terminus (aPP-C) or the N-terminus (aPP-N) was injected into fasted chickens and allowed to circulate for 3-120 min. At the end of the equilibration period, the anesthetized bird was perfused first with saline, then with Buoin's solution. Samples of fixed tissue from various organs were collected, weighed, and counted. Control experiments consisted of coinjection of unlabeled aPP to compete for receptors. The rate of disappearance of aPP-N from plasma was greater than that of aPP-C. Binding of aPP-N by spleen, duodenum, ileum, pancreas, and bone marrow was markedly reduced by coinjection of unlabeled aPP. A similar but less marked reduction in binding was found in liver and proventriculus. aPP-C gave less conclusive results. The maximal competitive effect of unlabeled PP could be achieved in most cases with 30 microgram unlabeled aPP. It is concluded that pancreas, duodenum, ileum, spleen, and bone marrow, and probably liver and proventriculus, are target organs for aPP in the chicken and that the C-terminal region of aPP is involved in receptor binding

The cellular transcription factor CREB corresponds to activating transcription factor 47 (ATF-47) and forms complexes with a group of polypeptides related to ATF-43.

Science.gov (United States)

Hurst, H C; Masson, N; Jones, N C; Lee, K A

1990-12-01

Promoter elements containing the sequence motif CGTCA are important for a variety of inducible responses at the transcriptional level. Multiple cellular factors specifically bind to these elements and are encoded by a multigene family. Among these factors, polypeptides termed activating transcription factor 43 (ATF-43) and ATF-47 have been purified from HeLa cells and a factor referred to as cyclic AMP response element-binding protein (CREB) has been isolated from PC12 cells and rat brain. We demonstrated that CREB and ATF-47 are identical and that CREB and ATF-43 form protein-protein complexes. We also found that the cis requirements for stable DNA binding by ATF-43 and CREB are different. Using antibodies to ATF-43 we have identified a group of polypeptides (ATF-43) in the size range from 40 to 43 kDa. ATF-43 polypeptides are related by their reactivity with anti-ATF-43, DNA-binding specificity, complex formation with CREB, heat stability, and phosphorylation by protein kinase A. Certain cell types vary in their ATF-43 complement, suggesting that CREB activity is modulated in a cell-type-specific manner through interaction with ATF-43. ATF-43 polypeptides do not appear simply to correspond to the gene products of the ATF multigene family, suggesting that the size of the ATF family at the protein level is even larger than predicted from cDNA-cloning studies.

Effect of double-tailed surfactant architecture on the conformation, self-assembly, and processing in polypeptide-surfactant complexes.

Science.gov (United States)

Junnila, Susanna; Hanski, Sirkku; Oakley, Richard J; Nummelin, Sami; Ruokolainen, Janne; Faul, Charl F J; Ikkala, Olli

2009-10-12

This work describes the solid-state conformational and structural properties of self-assembled polypeptide-surfactant complexes with double-tailed surfactants. Poly(L-lysine) was complexed with three dialkyl esters of phosphoric acid (i.e., phosphodiester surfactants), where the surfactant tail branching and length was varied to tune the supramolecular architecture in a facile way. After complexation with the branched surfactant bis(2-ethylhexyl) phosphate in an aqueous solution, the polypeptide chains adopted an alpha-helical conformation. These rod-like helices self-assembled into cylindrical phases with the amorphous alkyl tails pointing outward. In complexes with dioctyl phosphate and didodecyl phosphate, which have two linear n-octyl or n-dodecyl tails, respectively, the polypeptide formed antiparallel beta-sheets separated by alkyl layers, resulting in well-ordered lamellar self-assemblies. By heating, it was possible to trigger a partial opening of the beta-sheets and disruption of the lamellar phase. After repeated heating/cooling, all of these complexes also showed a glass transition between 37 and 50 degrees C. Organic solvent treatment and plasticization by overstoichiometric amount of surfactant led to structure modification in poly(L-lysine)-dioctyl phosphate complexes, PLL(diC8)(x) (x = 1.0-3.0). Here, the alpha-helical PLL is surrounded by the surfactants and these bottle-brush-like chains self-assemble in a hexagonal cylindrical morphology. As x is increased, the materials are clearly plasticized and the degree of ordering is improved: The stiff alpha-helical backbones in a softened surfactant matrix give rise to thermotropic liquid-crystalline phases. The complexes were examined by Fourier transform infrared spectroscopy, small- and wide-angle X-ray scattering, transmission electron microscopy, differential scanning calorimetry, polarized optical microscopy, and circular dichroism.

Skin peptide tyrosine-tyrosine, a member of the pancreatic polypeptide family: isolation, structure, synthesis, and endocrine activity.

Science.gov (United States)

Mor, A; Chartrel, N; Vaudry, H; Nicolas, P

1994-10-25

Pancreatic polypeptide, peptide tyrosine-tyrosine (PYY), and neuropeptide tyrosine (NPY), three members of a family of structurally related peptides, are mainly expressed in the endocrine pancreas, in endocrine cells of the gut, and in the brain, respectively. In the present study, we have isolated a peptide of the pancreatic polypeptide family from the skin of the South American arboreal frog Phyllomedusa bicolor. The primary structure of the peptide was established as Tyr-Pro-Pro-Lys-Pro-Glu-Ser-Pro-Gly-Glu10-Asp-Ala-Ser-Pro-Glu-Glu- Met-Asn- Lys-Tyr20-Leu-Thr-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu30-Val-Thr- Arg-Gln-Arg-Tyr-NH2 . This unusual peptide, named skin peptide tyrosine-tyrosine (SPYY), exhibits 94% similarity with PYY from the frog Rana ridibunda. A synthetic replicate of SPYY inhibits melanotropin release from perifused frog neurointermediate lobes in very much the same way as NPY. These results demonstrate the occurrence of a PYY-like peptide in frog skin. Our data also suggest the existence of a pituitary-skin regulatory loop in amphibians.

Transgenic rescue of adipocyte glucose-dependent insulinotropic polypeptide receptor expression restores high fat diet-induced body weight gain

DEFF Research Database (Denmark)

Ugleholdt, Randi; Pedersen, Jens; Bassi, Maria Rosaria

2011-01-01

that was similar between the groups. In contrast, glucose-dependent insulinotropic polypeptide-mediated insulin secretion does not seem to be important for regulation of body weight after high fat feeding. The study supports a role of the adipocyte GIPr in nutrient-dependent regulation of body weight and lean mass...

Primary structure of pancreatic polypeptide from four species of Perissodactyla (Przewalski's horse, zebra, rhino, tapir).

Science.gov (United States)

Henry, J S; Lance, V A; Conlon, J M

1991-12-01

Pancreatic polypeptide (PP) has been purified from extracts of the pancreas of four species of odd-toed ungulates (Perissodactyla): Przewalski's horse, mountain zebra, white rhinoceros, and mountain tapir. The amino acid sequence of Przewalski's horse pancreatic polypeptide was established as Ala-Pro-Met-Glu-Pro-Val-Tyr-Pro-Gly-Asp10-Asn- Ala-Thr-Pro-Glu-Gln-Met-Ala-Gln-Tyr20-Ala-Ala-Glu-Leu-Arg-Arg-Tyr- Ile-Asn-Met30 - Leu-Thr-Arg-Pro-Arg-Tyr.NH2. Zebra PP was identical to Przewalski's horse PP, rhinoceros PP contained three substitutions relative to the horse (Ser for Ala1, Leu for Met3, and Glu for Gln16), and tapir PP contained one substitution relative to the horse (Leu for Met3). On the basis of morphological characteristics and the fossil record, the rhinocerotids are classified with the tapirids in the suborder Ceratomorpha, whereas the horse and zebra belong to a separate suborder, Hippomorpha. On the basis of structural similarity of the PP molecules, however, it would appear that the tapir is more closely related to the horse than to the rhinoceros. These observations provide a further example of the need for extreme caution when inferring taxonomic or phylogenetic relationships between species from the structures of homologous peptides.

Plasma low-molecular-weight proteome profiling identified neuropeptide-Y as a prostate cancer biomarker polypeptide.

Science.gov (United States)

Ueda, Koji; Tatsuguchi, Ayako; Saichi, Naomi; Toyama, Atsuhiko; Tamura, Kenji; Furihata, Mutsuo; Takata, Ryo; Akamatsu, Shusuke; Igarashi, Masahiro; Nakayama, Masato; Sato, Taka-Aki; Ogawa, Osamu; Fujioka, Tomoaki; Shuin, Taro; Nakamura, Yusuke; Nakagawa, Hidewaki

2013-10-04

In prostate cancer diagnosis, PSA test has greatly contributed to the early detection of prostate cancer; however, expanding overdiagnosis and unnecessary biopsies have emerged as serious issues. To explore plasma biomarkers complementing the specificity of PSA test, we developed a unique proteomic technology QUEST-MS (Quick Enrichment of Small Targets for Mass Spectrometry). The QUEST-MS method based on 96-well formatted sequential reversed-phase chromatography allowing efficient enrichment of <20 kDa proteins quickly and reproducibly. Plasma from 24 healthy controls, 19 benign prostate hypertrophy patients, and 73 prostate cancer patients were purified with QUEST-MS and analyzed by LC/MS/MS. Among 153 057 nonredundant peptides, 189 peptides showed prostate cancer specific detection pattern, which included a neurotransmitter polypeptide neuropeptide-Y (NPY). We further validated the screening results by targeted multiple reaction monitoring technology using independent sample set (n = 110). The ROC curve analysis revealed that logistic regression-based combination of NPY, and PSA showed 81.5% sensitivity and 82.2% specificity for prostate cancer diagnosis. Thus QUEST-MS technology allowed comprehensive and high-throughput profiling of plasma polypeptides and had potential to effectively uncover very low abundant tumor-derived small molecules, such as neurotransmitters, peptide hormones, or cytokines.

De novo DESIGN AND SYNTHESIS OF AN ICE-BINDING, DENDRIMERIC, POLYPEPTIDE BASED ON INSECT ANTIFREEZE PROTEINS

Directory of Open Access Journals (Sweden)

Ricardo Vera Bravo

2011-12-01

Full Text Available A new strategy is presented for the designand synthesis of peptides that exhibitice-binding and antifreeze activity. Apennant-type dendrimer polypeptidescaffold combining an α-helical backbonewith four short β-strand branches wassynthesized in solid phase using Fmocchemistry in a divergent approach. The51-residue dendrimer was characterizedby reverse phase high performance liquidchromatography, mass spectrometry andcircular dichroism. Each β-strand branchcontained three overlapping TXT aminoacid repeats, an ice-binding motif foundin the ice-binding face of the sprucebudworm (Choristoneura fumiferanaand beetle (Tenebrio molitor antifreezeproteins. Ice crystals in the presence ofthe polypeptide monomer displayed flat,hexagonal plate morphology, similar tothat produced by weakly active antifreezeproteins. An oxidized dimeric form of thedendrimer polypeptide also produced flathexagonal ice crystals and was capableof inhibiting ice crystal growth upontemperature reduction, a phenomenontermed thermal hysteresis, a definingproperty of antifreeze proteins. Linkageof the pennant-type dendrimer to a trifunctionalcascade-type polypeptideproduced a trimeric macromolecule thatgave flat hexagonal ice crystals withhigher thermal hysteresis activity thanthe dimer or monomer and an ice crystal burst pattern similar to that producedby samples containing insect antifreezeproteins. This macromolecule was alsocapable of inhibiting ice recrystallization.

Enamel formation in vitro in mouse molar explants exposed to amelogenin polypeptides ATMP and LRAP on enamel development.

Science.gov (United States)

Ravindranath, Rajeswari M H; Devarajan, Asokan; Bringas, Pablo

2007-12-01

The enamel matrix contains amelogenin, leucine-rich amelogenin-polypeptide (LRAP), resulting from alternative splicing of the primary amelogenin-RNA transcript and tyrosine-rich amelogenin-polypeptide (TRAP), a proteolytic product of amelogenin. Presence of amelogenin-trityrosyl-motif peptide (ATMP) distinguishes TRAP from LRAP. The roles of these polypeptides in the formation of enamel remain to be elucidated. The mouse in vitro molar tooth-organ developed from bud stage (E16) was exposed to LRAP, ATMP, and mutated ATMP (T-ATMP, third proline replaced by threonine). The histology and morphometry of the explants on day-12 in culture was examined using Mallory's stain. Guanidine-HCl soluble protein concentrations of explants were compared. The enamel width and protein solubility indicate that the explant on day-12 is comparable to postnatal molar on day-3 in vivo. The enamel of both untreated explants as well as that in vivo is fuchinophilic (acid fuchsin, AF+). ATMP reduced the ameloblast-height, accumulated AF+ spherules at the apical end of ameloblasts, and disrupted enamel-dentin bonding. T-ATMP abrogated deposition of AF+ material on the aniline blue positive (AB+) enamel matrix. LRAP reduced ameloblast-height, increased the enamel-width without disruption (at 17.25 nmol) and increased the density of AF+ dentinal tubules. AF+ substance from the tubules is released onto the surface of the dentin. The Guanidine-HCl-soluble protein is elevated in ATMP-treated explants but decreased in LRAP-treated explants. Exogenous ATMP, T-ATMP and LRAP have divergent effects on developing enamel. Exogenous ATMP, but not LRAP, abrogates enamel-dentin bonding at 17.25 nmol. LRAP may play a role in the differentiation of ameloblasts, growth of enamel and formation of dentinal tubules.

A nontoxic polypeptide oligomer with a fungicide potency under agricultural conditions which is equal or greater than that of their chemical counterparts.

Directory of Open Access Journals (Sweden)

Sara Monteiro

Full Text Available There are literally hundreds of polypeptides described in the literature which exhibit fungicide activity. Tens of them have had attempted protection by patent applications but none, as far as we are aware, have found application under real agricultural conditions. The reasons behind may be multiple where the sensitivity to the Sun UV radiation can come in first place. Here we describe a multifunctional glyco-oligomer with 210 kDa which is mainly composed by a 20 kDa polypeptide termed Blad that has been previously shown to be a stable intermediary product of β-conglutin catabolism. This oligomer accumulates exclusively in the cotyledons of Lupinus species, between days 4 and 12 after the onset of germination. Blad-oligomer reveals a plethora of biochemical properties, like lectin and catalytic activities, which are not unusual per si, but are remarkable when found to coexist in the same protein molecule. With this vast range of chemical characteristics, antifungal activity arises almost as a natural consequence. The biological significance and potential technological applications of Blad-oligomer as a plant fungicide to agriculture, its uniqueness stems from being of polypeptidic in nature, and with efficacies which are either equal or greater than the top fungicides currently in the market are addressed.

INTERACTION BETWEEN DIFFERENT MOLECULAR FORMS OF IMMUNOGLOBULIN A AND RECOMBINANT DERIVATIVES POLYPEPTIDES OF BAC RECEPTOR PROTEINS FROM GROUP B STREPTOCOCCI

Directory of Open Access Journals (Sweden)

A. S. Korzhueva

2008-01-01

Full Text Available Abstract. The article concerns interactions between immunoglobulin A and recombinant P6, P7, P8 polypeptides, designed on the basis of externally localized Bac protein of the Group B streptococci, possessing IgA-binding activity.There is a current demand for immunochemical reagents that are strictly specific for IgA, in order to develop antigenic standards for detection of IgA levels in biological fluids, as well as for affinity purification of IgA and its fragments.To analyze an opportunity of the abovementioned application ways for these proteins, a special study was performed to assay an interaction capability of recombinant P6, P7, P8 polypeptides binding to Fc regions of different IgA forms (serum IgA, secretory IgA, subclasses of serum IgA – IgA1, IgA2. Selectivity of ligand binding was specially confirmed.It was found out that, among three presented polypeptides, the structure of recombinant P6 derivative proved to be optimal for IgA-binding ability of Bac protein.Structural features of IgA-binding fragments of Bac protein, i.e., binding site position on the IgA molecule (proximity to epitopes for three monoclonal antibodies, variability of the site structure, as well as resistance of binding site for P6, P7, P8 in IgA molecule against partial disulfide bonds reduction. (Med. Immunol., vol. 10, N 4-5, pp 327-336.

NMR experiments for resonance assignments of 13C, 15N doubly-labeled flexible polypeptides: Application to the human prion protein hPrP(23-230)

International Nuclear Information System (INIS)

Liu Aizhuo; Riek, Roland; Wider, Gerhard; Schroetter, Christine von; Zahn, Ralph; Wuethrich, Kurt

2000-01-01

A combination of three heteronuclear three-dimensional NMR experiments tailored for sequential resonance assignments in uniformly 15 N, 13 C-labeled flexible polypeptide chains is described. The 3D (H)N(CO-TOCSY)NH, 3D (H)CA(CO-TOCSY)NH and 3D (H)CBCA(CO-TOCSY)NH schemes make use of the favorable 15 N chemical shift dispersion in unfolded polypeptides, exploit the slow transverse 15 N relaxation rates of unfolded polypeptides in high resolution constant-time [ 1 H, 15 N]-correlation experiments, and use carbonyl carbon homonuclear isotropic mixing to transfer magnetization sequentially along the amino acid sequence. Practical applications are demonstrated with the 100-residue flexible tail of the recombinant human prion protein, making use of spectral resolution up to 0.6 Hz in the 15 N dimension, simultaneous correlation with the two adjacent amino acid residues to overcome problems associated with spectral overlap, and the potential of the presently described experiments to establish nearest-neighbor correlations across proline residues in the amino acid sequence

Elastin as a self-organizing biomaterial: use of recombinantly expressed human elastin polypeptides as a model for investigations of structure and self-assembly of elastin.

Science.gov (United States)

Keeley, Fred W; Bellingham, Catherine M; Woodhouse, Kimberley A

2002-02-28

Elastin is the major extracellular matrix protein of large arteries such as the aorta, imparting characteristics of extensibility and elastic recoil. Once laid down in tissues, polymeric elastin is not subject to turnover, but is able to sustain its mechanical resilience through thousands of millions of cycles of extension and recoil. Elastin consists of ca. 36 domains with alternating hydrophobic and cross-linking characteristics. It has been suggested that these hydrophobic domains, predominantly containing glycine, proline, leucine and valine, often occurring in tandemly repeated sequences, are responsible for the ability of elastin to align monomeric chains for covalent cross-linking. We have shown that small, recombinantly expressed polypeptides based on sequences of human elastin contain sufficient information to self-organize into fibrillar structures and promote the formation of lysine-derived cross-links. These cross-linked polypeptides can also be fabricated into membrane structures that have solubility and mechanical properties reminiscent of native insoluble elastin. Understanding the basis of the self-organizational ability of elastin-based polypeptides may provide important clues for the general design of self-assembling biomaterials.

Application of evolutionary algorithm methods to polypeptide folding: comparison with experimental results for unsolvated Ac-(Ala-Gly-Gly)5-LysH+

DEFF Research Database (Denmark)

Damsbo, Martin; Kinnear, Brian S; Hartings, Matthew R

2004-01-01

We present an evolutionary method for finding the low-energy conformations of polypeptides. The application, called FOLDAWAY,is based on a generic framework and uses several evolutionary operators as well as local optimization to navigate the complex energy landscape of polypeptides. It maintains...... mobility measurements. It has a flat energy landscape where helical and globular conformations have similar energies. FOLDAWAY locates several large groups of structures not found in previous molecular dynamics simulations for this peptide, including compact globular conformations, which are probably...... two complementary representations of the structures and uses the CHARMM force field for evaluating the energies. The method is applied to unsolvated Met-enkephalin and Ac-(Ala-Gly-Gly)(5)-Lys(+)H(+). Unsolvated Ac-(Ala-Gly-Gly)(5)-Lys(+)H(+) has been the object of recent experimental studies using ion...

Islet Amyloid Polypeptide: Structure, Function, and Pathophysiology

Directory of Open Access Journals (Sweden)

Rehana Akter

2016-01-01

Full Text Available The hormone islet amyloid polypeptide (IAPP, or amylin plays a role in glucose homeostasis but aggregates to form islet amyloid in type-2 diabetes. Islet amyloid formation contributes to β-cell dysfunction and death in the disease and to the failure of islet transplants. Recent work suggests a role for IAPP aggregation in cardiovascular complications of type-2 diabetes and hints at a possible role in type-1 diabetes. The mechanisms of IAPP amyloid formation in vivo or in vitro are not understood and the mechanisms of IAPP induced β-cell death are not fully defined. Activation of the inflammasome, defects in autophagy, ER stress, generation of reactive oxygen species, membrane disruption, and receptor mediated mechanisms have all been proposed to play a role. Open questions in the field include the relative importance of the various mechanisms of β-cell death, the relevance of reductionist biophysical studies to the situation in vivo, the molecular mechanism of amyloid formation in vitro and in vivo, the factors which trigger amyloid formation in type-2 diabetes, the potential role of IAPP in type-1 diabetes, the development of clinically relevant inhibitors of islet amyloidosis toxicity, and the design of soluble, bioactive variants of IAPP for use as adjuncts to insulin therapy.

Toughening of Thermoresponsive Arrested Networks of Elastin-Like Polypeptides To Engineer Cytocompatible Tissue Scaffolds.

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Glassman, Matthew J; Avery, Reginald K; Khademhosseini, Ali; Olsen, Bradley D

2016-02-08

Formulation of tissue engineering or regenerative scaffolds from simple bioactive polymers with tunable structure and mechanics is crucial for the regeneration of complex tissues, and hydrogels from recombinant proteins, such as elastin-like polypeptides (ELPs), are promising platforms to support these applications. The arrested phase separation of ELPs has been shown to yield remarkably stiff, biocontinuous, nanostructured networks, but these gels are limited in applications by their relatively brittle nature. Here, a gel-forming ELP is chain-extended by telechelic oxidative coupling, forming extensible, tough hydrogels. Small angle scattering indicates that the chain-extended polypeptides form a fractal network of nanoscale aggregates over a broad concentration range, accessing moduli ranging from 5 kPa to over 1 MPa over a concentration range of 5-30 wt %. These networks exhibited excellent erosion resistance and allowed for the diffusion and release of encapsulated particles consistent with a bicontinuous, porous structure with a broad distribution of pore sizes. Biofunctionalized, toughened networks were found to maintain the viability of human mesenchymal stem cells (hMSCs) in 2D, demonstrating signs of osteogenesis even in cell media without osteogenic molecules. Furthermore, chondrocytes could be readily mixed into these gels via thermoresponsive assembly and remained viable in extended culture. These studies demonstrate the ability to engineer ELP-based arrested physical networks on the molecular level to form reinforced, cytocompatible hydrogel matrices, supporting the promise of these new materials as candidates for the engineering and regeneration of stiff tissues.

Glucose-dependent insulinotropic polypeptide (GIP) receptor deletion leads to reduced bone strength and quality.

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Mieczkowska, Aleksandra; Irwin, Nigel; Flatt, Peter R; Chappard, Daniel; Mabilleau, Guillaume

2013-10-01

Bone is permanently remodeled by a complex network of local, hormonal and neuronal factors that affect osteoclast and osteoblast biology. In this context, a role for gastro-intestinal hormones has been proposed based on evidence that bone resorption dramatically falls after a meal. Glucose-dependent insulinotropic polypeptide (GIP) is one of the candidate hormones as its receptor, glucose-dependent insulinotropic polypeptide receptor (GIPR), is expressed in bone. In the present study we investigated bone strength and quality by three-point bending, quantitative x-ray microradiography, microCT, qBEI and FTIR in a GIPR knockout (GIPR KO) mouse model and compared with control wild-type (WT) animals. Animals with a deletion of the GIPR presented with a significant reduction in ultimate load (--11%), stiffness (-16%), total absorbed (-28%) and post-yield energies (-27%) as compared with WT animals. Furthermore, despite no change in bone outer diameter, the bone marrow diameter was significantly increased and as a result cortical thickness was significantly decreased by 20% in GIPR deficient animals. Bone resorption at the endosteal surface was significantly increased whilst bone formation was unchanged in GIPR deficient animals. Deficient animals also presented with a pronounced reduction in the degree of mineralization of bone matrix. Furthermore, the amount of mature cross-links of collagen matrix was significantly reduced in GIPR deficient animals and was associated with lowered intrinsic material properties. Taken together, these data support a positive effect of the GIPR on bone strength and quality. © 2013.

Iodine and tritium labelling of curarizing and cardiotoxic agents. Study of the conformation of toxic polypeptides extracted from snake venom

International Nuclear Information System (INIS)

Menez, Andre.

1977-01-01

A short review of present-day knowledge on the action mechanism of toxic snake venom polypeptides is followed by a study of the radioactive labelling of some toxic compounds. Those dealt with more especially are Naja nigricollis α toxin and Laticauda semifasciata b erabutoxin, then (+) tubocurarin, a non-peptidic curarizing alkaloid, and two cardiotoxic polypeptides: cytotoxin II and cardiotoxin γ extracted from the venom of Naja naja and Naja nigricollis respectively. The labelling principle is based on the specific fixation of one or more iodine atoms then tritium substitution of the halogen by catalytic hydrogenolysis. As predicted from titration of the aromatic groups the halogenation process, obtained by addition of iodine monochloride, takes place sometimes on the phenolic nuclei and sometimes on the imidazole nuclei, the position of which targets within each sequence has been identified. From results of the study of reactivity towards iodine combined with those of basic titration, the accessibility of several aromatic nuclei has also been defined. Each iodinated polypeptide is then hydrogenolysed in the presence of tritium gas giving a specific activity between 4 and 27 Ci/mmole according to the compound treated. In all cases the biological potential and physical properties of the radioactive material obtained by the above titration process remained intact. An example of the bonding kinetics of short toxins with the partially purified choligenic receptor is given in the special case of tritiated b erabutoxin. The affinity of this toxin for its receptor target is strong, though slightly less so than that of tritiated Naja nigricollis α toxin [fr

Expression and purification of short hydrophobic elastin-like polypeptides with maltose-binding protein as a solubility tag.

Science.gov (United States)

Bataille, Laure; Dieryck, Wilfrid; Hocquellet, Agnès; Cabanne, Charlotte; Bathany, Katell; Lecommandoux, Sébastien; Garbay, Bertrand; Garanger, Elisabeth

2015-06-01

Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. The recombinant expression of hydrophobic elastin-like polypeptides is often difficult because they possess low transition temperatures, and therefore form aggregates at sub-ambient temperatures. To circumvent this difficulty, we expressed in Escherichia coli three hydrophobic ELPs (VPGIG)n with variable lengths (n=20, 40, and 60) in fusion with the maltose-binding protein (MBP). Fusion proteins were soluble and yields of purified MBP-ELP ranged between 66 and 127mg/L culture. After digestion of the fusion proteins by enterokinase, the ELP moiety was purified by using inverse transition cycling. The purified fraction containing ELP40 was slightly contaminated by traces of undigested fusion protein. Purification of ELP60 was impaired because of co-purification of the MBP tag during inverse transition cycling. ELP20 was successfully purified to homogeneity, as assessed by gel electrophoresis and mass spectrometry analyses. The transition temperature of ELP20 was measured at 15.4°C in low salt buffer. In conclusion, this method can be used to produce hydrophobic ELP of low molecular mass. Copyright © 2015 Elsevier Inc. All rights reserved.

Bioinspired Star-Shaped Poly(l-lysine) Polypeptides: Efficient Polymeric Nanocarriers for the Delivery of DNA to Mesenchymal Stem Cells.

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Walsh, David P; Murphy, Robert D; Panarella, Angela; Raftery, Rosanne M; Cavanagh, Brenton; Simpson, Jeremy C; O'Brien, Fergal J; Heise, Andreas; Cryan, Sally-Ann

2018-05-07

The field of tissue engineering is increasingly recognizing that gene therapy can be employed for modulating in vivo cellular response thereby guiding tissue regeneration. However, the field lacks a versatile and biocompatible gene delivery platform capable of efficiently delivering transgenes to mesenchymal stem cells (MSCs), a cell type often refractory to transfection. Herein, we describe the extensive and systematic exploration of three architectural variations of star-shaped poly(l-lysine) polypeptide (star-PLL) with varying number and length of poly(l-lysine) arms as potential nonviral gene delivery vectors for MSCs. We demonstrate that star-PLL vectors are capable of self-assembling with pDNA to form stable, cationic nanomedicines. Utilizing high content screening, live cell imaging, and mechanistic uptake studies we confirm the intracellular delivery of pDNA by star-PLLs to MSCs is a rapid process, which likely proceeds via a clathrin-independent mechanism. We identify a star-PLL composition with 64 poly(l-lysine) arms and five l-lysine subunits per arm as a particularly efficient vector that is capable of delivering both reporter genes and the therapeutic transgenes bone morphogenetic protein-2 and vascular endothelial growth factor to MSCs. This composition facilitated a 1000-fold increase in transgene expression in MSCs compared to its linear analogue, linear poly(l-lysine). Furthermore, it demonstrated comparable transgene expression to the widely used vector polyethylenimine using a lower pDNA dose with significantly less cytotoxicity. Overall, this study illustrates the ability of the star-PLL vectors to facilitate efficient, nontoxic nucleic acid delivery to MSCs thereby functioning as an innovative nanomedicine platform for tissue engineering applications.

SM50 repeat-polypeptides self-assemble into discrete matrix subunits and promote appositional calcium carbonate crystal growth during sea urchin tooth biomineralization.

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Mao, Yelin; Satchell, Paul G; Luan, Xianghong; Diekwisch, Thomas G H

2016-01-01

The two major proteins involved in vertebrate enamel formation and echinoderm sea urchin tooth biomineralization, amelogenin and SM50, are both characterized by elongated polyproline repeat domains in the center of the macromolecule. To determine the role of polyproline repeat polypeptides in basal deuterostome biomineralization, we have mapped the localization of SM50 as it relates to crystal growth, conducted self-assembly studies of SM50 repeat polypeptides, and examined their effect on calcium carbonate and apatite crystal growth. Electron micrographs of the growth zone of Strongylocentrotus purpuratus sea urchin teeth documented a series of successive events from intravesicular mineral nucleation to mineral deposition at the interface between tooth surface and odontoblast syncytium. Using immunohistochemistry, SM50 was detected within the cytoplasm of cells associated with the developing tooth mineral, at the mineral secreting front, and adjacent to initial mineral deposits, but not in muscles and ligaments. Polypeptides derived from the SM50 polyproline alternating hexa- and hepta-peptide repeat region (SM50P6P7) formed highly discrete, donut-shaped self-assembly patterns. In calcium carbonate crystal growth studies, SM50P6P7 repeat peptides triggered the growth of expansive networks of fused calcium carbonate crystals while in apatite growth studies, SM50P6P7 peptides facilitated the growth of needle-shaped and parallel arranged crystals resembling those found in developing vertebrate enamel. In comparison, SM50P6P7 surpassed the PXX24 polypeptide repeat region derived from the vertebrate enamel protein amelogenin in its ability to promote crystal nucleation and appositional crystal growth. Together, these studies establish the SM50P6P7 polyproline repeat region as a potent regulator in the protein-guided appositional crystal growth that occurs during continuous tooth mineralization and eruption. In addition, our studies highlight the role of species

Is pancreatic polypeptide response to food ingestion a reliable index of vagal function in type 1 diabetes?

DEFF Research Database (Denmark)

Damholt, M B; Arlien-Soeborg, P; Hilsted, L

2006-01-01

The diagnosis of autonomic neuropathy in diabetic patients is based on cardiovascular reflex tests. Since cardiac function may be affected by arteriosclerosis and cardiomyopathy in type 1 diabetes mellitus, alternative tests reflecting vagal nerve function, in other organ systems, are needed....... In this study the pancreatic polypeptide (PP) response to a mixed meal was evaluated in healthy subjects and in recently diagnosed type 1 diabetic patients....

Vasoactive intestinal polypeptide (VIP) tissue distribution in the rat as measured by radioimmunoassay and by radioreceptorassay

International Nuclear Information System (INIS)

Besson, J.; Dupont, C.; Laburthe, M.; Bataille, D.; Rosselin, G.

1977-01-01

A new radioimmunoassay which allows the measurement of the rat vasoactive intestinal polypeptide, was performed. VIP is present in the whole digestive tract of rat, mainly between the duodenum and the colon. 1.5% of the total VIP is present in brain. The VIP-like immunoreactivity appears to correspond to biologically active molecule since a radioreceptorassay using liver plasma membranes as the target tissue, gives the same results as the radioimmunoassay [fr

Heterogeneity of rabbit endogenous pyrogens is not attributable to glycosylated variants of a single polypeptide chain.

OpenAIRE

Murphy, P A; Cebula, T A; Windle, B E

1981-01-01

Rabbit endogenous pyrogens were of about the same molecular size, but showed considerable heterogeneity of their isoelectric points. We attempted to show that this heterogeneity was attributable to variable glycosylation of a single polypeptide chain. When peritoneal exudate cells were stimulated to make pyrogens in the presence of 2-deoxy-D-glucose, there was a relatively trivial suppression of pyrogen release, and analysis by isoelectric focusing showed parallel inhibition of secretion of a...

Bovine pancreatic polypeptide (bPP) undergoes significant changes in conformation and dynamics upon binding to DPC micelles.

Science.gov (United States)

Lerch, Mirjam; Gafner, Verena; Bader, Reto; Christen, Barbara; Folkers, Gerd; Zerbe, Oliver

2002-10-04

The pancreatic polypeptide (PP), a 36-residue, C-terminally amidated polypeptide hormone is a member of the neuropeptide Y (NPY) family. Here, we have studied the structure and dynamics of bovine pancreatic polypeptide (bPP) when bound to DPC-micelles as a membrane-mimicking model as well as the dynamics of bPP in solution. The comparison of structure and dynamics of bPP in both states reveals remarkable differences. The overall correlation time of 5.08ns derived from the 15N relaxation data proves unambiguously that bPP in solution exists as a dimer. Therein, intermolecular as well as intramolecular hydrophobic interactions from residues of both the amphiphilic helix and of the back-folded N terminus contribute to the stability of the PP fold. The overall rigidity is well-reflected in positive values for the heteronuclear NOE for residues 4-34. The membrane-bound species displays a partitioning into a more flexible N-terminal region and a well-defined alpha-helical region comprising residues 17-31. The average RMSD value for residues 17-31 is 0.22(+/-0.09)A. The flexibility of the N terminus is compatible with negative values of the heteronuclear NOE observed for the N-terminal residues 4-12 and low values of the generalized order parameter S(2). The membrane-peptide interface was investigated by micelle-integrating spin-labels and H,2H exchange measurements. It is formed by those residues which make contacts between the C-terminal alpha-helix and the polyproline helix. In contrast to pNPY, also residues from the N terminus display spatial proximity to the membrane interface. Furthermore, the orientation of the C terminus, that presumably contains residues involved in receptor binding, is different in the two environments. We speculate that this pre-positioning of residues could be an important requirement for receptor activation. Moreover, we doubt that the PP fold is of functional relevance for binding at the Y(4) receptor.

Gastric inhibitory polypeptide does not inhibit gastric emptying in humans

DEFF Research Database (Denmark)

Meier, Juris J; Goetze, Oliver; Anstipp, Jens

2004-01-01

) = 0.15, P = 0.15 for intact GIP; r(2) = 0.21, P = 0.086 for total GIP). We conclude that gastric emptying does not appear to be influenced by GIP. The secretion of GIP after meal ingestion is not suppressed by its exogenous administration. The lack of effect of GIP on gastric emptying underlines......The insulinotropic gut hormone gastric inhibitory polypeptide (GIP) has been demonstrated to inhibit gastric acid secretion and was proposed to possess "enterogastrone" activity. GIP effects on gastric emptying have not yet been studied. Fifteen healthy male volunteers (23.9 +/- 3.3 yr, body mass....... Gastric emptying was calculated from the (13)CO(2) exhalation rates in breath samples collected over 360 min. Venous blood was drawn in 30-min intervals for the determination of glucose, insulin, C-peptide, and GIP (total and intact). Statistical calculations were made by use of repeated-measures ANOVA...

Pituitary adenylate cyclase-activating polypeptide: occurrence and relaxant effect in female genital tract

DEFF Research Database (Denmark)

Steenstrup, B R; Alm, P; Hannibal, J

1995-01-01

The distribution, localization, and smooth muscle effects of pituitary adenylate cyclase-activating polypeptide (PACAP) were studied in the human female genital tract. The concentrations of PACAP-38 and PACAP-27 were measured by radioimmunoassays, and both peptides were found throughout the genital...... was observed. The findings suggest a smooth muscle regulatory role of PACAP in the human female reproductive tract....... tract. The highest concentrations of PACAP-38 were detected in the ovary, the upper part of vagina, and the perineum. The concentrations of PACAP-27 were generally low, in some regions below the detection limit and in other regions 1 to 5% of the PACAP-38 concentrations. Immunocytochemistry revealed...

Aspects of structural landscape of human islet amyloid polypeptide

Energy Technology Data Exchange (ETDEWEB)

He, Jianfeng, E-mail: hjf@bit.edu.cn; Dai, Jin, E-mail: daijing491@gmail.com [School of Physics, Beijing Institute of Technology, Beijing 100081 (China); Li, Jing, E-mail: jinglichina@139.com [Institute of Biopharmaceutical Research, Yangtze River Pharmaceutical Group Beijing Haiyan Pharmaceutical Co., Ltd, Beijing 102206 (China); Peng, Xubiao, E-mail: xubiaopeng@gmail.com [Department of Physics and Astronomy, Uppsala University, P.O. Box 803, S-75108 Uppsala (Sweden); Niemi, Antti J., E-mail: Antti.Niemi@physics.uu.se [School of Physics, Beijing Institute of Technology, Beijing 100081 (China); Department of Physics and Astronomy, Uppsala University, P.O. Box 803, S-75108 Uppsala (Sweden); Laboratoire de Mathematiques et Physique Theorique CNRS UMR 6083, Fédération Denis Poisson, Université de Tours, Parc de Grandmont, F37200 Tours (France)

2015-01-28

The human islet amyloid polypeptide (hIAPP) co-operates with insulin to maintain glycemic balance. It also constitutes the amyloid plaques that aggregate in the pancreas of type-II diabetic patients. We have performed extensive in silico investigations to analyse the structural landscape of monomeric hIAPP, which is presumed to be intrinsically disordered. For this, we construct from first principles a highly predictive energy function that describes a monomeric hIAPP observed in a nuclear magnetic resonance experiment, as a local energy minimum. We subject our theoretical model of hIAPP to repeated heating and cooling simulations, back and forth between a high temperature regime where the conformation resembles a random walker and a low temperature limit where no thermal motions prevail. We find that the final low temperature conformations display a high level of degeneracy, in a manner which is fully in line with the presumed intrinsically disordered character of hIAPP. In particular, we identify an isolated family of α-helical conformations that might cause the transition to amyloidosis, by nucleation.

Cultivation of micro-algae in closed tubular reactor

Energy Technology Data Exchange (ETDEWEB)

Gudin, C.; Bernard, A.; Chaumont, D.

1983-11-01

A description is presented of the three culture pilot utilities in activity under natural light, including glass tubular solar collector (30 mm diameter) in which the microalgae culture circulates. The utility is controled automatically (thermal regulation, gaseous transfers, continuous culture organization). The tests were conducted for the production of polysaccharides (Porphyridium cruentum, chlamydomonas mexicana) or hydrocarbons (Botriococcus braunii).

Protective effect of topically applied polypeptide from Chlamys farreri against ultraviolet radiation-induced chronic skin damage in guinea pig

Science.gov (United States)

Chi, Mingliang; Cao, Pengli; Yu, Guoying; Zhu, Li; Wang, Yuejun; Wang, Chunbo

2003-12-01

Polypeptide from Chlamys farreri (PCF), a topical polypeptide isolated from Chlamys farreri, was used in this experiment aimed to investigate the photoprotective effect of PCF against chronic skin damage induced by ultraviolet A (UVA) and ultraviolet B (UVB) radiation. The chronic ultraviolet-irradiated guinea pig model was established, and visible changes in the skin including wrinkling, sagging and erythema were observed. Malondialdehyde (MDA) and antioxidant enzymes including superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) in the dorsal skin were determined using biochemical methods. The results showed: (1) PCF (5 % and 20%) could greatly protect the dorsal skin of guinea pig against wrinkling, sagging and erythema induced by UV radiation in a concentration-dependent manner. (2) PCF could reduce MDA formation in the dorsal skin caused by UV irradiation, while increasing the activities of SOD and GSH-px. (3) The differences among the PCF groups and UV model group were significant ( Psolar UV spectrum photoprotection; and that the antioxidant property of PCF might play a role in photoprotection.

Zwitterionic states in gas-phase polypeptide ions revealed by 157-nm ultra-violet photodissociation

DEFF Research Database (Denmark)

Kjeldsen, Frank; Silivra, Oleg A; Zubarev, Roman A

2006-01-01

carboxylic groups relative to competing COOH losses (45 Da) from neutral carboxylic groups. Loss of CO2 is a strong indication of the presence of a zwitterionic [(+)...(-)...(+)] salt bridge in the gas-phase polypeptide cation. This method provides a tool for studying, for example, the nature of binding...... of the fragment formation should be taken into account. Cations of Trp-cage proteins exist preferentially as zwitterions, with the deprotonation position divided between the Asp9 residue and the C terminus in the ratio 3:2. The majority of dications of the same molecule are not zwitterions. Furthermore, 157-nm...

Identification and characterization of human polyserase-3, a novel protein with tandem serine-protease domains in the same polypeptide chain

Directory of Open Access Journals (Sweden)

Garabaya Cecilia

2006-03-01

Full Text Available Abstract Background We have previously described the identification and characterization of polyserase-1 and polyserase-2, two human serine proteases containing three different catalytic domains within the same polypeptide chain. Polyserase-1 shows a complex organization and it is synthesized as a membrane-bound protein which can generate three independent serine protease domains as a consequence of post-translational processing events. The two first domains are enzymatically active. By contrast, polyserase-2 is an extracellular glycosylated protein whose three protease domains remain embedded in the same chain, and only the first domain possesses catalytic activity. Results Following our interest in the study of the human degradome, we have cloned a human liver cDNA encoding polyserase-3, a new protease with tandem serine protease domains in the same polypeptide chain. Comparative analysis of polyserase-3 with the two human polyserases described to date, revealed that this novel polyprotein is more closely related to polyserase-2 than to polyserase-1. Thus, polyserase-3 is a secreted protein such as polyserase-2, but lacks additional domains like the type II transmembrane motif and the low-density lipoprotein receptor module present in the membrane-anchored polyserase-1. Moreover, analysis of post-translational mechanisms operating in polyserase-3 maturation showed that its two protease domains remain as integral parts of the same polypeptide chain. This situation is similar to that observed in polyserase-2, but distinct from polyserase-1 whose protease domains are proteolytically released from the original chain to generate independent units. Immunolocalization studies indicated that polyserase-3 is secreted as a non-glycosylated protein, thus being also distinct from polyserase-2, which is a heavily glycosylated protein. Enzymatic assays indicated that recombinant polyserase-3 degrades the α-chain of fibrinogen as well as pro

Some studies on the composition and surface properties of oil bodies from the seed cotyledons of safflower (Carthamus tinctorius) and linseed (Linum ustatissimum).

Science.gov (United States)

Slack, C R; Bertaud, W S; Shaw, B D; Holland, R; Browse, J; Wright, H

1980-01-01

1. The average oil-body diameter in intact cells of developing linseed (Linum usitatissimum) and safflower (Carthamus tinctorius) cotyledons was similar (about 1.4 micrometer), and there was little change in size after oil bodies were isolated and repeatedly washed. 2. The glycerolipid composition of washed oil bodies from both developing and mature cotyledons of the two species was similar; oil bodies from ten different batches of cotyledons contained 4.3 +/- 0.16 mumol of 3-sn-phosphatidylcholine and 25.2 +/- 1.7 mumol of diacylglycerol per 1000 mumol of triacylglycerol. During four successive washings of a once-washed oil-body preparation, the proportion of diacylglycerol to triacylglycerol remained constant and that of 3-sn-phosphatidylcholine to triacylglycerol decreased by only 20%. 3. The protein content of thrice-washed oil bodies from the two species was similar, about 2.4% of the weight of glycerolipids, and appeared to be independent of the stage of cotyledon maturity. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that the protein of purified oil bodies from the two species consisted mainly of only four polypeptides and that two of the polypeptides from each species had apparent mol.wts. of 17500 and 15500. Similar patterns of polypeptides were obtained after the hydrolysis of the 15500-mol.wt. polypeptides from linseed and safflower oil bodies by Staphylococcus aureus V8 proteinase, whereas the proteolysis of the 17500-mol.wt. polypeptides from the two species produced different patterns of polypeptides. 4. The 3-sn-phosphatidylcholine in oil-body preparations was hydrolysed about 85% by bee-venom phospholipase A2 without any apparent coalescence of the oil bodies. Incubation with lipase from Rhizopus arrhizus caused rapid coalescence of the oil bodies, and this lipase appeared to initially hydrolyse diacylglycerols in preference to triacylglycerol. 5. Oil bodies from both species were almost completely dispersed in suspensions of

Structural stability of Amandin, a major allergen from almond (Prunus dulcis), and its acidic and basic polypeptides.

Science.gov (United States)

Albillos, Silvia M; Menhart, Nicholas; Fu, Tong-Jen

2009-06-10

Information relating to the resistance of food allergens to thermal and/or chemical denaturation is critical if a reduction in protein allergenicity is to be achieved through food-processing means. This study examined the changes in the secondary structure of an almond allergen, amandin, and its acidic and basic polypeptides as a result of thermal and chemical denaturation. Amandin ( approximately 370 kDa) was purified by cryoprecipitation followed by gel filtration chromatography and subjected to thermal (13-96 degrees C) and chemical (urea and dithiothreitol) treatments. Changes in the secondary structure of the protein were followed using circular dichroism spectroscopy. The secondary structure of the hexameric amandin did not undergo remarkable changes at temperatures up to 90 degrees C, although protein aggregation was observed. In the presence of a reducing agent, irreversible denaturation occurred with the following experimental values: T(m) = 72.53 degrees C (transition temperature), DeltaH = 87.40 kcal/mol (unfolding enthalpy), and C(p) = 2.48 kcal/(mol degrees C) (heat capacity). The concentration of urea needed to achieve 50% denaturation was 2.59 M, and the Gibbs free energy of chemical denaturation was calculated to be DeltaG = 3.82 kcal/mol. The basic and acidic polypeptides of amandin had lower thermal stabilities than the multimeric protein.

Micelles Formed by Polypeptide Containing Polymers Synthesized Via N-Carboxy Anhydrides and Their Application for Cancer Treatment

Directory of Open Access Journals (Sweden)

Dimitrios Skoulas

2017-06-01

Full Text Available The development of multifunctional polymeric materials for biological applications is mainly guided by the goal of achieving the encapsulation of pharmaceutical compounds through a self-assembly process to form nanoconstructs that control the biodistribution of the active compounds, and therefore minimize systemic side effects. Micelles are formed from amphiphilic polymers in a selective solvent. In biological applications, micelles are formed in water, and their cores are loaded with hydrophobic pharmaceutics, where they are solubilized and are usually delivered through the blood compartment. Even though a large number of polymeric materials that form nanocarrier delivery systems has been investigated, a surprisingly small subset of these technologies has demonstrated potentially curative preclinical results, and fewer have progressed towards commercialization. One of the most promising classes of polymeric materials for drug delivery applications is polypeptides, which combine the properties of the conventional polymers with the 3D structure of natural proteins, i.e., α-helices and β-sheets. In this article, the synthetic pathways followed to develop well-defined polymeric micelles based on polypeptides prepared through ring-opening polymerization (ROP of N-carboxy anhydrides are reviewed. Among these works, we focus on studies performed on micellar delivery systems to treat cancer. The review is limited to systems presented from 2000–2017.

A multiscale model to evaluate the efficacy of anticancer therapies based on chimeric polypeptide nanoparticles

Science.gov (United States)

Paiva, L. R.; Martins, M. L.

2011-01-01

A multiscale model for tumor growth and its chemotherapy using conjugate nanoparticles is presented, and the corresponding therapeutic outcomes are evaluated. It is found that doxorubicin assembled into chimeric polypeptide nanoparticles cannot eradicate either vascularized primary tumors or avascular micrometastasis even administrated at loads close to their maximum tolerated doses. Furthermore, an effective and safety treatment demands for conjugate nanoparticles targeted to the malignant cells with much higher specificity and affinity than those currently observed in order to leave most of the normal tissues unaffected and to ensure a fast intracellular drug accumulation.

Pituitary adenylate cyclase activating polypeptide induces vascular relaxation and inhibits non-vascular smooth muscle activity in the rabbit female genital tract

DEFF Research Database (Denmark)

Steenstrup, B R; Ottesen, B; Jørgensen, M

1994-01-01

In vitro effects of two bioactive forms of pituitary adenylate cyclase activating polypeptide (PACAP): PACAP-38 and PACAP-27 were studied on rabbit vascular and non-vascular smooth muscle. Segments of the ovarian artery and muscle strips from the fallopian tube were used. Two series of experiment...

Peptide binding specificity of the chaperone calreticulin

DEFF Research Database (Denmark)

Sandhu, N.; Duus, K.; Jorgensen, C.S.

2007-01-01

Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length and composit......Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length...... than 5 amino acids showed binding and a clear correlation with hydrophobicity was demonstrated for oligomers of different hydrophobic amino acids. Insertion of hydrophilic amino acids in a hydrophobic sequence diminished or abolished binding. In conclusion our results show that calreticulin has...

Short term aerobic exercise training increases postprandial pancreatic polypeptide but not peptide YY concentrations in obese individuals

OpenAIRE

Kanaley, Jill A.; Heden, Timothy D.; Liu, Ying; Whaley-Connell, Adam T.; Chockalingam, Anand; Dellsperger, Kevin C.; Fairchild, Timothy J.

2013-01-01

Objective Short-term exercise training improves glycemic control, but the effect of short-term training on postprandial satiety peptide responses or perceived satiety remains unknown. We tested the hypothesis that short-term aerobic exercise training (15 days) would alter postprandial pancreatic and gut peptide [pancreatic polypeptide (PP) and peptide YY (PYY)] responses and perceived appetite and satiety in obese individuals. Subjects Thirteen healthy obese men and women (age: 42±2 y; BMI: 3...

Coulomb repulsion in short polypeptides.

Science.gov (United States)

Norouzy, Amir; Assaf, Khaleel I; Zhang, Shuai; Jacob, Maik H; Nau, Werner M

2015-01-08

Coulomb repulsion between like-charged side chains is presently viewed as a major force that impacts the biological activity of intrinsically disordered polypeptides (IDPs) by determining their spatial dimensions. We investigated short synthetic models of IDPs, purely composed of ionizable amino acid residues and therefore expected to display an extreme structural and dynamic response to pH variation. Two synergistic, custom-made, time-resolved fluorescence methods were applied in tandem to study the structure and dynamics of the acidic and basic hexapeptides Asp6, Glu6, Arg6, Lys6, and His6 between pH 1 and 12. (i) End-to-end distances were obtained from the short-distance Förster resonance energy transfer (sdFRET) from N-terminal 5-fluoro-l-tryptophan (FTrp) to C-terminal Dbo. (ii) End-to-end collision rates were obtained for the same peptides from the collision-induced fluorescence quenching (CIFQ) of Dbo by FTrp. Unexpectedly, the very high increase of charge density at elevated pH had no dynamical or conformational consequence in the anionic chains, neither in the absence nor in the presence of salt, in conflict with the common view and in partial conflict with accompanying molecular dynamics simulations. In contrast, the cationic peptides responded to ionization but with surprising patterns that mirrored the rich individual characteristics of each side chain type. The contrasting results had to be interpreted, by considering salt screening experiments, N-terminal acetylation, and simulations, in terms of an interplay of local dielectric constant and peptide-length dependent side chain charge-charge repulsion, side chain functional group solvation, N-terminal and side chain charge-charge repulsion, and side chain-side chain as well as side chain-backbone interactions. The common picture that emerged is that Coulomb repulsion between water-solvated side chains is efficiently quenched in short peptides as long as side chains are not in direct contact with each

Characterization of Nora Virus Structural Proteins via Western Blot Analysis.

Science.gov (United States)

Ericson, Brad L; Carlson, Darby J; Carlson, Kimberly A

2016-01-01

Nora virus is a single stranded RNA picorna-like virus with four open reading frames (ORFs). The coding potentials of the ORFs are not fully characterized, but ORF3 and ORF4 are believed to encode the capsid proteins (VP3, VP4a, VP4b, and VP4c) comprising the virion. To determine the polypeptide composition of Nora virus virions, polypeptides from purified virus were compared to polypeptides detected in Nora virus infected Drosophila melanogaster. Nora virus was purified from infected flies and used to challenge mice for the production of antisera. ORF3, ORF4a, ORF4b, and ORF4c were individually cloned and expressed in E. coli; resultant recombinant proteins purified and were used to make monospecific antisera. Antisera were evaluated via Western blot against whole virus particles and Nora virus infected fly lysates. Viral purification yielded two particle types with densities of ~1.31 g/mL (empty particles) and ~1.33 g/mL (complete virions). Comparison of purified virus polypeptide composition to Nora virus infected D. melanogaster lysate showed the number of proteins in infected cell lysates is less than purified virus. Our results suggest the virion is composed of 6 polypeptides, VP3, VP4a, two forms of VP4b, and two forms of VP4c. This polypeptide composition is similar to other small RNA insect viruses.

Distinct localization of FMRFamide- and bovine pancreatic polypeptide-like material in the brain, retrocerebral complex and suboesophageal ganglion of the cockroach Periplaneta americana L

DEFF Research Database (Denmark)

Verhaert, P; Grimmelikhuijzen, C J; De Loof, A

1985-01-01

One bovine pancreatic polypeptide (BPP) antiserum and two FMRFamide antisera were applied in the peroxidase-antiperoxidase (PAP) immunohistochemical technique on a complete series of sections of brains, suboesophageal ganglia (SOG), corpora cardiaca (CC) and corpora allata of Periplaneta americana...... L. Double immunohistochemical staining demonstrated that the same perikarya and processes were stained by both the BPP and FMRFamide antisera. This was caused by cross-reaction of the BPP and FMRFamide antisera with common antigenic determinants as was shown by a number of solid-phase absorptions....... Application of a third FMRFamide antiserum, which was especially selected for its inability to react with bovine and avian pancreatic polypeptide, showed that more than half of the structures that were stained with the 'unspecific' BPP and FMRFamide antisera, contained material which was genuinely FMRFamide...

Lectin Domains of Polypeptide GalNAc Transferases Exhibit Glycopeptide Binding Specificity

DEFF Research Database (Denmark)

Pedersen, Johannes W; Bennett, Eric P; Schjoldager, Katrine T-B G

2011-01-01

UDP-GalNAc:polypeptide a-N-acetylgalactosaminyltransferases (GalNAc-Ts) constitute a family of up to 20 transferases that initiate mucin-type O-glycosylation. The transferases are structurally composed of catalytic and lectin domains. Two modes have been identified for the selection...... of glycosylation sites by GalNAc-Ts: confined sequence recognition by the catalytic domain alone, and concerted recognition of acceptor sites and adjacent GalNAc-glycosylated sites by the catalytic and lectin domains, respectively. Thus far, only the catalytic domain has been shown to have peptide sequence...... on sequences of mucins MUC1, MUC2, MUC4, MUC5AC, MUC6, and MUC7 as well as a random glycopeptide bead library, we examined the binding properties of four different lectin domains. The lectin domains of GalNAc-T1, -T2, -T3, and -T4 bound different subsets of small glycopeptides. These results indicate...

Tuning Thermoresponsive Properties of Cationic Elastin-like Polypeptides by Varying Counterions and Side-Chains.

Science.gov (United States)

Petitdemange, Rosine; Garanger, Elisabeth; Bataille, Laure; Bathany, Katell; Garbay, Bertrand; Deming, Timothy J; Lecommandoux, Sébastien

2017-05-17

We report the synthesis of methionine-containing recombinant elastin-like polypeptides (ELPs) of different lengths that contain periodically spaced methionine residues. These ELPs were chemoselectively alkylated at all methionine residues to give polycationic derivatives. Some of these samples were found to possess solubility transitions in water, where the temperature of these transitions varied with ELP concentration, nature of the methionine alkylating group, and nature of the sulfonium counterions. These studies show that introduction and controlled spacing of methionine sulfonium residues into ELPs can be used as a means both to tune their solubility transition temperatures in water using a variety of different parameters and to introduce new side-chain functionality.

Solid self-nanoemulsifying drug delivery systems for oral delivery of polypeptide-k: Formulation, optimization, in-vitro and in-vivo antidiabetic evaluation.

Science.gov (United States)

Garg, Varun; Kaur, Puneet; Singh, Sachin Kumar; Kumar, Bimlesh; Bawa, Palak; Gulati, Monica; Yadav, Ankit Kumar

2017-11-15

Development of self-nanoemulsifying drug delivery systems (SNEDDS) of polypeptide-k (PPK) is reported with the aim to achieve its oral delivery. Box-Behnken design (BBD) was adopted to develop and optimize the composition of SNEDDS. Oleoyl polyoxyl-6 glycerides (A), Tween 80 (B), and diethylene glycol monoethyl ether (C) were used as oil, surfactant and co-surfactant, respectively as independent variables. The effect of variation in their composition was observed on the mean droplet size (y1), polydispersity index (PDI) (y2), % drug loading (y3) and zeta potential (y4). As per the optimal design, seventeen SNEDDS prototypes were prepared. The optimized composition of SNEDDS formulation was 25% v/v Oleoyl polyoxyl-6 glycerides, 37% v/v Tween 80, 38% v/v diethylene glycol monoethyl ether, and 3% w/v PPK. The optimized formulation revealed values of y1, y2, y3, and y4 as 31.89nm, 0.16, 73.15%, and -15.65mV, respectively. Further the optimized liquid SNEDDS were solidified through spray drying using various hydrophilic and hydrophobic carriers. Among the various carriers, Aerosil 200 was found to provide desirable flow, compression, disintegration and dissolution properties. Both, liquid and solid-SNEDDS have shown release of >90% within 10min. The formulation was found stable with change in pH, dilution, temperature variation and freeze thaw cycles in terms of droplet size, zeta potential, drug precipitation and phase separation. Crystalline PPK was observed in amorphous state in solid SNEDDS when characterized through DSC and PXRD studies. The biochemical, hematological and histopathological results of streptozotocin induced diabetic rats shown promising antidiabetic potential of PPK loaded in SNEDDS at its both the doses (i.e. 400mg/kg and 800mg/kg) as compared to its naïve form at both the doses. The study revealed successful formulation of SNEDDS for oral delivery of PPK. Copyright © 2017 Elsevier B.V. All rights reserved.

In vitro gamma irradiation of some purified polypeptide hormones and their biological and radioimmunological activity

International Nuclear Information System (INIS)

Hromadova, M.; Macho, L.; Strbak, V.; Vigas, M.; Mikulaj, L.

1979-01-01

Some polypeptide hormones (adrenocorticotropin - ACTH, human and bovine growth hormone - GH, human menopausal gonadotropin - HMG, human luteinizing hormone - LH, and bovine thyrotropin - TSH) were irradiated either with 2.5 or 12.5 Mrad (1.1 Mrad/h) or both and their biological activity or immunoreactivity was tested within few days or 3 to 5 months after irradiation. Biological activity of irradiated ACTH (estimation of corticosterone released into medium by incubated adrenals - Saffran and Schally 1955) was not decreased in both time intervals tested. Ten days after irradiation of bovine GH no changes in biological activity (tibia test - Wilhelmi 1973) were found. No decrease of biological activity of irradiated HMG (augmentation of ovarian and uterine weight - Butt 1973) was found 4 months after irradiation and, finaly, no decrease of bovine TSH activity (radioiodine release from prelabelled thyroid in mice - McKenzie 1958) was found 2 to 30 days after irradiation with 2.5 Mrad, while a decrease was observed after 12.5 Mrad. Three to five months after irradiation, however, there was a decrease of biological activity after both doses. The immunological reactivity of irradiated HMG and LH did not differ from that of nonirradiated samples. The same was found with human GH after 2.5 Mrad, while a decrease of reactivity after 12.5 Mrad was detected. It was concluded that, in most of cases, the sterilizing dose of gamma radiation (2.5 Mrad) did not affect the biological activity of polypeptide hormones and that their sensitivity to irradiation appears to differ. (author)

Molecular Dynamics Simulation of Cholera Toxin A-1 Polypeptide

Directory of Open Access Journals (Sweden)

Badshah Syed Lal

2016-01-01

Full Text Available A molecular dynamics (MD simulation study of the enzymatic portion of cholera toxin; cholera toxin A-1 polypeptide (CTA1 was performed at 283, 310 and 323 K. From total energy analysis it was observed that this toxin is stable thermodynamically and these outcomes were likewise confirmed by root mean square deviations (RMSD investigations. The Cα root mean square fluctuation (RMSF examinations revealed that there are a number of residues inside CTA1, which can be used as target for designing and synthesizing inhibitory drugs, in order to inactivate cholera toxin inside the human body. The fluctuations in the radius of gyration and hydrogen bonding in CTA1 proved that protein unfolding and refolding were normal routine phenomena in its structure at all temperatures. Solvent accessible surface area study identified the hydrophilic nature of the CTA1, and due to this property it can be a potential biological weapon. The structural identification (STRIDE algorithm for proteins was successfully used to determine the partially disordered secondary structure of CTA1. On account of this partially disordered secondary structure, it can easily deceive the proteolytic enzymes of the endoplasmic reticulum of host cells.

Dual binding mode of the nascent polypeptide-associated complex reveals a novel universal adapter site on the ribosome.

Science.gov (United States)

Pech, Markus; Spreter, Thomas; Beckmann, Roland; Beatrix, Birgitta

2010-06-18

Nascent polypeptide-associated complex (NAC) was identified in eukaryotes as the first cytosolic factor that contacts the nascent polypeptide chain emerging from the ribosome. NAC is present as a homodimer in archaea and as a highly conserved heterodimer in eukaryotes. Mutations in NAC cause severe embryonically lethal phenotypes in mice, Drosophila melanogaster, and Caenorhabditis elegans. In the yeast Saccharomyces cerevisiae NAC is quantitatively associated with ribosomes. Here we show that NAC contacts several ribosomal proteins. The N terminus of betaNAC, however, specifically contacts near the tunnel exit ribosomal protein Rpl31, which is unique to eukaryotes and archaea. Moreover, the first 23 amino acids of betaNAC are sufficient to direct an otherwise non-associated protein to the ribosome. In contrast, alphaNAC (Egd2p) contacts Rpl17, the direct neighbor of Rpl31 at the ribosomal tunnel exit site. Rpl31 was also recently identified as a contact site for the SRP receptor and the ribosome-associated complex. Furthermore, in Escherichia coli peptide deformylase (PDF) interacts with the corresponding surface area on the eubacterial ribosome. In addition to the previously identified universal adapter site represented by Rpl25/Rpl35, we therefore refer to Rpl31/Rpl17 as a novel universal docking site for ribosome-associated factors on the eukaryotic ribosome.

Vasoactive intestinal polypeptide (VIP) innervation of the human eyelid glands.

Science.gov (United States)

Seifert, P; Spitznas, M

1999-06-01

This study was conducted to obtain morphological proof of innervating nerve fibres in the glands of the human eyelid (accessory lacrimal glands of Wolfring, meibomian glands, goblet cells, glands of Zeis, glands of Moll, sweat glands, glands of lanugo hair follicles) and identification of the secretomotorically active neuropeptide vasoactive intestinal polypeptide (VIP) as a common transmitter. Epoxy-embedded ultrathin sections of tissue samples from human eyelids were studied using electron microscopy. Paraffin sections fixed in Bouin-Hollande solution were immunostained with rabbit antiserum against VIP. With the electron microscope we were able to identify nerves in the glandular stroma of all the glands examined with the exception of goblet cells. Intraepithelial single axons were only seen in the parenchyma of Wolfring glands. The morphological findings corresponded with the immunological finding of VIP-positive, nerve-like structures in the same locations, with the exception of lanugo hair follicle glands, and goblet cells. Our findings indicate that the glands of the eyelids and main lacrimal gland represent a functional unit with VIP as a possible common stimulating factor. Copyright 1999 Academic Press.

Development of the kits for RIA simultaneous determination of polypeptide hormones

International Nuclear Information System (INIS)

Szybinski, Z.

1982-12-01

A simple and universal modification of chloramine T technique has been developed for the radioactive iodination of several polypeptide hormones such as insulin, human growth hormone (HGH), human TSH, synthetic human gastrin and beta-endorphine. The prepared products proved to have good immunoreactivity suitable for RIA purposes. The technique is inexpensive and quick. A new procedure has also been worked out utilizing horse myeloperoxidase in solid state as catalyser. The hormones iodinated with this technique show better parameters (e.g. longer stability, better binding to antibody, more favourable adsorption on dextran-coated charcoal); however the specific activities achieved were lower. The possibilities of simultaneous measurement of insulin and HGH have been studied. In this connection, a comparatively simple method for the determination of the endogenous anti-insulin antibodies was developed and used for the control of patients with diabetes and for the checking of new insulin preparations. However, the technique requires relatively sophisticated equipment and computerized calculations

CS5931, a Novel Polypeptide in Ciona savignyi, Represses Angiogenesis via Inhibiting Vascular Endothelial Growth Factor (VEGF and Matrix Metalloproteinases (MMPs

Directory of Open Access Journals (Sweden)

Ge Liu

2014-03-01

Full Text Available CS5931 is a novel polypeptide from Ciona savignyi with anticancer activities. Previous study in our laboratory has shown that CS5931 can induce cell death via mitochondrial apoptotic pathway. In the present study, we found that the polypeptide could inhibit angiogenesis both in vitro and in vivo. CS5931 inhibited the proliferation, migration and formation of capillary-like structures of HUVECs (Human Umbilical Vein Endothelial Cell in a dose-dependent manner. Additionally, CS5931 repressed spontaneous angiogenesis of the zebrafish vessels. Further studies showed that CS5931 also blocked vascular endothelial growth factor (VEGF production but without any effect on its mRNA expression. Moreover, CS5931 reduced the expression of matrix metalloproteinases (MMP-2 and MMP-9 both on protein and mRNA levels in HUVEC cells. We demonstrated that CS5931 possessed strong anti-angiogenic activity both in vitro and in vivo, possible via VEGF and MMPs. This study indicates that CS5931 has the potential to be developed as a novel therapeutic agent as an inhibitor of angiogenesis for the treatment of cancer.

Loss of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 and reduced O-glycosylation in colon carcinoma cells selected for hepatic metastasis

DEFF Research Database (Denmark)

Kato, Kentaro; Takeuchi, Hideyuki; Kanoh, Akira

2010-01-01

O-glycosylation of mucin is initiated by the attachment of N-acetyl-D-galactosamine (GalNAc) to serine or threonine residues in mucin core polypeptides by UDPGalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). It is not well understood how GalNAc attachment is regulated by multiple...... ppGalNAc-Ts in each cell. In the present study, the expression levels of murine ppGalNAc-Ts (mGalNAc-Ts), T1, T2, T3, T4, T6, and T7 were compared between mouse colon carcinoma colon 38 cells and variant SL4 cells, selected for their metastatic potentials, by using the competitive RT-PCR method....... The expression levels of mGalNAc-T1, T2, and T7 were slightly higher in the SL4 cells than in the colon 38 cells, whereas the expression level of mGalNAc-T3 in the SL4 cells was 1.5% of that in the colon 38 cells. Products of enzymatic incorporations of GalNAc residues into FITCPTTTPITTTTK peptide by the use...

Cellobiohydrolase I enzymes

Science.gov (United States)

Adney, William S; Himmel, Michael E; Decker, Stephen R; Knoshaug, Eric P; Nimlos, Mark R; Crowley, Michael F; Jeoh, Tina

2014-01-28

Provided herein is an isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide, wherein the mutations reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. Also provided herein is an isolated Cel7A polypeptide comprising increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The increased O-linked glycosylation is a result of the addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide. In some embodiments, the isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide further comprises increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The mutations in the catalytic domain reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. The addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide increases O-linked glycosylation of the isolated polypeptide. Further provided are compositions comprising such polypeptides and nucleic acids encoding such polypeptides. Still further provided are methods for making such polypeptides.

CNS-syndrome. Characterization of rat brain intermediate filaments

International Nuclear Information System (INIS)

Nedzvetskij, V.S.; Busygina, S.G.; Berezin, V.A.; Dvoretskij, A.I.

1990-01-01

A study was made of the effect of ionizing radiation on the content and polypeptide composition of filamentous and soluble glial fibrillary acidic protein (GFAP) in different regions of rat brain. Ionizing radiation was shown to decrease considerably the level of soluble GFAP in cerebral cortex, cerebellum, middle brain and hippocampus. Polypeptide composition of soluble GFAP detected by the immonublot-method was found to be changed considerably in different brain areas of irradiated animals

Protein Complexation and pH Dependent Release Using Boronic Acid Containing PEG-Polypeptide Copolymers.

Science.gov (United States)

Negri, Graciela E; Deming, Timothy J

2017-01-01

New poly(L-lysine)-b-poly(ethylene glycol) copolypeptides have been prepared, where the side-chain amine groups of lysine residues are modified to contain ortho-amine substituted phenylboronic acid, i.e., Wulff-type phenylboronic acid (WBA), groups to improve their pH responsive, carbohydrate binding properties. These block copolymers form nanoscale complexes with glycosylated proteins that are stable at physiological pH, yet dissociate and release the glycoproteins under acidic conditions, similar to those found in endosomal and lysosomal compartments within cells. These results suggest that WBA modified polypeptide copolymers are promising for further development as degradable carriers for intracellular protein delivery. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Angioplastic necrolytic migratory erythema. Unique association of necrolytic migratory erythema, extensive angioplasia, and high molecular weight glucagon-like polypeptide

International Nuclear Information System (INIS)

Franchimont, C.; Pierard, G.E.; Luyckx, A.S.; Gerard, J.; Lapiere, C.M.

1982-01-01

A diabetic patient developed necrolytic migratory erythema with extensive angioplasia and high molecular weight glucagon-like polypeptide. There was no associated neoplasm such as glucagonoma. Lesions in the skin were studied by standard optical microscopy and by radioautography after incorporation of tritiated thymidine. Alterations in the skin begin as focal necrosis in the epidermis and in epithelial structures of adnexa, followed by marked angioplasia and a superficial and deep perivascular dermatitis

Phycobilisome Mobility and Its Role in the Regulation of Light Harvesting in Red Algae

Czech Academy of Sciences Publication Activity Database

Kaňa, Radek; Kotabová, Eva; Lukeš, Martin; Papáček, Š.; Matonoha, Ctirad; Liu, L.N.; Prášil, Ondřej; Mullineaux, C.W.

2014-01-01

Roč. 165, č. 4 (2014), s. 1618-1631 ISSN 0032-0889 R&D Projects: GA ČR GAP501/12/0304; GA MŠk ED2.1.00/03.0110 Institutional support: RVO:61388971 ; RVO:67985807 Keywords : EXCITATION-ENERGY TRANSFER * CYANOBACTERIAL THYLAKOID MEMBRANE S * MICROALGA PORPHYRIDIUM-CRUENTUM Subject RIV: EE - Microbiology, Virology; BA - General Mathematics (UIVT-O) Impact factor: 6.841, year: 2014

Characterisation of the LH2 spectral variants produced by the photosynthetic purple sulphur bacterium Allochromatium vinosum.

Science.gov (United States)

Carey, Anne-Marie; Hacking, Kirsty; Picken, Nichola; Honkanen, Suvi; Kelly, Sharon; Niedzwiedzki, Dariusz M; Blankenship, Robert E; Shimizu, Yuuki; Wang-Otomo, Zheng-Yu; Cogdell, Richard J

2014-11-01

This study systematically investigated the different types of LH2 produced by Allochromatium (Alc.) vinosum, a photosynthetic purple sulphur bacterium, in response to variations in growth conditions. Three different spectral forms of LH2 were isolated and purified, the B800-820, B800-840 and B800-850 LH2 types, all of which exhibit an unusual split 800 peak in their low temperature absorption spectra. However, it is likely that more forms are also present. Relatively more B800-820 and B800-840 are produced under low light conditions, while relatively more B800-850 is produced under high light conditions. Polypeptide compositions of the three different LH2 types were determined by a combination of HPLC and TOF/MS. The B800-820, B800-840 and B800-850 LH2 types all have a heterogeneous polypeptide composition, containing multiple types of both α and β polypeptides, and differ in their precise polypeptide composition. They all have a mixed carotenoid composition, containing carotenoids of the spirilloxanthin series. In all cases the most abundant carotenoid is rhodopin; however, there is a shift towards carotenoids with a higher conjugation number in LH2 complexes produced under low light conditions. CD spectroscopy, together with the polypeptide analysis, demonstrates that these Alc. vinosum LH2 complexes are more closely related to the LH2 complex from Phs. molischianum than they are to the LH2 complexes from Rps. acidophila. Copyright © 2014. Published by Elsevier B.V.

Near infrared imaging-guided photodynamic therapy under an extremely low energy of light by galactose targeted amphiphilic polypeptide micelle encapsulating BODIPY-Br2.

Science.gov (United States)

Liu, Le; Ruan, Zheng; Li, Tuanwei; Yuan, Pan; Yan, Lifeng

2016-10-18

Near infrared (NIR) imaging-guided photodynamic therapy (PDT) is attractive, especially the utilization of one dye as both a photosensitizer and fluorescent probe, and the as-synthesized BODIPY-Br 2 molecule is a candidate. Here, a galactose targeted amphiphilic copolymer of a polypeptide was synthesized and its micelles work as nanocarriers for BODIPY for targeting the NIR imaging-guided PDT of hepatoma cancer cells. At the same time, BODIPY could light up the cytoplasm for real-time imaging and kill cancer cells when the light was switched on. In vitro tests performed on both HepG2 and HeLa cells confirmed that the as-prepared PMAGP-POEGMA-PLys-B micelles showed efficient cell suppression of the cells with galactose receptors in the presence of light under an extremely low energy density (6.5 J cm -2 ). This protocol highlights the potential of polypeptides as biodegradable carriers for NIR image-guided and confined targeting photodynamic therapy.

Design of a software for calculating isoelectric point of a polypeptide according to their net charge using the graphical programming language LabVIEW.

Science.gov (United States)

Tovar, Glomen

2018-01-01

A software to calculate the net charge and to predict the isoelectric point (pI) of a polypeptide is developed in this work using the graphical programming language LabVIEW. Through this instrument the net charges of the ionizable residues of the polypeptide chains of the proteins are calculated at different pH values, tabulated, pI is predicted and an Excel (-xls) type file is generated. In this work, the experimental values of the pIs (pI) of different proteins are compared with the values of the pIs (pI) calculated graphically, achieving a correlation coefficient (R) of 0.934746 which represents a good reliability for a p program can constitute an instrument applicable in the laboratory, facilitating the calculation to graduate students and junior researchers. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):39-46, 2018. © 2017 The International Union of Biochemistry and Molecular Biology.

Potent and selective mediators of cholesterol efflux

Energy Technology Data Exchange (ETDEWEB)

Bielicki, John K; Johansson, Jan

2015-03-24

The present invention provides a family of non-naturally occurring polypeptides having cholesterol efflux activity that parallels that of full-length apolipoproteins (e.g., Apo AI and Apo E), and having high selectivity for ABAC1 that parallels that of full-length apolipoproteins. The invention also provides compositions comprising such polypeptides, methods of identifying, screening and synthesizing such polypeptides, and methods of treating, preventing or diagnosing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation.

Peptide mediators of cholesterol efflux

Energy Technology Data Exchange (ETDEWEB)

Bielicki, John K.; Johansson, Jan

2013-04-09

The present invention provides a family of non-naturally occurring polypeptides having cholesterol efflux activity that parallels that of full-length apolipoproteins (e.g., Apo AI and Apo E), and having high selectivity for ABAC1 that parallels that of full-length apolipoproteins. The invention also provides compositions comprising such polypeptides, methods of identifying, screening and synthesizing such polypeptides, and methods of treating, preventing or diagnosing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation.

NMR structure of the glucose-dependent insulinotropic polypeptide fragment, GIP(1-30)amide

International Nuclear Information System (INIS)

Alana, Inigo; Hewage, Chandralal M.; G. Malthouse, J. Paul; Parker, Jeremy C.; Gault, Victor A.; O'Harte, Finbarr P.M.

2004-01-01

Glucose-dependent insulinotropic polypeptide is an incretin hormone that stimulates insulin secretion and reduces postprandial glycaemic excursions. The glucose-dependent action of GIP on pancreatic β-cells has attracted attention towards its exploitation as a potential drug for type 2 diabetes. Use of NMR or X-ray crystallography is vital to determine the three-dimensional structure of the peptide. Therefore, to understand the basic structural requirements for the biological activity of GIP, the solution structure of the major biologically active fragment, GIP(1-30)amide, was investigated by proton NMR spectroscopy and molecular modelling. The structure is characterised by a full length α-helical conformation between residues F 6 and A 28 . This structural information could play an important role in the design of therapeutic agents based upon GIP receptor agonists

Nucleophilic behavior of lysine-501 of the alpha-polypeptide of sodium and potassium ion activated adenosinetriphosphatase consistent with a role in binding adenosine triphosphate

International Nuclear Information System (INIS)

Xu, K.Y.; Kyte, J.

1989-01-01

An immunoadsorbent specific for the carboxy-terminal sequence -GAPER, which comprises residues 502-506 of the alpha-polypeptide of ovine sodium and potassium ion activated adenosinetriphosphatase [(Na+ + K+)-ATPase], was used to isolate the products of the reaction between the lysine immediately preceding this sequence in the intact protein and either [3H]acetic anhydride or fluorescein 5'-isothiocyanate. Changes in the apparent nucleophilicity of this lysine, Lys501, were observed with both reagents when ATP was bound by the intact, native enzyme poised in the E1 conformation or when the structure of the enzyme was changed from the E1 conformation into the E2-P conformation. With both reagents, a decrease of more than 4-fold in the yield of incorporation occurred during the former change, but a decrease of only 2-fold occurred during the latter. Because a much larger decrease occurred when ATP was bound in the absence of a conformational change than occurred when a major conformational change took place in the absence of the occupation of the active site, these changes in the incorporation of [3H]acetyl suggest that Lys501 from the alpha polypeptide is directly involved in binding ATP within the active site of (Na+ + K+)-ATPase. The immunochemical reactions between the specific polyclonal antibodies raised against the sequence-GAPER and denatured or enzymically active (Na+ + K+)-ATPase were also investigated. Western blots and the inhibition of enzymic activity caused by the antibody have shown that it can bind to both the denatured and the native form of the alpha-polypeptide, respectively

Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis

DEFF Research Database (Denmark)

Kong, Yun; Joshi, Hiren J; Schjoldager, Katrine Ter-Borch Gram

2015-01-01

N-acetylgalactosaminyltransferase (GalNAc)-type (mucin-type) O-glycosylation is an abundant and highly diverse modification of proteins. This type of O-glycosylation is initiated in the Golgi by a large family of up to 20 homologous polypeptide GalNAc-T isoenzymes that transfer GalNAc to Ser, Thr...... and possibly Tyr residues. These GalNAc residues are then further elongated by a large set of glycosyltransferases to build a variety of complex O-glycan structures. What determines O-glycan site occupancy is still poorly understood, although it is clear that the substrate specificities of individual...... isoenzymes and the repertoire of GalNAc-Ts in cells are key parameters. The GalNAc-T isoenzymes are differentially expressed in cells and tissues in principle allowing cells to produce unique O-glycoproteomes dependent on the specific subset of isoforms present. In vitro analysis of acceptor peptide...

Recombinant proteins as vaccines for protection against disease induced by infection with mink astrovirus

DEFF Research Database (Denmark)

2012-01-01

and polypeptides of the capsid protein of a novel mink astrovirus strain denoted DK7627. Such polynucleotides and polypeptides may be used for the production of vaccines against mink astrovirus which may induce pre-weaning diarrhoea in minks. The invention furthermore relates to vectors, host cells, compositions...

The dipeptidyl peptidase 4 inhibitor vildagliptin does not accentuate glibenclamide-induced hypoglycemia but reduces glucose-induced glucagon-like peptide 1 and gastric inhibitory polypeptide secretion

DEFF Research Database (Denmark)

El-Ouaghlidi, Andrea; Rehring, Erika; Holst, Jens Juul

2007-01-01

BACKGROUND/AIMS: Inhibition of dipeptidyl peptidase 4 by vildagliptin enhances the concentrations of the active form of the incretin hormones glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP). The present study asked whether vildagliptin accentuates glibenclamide-induced hy...

Thermal Synthesis of Polypeptides from N-Butyloxycarbonyl Oligopeptides Containing Aspartyl Residue at C-Terminus

Directory of Open Access Journals (Sweden)

Toratane Munegumi

2017-01-01

Full Text Available The thermal reactions of amino acids have been investigated for pure organic synthesis, materials preparation in industry, and prebiotic chemistry. N-t-Butyloxycarbonyl aspartic acid (Boc-Asp releases 2-butene and carbon dioxide upon heating without solvents. The resulting mixture of the free molten aspartic acid was dehydrated to give peptide bonds. This study describes the thermal reactions of N-t-butyloxycarbonyl peptides (Boc-Gly-L-Asp, Boc-L-Ala-L-Asp, Boc-L-Val-L-Asp, and Boc-Gly-Gly-L-Asp having an aspartic residue at the carboxyl terminus. The peptides were deprotected upon heating at a constant temperature between 110 and 170°C for 1 to 24 h to afford polypeptides in which the average molecular weight reached 7800.

β-Hairpin of Islet Amyloid Polypeptide Bound to an Aggregation Inhibitor

Science.gov (United States)

Mirecka, Ewa A.; Feuerstein, Sophie; Gremer, Lothar; Schröder, Gunnar F.; Stoldt, Matthias; Willbold, Dieter; Hoyer, Wolfgang

2016-01-01

In type 2 diabetes, the formation of islet amyloid consisting of islet amyloid polypeptide (IAPP) is associated with reduction in β-cell mass and contributes to the failure of islet cell transplantation. Rational design of inhibitors of IAPP amyloid formation has therapeutic potential, but is hampered by the lack of structural information on inhibitor complexes of the conformationally flexible, aggregation-prone IAPP. Here we characterize a β-hairpin conformation of IAPP in complex with the engineered binding protein β-wrapin HI18. The β-strands correspond to two amyloidogenic motifs, 12-LANFLVH-18 and 22-NFGAILS-28, which are connected by a turn established around Ser-20. Besides backbone hydrogen bonding, the IAPP:HI18 interaction surface is dominated by non-polar contacts involving hydrophobic side chains of the IAPP β-strands. Apart from monomers, HI18 binds oligomers and fibrils and inhibits IAPP aggregation and toxicity at low substoichiometric concentrations. The IAPP β-hairpin can serve as a molecular recognition motif enabling control of IAPP aggregation. PMID:27641459

Multiscale characterization of a chimeric biomimetic polypeptide for stem cell culture

International Nuclear Information System (INIS)

Sbrana, F; Vassalli, M; Fotia, C; Baldini, N; Ciapetti, G; Bracalello, A; Bochicchio, B; Marletta, G

2012-01-01

Mesenchymal stem cells have attracted great interest in the field of tissue engineering and regenerative medicine because of their multipotentiality and relative ease of isolation from adult tissues. The medical application of this cellular system requires the inclusion in a growth and delivery scaffold that is crucial for the clinical effectiveness of the therapy. In particular, the ideal scaffolding material should have the needed porosity and mechanical strength to allow a good integration with the surrounding tissues, but it should also assure high biocompatibility and full resorbability. For such a purpose, protein-inspired biomaterials and, in particular, elastomeric-derived polypeptides are playing a major role, in which they are expected to fulfil many of the biological and mechanical requirements. A specific chimeric protein, designed starting from elastin, resilin and collagen sequences, was characterized over different length scales. Single-molecule mechanics, aggregation properties and compatibility with human mesenchymal stem cells were tested, showing that the engineered compound is a good candidate as a stem cell scaffold to be used in tissue engineering applications. (paper)

Breakout character of islet amyloid polypeptide hydrophobic mutations at the onset of type-2 diabetes

Science.gov (United States)

Frigori, Rafael B.

2014-11-01

Toxic fibrillar aggregates of islet amyloid polypeptide (IAPP) appear as the physical outcome of a peptidic phase transition signaling the onset of type-2 diabetes mellitus in different mammalian species. In particular, experimentally verified mutations on the amyloidogenic segment 20-29 in humans, cats, and rats are highly correlated with the molecular aggregation propensities. Through a microcanonical analysis of the aggregation of IAPP20 -29 isoforms, we show that a minimalist one-bead hydrophobic-polar continuum model for protein interactions properly quantifies those propensities from free-energy barriers. Our results highlight the central role of sequence-dependent hydrophobic mutations on hot spots for stabilization, and thus for the engineering, of such biological peptides.

Cleavage sites in the polypeptide precursors of poliovirus protein P2-X

International Nuclear Information System (INIS)

Selmer, B.L.; Hanecak, R.; Anderson, C.W.; Wimmer, E.

1981-01-01

Partial amino-terminal sequence analysis has been performed on the three major polypeptide products (P2-3b, P2-5b, and P2-X) from the central region (P2) of the poliovirus polyprotein, and this analysis precisely locates the amino termini of these products with respect to the nucleotide sequence of the poliovirus RNA genome. Like most of the products of the replicase region (P3), the amino termini of P2-5b and P2-X are generated by cleavage between glutamine and glycine residues. Thus, P2-5b and P2-X are probably both produced by the action of a singly (virus-encoded.) proteinase. The amino terminus of P2-3b, on the other hand, is produced by a cleavage between the carboxy-terminal tyrosine of VP1 and the glycine encoded by nucleotides 3381-3383. This result may suggest that more than one proteolytic activity is required for the complete processing of the poliovirus polyprotein

Membrane prteins of herpes simplex infected cells. Immunological and biochemical studies

NARCIS (Netherlands)

Welling-Wester, Sijtske

1981-01-01

As a consequence of infection with herpes simplex virus (HSV), cells exhibit a number of alterations. One of these is expressed as a change in the polypeptide composition of the surface of the infected cells. In this study several methods used for the isolation of these polypeptides expressed on the

Oxidative stress status, antioxidant metabolism and polypeptide patterns in Juncus maritimus shoots exhibiting differential mercury burdens in Ria de Aveiro coastal lagoon (Portugal).

Science.gov (United States)

Anjum, Naser A; Duarte, Armando C; Pereira, Eduarda; Ahmad, Iqbal

2014-05-01

This study assessed the oxidative stress status, antioxidant metabolism and polypeptide patterns in salt marsh macrophyte Juncus maritimus shoots exhibiting differential mercury burdens in Ria de Aveiro coastal lagoon at reference and the sites with highest, moderate and the lowest mercury contamination. In order to achieve these goals, shoot-mercury burden and the responses of representative oxidative stress indices, and the components of both non-glutathione- and glutathione-based H2O2-metabolizing systems were analyzed and cross-talked with shoot-polypeptide patterns. Compared to the reference site, significant elevations in J. maritimus shoot mercury and the oxidative stress indices such as H2O2, lipid peroxidation, electrolyte leakage and reactive carbonyls were maximum at the site with highest followed by moderate and the lowest mercury contamination. Significantly elevated activity of non-glutathione-based H2O2-metabolizing enzymes such as ascorbate peroxidase and catalase accompanied the studied damage-endpoint responses, whereas the activity of glutathione-based H2O2-scavenging enzymes glutathione peroxidase and glutathione sulfo-transferase was inhibited. Concomitantly, significantly enhanced glutathione reductase activity and the contents of both reduced and oxidized glutathione were perceptible in high mercury-exhibiting shoots. It is inferred that high mercury-accrued elevations in oxidative stress indices were obvious, where non-glutathione-based H2O2-decomposing enzyme system was dominant over the glutathione-based H2O2-scavenging enzyme system. In particular, the glutathione-based H2O2-scavenging system failed to coordinate with elevated glutathione reductase which in turn resulted into increased pool of oxidized glutathione and the ratio of oxidized glutathione-to-reduced glutathione. The substantiation of the studied oxidative stress indices and antioxidant metabolism with approximately 53-kDa polypeptide warrants further studies.

Intestinal mucosa is a target tissue for pancreatic polypeptide

International Nuclear Information System (INIS)

Gilbert, W.R.; Kramer, J.L.; Frank, B.H.; Gingerich, R.L.

1986-01-01

Studies were carried out to identify mammalian tissues capable of specifically binding mammalian pancreatic polypeptide (PP). Bovine PP (bPP) radiolabeled with 125 I was purified by HPLC to yield [ 125 I]iodo-(Tyr-27) bPP. The label was injected into three pairs of fasted littermate dogs and allowed to circulate for 5 min. One of the dogs was a control which received an excess of unlabeled porcine PP to provide competition for receptor binding. Unbound bPP was removed by perfusion with Krebs-Ringer bicarbonate and the tissue fixed in situ with Karnovsky's fixative. Tissue samples from various organs were removed, weighed, and counted. The entire gastrointestinal tract demonstrated high levels of 125 I after injection of the labeled peptide. The duodenum, jejunum, ileum, and colon were the only tissues to exhibit specific binding of bPP. These tissues (mucosal and muscle layers) from experimental animals exhibited 31-76% higher binding than the corresponding tissues from the control animals. Sections of the gastrointestinal tract were scraped to separate the mucosal layer from the underlying muscle layer. The mucosal layer of the duodenum, jejunum, and ileum exhibited 145-162% increases in binding compared to the control animals. The muscle layer of these tissues demonstrated no significant increase. These findings demonstrate that mucosal layer of the small intestine is a target tissue for mammalian PP

Effects of gastric inhibitory polypeptide, glucagon-like peptide-1 and glucagon-like peptide-1 receptor agonists on Bone Cell Metabolism

DEFF Research Database (Denmark)

Hansen, Morten S S; Tencerova, Michaela; Frølich, Jacob

2018-01-01

The relationship between gut and skeleton is increasingly recognised as part of the integrated physiology of the whole organism. The incretin hormones gastric inhibitory polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are secreted from the intestine in response to nutrient intake and exhibi......-clinical investigations, clinical trials are needed to clarify if similar effects are present and clinically relevant in humans. This article is protected by copyright. All rights reserved....

Solvent deuteration enhancement of hydrophobicity: DSC study of the inverse temperature transition of elastin-based polypeptides

International Nuclear Information System (INIS)

Chihao Luan; Urry, D.W.

1991-01-01

As previously shown, the polypentapeptide of elastin, (Val 1 -Pro 2 -Gly 3 -Val 4 -Gly 5 ) n or simply poly(VPGVG), undergoes an inverse temperature transition which is seen macroscopically as a phase separation with a dense viscoelastic phase of about 60% water, 40% peptide by weight and which is characterized molecularly by increase in intra- and intermolecular order as evidenced by formation of specific hydrophobic contacts. Furthermore, from an extensive study of polypentapeptides of the composition poly[f x (VPGXG), f v (VPGVG)], where f x + f v = 1 and X is any of the amino acid residues with apolar (hydrophobic) side chains, it has been shown that the temperature of the transition decreases and the heat of the transition increases with increased hydrophobicity. In the present paper differential scanning calorimetry has been utilized to determine the effect of D 2 O on the temperature and heats of the inverse temperature transitions for poly(VPGVG), poly(IPGVG), poly(LPGVG), and poly(VPAVG) and in the latter case in the presence of 0.5 and 1.0 N NaCl and of 1,2, and 3 M urea. In all cases, the effect of D 2 O as compared to H 2 O is to lower the transition temperature about 2 C and to increase the heat of the transition about 10%, and this occurs also in the presence of NaCl, which itself lowers the temperature and increases the heat, and in the presence of urea, which itself raises the temperature and decreases the heat of the transition. It is concluded that the effect of replacement of H 2 O by D 2 O by D 2 O in these polypeptides is to effect a small but consistent increase in the expression of hydrophobicity

Mucin-type O-glycosylation is controlled by short- and long-range glycopeptide substrate recognition that varies among members of the polypeptide GalNAc transferase family

DEFF Research Database (Denmark)

Revoredo, Leslie; Wang, Shengjun; Bennett, Eric Paul

2016-01-01

A large family of UDP-GalNAc:polypeptide GalNAc transferases (ppGalNAc-Ts) initiates and defines sites of mucin-type Ser/Thr-O-GalNAc glycosylation. Family members have been classified into peptide- and glycopeptide-preferring subfamilies, although both families possess variable activities agains...

THE STRUCTURE, ORIGIN, ISOLATION, AND COMPOSITION OF THE TUBULAR MASTIGONEMES OF THE OCHROMONAS FLAGELLUM

Science.gov (United States)

Bouck, G. Benjamin

1971-01-01

The structure, assembly, and composition of the extracellular hairs (mastigonemes) of Ochromonas are detailed in this report. These mastigonemes form two lateral unbalanced rows, each row on opposite sides of the long anterior flagellum. Each mastigoneme consists of lateral filaments of two distinct sizes attached to a tubular shaft. The shaft is further differentiated into a basal region at one end and a group of from one to three terminal filaments at the free end. Mastigoneme ontogeny as revealed especially in deflagellated and regenerating cells appears to begin by assembly of the basal region and shaft within the perinuclear continuum. However, addition of lateral filaments to the shaft and extrusion of the mastigonemes to the cell surface is mediated by the Golgi complex. The ultimate distribution of mastigonemes on the flagellar surface seems to be the result of extrusion of mastigonemes near the base of the flagellum, and it is suggested that mastigonemes are then pulled up the flagellum as the axoneme elongates. Efforts to characterize mastigonemes biochemically after isolation and purification on cesium chloride (CsCl) followed by electrophoresis on acrylamide gels have demonstrated what appear to be a single major polypeptide and several differentially migrating carbohydrates. The polypeptide is not homologous with microtuble protein. The functionally anomalous role of mastigonemes in reversing flagellar thrust is discussed in relation to their distribution relative to flagellar anatomy and to the plane of flagellar undulations. PMID:5123323

Vasoactive intestinal polypeptide and peptide histidine methionine. Presence in human follicular fluid and effects on DNA synthesis and steroid secretion in cultured human granulosa/lutein cells

DEFF Research Database (Denmark)

Gräs, S; Ovesen, Per Glud; Andersen, A N

1994-01-01

Vasoactive intestinal polypeptide (VIP) and peptide histidine methionine (PHM) originate from the same precursor molecule, prepro VIP. In the present study we examined the concentrations of VIP and PHM in human follicular fluid and their effects on cultured human granulosa/lutein cells. Follicula...

Hepatic uptake of conjugated bile acids is mediated by both sodium taurocholate cotransporting polypeptide and organic anion transporting polypeptides and modulated by intestinal sensing of plasma bile acid levels in mice.

Science.gov (United States)

Slijepcevic, Davor; Roscam Abbing, Reinout L P; Katafuchi, Takeshi; Blank, Antje; Donkers, Joanne M; van Hoppe, Stéphanie; de Waart, Dirk R; Tolenaars, Dagmar; van der Meer, Jonathan H M; Wildenberg, Manon; Beuers, Ulrich; Oude Elferink, Ronald P J; Schinkel, Alfred H; van de Graaf, Stan F J

2017-11-01

The Na + -taurocholate cotransporting polypeptide (NTCP/SLC10A1) is believed to be pivotal for hepatic uptake of conjugated bile acids. However, plasma bile acid levels are normal in a subset of NTCP knockout mice and in mice treated with myrcludex B, a specific NTCP inhibitor. Here, we elucidated which transport proteins mediate the hepatic uptake of conjugated bile acids and demonstrated intestinal sensing of elevated bile acid levels in plasma in mice. Mice or healthy volunteers were treated with myrcludex B. Hepatic bile acid uptake kinetics were determined in wild-type (WT), organic anion transporting polypeptide (OATP) knockout mice (lacking Slco1a/1b isoforms), and human OATP1B1-transgenic mice. Effects of fibroblast growth factor 19 (FGF19) on hepatic transporter mRNA levels were assessed in rat hepatoma cells and in mice by peptide injection or adeno-associated virus-mediated overexpression. NTCP inhibition using myrcludex B had only moderate effects on bile acid kinetics in WT mice, but completely inhibited active transport of conjugated bile acid species in OATP knockout mice. Cholesterol 7α-hydroxylase Cyp7a1 expression was strongly down-regulated upon prolonged inhibition of hepatic uptake of conjugated bile acids. Fgf15 (mouse counterpart of FGF19) expression was induced in hypercholanemic OATP and NTCP knockout mice, as well as in myrcludex B-treated cholestatic mice, whereas plasma FGF19 was not induced in humans treated with myrcludex B. Fgf15/FGF19 expression was induced in polarized human enterocyte-models and mouse organoids by basolateral incubation with a high concentration (1 mM) of conjugated bile acids. NTCP and OATPs contribute to hepatic uptake of conjugated bile acids in mice, whereas the predominant uptake in humans is NTCP mediated. Enterocytes sense highly elevated levels of (conjugated) bile acids in the systemic circulation to induce FGF15/19, which modulates hepatic bile acid synthesis and uptake. (Hepatology 2017;66:1631-1643). �

Bovine pancreatic polypeptide as an antagonist of muscarinic cholinergic receptors

International Nuclear Information System (INIS)

Pan, G.Z.; Lu, L.; Qian, J.; Xue, B.G.

1987-01-01

In dispersed acini from rat pancreas, it was found that bovine pancreatic polypeptide (BPP) and its C-fragment hexapeptide amide (PP-6), at concentrations of 0.1 and 30 μM, respectively, could significantly inhibit amylase secretion stimulated by carbachol, and this inhibition by BPP was dose dependent. 45 Ca outflux induced by carbachol was also inhibited by BPP or PP-6, but they had no effect on cholecystokinin octapeptide- (CCK-8) or A23187-stimulated 45 Ca outflux. BPP was also capable of displacing the specific binding of [ 3 H]-quinuclidinyl benzilate to its receptors, and it possessed a higher affinity (K/sub i/35nM) than carbachol (K/sub i/ 1.8 μM) in binding with M-receptors. It is concluded from this study that BPP acts as an antagonist of muscarinic cholinergic receptors in rat pancreatic acini. In addition, BPP inhibited the potentiation of amylase secretion caused by the combination of carbachol plus secretin or vasoactive intestinal peptide. This may be a possible explanation of the inhibitory effect of BPP on secretin-induced pancreatic enzyme secretion shown in vivo, since pancreatic enzyme secretion stimulated by secretin under experimental conditions may be the result of potentiation of enzyme release produced by the peptide in combination with a cholinergic stimulant

Useful halophilic, thermostable and ionic liquids tolerant cellulases

Science.gov (United States)

Zhang, Tao; Datta, Supratim; Simmons, Blake A.; Rubin, Edward M.

2016-06-28

The present invention provides for an isolated or recombinant polypeptide comprising an amino acid sequence having at least 70% identity with the amino acid sequence of a Halorhabdus utahensis cellulase, such as Hu-CBH1, wherein said amino acid sequence has a halophilic thermostable and/or thermophilic cellobiohydrolase (CBH) activity. In some embodiments, the polypeptide has a CBH activity that is resistant to up to about 20% of ionic liquids. The present invention also provides for compositions comprising and methods using the isolated or recombinant polypeptide.

Methods of hydrolyzing a cellulose using halophilic, thermostable and ionic liquids tolerant cellulases

Energy Technology Data Exchange (ETDEWEB)

Zhang, Tao; Datta, Supratim; Simmons, Blake A.; Rubin, Edward M.

2018-01-09

The present invention provides for an isolated or recombinant polypeptide comprising an amino acid sequence having at least 70% identity with the amino acid sequence of a Halorhabdus utahensis cellulase, such as Hu-CBH1, wherein said amino acid sequence has a halophilic thermostable and/or thermophilic cellobiohydrolase (CBH) activity. In some embodiments, the polypeptide has a CBH activity that is resistant to up to about 20% of ionic liquids. The present invention also provides for compositions comprising and methods using the isolated or recombinant polypeptide.

Substrate specificities of three members of the human UDP-N-acetyl-alpha-D-galactosamine:Polypeptide N-acetylgalactosaminyltransferase family, GalNAc-T1, -T2, and -T3

DEFF Research Database (Denmark)

Wandall, H H; Hassan, H; Mirgorodskaya, E

1997-01-01

Mucin-type O-glycosylation is initiated by UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). The role each GalNAc-transferase plays in O-glycosylation is unclear. In this report we characterized the specificity and kinetic properties of three purified...

Cellulolytic enzymes, nucleic acids encoding them and methods for making and using them

Science.gov (United States)

Gray, Kevin A [San Diego, CA; Zhao, Lishan [Emeryville, CA; Cayouette, Michelle H [San Diego, CA

2012-01-24

The invention provides polypeptides having any cellulolytic activity, e.g., a cellulase activity, a endoglucanase, a cellobiohydrolase, a beta-glucosidase, a xylanase, a mannanse, a .beta.-xylosidase, an arabinofuranosidase, and/or an oligomerase activity, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. In one aspect, the invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. In one aspect, the invention provides polypeptides having an oligomerase activity, e.g., enzymes that convert recalcitrant soluble oligomers to fermentable sugars in the saccharification of biomass. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

Design of a Software for Calculating Isoelectric Point of a Polypeptide According to Their Net Charge Using the Graphical Programming Language LabVIEW

Science.gov (United States)

Tovar, Glomen

2018-01-01

A software to calculate the net charge and to predict the isoelectric point (pI) of a polypeptide is developed in this work using the graphical programming language LabVIEW. Through this instrument the net charges of the ionizable residues of the chains of the proteins are calculated at different pH values, tabulated, pI is predicted and an Excel…

Anticancer Activity of Cobra Venom Polypeptide, Cytotoxin-II, against Human Breast Adenocarcinoma Cell Line (MCF-7) via the Induction of Apoptosis

OpenAIRE

Ebrahim, Karim; Shirazi, Farshad H.; Vatanpour, Hosein; zare, Abas; Kobarfard, Farzad; Rabiei, Hadi

2014-01-01

Purpose Breast cancer is a significant health problem worldwide, accounting for a quarter of all cancer diagnoses in women. Current strategies for breast cancer treatment are not fully effective, and there is substantial interest in the identification of novel anticancer agents especially from natural products including toxins. Cytotoxins are polypeptides found in the venom of cobras and have various physiological effects. In the present study, the anticancer potential of cytotoxin-II against...

Programs as Polypeptides.

Science.gov (United States)

Williams, Lance R

2016-01-01

Object-oriented combinator chemistry (OOCC) is an artificial chemistry with composition devices borrowed from object-oriented and functional programming languages. Actors in OOCC are embedded in space and subject to diffusion; since they are neither created nor destroyed, their mass is conserved. Actors use programs constructed from combinators to asynchronously update their own states and the states of other actors in their neighborhoods. The fact that programs and combinators are themselves reified as actors makes it possible to build programs that build programs from combinators of a few primitive types using asynchronous spatial processes that resemble chemistry as much as computation. To demonstrate this, OOCC is used to define a parallel, asynchronous, spatially distributed self-replicating system modeled in part on the living cell. Since interactions among its parts result in the construction of more of these same parts, the system is strongly constructive. The system's high normalized complexity is contrasted with that of a simple composome.

l-Cystine-Crosslinked Polypeptide Nanogel as a Reduction-Responsive Excipient for Prostate Cancer Chemotherapy

Directory of Open Access Journals (Sweden)

Liang He

2016-01-01

Full Text Available Smart polymer nanogel-assisted drug delivery systems have attracted more and more attention in cancer chemotherapy because of their well-defined morphologies and pleiotropic functions in recent years. In this work, an l-cystine-crosslinked reduction-responsive polypeptide nanogel of methoxy poly(ethylene glycol-poly(l-phenylalanine-co-l-cystine (mPEG-P(LP-co-LC was employed as a smart excipient for RM-1 prostate cancer (PCa chemotherapy. Doxorubicin (DOX, as a regular chemotherapy drug, was embedded in the nanogel. The loading nanogel marked as NG/DOX was shown to exhibit glutathione (GSH-induced swelling and GSH-accelerated DOX release. Subsequently, NG/DOX showed efficient cellular uptake and proliferation inhibition. Furthermore, NG/DOX presented enhanced antitumor efficacy and security in an RM-1 PCa-grafted mouse model in vivo, indicating its great potential for clinical treatment.

Islet amyloid polypeptide and high hydrostatic pressure: towards an understanding of the fibrillization process

Science.gov (United States)

Lopes, D. H. J.; Smirnovas, V.; Winter, R.

2008-07-01

Type II Diabetes Mellitus is a disease which is characterized by peripheral insulin resistance coupled with a progressive loss of insulin secretion that is associated with a decrease in pancreatic islet β-cell mass and the deposition of amyloid in the extracellular matrix of β-cells, which lead to islet cell death. The principal component of the islet amyloid is a pancreatic hormone called islet amyloid polypeptide (IAPP). High-pressure coupled with FT-IR, CD, ThT fluorescence spectroscopic and AFM studies were carried out to reveal information on the aggregation pathway as well as the aggregate structure of IAPP. Our data indicate that IAPP pre-formed fibrils exhibit a strong polymorphism with heterogeneous structures very sensitive to high hydrostatic pressure, indicating a high percentage of ionic and hydrophobic interactions being responsible for the stability the IAPP fibrils.

Distribution and protective function of pituitary adenylate cyclase-activating polypeptide (PACAP in the retina

Directory of Open Access Journals (Sweden)

Tomoya eNakamachi

2012-11-01

Full Text Available Pituitary adenylate cyclase-activating polypeptide (PACAP, which is found in 27- or 38-amino acid forms, belongs to the VIP/glucagon/secretin family. PACAP and its three receptor subtypes are expressed in neural tissues, with PACAP known to exert a protective effect against several types of neural damage. The retina is considered to be part of the central nervous system, and retinopathy is a common cause of profound and intractable loss of vision. This review will examine the expression and morphological distribution of PACAP and its receptors in the retina, and will summarize the current state of knowledge regarding the protective effect of PACAP against different kinds of retinal damage, such as that identified in association with diabetes, ultraviolet light, hypoxia, optic nerve transection, and toxins. This article will also address PACAP-mediated protective pathways involving retinal glial cells.

Vasoactive intestinal polypeptide induces glycogenolysis in mouse cortical slices: a possible regulatory mechanism for the local control of energy metabolism.

OpenAIRE

Magistretti, P J; Morrison, J H; Shoemaker, W J; Sapin, V; Bloom, F E

1981-01-01

Mouse cerebral cortex slices will synthesize [3H]glycogen in vitro. Vasoactive intestinal polypeptide (VIP) stimulates the enzymatic breakdown of this [3H]glycogen. The concentration giving 50% of maximum effectiveness (EC50) is 26 nM. Under the same experimental conditions norepinephrine also induces a concentration-dependent [3H]glycogen hydrolysis with an EC50 of 500 nM. The effect of VIP is not mediated by the release of norepinephrine because it is not blocked by the noradrenergic antago...

Pancreatic polypeptide: Identification of target tissues and investigation of possible physiologic significance

International Nuclear Information System (INIS)

Shetzline, M.A.

1988-01-01

Pancreatic Polypeptide (PP) is a 36 amino acid peptide with hormonal properties but whose physiologic function remains unknown. In order to determine the function of this peptide we investigated potential target tissues using an in vivo radioreceptor assay. In vitro high concentrations of unlabeled hormone competitively inhibit low concentrations of labeled hormone from binding to receptors. Our in vivo studies indicate that, in the presence of concentrated unlabeled peptide, labeled PP distributes between the plasma and interstitial fluid. When saline rather than excess unlabeled PP is injected, the labeled peptide appears to distribute in a volume exceeding the combined plasma and interstitial fluid volume of tissue. The distribution volume which exceeds the anatomic extracellular volume and which is not present with excess unlabeled peptide, is the receptor compartment. With this assay we demonstrated in the rat specific and displaceable PP binding to the ductus choledochus, duodenum, ileum, and adrenal gland. In vitro rat adrenal cell membranes showed specific I-125 labeled PP binding. Specific binding also occurred in bovine cortical and medullary membranes

Design of monodisperse and well-defined polypeptide-based polyvalent inhibitors of anthrax toxin.

Science.gov (United States)

Patke, Sanket; Boggara, Mohan; Maheshwari, Ronak; Srivastava, Sunit K; Arha, Manish; Douaisi, Marc; Martin, Jacob T; Harvey, Ian B; Brier, Matthew; Rosen, Tania; Mogridge, Jeremy; Kane, Ravi S

2014-07-28

The design of polyvalent molecules, presenting multiple copies of a specific ligand, represents a promising strategy to inhibit pathogens and toxins. The ability to control independently the valency and the spacing between ligands would be valuable for elucidating structure-activity relationships and for designing potent polyvalent molecules. To that end, we designed monodisperse polypeptide-based polyvalent inhibitors of anthrax toxin in which multiple copies of an inhibitory toxin-binding peptide were separated by flexible peptide linkers. By tuning the valency and linker length, we designed polyvalent inhibitors that were over four orders of magnitude more potent than the corresponding monovalent ligands. This strategy for the rational design of monodisperse polyvalent molecules may not only be broadly applicable for the inhibition of toxins and pathogens, but also for controlling the nanoscale organization of cellular receptors to regulate signaling and the fate of stem cells. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A paclitaxel-loaded recombinant polypeptide nanoparticle outperforms Abraxane in multiple murine cancer models

Science.gov (United States)

Bhattacharyya, Jayanta; Bellucci, Joseph J.; Weitzhandler, Isaac; McDaniel, Jonathan R.; Spasojevic, Ivan; Li, Xinghai; Lin, Chao-Chieh; Chi, Jen-Tsan Ashley; Chilkoti, Ashutosh

2015-08-01

Packaging clinically relevant hydrophobic drugs into a self-assembled nanoparticle can improve their aqueous solubility, plasma half-life, tumour-specific uptake and therapeutic potential. To this end, here we conjugated paclitaxel (PTX) to recombinant chimeric polypeptides (CPs) that spontaneously self-assemble into ~60 nm near-monodisperse nanoparticles that increased the systemic exposure of PTX by sevenfold compared with free drug and twofold compared with the Food and Drug Administration-approved taxane nanoformulation (Abraxane). The tumour uptake of the CP-PTX nanoparticle was fivefold greater than free drug and twofold greater than Abraxane. In a murine cancer model of human triple-negative breast cancer and prostate cancer, CP-PTX induced near-complete tumour regression after a single dose in both tumour models, whereas at the same dose, no mice treated with Abraxane survived for >80 days (breast) and 60 days (prostate), respectively. These results show that a molecularly engineered nanoparticle with precisely engineered design features outperforms Abraxane, the current gold standard for PTX delivery.

Celluloytic enzymes, nucleic acids encoding them and methods for making and using them

Energy Technology Data Exchange (ETDEWEB)

Gray, Kevin A; Zhao, Lishan; Cayouette, Michelle H

2015-11-04

The invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

Celluloytic enzymes, nucleic acids encoding them and methods for making and using them

Science.gov (United States)

Gray, Kevin A.; Zhao, Lishan; Cayouette, Michelle H.

2015-09-08

The invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

Peptides having reduced toxicity that stimulate cholesterol efflux

Energy Technology Data Exchange (ETDEWEB)

Bielicki, John K.; Johansson, Jan; Danho, Waleed

2016-08-16

The present invention provides a family of non-naturally occurring polypeptides having cholesterol efflux activity that parallels that of full-length apolipoproteins (e.g., Apo AI and Apo E), and having high selectivity for ABCA1 that parallels that of full-length apolipoproteins. Further, the peptides of the invention have little or no toxicity when administered at therapeutic and higher doses. The invention also provides compositions comprising such polypeptides, methods of identifying, screening and synthesizing such polypeptides, and methods of treating, preventing or diagnosing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation.

Dock 'n roll: folding of a silk-inspired polypeptide into an amyloid-like beta solenoid.

Science.gov (United States)

Zhao, Binwu; Cohen Stuart, Martien A; Hall, Carol K

2016-04-20

Polypeptides containing the motif ((GA)mGX)n occur in silk and have a strong tendency to self-assemble. For example, polypeptides containing (GAGAGAGX)n, where X = G or H have been observed to form filaments; similar sequences but with X = Q have been used in the design of coat proteins (capsids) for artificial viruses. The structure of the (GAGAGAGX)m filaments has been proposed to be a stack of peptides in a β roll structure with the hydrophobic side chains pointing outwards (hydrophobic shell). Another possible configuration, a β roll or β solenoid structure which has its hydrophobic side chains buried inside (hydrophobic core) was, however, overlooked. We perform ground state analysis as well as atomic-level molecular dynamics simulations, both on single molecules and on two-molecule stacks of the silk-inspired sequence (GAGAGAGQ)10, to decide whether the hydrophobic core or the hydrophobic shell configuration is the most stable one. We find that a stack of two hydrophobic core molecules is energetically more favorable than a stack of two hydrophobic shell molecules. A shell molecule initially placed in a perfect β roll structure tends to rotate its strands, breaking in-plane hydrogen bonds and forming out-of-plane hydrogen bonds, while a core molecule stays in the β roll structure. The hydrophobic shell structure has type II' β turns whereas the core configuration has type II β turns; only the latter secondary structure agrees well with solid-state NMR experiments on a similar sequence (GA)15. We also observe that the core stack has a higher number of intra-molecular hydrogen bonds and a higher number of hydrogen bonds between stack and water than the shell stack. Hence, we conclude that the hydrophobic core configuration is the most likely structure. In the stacked state, each peptide has more intra-molecular hydrogen bonds than a single folded molecule, which suggests that stacking provides the extra stability needed for molecules to reach the folded

A plant small polypeptide is a novel component of DNA-binding protein phosphatase 1-mediated resistance to plum pox virus in Arabidopsis.

Science.gov (United States)

Castelló, María José; Carrasco, Jose Luis; Navarrete-Gómez, Marisa; Daniel, Jacques; Granot, David; Vera, Pablo

2011-12-01

DNA-binding protein phosphatases (DBPs) have been identified as a novel class of plant-specific regulatory factors playing a role in plant-virus interactions. NtDBP1 from tobacco (Nicotiana tabacum) was shown to participate in transcriptional regulation of gene expression in response to virus infection in compatible interactions, and AtDBP1, its closest relative in the model plant Arabidopsis (Arabidopsis thaliana), has recently been found to mediate susceptibility to potyvirus, one of the most speciose taxa of plant viruses. Here, we report on the identification of a novel family of highly conserved small polypeptides that interact with DBP1 proteins both in tobacco and Arabidopsis, which we have designated DBP-interacting protein 2 (DIP2). The interaction of AtDIP2 with AtDBP1 was demonstrated in vivo by bimolecular fluorescence complementation, and AtDIP2 was shown to functionally interfere with AtDBP1 in yeast. Furthermore, reducing AtDIP2 gene expression leads to increased susceptibility to the potyvirus Plum pox virus and to a lesser extent also to Turnip mosaic virus, whereas overexpression results in enhanced resistance. Therefore, we describe a novel family of conserved small polypeptides in plants and identify AtDIP2 as a novel host factor contributing to resistance to potyvirus in Arabidopsis.

Bio-inspired synthesis of hybrid silica nanoparticles templated from elastin-like polypeptide micelles

Science.gov (United States)

Han, Wei; MacEwan, Sarah R.; Chilkoti, Ashutosh; López, Gabriel P.

2015-07-01

The programmed self-assembly of block copolymers into higher order nanoscale structures offers many attractive attributes for the development of new nanomaterials for numerous applications including drug delivery and biosensing. The incorporation of biomimetic silaffin peptides in these block copolymers enables the formation of hybrid organic-inorganic materials, which can potentially enhance the utility and stability of self-assembled nanostructures. We demonstrate the design, synthesis and characterization of amphiphilic elastin-like polypeptide (ELP) diblock copolymers that undergo temperature-triggered self-assembly into well-defined spherical micelles. Genetically encoded incorporation of the silaffin R5 peptide at the hydrophilic terminus of the diblock ELP leads to presentation of the silaffin R5 peptide on the coronae of the micelles, which results in localized condensation of silica and the formation of near-monodisperse, discrete, sub-100 nm diameter hybrid ELP-silica particles. This synthesis method, can be carried out under mild reaction conditions suitable for bioactive materials, and will serve as the basis for the development and application of functional nanomaterials. Beyond silicification, the general strategies described herein may also be adapted for the synthesis of other biohybrid nanomaterials as well.The programmed self-assembly of block copolymers into higher order nanoscale structures offers many attractive attributes for the development of new nanomaterials for numerous applications including drug delivery and biosensing. The incorporation of biomimetic silaffin peptides in these block copolymers enables the formation of hybrid organic-inorganic materials, which can potentially enhance the utility and stability of self-assembled nanostructures. We demonstrate the design, synthesis and characterization of amphiphilic elastin-like polypeptide (ELP) diblock copolymers that undergo temperature-triggered self-assembly into well

An engineered polypeptide around nano-sized manganese-calcium oxide: copying plants for water oxidation.

Science.gov (United States)

Najafpour, Mohammad Mahdi; Ghobadi, Mohadeseh Zarei; Sarvi, Bahram; Haghighi, Behzad

2015-09-14

Synthesis of new efficient catalysts inspired by Nature is a key goal in the production of clean fuel. Different compounds based on manganese oxide have been investigated in order to find their water-oxidation activity. Herein, we introduce a novel engineered polypeptide containing tyrosine around nano-sized manganese-calcium oxide, which was shown to be a highly active catalyst toward water oxidation at low overpotential (240 mV), with high turnover frequency of 1.5 × 10(-2) s(-1) at pH = 6.3 in the Mn(III)/Mn(IV) oxidation range. The compound is a novel structural and efficient functional model for the water-oxidizing complex in Photosystem II. A new proposed clever strategy used by Nature in water oxidation is also discussed. The new model of the water-oxidizing complex opens a new perspective for synthesis of efficient water-oxidation catalysts.

Differential expression of isoproterenol-induced salivary polypeptides in two mouse strains that are congenic for the H-2 histocompatibility gene complex.

Science.gov (United States)

López Solís, Remigio O; Weis, Ulrike Kemmerling; Ceballos, Alicia Ramos; Salas, Gustavo Hoecker

2003-12-01

Two inbred mouse strains, A/Snell and A.Swiss, which were produced as congenic with regard to the H-2 histocompatibility gene complex, are homozygous for two different groups of isoproterenol-induced salivary polypeptides (IISP). These polypeptides, which have been considered as markers of the hypertrophic growth of the parotid acinar cells, are members of the complex family of salivary proline-rich proteins (PRP) on the basis of both their massive accumulation in the parotid acinar cells in response to chronic isoproterenol, secretory character, high solubility in trichloroacetic acid and metachromatic staining by Coomassie blue. IISP expressed in both mouse strains were identified by unidimensional SDS-polyacrylamide electrophoresis and Coomassie blue staining both in parotid gland homogenates and in whole salivas obtained from mice repeatedly stimulated at 24-h intervals with isoproterenol. Parotid glands from 40 mice (20 A/Snell and 20 A.Swiss) and salivas from 270 mice (200 A/Snell and 70 A.Swiss) were analyzed. One of the congenic strains (A/Snell) expressed five IISP (Mr 65, 61, 51.5, 38, and 37 kDa) and the other strain (A.Swiss) expressed six IISP (Mr 59, 57, 54.5, 46, 36, and 34 kDa). No inter-individual intra-strain variations were observed, thus defining strain-associated patterns of IISP (PRP). Copyright 2003 Wiley-Liss, Inc.

Association of Sendai virion envelope and a mouse surface membrane polypeptide on newly infected cells: lack of association with H-2K/D or alteration of viral immunogenicity

International Nuclear Information System (INIS)

Zarling, D.A.; Miskimen, J.A.; Fan, D.P; Fujimoto, E.K.; Smith, P.K.

1982-01-01

The reagent N-succinimidyl 4-azidophenyl-1,3'-dithiopropionate (SADP) was synthesized and then coupled to purified Sendai virions by the amino-reactive end of the SADP molecule. This SADP-coupled virus was fused into the membranes of surface radioiodinated P815 cells, and target structures were allowed to form. Next, the photosensitive group on SADP was activated with ultraviolet light to covalently couple the viral proteins to any neighboring cell surface proteins. The cellular neighbors were isolated from detergent extracts of membrane proteins after immunoprecipitation with antibody specific for Sendai virion proteins. The covalent cross-links between the nonradioactive Sendai proteins and the radioiodinated cellular polypeptide neighbors were broken, and the host cell polypeptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by autoradiography. One of these neighboring cellular proteins had an apparent m.w. of 17,000, and none was found with the characteristic size and tryptic map of either the H-2K or D gene products. Thus, the H-2K or D proteins are unlikely to be SADP- detectable neighbors of Sendai viral antigens recognized by CTL. In further experiments, the complexes of Sendai virion proteins crosslinked to cellular polypeptide neighbors were isolated from the membrane of newly infected cells and were shown to be able to stimulate CTL in vitro with approximately the same efficiency as uncross-linked Sendai virion proteins. Thus, Sendai viral proteins in the membrane of newly infected cells do not appear to be in highly immunogenic complexes with either H-2K/D or any other cellular proteins

Experiments and strategies for the assignment of fully13 C/15N-labelled polypeptides by solid state NMR

International Nuclear Information System (INIS)

Straus, Suzana K.; Bremi, Tobias; Ernst, Richard R.

1998-01-01

High-resolution heteronuclear NMR correlation experiments and strategies are proposed for the assignment of fully 13 C/ 15 N-labelled polypeptides in the solid state. By the combination of intra-residue and inter-residue 13 C- 15 N correlation experiments with 13 C- 13 C spin-diffusion studies, it becomes feasible to partially assign backbone and side-chain resonances in solid proteins. The performance of sequences using 15 N instead of 13 C detection is evaluated regarding sensitivity and resolution for a labelled dipeptide (L-Val-L-Phe). The techniques are used for a partial assignment of the 15 N and 13 C resonances in human ubiquitin

Star Polymers Reduce Islet Amyloid Polypeptide Toxicity via Accelerated Amyloid Aggregation.

Science.gov (United States)

Pilkington, Emily H; Lai, May; Ge, Xinwei; Stanley, William J; Wang, Bo; Wang, Miaoyi; Kakinen, Aleksandr; Sani, Marc-Antonie; Whittaker, Michael R; Gurzov, Esteban N; Ding, Feng; Quinn, John F; Davis, Thomas P; Ke, Pu Chun

2017-12-11

Protein aggregation into amyloid fibrils is a ubiquitous phenomenon across the spectrum of neurodegenerative disorders and type 2 diabetes. A common strategy against amyloidogenesis is to minimize the populations of toxic oligomers and protofibrils by inhibiting protein aggregation with small molecules or nanoparticles. However, melanin synthesis in nature is realized by accelerated protein fibrillation to circumvent accumulation of toxic intermediates. Accordingly, we designed and demonstrated the use of star-shaped poly(2-hydroxyethyl acrylate) (PHEA) nanostructures for promoting aggregation while ameliorating the toxicity of human islet amyloid polypeptide (IAPP), the peptide involved in glycemic control and the pathology of type 2 diabetes. The binding of PHEA elevated the β-sheet content in IAPP aggregates while rendering a new morphology of "stelliform" amyloids originating from the polymers. Atomistic molecular dynamics simulations revealed that the PHEA arms served as rodlike scaffolds for IAPP binding and subsequently accelerated IAPP aggregation by increased local peptide concentration. The tertiary structure of the star nanoparticles was found to be essential for driving the specific interactions required to impel the accelerated IAPP aggregation. This study sheds new light on the structure-toxicity relationship of IAPP and points to the potential of exploiting star polymers as a new class of therapeutic agents against amyloidogenesis.

Glucagon-like peptide-2, but not glucose-dependent insulinotropic polypeptide, stimulates glucagon release in patients with type 1 diabetes

DEFF Research Database (Denmark)

Christensen, Mikkel; Knop, Filip K; Vilsbøll, Tina

2010-01-01

This study investigated the glucagon-releasing properties of the hormones glucagon-like peptide-2 (GLP-2) and glucose-dependent insulinotropic polypeptide (GIP) in 8 patients with type 1 diabetes mellitus (T1DM) without paracrine intraislet influence of insulin (C-peptide negative following a 5 g...... intravenous arginine stimulation; on study days only treated with basal insulin substitution). On 3 study days, 180-minute two-step glucose clamps were performed. Plasma glucose (PG) was clamped at fasting values, with a mean of 7.4+/-0.5 mM in the first 90 min (period 1) and raised 1.5 times the fasting...

The proteins of the grape (Vitis vinifera L.) seed endosperm: fractionation and identification of the major components.

Science.gov (United States)

Gazzola, Diana; Vincenzi, Simone; Gastaldon, Luca; Tolin, Serena; Pasini, Gabriella; Curioni, Andrea

2014-07-15

In the present study, grape (Vitis vinifera L.) seed endosperm proteins were characterized after sequential fractionation, according to a modified Osborne procedure. The salt-soluble fraction (albumins and globulins) comprised the majority (58.4%) of the total extracted protein. The protein fractions analysed by SDS-PAGE showed similar bands, indicating different solubility of the same protein components. SDS-PAGE in non-reducing and reducing conditions revealed the polypeptide composition of the protein bands. The main polypeptides, which were similar in all the grape varieties analysed, were identified by LC-MS/MS as homologous to the 11S globulin-like seed storage proteins of other plant species, while a monomeric 43 kDa protein presented high homology with the 7S globulins of legume seeds. The results provide new insights about the identity, structure and polypeptide composition of the grape seed storage proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

Causative factors for formation of toxic islet amyloid polypeptide oligomer in type 2 diabetes mellitus

Directory of Open Access Journals (Sweden)

Jeong HR

2015-11-01

Full Text Available Hye Rin Jeong, Seong Soo A AnDepartment of Bionano Technology, Gachon Medical Research Institute, Gachon University, Gyeonggi-do, Republic of KoreaAbstract: Human islet amyloid polypeptide (h-IAPP is a peptide hormone that is synthesized and cosecreted with insulin from insulin-secreting pancreatic β-cells. Recently, h-IAPP was proposed to be the main component responsible for the cytotoxic pancreatic amyloid deposits in patients with type 2 diabetes mellitus (T2DM. Since the causative factors of IAPP (or amylin oligomer aggregation are not fully understood, this review will discuss the various forms of h-IAPP aggregation. Not all forms of IAPP aggregates trigger the destruction of β-cell function and loss of β-cell mass; however, toxic oligomers do trigger these events. Once these toxic oligomers form under abnormal metabolic conditions in T2DM, they can lead to cell disruption by inducing cell membrane destabilization. In this review, the various factors that have been shown to induce toxic IAPP oligomer formation will be presented, as well as the potential mechanism of oligomer and fibril formation from pro-IAPPs. Initially, pro-IAPPs undergo enzymatic reactions to produce the IAPP monomers, which can then develop into oligomers and fibrils. By this mechanism, toxic oligomers could be generated by diverse pathway components. Thus, the interconnections between factors that influence amyloid aggregation (eg, absence of PC2 enzyme, deamidation, reduction of disulfide bonds, environmental factors in the cell, genetic mutations, copper metal ions, and heparin will be presented. Hence, this review will aid in understanding the fundamental causative factors contributing to IAPP oligomer formation and support studies for investigating novel T2DM therapeutic approaches, such as the development of inhibitory agents for preventing oligomerization at the early stages of diabetic pathology.Keywords: amyloid aggregation, causative factor, IAPP, islet

Dock ’n Roll: Folding of a Silk-Inspired Polypeptide into an Amyloid-like Beta Solenoid

Science.gov (United States)

Zhao, Binwu; Cohen Stuart, Martien A.; Hall, Carol K.

2016-01-01

Polypeptides containing the motif ((GA)mGX)n occur in silk (we refer to them as ‘silk-like’) and have a strong tendency to self-assemble. For example, polypeptides containing (GAGAGAGX)n, where X = G or H have been observed to form filaments; similar sequences but with X = Q have been used in the design of coat proteins (capsids) for artificial viruses. The structure of the (GAGAGAGX)m filaments has been proposed to be a stack of peptides in a β roll structure with the hydrophobic side chains pointing outwards (hydrophobic shell). Another possible configuration, a β roll or β solenoid structure which has its hydrophobic side chains buried inside (hydrophobic core) was, however, overlooked. We perform ground state analysis as well as atomic-level molecular dynamics simulations, both on single molecules and on two-molecule stacks of the silk-inspired sequence (GAGAGAGQ)10, to decide whether the hydrophobic core or the hydrophobic shell configuration is the most stable one. We find that a stack of two hydrophobic core molecules is energetically more favorable than a stack of two shell molecules. A shell molecule initially placed in a perfect β roll structure tends to rotate its strands, breaking in-plane hydrogen bonds and forming out-of-plane hydrogen bonds, while a core molecule stays in the β roll structure. The hydrophobic shell structure has type II’ β turns whereas the core configuration has type II β turns; only the latter secondary structure agrees well with solid-state NMR experiments on a similar sequence (GA)15. We also observe that the core stack has a higher number of intra-molecular hydrogen bonds and a higher number of hydrogen bonds between stack and water than the shell stack. Hence, we conclude that the hydrophobic core configuration is the most likely structure. In the stacked state, each peptide has more intra-molecular hydrogen bonds than a single folded molecule, which suggests that stacking provides the extra stability needed for

Glucose-Dependent Insulinotropic Polypeptide Mitigates 6-OHDA-Induced Behavioral Impairments in Parkinsonian Rats

Science.gov (United States)

Yu, Yu-Wen; Hsueh, Shih-Chang; Lai, Jing-Huei; Chen, Yen-Hua; Kang, Shuo-Jhen; Hsieh, Tsung-Hsun; Hoffer, Barry J.; Li, Yazhou; Greig, Nigel H.; Chiang, Yung-Hsiao

2018-01-01

In the present study, the effectiveness of glucose-dependent insulinotropic polypeptide (GIP) was evaluated by behavioral tests in 6-hydroxydopamine (6-OHDA) hemi-parkinsonian (PD) rats. Pharmacokinetic measurements of GIP were carried out at the same dose studied behaviorally, as well as at a lower dose used previously. GIP was delivered by subcutaneous administration (s.c.) using implanted ALZET micro-osmotic pumps. After two days of pre-treatment, male Sprague Dawley rats received a single unilateral injection of 6-OHDA into the medial forebrain bundle (MFB). The neuroprotective effects of GIP were evaluated by apomorphine-induced contralateral rotations, as well as by locomotor and anxiety-like behaviors in open-field tests. Concentrations of human active and total GIP were measured in plasma during a five-day treatment period by ELISA and were found to be within a clinically translatable range. GIP pretreatment reduced behavioral abnormalities induced by the unilateral nigrostriatal dopamine (DA) lesion produced by 6-OHDA, and thus may be a novel target for PD therapeutic development. PMID:29641447

Glucose-Dependent Insulinotropic Polypeptide Mitigates 6-OHDA-Induced Behavioral Impairments in Parkinsonian Rats

Directory of Open Access Journals (Sweden)

Yu-Wen Yu

2018-04-01

Full Text Available In the present study, the effectiveness of glucose-dependent insulinotropic polypeptide (GIP was evaluated by behavioral tests in 6-hydroxydopamine (6-OHDA hemi-parkinsonian (PD rats. Pharmacokinetic measurements of GIP were carried out at the same dose studied behaviorally, as well as at a lower dose used previously. GIP was delivered by subcutaneous administration (s.c. using implanted ALZET micro-osmotic pumps. After two days of pre-treatment, male Sprague Dawley rats received a single unilateral injection of 6-OHDA into the medial forebrain bundle (MFB. The neuroprotective effects of GIP were evaluated by apomorphine-induced contralateral rotations, as well as by locomotor and anxiety-like behaviors in open-field tests. Concentrations of human active and total GIP were measured in plasma during a five-day treatment period by ELISA and were found to be within a clinically translatable range. GIP pretreatment reduced behavioral abnormalities induced by the unilateral nigrostriatal dopamine (DA lesion produced by 6-OHDA, and thus may be a novel target for PD therapeutic development.

Genetically encoded lipid-polypeptide hybrid biomaterials that exhibit temperature-triggered hierarchical self-assembly

Science.gov (United States)

Mozhdehi, Davoud; Luginbuhl, Kelli M.; Simon, Joseph R.; Dzuricky, Michael; Berger, Rüdiger; Varol, H. Samet; Huang, Fred C.; Buehne, Kristen L.; Mayne, Nicholas R.; Weitzhandler, Isaac; Bonn, Mischa; Parekh, Sapun H.; Chilkoti, Ashutosh

2018-05-01

Post-translational modification of proteins is a strategy widely used in biological systems. It expands the diversity of the proteome and allows for tailoring of both the function and localization of proteins within cells as well as the material properties of structural proteins and matrices. Despite their ubiquity in biology, with a few exceptions, the potential of post-translational modifications in biomaterials synthesis has remained largely untapped. As a proof of concept to demonstrate the feasibility of creating a genetically encoded biohybrid material through post-translational modification, we report here the generation of a family of three stimulus-responsive hybrid materials—fatty-acid-modified elastin-like polypeptides—using a one-pot recombinant expression and post-translational lipidation methodology. These hybrid biomaterials contain an amphiphilic domain, composed of a β-sheet-forming peptide that is post-translationally functionalized with a C14 alkyl chain, fused to a thermally responsive elastin-like polypeptide. They exhibit temperature-triggered hierarchical self-assembly across multiple length scales with varied structure and material properties that can be controlled at the sequence level.

Role of pancreatic polypeptide in the regulation of pancreatic exocrine secretion in dogs

International Nuclear Information System (INIS)

Shiratori, Keiko; Lee, K.Y.; Chang, Tamin; Jo, Y.H.; Coy, D.H.; Chey, W.Y.

1988-01-01

The effect of intravenous infusion of synthetic human pancreatic polypeptide (HPP) or a rabbit anti-PP serum on pancreatic exocrine secretion was studied in 10 dogs with gastric and Thomas duodenal cannulas. The infusion of HPP, achieved a plasma PP concentration that mimicked the peak plasma concentration of PP in both interdigestive and postprandial states. This dose of HPP significantly inhibited pancreatic secretion in the interdigestive state. By contrast, immunoneutralization of circulating PP by a rabbit anti-PP serum resulted in significant increases in both interdigestive and postprandial pancreatic secretion, including water, bicarbonate, and protein. The increase in the pancreatic secretion paralleled a decrease in circulating PP level, which lasted for as long as 5 days. Furthermore, the anti-PP serum blocked the inhibitory action of exogenous HPP on pancreatic exocrine secretion. The present study indicates that endogenous PP plays a significant role in the regulation of the pancreatic exocrine secretion in both interdigestive and digestive states. Thus the authors conclude that PP is another hormone regulating pancreatic exocrine secretion in dogs

Changes in the infrared microspectroscopic characteristics of DNA caused by cationic elements, different base richness and single-stranded form.

Directory of Open Access Journals (Sweden)

Maria Luiza S Mello

Full Text Available BACKGROUND: The infrared (IR analysis of dried samples of DNA and DNA-polypeptide complexes is still scarce. Here we have studied the FT-IR profiles of these components to further the understanding of the FT-IR signatures of chromatin and cell nuclei. METHODOLOGY/PRINCIPAL FINDINGS: Calf thymus and salmon testis DNA, and complexes of histone H1, protamine, poly-L-lysine and poly-L-arginine (histone-mimic macromolecules with DNA were analyzed in an IR microspectroscope equipped with an attenuated total reflection diamond objective and Grams software. Conditions including polypeptides bound to the DNA, DNA base composition, and single-stranded form were found to differently affect the vibrational characteristics of the chemical groups (especially, PO(2(- in the nucleic acid. The antisymmetric stretching (ν(as of the DNA PO(2(- was greater than the symmetric stretching (ν(s of these groups and increased in the polypeptide-DNA complexes. A shift of the ν(as of the DNA PO(2(- to a lower frequency and an increased intensity of this vibration were induced especially by lysine-rich histones. Lysine richness additionally contributed to an increase in the vibrational stretching of the amide I group. Even in simple molecules such as inorganic phosphates, the vibrational characteristics of the phosphate anions were differently affected by different cations. As a result of the optimization of the DNA conformation by binding to arginine-rich polypeptides, enhancements of the vibrational characteristics in the FT-IR fingerprint could be detected. Although different profiles were obtained for the DNA with different base compositions, this situation was no longer verified in the polypeptide-DNA complexes and most likely in isolated chromatin or cell nuclei. However, the ν(as PO(2(-/ν(s PO(2(- ratio could discriminate DNA with different base compositions and DNA in a single-stranded form. CONCLUSIONS/SIGNIFICANCE: FT-IR spectral profiles are a valuable tool

Degradation of surface-labeled hepatoma membrane polypeptides: effect of inhibitors

International Nuclear Information System (INIS)

Hare, J.F.; Huston, M.

1984-01-01

When their membrane proteins were labeled with 125I by lactoperoxidase, dividing hepatoma cells lost radioactivity to the medium in a biphasic manner (T1/2 . 16-26 h, greater than 40 h). Lysosomotropic weak bases, chloroquine, and NH4Cl inhibited the rapid phase by 59%. More than 50% of the radioactivity which accumulates in the media from dividing cells during the first 4 h after labeling was trichloroacetic acid-soluble, and was identified as iodotyrosine. Iodotyrosine release from labeled membrane proteins was 60-71% inhibited by lysosomotropic agents chloroquine and NH4Cl as well as the sodium-proton ionophore, monensin. The inhibitory effect of NH4Cl and monensin was reversible. Inhibitors of microtubule and microfilament function and transglutamination had no effect on release of iodotyrosine to the medium, but trypsin-like protease inhibitors, p-aminobenzamidine, tosyl-L-lysine/chloromethylketone, and phenylmethylsulfonyl fluoride, as well as the cathepsin B inhibitor, leupeptin, inhibited by 21-24%. Iodotyrosine release showed a biphasic Arrhenius plot with an activation energy of 17 kcal/mol above but 27 kcal/mol below 20 degrees C. These results indicate that cell membrane polypeptides require a temperature-limiting event as well as passage through an ion-sensitive compartment prior to their complete degradation to constituent amino acids. In contrast to other lysosomal-mediated events, however, iodinated membrane proteins of dividing cells are degraded in a manner insensitive to agents which disrupt the cytoskeleton

Hyperglycemia acutely lowers the postprandial excursions of glucagon-like Peptide-1 and gastric inhibitory polypeptide in humans

DEFF Research Database (Denmark)

Vollmer, Kirsten; Gardiwal, Husai; Menge, Bjoern A

2009-01-01

INTRODUCTION: Impaired secretion of glucagon-like peptide 1 (GLP-1) has been suggested to contribute to the deficient incretin effect in patients with type 2 diabetes. It is unclear whether this is a primary defect or a consequence of the hyperglycemia in type 2 diabetes. We examined whether acute...... hyperglycemia reduces the postprandial excursions of gastric inhibitory polypeptide (GIP) and GLP-1, and if so, whether this can be attributed to changes in gastric emptying. PATIENTS AND METHODS: Fifteen nondiabetic individuals participated in a euglycemic clamp and a hyperglycemic clamp experiment, carried...... the hyperglycemic clamp experiments and 83 +/- 3 mg/dl during the euglycemia (P hyperglycemia, but meal ingestion led to a decline in glucose requirements in both experiments (P

Identification of the polypeptides encoded in the unassigned reading frames 2, 4, 4L, and 5 of human mitochondrial DNA

International Nuclear Information System (INIS)

Mariottini, P.; Chomyn, A.; Riley, M.; Cottrell, B.; Doolittle, R.F.; Attardi, G.

1986-01-01

In previous work, antibodies prepared against chemically synthesized peptides predicted from the DNA sequence were used to identify the polypeptides encoded in three of the eight unassigned reading frames (URFs) of human mitochondrial DNA (mtDNA). In the present study, this approach has been extended to other human mtDNA URFs. In particular, antibodies directed against the NH 2 -terminal octapeptide of the putative URF2 product specifically precipitated component 11 of the HeLa cell mitochondrial translation products, the reaction being inhibited by the specific peptide. Similarly, antibodies directed against the COOH-terminal nonapeptide of the putative URF4 product reacted specifically with components 4 and 5, and antibodies against a COOH-terminal heptapeptide of the presumptive URF4L product reacted specifically with component 26. Antibodies against the NH 2 -terminal heptapeptide of the putative product of URF5 reacted with component 1, but only to a marginal extent; however, the results of a trypsin fingerprinting analysis of component 1 point strongly to this component as being the authentic product of URF5. The polypeptide assignments to the mtDNA URFs analyzed here are supported by the relative electrophoretic mobilities of proteins 11, 4-5, 26, and 1, which are those expected for the molecular weights predicted from the DNA sequence for the products of URF2, URF4, URF4L, and URF5, respectively. With the present assignment, seven of the eight human mtDNA URFs have been shown to be expressed in HeLa cells

Binary polypeptide system for permanent and oriented protein immobilization

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Bailes Julian

2010-05-01

Full Text Available Abstract Background Many techniques in molecular biology, clinical diagnostics and biotechnology rely on binary affinity tags. The existing tags are based on either small molecules (e.g., biotin/streptavidin or glutathione/GST or peptide tags (FLAG, Myc, HA, Strep-tag and His-tag. Among these, the biotin-streptavidin system is most popular due to the nearly irreversible interaction of biotin with the tetrameric protein, streptavidin. The major drawback of the stable biotin-streptavidin system, however, is that neither of the two tags can be added to a protein of interest via recombinant means (except for the Strep-tag case leading to the requirement for chemical coupling. Results Here we report a new immobilization system which utilizes two monomeric polypeptides which self-assemble to produce non-covalent yet nearly irreversible complex which is stable in strong detergents, chaotropic agents, as well as in acids and alkali. Our system is based on the core region of the tetra-helical bundle known as the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex. This irreversible protein attachment system (IPAS uses either a shortened syntaxin helix and fused SNAP25-synaptobrevin or a fused syntaxin-synaptobrevin and SNAP25 allowing a two-component system suitable for recombinant protein tagging, capture and immobilization. We also show that IPAS is suitable for use with traditional beads and chromatography, planar surfaces and Biacore, gold nanoparticles and for protein-protein interaction in solution. Conclusions IPAS offers an alternative to chemical cross-linking, streptavidin-biotin system and to traditional peptide affinity tags and can be used for a wide range of applications in nanotechnology and molecular sciences.

Identification of dehydrin-like proteins responsive to chilling in floral buds of blueberry (Vaccinium, section Cyanococcus).

Science.gov (United States)

Muthalif, M M; Rowland, L J

1994-04-01

The level of three major polypeptides of 65, 60, and 14 kD increased in response to chilling unit accumulation in floral buds of a woody perennial, blueberry (Vaccinium, section Cynaococcus). The level of the polypeptides increased most dramatically within 300 h of chilling and decreased to the prechilling level with the initiation of budbreak. Cold-hardiness levels were assessed for dormant buds of Vaccinium corymbosum and Vaccinium ashei after different chilling treatments until the resumption of growth. These levels coincided with the level of the chilling-responsive polypeptides. Like some other previously described cold-induced proteins in annual plants, the level of the chilling-induced polypeptides also increased in leaves in response to cold treatment; the chilling-induced polypeptides were heat stable, resisting aggregation after incubation at 95 degrees C for 15 min. By fractionating bud proteins first by isoelectric point (pI) and then by molecular mass, the pI values of the 65- and 60-kD polypeptides were found to be 7.5 to 8.0 and the pI value of the 14-kD polypeptide was judged to be 8.5. Purification of the 65- and 60-kD polypeptides, followed by digestion with endoproteinase Lys-C and sequencing of selected fragments, revealed similarities in amino acid composition between the 65- and 60-kD polypeptides and dehydrins. Indeed, antiserum to the lysine-rich consensus sequence EKKGIMDKIKEKLPG of dehydrin proteins cross-reacted to all three of the major chilling-responsive polypeptides of blueberry, identifying these as dehydrins or dehydrin-like proteins.

Reaction mechanisms in the radiolysis of peptides, polypeptides and proteins II reactions at side-chain loci in model systems

International Nuclear Information System (INIS)

Garrison, W.M.

1983-11-01

The major emphasis in radiation biology at the molecular level has been on the nucleic acid component of the nucleic acid-protein complex because of its primary genetic importance. But there is increasing evidence that radiation damage to the protein component also has important biological implications. Damage to capsid protein now appears to be a major factor in the radiation inactivation of phage and other viruses. And, there is increasing evidence that radiation-chemical change in the protein component of chromation leads to changes in the stability of the repressor-operator complexes involved in gene expression. Knowledge of the radiation chemistry of protein is also of importance in other fields such as the application of radiation sterilization to foods and drugs. Recent findings that a class of compounds, the α,α'-diaminodicarboxylic acids, not normally present in food proteins, are formed in protein radiolysis is of particular significance since certain of their peptide derivatives have been showing to exhibit immunological activity. The purpose of this review is to bring together and to correlate our present knowledge of products and mechanisms in the radiolysis of peptides, polypeptides and proteins both aqueous and solid-state. In part 1 we presented a discussion of the radiation-induced reactions of the peptide main-chain in model peptide and polypeptide systems. Here in part 2 the emphasis is on the competing radiation chemistry at side-chain loci of peptide derivatives of aliphatic, aromatic-unsaturated and sulfur-containing amino acids in similar systems. Information obtained with the various experimental techniques of product analysis, competition kinetics, spin-trapping, pulse radiolysis, and ESR spectroscopy are included

Elastin-like polypeptide switches: A design strategy to detect multimeric proteins.

Science.gov (United States)

Dhandhukia, Jugal P; Brill, Dab A; Kouhi, Aida; Pastuszka, Martha K; MacKay, J Andrew

2017-09-01

Elastin-Like Polypeptides (ELPs) reversibly phase separate in response to changes in temperature, pressure, concentration, pH, and ionic species. While powerful triggers, biological microenvironments present a multitude of more specific biological cues, such as antibodies, cytokines, and cell-surface receptors. To develop better biosensors and bioresponsive drug carriers, rational strategies are required to sense and respond to these target proteins. We recently reported that noncovalent association of two ELP fusion proteins to a "chemical inducer of dimerization" small molecule (1.5 kDa) induces phase separation at physiological temperatures. Having detected a small molecule, here we present the first evidence that ELP multimerization can also detect a much larger (60 kDa) protein target. To demonstrate this strategy, ELPs were biotinylated at their amino terminus and mixed with tetrameric streptavidin. At a stoichiometric ratio of [4:1], two to three biotin-ELPs associate with streptavidin into multimeric complexes with an apparent K d of 5 nM. The increased ELP density around a streptavidin core strongly promotes isothermal phase separation, which was tuned to occur at physiological temperature. This phase separation reverses upon saturation with excess streptavidin, which only favors [1:1] complexes. Together, these findings suggest that ELP association with multimeric biomolecules is a viable strategy to deliberately engineer ELPs that respond to multimeric protein substrates. © 2017 The Protein Society.

Polypeptide structure of a human dermal fibroblast-activating factor (FAF) derived from the U937 cultured line of human monocyte-like cells

International Nuclear Information System (INIS)

Cooke, M.P.; Allar, W.J.; Goetzl, E.J.; Dohlman, J.G.

1986-01-01

Six liter batches of 1 x 10 6 U937 cells/ml of serum-free RPMI medium were incubated with 100 ng/ml of phorbol myristate acetate for 48 hr at 37 0 C in 5% CO 2 in air to generate FAFs, as quantified by the stimulation of uptake of [ 3 H]thymidine by quiescent human dermal fibroblasts. Filtration of the supernatants on Sephadex G-75 resolved two FAFs of approximately 40,000 and 10-13,000 daltons. The latter principle was purified to homogeneity by sequential Sephadex G-50 filtration, revealing an apparent m.w. of 7-8000, Mono-Q FPLC anion-exchange chromatography with a linear gradient from 20 mM Tris-HCl (pH 8.3) to 0.5 M NaCl-20 mM Tris-HCl in 30 min, and two cycles of high-performance liquid chromatography (HPLC) on a 300 A pore 10 μm C4 column at 1 ml/min with 0.05% trifluoroacetic acid (TFA) in water to 30:70 (v:v) and then to 60:40 (v:v) acetonitrile: 0.05% TFA linearly in 15 min and 30 min, respectively, The FAF activity eluted from HPLC in a sharp peak of O.D. 215 nm at 45% acetonitrile. Analyses of amino acid composition of the highly purified 7-8000 dalton FAF-U937 revealed 37% hydrophobic, 14% basic, and 21% acidic or amide residues, as well as one tryrosine and one methionine. This U937 cell-derived FAF appears to be a unique acidic polypeptide growth factor

Glucose-dependent insulinotropic polypeptide has impaired effect on abdominal, subcutaneous adipose tissue metabolism in obese subjects

DEFF Research Database (Denmark)

Asmar, M; Simonsen, L; Arngrim, N

2013-01-01

OBJECTIVE: Glucose-dependent insulinotropic polypeptide (GIP) appears to have a role in lipid metabolism. Recently, we showed that GIP in combination with hyperinsulinemia and hyperglycemia increases triglyceride uptake in abdominal, subcutaneous adipose tissue in lean humans. It has been suggested...... that increased GIP secretion in obesity will promote lipid deposition in adipose tissue. In light of the current attempts to employ GIP antagonists in the treatment and prevention of human obesity, the present experiments were performed in order to elucidate whether the adipose tissue lipid metabolism would...... to an oral glucose challenge: (i) NGT and (ii) IGT. Abdominal, subcutaneous adipose tissue lipid metabolism was studied by conducting measurements of arteriovenous concentrations of metabolites and regional adipose tissue blood flow (ATBF) during GIP (1.5 pmol kg(-1) min(-1)) in combination with a HI...

Zn(OAc2-Catalyzing Ring-Opening Polymerization of N-Carboxyanhydrides for the Synthesis of Well-Defined Polypeptides

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Yanzhao Nie

2018-03-01

Full Text Available Despite notable progress, the fabrication of well-defined polypeptides via controlled ring-opening polymerization (ROP of α-amino acid N-carboxyanhydrides (NCAs using convenient catalysts under mild conditions in a relatively short polymerization time is still challenging. Herein, an easily obtained catalyst system composed of zinc acetate and aniline was explored to mediate the fast ROP of γ-benzyl-l-glutamate-N-carboxyanhydride (BLG-NCA monomer, to produce poly(γ-benzyl-l-glutamates (PBLGs with controllable molecular weights and narrow dispersity. Considering the excellent cooperative action of zinc acetate and a broad scope of aniline derivatives with different functional groups to control ROP of BLG-NCA, this method may offer a useful platform enabling the rapid generation of end-functionalized PBLG and block copolymers for numerous biomedical applications.

Genipin-cross-linked poly(L-lysine)-based hydrogels: synthesis, characterization, and drug encapsulation.

Science.gov (United States)

Wang, Steven S S; Hsieh, Ping-Lun; Chen, Pei-Shan; Chen, Yu-Tien; Jan, Jeng-Shiung

2013-11-01

Genipin-cross-linked hydrogels composed of biodegradable and pH-sensitive cationic poly(L-lysine) (PLL), poly(L-lysine)-block-poly(L-alanine) (PLL-b-PLAla), and poly(L-lysine)-block-polyglycine (PLL-b-PGly) polypeptides were synthesized, characterized, and used as carriers for drug delivery. These polypeptide hydrogels can respond to pH-stimulus and their gelling and mechanical properties, degradation rate, and drug release behavior can be tuned by varying polypeptide composition and cross-linking degree. Comparing with natural polymers, the synthetic polypeptides with well-defined chain length and composition can warrant the preparation of the hydrogels with tunable properties to meet the criteria for specific biomedical applications. These hydrogels composed of natural building blocks exhibited good cell compatibility and enzyme degradability and can support cell attachment/proliferation. The evaluation of these hydrogels for in vitro drug release revealed that the controlled release profile was a biphasic pattern with a mild burst release and a moderate release rate thereafter, suggesting the drug molecules were encapsulated inside the gel matrix. With the versatility of polymer chemistry and conjugation of functional moieties, it is expected these hydrogels can be useful for biomedical applications such as polymer therapeutics and tissue engineering. Copyright © 2013 Elsevier B.V. All rights reserved.

Cell proliferation and migration are modulated by Cdk-1-phosphorylated endothelial-monocyte activating polypeptide II.

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Margaret A Schwarz

Full Text Available Endothelial-Monocyte Activating Polypeptide (EMAP II is a secreted protein with well-established anti-angiogenic activities. Intracellular EMAP II expression is increased during fetal development at epithelial/mesenchymal boundaries and in pathophysiologic fibroproliferative cells of bronchopulmonary dysplasia, emphysema, and scar fibroblast tissue following myocardial ischemia. Precise function and regulation of intracellular EMAP II, however, has not been explored to date.Here we show that high intracellular EMAP II suppresses cellular proliferation by slowing progression through the G2M cell cycle transition in epithelium and fibroblast. Furthermore, EMAP II binds to and is phosphorylated by Cdk1, and exhibits nuclear/cytoplasmic partitioning, with only nuclear EMAP II being phosphorylated. We observed that extracellular secreted EMAP II induces endothelial cell apoptosis, where as excess intracellular EMAP II facilitates epithelial and fibroblast cells migration.Our findings suggest that EMAP II has specific intracellular effects, and that this intracellular function appears to antagonize its extracellular anti-angiogenic effects during fetal development and pulmonary disease progression.

Practical multipeptide synthesis: dedicated software for the definition of multiple, overlapping peptides covering polypeptide sequences.

Science.gov (United States)

Heegaard, P M; Holm, A; Hagerup, M

1993-01-01

A personal computer program for the conversion of linear amino acid sequences to multiple, small, overlapping peptide sequences has been developed. Peptide lengths and "jumps" (the distance between two consecutive overlapping peptides) are defined by the user. To facilitate the use of the program for parallel solid-phase chemical peptide syntheses for the synchronous production of multiple peptides, amino acids at each acylation step are laid out by the program in a convenient standard multi-well setup. Also, the total number of equivalents, as well as the derived amount in milligrams (depend-ending on user-defined equivalent weights and molar surplus), of each amino acid are given. The program facilitates the implementation of multipeptide synthesis, e.g., for the elucidation of polypeptide structure-function relationships, and greatly reduces the risk of introducing mistakes at the planning step. It is written in Pascal and runs on any DOS-based personal computer. No special graphic display is needed.

Pancreatic polypeptide is involved in the regulation of body weight in pima Indian male subjects

DEFF Research Database (Denmark)

Koska, Juraj; DelParigi, Angelo; de Courten, Barbora

2004-01-01

Pancreatic polypeptide (PP) is released from the pancreas in response to a meal. In humans, low-circulating PP levels have been observed in obesity, and administration of pharmacological doses of PP has been shown to decrease food intake. The aim of the present study was to investigate whether low...... circulating PP is associated with weight gain in Pima Indians. Plasma PP concentrations were measured after an overnight fast and 30 min after a standardized mixed meal in 33 nondiabetic male subjects who had a follow-up visit 4.9 +/- 2.5 years later. Cross-sectionally, fasting and postprandial PP levels were...... negatively associated with body size and adiposity. Prospectively, the change in PP response to the meal was negatively associated with the change in body weight (r = -0.53, P = 0.002). In contrast, a high fasting PP level was positively associated with change in body weight (r = 0.45, P = 0...

The 75-kilodalton cytoplasmic Chlamydia trachomatis L2 polypeptide is a DnaK-like protein

DEFF Research Database (Denmark)

Birkelund, Svend; Lundemose, AG; Christiansen, Gunna

1990-01-01

,980-base-pair open reading frame revealed 94% homology with a 75-kilodalton protein from C. trachomatis serovar D and 57% homology with the DnaK proteins of E. coli and of Bacillus megaterium, while amino acid homology with human heat shock protein 70 (hsp70) was 42%. The promoter region was identified......The gene coding for the 75-kilodalton cytoplasmic Chlamydia trachomatis L2 polypeptide has been cloned in Escherichia coli, and the nucleotide sequence has been determined. The cloned DNA fragment contained the coding region as well as the putative promoter. The deduced amino acid sequence of the 1...... by computer search and by primer extension of mRNA synthesized in recombinant E. coli. The promoter region which differed from the putative promoter region in serovar D was shown to be a mixed promoter type in which the -10 region showed a regular TATA box configuration while the -35 region showed high...

Quantifying polypeptide conformational space: sensitivity to conformation and ensemble definition.

Science.gov (United States)

Sullivan, David C; Lim, Carmay

2006-08-24

Quantifying the density of conformations over phase space (the conformational distribution) is needed to model important macromolecular processes such as protein folding. In this work, we quantify the conformational distribution for a simple polypeptide (N-mer polyalanine) using the cumulative distribution function (CDF), which gives the probability that two randomly selected conformations are separated by less than a "conformational" distance and whose inverse gives conformation counts as a function of conformational radius. An important finding is that the conformation counts obtained by the CDF inverse depend critically on the assignment of a conformation's distance span and the ensemble (e.g., unfolded state model): varying ensemble and conformation definition (1 --> 2 A) varies the CDF-based conformation counts for Ala(50) from 10(11) to 10(69). In particular, relatively short molecular dynamics (MD) relaxation of Ala(50)'s random-walk ensemble reduces the number of conformers from 10(55) to 10(14) (using a 1 A root-mean-square-deviation radius conformation definition) pointing to potential disconnections in comparing the results from simplified models of unfolded proteins with those from all-atom MD simulations. Explicit waters are found to roughen the landscape considerably. Under some common conformation definitions, the results herein provide (i) an upper limit to the number of accessible conformations that compose unfolded states of proteins, (ii) the optimal clustering radius/conformation radius for counting conformations for a given energy and solvent model, (iii) a means of comparing various studies, and (iv) an assessment of the applicability of random search in protein folding.

Interpretation of sequential measurements of cancer antigen 125 (CA 125), carcinoembryonic antigen (CEA), and tissue polypeptide antigen (TPA) based on analytical imprecision and biological variation in the monitoring of ovarian cancer

DEFF Research Database (Denmark)

Tuxen, Malgorzata K.; Sölétormos, G; Petersen, P H

2001-01-01

The main objective with cancer antigen 125 (CA 125), carcinoembryonic antigen (CEA), and tissue polypeptide antigen (TPA) monitoring of ovarian cancer patients is to detect an early change of disease activity with high reliability. We hypothesized that a monitoring scheme for ovarian cancer patie...

The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc: lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation

DEFF Research Database (Denmark)

Wandall, Hans H; Irazoqui, Fernando; Tarp, Mads Agervig

2007-01-01

Initiation of mucin-type O-glycosylation is controlled by a large family of UDP GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Most GalNAc-transferases contain a ricin-like lectin domain in the C-terminal end, which may confer GalNAc-glycopeptide substrate specificit...

Radioimmunoassay of vasoactive intestinal polypeptide (VIP) in plasma. [/sup 125/I tracer techniques

Energy Technology Data Exchange (ETDEWEB)

Fahrenkrug, J.; Schaffalitzky de Muckadell, O.B.

1977-06-01

A sensitive and specific radioimmunoassay for vasoactive intestinal polypeptide (VIP) has been developed, which can detect 3.3 pmol x L/sup -1/ of the peptide in plasma. Antisera to highly purified porcine VIP coupled to albumin were raised in eight rabbits. The final dilution, the avidity, and the specificity of each antiserum were determined. /sup 125/I-VIP served as label, and highly purified porcine VIP was used as standard. Separation of antibody-bound and free VIP was achieved by plasma-coated charcoal. Nonspecific interference with the assay system was excluded by extraction of plasm samples with ethanol. The reliability of the assay was investigated by recovery experiments, by serial dilution of plasma samples with high concentration of endogenous VIP, and by immunosorption. The within-and between assay reproducibility at a concentration of 18.3 pmol x L/sup -1/ was 1.6 and 2.3 pmol x L/sup -1/ (1 S.D.), respectively. Median fasting concentration of VIP in plasma from 74 normal subjects was 7.3 pmol x L/sup -1/ (range: 0 to 20.0 pmol x L/sup -1/).

Interactions between crude drug extracts used in Japanese traditional Kampo medicines and organic anion-transporting polypeptide 2B1.

Science.gov (United States)

Iijima, Rie; Watanabe, Tomoki; Ishiuchi, Kan'ichiro; Matsumoto, Takashi; Watanabe, Junko; Makino, Toshiaki

2018-03-25

The use of herbal medicines has become popular worldwide, and the information on drug interactions between herbal medicines and chemical drugs is needed. We screened the inhibitory effects of crude drugs used in Kampo medicines used in Japan on organic anion-transporting polypeptide (OATP) 2B1 to predict potential interactions between Kampo medicines and chemical drugs used together. We chose 98 kinds of crude drugs frequently used as ingredients of Kampo formulations in Japan and prepared their boiling water extracts. We then screened their inhibitory effects on OATP2B1 by measuring the uptake of estrone 3-sulphate (E3S) by HEK293 cells stably expressing OATP2B1. At the concentration of 100µg/ml, the extracts prepared from 12 kinds of crude drugs, Scuteralliae Radix, Arecae Semen, Aurantii Fructus Immaturus, Perillae Herba, Panacis Japonici Rhizoma, Moutan Cortex, Polygalae Radix, Rhei Rhizoma, Cannabis Fructus, Chrysanthemi Flos, Eriobotryae Folium, and Querci Cortex, suppressed the function of OATP2B1 by less than 20%. The extract of bofutsushosan, a representative Kampo formulation, inhibited OATP2B1 function with sufficient levels to suppress absorption of OATP2B1 substrates in clinics. We further evaluated the inhibitory effects of several ingredients containing Rhei Rhizoma, Perillae Herba, and Moutan Cortex on OATP2B1. Because of crude drugs used in Kampo medicines might suppress absorption of OATP2B1 substrates, these results may contribute to the safe and effective use of Kampo medicine in clinics. A list of abbreviations: EC, (-)-epicatechin; ECG, epicatechin gallate; EGC, epigallocatechin; EGCG, Epigallocatechin gallate; FBS, fetal bovine serum; grapefruit juice; HEK293, Human embryonic kidney; IC 50, The half inhibitory concentration; OATP, organic anion-transporting polypeptide; β-PGG, penta-O-galloyl-β-D-glucose; t.i.d, 3 times a day. Copyright © 2017 Elsevier B.V. All rights reserved.

The blunted effect of glucose-dependent insulinotropic polypeptide in subcutaneous abdominal adipose tissue in obese subjects is partly reversed by weight loss

DEFF Research Database (Denmark)

Asmar, M; Arngrim, N; Simonsen, L.

2016-01-01

BACKGROUND: Glucose-dependent insulinotropic polypeptide (GIP) appears to have impaired effect on subcutaneous abdominal adipose tissue metabolism in obese subjects. The aim of the present study was to examine whether weight loss may reverse the impaired effect of GIP on subcutaneous abdominal...... adipose tissue in obese subjects. METHODS: Five obese males participated in a 12-week weight loss program, which consisted of caloric restriction (800 Cal day(-)(1)) followed by 4 weeks of weight-maintenance diet. Before and after weight loss, subcutaneous adipose tissue lipid metabolism was studied...... after weight loss, Pobese subjects, weight...

Effect of vasoactive intestinal polypeptide and somatostatin on secretion of epidermal growth factor and bicarbonate from Brunner's glands

DEFF Research Database (Denmark)

Poulsen, Steen Seier

1984-01-01

The effect of VIP and somatostatin on secretion of epidermal growth factor and bicarbonate from Brunner's glands was investigated in the rat. Vasoactive intestinal polypeptide infused in doses of 10 and 100 ng/kg/h significantly increased epidermal growth factor and bicarbonate output......, but the concentrations did not change. Somatostatin infused at doses of 1, 10, 100 and 1000 ng/kg/h against a background of VIP 100 ng/kg/h inhibited in dose-dependent fashion the stimulated epidermal growth factor and bicarbonate outputs from rat Brunner's gland pouches. Also basal secretion was inhibited...... growth factor and bicarbonate from Brunner's glands, an effect which is inhibited by somatostatin. A possible role for somatostatin in the control of Brunner's gland secretion is suggested....

Pituitary adenylate cyclase activating polypeptide in stress-related disorders: data convergence from animal and human studies.

Science.gov (United States)

Hammack, Sayamwong E; May, Victor

2015-08-01

The maladaptive expression and function of several stress-associated hormones have been implicated in pathological stress and anxiety-related disorders. Among these, recent evidence has suggested that pituitary adenylate cyclase activating polypeptide (PACAP) has critical roles in central neurocircuits mediating stress-related emotional behaviors. We describe the PACAPergic systems, the data implicating PACAP in stress biology, and how altered PACAP expression and signaling may result in psychopathologies. We include our work implicating PACAP signaling within the bed nucleus of the stria terminalis in mediating the consequences of stressor exposure and relatedly, describe more recent studies suggesting that PACAP in the central nucleus of the amygdala may impact the emotional aspects of chronic pain states. In aggregate, these results are consistent with data suggesting that PACAP dysregulation is associated with posttraumatic stress disorder in humans. Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Immunohistochemical localization of glucagon and pancreatic polypeptide on rat endocrine pancreas: coexistence in rat islet cells

Directory of Open Access Journals (Sweden)

YH Huang

2009-08-01

Full Text Available We used immunofluorescence double staining method to investigate the cellular localization of glucagon and pancreatic polypeptide (PP in rat pancreatic islets. The results showed that both A-cells (glucagon-secreting cells and PP-cells (PPsecreting cells were located in the periphery of the islets. However, A-cells and PP-cells had a different regional distribution. Most of A-cells were located in the splenic lobe but a few of them were in the duodenal lobe of the pancreas. In contrast, the majority of PP-cells were found in the duodenal lobe and a few of them were in the splenic lobe of the pancreas. Furthermore, we found that 67.74% A-cells had PP immunoreactivity, 70.92% PP-cells contained glucagon immunoreactivity with immunofluorescence double staining. Our data support the concept of a common precursor stem cell for pancreatic hormone-producing cells.

Sustained release of antibiotics from injectable and thermally responsive polypeptide depots.

Science.gov (United States)

Adams, Samuel B; Shamji, Mohammed F; Nettles, Dana L; Hwang, Priscilla; Setton, Lori A

2009-07-01

Biodegradable polymeric scaffolds are of interest for delivering antibiotics to local sites of infection in orthopaedic applications, such as bone and diarthrodial joints. The objective of this study was to develop a biodegradable scaffold with ease of drug loading in aqueous solution, while providing for drug depot delivery via syringe injection. Elastin-like polypeptides (ELPs) were used for this application, biopolymers of repeating pentapeptide sequences that were thermally triggered to undergo in situ depot formation at body temperature. ELPs were modified to enable loading with the antibiotics, cefazolin, and vancomycin, followed by induction of the phase transition in vitro. Cefazolin and vancomycin concentrations were monitored, as well as bioactivity of the released antibiotics, to test an ability of the ELP depot to provide for prolonged release of bioactive drugs. Further tests of formulation viscosity were conducted to test suitability as an injectable drug carrier. Results demonstrate sustained release of therapeutic concentrations of bioactive antibiotics by the ELP, with first-order time constants for drug release of approximately 25 h for cefazolin and approximately 500 h for vancomycin. These findings illustrate that an injectable, in situ forming ELP depot can provide for sustained release of antibiotics with an effect that varies across antibiotic formulation. ELPs have important advantages for drug delivery, as they are known to be biocompatible, biodegradable, and elicit no known immune response. These benefits suggest distinct advantages over currently used carriers for antibiotic drug delivery in orthopedic applications. (c) 2008 Wiley Periodicals, Inc.

Removal of uranyl ions from residual waters using some algae types

International Nuclear Information System (INIS)

Cecal, Al.; Rudic, V.; Gulea, A.; Palamaru, I.; Humelnicu, D.; Salaru, V.V.; Popa, K.

1997-01-01

This paper deals with a study on the bioaccumulation of uranyl ions resulted from residual effluents, by means of some microbiological collectors: Scenedesmus quadricauda, Anabaena karakumica, Calothrix brevissima, Penicillium sp, as well as the Glucide extract of Porphyridium cruentum, in several experimental conditions. The retaining degree of the bioaccumulated uranyl ions, as well as the leaching degree, in HCl and H 2 O media, of the same ions previously retained on algae, were karakumica >Penicillium sp> Calothrix brevissima. The leaching effect of bioaccumulated uranyl ions is higher in hydrochloric acid then in water. (authors)

Histopatología de Ustilaginales (carbones) en Poaceas de los géneros Sorghum, Bromus y Glyceria

OpenAIRE

Astiz Gassó, Marta Mónica

2017-01-01

Sporisorium cruentum (Kühn.), es el agente causal de enfermedades en el género Sorghum incluyendo Sorghum halepense (L.) Pers.. El presente trabajo tuvo por objeto analizar el desarrollo y la evolución del patógeno en los tejidos del hospedante, y evaluar las implicancias de la enfermedad en el crecimiento vegetativo de los rizomas del sorgo de alepo. Para determinar la presencia del hongo se realizaron cortes histológicos del hospedante que se observaron por medio de microscopía óptica (MO) ...

Some Gram-negative Lipoproteins Keep Their Surface Topology When Transplanted from One Species to Another and Deliver Foreign Polypeptides to the Bacterial Surface*

Science.gov (United States)

Fantappiè, Laura; Irene, Carmela; De Santis, Micaela; Armini, Alessandro; Gagliardi, Assunta; Tomasi, Michele; Parri, Matteo; Cafardi, Valeria; Bonomi, Serena; Ganfini, Luisa; Zerbini, Francesca; Zanella, Ilaria; Carnemolla, Chiara; Bini, Luca; Grandi, Alberto; Grandi, Guido

2017-01-01

In Gram-negative bacteria, outer membrane-associated lipoproteins can either face the periplasm or protrude out of the bacterial surface. The mechanisms involved in lipoprotein transport through the outer membrane are not fully elucidated. Some lipoproteins reach the surface by using species-specific transport machinery. By contrast, a still poorly characterized group of lipoproteins appears to always cross the outer membrane, even when transplanted from one organism to another. To investigate such lipoproteins, we tested the expression and compartmentalization in E. coli of three surface-exposed lipoproteins, two from Neisseria meningitidis (Nm-fHbp and NHBA) and one from Aggregatibacter actinomycetemcomitans (Aa-fHbp). We found that all three lipoproteins were lipidated and compartmentalized in the E. coli outer membrane and in outer membrane vesicles. Furthermore, fluorescent antibody cell sorting analysis, proteolytic surface shaving, and confocal microscopy revealed that all three proteins were also exposed on the surface of the outer membrane. Removal or substitution of the first four amino acids following the lipidated cysteine residue and extensive deletions of the C-terminal regions in Nm-fHbp did not prevent the protein from reaching the surface of the outer membrane. Heterologous polypeptides, fused to the C termini of Nm-fHbp and NHBA, were efficiently transported to the E. coli cell surface and compartmentalized in outer membrane vesicles, demonstrating that these lipoproteins can be exploited in biotechnological applications requiring Gram-negative bacterial surface display of foreign polypeptides. PMID:28483926

[Peroxide modification of membranes and isomorphic composition of cytochrome P-450 of rat liver microsomes during antioxidant deficiency].

Science.gov (United States)

Gubskiy, Iu I; Paramonova, G I; Boldeskul, A E; Primak, R G; Bogdanova, L A; Zadorina, O V; Litvinova, N V

1992-01-01

Lipid peroxidation (LPO), physico-chemical properties of the membranes and isoformic composition of microsomal cytochrome P-450 from the rat liver were studied under conditions of antioxidant insufficiency (AOI) which was modelled by exclusion of alpha-tocopherol from the animals' ration. An insignificant accumulation of microsomal diene conjugates and schiff bases against a sharp increase of the ability to the prooxidant stimulated LPO in vitro took place. A significant decrease of membrane lipid microviscosity and a change in surface properties of microsomal membranes of rats with AOI was determined. Absence of alpha-tocopherol in the ration was accompanied by a significant change in the content of separate isoforms of cytochrome P-450 exhibited in growth of a polypeptide with m. w. 54 kDa and the lowering of proteins with m. w. 48 and 50 kDa. Less intensive quenching of tryptophan fluorescence by acrylamide was also revealed, which testified to a lower accessibility of the quencher to membrane proteins or their fluorophore sites. Modification of lipid composition and of physicochemical properties of the rat liver membrane microsomes which was observed at AOI was significantly correlated by pretreatment with the antioxidant 4-methyl-2,6-ditretbutylphenol (ionol).

The multi-epitope polypeptide approach in HIV-1 vaccine development.

Science.gov (United States)

Cano, C A

1999-11-01

The application of a preventive HIV vaccine is the only hope for most developing countries to halt the AIDS pandemic. A project aimed to develop a preventive AIDS vaccine is being carried out since 1992 by three Cuban research institutions: Centro de Ingeniería Genética y Biotecnologia de La Habana, Instituto de Medicina Tropical 'Pedro Kouri' and Laboratorio de Investigaciones de SIDA de La Habana. The project includes two main strategies: (a) generation of recombinant multi-epitope polypeptides (MEPs) bearing several copies of the V3 loop from different HIV-1 isolates; and (b) development of immunogens capable of inducing a cytotoxic T cell response (CTL) specific for human immunodeficiency virus type 1 (HIV-1) antigens. This article summarizes the work in the first of these strategies. Based on the sequence of the V3 loop of HIV-1 we constructed a series of MEPs and evaluated their immunogenicity in mice, rabbits and macaques. The MEP TAB9, containing six V3 epitopes from isolates LR10, JY1, RF, MN, BRVA and IIIB, was selected together with the oil adjuvant Montanide ISA720 (SEPPIC, France) to perform a Phase I clinical trial in HIV seronegative Cuban volunteers. The trial was double blinded, randomized, and fulfilled all ethical and regulatory requirements. All TAB9 vaccinated volunteers developed a strong immune response and neutralizing antibodies were observed in the 50% of the subjects. However the second and third inoculations of the vaccine were not well tolerated because transient severe local reactions appeared in some individuals. A new formulation of TAB9 is currently in pre-clinical studies and is expected to enter clinical trials in 1999.

The cellular transcription factor CREB corresponds to activating transcription factor 47 (ATF-47) and forms complexes with a group of polypeptides related to ATF-43.

OpenAIRE

Hurst, H C; Masson, N; Jones, N C; Lee, K A

1990-01-01

Promoter elements containing the sequence motif CGTCA are important for a variety of inducible responses at the transcriptional level. Multiple cellular factors specifically bind to these elements and are encoded by a multigene family. Among these factors, polypeptides termed activating transcription factor 43 (ATF-43) and ATF-47 have been purified from HeLa cells and a factor referred to as cyclic AMP response element-binding protein (CREB) has been isolated from PC12 cells and rat brain. We...

Proghrelin-derived peptides influence the secretion of insulin, glucagon, pancreatic polypeptide and somatostatin: a study on isolated islets from mouse and rat pancreas

DEFF Research Database (Denmark)

Qader, S.S.; Hakanson, R.; Lundquist, I.

2008-01-01

ghrelin, and to the 23-amino acid peptide obestatin, claimed to be a physiological opponent of acyl ghrelin. This study examines the effects of the proghrelin products, alone and in combinations, on the secretion of insulin, glucagon, pancreatic polypeptide (PP) and somatostatin from isolated islets...... times higher concentration than acyl ghrelin (corresponding to the ratio of the two peptides in circulation), desacyl ghrelin abolished the effects of acyl ghrelin but not those of obestatin. Acyl ghrelin and obestatin affected the secretion of glucagon, PP and somatostatin at physiologically relevant...

Distribution of Pancreatic Polypeptide-secreting Endocrine Cells in Nondiabetic and Diabetic Cases.

Science.gov (United States)

Śliwińska-Mossoń, Mariola; Milnerowicz, Halina

2017-07-01

The aim of the study was to demonstrate the effects of cigarette smoking and ongoing inflammation in chronic pancreatitis on the functioning of pancreatic polypeptide (PP)-secreting cells and to determine the relationship between the occurrence of an increased number of PP cells in the pancreas, the change in their location, and the intensity of their inflammatory changes in the course of pancreatitis and diabetes. Samples of tissues from healthy persons and from patients were verified histopathologically, and then PP was localized by immunohistochemical staining using the monoclonal anti-human PP antibody. The histopathologic evaluation of the hormone expression intensity in tissue sections was carried out using the semiquantitative method and was calculated with digital image analysis. The present study showed a very strong PP expression in the pancreatic tissue (especially in the head of the pancreas) derived from smoking patients with diabetes. The increase in the percentage of cells in the PP islets, between the acinar cells in smoking patients with diabetes and a statistically significant increase in the expression of PP, indicates a pancreatic endocrine dysfunction and suggests that cigarette smoking has a negative impact on the organ's efficiency. Because of its properties, the PP appears to be a useful marker of the endocrine insufficiency of the pancreas and a specific prognostic parameter of developing diabetes due to chronic pancreatitis.

Investigation and characterization of receptors for pituitary adenylate cyclase-activating polypeptide in human brain by radioligand binding and chemical cross-linking

International Nuclear Information System (INIS)

Suda, K.; Smith, D.M.; Ghatei, M.A.; Murphy, J.K.; Bloom, S.R.

1991-01-01

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel peptide of hypothalamic origin which increases adenylate cyclase activity in rat anterior pituitary cell cultures. The 38-amino acid peptide shows a close sequence homology to vasoactive intestinal peptide (VIP). Binding sites for PACAP in membranes from postmortem human brain tissue were studied using [ 125 I]PACAP27 as the radioligand. High specific binding sites (amount of specific binding measured at 0.25 nM [ 125 I]PACAP27 in femtomoles per mg protein +/- SEM; n = 4) were present in hypothalamus (344.5 +/- 13.0), brain stem (343.0 +/- 29.3), cerebellum (292.0 +/- 21.1), cortex (259.6 +/- 19.8), and basal ganglia (259.2 +/- 50.3). Specific binding sites in pituitary, although present, were less abundant (35.0 +/- 8.9). Binding of [ 125 I]PACAP27 was reversible and time, pH, and temperature dependent. Despite the homology with VIP, VIP was a poor inhibitor of [ 125 I]PACAP27 binding (IC50, greater than 1 microM) compared with PACAP27 (IC50, 0.5-1.3 nM) and PACAP38 (IC50, 0.2-1.3 nM). Scatchard plots of [ 125 I]PACAP27 binding showed the presence of both high and lower affinity sites. Chemical cross-linking of PACAP-binding sites revealed that [ 125 I]PACAP27 was bound to polypeptide chains of 67,000 and 48,000 mol wt. Thus, we have demonstrated the presence of PACAP-specific receptors in human brain which are not VIP receptors. This opens the possibility of PACAP functioning as a novel neurotransmitter/neuromodulator in human brain

The pathophysiology of diabetes involves a defective amplification of the late-phase insulin response to glucose by glucose-dependent insulinotropic polypeptide-regardless of etiology and phenotype

DEFF Research Database (Denmark)

Vilsbøll, Tina; Knop, F K; Krarup, T

2003-01-01

diabetic patients. We studied (six in each group): 1) patients with diabetes mellitus secondary to chronic pancreatitis; 2) lean type 2 diabetic patients (body mass index ... incretin hormone, glucose-dependent insulinotropic polypeptide (GIP), is seen in these patients. The aim of the present investigation was to evaluate plasma insulin and C-peptide responses to GLP-1 and GIP in five groups of diabetic patients with etiology and phenotype distinct from the obese type 2...

Effect of apolipoprotein B mRNA-editing catalytic polypeptide-like protein-3G in cervical cancer.

Science.gov (United States)

Xu, Yanhua; Leng, Junhong; Xue, Fang; Dong, Ruiqian

2015-01-01

Cervical cancer is one of the most common gynecologic cancers. The role of apolipoprotein B mRNA-editing catalytic polypeptide-like protein-3G (APCBEC-3G) in cervical cancer has yet to be elucidated. This study intends to explore the effect of APCBEC-3G on cervical cancer cell proliferation and invasion. In vitro, the cervical cancer cell line Hela was transfected by APCBEC-3G plasmid. The mRNA and protein expression levels of APCBEC-3G were detected by Real-time PCR and Western blot, respectively. Cervical cancer cell proliferation was determined by MTT. Transwell assay was applied to measure the effect of APCBEC-3G on cell invasion. APCBEC-3G mRNA and protein increased significantly after transfection (P3G serves as a suppressor of cervical cancer cell proliferation and invasion. Our research provides theoretical basis for further investigation APOBEC-3G effect in cervical cancer occurrence and development.

Engineered aggregation inhibitor fusion for production of highly amyloidogenic human islet amyloid polypeptide.

Science.gov (United States)

Mirecka, Ewa Agnieszka; Gremer, Lothar; Schiefer, Stephanie; Oesterhelt, Filipp; Stoldt, Matthias; Willbold, Dieter; Hoyer, Wolfgang

2014-12-10

Human islet amyloid polypeptide (IAPP) is the major component of pancreatic amyloid deposits in type 2 diabetes. The structural conversion of IAPP from a monomeric state into amyloid assemblies is the subject of intense research. Recombinant production of IAPP is, however, difficult due to its extreme aggregation propensity. Here we describe a novel strategy for expression of IAPP in Escherichia coli, based on an engineered protein tag, which sequesters IAPP monomers and prevents IAPP aggregation. The IAPP-binding protein HI18 was selected by phage display from a β-wrapin library. Fusion of HI18 to IAPP enabled the soluble expression of the construct. IAPP was cleaved from the fusion construct and purified to homogeneity with a yield of 3mg of isotopically labeled peptide per liter of culture. In the monomeric state, IAPP was largely disordered as evidenced by far-UV CD and liquid-state NMR spectroscopy but competent to form amyloid fibrils according to atomic force microscopy. These results demonstrate the ability of the engineered β-wrapin HI18 for shielding the hydrophobic sequence of IAPP during expression and purification. Fusion of aggregation-inhibiting β-wrapins is a suitable approach for the recombinant production of aggregation-prone proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

Pituitary Adenylate Cyclase-Activating Polypeptide Reverses Ammonium Metavanadate-Induced Airway Hyperresponsiveness in Rats

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Mounira Tlili

2015-01-01

Full Text Available The rate of atmospheric vanadium is constantly increasing due to fossil fuel combustion. This environmental pollution favours vanadium exposure in particular to its vanadate form, causing occupational bronchial asthma and bronchitis. Based on the well admitted bronchodilator properties of the pituitary adenylate cyclase-activating polypeptide (PACAP, we investigated the ability of this neuropeptide to reverse the vanadate-induced airway hyperresponsiveness in rats. Exposure to ammonium metavanadate aerosols (5 mg/m3/h for 15 minutes induced 4 hours later an array of pathophysiological events, including increase of bronchial resistance and histological alterations, activation of proinflammatory alveolar macrophages, and increased oxidative stress status. Powerfully, PACAP inhalation (0.1 mM for 10 minutes alleviated many of these deleterious effects as demonstrated by a decrease of bronchial resistance and histological restoration. PACAP reduced the level of expression of mRNA encoding inflammatory chemokines (MIP-1α, MIP-2, and KC and cytokines (IL-1α and TNF-α in alveolar macrophages and improved the antioxidant status. PACAP reverses the vanadate-induced airway hyperresponsiveness not only through its bronchodilator activity but also by counteracting the proinflammatory and prooxidative effects of the metal. Then, the development of stable analogs of PACAP could represent a promising therapeutic alternative for the treatment of inflammatory respiratory disorders.

Maternally Sequestered Therapeutic Polypeptides – A New Approach for the Management of Preeclampsia

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Eric eGeorge

2014-09-01

Full Text Available The last several decades have seen intensive research into the molecular mechanisms underlying the symptoms of preeclampsia. While the underlying cause of preeclampsia is believed to be defective placental development and resulting placental ischemia, it is only recently that the links between the ischemic placenta and maternal symptomatic manifestation have been elucidated. Several different pathways have been implicated in the development of the disorder; most notably production of the anti-angiogenic protein sFlt-1, induction of auto-immunity and inflammation, and production of reactive oxygen species. While the molecular mechanisms are becoming clearer, translating that knowledge into effective therapeutics has proven elusive. Here we describe a number of peptide based therapies we have developed to target theses pathways, and which are currently being tested in preclinical models. These therapeutics are based on a synthetic polymeric carrier elastin-like polypeptide (ELP, which can be synthesized in various sequences and sizes to stabilize the therapeutic peptide and avoid crossing the placental interface. This prevents fetal exposure and potential developmental effects. The therapeutics designed will target known pathogenic pathways, and the ELP carrier could prove to be a versatile delivery system for administration of a variety of therapeutics during pregnancy.

Production and characterization of monoclonal antibodies that discriminate among individual S100 polypeptides

International Nuclear Information System (INIS)

Van Eldik, L.J.

1984-01-01

The term S100 refers to a heterogeneous fraction of low molecular weight, acidic, calcium binding proteins. The S100 fraction is a mixture of polypeptides, only some of which have been isolated and characterized. The amino acid sequences of two S100 proteins from bovine brain, S100α and S100β, have been determined. The physiological functions of the S100 proteins are not known. Although assay of immunoreactive S100 has been used clinically to screen tumors of neural origin, as an index of cell injury in various disorders, and as an index of malignancy, most of the antisera used in previous studies react with more than one protein in the S100 fraction. Even the currently available monoclonal antibodies against S100 (2-4) do not appear to measure the individual S100α and S100β components. In order to unequivocally interpret studies on the localization of S100 and its potential alterations in various disease states, and on the validity of S100 immunoreactivity as a diagnostic tool for tumor diagnosis, it would be useful to have antibodies that discriminate among the individual S100 components. The authors report here the production of monoclonal antibodies that appear to be specific for S100β

ThPOD3, a truncated polypeptide from Tamarix hispida, conferred drought tolerance in Escherichia coli.

Science.gov (United States)

Guo, Xiao-Hong; Jiang, Jing; Wang, Bai-Chen; Li, Hui-Yu; Wang, Yu-Cheng; Yang, Chuan-Ping; Liu, Gui-Feng

2010-03-01

The ThPOD1 gene encodes a peroxidase and was isolated from a Tamarix hispida NaCl-stress root cDNA library. We found that ThPOD1 expression could be induced by abiotic stresses such as cold, salt, drought and exogenous abscisic acid. These findings suggested that ThPOD1 might be involved in the plant response to environmental stresses and ABA treatment. To elucidate the function of this gene, recombinant plasmids expressing full-length ThPOD1 as well as ThPOD2 (aa 41-337), and ThPOD3 (aa 73-337) truncated polypeptides were constructed. SDS-PAGE and Western blot analyses of the fusion proteins revealed that the molecular weights of ThPOD1, ThPOD2 and ThPOD3 were approximately 57, approximately 50 and approximately 47 kDa, respectively. Stress assays of E. coli treated with the recombinant plasmids indicated that ThPOD3 could improve resistance to drought stress. This finding could potentially be used to improve plant tolerance to drought stress via gene transfer.

Neurofilament heavy polypeptide regulates the Akt-beta-catenin pathway in human esophageal squamous cell carcinoma.

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Myoung Sook Kim

2010-02-01

Full Text Available Aerobic glycolysis and mitochondrial dysfunction are common features of aggressive cancer growth. We observed promoter methylation and loss of expression in neurofilament heavy polypeptide (NEFH in a significant proportion of primary esophageal squamous cell carcinoma (ESCC samples that were of a high tumor grade and advanced stage. RNA interference-mediated knockdown of NEFH accelerated ESCC cell growth in culture and increased tumorigenicity in vivo, whereas forced expression of NEFH significantly inhibited cell growth and colony formation. Loss of NEFH caused up-regulation of pyruvate kinase-M2 type and down-regulation of pyruvate dehydrogenase, via activation of the Akt/beta-catenin pathway, resulting in enhanced aerobic glycolysis and mitochondrial dysfunction. The acceleration of glycolysis and mitochondrial dysfunction in NEFH-knockdown cells was suppressed in the absence of beta-catenin expression, and was decreased by the treatment of 2-Deoxyglucose, a glycolytic inhibitor, or API-2, an Akt inhibitor. Loss of NEFH activates the Akt/beta-catenin pathway and increases glycolysis and mitochondrial dysfunction. Cancer cells with methylated NEFH can be targeted for destruction with specific inhibitors of deregulated downstream pathways.

Conformations of islet amyloid polypeptide monomers in a membrane environment: implications for fibril formation.

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Mojie Duan

Full Text Available The amyloid fibrils formed by islet amyloid polypeptide (IAPP are associated with type II diabetes. One of the proposed mechanisms of the toxicity of IAPP is that it causes membrane damage. The fatal mutation of S20G human IAPP was reported to lead to early onset of type II diabetes and high tendency of amyloid formation in vitro. Characterizing the structural features of the S20G mutant in its monomeric state is experimentally difficult because of its unusually fast aggregation rate. Computational work complements experimental studies. We performed a series of molecular dynamics simulations of the monomeric state of human variants in the membrane. Our simulations are validated by extensive comparisons with experimental data. We find that a helical disruption at His18 is common to both human variants. An L-shaped motif of S20G mutant is observed in one of the conformational families. This motif that bends at His18 resembles the overall topology of IAPP fibrils. The conformational preorganization into the fibril-like topology provides a possible explanation for the fast aggregation rate of S20G IAPP.

Intersegment interactions and helix-coil transition within the generalized model of polypeptide chains approach

Science.gov (United States)

Badasyan, A. V.; Hayrapetyan, G. N.; Tonoyan, Sh. A.; Mamasakhlisov, Y. Sh.; Benight, A. S.; Morozov, V. F.

2009-09-01

The generalized model of polypeptide chains is extended to describe the helix-coil transition in a system comprised of two chains interacting side-by-side. The Hamiltonian of the model takes into account four possible types of interactions between repeated units of the two chains, i.e., helix-helix, helix-coil, coil-helix, and coil-coil. Analysis reveals when the energy Ihh+Icc of (h-h, c-c) interactions overwhelms the energy Ihc+Ich of mixed (h-c, c-h) interactions, the correlation length rises substantially, resulting in narrowing of the transition interval. In the opposite case, when Ihh+Icc

A New in Vitro Anti-Tumor Polypeptide Isolated from Arca inflata

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Jian Xu

2013-12-01

Full Text Available A new in vitro anti-tumor polypeptide, coded as J2-C3, was isolated from Arca inflata Reeve and purified by diethyl-aminoethanol (DEAE-sepharose Fast Flow anion exchange and phenyl sepharose CL-4B hydrophobic chromatography. J2-C3 was identified to be a homogeneous compound by native polyacrylamide gel electrophoresis (Native-PAGE. The purity of J2-C3 was over 99% in reversed phase-high performance liquid chromatography (RP-HPLC. The molecular weight was determined as 20,538.0 Da by electrospray-ionization mass spectrometry (ESI-MS/MS. J2-C3 was rich in Glx (Gln + Glu, Lys, and Asx (Asp + Asn according to amino acid analysis. Four partial amino acid sequences of this peptide were determined as L/ISMEDVEESR, KNGMHSI/LDVNHDGR, AMKI/LI/LNPKKGI/LVPR and AMGAHKPPKGNEL/IGHR via MALDI-TOF/TOF-MS and de novo sequencing. Secondary structural analysis by CD spectroscopy revealed that J2-C3 had the α-helix (45.2%, β-sheet (2.9%, β-turn (26.0% and random coil (25.9%. The anti-tumor effect of J2-C3 against human tumor cells was measured by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay, and the IC50 values of J2-C3 were 65.57, 93.33 and 122.95 µg/mL against A549, HT-29 and HepG2 cell lines, respectively. Therefore, J2-C3 might be developed as a potential anti-tumor agent.

Proteome analysis of muscadine grape leaves

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Sheikh M Basha

2009-04-01

Full Text Available Sheikh M Basha1, Ramesh Katam1, Hemanth Vasanthaiah1, Frank Matta21Center for Viticulture and Small Fruit Research, Florida A and M University, Tallahassee, FL, USA; 2Plant and Soil Science Department, Mississippi State University, Mississippi State, MS, USAAbstract: Muscadine grapes are native to the southeastern United States and are used for making wine and consumed as fresh fruit. Grape berries, as ‘sink organs,’ rely on the use of available carbohydrate resources produced by photosynthesis to support their development and composition. A high throughput two-dimensional gel electrophoresis (2-DE was conducted on muscadine (Vitis rotundifolia grape leaf proteins to document complexity in their composition and to determine protein identity and function for enhancing photosynthetic efficiency of muscadine grape. 2-DE resolved muscadine leaf proteins into >258 polypeptides with pIs between 3.5 and 8.0 and molecular weight between 12,000 to 15,0000 Daltons. The consistently expressed proteins were excised and subjected to sequencing. Homology search of protein sequences showed 84% identity with Viridi plantae database. Identity of some of these proteins included RuBisCO, glutamine synthetase, pathogenesis-related protein, glyoxisomal malate dehydrogenase, ribonucleoprotein, chloroplast precursor, oxygen evolving enhancer protein. Comparative analysis of 10 muscadine cultivars showed quantitative differences in expression of 39 polypeptides among these genotypes. The results suggested that the polypeptide composition of muscadine grape leaf is complex, and polypeptide number and amount vary widely among muscadine genotypes, and these variations may be responsible for differences in their physiology, berry and stress tolerance characteristics.Keywords: grapevine, leaves, muscadine, proteins, sequencing, 2-DE

Organic anion transporting polypeptide 1B transporters modulate hydroxyurea pharmacokinetics.

Science.gov (United States)

Walker, Aisha L; Lancaster, Cynthia S; Finkelstein, David; Ware, Russell E; Sparreboom, Alex

2013-12-15

Hydroxyurea is currently the only FDA-approved drug that ameliorates the pathophysiology of sickle cell anemia. Unfortunately, substantial interpatient variability in the pharmacokinetics (PK) of hydroxyurea may result in variation of the drug's efficacy. However, little is known about mechanisms that modulate hydroxyurea PK. Recent in vitro studies identifying hydroxyurea as a substrate for organic anion transporting polypeptide (OATP1B) transporters prompted the current investigation assessing the role of OATP1B transporters in modulating hydroxyurea PK. Using wild-type and Oatp1b knockout (Oatp1b(-/-)) mice, hydroxyurea PK was analyzed in vivo by measuring [(14)C]hydroxyurea distribution in plasma, kidney, liver, urine, or the exhaled (14)CO2 metabolite. Plasma levels were significantly reduced by 20% in Oatp1b(-/-) mice compared with wild-type (area under the curve of 38.64 or 48.45 μg·h(-1)·ml(-1), respectively) after oral administration, whereas no difference was observed between groups following intravenous administration. Accumulation in the kidney was significantly decreased by twofold in Oatp1b(-/-) mice (356.9 vs. 748.1 pmol/g), which correlated with a significant decrease in urinary excretion. Hydroxyurea accumulation in the liver was also decreased (136.6 vs. 107.3 pmol/g in wild-type or Oatp1b(-/-) mice, respectively) correlating with a decrease in exhaled (14)CO2. These findings illustrate that deficiency of Oatp1b transporters alters the absorption, distribution, and elimination of hydroxyurea thus providing the first in vivo evidence that cell membrane transporters may play a significant role in modulating hydroxyurea PK. Future studies to investigate other transporters and their role in hydroxyurea disposition are warranted for understanding the sources of variation in hydroxyurea's PK.

Some Gram-negative Lipoproteins Keep Their Surface Topology When Transplanted from One Species to Another and Deliver Foreign Polypeptides to the Bacterial Surface.

Science.gov (United States)

Fantappiè, Laura; Irene, Carmela; De Santis, Micaela; Armini, Alessandro; Gagliardi, Assunta; Tomasi, Michele; Parri, Matteo; Cafardi, Valeria; Bonomi, Serena; Ganfini, Luisa; Zerbini, Francesca; Zanella, Ilaria; Carnemolla, Chiara; Bini, Luca; Grandi, Alberto; Grandi, Guido

2017-07-01

In Gram-negative bacteria, outer membrane-associated lipoproteins can either face the periplasm or protrude out of the bacterial surface. The mechanisms involved in lipoprotein transport through the outer membrane are not fully elucidated. Some lipoproteins reach the surface by using species-specific transport machinery. By contrast, a still poorly characterized group of lipoproteins appears to always cross the outer membrane, even when transplanted from one organism to another. To investigate such lipoproteins, we tested the expression and compartmentalization in E. coli of three surface-exposed lipoproteins, two from Neisseria meningitidis (Nm-fHbp and NHBA) and one from Aggregatibacter actinomycetemcomitans (Aa-fHbp). We found that all three lipoproteins were lipidated and compartmentalized in the E. coli outer membrane and in outer membrane vesicles. Furthermore, fluorescent antibody cell sorting analysis, proteolytic surface shaving, and confocal microscopy revealed that all three proteins were also exposed on the surface of the outer membrane. Removal or substitution of the first four amino acids following the lipidated cysteine residue and extensive deletions of the C-terminal regions in Nm-fHbp did not prevent the protein from reaching the surface of the outer membrane. Heterologous polypeptides, fused to the C termini of Nm-fHbp and NHBA, were efficiently transported to the E. coli cell surface and compartmentalized in outer membrane vesicles, demonstrating that these lipoproteins can be exploited in biotechnological applications requiring Gram-negative bacterial surface display of foreign polypeptides. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A composite model including visfatin, tissue polypeptide-specific antigen, hyaluronic acid, and hematological variables for the diagnosis of moderate-to-severe fibrosis in nonalcoholic fatty liver disease: a preliminary study.

Science.gov (United States)

Chwist, Alina; Hartleb, Marek; Lekstan, Andrzej; Kukla, Michał; Gutkowski, Krzysztof; Kajor, Maciej

2014-01-01

Histopathological risk factors for end-stage liver failure in patients with nonalcoholic fatty liver disease (NAFLD) include nonalcoholic steatohepatitis (NASH) and advanced liver fibrosis. There is a need for noninvasive diagnostic methods for these 2 conditions. The aim of this study was to investigate new laboratory variables with a predictive potential to detect advanced fibrosis (stages 2 and 3) in NAFLD. The study involved 70 patients with histologically proven NAFLD of varied severity. Additional laboratory variables included zonulin, haptoglobin, visfatin, adiponectin, leptin, tissue polypeptide-specific antigen (TPSA), hyaluronic acid, and interleukin 6. Patients with NASH (NAFLD activity score of ≥5) had significantly higher HOMA-IR values and serum levels of visfatin, haptoglobin, and zonulin as compared with those without NASH on histological examination. Advanced fibrosis was found in 16 patients (22.9%) and the risk factors associated with its prevalence were age, the ratio of erythrocyte count to red blood cell distribution width, platelet count, and serum levels of visfatin and TPSA. Based on these variables, we constructed a scoring system that differentiated between NAFLD patients with and without advanced fibrosis with a sensitivity of 75% and specificity of 100% (area under the receiver operating characteristic curve, 0.93). The scoring system based on the above variables allows to predict advanced fibrosis with high sensitivity and specificity. However, its clinical utility should be verified in further studies involving a larger number of patients.

Rescue of Drosophila Melanogaster l(2)35Aa lethality is only mediated by polypeptide GalNAc-transferase pgant35A, but not by the evolutionary conserved human ortholog GalNAc-transferase-T11

DEFF Research Database (Denmark)

Bennett, Eric P; Chen, Ya-Wen; Schwientek, Tilo

2010-01-01

The Drosophila l(2)35Aa gene encodes a UDP-N-acetylgalactosamine: Polypeptide N-acetylgalactosaminyltransferase, essential for embryogenesis and development (J. Biol. Chem. 277, 22623-22638; J. Biol. Chem. 277, 22616-22). l(2)35Aa, also known as pgant35A, is a member of a large evolutionarily con...

Structure and pharmacological actions of phyllocaerulein, a caerulein-like nonapeptide: its occurrence in extracts of the skin of Phyllomedusa sauvagei and related Phyllomedusa species.

Science.gov (United States)

Anastasi, A; Bertaccini, G; Cei, J M; De Caro, G; Erspamer, V; Impicciatore, M

1969-09-01

1. The South American amphibian Phyllomedusa sauvagei contains in its skin large amounts of a polypeptide closely resembling caerulein in its pharmacological actions. This polypeptide, called phyllocaerulein, was obtained in a pure form, and upon acid hydrolysis, enzymic digestion and end-group determination experiments it proved to be a nonapeptide of the following composition Pyr-Glu-Tyr(SO(3)H)-Thr-Gly-Trp-Met-Asp-Phe-NH(2)It may be seen that caerulein and phyllocaerulein have in common the C-terminal heptapeptide and the N-terminal pyroglutamyl residue.2. Phyllocaerulein is indistinguishable from caerulein even in parallel bioassay. However, the former polypeptide seems to be somewhat more potent than the latter on all the preparations tested.3. In different batches of Phyllomedusa sauvagei skin the phyllocaerulein content ranged between 150 and 600 mug/g of fresh tissue.Phyllocaerulein or similar polypeptides occur also in the skin of several other Phyllomedusa species, among which are Phyll. burmeisteri, Phyll. dachnicolor, Phyll, helenae, Phyll. annae, Phyll. callidryas and Phyll. bicolor.4. The qualitative identification and quantitative estimation of caerulein-like polypeptides in crude skin extracts may be complicated by the concomitant occurrence of other active polypeptides. These, however, are poorly effective on some test preparations which seem to respond selectively to caerulein.5. Like that of caerulein, the biological significance of phyllocaerulein is completely obscure.

Structure and pharmacological actions of phyllocaerulein, a caerulein-like nonapeptide

Science.gov (United States)

Anastasi, A.; Bertaccini, G.; Cei, J. M.; De Caro, G.; Erspamer, V.; Impicciatore, M.

1969-01-01

1. The South American amphibian Phyllomedusa sauvagei contains in its skin large amounts of a polypeptide closely resembling caerulein in its pharmacological actions. This polypeptide, called phyllocaerulein, was obtained in a pure form, and upon acid hydrolysis, enzymic digestion and end-group determination experiments it proved to be a nonapeptide of the following composition Pyr-Glu-Tyr(SO3H)-Thr-Gly-Trp-Met-Asp-Phe-NH2 It may be seen that caerulein and phyllocaerulein have in common the C-terminal heptapeptide and the N-terminal pyroglutamyl residue. 2. Phyllocaerulein is indistinguishable from caerulein even in parallel bioassay. However, the former polypeptide seems to be somewhat more potent than the latter on all the preparations tested. 3. In different batches of Phyllomedusa sauvagei skin the phyllocaerulein content ranged between 150 and 600 μg/g of fresh tissue. Phyllocaerulein or similar polypeptides occur also in the skin of several other Phyllomedusa species, among which are Phyll. burmeisteri, Phyll. dachnicolor, Phyll, helenae, Phyll. annae, Phyll. callidryas and Phyll. bicolor. 4. The qualitative identification and quantitative estimation of caerulein-like polypeptides in crude skin extracts may be complicated by the concomitant occurrence of other active polypeptides. These, however, are poorly effective on some test preparations which seem to respond selectively to caerulein. 5. Like that of caerulein, the biological significance of phyllocaerulein is completely obscure. PMID:5824931

Normal secretion and action of the gut incretin hormones glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide in young men with low birth weight

DEFF Research Database (Denmark)

Schou, Jakob Hagen; Pilgaard, Kasper; Vilsbøll, Tina

2005-01-01

polypeptide (GIP) in young LBW men (n = 24) and matched normal birth weight controls (NBW) (n = 25). RESULTS: LBW subjects were 5 cm shorter but had a body mass index similar to NBW. LBW subjects had significantly elevated fasting and postprandial plasma glucose, as well as postprandial (standard meal test......CONTEXT: Low birth weight (LBW) is associated with increased risk of type 2 diabetes mellitus. An impaired incretin effect was reported previously in type 2 diabetic patients. OBJECTIVE: We studied the secretion and action of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic...

Multi-functional roles for the polypeptide transport associated domains of Toc75 in chloroplast protein import

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Paila, Yamuna D; Richardson, Lynn GL; Inoue, Hitoshi; Parks, Elizabeth S; McMahon, James; Inoue, Kentaro; Schnell, Danny J

2016-01-01

Toc75 plays a central role in chloroplast biogenesis in plants as the membrane channel of the protein import translocon at the outer envelope of chloroplasts (TOC). Toc75 is a member of the Omp85 family of bacterial and organellar membrane insertases, characterized by N-terminal POTRA (polypeptide-transport associated) domains and C-terminal membrane-integrated β-barrels. We demonstrate that the Toc75 POTRA domains are essential for protein import and contribute to interactions with TOC receptors, thereby coupling preprotein recognition at the chloroplast surface with membrane translocation. The POTRA domains also interact with preproteins and mediate the recruitment of molecular chaperones in the intermembrane space to facilitate membrane transport. Our studies are consistent with the multi-functional roles of POTRA domains observed in other Omp85 family members and demonstrate that the domains of Toc75 have evolved unique properties specific to the acquisition of protein import during endosymbiotic evolution of the TOC system in plastids. DOI: http://dx.doi.org/10.7554/eLife.12631.001 PMID:26999824

An acetylation site in lectin domain modulates the biological activity of polypeptide GalNAc-transferase-2

DEFF Research Database (Denmark)

Zlocowski, Natacha; Lorenz, Virginia; Bennett, Eric Paul

2013-01-01

Abstract Polypeptide GalNAc-transferases (ppGalNAc-Ts) are a family of enzymes that catalyze the initiation of mucin-type O-glycosylation. All ppGalNAc-T family members contain a common (QXW)3 motif which is present in R-type lectin group. Acetylation site K521 is part of the QKW motif of ß......-trefoil in the lectin domain of ppGalNAc-T2. We used a combination of acetylation and site-directed mutagenesis approaches to examine the functional role of K521 in ppGalNAc-T2. Binding assays of non-acetylated and acetylated forms of the mutant ppGalNAc-T2K521Q to various naked and aGalNAc-glycosylated mucin peptides...... indicated that degree of interaction of lectin domain with aGalNAc depends on the peptide sequence of mucin. Studies of inhibitory effect of various carbohydrates on interactions of ppGalNAc-T2 with MUC1aGalNAc indicate that point K521Q mutation enhance the carbohydrate specificity of lectin domain for aGalNAc...

Vasoactive intestinal polypeptide excites medial pontine reticular formation neurons in the brainstem rapid eye movement sleep-induction zone

DEFF Research Database (Denmark)

Kohlmeier, Kristi Anne; Reiner, P B

1999-01-01

Although it has long been known that microinjection of the cholinergic agonist carbachol into the medial pontine reticular formation (mPRF) induces a state that resembles rapid eye movement (REM) sleep, it is likely that other transmitters contribute to mPRF regulation of behavioral states. A key...... candidate is the peptide vasoactive intestinal polypeptide (VIP), which innervates the mPRF and induces REM sleep when injected into this region of the brainstem. To begin understanding the cellular mechanisms underlying this phenomenon, we examined the effects of VIP on mPRF cells using whole-cell patch...... conclude that VIP excites mPRF neurons by activation of a sodium current. This effect is mediated at least in part by G-protein stimulation of adenylyl cyclase, cAMP, and protein kinase A. These data suggest that VIP may play a physiological role in REM induction by its actions on mPRF neurons....

Membrane Disordering is not Sufficient for Membrane Permeabilization by Islet Amyloidogenic Polypeptide: Studies of IAPP(20-29) Fragments

Science.gov (United States)

Brender, Jeffrey R.; Heyl, Deborah L.; Samisetti, Shyamprasad; Kotler, Samuel A.; Osborne, Joshua M.; Pesaru, Ranadheer R.; Ramamoorthy, Ayyalusamy

2013-01-01

A key factor in the development of type II diabetes is the loss of insulin-producing beta-cells. Human islet amyloid polypeptide protein (human-IAPP) is believed to play a crucial role in this process by forming small aggregates that exhibit toxicity by disrupting the cell membrane. The actual mechanism of membrane disruption is complex and appears to involve an early component before fiber formation and later component associated with fiber formation on the membrane. By comparing the peptide-lipid interactions derived from solid-state NMR experiments of two IAPP fragments that bind the membrane and cause membrane disordering to IAPP derived peptides known to cause significant early membrane permeabilization, we show here that membrane disordering is not likely to be sufficient by itself to cause the early membrane permeabilization observed by IAPP, and may play a lesser role in IAPP membrane disruption than expected. PMID:23493863

Cholinergic signaling mediates the effects of xenin-25 on secretion of pancreatic polypeptide but not insulin or glucagon in humans with impaired glucose tolerance.

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Songyan Wang

Full Text Available We previously demonstrated that infusion of an intestinal peptide called xenin-25 (Xen amplifies the effects of glucose-dependent insulinotropic polypeptide (GIP on insulin secretion rates (ISRs and plasma glucagon levels in humans. However, these effects of Xen, but not GIP, were blunted in humans with type 2 diabetes. Thus, Xen rather than GIP signaling to islets fails early during development of type 2 diabetes. The current crossover study determines if cholinergic signaling relays the effects of Xen on insulin and glucagon release in humans as in mice. Fasted subjects with impaired glucose tolerance were studied. On eight separate occasions, each person underwent a single graded glucose infusion- two each with infusion of albumin, Xen, GIP, and GIP plus Xen. Each infusate was administered ± atropine. Heart rate and plasma glucose, insulin, C-peptide, glucagon, and pancreatic polypeptide (PP levels were measured. ISRs were calculated from C-peptide levels. All peptides profoundly increased PP responses. From 0 to 40 min, peptide(s infusions had little effect on plasma glucose concentrations. However, GIP, but not Xen, rapidly and transiently increased ISRs and glucagon levels. Both responses were further amplified when Xen was co-administered with GIP. From 40 to 240 min, glucose levels and ISRs continually increased while glucagon concentrations declined, regardless of infusate. Atropine increased resting heart rate and blocked all PP responses but did not affect ISRs or plasma glucagon levels during any of the peptide infusions. Thus, cholinergic signaling mediates the effects of Xen on insulin and glucagon release in mice but not humans.

Amino Acid Composition, Molecular Weight Distribution and Gel Electrophoresis of Walnut (Juglans regia L. Proteins and Protein Fractionations

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Xiaoying Mao

2014-01-01

Full Text Available As a by-product of oil production, walnut proteins are considered as an additional source of plant protein for human food. To make full use of the protein resource, a comprehensive understanding of composition and characteristics of walnut proteins are required. Walnut proteins have been fractionated and characterized in this study. Amino acid composition, molecular weight distribution and gel electrophoresis of walnut proteins and protein fractionations were analyzed. The proteins were sequentially separated into four fractions according to their solubility. Glutelin was the main component of the protein extract. The content of glutelin, albumin, globulin and prolamin was about 72.06%, 7.54%, 15.67% and 4.73% respectively. Glutelin, albumin and globulin have a balanced content of essential amino acids, except for methionine, with respect to the FAO pattern recommended for adults. SDS-PAGE patterns of albumin, globulin and glutelin showed several polypeptides with molecular weights 14.4 to 66.2 kDa. The pattern of walnut proteins in two-dimension electrophoresis (2-DE showed that the isoelectric point was mainly in the range of 4.8–6.8. The results of size exclusion chromatogram indicated molecular weight of the major components of walnut proteins were between 3.54 and 81.76 kDa.

The polypeptide in Chlamys farreri can protect human dermal fibroblasts from ultraviolet B damage

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Zhang, Yujiang; Zhan, Songmei; Cao, Pengli; Liu, Ning; Chen, Xuehong; Wang, Yuejun; Wang, Chunbo

2005-09-01

To investigate the effect of polypeptide from Chlamys farreri (PCF) on NHDF in vitro, we modeled oxidative damage on normal human dermal fibroblasts (NHDF) exposed to ultraviolet B (UVB). In this study, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) were tested to measure cell viability. Enzymes including superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) and xanthine oxidase (XOD) were determined biochemically. Total antioxidative capacity (T-AOC) and anti-superoxide anion capacity (A-SAC) were also determined. Ultrastructure of fibroblasts was observed under transmission electron microscope. The results showed that: UVB (1.176×10-4 J/cm2) suppressed the growth of fibroblasts and the introduction of PCF (0.25% 1%) before UVB reduced the suppression in a concentration-dependent manner. PCF could enhance the activities of SOD, GSH-PX and T-AOC as well as A-SAC. Also PCF could inhibit XOD activity, while it did not affect CAT activity. Ultrastructure of fibroblasts were damaged after UVB irradiation, concentration-dependent PCF reduced the destructive effect of UVB on cells. These results indicated that PCF can protect human dermal fibroblasts from being harmed by UVB irradiation via its antioxidant proerty.

Integrin-binding elastin-like polypeptide as an in situ gelling delivery matrix enhances the therapeutic efficacy of adipose stem cells in healing full-thickness cutaneous wounds.

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Choi, Seong-Kyoon; Park, Jin-Kyu; Kim, Jung-Hee; Lee, Kyeong-Min; Kim, Enjoo; Jeong, Kyu-Shik; Jeon, Won Bae

2016-09-10

One crucial issue in stem cell therapy used for tissue repair is often the lack of selective carriers to deliver stem cells to the site of injury where the native extracellular matrix is pathologically damaged or lost. Therefore, it is necessary to develop a biomaterial that is permissive to stem cells and is suitable to replace injured or missing matrix. The major aim of this study is to investigate the potential of an RGD-containing elastin-like polypeptide (REP) with the structure TGPG[VGRGD(VGVPG)6]20WPC to engraft adipose stem cells (ASC) to full-thickness excisional wounds in mice. We implanted REP into the wound defects via body temperature-induced in situ aggregation. Engrafted REP exhibited a half-life of 2.6days in the wounds and did not elicit any pathological immune responses. REP itself significantly accelerated wound closure and reepithelialization and upregulated the expression of dermal tissue components. A combined administration of REP and ASC formed a hydrogel-like ASC/REP composite, which provided better neovascularization than the use of ASCs alone and increased the viability of transplanted ASC, improving overall wound healing. In vitro and in vivo mechanistic investigations suggested that REP enhances ASC survival at least in part via the Fak/Src adhesion-induced upregulation of Mek/Erk and PI3K/Akt survival pathways. We conclude that REP is a promising therapeutic agent for the improvement of stem cell-based therapy for enhanced tissue regeneration and repair. Copyright © 2016 Elsevier B.V. All rights reserved.

Ester-Mediated Amide Bond Formation Driven by Wet-Dry Cycles: A Possible Path to Polypeptides on the Prebiotic Earth.

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Forsythe, Jay G; Yu, Sheng-Sheng; Mamajanov, Irena; Grover, Martha A; Krishnamurthy, Ramanarayanan; Fernández, Facundo M; Hud, Nicholas V

2015-08-17

Although it is generally accepted that amino acids were present on the prebiotic Earth, the mechanism by which α-amino acids were condensed into polypeptides before the emergence of enzymes remains unsolved. Here, we demonstrate a prebiotically plausible mechanism for peptide (amide) bond formation that is enabled by α-hydroxy acids, which were likely present along with amino acids on the early Earth. Together, α-hydroxy acids and α-amino acids form depsipeptides-oligomers with a combination of ester and amide linkages-in model prebiotic reactions that are driven by wet-cool/dry-hot cycles. Through a combination of ester-amide bond exchange and ester bond hydrolysis, depsipeptides are enriched with amino acids over time. These results support a long-standing hypothesis that peptides might have arisen from ester-based precursors. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthetic bovine proline-rich-polypeptides generate hydroxyl radicals and fail to protect dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurotoxicity in mice.

Science.gov (United States)

Knaryan, Varduhi H; Samantaray, Supriti; Varghese, Merina; Srinivasan, Ambika; Galoyan, Armen A; Mohanakumar, Kochupurackal P

2006-08-01

Proline-rich-polypeptides (PRPs) isolated from bovine hypothalamus have been shown to render protection against neuronal injury of the brain and spinal cord. We examined two PRPs containing 15 and 10 amino acid residues (PRP-1 and PRP-4 synthetic polypeptide) for their effect, if any, on dopaminergic neuronal damage caused by the parkinsonian neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Effects of these PRPs on hydroxyl radical ((*)OH) generation in a Fenton-like reaction as well as from isolated mitochondria were monitored, employing a sensitive salicylate hydroxylation procedure. Balb/c mice treated (i.p., twice, 16 h apart) with MPTP (30 mg/kg) or PRP-1 (1.6 mg/kg), but not PRP-4 (1.6 mg/kg) showed significant loss of striatal dopamine and norepinephrine as assayed by an HPLC-electrochemical procedure. Pretreatment with the PRPs, 30 min prior to the neurotoxin administration failed to attenuate MPTP-induced striatal dopamine or norepinephrine depletion, but significantly attenuated the MPTP-induced decrease in dopamine turnover. A significant increase in the generation of (*)OH by the PRPs in a Fenton-like reaction or from isolated mitochondria suggests their pro-oxidant action, and explains their failure to protect against MPTP-induced parkinsonism in mice.

Characterization of plasma menbrane polypeptides of trypanosoma from bats Caracterização de polipeptídeos de membrana plasmática de tripanosomas de morcegos

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R. T. Pinho

1989-03-01

Full Text Available Cell surface proteins of Trypanosoma dionisii, Trypanosoma vespertilionis and Trypanosoma sp. (M238 were radiodinated and their distribution both in the detergent-poor (DPP and dertergent-enriched phase (DRP was studied using a phase separation technique in Triton X-114 as well as polyacrylamide gel electrophoresis in sodium dodecyl sulphate (SDS-PAGE. Significant differences were observed in the proteins present in the DRP when the three species of trypanosoma were compared. Two major bands with 88 and 70 KDa were observed in T. sp. (M238 but were not detectable in T. dionisii and T. vespertilionis. Three polypeptides whith 96, 77 and 60 KDa were identified in the DRP of T. vespertilionis. Three major bands with 84, 72 and 60 KDa were observed in the DRP of T. dionisii. Two polypeptides with 34-36 KDa present in the DPP, were observed in the three Trypanosome species analyzed. Our observations show that T. sp. (M238 has characteristic surface polypeptides not found in T. vespertilionis.As proteínas de superfície de Trypanosoma dionisii, Trypanosoma vespertilionis e Trypanosoma sp. (M238 foram radiodinados e sua distribuição na fase rica em detergente (DRP e fase pobre em detergente (DPP foram estudadas pela técnica de separação de fases com Triton X-114 e por eletroforese em gel e policrilamida em presença de dodecil sulfato de sódio (SDS-PAGE. Foram observadas diferenças significativas nas prote��nas presentes na DRP quando a três espécies de tripanosomas foram comparadas. Duas bandas com 88 e 70 KDa foram observadas em T. sp. (M238 e não foram detectadas em T. dionisii e t. vespertilionis. Três polípeptídeos com 96, 77 e 60 KDa foram identificados na fase DRP de T. vespertilionis. Três bandas com 84, 72 e 60 KDa foram visualizadas na fase DRP de T. dionisii. Dois polipeptídeos com 34-36 KDA presentes na fase DPP, foram observados nas três espécies de tripanosomas analisadas. Nossas observações mostraram que T. sp. (M

Chaperones ameliorate beta cell dysfunction associated with human islet amyloid polypeptide overexpression.

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Lisa Cadavez

Full Text Available In type 2 diabetes, beta-cell dysfunction is thought to be due to several causes, one being the formation of toxic protein aggregates called islet amyloid, formed by accumulations of misfolded human islet amyloid polypeptide (hIAPP. The process of hIAPP misfolding and aggregation is one of the factors that may activate the unfolded protein response (UPR, perturbing endoplasmic reticulum (ER homeostasis. Molecular chaperones have been described to be important in regulating ER response to ER stress. In the present work, we evaluate the role of chaperones in a stressed cellular model of hIAPP overexpression. A rat pancreatic beta-cell line expressing hIAPP exposed to thapsigargin or treated with high glucose and palmitic acid, both of which are known ER stress inducers, showed an increase in ER stress genes when compared to INS1E cells expressing rat IAPP or INS1E control cells. Treatment with molecular chaperone glucose-regulated protein 78 kDa (GRP78, also known as BiP or protein disulfite isomerase (PDI, and chemical chaperones taurine-conjugated ursodeoxycholic acid (TUDCA or 4-phenylbutyrate (PBA, alleviated ER stress and increased insulin secretion in hIAPP-expressing cells. Our results suggest that the overexpression of hIAPP induces a stronger response of ER stress markers. Moreover, endogenous and chemical chaperones are able to ameliorate induced ER stress and increase insulin secretion, suggesting that improving chaperone capacity can play an important role in improving beta-cell function in type 2 diabetes.

Pituitary Adenylate-Cyclase Activating Polypeptide Regulates Hunger- and Palatability-Induced Binge Eating

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Matthew M. Hurley

2016-08-01

Full Text Available While pituitary adenylate cyclase activating polypeptide (PACAP signaling in the hypothalamic ventromedial nuclei (VMN has been shown to regulate feeding, a challenge in unmasking a role for this peptide in obesity is that excess feeding can involve numerous mechanisms including homeostatic (hunger and hedonic-related (palatability drives. In these studies, we first isolated distinct feeding drives by developing a novel model of binge behavior in which homeostatic-driven feeding was temporally separated from feeding driven by food palatability. We found that stimulation of the VMN, achieved by local microinjections of AMPA, decreased standard chow consumption in food-restricted rats (e.g., homeostatic feeding; surprisingly, this manipulation failed to alter palatable food consumption in satiated rats (e.g., hedonic feeding. In contrast, inhibition of the nucleus accumbens (NAc, through local microinjections of GABA receptor agonists baclofen and muscimol, decreased hedonic feeding without altering homeostatic feeding. PACAP microinjections produced the site-specific changes in synaptic transmission needed to decrease feeding via VMN or NAc circuitry. PACAP into the NAc mimicked the actions of GABA agonists by reducing hedonic feeding without altering homeostatic feeding. In contrast, PACAP into the VMN mimicked the actions of AMPA by decreasing homeostatic feeding without affecting hedonic feeding. Slice electrophysiology recordings verified PACAP excitation of VMN neurons and inhibition of NAc neurons. These data suggest that the VMN and NAc regulate distinct circuits giving rise to unique feeding drives, but that both can be regulated by the neuropeptide PACAP to potentially curb excessive eating stemming from either drive.

Controlling the Size and Shape of the Elastin-Like Polypeptide based Micelles

Science.gov (United States)

Streletzky, Kiril; Shuman, Hannah; Maraschky, Adam; Holland, Nolan

Elastin-like polypeptide (ELP) trimer constructs make reliable environmentally responsive micellar systems because they exhibit a controllable transition from being water-soluble at low temperatures to aggregating at high temperatures. It has been shown that depending on the specific details of the ELP design (length of the ELP chain, pH and salt concentration) micelles can vary in size and shape between spherical micelles with diameter 30-100 nm to elongated particles with an aspect ratio of about 10. This makes ELP trimers a convenient platform for developing potential drug delivery and bio-sensing applications as well as for understanding micelle formation in ELP systems. Since at a given salt concentration, the headgroup area for each foldon should be constant, the size of the micelles is expected to be proportional to the volume of the linear ELP available per foldon headgroup. Therefore, adding linear ELPs to a system of ELP-foldon should result in changes of the micelle volume allowing to control micelle size and possibly shape. The effects of addition of linear ELPs on size, shape, and molecular weight of micelles at different salt concentrations were studied by a combination of Dynamic Light Scattering and Static Light Scattering. The initial results on 50 µM ELP-foldon samples (at low salt) show that Rh of mixed micelles increases more than 5-fold as the amount of linear ELP raised from 0 to 50 µM. It was also found that a given mixture of linear and trimer constructs has two temperature-based transitions and therefore displays three predominant size regimes.

Vasoactive intestinal polypeptide and peptide histidine methionine. Presence in human follicular fluid and effects on DNA synthesis and steroid secretion in cultured human granulosa/lutein cells

DEFF Research Database (Denmark)

Gräs, S; Ovesen, P; Andersen, A N

1994-01-01

Vasoactive intestinal polypeptide (VIP) and peptide histidine methionine (PHM) originate from the same precursor molecule, prepro VIP. In the present study we examined the concentrations of VIP and PHM in human follicular fluid and their effects on cultured human granulosa/lutein cells. Follicular....../l, respectively. VIP at a concentration of 10 nmol/l caused a significant increase in [3H]thymidine incorporation, and at 1000 nmol/l a significant increase in oestradiol secretion was observed. VIP had no effect on progesterone secretion. PHM at the concentrations tested did not influence any of the activities...

A Biodegradable and Proteolipid Bone Repair Composite,

Science.gov (United States)

1983-11-10

that the positive bone healing response engen - dered in experimental animals from the copolymer of PLA and PGA may be a con- sequence of several factors...residues interaction with host organic matrix could function as a mechanism engendering release from the matrix of certain polypeptides, such as bone...34 Calcif Tissue Int, 34:376-381, 1982. 19. Wuthier RE, "A Review of the Primary Mechanism of Enchondral Calcification with Special Emphasis on the

Reversible end-to-end assembly of gold nanorods using a disulfide-modified polypeptide

International Nuclear Information System (INIS)

Walker, David A; Gupta, Vinay K

2008-01-01

Directing the self-assembly of colloidal particles into nanostructures is of great interest in nanotechnology. Here, reversible end-to-end assembly of gold nanorods (GNR) is induced by pH-dependent changes in the secondary conformation of a disulfide-modified poly(L-glutamic acid) (SSPLGA). The disulfide anchoring group drives chemisorption of the polyacid onto the end of the gold nanorods in an ethanolic solution. A layer of poly(vinyl pyrrolidone) is adsorbed on the positively charged, surfactant-stabilized GNR to screen the surfactant bilayer charge and provide stability for dispersion of the GNR in ethanol. For comparison, irreversible end-to-end assembly using a bidentate ligand, namely 1,6-hexanedithiol, is also performed. Characterization of the modified GNR and its end-to-end linking behavior using SSPLGA and hexanedithiol is performed using dynamic light scattering (DLS), UV-vis absorption spectroscopy and transmission electron microscopy (TEM). Experimental results show that, in a colloidal solution of GNR-SSPLGA at a pH∼3.5, where the PLGA is in an α-helical conformation, the modified GNR self-assemble into one-dimensional nanostructures. The linking behavior can be reversed by increasing the pH (>8.5) to drive the conformation of the polypeptide to a random coil and this reversal with pH occurs rapidly within minutes. Cycling the pH multiple times between low and high pH values can be used to drive the formation of the nanostructures of the GNR and disperse them in solution.

Presence and Effects of Pituitary Adenylate Cyclase Activating Polypeptide Under Physiological and Pathological Conditions in the Stomach

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Dora Reglodi

2018-03-01

Full Text Available Pituitary adenylate cyclase activating polypeptide (PACAP is a multifunctional neuropeptide with widespread occurrence throughout the body including the gastrointestinal system. In the small and large intestine, effects of PACAP on cell proliferation, secretion, motility, gut immunology and blood flow, as well as its importance in bowel inflammatory reactions and cancer development have been shown and reviewed earlier. However, no current review is available on the actions of PACAP in the stomach in spite of numerous data published on the gastric presence and actions of the peptide. Therefore, the aim of the present review is to summarize currently available data on the distribution and effects of PACAP in the stomach. We review data on the localization of PACAP and its receptors in the stomach wall of various mammalian and non-mammalian species, we then give an overview on PACAP’s effects on secretion of gastric acid and various hormones. Effects on cell proliferation, differentiation, blood flow and gastric motility are also reviewed. Finally, we outline PACAP’s involvement and changes in various human pathological conditions.

Influence of Aluminium and EGCG on Fibrillation and Aggregation of Human Islet Amyloid Polypeptide

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Zhi-Xue Xu

2016-01-01

Full Text Available The abnormal fibrillation of human islet amyloid polypeptide (hIAPP has been implicated in the development of type II diabetes. Aluminum is known to trigger the structural transformation of many amyloid proteins and induce the formation of toxic aggregate species. The (−-epigallocatechin gallate (EGCG is considered capable of binding both metal ions and amyloid proteins with inhibitory effect on the fibrillation of amyloid proteins. However, the effect of Al(III/EGCG complex on hIAPP fibrillation is unclear. In the present work, we sought to view insight into the structures and properties of Al(III and EGCG complex by using spectroscopic experiments and quantum chemical calculations and also investigated the influence of Al(III and EGCG on hIAPP fibrillation and aggregation as well as their combined interference on this process. Our studies demonstrated that Al(III could promote fibrillation and aggregation of hIAPP, while EGCG could inhibit the fibrillation of hIAPP and lead to the formation of hIAPP amorphous aggregates instead of the ordered fibrils. Furthermore, we proved that the Al(III/EGCG complex in molar ratio of 1 : 1 as Al(EGCG(H2O2 could inhibit the hIAPP fibrillation more effectively than EGCG alone. The results provide the invaluable reference for the new drug development to treat type II diabetes.

Interaction between amyloid beta peptide and an aggregation blocker peptide mimicking islet amyloid polypeptide.

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Nasrollah Rezaei-Ghaleh

Full Text Available Assembly of amyloid-beta peptide (Aβ into cytotoxic oligomeric and fibrillar aggregates is believed to be a major pathologic event in Alzheimer's disease (AD and interfering with Aβ aggregation is an important strategy in the development of novel therapeutic approaches. Prior studies have shown that the double N-methylated analogue of islet amyloid polypeptide (IAPP IAPP-GI, which is a conformationally constrained IAPP analogue mimicking a non-amyloidogenic IAPP conformation, is capable of blocking cytotoxic self-assembly of Aβ. Here we investigate the interaction of IAPP-GI with Aβ40 and Aβ42 using NMR spectroscopy. The most pronounced NMR chemical shift changes were observed for residues 13-20, while residues 7-9, 15-16 as well as the C-terminal half of Aβ--that is both regions of the Aβ sequence that are converted into β-strands in amyloid fibrils--were less accessible to solvent in the presence of IAPP-GI. At the same time, interaction of IAPP-GI with Aβ resulted in a concentration-dependent co-aggregation of Aβ and IAPP-GI that was enhanced for the more aggregation prone Aβ42 peptide. On the basis of the reduced toxicity of the Aβ peptide in the presence of IAPP-GI, our data are consistent with the suggestion that IAPP-GI redirects Aβ into nontoxic "off-pathway" aggregates.

Evaluation of Urinary Nuclear Matrix Protein-22 as Tumor Marker Versus Tissue Polypeptide Specific Antigen in Bilharzial and Bladder Cancer

International Nuclear Information System (INIS)

Ahmed, W.A.; El-Kabany, H.

2004-01-01

Urinary nuclear matrix protein-22 (NMP-22) and tissue polypeptide specific antigen (TPS) were determined as potential marker for early detection of bladder tumors in patients with high risk (Bilharzial-patients), monitoring and follow up bladder cancer patients. The objective was to determine sensitivity and specificity of markers for bilharzial and cancer lesions. The levels of two parameters were determined pre and post operation. A total of 110 individuals, 20 healthy, 20 bilharzial patients and 70 bladder cancer patients with confirmed diagnosis were investigated. Urine samples were assayed for NMP-22 and TPS test kits. Some bladder cancer patients were selected to follow up. NMP-22 showed highly significant increase (P,0.001) more than TPS (P<0.01) in bladder cancer patients when compared with bilharzial and control group. Overall sensitivity is 7.8% for TPS and 98.5% for NMP-22

Human Immunodeficiency Virus type 1 group M consensus and mosaic envelope glycoproteins

Science.gov (United States)

Korber, Bette T.; Fischer, William; Liao, Hua-Xin; Haynes, Barton F.; Letvin, Norman; Hahn, Beatrice H.

2017-11-21

The disclosure relates to nucleic acids mosaic clade M HIV-1 Env polypeptides and to compositions and vectors comprising same. The nucleic acids are suitable for use in inducing an immune response to HIV-1 in a human.

Sequence-Dependent Self-Assembly and Structural Diversity of Islet Amyloid Polypeptide-Derived β-Sheet Fibrils

International Nuclear Information System (INIS)

Wang, Shih-Ting; Lin, Yiyang; Spencer, Ryan K.; Thomas, Michael R.; Nguyen, Andy I.

2017-01-01

Determining the structural origins of amyloid fibrillation is essential for understanding both the pathology of amyloidosis and the rational design of inhibitors to prevent or reverse amyloid formation. In this work, the decisive roles of peptide structures on amyloid self-assembly and morphological diversity were investigated by the design of eight amyloidogenic peptides derived from islet amyloid polypeptide. Among the segments, two distinct morphologies were highlighted in the form of twisted and planar (untwisted) ribbons with varied diameters, thicknesses, and lengths. In particular, transformation of amyloid fibrils from twisted ribbons into untwisted structures was triggered by substitution of the C-terminal serine with threonine, where the side chain methyl group was responsible for the distinct morphological change. This effect was confirmed following serine substitution with alanine and valine and was ascribed to the restriction of intersheet torsional strain through the increased hydrophobic interactions and hydrogen bonding. We also studied the variation of fibril morphology (i.e., association and helicity) and peptide aggregation propensity by increasing the hydrophobicity of the peptide side group, capping the N-terminus, and extending sequence length. Lastly, we anticipate that our insights into sequence-dependent fibrillation and morphological diversity will shed light on the structural interpretation of amyloidogenesis and development of structure-specific imaging agents and aggregation inhibitors.

Exon organization of the mouse entactin gene corresponds to the structural domains of the polypeptide and has regional homology to the low-density lipoprotein receptor gene

DEFF Research Database (Denmark)

Durkin, M E; Wewer, U M; Chung, A E

1995-01-01

of the mouse entactin gene closely corresponds to the organization of the polypeptide into distinct structural and functional domains. The two amino-terminal globular domains are encoded by three exons each. Single exons encode the two protease-sensitive, O-glycosylated linking regions. The six EGF......Entactin is a widespread basement membrane protein of 150 kDa that binds to type IV collagen and laminin. The complete exon-intron structure of the mouse entactin gene has been determined from lambda genomic DNA clones. The gene spans at least 65 kb and contains 20 exons. The exon organization...

O-Glycosylation Modulates Proprotein Convertase Activation of Angiopoietin-like Protein 3: POSSIBLE ROLE OF POLYPEPTIDE GalNAc-TRANSFERASE-2 IN REGULATION OF CONCENTRATIONS OF PLASMA LIPIDS

DEFF Research Database (Denmark)

Schjoldager, Katrine Ter-Borch Gram; Vester-Christensen, Malene B; Bennett, Eric Paul

2010-01-01

immediately C-terminal (TT(226)). We developed an in vivo model system in CHO ldlD cells that was used to show that O-glycosylation in the processing site blocked processing of ANGPTL3. Genome-wide SNP association studies have identified the polypeptide GalNAc-transferase gene, GALNT2, as a candidate gene...... for low HDL and high triglyceride blood levels. We hypothesized that the GalNAc-T2 transferase performed critical O-glycosylation of proteins involved in lipid metabolism. Screening of a panel of proteins known to affect lipid metabolism for potential sites glycosylated by GalNAc-T2 led to identification...

Convergent synthesis of proteins by kinetically controlled ligation

Science.gov (United States)

Kent, Stephen; Pentelute, Brad; Bang, Duhee; Johnson, Erik; Durek, Thomas

2010-03-09

The present invention concerns methods and compositions for synthesizing a polypeptide using kinetically controlled reactions involving fragments of the polypeptide for a fully convergent process. In more specific embodiments, a ligation involves reacting a first peptide having a protected cysteyl group at its N-terminal and a phenylthioester at its C-terminal with a second peptide having a cysteine residue at its N-termini and a thioester at its C-termini to form a ligation product. Subsequent reactions may involve deprotecting the cysteyl group of the resulting ligation product and/or converting the thioester into a thiophenylester.

Removal of uranyl ions from residual waters using some algae types

International Nuclear Information System (INIS)

Cecal, A.; Palamaru, I.; Humelnicu, D.; Popa, K.; Salaru, V.V.; Rudic, V.; Gulea, A.

1999-01-01

This paper deals with a study on the bioaccumulation of uranyl ions resulted from residual effluents by means of some microbiological collectors: Scenedesmus quadricauda, Anabaena karakumica, Calothrix brevissima, Penicillinium sp, as well as the Glucid extract of Porphyridium cruentum, under various experimental conditions. The retaining degree of the bioaccumulated uranyl ions, as well as the leaching degree, in HCl and H 2 O media, of the same ions previously retained on algae were established. The retaining degree decreases in the series: Scenedesmus quadricauda > Anabaena karakumica > Penicillinium sp > Calothrix brevissima. The leaching effect of bioaccumulated uranyl ions is higher in hydrochloric acid than in water. (author)

High molecular weight PEGylation of human pancreatic polypeptide at position 22 improves stability and reduces food intake in mice

DEFF Research Database (Denmark)

Thieme, V; Jolly, N; Madsen, A N

2016-01-01

BACKGROUND AND PURPOSE: Human pancreatic polypeptide (hPP) is known to suppress appetite and food intake, thereby representing a potential therapeutic approach against obesity and associated metabolic disorders. The aim of this study was to improve hPP stability by covalent PEGylation with diverse...... fasting-induced food intake and bioavailability. KEY RESULTS: In human epithelia and colonic mucosal preparations, activity of the modified hPP peptides depended on the core sequence and latency of the peptides was related to PEG size. Peptides modified with a 22 kDa PEG (PEG22) remained intact in blood...... plasma and on incubation with liver homogenates for more than 96 h. Finally, hPP2-36 , [K(22) (PEG22)]hPP2-36 and [K(22) (PEG22),Q(34) ]hPP significantly reduced cumulative food intake in mice over 16 h after s.c. administration. CONCLUSIONS AND IMPLICATIONS: Modification with PEG22 at position 22...

Comparative assessment of in vitro release kinetics of calcitonin polypeptide from biodegradable microspheres.

Science.gov (United States)

Prabhu, Sunil; Sullivan, Jennifer L; Betageri, Guru V

2002-01-01

The objective of our study was to compare the in vitro release kinetics of a sustained-release injectable microsphere formulation of the polypeptide drug, calcitonin (CT), to optimize the characteristics of drug release from poly-(lactide-co-glycolide) (PLGA) copolymer biodegradable microspheres. A modified solvent evaporation and double emulsion technique was used to prepare the microspheres. Release kinetic studies were carried out in silanized tubes and dialysis bags, whereby microspheres were suspended and incubated in phosphate buffered saline, sampled at fixed intervals, and analyzed for drug content using a modified Lowry protein assay procedure. An initial burst was observed whereby about 50% of the total dose of the drug was released from the microspheres within 24 hr and 75% within 3 days. This was followed by a period of slow release over a period of 3 weeks in which another 10-15% of drug was released. Drug release from the dialysis bags was more gradual, and 50% CT was released only after 4 days and 75% after 12 days of release. Scanning electron micrographs revealed spherical particles with channel-like structures and a porous surface after being suspended in an aqueous solution for 5 days. Differential scanning calorimetric studies revealed that CT was present as a mix of amorphous and crystalline forms within the microspheres. Overall, these studies demonstrated that sustained release of CT from PLGA microspheres over a 3-week period is feasible and that release of drug from dialysis bags was more predictable than from tubes.

Acid dissociation constant and apparent nucleophilicity of lysine-501 of the alpha-polypeptide of sodium and potassium ion activated adenosinetriphosphatase

International Nuclear Information System (INIS)

Xu, K.Y.

1989-01-01

A combination of competitive labeling with [ 3 H]acetic anhydride and immunoaffinity chromatography is described that permits the assignment of the acid dissociation constant and the absolute nucleophilicity of individual lysines in a native enzyme. The acid dissociation constant of lysine-501 of the alpha-polypeptide in native (Na+ + K+)-ATPase was determined. This lysine had a normal pKa of 10.4. The rate constant for the reaction of the free base of lysine-501 with acetic anhydride at 10 degrees C is 400 M-1 s-1. This value is only 30% that for a fully accessible lysine in a protein. The lower than normal apparent nucleophilicity suggests that lysine-501 is hindered from reacting with its intrinsic nucleophilicity by the tertiary structure of the enzyme and is consistent with its location within a pocket that forms the active site upon the surface of the native protein

Xylitol vs glucose: Effect on the rate of gastric emptying and motilin, insulin, and gastric inhibitory polypeptide release

International Nuclear Information System (INIS)

Salminen, E.K.; Salminen, S.J.; Porkka, L.; Kwasowski, P.; Marks, V.; Koivistoinen, P.E.

1989-01-01

The effect of xylitol and glucose on the rate of gastric emptying and intestinal transit and on motilin, gastric inhibitory polypeptide (GIP), and insulin release were studied in human volunteers. A single oral dose of 200 mL water containing 30 g glucose or 30 g xylitol, mixed with a 99m technetium-tin (99mTc-Sn) colloid, was used. Similar dosing without the label was used in motilin, GIP, and insulin studies. Xylitol decreased the rate of gastric emptying but concomitantly accelerated intestinal transit compared with glucose. The half-times for gastric emptying were 77.5 +/- 4.6 and 39.8 +/- 3.4 min after ingestion of xylitol and glucose solutions, respectively. Glucose suppressed motilin and stimulated GIP secretion; xylitol stimulated motilin secretion but had no effect on GIP, which is currently the main candidate for the role of enterogastrone. The accelerated intestinal transit and increase in plasma motilin observed after xylitol ingestion were thought to be causally related to the diarrhea and gastrointestinal discomfort produced by it

Pituitary adenylate cyclase-activating polypeptide type 1 (PAC1) receptor is expressed during embryonic development of the earthworm.

Science.gov (United States)

Boros, Akos; Somogyi, Ildikó; Engelmann, Péter; Lubics, Andrea; Reglodi, Dóra; Pollák, Edit; Molnár, László

2010-03-01

Pituitary adenylate cyclase activating polypeptide (PACAP)-like molecules have been shown to be present in cocoon albumin and in Eisenia fetida embryos at an early developmental stage (E1) by immunocytochemistry and radioimmunoassay. Here, we focus on detecting the stage at which PAC1 receptor (PAC1R)-like immunoreactivity first appears in germinal layers and structures, e.g., various parts of the central nervous system (CNS), in developing earthworm embryos. PAC1R-like immunoreactivity was revealed by Western blot and Far Western blot as early as the E2 developmental stage, occurring in the ectoderm and later in specific neurons of the developing CNS. Labeled CNS neurons were first seen in the supraesophageal ganglion (brain) and subsequently in the subesophageal and ventral nerve cord ganglia. Ultrastructurally, PAC1Rs were located mainly on plasma membranes and intracellular membranes, especially on cisternae of the endoplasmic reticulum. Therefore, PACAP-like compounds probably influence the differentiation of germinal layers (at least the ectoderm) and of some neurons and might act as signaling molecules during earthworm embryonic development.

Diagnostic value of tissue polypeptide specific antigen in serum for patients with pancreatic carcinoma

International Nuclear Information System (INIS)

Lu Yun; Liu Xingdang; Li Ding; Dei Shouzhen

2009-01-01

Objective: To compare the diagnostic value of tissue polypeptide specific antigen (TPS) in serum, with the carbohydrate antigens (CA) 19-9, CA50, CA125 and CA242 in pancreatic carcinoma. Methods: Serum TPS was measured with enzyme linked immunosorbent assay (ELISA). CA19-9 and CA125 were measured with chemiluminescent immunoassay. CAS0 and CA242 were measured with immunoradiometric assay in 33 patients with pancreatitis, 34 patients with pathologically proven pancreatic carcinoma, and 35 patients with non-pancreatic malignancies. Statistic analysis was carried out with SPSS 9.0 software. Results: Patients with pancreatic carcinoma had relatively higher levels of TPS [(386.5 ± 315. 1) U/L] and CA19-9 [(10 820.9 ± 389.7) kU/L] when compared with patients with pancreatitis [(86. 2 ± 28.1) U/L and (61.5 ± 24.7) kU/L, respectively; F = 936. 42, P < 0. 001 ; F = 2217. 09, P < 0. 001], with a sensitivity and a specificity of 70.6% (48/68) and 57.4% (39/68), respectively, for TPS, and 82.4% (28/34) and 77.9% (53/68), respectively, for CA19-9. Diagnostic performance was further improved when TPS was assayed in combination with CA19-9, CA50, CA125 and CA242. Conclusion: Serum TPS has an incremental value in complementing CA19-9 in the diagnosis of pancreatic carcinoma. (authors)

Characterization of Pea Vicilin. 1. Denoting Convicilin as the α-Subunit of the Pisum Vicilin Family

NARCIS (Netherlands)

O'Kane, F.E.; Happe, R.P.; Vereijken, J.M.; Gruppen, H.; Boekel, M.A.J.S. van

2004-01-01

Vicilin, a major globulin protein of pea that has been described as "extremely heterogeneous in terms of its polypeptide composition", was extracted from pea flour under alkaline conditions and subsequently fractionated by salt under acid conditions. This procedure induced the separation of vicilin

Anticoagulant and calcium-binding properties of high molecular weight derivatives of human fibrinogen, produced by plasmin (fragments X)

NARCIS (Netherlands)

Nieuwenhuizen, W.; Gravesen, M.

1981-01-01

Early plasmin degradation products (X fragments) of human fibrinogen were prepared in the presence of calcium-ions or EGTA, and purified on Sepharose 6B-CL. X fragments were characterized with respect to amino-terminal amino acids, polypeptide-chain composition, anticlotting properties and

High plasma levels of islet amyloid polypeptide in young with new-onset of type 1 diabetes mellitus.

Directory of Open Access Journals (Sweden)

Johan F Paulsson

Full Text Available AIMS/HYPOTHESIS: Islet amyloid polypeptide (IAPP is a beta cell hormone secreted together with insulin upon glucose stimulation. IAPP participates in normal glucose regulation, but IAPP is also known for its ability to misfold and form islet amyloid. Amyloid fibrils form through smaller cell toxic intermediates and deposited amyloid disrupts normal islet architecture. Even though IAPP and amyloid formation are much discussed in type 2 diabetes, our aim was to study the significance of IAPP in type 1 diabetes. RESULTS: Plasma IAPP levels in children and adolescents with newly diagnosed type 1 diabetes (n = 224 were analysed and concentrations exceeding 100 pmol/L (127.2-888.7 pmol/L were found in 11% (25/224. The IAPP increase did not correlate with C-peptide levels. CONCLUSIONS/INTERPRETATION: Plasma levels of IAPP and insulin deviate in a subpopulation of young with newly-diagnosed type 1 diabetes. The determined elevated levels of IAPP might increase the risk for IAPP misfolding and formation of cell toxic amyloid in beta cells. This finding add IAPP-aggregation to the list over putative pathological factors causing type 1 diabetes.

Therapeutic potential of the original incretin hormone glucose-dependent insulinotropic polypeptide: diabetes, obesity, osteoporosis and Alzheimer's disease?

Science.gov (United States)

Irwin, Nigel; Gault, Victor; Flatt, Peter R

2010-09-01

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone that potentiates nutrient-induced insulin release. To date, the physiological importance of GIP has received much less attention than its younger sister incretin hormone glucagon-like peptide-1. Thus, it is worthwhile to refocus on this important and somewhat neglected incretin hormone. The potential role of GIP as a treatment option for type 2 diabetes is highlighted. Furthermore, the use of GIP as a new therapeutic option for obesity, osteoporosis and cognitive impairment is also considered. Long-acting GIP receptor agonists offer a potential new class of antidiabetic drugs. Furthermore, recent observations suggest an as yet untapped potential for GIP agonists in the treatment of osteoporosis and cognitive impairment. In addition, GIP is known to play a role in lipid metabolism and fat deposition. Accordingly, both genetic and chemical ablation of GIP signalling in mice with obesity-diabetes can protect against, or reverse, many of the obesity-associated metabolic disturbances. This review focuses on preclinical data generated to date. GIP-based therapeutics have potential for the treatment of type 2 diabetes and obesity, with the possibility of further beneficial actions in osteoporosis and cognitive decline.

Functional conservation of the hydrophobic domain of polypeptide 3AB between human rhinovirus and poliovirus

International Nuclear Information System (INIS)

Towner, Jonathan S.; Brown, David M.; Nguyen, Joseph H.C.; Semler, Bert L.

2003-01-01

In this study we exchanged portions of the poliovirus type 1 (PV1) hydrophobic domain within the membrane-associated polypeptide 3AB for the analogous sequences from human rhinovirus 14 (HRV14). The sequence exchanges were based upon a previous report in which the 22 amino acid hydrophobic region was subdivided into two domains, I and II, the latter of which was shown to be required for membrane association (J. Biol. Chem. 271 (1996), 26810). Using these divisions, the HRV14 sequences were cloned into the complete poliovirus type 1 cDNA sequence. RNAs transcribed from these cDNAs were transfected into HeLa cell monolayers and used in HeLa cell-free translation/replication assays. The data indicated that 3AB sequences from PV1 and HRV14 are interchangeable; however, the substitutions cause a range of significant RNA replication defects, and in some cases, protein processing defects. Following transfection of RNAs encoding the domain substitutions into HeLa cell monolayers, virus isolates were harvested, and the corresponding viral RNAs were sequenced. The sequence data revealed that for the carboxy-terminal domain substitutions (domain II), multiple nucleotide changes were identified in the first, second, and third positions of different codons. In addition, the data indicated that for one of the PV1/HRV14 chimeras to replicate, compensatory mutations within poliovirus protein 2B may be required

PR-957, a selective inhibitor of immunoproteasome subunit low-MW polypeptide 7, attenuates experimental autoimmune neuritis by suppressing Th17-cell differentiation and regulating cytokine production.

Science.gov (United States)

Liu, Haijie; Wan, Chunxiao; Ding, Yanan; Han, Ranran; He, Yating; Xiao, Jinting; Hao, Junwei

2017-04-01

Experimental autoimmune neuritis (EAN) is a CD4 + T-cell-mediated autoimmune inflammatory demyelinating disease of the peripheral nervous system. It has been replicated in an animal model of human inflammatory demyelinating polyradiculoneuropathy, Guillain-Barré syndrome. In this study, we evaluated the therapeutic efficacy of a selective inhibitor of the immunoproteasome subunit, low-MW polypeptide 7 (PR-957) in rats with EAN. Our results showed that PR-957 significantly delayed onset day, reduced severity and shortened duration of EAN, and alleviated demyelination and inflammatory infiltration in sciatic nerves. In addition to significantly regulating expression of the cytokine profile, PR-957 treatment down-regulated the proportion of proinflammatory T-helper (T h )17 cells in sciatic nerves and spleens of rats with EAN. Data presented show the role of PR-957 in the signal transducer and activator of transcription 3 (STAT3) pathway. PR-957 not only decreased expression of IL-6 and IL-23 but also led to down-regulation of STAT3 phosphorylation in CD4 + T cells. Regulation of the STAT3 pathway led to a reduction in retinoid-related orphan nuclear receptor γ t and IL-17 production. Furthermore, reduction of STAT3 phosphorylation may have directly suppressed T h 17-cell differentiation. Therefore, our study demonstrates that PR-957 could potently alleviate inflammation in rats with EAN and that it may be a likely candidate for treating Guillain-Barré syndrome.-Liu, H., Wan, C., Ding, Y., Han, R., He, Y., Xiao, J., Hao, J. PR-957, a selective inhibitor of immunoproteasome subunit low-MW polypeptide 7, attenuates experimental autoimmune neuritis by suppressing T h 17-cell differentiation and regulating cytokine production. © FASEB.

Heterogeneity of rabbit endogenous pyrogens is not attributable to glycosylated variants of a single polypeptide chain.

Science.gov (United States)

Murphy, P A; Cebula, T A; Windle, B E

1981-10-01

Rabbit endogenous pyrogens were of about the same molecular size, but showed considerable heterogeneity of their isoelectric points. We attempted to show that this heterogeneity was attributable to variable glycosylation of a single polypeptide chain. When peritoneal exudate cells were stimulated to make pyrogens in the presence of 2-deoxy-D-glucose, there was a relatively trivial suppression of pyrogen release, and analysis by isoelectric focusing showed parallel inhibition of secretion of all the forms of endogenous pyrogen. When cells were stimulated in the presence of 3H-labeled amino acids and 14C-labeled glucosamine or glucose, the purified pyrogens were labeled with 3H but not with 14C. Macrophage membrane preparations were made which contained glycosyl transferases and could transfer sugar residues from sugar nucleotides to deglycosylated fetuin. These macrophage membrane preparations did not transfer sugars to the pI 7.3 endogenous pyrogen. Treatment of endogenous pyrogens with neuraminidase or with periodate produced no evidence suggesting that the pyrogens were glycosylated. Last, endogenous pyrogens did not bind to any of four lectins with different carbohydrate specificities. This evidence suggests that the heterogeneity of rabbit endogenous pyrogens is not attributable to glycosylation and must have some other cause.

Effects of gamma irradiation on chemical composition and ruminal protein degradation of canola meal

Science.gov (United States)

Shawrang, P.; Nikkhah, A.; Zare-Shahneh, A.; Sadeghi, A. A.; Raisali, G.; Moradi-Shahrebabak, M.

2008-07-01

Gamma irradiation of canola meal (at doses of 25, 50 and 75 kGy) could alter its ruminal protein degradation characteristics by cross-linking of the polypeptide chains. This processing resulted in decrease (linear effect, Pruminal protein degradation and increase (linear effect, Pruminant nutrition.

Unraveling origins of the heterogeneous curvature dependence of polypeptide interactions with carbon nanostructures.

Science.gov (United States)

Jana, Asis K; Tiwari, Mrityunjay K; Vanka, Kumar; Sengupta, Neelanjana

2016-02-17

Emerging nanotechnology has rapidly broadened interfacial prospects of biological molecules with carbon nanomaterials (CNs). A prerequisite for effectively harnessing such hybrid materials is a multi-faceted understanding of their complex interfacial interactions as functions of the physico-chemical characteristics and the surface topography of the individual components. In this article, we address the origins of the curvature dependence of polypeptide adsorption on CN surfaces (CNSs), a phenomenon bearing an acute influence upon the behavior and activity of CN-protein conjugates. Our benchmark molecular dynamics (MD) simulations with the amphiphilic full-length amyloid beta (Aβ) peptide demonstrate that protein adsorption is strongest on the concave (inner) CN surface, weakest on the convex (outer) surface, and intermediary on the planar surface, in agreement with recent experimental reports. The curvature effects, however, are found to manifest non-uniformly between the amino acid subtypes. To understand the underlying interplay of the chemical nature of the amino acids and surface topography of the CNs, we performed high-level quantum chemical (QM) calculations with amino acid analogs (AAA) representing their five prominent classes, and convex, concave and planar CN fragments. Molecular electrostatic potential maps reveal pronounced curvature dependence in the mixing of electron densities, and a resulting variance in the stabilization of the non-covalently bound molecular complexes. Interestingly, our study revealed that the interaction trends of the high-level QM calculations were captured well by the empirical force field. The findings in this study have important bearing upon the design of carbon based bio-nanomaterials, and additionally, provide valuable insights into the accuracy of various computational techniques for probing non-bonded interfacial interactions.

Drosophila Melanogaster as a Model System for Studies of Islet Amyloid Polypeptide Aggregation

Science.gov (United States)

Schultz, Sebastian Wolfgang; Nilsson, K. Peter R.; Westermark, Gunilla Torstensdotter

2011-01-01

Background Recent research supports that aggregation of islet amyloid polypeptide (IAPP) leads to cell death and this makes islet amyloid a plausible cause for the reduction of beta cell mass, demonstrated in patients with type 2 diabetes. IAPP is produced by the beta cells as a prohormone, and proIAPP is processed into IAPP by the prohormone convertases PC1/3 and PC2 in the secretory granules. Little is known about the pathogenesis for islet amyloid and which intracellular mechanisms are involved in amyloidogenesis and induction of cell death. Methodology/Principal Findings We have established expression of human proIAPP (hproIAPP), human IAPP (hIAPP) and the non-amyloidogenic mouse IAPP (mIAPP) in Drosophila melanogaster, and compared survival of flies with the expression driven to different cell populations. Only flies expressing hproIAPP in neurons driven by the Gal4 driver elavC155,Gal4 showed a reduction in lifespan whereas neither expression of hIAPP or mIAPP influenced survival. Both hIAPP and hproIAPP expression caused formation of aggregates in CNS and fat body region, and these aggregates were both stained by the dyes Congo red and pFTAA, both known to detect amyloid. Also, the morphology of the highly organized protein granules that developed in the fat body of the head in hIAPP and hproIAPP expressing flies was characterized, and determined to consist of 15.8 nm thick pentagonal rod-like structures. Conclusions/Significance These findings point to a potential for Drosophila melanogaster to serve as a model system for studies of hproIAPP and hIAPP expression with subsequent aggregation and developed pathology. PMID:21695120

Pancreatic Polypeptide Cell Proliferation in the Pancreas and Duodenum Coexisting in a Patient With Pancreatic Adenocarcinoma Treated With a GLP-1 Analog.

Science.gov (United States)

Talmon, Geoffrey A; Wren, J David; Nguyen, Christophe L; Pour, Parviz M

2017-07-01

A partial pancreaticogastrodudenectomy was performed on a 66-year old man with type 2 diabetes mellitus because of an invasive, moderately differentiated adenocarcinoma in the head of the pancreas. In the adjacent grossly normal tissue of the uncinate process, there was a massive proliferation of pancreatic polypeptide (PP) cells confined to this region and showed invasive pattern. Strikingly, in the heaped area of his duodenum, there was a strikingly large number of PP, glucagon, a few insulin cells in a mini-islet-like patterns composed of glucagon and insulin cells. Among the etiological factors, the possible long-lasting effects of the GLP-1 analog, with which the patient was treated, are discussed. This is the first report in the literature of both the coexistence of a pancreatic adenocarcinoma and invasive PPoma and the occurrence of PP and insulin cells in human duodenal mucosa.

Polypeptide nanogels with hydrophobic moieties in the cross-linked ionic cores: Synthesis, characterization and implications for anticancer drug delivery

Science.gov (United States)

Kim, Jong Oh; Oberoi, Hardeep S.; Desale, Swapnil; Kabanov, Alexander V.; Bronich, Tatiana K.

2014-01-01

Polymer nanogels have gained considerable attention as a potential platform for drug delivery applications. Here we describe the design and synthesis of novel polypeptide-based nanogels with hydrophobic moieties in the cross-linked ionic cores. Diblock copolymer, poly(ethylene glycol)-b-poly(L-glutamic acid), hydrophobically modified with L-phenylalanine methyl ester moieties was used for controlled template synthesis of nanogels with small size (ca. 70 nm in diameter) and narrow particle size distribution. Steady-state and time-resolved fluorescence studies using coumarin C153 indicated the existence of hydrophobic domains in the ionic cores of the nanogels. Stable doxorubicin-loaded nanogels were prepared at high drug capacity (30 w/w%). We show that nanogels are enzymatically-degradable leading to accelerated drug release under simulated lysosomal acidic pH. Furthermore, we demonstrate that the nanogel-based formulation of doxorubicin is well tolerated and exhibit an improved antitumor activity compared to a free doxorubicin in an ovarian tumor xenograft mouse model. Our results signify the point to a potential of these biodegradable nanogels as attractive carriers for delivery of chemotherapeutics. PMID:23998716

Rapid and simple purification of elastin-like polypeptides directly from whole cells and cell lysates by organic solvent extraction.

Science.gov (United States)

VerHeul, Ross; Sweet, Craig; Thompson, David H

2018-03-26

Elastin-like polypeptides (ELP) are a well-known class of proteins that are being increasingly utilized in a variety of biomedical applications, due to their beneficial physicochemical properties. A unifying feature of ELP is their demonstration of a sequence tunable inverse transition temperature (Tt) that enables purification using a simple, straightforward process called inverse transition cycling (ITC). Despite the utility of ITC, the process is inherently limited to ELP with an experimentally accessible Tt. Since the underlying basis for the ELP Tt is related to its high overall hydrophobicity, we anticipated that ELP would be excellent candidates for purification by organic extraction. We report the first method for rapidly purifying ELP directly from whole E. coli cells or clarified lysates using pure organic solvents and solvent mixtures, followed by aqueous back extraction. Our results show that small ELP and a large ELP-fusion protein can be isolated in high yield from whole cells or cell lysates with greater than 95% purity in less than 30 min and with very low levels of LPS and DNA contamination.

Impact of acute stress on antimicrobial polypeptides mRNA copy number in several tissues of marine sea bass (Dicentrarchus labrax

Directory of Open Access Journals (Sweden)

Terova Genciana

2011-12-01

Full Text Available Abstract Background In comparison to higher vertebrates, fish are thought to rely heavily on innate immune system for initial protection against pathogen invasion because their acquired immune system displays a considerably poor immunological memory, and short-lived secondary response. The endogenous antimicrobial polypeptides (AMPPs directly and rapidly killing pathogens such as bacteria, fungi, parasites, and viruses are included within the realm of innate defenses. In addition to piscidins, AMPPs that in recent years have been shown to be commonly linked to innate defense, are histones and their polypeptide fragments, and peptides derived from the respiratory protein hemoglobin. There is evidence that a number of stresses lead to significant regulation of AMPPs and thus their monitoring could be a highly sensitive measure of health status and risk of an infectious disease outbreak, which is a major impediment to the continued success of virtually all aquaculture enterprises and is often the most significant cause of economic losses. Results We firstly isolated and deposited in Genbank database the cDNA sequences encoding for hemoglobin-β-like protein (Hb-LP [GeneBank: JN410659], H2B histone-like protein 1 (HLP1 GenBank: JN410660], and HLP2 [GenBank: JN410661]. The "de novo" prediction of the three-dimensional structures for each protein is presented. Phylogenetic trees were constructed on Hb-LP, HLP1, and HLP2 sequences of sea bass and those of other teleost, avian, reptiles, amphibian and mammalian species. We then used real time RT-PCR technology to monitor for the first time in sea bass, dynamic changes in mRNA copy number of Hb-LP, HLP1, HLP2, and dicentracin in gills, skin, eyes, stomach and proximal intestine in response to acute crowding/confinement stress. We showed that acute crowding stress induces an increase in the expression levels of the aforementioned genes, in gills and skin of sea bass, but not in other tissues, and that

Polypeptide-based nanogels co-encapsulating a synergistic combination of doxorubicin with 17-AAG show potent anti-tumor activity in ErbB2-driven breast cancer models.

Science.gov (United States)

Desale, Swapnil S; Raja, Srikumar M; Kim, Jong Oh; Mohapatra, Bhopal; Soni, Kruti S; Luan, Haitao; Williams, Stetson H; Bielecki, Timothy A; Feng, Dan; Storck, Matthew; Band, Vimla; Cohen, Samuel M; Band, Hamid; Bronich, Tatiana K

2015-06-28

ErbB2-driven breast cancers constitute 20-25% of the cases diagnosed within the USA. The humanized anti-ErbB2 monoclonal antibody, Trastuzumab (Herceptin™; Genentech), with chemotherapy is the current standard of treatment. Novel agents and strategies continue to be explored, given the challenges posed by Trastuzumab-resistance development in most patients. The HSP90 inhibitor, 17-allylaminodemethoxygeldanamycin (17-AAG), which induces ErbB2 degradation and attenuates downstream oncogenic signaling, is one such agent that showed significant promise in early phase I and II clinical trials. Its low water solubility, potential toxicities and undesirable side effects observed in patients, partly due to the Cremophor-based formulation, have been discouraging factors in the advancement of this promising drug into clinical use. Encapsulation of 17-AAG into polymeric nanoparticle formulations, particularly in synergistic combination with conventional chemotherapeutics, represents an alternative approach to overcome these problems. Herein, we report an efficient co-encapsulation of 17-AAG and doxorubicin, a clinically well-established and effective modality in breast cancer treatment, into biodegradable and biocompatible polypeptide-based nanogels. Dual drug-loaded nanogels displayed potent cytotoxicity in a breast cancer cell panel and exerted selective synergistic anticancer activity against ErbB2-overexpressing breast cancer cell lines. Analysis of ErbB2 degradation confirmed efficient 17-AAG release from nanogels with activity comparable to free 17-AAG. Furthermore, nanogels containing both 17-AAG and doxorubicin exhibited superior antitumor efficacy in vivo in an ErbB2-driven xenograft model compared to the combination of free drugs. These studies demonstrate that polypeptide-based nanogels can serve as novel nanocarriers for encapsulating 17-AAG along with other chemotherapeutics, providing an opportunity to overcome solubility issues and thereby exploit its full

In situ targeting TEM8 via immune response and polypeptide recognition by wavelength-modulated surface plasmon resonance biosensor

Science.gov (United States)

Wang, Yimin; Luo, Zewei; Liu, Kunping; Wang, Jie; Duan, Yixiang

2016-01-01

There is an increasing interest in real-time and in situ monitoring of living cell activities in life science and medicine. This paper reports a whole cell sensing protocol over the interface of Au film coupled in a wavelength-modulated surface plasmon resonance (WMSPR) biosensor. With dual parabolic mirrors integrated in the sensor, the compact and miniaturized instrument shows satisfactory refractive index sensitivity (2220 nm/RIU) and a high resolution of resonance wavelength shift of 0.3 nm to liquid samples. The affinity interactions between the biomarker of human tumor endothelial marker 8 (TEM8) and antibody (Ab) or specific polypeptide (PEP) were firstly introduced to WMSPR biosensor analysis. Both the interaction events of Ab-cell and PEP-cell over the Au film interface can be recognized by the sensor and the balance time of interactions is about 20 min. The concentration range of Ab for quantitative monitoring of the TEM8 expression on human colon carcinoma SW620 cells was investigated. The present low-cost and time-saving method provides a time resolution of binding specificity between Ab/PEP and TEM8 for real-time analysis of antigen on living tumor cell surface. PMID:26822761

A positive 111in-pentetreotide scan in a patient with a pancreatic polypeptide secreting tumour

International Nuclear Information System (INIS)

Stanton, K.; Cehic, G.

2003-01-01

Full text: A 55-year-old male presented to our department with a known polypeptide secreting pancreatic tumour. An 111 In-pentetreotide scan (OctreoScan) was performed to determine whether the tumour expressed somatostatin receptors (SR) and thereby aid in therapy planning. 120 MBq 111 In-pentetreotide was administered intravenously. Images were acquired at 4 and 30 hours. Whole body images were acquired with spot views and tomography of the liver at 30 hours. Images showed intense uptake of the tracer in the lobular midline pancreatic mass. There was also uptake in multiple liver metastases. 111 In-pentetreotide is a synthetic somatostatin analogue and its uptake demonstrates the presence of SR on tumour cells, especially those of a neuro-endocrine nature. A 123 I Metaiodobenzylguanidine (MIBG) scan was also performed to determine whether the more widely available MIBG therapy would be appropriate for this patient. This scan was negative. The patient has received 3 cycles of chemotherapy with Streptozotocin and 5-fluorouracil. He has had a good partial response to therapy as demonstrated on CT scan. The patient is currently clinically well, his symptoms have resolved and weight stabilised. Good biochemical response to chemotherapy is indicated by halved pancreatic peptide levels. To date chemotherapy has been the mainstay of therapy for neuroendocrine tumours. Radioimmunotherapy (targeted to SR positive tumours) is currently being investigated as a therapy alternative and may be a future treatment option. Copyright (2003) The Australian and New Zealand Society of Nuclear Medicine Inc

Prebiotic Fiber Increases Hepatic Acetyl CoA Carboxylase Phosphorylation and Suppresses Glucose-Dependent Insulinotropic Polypeptide Secretion More Effectively When Used with Metformin in Obese Rats1,2

Science.gov (United States)

Pyra, Kim A.; Saha, Dolan C.; Reimer, Raylene A.

2013-01-01

Independently, metformin (MET) and the prebiotic, oligofructose (OFS), have been shown to increase glucagon-like peptide (GLP-1) secretion. Our objective was to determine whether using OFS as an adjunct with MET augments GLP-1 secretion in obese rats. Male, diet-induced obese Sprague Dawley rats were randomized to: 1) high-fat/-sucrose diet [HFHS; control (C); 20% fat, 50% sucrose wt:wt]; 2) HFHS+10% OFS (OFS); 3) HFHS + MET [300 mg/kg/d (MET)]; 4) HFHS+10% OFS+MET (OFS +MET). Body composition, glycemia, satiety hormones, and mechanisms related to dipeptidyl peptidase 4 (DPP4) activity in plasma, hepatic AMP-activated protein kinase (AMPK; Western blots), and gut microbiota (qPCR) were examined. Direct effects of MET and SCFA were examined in human enteroendocrine cells. The interaction between OFS and MET affected fat mass, hepatic TG, secretion of glucose-dependent insulinotropic polypeptide (GIP) and leptin, and AMPKα2 mRNA and phosphorylated acetyl CoA carboxylase (pACC) levels (P < 0.05). Combined, OFS and MET reduced GIP secretion to a greater extent than either treatment alone (P < 0.05). The hepatic pACC level was increased by OFS+MET by at least 50% above all other treatments, which did not differ from each other (P < 0.05). OFS decreased plasma DPP4 activity (P < 0.001). Cecal Bifidobacteria (P < 0.001) were markedly increased and C. leptum decreased (P < 0.001) with OFS consumption. In human enteroendocrine cells, the interaction between MET and SCFA affected GLP-1 secretion (P < 0.04) but was not associated with higher GLP-1 than the highest individual doses. In conclusion, the combined actions of OFS and MET were associated with important interaction effects that have the potential to improve metabolic outcomes associated with obesity. PMID:22223580

Human elastin polypeptides improve the biomechanical properties of three-dimensional matrices through the regulation of elastogenesis.

Science.gov (United States)

Boccafoschi, Francesca; Ramella, Martina; Sibillano, Teresa; De Caro, Liberato; Giannini, Cinzia; Comparelli, Roberto; Bandiera, Antonella; Cannas, Mario

2015-03-01

The replacement of diseased tissues with biological substitutes with suitable biomechanical properties is one of the most important goal in tissue engineering. Collagen represents a satisfactory choice for scaffolds. Unfortunately, the lack of elasticity represents a restriction to a wide use of collagen for several applications. In this work, we studied the effect of human elastin-like polypeptide (HELP) as hybrid collagen-elastin matrices. In particular, we studied the biomechanical properties of collagen/HELP scaffolds considering several components involved in ECM remodeling (elastin, collagen, fibrillin, lectin-like receptor, metalloproteinases) and cell phenotype (myogenin, myosin heavy chain) with particular awareness for vascular tissue engineering applications. Elastin and collagen content resulted upregulated in collagen-HELP matrices, even showing an improved structural remodeling through the involvement of proteins to a ECM remodeling activity. Moreover, the hybrid matrices enhanced the contractile activity of C2C12 cells concurring to improve the mechanical properties of the scaffold. Finally, small-angle X-ray scattering analyses were performed to enable a very detailed analysis of the matrices at the nanoscale, comparing the scaffolds with native blood vessels. In conclusion, our work shows the use of recombinant HELP, as a very promising complement able to significantly improve the biomechanical properties of three-dimensional collagen matrices in terms of tensile stress and elastic modulus. © 2014 Wiley Periodicals, Inc.

Effects of pituitary adenylate cyclase activating polypeptide in the urinary system, with special emphasis on its protective effects in the kidney.

Science.gov (United States)

Reglodi, Dora; Kiss, Peter; Horvath, Gabriella; Lubics, Andrea; Laszlo, Eszter; Tamas, Andrea; Racz, Boglarka; Szakaly, Peter

2012-04-01

Pituitary adenylate cyclase activating polypeptide (PACAP) is a widespread neuropeptide with diverse effects in the nervous system and peripheral organs. One of the most well-studied effects of PACAP is its cytoprotective action, against different harmful stimuli in a wide variety of cells and tissues. PACAP occurs in the urinary system, from the kidney to the lower urinary tract. The present review focuses on the nephroprotective effects of PACAP and summarizes data obtained regarding the protective effects of PACAP in different models of kidney pathologies. In vitro data show that PACAP protects tubular cells against oxidative stress, myeloma light chain, cisplatin, cyclosporine-A and hypoxia. In vivo data provide evidence for its protective effects in ischemia/reperfusion, cisplatin, cyclosporine-A, myeloma kidney injury, diabetic nephropathy and gentamicin-induced kidney damage. Results accumulated on the renoprotective effects of PACAP suggest that PACAP is an emerging candidate for treatment of human kidney pathologies. Copyright © 2011 Elsevier Ltd. All rights reserved.

Parameters affecting the inhibition of Candida albicans GDH 2023 and GRI 2773 blastospore viability by purified synthetic salivary histidine-rich polypeptides.

Science.gov (United States)

Santarpia, R P; Cho, M I; Pollock, J J

1990-08-01

Purified synthetic salivary histidine-rich polypeptides, HRPs 2, 3, 4, 5, and 6, were observed to inhibit Candida albicans blastospore viability at yeast cell concentrations ranging from 10(2) to greater than 10(6) colony forming units per ml. Among the HRPs, HRP-4 was the best inhibitor with significant killing activity noted at a peptide concentration of 0.5 microgram per ml. Antifungal potency under growth conditions was observed to be dependent upon pH. In contrast, killing did not vary throughout the pH range tested under non-growth conditions. Electron microscopy results demonstrated HRP damage at pH 5 which appeared to be initiated at the membrane. At pH 7.4, micrographs revealed clear evidence of intracellular destruction suggesting more extensive damage at neutral as compared to acidic pH. These results suggest that within the changing realm of the oral cavity, the HRPs would be expected to be potent killers of C. albicans.

A novel photoaffinity ligand for the phencyclidine site of the N-methyl-D-aspartate receptor labels a Mr 120,000 polypeptide

International Nuclear Information System (INIS)

Sonders, M.S.; Barmettler, P.; Lee, J.A.; Kitahara, Y.; Keana, J.F.; Weber, E.

1990-01-01

A radiolabeled photoaffinity ligand has been developed for the N-methyl-D-aspartate (NMDA)-preferring excitatory amino acid receptor complex. [3H]3-Azido-(5S, 10R)(+)-5-methyl-10,11-dihydro-5H- dibenzo[a,d]cyclohepten-5,10-imine [3H]3-azido-MK-801 demonstrated nearly identical affinity, density of binding sites, selectivity, pH sensitivity, and pharmacological profile in reversible binding assays with guinea pig brain homogenates to those displayed by its parent compound, MK-801. When employed in a photo-labeling protocol designed to optimize specific incorporation, [3H]3-azido-MK-801 labeled a single protein band which migrated in sodium dodecyl sulfate-polyacrylamide gels with Mr = 120,000. Incorporation of tritium into this band was completely inhibited when homogenates and [3H]3-azido-MK-801 were coincubated with 10 microM phencyclidine. These data suggest that the phencyclidine site of the NMDA receptor complex is at least in part comprised of a Mr = 120,000 polypeptide

Recent Insights in Islet Amyloid Polypeptide-Induced Membrane Disruption and Its Role in β-Cell Death in Type 2 Diabetes Mellitus

Directory of Open Access Journals (Sweden)

Lucie Khemtémourian

2008-01-01

Full Text Available The presence of fibrillar protein deposits (amyloid of human islet amyloid polypeptide (hIAPP in the pancreatic islets of Langerhans is thought to be related to death of the insulin-producing islet β-cells in type 2 diabetes mellitus (DM2. The mechanism of hIAPP-induced β-cell death is not understood. However, there is growing evidence that hIAPP-induced disruption of β-cell membranes is the cause of hIAPP cytotoxicity. Amyloid cytotoxicity by membrane damage has not only been suggested for hIAPP, but also for peptides and proteins related to other misfolding diseases, like Alzheimer’s disease, Parkinson’s disease, and prion diseases. Here we review the interaction of hIAPP with membranes, and discuss recent progress in the field, with a focus on hIAPP structure and on the proposed mechanisms of hIAPP-induced membrane damage in relation to β-cell death in DM2.

Isoform-specific regulation of osteogenic factors by polypeptide N-Acetylgalactosaminyltransferases 1 and 4

International Nuclear Information System (INIS)

Tang, Juan; Zheng, Hanxi; Chen, Ling; Gao, Shangshang; Shi, Xiaorui; Liu, Jingjing; Xu, Lan

2017-01-01

The family of UDP-GalNAc polypeptide: N-Acetylgalactosaminlytransfersases (ppGalNAcTs) catalyzes the initial step of O-linked protein glycosylation. Mucin-type O-glycoproteins are abundant in the bone and may play an important role in osteogenesis. Herein, we examined the effects of ppGalNAc-T isoforms on osteogenesis of MC3T3-E1 pre-osteoblasts. We found that ppGalNAc-T1 and -T4 isoforms were highly expressed during osteogenesis of MC3T3-E1 and their knockdown by short hairpin RNA (shRNA) decreased osteoblast formation and bone mineralization. Knockdown of ppGalNAc-T1 or -T4 decreased mRNA and protein levels of bone sialoprotein (BSP). Knockdown of ppGalNAc-T1decreased mRNA levels of osteocalcin (OC), osteoprotegerin (OPG). Knockdown ofppGalNAc-T4 isoform decreased mRNA levels of OC, OPG and vitamin D receptor (VDR). While knockdown of T1 or T4 isoforms did not change the expression of osteopontin (OPN), COLLI, receptor activator for nuclear factor-κB ligand (RANKL) and transforming growth factor-β (TGF-β). Our results demonstrated that the ppGalNAc-T4 was highly expressed in MC3T3-E1 cells during osteogenesis for the first time. We also found that ppGalNAc-T1 and -T4 affected the expression of different osteogenic factors, suggesting distinct roles ppGalNAc-T isoformsplay in regulating osteogenesis in vitro. - Highlights: • ppGalNAc-T1 and T4 are highly expressed during MC3T3 cell osteogenesis. • Knockdown of ppGalNAc-T1 and -T4 decreases osteogenic differentiation and mineralization. • Expression of osteogenic factors are differentially affected by decreased ppGalNAc-T1 and -T4 expression.

De novo expression of human polypeptide N-acetylgalactosaminyltransferase 6 (GalNAc-T6) in colon adenocarcinoma inhibits the differentiation of colonic epithelium

DEFF Research Database (Denmark)

Lavrsen, Kirstine; Dabelsteen, Sally; Vakhrushev, Sergey Y

2018-01-01

Aberrant expression of O-glycans is a hallmark of epithelial cancers. Mucin type O-glycosylation is initiated by a large family of UDP-GalNAc:polypeptide N-acetyl-galactosaminyltransferases (GalNAc-Ts), that target different proteins and are differentially expressed in cells and organs. Here we...... investigated the expression patterns of all of the GalNAc-Ts in colon cancer by analysing transcriptomic data. We found that GalNAc-T6 was highly upregulated in colon adenocarcinomas but absent in normal-appearing adjacent colon tissue. The results were verified by immunohistochemistry, suggesting that Gal......NAc-T6 plays a role in colon carcinogenesis. To investigate the function of GalNAc-T6 in colon cancer, we used precise gene targeting to produce isogenic colon cancer cell lines with a knockout/-rescue system for GalNAc-T6. GalNAc-T6 expression was associated with a cancer-like, dysplastic growth pattern...

Comprehensive behavioral analysis of pituitary adenylate cyclase-activating polypeptide (PACAP knockout mice

Directory of Open Access Journals (Sweden)

Satoko eHattori

2012-10-01

Full Text Available Pituitary adenylate cyclase-activating polypeptide (PACAP is a neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. PACAP is widely expressed throughout the brain and exerts its functions through the PACAP-specific receptor (PAC1. Recent studies reveal that genetic variants of the PACAP and PAC1 genes are associated with mental disorders, and several behavioral abnormalities of PACAP knockout (KO mice are reported. However, an insufficient number of backcrosses was made using PACAP KO mice on the C57BL/6J background due to their postnatal mortality. To elucidate the effects of PACAP on neuropsychiatric function, the PACAP gene was knocked out in F1 hybrid mice (C57BL/6J x 129SvEv for appropriate control of the genetic background. The PACAP KO mice were then subjected to a behavioral test battery. PACAP deficiency had no significant effects on neurological screen. As shown previously, the mice exhibited significantly increased locomotor activity in a novel environment and abnormal anxiety-like behavior, while no obvious differences between genotypes were shown in home cage activity. In contrast to previous reports, the PACAP KO mice showed normal prepulse inhibition and slightly decreased depression-like behavior. Previous study demonstrates that the social interaction in a resident-intruder test was decreased in PACAP KO mice. On the other hand, we showed that PACAP KO mice exhibited increased social interaction in Crawley’s three-chamber social approach test, although PACAP KO had no significant impact on social interaction in a home cage. PACAP KO mice also exhibited mild performance deficit in working memory in an eight-arm radial maze and the T-maze, while they did not show any significant abnormalities in the left-right discrimination task in the T-maze. These results suggest that PACAP has an important role in the regulation of locomotor activity, social behavior, anxiety-like behavior and, potentially

Investigation of light-induced conformation changes in spiropyran-modified succinylated poly(L-lysine).

Science.gov (United States)

Cooper, T M; Stone, M O; Natarajan, L V; Crane, R L

1995-08-01

To determine the maximum range of coupling between side-chain photochromism and polypeptide conformation change, we modified the carboxylate side chains of succinylated poly(L-lysine) with a spiropyran to form polypeptide I. The extent of modification was determined to be 35.5%. The spacer group length between the polypeptide alpha-carbon and the dye was 12 atoms, providing minimum polypeptide-dye interaction. Conformation changes were monitored by circular dichroism as a function of light adaptation and solvent composition (hexafluoroisopropanol [HFIP] vs trifluoroethanol [TFE]). Under all solvent compositions, the dark-adapted dye was in the merocyanine form. Light adaptation by visible light converted the dye to the spiropyran form. When dissolved in TFE, I adopted a helical conformation insensitive to light adaptation. With increasing percentage HFIP, a solvent-induced helix-to-coil transition was observed around 80% (vol/vol) HFIP. At 100% HFIP, both light- and dark-adapted forms of I were in the coil state. Near the midpoint of the solvent-induced helix-to-coil transition, light adaptation caused conformation changes. Applying helix-to-coil transition theory, we measured a statistically significant difference in coil segment-HFIP binding constant for light- vs dark-adapted solutions (6.38 +/- 0.03 M-1 vs 6.56 +/- 0.03 M-1), but not for the nucleation parameter sigma (1.2 +/- 0.4 10(-3) vs 1.3 +/- 0.3 x 10(-3). The small binding constant difference translated to a light-induced binding energy difference of 17 cal/mol/monomer. Near the midpoint of the helix-to-coil transition, collective interactions between monomer units made possible the translation of a small energy difference (less than RT) into large macromolecular conformation changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Luteinizing hormone-stimulated pituitary adenylate cyclase-activating polypeptide system and its role in progesterone production in human luteinized granulosa cells.

Science.gov (United States)

Park, Hyun-Jeong; Choi, Bum-Chae; Song, Sang-Jin; Lee, Dong-Sik; Roh, Jaesook; Chun, Sang-Young

2010-01-01

The present study examined the gonadotropin regulation of pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP type I receptor (PAC(1)-R) expression, and its role in progesterone production in the human luteinized granulosa cells. The stimulation of both PACAP and PAC(1)-R mRNA levels by LH was detected using a competitive reverse transcription-polymerase chain reaction (RT-PCR). PACAP transcript was stimulated by LH reaching maximum levels at 12 hours in a dose dependent manner. LH treatment also stimulated PAC(1)-R mRNA levels within 24 hours. Addition of PACAP-38 (10(-7) M) as well as LH significantly stimulated progesterone production during 48 hours culture. Furthermore, co-treatment with PACAP antagonist partially inhibited LH-stimulated progesterone production. Treatment with vasoactive intestinal peptide, however, did not affect progesterone production. Taken together, the present study demonstrates that LH causes a transient stimulation of PACAP and PAC(1)-R expression and that PACAP stimulates progesterone production in the human luteinized granulosa cells, suggesting a possible role of PACAP as a local ovarian regulator in luteinization.

The effect of pancreatic polypeptide and peptide YY on pancreatic blood flow and pancreatic exocrine secretion in the anesthetized dog

International Nuclear Information System (INIS)

DeMar, A.R.; Lake, R.; Fink, A.S.

1991-01-01

Pancreatic polypeptide (PP) and peptide YY (PYY) are inhibitors of pancreatic exocrine secretion in vivo but not in vitro, which suggests intermediate mechanisms of action. To examine the role of pancreatic blood flow in these inhibitory effects, xenon-133 gas clearance was used to measure pancreatic blood flow while simultaneously measuring pancreatic exocrine secretion. PP or PYY (400 pmol/kg/h) was administered during the intermediate hour of a 3-h secretin (125 ng/kg/h)/cholecystokinin octapeptide (CCK-8) (50 ng/kg/h) infusion. Exocrine secretion and pancreatic blood flow during the PP or PYY hours were compared with that observed in the first and third hours of the secretin/CCK-8 infusion. PP and PYY significantly inhibited secretin/CCK-8-induced pancreatic exocrine secretion. In addition, PYY (but not PP) significantly reduced pancreatic blood flow during secretin/CCK-8 stimulation. Nevertheless, there was no correlation between pancreatic blood flow and bicarbonate or protein outputs. It is concluded that changes in pancreatic blood flow do not mediate the inhibitory effects of PP or PYY on the exocrine pancreas

Ligand-Induced Cross-Linking of Z-Elastin-like Polypeptide-Functionalized E2 Protein Nanoparticles for Enhanced Affinity Precipitation of Antibodies.

Science.gov (United States)

Swartz, Andrew R; Sun, Qing; Chen, Wilfred

2017-05-08

Affinity precipitation is an ideal alternative to chromatography for antibody purification because it combines the high selectivity of an affinity ligand with the operational benefits of precipitation. However, the widespread use of elastin-like polypeptide (ELP) capture scaffolds for antibody purification has been hindered by the high salt concentrations and temperatures necessary for efficient ELP aggregation. In this paper, we employed a tandem approach to enhance ELP aggregation by enlarging the dimension of the capturing scaffold and by creating IgG-triggered scaffold cross-linking. This was accomplished by covalently conjugating the Z-domain-ELP (Z-ELP) capturing scaffold to a 25 nm diameter E2 protein nanocage using Sortase A ligation. We demonstrated the isothermal recovery of IgG in the virtual absence of salt due to the significantly increased scaffold dimension and cross-linking from multivalent IgG-E2 interactions. Because IgG cross-linking is reversible at low pH, it may be feasible to achieve a high yielding IgG purification by isothermal phase separation using a simple pH trigger.

Endothelial-monocyte activating polypeptide II alters fibronectin based endothelial cell adhesion and matrix assembly via alpha5 beta1 integrin

International Nuclear Information System (INIS)

Schwarz, Margaret A.; Zheng, Hiahua; Liu, Jie; Corbett, Siobhan; Schwarz, Roderich E.

2005-01-01

Mature Endothelial-Monocyte Activating Polypeptide (mEMAP) II functions as a potent antiangiogenic peptide. Although the anti-tumor effect of mEMAP II has been described, little is known regarding its mechanism of action. Observations that mEMAP II induced apoptosis only in a subset of migrating and proliferating endothelial cells (EC) suggests a targeted effect on cells engaged in angiogenic activities which are known to rely upon cell adhesion and migration. Indeed, we demonstrate that mEMAP II inhibited fibronectin (FN) dependent microvascular EC (MEC) adhesion and spreading and we show that this depends upon the alpha5 beta1 integrin. Immunofluorescence analysis demonstrated that mEMAP II-dependent blockade of FN-alpha5 beta1 interactions was associated with disassembly of both actin stress fiber networks and FN matrix. These findings suggest that mEMAP II blocks MEC adhesion and spreading on fibronectin, via a direct interaction with the integrin alpha5 beta1, thus implicating that alpha5 integrin may be a mediator of mEMAP II's antiangiogenic function

Vibrational infrared and Raman spectra of polypeptides: Fragments-in-fragments within molecular tailoring approach

Energy Technology Data Exchange (ETDEWEB)

Sahu, Nityananda; Gadre, Shridhar R., E-mail: gadre@iitk.ac.in [Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur 208 016 (India)

2016-03-21

The present work reports the calculation of vibrational infrared (IR) and Raman spectra of large molecular systems employing molecular tailoring approach (MTA). Further, it extends the grafting procedure for the accurate evaluation of IR and Raman spectra of large molecular systems, employing a new methodology termed as Fragments-in-Fragments (FIF), within MTA. Unlike the previous MTA-based studies, the accurate estimation of the requisite molecular properties is achieved without performing any full calculations (FC). The basic idea of the grafting procedure is implemented by invoking the nearly basis-set-independent nature of the MTA-based error vis-à-vis the respective FCs. FIF has been tested out for the estimation of the above molecular properties for three isomers, viz., β-strand, 3{sub 10}- and α-helix of acetyl(alanine){sub n}NH{sub 2} (n = 10, 15) polypeptides, three conformers of doubly protonated gramicidin S decapeptide and trpzip2 protein (PDB id: 1LE1), respectively, employing BP86/TZVP, M06/6-311G**, and M05-2X/6-31G** levels of theory. For most of the cases, a maximum difference of 3 cm{sup −1} is achieved between the grafted-MTA frequencies and the corresponding FC values. Further, a comparison of the BP86/TZVP level IR and Raman spectra of α-helical (alanine){sub 20} and its N-deuterated derivative shows an excellent agreement with the existing experimental spectra. In view of the requirement of only MTA-based calculations and the ability of FIF to work at any level of theory, the current methodology provides a cost-effective solution for obtaining accurate spectra of large molecular systems.

Composites

International Nuclear Information System (INIS)

Kasen, M.B.

1983-01-01

This chapter discusses the roles of composite laminates and aggregates in cryogenic technology. Filamentary-reinforced composites are emphasized because they are the most widely used composite materials. Topics considered include composite systems and terminology, design and fabrication, composite failure, high-pressure reinforced plastic laminates, low-pressure reinforced plastics, reinforced metals, selectively reinforced structures, the effect of cryogenic temperatures, woven-fabric and random-mat composites, uniaxial fiber-reinforced composites, composite joints in cryogenic structures, joining techniques at room temperature, radiation effects, testing laminates at cryogenic temperatures, static and cyclic tensile testing, static and cyclic compression testing, interlaminar shear testing, secondary property tests, and concrete aggregates. It is suggested that cryogenic composite technology would benefit from the development of a fracture mechanics model for predicting the fitness-for-purpose of polymer-matrix composite structures

Polypeptide N-Acetylgalactosaminyltransferase 13 Contributes to Neurogenesis via Stabilizing the Mucin-type O-Glycoprotein Podoplanin.

Science.gov (United States)

Xu, Yingjiao; Pang, Wenjie; Lu, Jishun; Shan, Aidong; Zhang, Yan

2016-11-04

Mucin-type O-glycosylation is initiated by an evolutionarily conserved family of polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). Previously, it was reported that ppGalNAc-T13 is restrictively expressed at a high level in the brain. Here we provide evidence for the critical role of ppGalNAc-T13 in neural differentiation. In detail, we show that the expression of ppGalNAc-T13 was dramatically up-regulated during early neurogenesis in mouse embryonic brains. Similar changes were also observed in cell models of neuronal differentiation by using either primary mouse cortical neural precursor cells or murine embryonal carcinoma P19 cells. Knockout of ppGalNAc-T13 in P19 cells suppressed not only neural induction but also neuronal differentiation. These effects are at least partly mediated by the mucin-type O-glycoprotein podoplanin (PDPN), as knockdown of PDPN led to a similar inhibition of neuronal differentiation and PDPN was significantly reduced at the posttranscriptional level after ppGalNAc-T13 knockout. Further data demonstrate that PDPN acts as a substrate of ppGalNAc-T13 and that the ppGalNAc-T13-mediated O-glycosylation on PDPN is important for its stability. Taken together, this study suggests that ppGalNAc-T13 contributes to neuronal differentiation through glycosylating and stabilizing PDPN, which provides insights into the regulatory roles of O-glycosylation in mammalian neural development. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Light Scattering Study of Mixed Micelles Made from Elastin-Like Polypeptide Linear Chains and Trimers

Science.gov (United States)

Terrano, Daniel; Tsuper, Ilona; Maraschky, Adam; Holland, Nolan; Streletzky, Kiril

Temperature sensitive nanoparticles were generated from a construct (H20F) of three chains of elastin-like polypeptides (ELP) linked to a negatively charged foldon domain. This ELP system was mixed at different ratios with linear chains of ELP (H40L) which lacks the foldon domain. The mixed system is soluble at room temperature and at a transition temperature (Tt) will form swollen micelles with the hydrophobic linear chains hidden inside. This system was studied using depolarized dynamic light scattering (DDLS) and static light scattering (SLS) to determine the size, shape, and internal structure of the mixed micelles. The mixed micelle in equal parts of H20F and H40L show a constant apparent hydrodynamic radius of 40-45 nm at the concentration window from 25:25 to 60:60 uM (1:1 ratio). At a fixed 50 uM concentration of the H20F, varying H40L concentration from 5 to 80 uM resulted in a linear growth in the hydrodynamic radius from about 11 to about 62 nm, along with a 1000-fold increase in VH signal. A possible simple model explaining the growth of the swollen micelles is considered. Lastly, the VH signal can indicate elongation in the geometry of the particle or could possibly be a result from anisotropic properties from the core of the micelle. SLS was used to study the molecular weight, and the radius of gyration of the micelle to help identify the structure and morphology of mixed micelles and the tangible cause of the VH signal.

Localization of three human polypeptide GalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylation throughout the Golgi apparatus

DEFF Research Database (Denmark)

Röttger, S; White, J; Wandall, H H

1998-01-01

O-glycosylation of proteins is initiated by a family of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactos-aminyltransferases (GalNAc-T). In this study, we have localized endogenous and epitope-tagged human GalNAc-T1, -T2 and -T3 to the Golgi apparatus in HeLa cells by subcellular fractionation......, immunofluorescence and immunoelectron microscopy. We show that all three GalNAc-transferases are concentrated about tenfold in Golgi stacks over Golgi associated tubular-vesicular membrane structures. Surprisingly, we find that GalNAc-T1, -T2 and -T3 are present throughout the Golgi stack suggesting that initiation...... of O-glycosylation may not be restricted to the cis Golgi, but occur at multiple sites within the Golgi apparatus. GalNAc-T1 distributes evenly across the Golgi stack whereas GalNAc-T2 and -T3 reside preferentially on the trans side and in the medial part of the Golgi stack, respectively. Moreover, we...

Tumor markers cancer antigen 15.3, carcinoembryonic antigen, and tissue polypeptide antigen for monitoring metastatic breast cancer during first-line chemotherapy and follow-up

DEFF Research Database (Denmark)

Sölétormos, G; Nielsen, D; Schiøler, V

1996-01-01

progressive disease, the median positive lead time was 35 days during therapy and 76 days during follow-up. Tumor marker assessment may document that a therapy is effective and ought to be continued in spite of adverse toxic effects, and that a treatment is ineffective and should be stopped to prevent......We investigated whether model systems integrating stochastic variation into criteria for marker assessment could be used for monitoring metastatic breast cancer. A total of 3989 serum samples was obtained from 204 patients receiving first-line chemotherapy and from 112 of these patients during...... follow-up. Each sample was analyzed for cancer antigen 15.3, carcinoembryonic antigen, and tissue polypeptide antigen. The efficiency for identifying progression and nonprogression was 94% during therapy and 85% during follow-up, with no false-positive marker results for progressive disease. At clinical...

Disturbed α-Cell Function in Mice with β-Cell Specific Overexpression of Human Islet Amyloid Polypeptide

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Bo Ahrén

2008-01-01

Full Text Available Exogenous administration of islet amyloid polypeptide (IAPP has been shown to inhibit both insulin and glucagon secretion. This study examined α-cell function in mice with β-cell specific overexpression of human IAPP (hIAPP after an oral protein gavage (75 mg whey protein/mouse. Baseline glucagon levels were higher in transgenic mice (41±4.0 pg/mL, n=6 than in wildtype animals (19±5.1 pg/mL, n=5, P=.015. In contrast, the glucagon response to protein was impaired in transgenic animals (21±2.7 pg/mL in transgenic mice versus 38±5.7 pg/mL in wildtype mice at 15 minutes; P=.027. Baseline insulin levels did not differ between the groups, while the insulin response, as the glucagon response, was impaired after protein challenge (P=.018. Glucose levels were not different between the groups and did not change significantly after protein gavage. Acetaminophen was given through gavage to the animals (2 mg/mouse to estimate gastric emptying. The plasma acetaminophen profile was similar in the two groups of mice. We conclude that disturbances in glucagon secretion exist in mice with β-cell specific overexpression of human IAPP, which are not secondary to changes in gastric emptying. The reduced glucagon response to protein challenge may reflect a direct inhibitory influence of hIAPP on glucagon secretion.

The effects of isatin (indole-2, 3-dione on pituitary adenylate cyclase-activating polypeptide-induced hyperthermia in rats

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Tóth Gábor

2002-02-01

Full Text Available Abstract Background Previous studies have demonstrated that centrally administered natriuretic peptides and pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38 have hyperthermic properties. Isatin (indole-2, 3-dione is an endogenous indole that has previously been found to inhibit hyperthermic effects of natriuretic peptides. In this study the aim was to investigate the effects of isatin on thermoregulatory actions of PACAP-38, in rats. Results One μg intracerebroventricular (icv. injection of PACAP-38 had hyperthermic effect in male, Wistar rats, with an onset of the effect at 2 h and a decline by the 6th h after administration. Intraperitoneal (ip. injection of different doses of isatin (25-50 mg/kg significantly decreased the hyperthermic effect of 1 μg PACAP-38 (icv., whereas 12.5 mg/kg isatin (ip. had no inhibiting effect. Isatin alone did not modify the body temperature of the animals. Conclusion The mechanisms that participate in the mediation of the PACAP-38-induced hyperthermia may be modified by isatin. The capability of isatin to antagonize the hyperthermia induced by all members of the natriuretic peptide family and by PACAP-38 makes it unlikely to be acting directly on receptors for natriuretic peptides or on those for PACAP in these hyperthermic processes.

Decontamination of radioactive liquid wastes by hydrophytic vegetal organisms

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Cecal, Al; Popa, K.; Potoroaca, V.; Melniciuc-Puica, N.

2001-01-01

Bioaccumulation of some radioactive ions from contaminated waste solutions, on hydrophytic vegetal organisms is discussed. In order to follow the distribution of radioactive ions 137 Cs + , 60 Co 2+ and 51 Cr 3+ in various cell components extracted from Spirulina platensis, Porphiridium cruentum, Scenedesmus quadricauda, Lemna minor, Elodea canadensis, Pistia stratiotes and Riccia fluitans, the plants were cultivated in radioactive solutions. The resulting complexes were extracted with acetone or acetic acid and separated chromatographically. The results show an intense activity of the polysaccharide and lipoid fractions in the bioaccumulation process. The bioaccumulation varies in the series: Spirulina > Scenedesmus > Porphiridium > Riccia > Pistia > Lemna ≥ Elodea for 137 Cs + and 60 Co 2+ ; Spirulina > Porphiridium > Scenedesmus > Riccia > Pistia > Lemna > Elodea for 51 Cr 3+ . (author)

Pituitary adenylate cyclase activating polypeptide modulates catecholamine storage and exocytosis in PC12 cells.

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Yan Dong

Full Text Available A number of efforts have been made to understand how pituitary adenylate cyclase activating polypeptide (PACAP functions as a neurotrophic and neuroprotective factor in Parkinson's disease (PD. Recently its effects on neurotransmission and underlying mechanisms have generated interest. In the present study, we investigate the effects of PACAP on catecholamine storage and secretion in PC12 cells with amperometry and transmission electron microscopy (TEM. PACAP increases quantal release induced by high K+ without significantly regulating the frequency of vesicle fusion events. TEM data indicate that the increased volume of the vesicle is mainly the result of enlargement of the fluidic space around the dense core. Moreover, the number of docked vesicles isn't modulated by PACAP. When cells are acutely treated with L-DOPA, the vesicular volume and quantal release both increase dramatically. It is likely that the characteristics of amperometric spikes from L-DOPA treated cells are associated with increased volume of individual vesicles rather than a direct effect on the mechanics of exocytosis. Treatment with PACAP versus L-DOPA results in different profiles of the dynamics of exocytosis. Release via the fusion pore prior to full exocytosis was observed with the same frequency following treatment with PACAP and L-DOPA. However, release events have a shorter duration and higher average current after PACAP treatment compared to L-DOPA. Furthermore, PACAP reduced the proportion of spikes having rapid decay time and shortened the decay time of both fast and slow spikes. In contrast, the distributions of the amperometric spike decay for both fast and slow spikes were shifted to longer time following L-DOPA treatment. Compared to L-DOPA, PACAP may produce multiple favorable effects on dopaminergic neurons, including protecting dopaminergic neurons against neurodegeneration and potentially regulating dopamine storage and release, making it a promising

No association between the protein tyrosine phosphatase, receptor-type, Z Polypeptide 1 (PTPRZ1) gene and schizophrenia in the Japanese population.

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Ito, Yoshihito; Yamada, Shinnosuke; Takahashi, Nagahide; Saito, Shinichi; Yoshimi, Akira; Inada, Toshiya; Noda, Yukihiro; Ozaki, Norio

2008-10-05

NRG1-ERBB signaling influences the risk for schizophrenia pathology. A recent study has reported that MAGI1, MAGI2, and protein tyrosine phosphatase, receptor-type, Z polypeptide 1 (PTPRZ1; located on 7q31.3) gene products regulate the NRG1-ERBB4 signaling pathway, and PTPRZ1 is associated with schizophrenia in a Caucasian population. By applying a gene-based association concept, we analyzed any association between PTPRZ1 tagging SNPs and schizophrenia in the Japanese population (576 schizophrenics and 768 controls). After linkage disequilibrium analysis, 29 single nucleotide polymorphisms (SNPs) were genotyped using a 5'-exonuclease allelic discrimination assay. We found a significant association of one tagging SNP in a genotype-wise analysis (P = 0.007); however, this might be resulted from type I error due to multiple testing (P = 0.17 after SNPSpD correction). No association was observed between schizophrenic patients and controls in either allelic, genotypic, or haplotypic analyses. Our results therefore suggest that PTPRZ1 is unlikely to be related to the development of schizophrenia in the Japanese population.

Human Islet Amyloid Polypeptide Transgenic Mice: In Vivo and Ex Vivo Models for the Role of hIAPP in Type 2 Diabetes Mellitus

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J. W. M. Höppener

2008-01-01

Full Text Available Human islet amyloid polypeptide (hIAPP, a pancreatic islet protein of 37 amino acids, is the main component of islet amyloid, seen at autopsy in patients with type 2 diabetes mellitus (DM2. To investigate the roles of hIAPP and islet amyloid in DM2, we generated transgenic mice expressing hIAPP in their islet beta cells. In this study, we found that after a long-term, high-fat diet challenge islet amyloid was observed in only 4 of 19 hIAPP transgenic mice. hIAPP transgenic females exhibited severe glucose intolerance, which was associated with a downregulation of GLUT-2 mRNA expression. In isolated islets from hIAPP males cultured for 3 weeks on high-glucose medium, the percentage of amyloid containing islets increased from 5.5% to 70%. This ex vivo system will allow a more rapid, convenient, and specific study of factors influencing islet amyloidosis as well as of therapeutic strategies to interfere with this pathological process.

Sampling the Denatured State of Polypeptides in Water, Urea, and Guanidine Chloride to Strict Equilibrium Conditions with the Help of Massively Parallel Computers.

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Meloni, Roberto; Camilloni, Carlo; Tiana, Guido

2014-02-11

The denatured state of polypeptides and proteins, stabilized by chemical denaturants like urea and guanidine chloride, displays residual secondary structure when studied by nuclear-magnetic-resonance spectroscopy. However, these experimental techniques are weakly sensitive, and thus molecular-dynamics simulations can be useful to complement the experimental findings. To sample the denatured state, we made use of massively-parallel computers and of a variant of the replica exchange algorithm, in which the different branches, connected with unbiased replicas, favor the formation and disruption of local secondary structure. The algorithm is applied to the second hairpin of GB1 in water, in urea, and in guanidine chloride. We show with the help of different criteria that the simulations converge to equilibrium. It results that urea and guanidine chloride, besides inducing some polyproline-II structure, have different effect on the hairpin. Urea disrupts completely the native region and stabilizes a state which resembles a random coil, while guanidine chloride has a milder effect.

Effects of velvet antler polypeptide on sexual behavior and testosterone synthesis in aging male mice.

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Zang, Zhi-Jun; Tang, Hong-Feng; Tuo, Ying; Xing, Wei-Jie; Ji, Su-Yun; Gao, Yong; Deng, Chun-Hua

2016-01-01

Twenty-four-month-old male C57BL/6 mice with low serum testosterone levels were used as a late-onset hypogonadism (LOH) animal model for examining the effects of velvet antler polypeptide (VAP) on sexual function and testosterone synthesis. These mice received VAP for 5 consecutive weeks by daily gavage at doses of 100, 200, or 300 mg kg-1 body weight per day (n = 10 mice per dose). Control animals (n = 10) received the same weight-based volume of vehicle. Sexual behavior and testosterone levels in serum and interstitial tissue of testis were measured after the last administration of VAP. Furthermore, to investigate the mechanisms of how VAP affects sexual behavior and testosterone synthesis in vivo, the expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) in Leydig cells was also measured by immunofluorescence staining and quantitative real-time PCR. As a result, VAP produced a significant improvement in the sexual function of these aging male mice. Serum testosterone level and intratesticular testosterone (ITT) concentration also increased in the VAP-treated groups. The expression of StAR, P450scc, and 3β-HSD was also found to be enhanced in the VAP-treated groups compared with the control group. Our results suggested that VAP was effective in improving sexual function in aging male mice. The effect of velvet antler on sexual function was due to the increased expression of several rate-limiting enzymes of testosterone synthesis (StAR, P450scc, and 3β-HSD) and the following promotion of testosterone synthesis in vivo.

Gastric inhibitory polypeptide receptor: association analyses for obesity of several polymorphisms in large study groups

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Rief Winfried

2009-03-01

Full Text Available Abstract Background Gastric inhibitory polypeptide (GIP is postulated to be involved in type 2 diabetes mellitus and obesity. It exerts its function through its receptor, GIPR. We genotyped three GIPR SNPs (rs8111428, rs2302382 and rs1800437 in German families with at least one obese index patient, two case-control studies and two cross-sectional population-based studies. Methods Genotyping was performed by MALDI-TOF, ARMS-PCR and RFLP. The family-study: 761 German families with at least one extremely obese child or adolescent (n = 1,041 and both parents (n = 1,522. Case-control study: (a German obese children (n = 333 and (b obese adults (n = 987 in comparison to 588 adult lean controls. The two cross-sectional population-based studies: KORA (n = 8,269 and SHIP (n = 4,310. Results We detected over-transmission of the A-allele of rs2302382 in the German families (pTDT-Test = 0.0089. In the combined case-control sample, we estimated an odd ratio of 1.54 (95%CI 1.09;2.19, pCA-Test = 0.014 for homozygotes of the rs2302382 A-allele compared to individuals with no A-allele. A similar trend was found in KORA where the rs2302382 A-allele led to an increase of 0.12 BMI units (p = 0.136. In SHIP, however, the A-allele of rs2302382 was estimated to contribute an average decrease of 0.27 BMI units (p-value = 0.031. Conclusion Our data suggest a potential relevance of GIPR variants for obesity. However, additional studies are warranted in light of the conflicting results obtained in one of the two population-based studies.

Pituitary adenylate cyclase-activating polypeptide stimulates glucose production via the hepatic sympathetic innervation in rats.

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Yi, Chun-Xia; Sun, Ning; Ackermans, Mariette T; Alkemade, Anneke; Foppen, Ewout; Shi, Jing; Serlie, Mireille J; Buijs, Ruud M; Fliers, Eric; Kalsbeek, Andries

2010-07-01

The unraveling of the elaborate brain networks that control glucose metabolism presents one of the current challenges in diabetes research. Within the central nervous system, the hypothalamus is regarded as the key brain area to regulate energy homeostasis. The aim of the present study was to investigate the hypothalamic mechanism involved in the hyperglycemic effects of the neuropeptide pituitary adenylyl cyclase-activating polypeptide (PACAP). Endogenous glucose production (EGP) was determined during intracerebroventricular infusions of PACAP-38, vasoactive intestinal peptide (VIP), or their receptor agonists. The specificity of their receptors was examined by coinfusions of receptor antagonists. The possible neuronal pathway involved was investigated by 1) local injections in hypothalamic nuclei, 2) retrograde neuronal tracing from the thoracic spinal cord to hypothalamic preautonomic neurons together with Fos immunoreactivity, and 3) specific hepatic sympathetic or parasympathetic denervation to block the autonomic neuronal input to liver. Intracerebroventricular infusion of PACAP-38 increased EGP to a similar extent as a VIP/PACAP-2 (VPAC2) receptor agonist, and intracerebroventricular administration of VIP had significantly less influence on EGP. The PACAP-38 induced increase of EGP was significantly suppressed by preinfusion of a VPAC2 but not a PAC1 receptor antagonist, as well as by hepatic sympathetic but not parasympathetic denervation. In the hypothalamus, Fos immunoreactivity induced by PACAP-38 was colocalized within autonomic neurons in paraventricular nuclei projecting to preganglionic sympathetic neurons in the spinal cord. Local infusion of PACAP-38 directly into the PVN induced a significant increase of EGP. This study demonstrates that PACAP-38 signaling via sympathetic preautonomic neurons located in the paraventricular nucleus is an important component in the hypothalamic control of hepatic glucose production.

Uptake of 14C-labelled chloroquine and an 125I-labelled chloroquine analogue in some polypeptide hormone producing cell systems

International Nuclear Information System (INIS)

Dencker, L.; Lindquist, N.G.; Tjaelve, H.

1976-01-01

After the injection of 14 C-labelled chloroquine and the 125 I-labelled chloroquine analogue 4-(3 1 -dimethylaminopropylamino)-7-iodoquinoline [ 125 I]DAPQ into mice, rats and a monkey the distribution of the radioactivity was studied by autoradiographical methods. A high and persistent uptake occurred in some endocrine cell systems, such as the pancreatic islets, the hypophysis, the adrenal medulla and the thyroid (in cells that were probably identical with the parafollicular cells). The melanin-containing tissues were the only ones which showed a higher uptake and retention of radioactivity. The above mentioned endocrine cells and the melanocytes have a common embryological origin and common morphological and cytochemical characteristics. They have been called the APUD (Amine Precursor Uptake and Decarboxylation)-cell series. It is proposed that the polypeptide hormone producing cells and the melanocytes may use a similar mechanism for accumulating chloroquine and (as shown earlier) also some other drugs such as nicotine, alprenolol, local anesthetics and atropine. These drugs however, accumulate stronger within the melanocytes and become bound to the melanin for a long time. The ability to accumulate these drugs may be considered another characteristic of the APUD-cell series. (author)

Marine Collagen Peptides from the Skin of Nile Tilapia (Oreochromis niloticus): Characterization and Wound Healing Evaluation.

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Hu, Zhang; Yang, Ping; Zhou, Chunxia; Li, Sidong; Hong, Pengzhi

2017-03-30

Burns can cause tremendous economic problems associated with irreparable harm to patients and their families. To characterize marine collagen peptides (MCPs) from the skin of Nile tilapia ( Oreochromis niloticus ), molecular weight distribution and amino acid composition of MCPs were determined, and Fourier transform infrared spectroscopy (FTIR) was used to analyze the chemical structure. Meanwhile, to evaluate the wound healing activity, in vitro and in vivo experiments were carried out. The results showed that MCPs prepared from the skin of Nile tilapia by composite enzymatic hydrolysis were composed of polypeptides with different molecular weights and the contents of polypeptides with molecular weights of less than 5 kDa accounted for 99.14%. From the amino acid composition, the majority of residues, accounting for over 58% of the total residues in MCPs, were hydrophilic. FTIR indicated that the main molecular conformations inside MCPs were random coil. In vitro scratch assay showed that there were significant effects on the scratch closure by the treatment of MCPs with the concentration of 50.0 μg/mL. In the experiments of deep partial-thickness scald wound in rabbits, MCPs could enhance the process of wound healing. Therefore, MCPs from the skin of Nile tilapia ( O. niloticus ) have promising applications in wound care.

Single-molecule spectroscopy reveals that individual low-light LH2 complexes from Rhodopseudomonas palustris 2.1.6. have a heterogeneous polypeptide composition.

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Brotosudarmo, Tatas H P; Kunz, Ralf; Böhm, Paul; Gardiner, Alastair T; Moulisová, Vladimíra; Cogdell, Richard J; Köhler, Jürgen

2009-09-02

Rhodopseudomonas palustris belongs to the group of purple bacteria that have the ability to produce LH2 complexes with unusual absorption spectra when they are grown at low-light intensity. This ability is often related to the presence of multiple genes encoding the antenna apoproteins. Here we report, for the first time to our knowledge, direct evidence that individual low-light LH2 complexes have a heterogeneous alphabeta-apoprotein composition that modulates the site energies of Bchl a molecules, producing absorption bands at 800, 820, and 850 nm. The arrangement of the Bchl a molecules in the "tightly coupled ring" can be modeled by nine alphabeta-Bchls dimers, such that the Bchls bound to six alphabeta-pairs have B820-like site energies and the remaining Bchl a molecules have B850-like site energies. Furthermore, the experimental data can only be satisfactorily modeled when these six alphabeta-pairs with B820 Bchl a molecules are distributed such that the symmetry of the assembly is reduced to C(3). It is also clear from the measured single-molecule spectra that the energies of the electronically excited states in the mixed B820/850 ring are mainly influenced by diagonal disorder.

Gibberellin-induced changes in the populations of translatable mRNAs and accumulated polypeptides in dwarfs of maize and pea

International Nuclear Information System (INIS)

Chory, J.; Voytas, D.F.; Olszewski, N.E.; Ausubel, F.M.

1987-01-01

Two-dimensional gel electrophoresis was used to characterize the molecular mechanism of gibberellin-induced stem elongation in maize and pea. Dwarf mutants of maize and pea lack endogenous gibberellin (GA 1 ) but become phenotypically normal with exogenous applications of this hormone. Sections from either etiolated maize or green pea seedlings were incubated in the presence of [ 35 S] methionine for 3 hours with or without gibberellin. Labeled proteins from soluble and particulate fractions were analyzed by two-dimensional gel electrophoresis and specific changes in the patterns of protein synthesis were observed upon treatment with gibberellin. Polyadenylated mRNAs from etiolated or green maize shoots and green pea epicotyls treated or not with gibberellin (a 0.5 to 16 hour time course) were assayed by translation in a rabbit reticulocyte extract and separation of products by two-dimensional gel electrophoresis. Both increases and decreases in the levels of specific polypeptides were seen for pea and corn, and these changes were observed within 30 minutes of treatment with gibberellin. Together, these data indicate that gibberellin induces changes in the expression of a subset of gene products within elongating dwarfs. This may be due to changes in transcription rate, mRNA stability, or increased efficiency of translation of certain mRNAs

Composite gravity and composite supergravity

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Lukierski, J.

1982-09-01

It is known that the composite YM H-gauge theory can be constructed from σ-fields taking values in a symmetric Riemannian space G/H. We extend such a framework to graded σ-fields taking values in supercosets. We show that from supercoset σ-fields one can construct composite gravity, and from supercoset σ-superfields the composite supergravity models. (author)

Dual-layered nanogel-coated hollow lipid/polypeptide conjugate assemblies for potential pH-triggered intracellular drug release.

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Wen-Hsuan Chiang

Full Text Available To achieve effective intracellular anticancer drug delivery, the polymeric vesicles supplemented with the pH-responsive outlayered gels as a delivery system of doxorubicin (DOX were developed from self-assembly of the lipid/polypeptide adduct, distearin grafted poly(γ-glutamic acid (poly(γ-GA, followed by sequential deposition of chitosan and poly(γ-GA-co-γ-glutamyl oxysuccinimide-g-monomethoxy poly(ethylene glycol in combination with in situ covalent cross-linking on assembly surfaces. The resultant gel-caged polymeric vesicles (GCPVs showed superior performance in regulating drug release in response to the external pH change. Under typical physiological conditions (pH 7.4 and 37 °C at which the γ-GA/DOX ionic pairings remained mostly undisturbed, the dense outlayered gels of GCPVs significantly reduced the premature leakage of the uncomplexed payload. With the environmental pH being reduced from pH 7.4 to 4.7, the drug liberation was appreciably promoted by the massive disruption of the ionic γ-GA/DOX complexes along with the significant swelling of nanogel layers upon the increased protonation of chitosan chain segments. After being internalized by HeLa cells via endocytosis, GCPVs exhibited cytotoxic effect comparable to free DOX achieved by rapidly releasing the payload in intracellular acidic endosomes and lysosomes. This strongly implies the great promise of such unique GCPVs as an intracellular drug delivery carrier for potential anticancer treatment.

Kynurenic Acid Inhibits the Electrical Stimulation Induced Elevated Pituitary Adenylate Cyclase-Activating Polypeptide Expression in the TNC

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Tamás Körtési

2018-01-01

Full Text Available BackgroundMigraine is a primary headache of imprecisely known mechanism, but activation of the trigeminovascular system (TS appears to be essential during the attack. Intensive research has recently focused on pituitary adenylate cyclase-activating polypeptide (PACAP and the kynurenine systems as potential pathogenic factors.AimWe investigated the link between these important mediators and the effects of kynurenic acid (KYNA and its synthetic analog (KYNA-a on PACAP expression in the rat trigeminal nucleus caudalis (TNC in a TS stimulation model related to migraine mechanisms.MethodsAdult male Sprague-Dawley rats were pretreated with KYNA, KYNA-a, the NMDA receptor antagonist MK-801, or saline (vehicle. Next, the trigeminal ganglion (TRG was electrically stimulated, the animals were transcardially perfused following 180 min, and the TNC was removed. In the TNC samples, 38 amino acid form of PACAP (PACAP1–38-like radioimmunoactivity was measured by radioimmunoassay, the relative optical density of preproPACAP was assessed by Western blot analysis, and PACAP1–38 mRNA was detected by real-time PCR.Results and conclusionElectrical TRG stimulation resulted in significant increases of PACAP1–38-LI, preproPACAP, and PACAP1–38 mRNA in the TNC. These increases were prevented by the pretreatments with KYNA, KYNA-a, and MK-801. This is the first study to provide evidence for a direct link between PACAP and the kynurenine system during TS activation.

Multitriggered Tumor-Responsive Drug Delivery Vehicles Based on Protein and Polypeptide Coassembly for Enhanced Photodynamic Tumor Ablation.

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Zhang, Ning; Zhao, Fenfang; Zou, Qianli; Li, Yongxin; Ma, Guanghui; Yan, Xuehai

2016-11-01

Tumor-responsive nanocarriers are highly valuable and demanded for smart drug delivery particularly in the field of photodynamic therapy (PDT), where a quick release of photosensitizers in tumors is preferred. Herein, it is demonstrated that protein-based nanospheres, prepared by the electrostatic assembly of proteins and polypeptides with intermolecular disulfide cross-linking and surface polyethylene glycol coupling, can be used as versatile tumor-responsive drug delivery vehicles for effective PDT. These nanospheres are capable of encapsulation of various photosensitizers including Chlorin e6 (Ce6), protoporphyrin IX, and verteporfin. The Chlorin e6-encapsulated nanospheres (Ce6-Ns) are responsive to changes in pH, redox potential, and proteinase concentration, resulting in multitriggered rapid release of Ce6 in an environment mimicking tumor tissues. In vivo fluorescence imaging results indicate that Ce6-Ns selectively accumulate near tumors and the quick release of Ce6 from Ce6-Ns can be triggered by tumors. In tumors the fluorescence of released Ce6 from Ce6-Ns is observed at 0.5 h postinjection, while in normal tissues the fluorescence appeared at 12 h postinjection. Tumor ablation is demonstrated by in vivo PDT using Ce6-Ns and the biocompatibility of Ce6-Ns is evident from the histopathology imaging, confirming the enhanced in vivo PDT efficacy and the biocompatibility of the assembled drug delivery vehicles. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Targeted Transgenic Overexpression of Mitochondrial Thymidine Kinase (TK2) Alters Mitochondrial DNA (mtDNA) and Mitochondrial Polypeptide Abundance

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Hosseini, Seyed H.; Kohler, James J.; Haase, Chad P.; Tioleco, Nina; Stuart, Tami; Keebaugh, Erin; Ludaway, Tomika; Russ, Rodney; Green, Elgin; Long, Robert; Wang, Liya; Eriksson, Staffan; Lewis, William

2007-01-01

Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-γ. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK1) were used to study NRTI mitochondrial toxicity. Echocardiography and nuclear magnetic resonance imaging defined cardiac performance and structure. TK gene copy and enzyme activity, mitochondrial (mt) DNA and polypeptide abundance, succinate dehydrogenase and cytochrome oxidase histochemistry, and electron microscopy correlated with transgenesis, mitochondrial structure, and biogenesis. Antiretroviral combinations simulated therapy. Untreated hTK1 or TK2 TGs exhibited normal left ventricle mass. In TK2 TGs, cardiac TK2 gene copy doubled, activity increased 300-fold, and mtDNA abundance doubled. Abundance of the 17-kd subunit of complex I, succinate dehydrogenase histochemical activity, and cristae density increased. NRTIs increased left ventricle mass 20% in TK2 TGs. TK activity increased 3 logs in hTK1 TGs, but no cardiac phenotype resulted. NRTIs abrogated functional effects of transgenically increased TK2 activity but had no effect on TK2 mtDNA abundance. Thus, NRTI mitochondrial phosphorylation by TK2 is integral to clinical NRTI mitochondrial toxicity. PMID:17322372

Abnormal iron metabolism and oxidative stress in mice expressing a mutant form of the ferritin light polypeptide gene

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Barbeito, Ana G.; Garringer, Holly J.; Baraibar, Martin A.; Gao, Xiaoying; Arredondo, Miguel; Núñez, Marco T.; Smith, Mark A.; Ghetti, Bernardino; Vidal, Ruben

2009-01-01

Insertional mutations in exon 4 of the ferritin light chain (FTL) gene are associated with hereditary ferritinopathy (HF) or neuroferritinopathy, an autosomal dominant neurodegenerative disease characterized by progressive impairment of motor and cognitive functions. To determine the pathogenic mechanisms by which mutations in FTL lead to neurodegeneration, we investigated iron metabolism and markers of oxidative stress in the brain of transgenic (Tg) mice that express the mutant human FTL498-499InsTC cDNA. Compared with wild-type mice, brain extracts from Tg (FTL-Tg) mice showed an increase in the cytoplasmic levels of both FTL and ferritin heavy chain polypeptides, a decrease in the protein and mRNA levels of transferrin receptor-1, and a significant increase in iron levels. Transgenic mice also showed the presence of markers for lipid peroxidation, protein carbonyls, and nitrone–protein adducts in the brain. However, gene expression analysis of iron management proteins in the liver of Tg mice indicates that the FTL-Tg mouse liver is iron deficient. Our data suggest that disruption of iron metabolism in the brain has a primary role in the process of neurodegeneration in HF and that the pathogenesis of HF is likely to result from a combination of reduction in iron storage function and enhanced toxicity associated with iron-induced ferritin aggregates in the brain. PMID:19519778

Mast cell chymase reduces the toxicity of Gila monster venom, scorpion venom, and vasoactive intestinal polypeptide in mice

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Akahoshi, Mitsuteru; Song, Chang Ho; Piliponsky, Adrian M.; Metz, Martin; Guzzetta, Andrew; Åbrink, Magnus; Schlenner, Susan M.; Feyerabend, Thorsten B.; Rodewald, Hans-Reimer; Pejler, Gunnar; Tsai, Mindy; Galli, Stephen J.

2011-01-01

Mast cell degranulation is important in the pathogenesis of anaphylaxis and allergic disorders. Many animal venoms contain components that can induce mast cell degranulation, and this has been thought to contribute to the pathology and mortality caused by envenomation. However, we recently reported evidence that mast cells can enhance the resistance of mice to the venoms of certain snakes and that mouse mast cell–derived carboxypeptidase A3 (CPA3) can contribute to this effect. Here, we investigated whether mast cells can enhance resistance to the venom of the Gila monster, a toxic component of that venom (helodermin), and the structurally similar mammalian peptide, vasoactive intestinal polypeptide (VIP). Using 2 types of mast cell–deficient mice, as well as mice selectively lacking CPA3 activity or the chymase mouse mast cell protease-4 (MCPT4), we found that mast cells and MCPT4, which can degrade helodermin, can enhance host resistance to the toxicity of Gila monster venom. Mast cells and MCPT4 also can limit the toxicity associated with high concentrations of VIP and can reduce the morbidity and mortality induced by venoms from 2 species of scorpions. Our findings support the notion that mast cells can enhance innate defense by degradation of diverse animal toxins and that release of MCPT4, in addition to CPA3, can contribute to this mast cell function. PMID:21926462

Enantiodiscrimination of flexible cyclic solutes using NMR spectroscopy in polypeptide chiral mesophases: investigation of cis-decalin and THF.

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Aroulanda, Christie; Lafon, Olivier; Lesot, Philippe

2009-08-06

The conformational dynamics and orientational behavior of two model cyclic molecules, cis-decalin (cis-dec) and tetrahydrofurane (THF), dissolved in weakly ordering, polypeptidic chiral liquid crystals (CLCs) are theoretically discussed and experimentally investigated using deuterium and carbon-13 NMR spectroscopies. The analysis of enantiomeric and enantiotopic discriminations in these compounds is shown to depend on the rate of conformational exchange regime, slow or fast. The slow exchange regime is illustrated through the case of cis-dec at low temperature (243 K). We show that the deuterium NMR spectra in this regime can be qualitatively and quantitatively interpreted by restricting the conformational pathway of cis-dec to two enantiomeric conformers of C(2)-symmetry. The orientational order parameters of these interconverting enantiomers are calculated by matching the (2)H quadrupolar splittings with calculated conformer structures. The fast exchange regime is investigated through the examples of cis-dec at high temperature (356 K) and THF at room temperature (300 K). The (2)H NMR spectra above the coalescence temperature are analyzed by introducing the concept of "average molecular structure". This fictitious structure allows easily identifying NMR equivalences of solutes dissolved in CLC. However, it cannot be applied to determine consistent orientational order parameters. This study emphasizes that enantiotopic discriminations observed for flexible molecules in the fast exchange regime can be quantitatively interpreted only by considering the orientational order of each conformer.

Process conditions and volumetric composition in composites

DEFF Research Database (Denmark)

Madsen, Bo

2013-01-01

The obtainable volumetric composition in composites is linked to the gravimetric composition, and it is influenced by the conditions of the manufacturing process. A model for the volumetric composition is presented, where the volume fractions of fibers, matrix and porosity are calculated...... as a function of the fiber weight fraction, and where parameters are included for the composite microstructure, and the fiber assembly compaction behavior. Based on experimental data of composites manufactured with different process conditions, together with model predictions, different types of process related...... effects are analyzed. The applied consolidation pressure is found to have a marked effect on the volumetric composition. A power-law relationship is found to well describe the found relations between the maximum obtainable fiber volume fraction and the consolidation pressure. The degree of fiber...

Participation of Glutamate-354 of the CP43 Polypeptide in the Ligation of Mn and the Binding of Substrate Water in Photosystem II

Energy Technology Data Exchange (ETDEWEB)

Service, Rachel; Yano, Junko; McConnell, Iain; Hwang, Hong Jin; Niks, Dimitri; Hille, Russ; Wydrzynski, Tom; Burnap, Robert; Hillier, Warwick; Debus, Richard

2010-09-30

In the current X-ray crystallographic structural models of photosystem II, Glu354 of the CP43 polypeptide is the only amino acid ligand of the oxygen-evolving Mn4Ca cluster that is not provided by the D1 polypeptide. To further explore the influence of this structurally unique residue on the properties of the Mn4Ca cluster, the CP43-E354Q mutant of the cyanobacterium Synechocystis sp. PCC 6803 was characterized with a variety of biophysical and spectroscopic methods, including polarography, EPR, X-ray Absorption, FTIR, and mass spectrometry. The kinetics of oxygen release in the mutant were essentially unchanged from those in wild-type. In addition, the oxygen flash-yields exhibited normal period-four oscillations having normal S state parameters, although the yields were lower, correlating with the mutant?s lower steady-state rate (approx. 20percent compared to wild-type). Experiments conducted with H218O showed that the fast and slow phases of substrate water exchange in CP43-E354Q thylakoid membranes were accelerated 8.5- and 1.8-fold, respectively, in the S3 state compared to wild-type. Purified oxygen-evolving CP43-E354Q PSII core complexes exhibited a slightly altered S1 state Mn-EXAFS spectrum, a slightly altered S2 state multiline EPR signal, a substantially altered S2-minus-S1 FTIR difference spectrum, and an unusually long lifetime for the S2 state (> 10 hours) in a substantial fraction of reaction centers. In contrast, the S2 state Mn-EXAFS spectrum was nearly indistinguishable from that of wild-type. The S2-minus-S1 FTIR difference spectrum showed alterations throughout the amide and carboxylate stretching regions. Global labeling with 15N and specific labeling with L-[1-13C]alanine revealed that the mutation perturbs both amide II and carboxylate stretching modes and shifts the symmetric carboxylate stretching modes of the ?-COO? group of D1-Ala344 (the C-terminus of the D1 polypeptide) to higher frequencies by 3 ? 4 cm-1 in both the S1 and S2 states

Human Islet Amyloid Polypeptide Fibril Binding to Catalase: A Transmission Electron Microscopy and Microplate Study

Directory of Open Access Journals (Sweden)

Nathaniel G. N. Milton

2010-01-01

Full Text Available The diabetes-associated human islet amyloid polypeptide (IAPP is a 37-amino-acid peptide that forms fibrils in vitro and in vivo. Human IAPP fibrils are toxic in a similar manner to Alzheimer's amyloid-β (Aβ and prion protein (PrP fibrils. Previous studies have shown that catalase binds to Aβ fibrils and appears to recognize a region containing the Gly-Ala-Ile-Ile sequence that is similar to the Gly-Ala-Ile-Leu sequence found in human IAPP residues 24-27. This study presents a transmission electron microscopy (TEM—based analysis of fibril formation and the binding of human erythrocyte catalase to IAPP fibrils. The results show that human IAPP 1-37, 8-37, and 20-29 peptides form fibrils with diverse and polymorphic structures. All three forms of IAPP bound catalase, and complexes of IAPP 1-37 or 8-37 with catalase were identified by immunoassay. The binding of biotinylated IAPP to catalase was high affinity with a KD of 0.77nM, and could be inhibited by either human or rat IAPP 1-37 and 8-37 forms. Fibrils formed by the PrP 118-135 peptide with a Gly-Ala-Val-Val sequence also bound catalase. These results suggest that catalase recognizes a Gly-Ala-Ile-Leu—like sequence in amyloid fibril-forming peptides. For IAPP 1-37 and 8-37, the catalase binding was primarily directed towards fibrillar rather than ribbon-like structures, suggesting differences in the accessibility of the human IAPP 24-27 Gly-Ala-Ile-Leu region. This suggests that catalase may be able to discriminate between different structural forms of IAPP fibrils. The ability of catalase to bind IAPP, Aβ, and PrP fibrils demonstrates the presence of similar accessible structural motifs that may be targets for antiamyloid therapeutic development.

The effect of gastric inhibitory polypeptide on intestinal glucose absorption and intestinal motility in mice

Energy Technology Data Exchange (ETDEWEB)

Ogawa, Eiichi [Department of Diabetes and Clinical Nutrition, Graduate School of Medicine, Kyoto University (Japan); Hosokawa, Masaya [Department of Diabetes and Clinical Nutrition, Graduate School of Medicine, Kyoto University (Japan); Faculty of Human Sciences, Tezukayama Gakuin University, Osaka (Japan); Harada, Norio; Yamane, Shunsuke; Hamasaki, Akihiro; Toyoda, Kentaro; Fujimoto, Shimpei; Fujita, Yoshihito; Fukuda, Kazuhito [Department of Diabetes and Clinical Nutrition, Graduate School of Medicine, Kyoto University (Japan); Tsukiyama, Katsushi; Yamada, Yuichiro [Department of Diabetes and Clinical Nutrition, Graduate School of Medicine, Kyoto University (Japan); Department of Internal Medicine, Division of Endocrinology, Diabetes and Geriatric Medicine, Akita University School of Medicine, Akita (Japan); Seino, Yutaka [Department of Diabetes and Clinical Nutrition, Graduate School of Medicine, Kyoto University (Japan); Kansai Electric Power Hospital, Osaka (Japan); Inagaki, Nobuya, E-mail: inagaki@metab.kuhp.kyoto-u.ac.jp [Department of Diabetes and Clinical Nutrition, Graduate School of Medicine, Kyoto University (Japan); CREST of Japan Science and Technology Cooperation (JST), Kyoto (Japan)

2011-01-07

Research highlights: {yields} Exogenous GIP inhibits intestinal motility through a somatostatin-mediated pathway. {yields} Exogenous GIP inhibits intestinal glucose absorption by reducing intestinal motility. {yields} The GIP-receptor-mediated action in intestine does not involve in GLP-1-mediated pathway. -- Abstract: Gastric inhibitory polypeptide (GIP) is released from the small intestine upon meal ingestion and increases insulin secretion from pancreatic {beta} cells. Although the GIP receptor is known to be expressed in small intestine, the effects of GIP in small intestine are not fully understood. This study was designed to clarify the effect of GIP on intestinal glucose absorption and intestinal motility. Intestinal glucose absorption in vivo was measured by single-pass perfusion method. Incorporation of [{sup 14}C]-glucose into everted jejunal rings in vitro was used to evaluate the effect of GIP on sodium-glucose co-transporter (SGLT). Motility of small intestine was measured by intestinal transit after oral administration of a non-absorbed marker. Intraperitoneal administration of GIP inhibited glucose absorption in wild-type mice in a concentration-dependent manner, showing maximum decrease at the dosage of 50 nmol/kg body weight. In glucagon-like-peptide-1 (GLP-1) receptor-deficient mice, GIP inhibited glucose absorption as in wild-type mice. In vitro examination of [{sup 14}C]-glucose uptake revealed that 100 nM GIP did not change SGLT-dependent glucose uptake in wild-type mice. After intraperitoneal administration of GIP (50 nmol/kg body weight), small intestinal transit was inhibited to 40% in both wild-type and GLP-1 receptor-deficient mice. Furthermore, a somatostatin receptor antagonist, cyclosomatostatin, reduced the inhibitory effect of GIP on both intestinal transit and glucose absorption in wild-type mice. These results demonstrate that exogenous GIP inhibits intestinal glucose absorption by reducing intestinal motility through a somatostatin

The effect of gastric inhibitory polypeptide on intestinal glucose absorption and intestinal motility in mice

International Nuclear Information System (INIS)

Ogawa, Eiichi; Hosokawa, Masaya; Harada, Norio; Yamane, Shunsuke; Hamasaki, Akihiro; Toyoda, Kentaro; Fujimoto, Shimpei; Fujita, Yoshihito; Fukuda, Kazuhito; Tsukiyama, Katsushi; Yamada, Yuichiro; Seino, Yutaka; Inagaki, Nobuya

2011-01-01

Research highlights: → Exogenous GIP inhibits intestinal motility through a somatostatin-mediated pathway. → Exogenous GIP inhibits intestinal glucose absorption by reducing intestinal motility. → The GIP-receptor-mediated action in intestine does not involve in GLP-1-mediated pathway. -- Abstract: Gastric inhibitory polypeptide (GIP) is released from the small intestine upon meal ingestion and increases insulin secretion from pancreatic β cells. Although the GIP receptor is known to be expressed in small intestine, the effects of GIP in small intestine are not fully understood. This study was designed to clarify the effect of GIP on intestinal glucose absorption and intestinal motility. Intestinal glucose absorption in vivo was measured by single-pass perfusion method. Incorporation of [ 14 C]-glucose into everted jejunal rings in vitro was used to evaluate the effect of GIP on sodium-glucose co-transporter (SGLT). Motility of small intestine was measured by intestinal transit after oral administration of a non-absorbed marker. Intraperitoneal administration of GIP inhibited glucose absorption in wild-type mice in a concentration-dependent manner, showing maximum decrease at the dosage of 50 nmol/kg body weight. In glucagon-like-peptide-1 (GLP-1) receptor-deficient mice, GIP inhibited glucose absorption as in wild-type mice. In vitro examination of [ 14 C]-glucose uptake revealed that 100 nM GIP did not change SGLT-dependent glucose uptake in wild-type mice. After intraperitoneal administration of GIP (50 nmol/kg body weight), small intestinal transit was inhibited to 40% in both wild-type and GLP-1 receptor-deficient mice. Furthermore, a somatostatin receptor antagonist, cyclosomatostatin, reduced the inhibitory effect of GIP on both intestinal transit and glucose absorption in wild-type mice. These results demonstrate that exogenous GIP inhibits intestinal glucose absorption by reducing intestinal motility through a somatostatin-mediated pathway rather

Evaluation of a biomimetic 3D substrate based on the Human Elastin-like Polypeptides (HELPs) model system for elastolytic activity detection.

Science.gov (United States)

Corich, Lucia; Busetti, Marina; Petix, Vincenzo; Passamonti, Sabina; Bandiera, Antonella

2017-08-10

Elastin is a fibrous protein that confers elasticity to tissues such as skin, arteries and lung. It is extensively cross-linked, highly hydrophobic and insoluble. Nevertheless, elastin can be hydrolysed by bacterial proteases in infectious diseases, resulting in more or less severe tissue damage. Thus, development of substrates able to reliably and specifically detect pathogen-secreted elastolytic activity is needed to improve the in vitro evaluation of the injury that bacterial proteases may provoke. In this work, two human biomimetic elastin polypeptides, HELP and HELP1, as well as the matrices derived from HELP, have been probed as substrates for elastolytic activity detection. Thirty strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients were analyzed in parallel with standard substrates, to detect proteolytic and elastolytic activity. Results point to the HELP-based 3D matrix as an interesting biomimetic model of elastin to assess bacterial elastolytic activity in vitro. Moreover, this model substrate enables to further elucidate the mechanism underlying elastin degradation at molecular level, as well as to develop biomimetic material-based devices responsive to external stimuli. Copyright © 2017 Elsevier B.V. All rights reserved.