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.

Development of elastin-like recombinamer films with antimicrobial activity

DEFF Research Database (Denmark)

Costa, André; Machado, Raul; Ribeiro, Artur

2015-01-01

In the present work we explored the ABP-CM4 peptide properties from Bombyx mori for the creation of biopolymers with broad antimicrobial activity. An antimicrobial recombinant protein-based polymer (rPBP) was designed by cloning the DNA sequence coding for ABP-CM4 in frame with the N......-terminus of the elastin-like recombinamer consisting of 200 repetitions of the pentamer VPAVG, here named A200. The new rPBP, named CM4-A200, was purified via a simplified nonchromatographic method, making use of the thermoresponsive behavior of the A200 polymer. ABP-CM4 peptide was also purified through...... the incorporation of a formic acid cleavage site between the peptide and the A200 sequence. In soluble state the antimicrobial activity of both CM4-A200 polymer and ABP-CM4 peptide was poorly effective. However, when the CM4-A200 polymer was processed into free-standing films high antimicrobial activity against...

Nucleation and growth of elastin-like peptide fibril multilayers: an in situ atomic force microscopy study

International Nuclear Information System (INIS)

Yang Guocheng; Yip, Christopher M; Wong, Michael K; Lin, Lauren E

2011-01-01

Controlling how molecules assemble into complex supramolecular architectures requires careful consideration of the subtle inter- and intra-molecular interactions that control their association. This is particularly crucial in the context of assembly at interfaces, where both surface chemistry and structure can play a role in directing structure formation. We report here the results of a study into the self-assembly of the elastin-like peptide EP I on structurally modified highly ordered pyrolytic graphite, including the role of spatial confinement on fibril nucleation and the growth of oriented fibril multilayers. In situ atomic force microscopy performed in fluid and at elevated temperature provided direct evidence of frustrated fibril nuclei and oriented growth of independent fibril domains. These results portend the application of this in situ strategy for studies of the nucleation and growth mechanisms of other fibril- and amyloid-forming proteins.

Thermoresponsive self-assembly of short elastin-like polypentapeptides and their poly(ethylene glycol) derivatives

Czech Academy of Sciences Publication Activity Database

Pechar, Michal; Brus, Jiří; Kostka, Libor; Koňák, Čestmír; Urbanová, Martina; Šlouf, Miroslav

2007-01-01

Roč. 7, č. 1 (2007), s. 56-69 ISSN 1616-5187 R&D Projects: GA ČR GA204/05/2255; GA AV ČR IAA100500501 Institutional research plan: CEZ:AV0Z40500505 Keywords : elastin -like peptides * self-assembly * poly(ethylene glycol) Subject RIV: CD - Macromolecular Chemistry Impact factor: 2.831, year: 2007

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.

Prophylactic effects of elastin peptide derived from the bulbus arteriosus of fish on vascular dysfunction in spontaneously hypertensive rats.

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Takemori, Kumiko; Yamamoto, Ei; Ito, Hiroyuki; Kometani, Takashi

2015-01-01

To determine the prophylactic effects of an elastin peptide derived from the bulbus arteriosus of bonitos and prolylglycine (PG), a degradation product of elastin peptide, on vascular dysfunction in spontaneously hypertensive rats (SHRs). Male 15-week-old SHR/Izm rats were fed without (control group) or with elastin peptide (1 g/kg body weight) for 5 weeks (EP group), or were infused via an osmotic mini-pump for 4 weeks with PG (PG group) or saline (control group). Using thoracic aortas, we assessed endothelial changes by scanning electron microscopy. Vascular reactivity (contraction and relaxation) and pressure-induced distension was compared. mRNA production levels of endothelial nitric oxide synthase (eNOS) and intercellular adhesion molecule-1 (ICAM-1) were investigated by real-time-polymerase chain reaction. Aortas of the EP group displayed limited endothelial damage compared with that in the control group. Under treatment of SHRs with elastin peptide, the effect of phenylephrine returned closer to the normal level observed in normotensive Wistar-Kyoto (WKY/Izm) rats. mRNA production of eNOS (but not ICAM-1) was greater in the EP group than in the control group. Endothelial damage was suppressed and pressure-induced vascular distension was greater in the PG group than in the corresponding control group. These results suggest that elastin peptide from bonitos elicits prophylactic affects hypertension-associated vascular dysfunction by targeting the eNOS signaling pathway. PG may be a key mediator of the beneficial effects of elastin peptide. Copyright © 2014 Elsevier Inc. All rights reserved.

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

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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.

In vitro cross-linking of elastin peptides and molecular characterization of the resultant biomaterials

DEFF Research Database (Denmark)

Heinz, Andrea; Ruttkies, Christoph K H; Jahreis, Günther

2013-01-01

-link desmosine or isodesmosine was unexpected, however, could be confirmed by tandem mass spectrometry and molecular dynamics simulations. CONCLUSIONS: The study demonstrated that it is possible to produce biopolymers containing polyfunctional cross-links characteristic of mature elastin from small elastin......BACKGROUND: Elastin is a vital protein and the major component of elastic fibers which provides resilience to many vertebrate tissues. Elastin's structure and function are influenced by extensive cross-linking, however, the cross-linking pattern is still unknown. METHODS: Small peptides containing...... and the insoluble polymers, after digestion with pancreatic elastase or trypsin, were furthermore comprehensively characterized on the molecular level using MALDI-TOF/TOF mass spectrometry. RESULTS: MS(2) data was used to develop the software PolyLinX, which is able to sequence not only linear and bifunctionally...

Elastin-derived peptides are new regulators of insulin resistance development in mice

DEFF Research Database (Denmark)

Blaise, Sébastien; Romier, Béatrice; Kawecki, Charlotte

2013-01-01

. In the current study, we show that elastin-derived peptides (EDPs) may be involved in the development of insulin resistance (IRES) in mice. In chow-fed mice, acute or chronic intravenous injections of EDPs induced hyperglycemic effects associated with glucose uptake reduction and IRES in skeletal muscle, liver......, and adipose tissue. Based on in vivo, in vitro, and in silico approaches, we propose that this IRES is due to interaction between the insulin receptor (IR) and the neuraminidase-1 subunit of the elastin receptor complex triggered by EDPs. This interplay was correlated with decreased sialic acid levels...

Neutrophil elastase and elastin-derived peptides in BAL fluid and emphysematous changes on CT scans

International Nuclear Information System (INIS)

Betsuyaku, Tomoko; Nishimura, Masaharu; Yoshioka, Aya; Takeyabu, Kimihiro; Miyamoto, Kenji; Kawakami, Yoshikazu

1996-01-01

We examined the relationship between neutrophil elastase, elastin-derived peptides in bronchoalveolar lavage (BAL) fluid, and the development of pulmonary emphysema. The level of neutrophil elastase was higher in asymptomatic current smokers with emphysematous changes on computed tomographic scans than in current smokers without emphysematous changes, and was found to be correlated with the level of elastin-derived peptides in BAL fluid. Subjects with high levels of neutrophil elastase in BAL fluid had faster annual declines in FEV 1 . We conclude that the level of neutrophil elastase in BAL fluid can be used to differentiate asymptomatic cigarette smokers who are at risk for pulmonary emphysema from those who are not. (author)

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

Induction and regulation of murine emphysema by elastin peptides.

Science.gov (United States)

Sellami, Mehdi; Meghraoui-Kheddar, Aïda; Terryn, Christine; Fichel, Caroline; Bouland, Nicole; Diebold, Marie-Daniele; Guenounou, Moncef; Héry-Huynh, Stéphanie; Le Naour, Richard

2016-01-01

Emphysema is the major component of chronic obstructive pulmonary disease (COPD). During emphysema, elastin breakdown in the lung tissue originates from the release of large amounts of elastase by inflammatory cells. Elevated levels of elastin-derived peptides (EP) reflect massive pulmonary elastin breakdown in COPD patients. Only the EP containing the GXXPG conformational motif with a type VIII β-turn are elastin receptor ligands inducing biological activities. In addition, the COOH-terminal glycine residue of the GXXPG motif seems a prerequisite to the biological activity. In this study, we endotracheally instilled C57BL/6J mice with GXXPG EP and/or COOH-terminal glycine deleted-EP whose sequences were designed by molecular dynamics and docking simulations. We investigated their effect on all criteria associated with the progression of murine emphysema. Bronchoalveolar lavages were recovered to analyze cell profiles by flow cytometry and lungs were prepared to allow morphological and histological analysis by immunostaining and confocal microscopy. We observed that exposure of mice to EP elicited hallmark features of emphysema with inflammatory cell accumulation associated with increased matrix metalloproteinases and desmosine expression and of remodeling of parenchymal tissue. We also identified an inactive COOH-terminal glycine deleted-EP that retains its binding-activity to EBP and that is able to inhibit the in vitro and in vivo activities of emphysema-inducing EP. This study demonstrates that EP are key actors in the development of emphysema and that they represent pharmacological targets for an alternative treatment of emphysema based on the identification of EP analogous antagonists by molecular modeling studies. Copyright © 2016 the American Physiological Society.

Elastin binds to a multifunctional 67-kilodalton peripheral membrane protein

International Nuclear Information System (INIS)

Mecham, R.P.; Hinek, A.; Entwistle, R.; Wrenn, D.S.; Griffin, G.L.; Senior, R.M.

1989-01-01

Elastin binding proteins from plasma membranes of elastin-producing cells were isolated by affinity chromatography on immobilized elastin peptides. Three proteins of 67, 61, and 55 kDa were released from the elastin resin by guanidine/detergent, soluble elastin peptides, synthetic peptide VGVAPG, or galactoside sugars, but not by synthetic RGD-containing peptide or sugars not related to galactose. All three proteins incorporated radiolabel upon extracellular iodination and contained [ 3 H]leucine following metabolic labeling, confirming that each is a synthetic product of the cell. The 67-kDa protein could be released from the cell surface with lactose-containing buffers, whereas solubilization of the 61- and 55-kDa components required the presence of detergent. Although all three proteins were retained on elastin affinity columns, the 61- and 55-kDa components were retained only in the presence of 67-kDa protein, suggesting that the 67-kDa protein binds elastin and the 61- and 55-kDa proteins bind to the 67-kDa protein. The authors propose that the 67-, 61-, and 55-kDa proteins constitute an elastin-receptor complex that forms a transmembrane link between the extracellular matrix and the intracellular compartment

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

Elastin in the Liver

Directory of Open Access Journals (Sweden)

Jiri Kanta

2016-10-01

Full Text Available A characteristic feature of liver cirrhosis is the accumulation of large amounts of connective tissue with the prevailing content of type I collagen. Elastin is a minor connective tissue component in normal liver but it is actively synthesized by hepatic stellate cells and portal fibroblasts in diseased liver. The accumulation of elastic fibers in later stages of liver fibrosis may contribute to the decreasing reversibility of the disease with advancing time. Elastin is formed by polymerization of tropoelastin monomers. It is an amorphous protein highly resistant to the action of proteases that forms the core of elastic fibers. Microfibrils surrounding the core are composed of fibrillins that bind a number of proteins involved in fiber formation. They include microfibril-associated glycoproteins (MAGPs, microfibrillar-associated proteins (MFAPs and fibulins. Lysyl oxidase (LOX and lysyl oxidase-like proteins (LOXLs are responsible for tropoelastin cross-linking and polymerization. TGF-β complexes attached to microfibrils release this cytokine and influence the behavior of the cells in the neighborhood. The role of TGF-β as the main profibrotic cytokine in the liver is well-known and the release of the cytokines of TGF-β superfamily from their storage in elastic fibers may affect the course of fibrosis. Elastic fibres are often studied in the tissues where they provide elasticity and resilience but their role is no longer viewed as purely mechanical. Tropoelastin, elastin polymer and elastin peptides resulting from partial elastin degradation influence fibroblastic and inflammatory cells as well as angiogenesis. A similar role may be performed by elastin in the liver. This article reviews the results of the research of liver elastic fibers on the backgound of the present knowledge of elastin biochemistry and physiology. The regulation of liver elastin synthesis and degradation may be important for the outcome of liver fibrosis.

Elastin-Like Recombinamers As Smart Drug Delivery Systems.

Science.gov (United States)

Arias, F Javier; Santos, Mercedes; Ibanez-Fonseca, Arturo; Pina, Maria Jesus; Serrano, Sofía

2018-02-19

Drug delivery systems that are able to control the release of bioactive molecules and designed to carry drugs to target sites are of particular interest for tissue therapy. Moreover, systems comprising materials that can respond to environmental stimuli and promote self-assembly and higher order supramolecular organization are especially useful in the biomedical field. Objetive: This review focuses on biomaterials suitable for this purpose and that include elastin-like recombinamers (ELRs), a class of proteinaceous polymers bioinspired by natural elastin, designed using recombinant technologies. The self-assembly and thermoresponsive behaviour of these systems, along with their biodegradability, biocompatibility and well-defined composition as a result of their tailormade design, make them particularly attractive for controlled drug delivery. ELR-based delivery systems that allow targeted delivery are reviewed, especially ELR-drug recombinant fusion constructs, ELR-drug systems chemically bioconjugated in their monomeric and soluble forms, and drug encapsulation by nanoparticle-forming ELRs. Subsequently, the review focuses on those drug carriers in which smart release is triggered by pH or temperature with a particular focus on cancer treatments. Systems for controlled drug release based on depots and hydrogels that act as both a support and reservoir in which drugs can be stored will be described, and their applications in drug delivery discussed. Finally, smart drug-delivery systems not based on ELRs, including those comprising proteins, synthetic polymers and non-polymeric systems, will also be briefly discussed. Several different constructions based on ELRs are potential candidates for controlled drug delivery to be applied in advanced biomedical treatments. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Regulation of elastin synthesis in developing sheep nuchal ligament by elastin mRNA levels

International Nuclear Information System (INIS)

Davidson, J.M.; Smith, K.; Shibahara, S.; Tolstoshev, P.; Crystal, R.G.

1982-01-01

Levels of elastin production in explant culture of fetal sheep nuchal ligament and corresponding levels of translatable elastin mRNA were determined in parallel studies during a period of rapid growth of the embryo. The identity of the explant culture and cell-free proucts was confirmed by peptide mapping, immunoprecipitation, and the characteristic lack of histidine and methionine. Elastin production was quantitated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and radioimmune precipitation. The translation products could be labeled with methionine only when NH 2 -terminally donated as f-Met-tRNA/sup Met//sub f/. Explant cultures showed a large rise in elastin production from 70 days after conception to 150 days after conception. Cell free translation of RNA demonstrated a parallel in elastin mRNA levels and in elastin mRNA per cell. It appears, therefore, that the marked emphasis the differentiating muchal ligament places on elastin production is modulated, at least in part, by the quantities of available elastin in mRNA

Soluble expression and purification of the recombinant bioactive peptide precursor BPP-1 in Escherichia coli using a cELP-SUMO dual fusion system.

Science.gov (United States)

Rao, Shengqi; Zang, Xiangyu; Yang, Zhenquan; Gao, Lu; Yin, Yongqi; Fang, Weiming

2016-02-01

A bioactive peptide precursor (BPP-1, 14.3 kDa/115AA), a newly designed polypeptide that may exert a potential antihypertensive effect in vivo, is composed of many different ACE inhibitory peptides and antioxidant peptides tandemly linked according to the restriction sites of gastrointestinal proteases. In this report, we present a novel method to obtain soluble BPP-1 in Escherichia coli using cationic elastin-like polypeptide and SUMO (cELP-SUMO) tags. The cELP-SUMO-tagged fusion protein was expressed in soluble form at 20 °C for 20 h. After purification based on the inverse transition cycling (ITC) method, the purified cELP-SUMO-CFPP fusion protein was subsequently cleaved by a SUMO protease to release the mature BPP-1. After a subsequent simple salt precipitation process, approximately 167.2 mg of recombinant BPP-1 was obtained from 1 l of bacterial culture with at least 92% purity. The molecular mass (Mr) of the recombinant BPP-1 was confirmed by MALDI-TOF MS to equal 14,347. The purified BPP-1 was subjected to simulated gastrointestinal digestion, and the resulting hydrolysates exhibited notable ACE inhibitory and antioxidant activities in vitro. This report provides the first description of the soluble production of a bioactive peptide multimer with potential ACE inhibitory and antioxidant activities in E. coli using a cELP-SUMO tag. Copyright © 2015 Elsevier Inc. All rights reserved.

Hydroxyapatite and Calcified Elastin Induce Osteoblast-like Differentiation in Rat Aortic Smooth Muscle Cells

Science.gov (United States)

Lei, Yang; Sinha, Aditi; Nosoudi, Nasim; Grover, Ankit; Vyavahare, Naren

2014-01-01

Vascular calcification can be categorized into two different types. Intimal calcification related to atherosclerosis and elastin-specific medial arterial calcification (MAC). Osteoblast-like differentiation of vascular smooth muscle cells (VSMCs) has been shown in both types; however, how this relates to initiation of vascular calcification is unclear. We hypothesize that the initial deposition of hydroxyapatite-like mineral in MAC occurs on degraded elastin first and that causes osteogenic transformation of VSMCs. To test this, rat aortic smooth muscle cells (RASMCs) were cultured on hydroxyapatite crystals and calcified aortic elastin. Using RT-PCR and specific protein assays, we demonstrate that RASMCs lose their smooth muscle lineage markers like alpha smooth muscle actin (SMA) and myosin heavy chain (MHC) and undergo chondrogenic/osteogenic transformation. This is indicated by an increase in the expression of typical chondrogenic proteins such as aggrecan, collagen type II alpha 1(Col2a1) and bone proteins such as runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN). Furthermore, when calcified conditions are removed, cells return to their original phenotype. Our data supports the hypothesis that elastin degradation and calcification precedes VSMCs' osteoblast-like differentiation. PMID:24447384

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

Directory of Open Access Journals (Sweden)

Tomoyuki Koga

2015-01-01

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

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.

Paramyxovirus F1 protein has two fusion peptides: implications for the mechanism of membrane fusion.

Science.gov (United States)

Peisajovich, S G; Samuel, O; Shai, Y

2000-03-10

Viral fusion proteins contain a highly hydrophobic segment, named the fusion peptide, which is thought to be responsible for the merging of the cellular and viral membranes. Paramyxoviruses are believed to contain a single fusion peptide at the N terminus of the F1 protein. However, here we identified an additional internal segment in the Sendai virus F1 protein (amino acids 214-226) highly homologous to the fusion peptides of HIV-1 and RSV. A synthetic peptide, which includes this region, was found to induce membrane fusion of large unilamellar vesicles, at concentrations where the known N-terminal fusion peptide is not effective. A scrambled peptide as well as several peptides from other regions of the F1 protein, which strongly bind to membranes, are not fusogenic. The functional and structural characterization of this active segment suggest that the F1 protein has an additional internal fusion peptide that could participate in the actual fusion event. The presence of homologous regions in other members of the same family suggests that the concerted action of two fusion peptides, one N-terminal and the other internal, is a general feature of paramyxoviruses. Copyright 2000 Academic Press.

Collagen like peptide bioconjugates for targeted drug delivery applications

Science.gov (United States)

Luo, Tianzhi

the coil/globule conformational transition of the PDEGMEMA building block above its LCST with stabilization of the nanostructures by the hydrophilic CLP. To the best of our knowledge, this is the first report on such assembled nanostructures from collagen-like peptide containing copolymers. Due to the strong propensity for CLPs to bind to natural collagen via strand invasion processes, these nanosized vesicles may be used as drug carriers for targeted delivery. In addition to synthetic polymers, the collagen like peptide is then conjugated with a thermoresponsive elastin-like peptide (ELP). The resulting ELP-CLP diblock conjugates show a remarkable reduction in the inverse transition temperature of the ELP domain, attributed to the anchoring effect of the CLP triple helix. The lower transition temperature of the conjugate enables facile formation of well-defined vesicles at physiological temperature and the unexpected resolubilization of the vesicles at elevated temperatures upon unfolding of the CLP domain. Given the ability of CLPs to modify collagens, this work provides not only a simple and versatile avenue for controlling the inverse transition behavior of elastin-like peptides, but also suggest future opportunities for these thermoresponsive nanostructures in biologically relevant environments. In the last section, the potential of using the ELP-CLP nanoparticles as drug delivery vehicles for targeting collagen containing matrices is evaluated. A sustained release of clinically relevant amount of encapsulated modelled drug is achieved within three weeks, followed by a thermally controlled burst release. As expected, the ELP-CLP nanoparticles show strong retention on collagen substrate, via specific binding through collagen triple helix hybridization. Additionally, cell viability and proliferation studies using fibroblasts and chondrocytes suggest the nanoparticles are non-cytotoxic. Additionally, almost no TNF-alpha expression from macrophages is observed

Degradation of tropoelastin and skin elastin by neprilysin

DEFF Research Database (Denmark)

Mora Huertas, Angela C.; Schmelzer, Christian E. H.; Luise, Chiara

2018-01-01

was to investigate the degradation of fibrillar skin elastin by neprilysin and the influence of the donor's age on the degradation process using mass spectrometry and bioinformatics approaches. The results showed that cleavage by neprilysin is dependent on previous damage of elastin. While neprilysin does not cleave...... young and intact skin elastin well, it degrades elastin fibers from older donors, which may further promote aging processes. With regards to the cleavage behavior of neprilysin, a strong preference for Gly at P1 was found, while Gly, Ala and Val were well accepted at P1' upon cleavage of tropoelastin...... and skin elastin. The results of the study indicate that the progressive release of bioactive elastin peptides by neprilysin upon skin aging may enhance local tissue damage and accelerate extracellular matrix aging processes....

Elastin hydrolysate derived from fish enhances proliferation of human skin fibroblasts and elastin synthesis in human skin fibroblasts and improves the skin conditions.

Science.gov (United States)

Shiratsuchi, Eri; Nakaba, Misako; Yamada, Michio

2016-03-30

Recent studies have shown that certain peptides significantly improve skin conditions, such as skin elasticity and the moisture content of the skin of healthy woman. This study aimed to investigate the effects of elastin hydrolysate on human skin. Proliferation and elastin synthesis were evaluated in human skin fibroblasts exposed to elastin hydrolysate and proryl-glycine (Pro-Gly), which is present in human blood after elastin hydrolysate ingestion. We also performed an ingestion test with elastin hydrolysate in humans and evaluated skin condition. Elastin hydrolysate and Pro-Gly enhanced the proliferation of fibroblasts and elastin synthesis. Maximal proliferation response was observed at 25 ng mL(-1) Pro-Gly. Ingestion of elastin hydrolysate improved skin condition, such as elasticity, number of wrinkles, and blood flow. Elasticity improved by 4% in the elastin hydrolysate group compared with 2% in the placebo group. Therefore, elastin hydrolysate activates human skin fibroblasts and has beneficial effects on skin conditions. © 2015 Society of Chemical Industry.

Molecular-level insights into aging processes of skin elastin.

Science.gov (United States)

Mora Huertas, Angela C; Schmelzer, Christian E H; Hoehenwarter, Wolfgang; Heyroth, Frank; Heinz, Andrea

2016-01-01

Skin aging is characterized by different features including wrinkling, atrophy of the dermis and loss of elasticity associated with damage to the extracellular matrix protein elastin. The aim of this study was to investigate the aging process of skin elastin at the molecular level by evaluating the influence of intrinsic (chronological aging) and extrinsic factors (sun exposure) on the morphology and susceptibility of elastin towards enzymatic degradation. Elastin was isolated from biopsies derived from sun-protected or sun-exposed skin of differently aged individuals. The morphology of the elastin fibers was characterized by scanning electron microscopy. Mass spectrometric analysis and label-free quantification allowed identifying differences in the cleavage patterns of the elastin samples after enzymatic digestion. Principal component analysis and hierarchical cluster analysis were used to visualize differences between the samples and to determine the contribution of extrinsic and intrinsic aging to the proteolytic susceptibility of elastin. Moreover, the release of potentially bioactive peptides was studied. Skin aging is associated with the decomposition of elastin fibers, which is more pronounced in sun-exposed tissue. Marker peptides were identified, which showed an age-related increase or decrease in their abundances and provide insights into the progression of the aging process of elastin fibers. Strong age-related cleavage occurs in hydrophobic tropoelastin domains 18, 20, 24 and 26. Photoaging makes the N-terminal and central parts of the tropoelastin molecules more susceptible towards enzymatic cleavage and, hence, accelerates the age-related degradation of elastin. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

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.

Aortic microcalcification is associated with elastin fragmentation in Marfan syndrome.

Science.gov (United States)

Wanga, Shaynah; Hibender, Stijntje; Ridwan, Yanto; van Roomen, Cindy; Vos, Mariska; van der Made, Ingeborg; van Vliet, Nicole; Franken, Romy; van Riel, Luigi Amjg; Groenink, Maarten; Zwinderman, Aeilko H; Mulder, Barbara Jm; de Vries, Carlie Jm; Essers, Jeroen; de Waard, Vivian

2017-11-01

Marfan syndrome (MFS) is a connective tissue disorder in which aortic rupture is the major cause of death. MFS patients with an aortic diameter below the advised limit for prophylactic surgery (elastin fragments play a causal role in aortic calcification in MFS, and that microcalcification serves as a marker for aortic disease severity. To address this hypothesis, we analysed MFS patient and mouse aortas. MFS patient aortic tissue showed enhanced microcalcification in areas with extensive elastic lamina fragmentation in the media. A causal relationship between medial injury and microcalcification was revealed by studies in vascular smooth muscle cells (SMCs); elastin peptides were shown to increase the activity of the calcification marker alkaline phosphatase (ALP) and reduce the expression of the calcification inhibitor matrix GLA protein in human SMCs. In murine Fbn1 C1039G/+ MFS aortic SMCs, Alpl mRNA and activity were upregulated as compared with wild-type SMCs. The elastin peptide-induced ALP activity was prevented by incubation with lactose or a neuraminidase inhibitor, which inhibit the elastin receptor complex, and a mitogen-activated protein kinase kinase-1/2 inhibitor, indicating downstream involvement of extracellular signal-regulated kinase-1/2 (ERK1/2) phosphorylation. Histological analyses in MFS mice revealed macrocalcification in the aortic root, whereas the ascending aorta contained microcalcification, as identified with the near-infrared fluorescent bisphosphonate probe OsteoSense-800. Significantly, microcalcification correlated strongly with aortic diameter, distensibility, elastin breaks, and phosphorylated ERK1/2. In conclusion, microcalcification co-localizes with aortic elastin degradation in MFS aortas of humans and mice, where elastin-derived peptides induce a calcification process in SMCs via the elastin receptor complex and ERK1/2 activation. We propose microcalcification as a novel imaging marker to monitor local elastin degradation and

Development of short and highly potent self-assembling elastin-derived pentapeptide repeats containing aromatic amino acid residues.

Science.gov (United States)

Taniguchi, Suguru; Watanabe, Noriko; Nose, Takeru; Maeda, Iori

2016-01-01

Tropoelastin is the primary component of elastin, which forms the elastic fibers that make up connective tissues. The hydrophobic domains of tropoelastin are thought to mediate the self-assembly of elastin into fibers, and the temperature-mediated self-assembly (coacervation) of one such repetitive peptide sequence (VPGVG) has been utilized in various bio-applications. To elucidate a mechanism for coacervation activity enhancement and to develop more potent coacervatable elastin-derived peptides, we synthesized two series of peptide analogs containing an aromatic amino acid, Trp or Tyr, in addition to Phe-containing analogs and tested their functional characteristics. Thus, position 1 of the hydrophobic pentapeptide repeat of elastin (X(1)P(2)G(3)V(4)G(5)) was substituted by Trp or Tyr. Eventually, we acquired a novel, short Trp-containing elastin-derived peptide analog (WPGVG)3 with potent coacervation ability. From the results obtained during this process, we determined the importance of aromaticity and hydrophobicity for the coacervation potency of elastin-derived peptide analogs. Generally, however, the production of long-chain synthetic polypeptides in quantities sufficient for commercial use remain cost-prohibitive. Therefore, the identification of (WPGVG)3, which is a 15-mer short peptide consisting simply of five natural amino acids and shows temperature-dependent self-assembly activity, might serve as a foundation for the development of various kinds of biomaterials. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

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'.

Molecular-level insights into aging processes of skin elastin

DEFF Research Database (Denmark)

Mora Huertas, Angela C; Schmelzer, Christian E H; Hoehenwarter, Wolfgang

2016-01-01

Skin aging is characterized by different features including wrinkling, atrophy of the dermis and loss of elasticity associated with damage to the extracellular matrix protein elastin. The aim of this study was to investigate the aging process of skin elastin at the molecular level by evaluating...... the influence of intrinsic (chronological aging) and extrinsic factors (sun exposure) on the morphology and susceptibility of elastin towards enzymatic degradation. Elastin was isolated from biopsies derived from sun-protected or sun-exposed skin of differently aged individuals. The morphology of the elastin...... pronounced in sun-exposed tissue. Marker peptides were identified, which showed an age-related increase or decrease in their abundances and provide insights into the progression of the aging process of elastin fibers. Strong age-related cleavage occurs in hydrophobic tropoelastin domains 18, 20, 24 and 26...

Multimerized CHR-derived peptides as HIV-1 fusion inhibitors.

Science.gov (United States)

Nomura, Wataru; Hashimoto, Chie; Suzuki, Takaharu; Ohashi, Nami; Fujino, Masayuki; Murakami, Tsutomu; Yamamoto, Naoki; Tamamura, Hirokazu

2013-08-01

To date, several HIV-1 fusion inhibitors based on the carboxy-terminal leucine/isoleucine heptad repeat (CHR) region of an HIV-1 envelope protein gp41 have been discovered. We have shown that a synthetic peptide mimetic of a trimer form of the CHR-derived peptide C34 has potent inhibitory activity against the HIV-1 fusion mechanism, compared to a monomer C34 peptide. The present study revealed that a dimeric form of C34 is evidently structurally critical for fusion inhibitors, and that the activity of multimerized CHR-derived peptides in fusion inhibition is affected by the properties of the unit peptides C34, SC34EK, and T20. The fluorescence-based study suggested that the N36-interactive sites of the C34 trimer, including hydrophobic residues, are exposed outside the trimer and that trimerization of C34 caused a remarkable increase in fusion inhibitory activity. The present results could be useful in the design of fusion inhibitors against viral infections which proceed via membrane fusion with host cells. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

Mechanical, structural, and dynamical modifications of cholesterol exposed porcine aortic elastin.

Science.gov (United States)

Bilici, Kubra; Morgan, Steven W; Silverstein, Moshe C; Wang, Yunjie; Sun, Hyung Jin; Zhang, Yanhang; Boutis, Gregory S

2016-11-01

Elastin is a protein of the extracellular matrix that contributes significantly to the elasticity of connective tissues. In this study, we examine dynamical and structural modifications of aortic elastin exposed to cholesterol by NMR spectroscopic and relaxation methodologies. Macroscopic measurements are also presented and reveal that cholesterol treatment may cause a decrease in the stiffness of tissue. 2 H NMR relaxation techniques revealed differences between the relative populations of water that correlate with the swelling of the tissue following cholesterol exposure. 13 C magic-angle-spinning NMR spectroscopy and relaxation methods indicate that cholesterol treated aortic elastin is more mobile than control samples. Molecular dynamics simulations on a short elastin repeat VPGVG in the presence of cholesterol are used to investigate the energetic and entropic contributions to the retractive force, in comparison to the same peptide in water. Peptide stiffness is observed to reduce following cholesterol exposure due to a decrease in the entropic force. Copyright © 2016 Elsevier B.V. All rights reserved.

Peptide-Mediated Liposome Fusion: The Effect of Anchor Positioning

Directory of Open Access Journals (Sweden)

Niek S. A. Crone

2018-01-01

Full Text Available A minimal model system for membrane fusion, comprising two complementary peptides dubbed “E” and “K” joined to a cholesterol anchor via a polyethyleneglycol spacer, has previously been developed in our group. This system promotes the fusion of large unilamellar vesicles and facilitates liposome-cell fusion both in vitro and in vivo. Whilst several aspects of the system have previously been investigated to provide an insight as to how fusion is facilitated, anchor positioning has not yet been considered. In this study, the effects of placing the anchor at either the N-terminus or in the center of the peptide are investigated using a combination of circular dichroism spectroscopy, dynamic light scattering, and fluorescence assays. It was discovered that anchoring the “K” peptide in the center of the sequence had no effect on its structure, its ability to interact with membranes, or its ability to promote fusion, whereas anchoring the ‘E’ peptide in the middle of the sequence dramatically decreases fusion efficiency. We postulate that anchoring the ‘E’ peptide in the middle of the sequence disrupts its ability to form homodimers with peptides on the same membrane, leading to aggregation and content leakage.

The dengue virus type 2 envelope protein fusion peptide is essential for membrane fusion

International Nuclear Information System (INIS)

Huang, Claire Y.-H.; Butrapet, Siritorn; Moss, Kelly J.; Childers, Thomas; Erb, Steven M.; Calvert, Amanda E.; Silengo, Shawn J.; Kinney, Richard M.; Blair, Carol D.; Roehrig, John T.

2010-01-01

The flaviviral envelope (E) protein directs virus-mediated membrane fusion. To investigate membrane fusion as a requirement for virus growth, we introduced 27 unique mutations into the fusion peptide of an infectious cDNA clone of dengue 2 virus and recovered seven stable mutant viruses. The fusion efficiency of the mutants was impaired, demonstrating for the first time the requirement for specific FP AAs in optimal fusion. Mutant viruses exhibited different growth kinetics and/or genetic stabilities in different cell types and adult mosquitoes. Virus particles could be recovered following RNA transfection of cells with four lethal mutants; however, recovered viruses could not re-infect cells. These viruses could enter cells, but internalized virus appeared to be retained in endosomal compartments of infected cells, thus suggesting a fusion blockade. Mutations of the FP also resulted in reduced virus reactivity with flavivirus group-reactive antibodies, confirming earlier reports using virus-like particles.

Determination of the minimal fusion peptide of bovine leukemia virus gp30

International Nuclear Information System (INIS)

Lorin, Aurelien; Lins, Laurence; Stroobant, Vincent; Brasseur, Robert; Charloteaux, Benoit

2007-01-01

In this study, we determined the minimal N-terminal fusion peptide of the gp30 of the bovine leukemia virus on the basis of the tilted peptide theory. We first used molecular modelling to predict that the gp30 minimal fusion peptide corresponds to the 15 first residues. Liposome lipid-mixing and leakage assays confirmed that the 15-residue long peptide induces fusion in vitro and that it is the shortest peptide inducing optimal fusion since longer peptides destabilize liposomes to the same extent but not shorter ones. The 15-residue long peptide can thus be considered as the minimal fusion peptide. The effect of mutations reported in the literature was also investigated. Interestingly, mutations related to glycoproteins unable to induce syncytia in cell-cell fusion assays correspond to peptides predicted as non-tilted. The relationship between obliquity and fusogenicity was also confirmed in vitro for one tilted and one non-tilted mutant peptide

Isolation of intact elastin fibers devoid of microfibrils.

NARCIS (Netherlands)

Daamen, W.F.; Hafmans, T.G.M.; Veerkamp, J.H.; Kuppevelt, A.H.M.S.M. van

2005-01-01

Purification protocols for elastin generally result in greatly damaged elastin fibers and this likely influences the biological response. We here describe a novel protocol for the isolation of elastin whereby the fibers stay intact, and introduce the term "elastin fiber" for intact elastic fibers

Biophysical characterization of a de novo elastin

Science.gov (United States)

Greenland, Kelly Nicole

Natural human elastin is found in tissue such as the lungs, arteries, and skin. This protein is formed at birth with no mechanism present to repair or supplement the initial quantity formed. As a result, the functionality and durability of elastin's elasticity is critically important. To date, the mechanics of this ability to stretch and recoil is not fully understood. This study utilizes de novo protein design to create a small library of simplistic versions of elastin-like proteins, demonstrate the elastin-like proteins, maintain elastin's functionality, and inquire into its structure using solution nuclear magnetic resonance (NMR). Elastin is formed from cross-linked tropoelastin. Therefore, the first generation of designed proteins consisted of one protein that utilized homogony of interspecies tropoelastin by using three common domains, two hydrophobic and one cross-linking domains. Basic modifications were made to open the hydrophobic region and also to make the protein easier to purify and characterize. The designed protein maintained its functionality, self-aggregating as the temperature increased. Uniquely, the protein remained self-aggregated as the temperature returned below the critical transition temperature. Self-aggregation was additionally induced by increasing salt concentrations and by modifying the pH. The protein appeared to have little secondary structure when studied with solution NMR. These results fueled a second generation of designed elastin-like proteins. This generation contained variations designed to study the cross-linking domain, one specific hydrophobic domain, and the effect of the length of the elastin-like protein. The cross-linking domain in one variation has been significantly modified while the flanking hydrophobic domains have remained unchanged. This characterization of this protein will answer questions regarding the specificity of the homologous nature of the cross-linking domain of tropoelastin across species. A second

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.

Fusion peptides from oncogenic chimeric proteins as putative specific biomarkers of cancer.

Science.gov (United States)

Conlon, Kevin P; Basrur, Venkatesha; Rolland, Delphine; Wolfe, Thomas; Nesvizhskii, Alexey I; MacCoss, Michael J; Lim, Megan S; Elenitoba-Johnson, Kojo S J

2013-10-01

Chromosomal translocations encoding chimeric fusion proteins constitute one of the most common mechanisms underlying oncogenic transformation in human cancer. Fusion peptides resulting from such oncogenic chimeric fusions, though unique to specific cancer subtypes, are unexplored as cancer biomarkers. Here we show, using an approach termed fusion peptide multiple reaction monitoring mass spectrometry, the direct identification of different cancer-specific fusion peptides arising from protein chimeras that are generated from the juxtaposition of heterologous genes fused by recurrent chromosomal translocations. Using fusion peptide multiple reaction monitoring mass spectrometry in a clinically relevant scenario, we demonstrate the specific, sensitive, and unambiguous detection of a specific diagnostic fusion peptide in clinical samples of anaplastic large cell lymphoma, but not in a diverse array of benign lymph nodes or other forms of primary malignant lymphomas and cancer-derived cell lines. Our studies highlight the utility of fusion peptides as cancer biomarkers and carry broad implications for the use of protein biomarkers in cancer detection and monitoring.

3D silicon doped hydroxyapatite scaffolds decorated with Elastin-like Recombinamers for bone regenerative medicine.

Science.gov (United States)

Vila, Mercedes; García, Ana; Girotti, Alessandra; Alonso, Matilde; Rodríguez-Cabello, Jose Carlos; González-Vázquez, Arlyng; Planell, Josep A; Engel, Elisabeth; Buján, Julia; García-Honduvilla, Natalio; Vallet-Regí, María

2016-11-01

The current study reports on the manufacturing by rapid prototyping technique of three-dimensional (3D) scaffolds based on silicon substituted hydroxyapatite with Elastin-like Recombinamers (ELRs) functionalized surfaces. Silicon doped hydroxyapatite (Si-HA), with Ca 10 (PO 4 ) 5.7 (SiO 4 ) 0.3 (OH) 1.7 h 0.3 nominal formula, was surface functionalized with two different types of polymers designed by genetic engineering: ELR-RGD that contain cell attachment specific sequences and ELR-SN A 15/RGD with both hydroxyapatite and cells domains that interact with the inorganic phase and with the cells, respectively. These hybrid materials were subjected to in vitro assays in order to clarify if the ELRs coating improved the well-known biocompatible and bone regeneration properties of calcium phosphates materials. The in vitro tests showed that there was a total and homogeneous colonization of the 3D scaffolds by Bone marrow Mesenchymal Stromal Cells (BMSCs). In addition, the BMSCs were viable and able to proliferate and differentiate into osteoblasts. Bone tissue engineering is an area of increasing interest because its main applications are directly related to the rising life expectancy of the population, which promotes higher rates of several bone pathologies, so innovative strategies are needed for bone tissue regeneration therapies. Here we use the rapid prototyping technology to allow moulding ceramic 3D scaffolds and we use different bio-polymers for the functionalization of their surfaces in order to enhance the biological response. Combining the ceramic material (silicon doped hydroxyapatite, Si-HA) and the Elastin like Recombinamers (ELRs) polymers with the presence of the integrin-mediate adhesion domain alone or in combination with SNA15 peptide that possess high affinity for hydroxyapatite, provided an improved Bone marrow Mesenchymal Stromal Cells (BMSCs) differentiation into osteoblastic linkage. Copyright © 2016 Acta Materialia Inc. Published by Elsevier

Circulating Anti-Elastin Antibody Levels and Arterial Disease Characteristics: Associations with Arterial Stiffness and Atherosclerosis.

Science.gov (United States)

Lee, Seung-Hyun; Shin, Kihyuk; Park, Sungha; Kang, Seok-Min; Choi, Donghoon; Lee, Seung-Hyo; Lee, Sang-Hak

2015-11-01

Elastin is a major arterial structural protein, and elastin-derived peptides are related to arterial change. We previously reported on a novel assay developed using aortic elastin peptides; however, its clinical implications remain unclear. In this study, we assessed whether anti-elastin antibody titers reflect the risk of coronary artery disease (CAD) or its characteristics. We included 174 CAD patients and 171 age- and sex-matched controls. Anti-elastin antibody titers were quantified by enzyme-linked immunosorbent assay. Parameters of arterial stiffness, including the augmentation index (AI) and heart-to-femoral pulse wave velocity (hfPWV), were measured non-invasively. The clinical and angiographic characteristics of CAD patients were also evaluated. Associations between anti-elastin levels and vascular characteristics were examined by linear regression analysis. The median blood level of anti-elastin was significantly lower in the CAD group than in the controls [197 arbitrary unit (a.u.) vs. 63 a.u., pelastin were significantly lower in men and in subjects with hypertension, diabetes mellitus, hyperlipidemia, or high hfPWV. Nevertheless, anti-elastin levels were not dependent on atherothrombotic events or the angiographic severity of CAD. In a multivariate analysis, male sex (β=-0.38, pelastin levels. Lower levels of anti-elastin are related to CAD. The association between antibody titers and CAD is linked to arterial stiffness rather than the advancement of atherosclerosis.

Simulation Studies of LCST-like Phase Transitions in Elastin-like Polypeptides (ELPs) and Conjugates of ELP with Rigid Macromolecules

Science.gov (United States)

Condon, Joshua; Martin, Tyler; Jayaraman, Arthi

We use atomistic (AA) and coarse-grained (CG) molecular dynamics simulations to elucidate the thermodynamic driving forces governing lower critical solution temperature (LCST)-like phase transition exhibited by elastin-like peptides (ELPs) and conjugates of ELP with other macromolecules. In the AA simulations, we study ELP oligomers in explicit water, and mark the transition as the temperature at which they undergo a change in ``hydration'' state. While AA simulations are restricted to small systems of short ELPs and do not capture the chain aggregation observed in experiments of ELPs, they guide the phenomenological CG model development by highlighting the solvent induced polymer-polymer effective interactions with changing temperature. In the CG simulations, we capture the LCST polymer aggregation by increasing polymer-polymer effective attractive interactions in an implicit solvent. We examine the impact of conjugating a block of LCST polymer to another rigid unresponsive macromolecular block on the LCST-like transition. We find that when multiple LCST polymers are conjugated to a rigid polymer block, increased crowding of the LCST polymers shifts the onset of chain aggregation to smaller effective polymer-polymer attraction compared to the free LCST polymers. These simulation results provide guidance on the design of conjugated bio-mimetic thermoresponsive materials, and shape the fundamental understanding of the impact of polymer crowding on phase behavior in thermoresponsive LCST polymer systems.

Vascular nanomedicine: Site specific delivery of elastin stabilizing therapeutics to damaged arteries

Science.gov (United States)

Sinha, Aditi

Elastin, a structural protein in the extra-cellular matrix, plays a critical role in the normal functioning of blood vessels. Apart from performing its primary function of providing resilience to arteries, it also plays major role in regulating cell-cell and cell-matrix interactions, response to injury, and morphogenesis. Medial arterial calcification (MAC) and abdominal aortic aneurysm (AAA) are two diseases where the structural and functional integrity of elastin is severely compromised. Although the clinical presentation of MAC and AAA differ, they have one common underlying causative mechanism---pathological degradation of elastin. Hence prevention of elastin degradation in the early stages of MAC and AAA can mitigate, partially if not wholly, the fatal consequences of both the diseases. The work presented here is motivated by the overwhelming statistics of people afflicted by elastin associated cardiovascular diseases and the unavailability of cure for the same. Overall goal of our research is to understand role of elastin degradation in cardiovascular diseases and to develop a targeted vascular drug delivery system that is minimally invasive, biodegradable, and non-toxic, that prevents elastin from degradation. Our hope is that such treatment will also help regenerate elastin, thereby providing a multi-fold treatment option for elasto-degenerative vascular diseases. For this purpose, we have first confirmed the combined role of degraded elastin and hyperglycemia in the pathogenesis of MAC. We have shown that in the absence of degraded elastin and TGF-beta1 (abundantly present in diabetic arteries) vascular smooth muscle cells maintain their homeostatic state, regardless of environmental glucose concentrations. However simultaneous exposure to glucose, elastin peptides and TGF-beta1 causes the pathological transgenesis of vascular cells to osteoblast-like cells. We show that plant derived polyphenols bind to vascular elastin with great affinity resulting in

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.

Synthetic ligands of the elastin receptor induce elastogenesis in human dermal fibroblasts via activation of their IGF-1 receptors.

Science.gov (United States)

Qa'aty, Nour; Vincent, Matthew; Wang, Yanting; Wang, Andrew; Mitts, Thomas F; Hinek, Aleksander

2015-12-01

We have previously reported that a mixture of peptides obtained after chemical or enzymatic degradation of bovine elastin, induced new elastogenesis in human skin. Now, we investigated the elastogenic potential of synthetic peptides mimicking the elastin-derived, VGVAPG sequence, IGVAPG sequence that we found in the rice bran, and a similar peptide, VGVTAG that we identified in the IGF-1-binding protein-1 (IGFBP-1). We now demonstrate that treatment with each of these xGVxxG peptides (recognizable by the anti-elastin antibody), up-regulated the levels of elastin-encoding mRNA, tropoelastin protein, and the deposition of new elastic fibers in cultures of human dermal fibroblasts and in cultured explants of human skin. Importantly, we found that such induction of new elastogenesis may involve two parallel signaling pathways triggered after activation of IGF-1 receptor. In the first one, the xGVxxG peptides interact with the cell surface elastin receptor, thereby causing the downstream activation of the c-Src kinase and a consequent cross-activation of the adjacent IGF-1R, even in the absence of its principal ligand. In the second pathway their hydrophobic association with the N-terminal domain (VGVTAG) of the serum-derived IGFBP-1 induces conformational changes of this IGF-1 chaperone allowing for the release of its cargo and a consequent ligand-specific phosphorylation of IGF-1R. We present a novel, clinically relevant mechanism in which products of partial degradation of dermal elastin may stimulate production of new elastic fibers by dermal fibroblasts. Our findings particularly encourage the use of biologically safe synthetic xGVxxG peptides for regeneration of the injured or aged human skin. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Modulated growth, stability and interactions of liquid-like coacervate assemblies of elastin.

Science.gov (United States)

Muiznieks, Lisa D; Cirulis, Judith T; van der Horst, Astrid; Reinhardt, Dieter P; Wuite, Gijs J L; Pomès, Régis; Keeley, Fred W

2014-06-01

Elastin self-assembles from monomers into polymer networks that display elasticity and resilience. The first major step in assembly is a liquid-liquid phase separation known as coacervation. This process represents a continuum of stages from initial phase separation to early growth of droplets by coalescence and later "maturation" leading to fiber formation. Assembly of tropoelastin-rich globules is on pathway for fiber formation in vivo. However, little is known about these intermediates beyond their size distribution. Here we investigate the contribution of sequence and structural motifs from full-length tropoelastin and a set of elastin-like polypeptides to the maturation of coacervate assemblies, observing their growth, stability and interaction behavior, and polypeptide alignment within matured globules. We conclude that maturation is driven by surface properties, leading to stabilization of the interface between the hydrophobic interior and aqueous solvent, potentially through structural motifs, and discuss implications for droplet interactions in fiber formation. Copyright © 2014. Published by Elsevier B.V.

Small surfactant-like peptides can drive soluble proteins into active aggregates

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Zhou Bihong

2012-01-01

Full Text Available Abstract Background Inactive protein inclusion bodies occur commonly in Escherichia coli (E. coli cells expressing heterologous proteins. Previously several independent groups have found that active protein aggregates or pseudo inclusion bodies can be induced by a fusion partner such as a cellulose binding domain from Clostridium cellulovorans (CBDclos when expressed in E. coli. More recently we further showed that a short amphipathic helical octadecapeptide 18A (EWLKAFYEKVLEKLKELF and a short beta structure peptide ELK16 (LELELKLKLELELKLK have a similar property. Results In this work, we explored a third type of peptides, surfactant-like peptides, for performing such a "pulling-down" function. One or more of three such peptides (L6KD, L6K2, DKL6 were fused to the carboxyl termini of model proteins including Aspergillus fumigatus amadoriase II (AMA, all three peptides were used, Bacillus subtilis lipase A (LipA, only L6KD was used, hereinafter the same, Bacillus pumilus xylosidase (XynB, and green fluorescent protein (GFP, and expressed in E. coli. All fusions were found to predominantly accumulate in the insoluble fractions, with specific activities ranging from 25% to 92% of the native counterparts. Transmission electron microscopic (TEM and confocal fluorescence microscopic analyses confirmed the formation of protein aggregates in the cell. Furthermore, binding assays with amyloid-specific dyes (thioflavin T and Cong red to the AMA-L6KD aggregate and the TEM analysis of the aggregate following digestion with protease K suggested that the AMA-L6KD aggregate may contain structures reminiscent of amyloids, including a fibril-like structure core. Conclusions This study shows that the surfactant-like peptides L6KD and it derivatives can act as a pull-down handler for converting soluble proteins into active aggregates, much like 18A and ELK16. These peptide-mediated protein aggregations might have important implications for protein aggregation in

The action of neutrophil serine proteases on elastin and its precursor

DEFF Research Database (Denmark)

Heinz, Andrea; Jung, Michael C; Jahreis, Günther

2012-01-01

This study aimed to investigate the degradation of the natural substrates tropoelastin and elastin by the neutrophil-derived serine proteases human leukocyte elastase (HLE), proteinase 3 (PR3) and cathepsin G (CG). Focus was placed on determining their cleavage site specificities using mass...... spectrometric techniques. Moreover, the release of bioactive peptides from elastin by the three proteases was studied. Tropoelastin was comprehensively degraded by all three proteases, whereas less cleavage occurred in mature cross-linked elastin. An analysis of the cleavage site specificities of the three...... proteases in tropoelastin and elastin revealed that HLE and PR3 similarly tolerate hydrophobic and/or aliphatic amino acids such as Ala, Gly and Val at P(1), which are also preferred by CG. In addition, CG prefers the bulky hydrophobic amino acid Leu and accepts the bulky aromatic amino acids Phe and Tyr...

Electrospun silk-elastin-like fibre mats for tissue engineering applications

International Nuclear Information System (INIS)

Machado, Raul; Da Costa, André; Padrão, Jorge; Gomes, Andreia; Casal, Margarida; Sencadas, Vitor; Costa, Carlos M; Lanceros-Méndez, Senentxu; Garcia-Arévalo, Carmen; Rodríguez-Cabello, José Carlos

2013-01-01

Protein-based polymers are present in a wide variety of organisms fulfilling structural and mechanical roles. Advances in protein engineering and recombinant DNA technology allow the design and production of recombinant protein-based polymers (rPBPs) with an absolute control of its composition. Although the application of recombinant proteins as biomaterials is still an emerging technology, the possibilities are limitless and far superior to natural or synthetic materials, as the complexity of the structural design can be fully customized. In this work, we report the electrospinning of two new genetically engineered silk-elastin-like proteins (SELPs) consisting of alternate silk- and elastin-like blocks. Electrospinning was performed with formic acid and aqueous solutions at different concentrations without addition of further agents. The size and morphology of the electrospun structures was characterized by scanning electron microscopy showing its dependence on the concentration and solvent used. Treatment with methanol-saturated air was employed to stabilize the structure and promote water insolubility through a time-dependent conversion of random coils into β-sheets (FTIR). The resultant methanol-treated electrospun mats were characterized for swelling degree (570–720%), water vapour transmission rate (1083 g/m 2 /day) and mechanical properties (modulus of elasticity ∼126 MPa). Furthermore, the methanol-treated SELP fibre mats showed no cytotoxicity and were able to support adhesion and proliferation of normal human skin fibroblasts. Adhesion was characterized by a filopodia-mediated mechanism. These results demonstrate that SELP fibre mats can provide promising solutions for the development of novel biomaterials suitable for tissue engineering applications. (paper)

Insights into the degradation of human elastin by matrilysin-1

DEFF Research Database (Denmark)

Heinz, Andrea; Taddese, Samuel; Sippl, Wolfgang

2011-01-01

Human matrilysin-1 (MMP-7) is one of the most potent elastases besides macrophage elastase in the family of matrix metalloproteinases (MMPs). It has been reported to provide macrophages with the highest elastinolytic capacity and plays key roles in diseases such as emphysema and cancer. Describin...... into the degradation of human elastin by MMP-7 and may aid in the development of approaches to treat elastin-degrading diseases.......Human matrilysin-1 (MMP-7) is one of the most potent elastases besides macrophage elastase in the family of matrix metalloproteinases (MMPs). It has been reported to provide macrophages with the highest elastinolytic capacity and plays key roles in diseases such as emphysema and cancer. Describing...... the enzymatic turnover of matrix components helps to understand the molecular basis of disease processes. Hence, in this work, the cleavage behavior of MMP-7 with respect to its natural substrate human elastin was investigated using mass spectrometric (MS) techniques and molecular modeling. Elastin peptides...

Co-delivery of a growth factor and a tissue-protective molecule using elastin biopolymers accelerates wound healing in diabetic mice.

Science.gov (United States)

Devalliere, Julie; Dooley, Kevin; Hu, Yong; Kelangi, Sarah S; Uygun, Basak E; Yarmush, Martin L

2017-10-01

Growth factor therapy is a promising approach for chronic diabetic wounds, but strategies to efficiently and cost-effectively deliver active molecules to the highly proteolytic wound environment remain as major obstacles. Here, we re-engineered keratinocyte growth factor (KGF) and the cellular protective peptide ARA290 into a protein polymer suspension with the purpose of increasing their proteolytic resistance, thus their activity in vivo. KGF and ARA290 were fused with elastin-like peptide (ELP), a protein polymer derived from tropoelastin, that confers the ability to separate into a colloidal suspension of liquid-like coacervates. ELP fusion did not diminish peptides activities as demonstrated by ability of KGF-ELP to accelerate keratinocyte proliferation and migration, and ARA290-ELP to protect cells from apoptosis. We examined the healing effect of ARA290-ELP and KGF-ELP alone or in combination, in a full-thickness diabetic wound model. In this model, ARA290-ELP was found to accelerate healing, notably by increasing angiogenesis in the wound bed. We further showed that co-delivery of ARA290 and KGF, with the 1:4 KGF-ELP to ARA290-ELP ratio, was the most effective wound treatment with the fastest healing rate, the thicker granulation tissue and regenerated epidermis after 28 days. Overall, this study shows that ARA290-ELP and KGF-ELP constitute promising new therapeutics for treatment of chronic wounds. Copyright © 2017 Elsevier Ltd. All rights reserved.

Protein-induced fusion can be modulated by target membrane lipids through a structural switch at the level of the fusion peptide

NARCIS (Netherlands)

Pecheur, EI; Martin, [No Value; Bienvenue, A; Ruysschaert, JM; Hoekstra, D

2000-01-01

Regulatory features of protein-induced membrane fusion are largely unclear, particularly at the level of the fusion peptide. Fusion peptides being part of larger protein complexes, such investigations are met with technical limitations. Here, we show that the fusion activity of influenza virus or

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.

A fusion-inhibiting peptide against Rift Valley fever virus inhibits multiple, diverse viruses.

Directory of Open Access Journals (Sweden)

Jeffrey W Koehler

Full Text Available For enveloped viruses, fusion of the viral envelope with a cellular membrane is critical for a productive infection to occur. This fusion process is mediated by at least three classes of fusion proteins (Class I, II, and III based on the protein sequence and structure. For Rift Valley fever virus (RVFV, the glycoprotein Gc (Class II fusion protein mediates this fusion event following entry into the endocytic pathway, allowing the viral genome access to the cell cytoplasm. Here, we show that peptides analogous to the RVFV Gc stem region inhibited RVFV infectivity in cell culture by inhibiting the fusion process. Further, we show that infectivity can be inhibited for diverse, unrelated RNA viruses that have Class I (Ebola virus, Class II (Andes virus, or Class III (vesicular stomatitis virus fusion proteins using this single peptide. Our findings are consistent with an inhibition mechanism similar to that proposed for stem peptide fusion inhibitors of dengue virus in which the RVFV inhibitory peptide first binds to both the virion and cell membranes, allowing it to traffic with the virus into the endocytic pathway. Upon acidification and rearrangement of Gc, the peptide is then able to specifically bind to Gc and prevent fusion of the viral and endocytic membranes, thus inhibiting viral infection. These results could provide novel insights into conserved features among the three classes of viral fusion proteins and offer direction for the future development of broadly active fusion inhibitors.

Comparison of five procedures for the purification of insoluble elastin.

NARCIS (Netherlands)

Daamen, W.F.; Hafmans, T.G.M.; Veerkamp, J.H.; Kuppevelt, A.H.M.S.M. van

2001-01-01

Elastin is an insoluble, highly cross-linked protein, providing elasticity to organs like lung. aorta, and ligaments. Despite its remarkable mechanical properties. elastin has found little use as a biomaterial. Purification of intact elastin from elastic fibres presents a major challenge, among

Polymorphisms in the human tropoelastin gene modify in vitro self-assembly and mechanical properties of elastin-like polypeptides.

Directory of Open Access Journals (Sweden)

David He

Full Text Available Elastin is a major structural component of elastic fibres that provide properties of stretch and recoil to tissues such as arteries, lung and skin. Remarkably, after initial deposition of elastin there is normally no subsequent turnover of this protein over the course of a lifetime. Consequently, elastic fibres must be extremely durable, able to withstand, for example in the human thoracic aorta, billions of cycles of stretch and recoil without mechanical failure. Major defects in the elastin gene (ELN are associated with a number of disorders including Supravalvular aortic stenosis (SVAS, Williams-Beuren syndrome (WBS and autosomal dominant cutis laxa (ADCL. Given the low turnover of elastin and the requirement for the long term durability of elastic fibres, we examined the possibility for more subtle polymorphisms in the human elastin gene to impact the assembly and long-term durability of the elastic matrix. Surveys of genetic variation resources identified 118 mutations in human ELN, 17 being non-synonymous. Introduction of two of these variants, G422S and K463R, in elastin-like polypeptides as well as full-length tropoelastin, resulted in changes in both their assembly and mechanical properties. Most notably G422S, which occurs in up to 40% of European populations, was found to enhance some elastomeric properties. These studies reveal that even apparently minor polymorphisms in human ELN can impact the assembly and mechanical properties of the elastic matrix, effects that over the course of a lifetime could result in altered susceptibility to cardiovascular disease.

Temperature induced conformational transitions of elastin-like polypentapeptides studied by Raman and NMR spectroscopy

Czech Academy of Sciences Publication Activity Database

Dybal, Jiří; Schmidt, Pavel; Kurková, Dana; Kříž, Jaroslav; Rodríguez-Cabello, J. C.; Alonso, M.

2002-01-01

Roč. 16, 3-4 (2002), s. 251-255 ISSN 0712-4813 R&D Projects: GA ČR GA203/00/1320; GA AV ČR IAA4050208 Institutional research plan: CEZ:AV0Z4050913 Keywords : quantum chemical calculations * elastin -like polypentapeptides * Raman spectra Subject RIV: CD - Macromolecular Chemistry Impact factor: 0.567, year: 2002

Early and late HIV-1 membrane fusion events are impaired by sphinganine lipidated peptides that target the fusion site.

Science.gov (United States)

Klug, Yoel A; Ashkenazi, Avraham; Viard, Mathias; Porat, Ziv; Blumenthal, Robert; Shai, Yechiel

2014-07-15

Lipid-conjugated peptides have advanced the understanding of membrane protein functions and the roles of lipids in the membrane milieu. These lipopeptides modulate various biological systems such as viral fusion. A single function has been suggested for the lipid, binding to the membrane and thus elevating the local concentration of the peptide at the target site. In the present paper, we challenged this argument by exploring in-depth the antiviral mechanism of lipopeptides, which comprise sphinganine, the lipid backbone of DHSM (dihydrosphingomyelin), and an HIV-1 envelope-derived peptide. Surprisingly, we discovered a partnership between the lipid and the peptide that impaired early membrane fusion events by reducing CD4 receptor lateral diffusion and HIV-1 fusion peptide-mediated lipid mixing. Moreover, only the joint function of sphinganine and its conjugate peptide disrupted HIV-1 fusion protein assembly and folding at the later fusion steps. Via imaging techniques we revealed for the first time the direct localization of these lipopeptides to the virus-cell and cell-cell contact sites. Overall, the findings of the present study may suggest lipid-protein interactions in various biological systems and may help uncover a role for elevated DHSM in HIV-1 and its target cell membranes.

Fluorescence, aggregation properties and FT-IR microspectroscopy of elastin and collagen fibers.

Science.gov (United States)

Vidal, Benedicto de Campos

2014-10-01

Histological and histochemical observations support the hypothesis that collagen fibers can link to elastic fibers. However, the resulting organization of elastin and collagen type complexes and differences between these materials in terms of macromolecular orientation and frequencies of their chemical vibrational groups have not yet been solved. This study aimed to investigate the macromolecular organization of pure elastin, collagen type I and elastin-collagen complexes using polarized light DIC-microscopy. Additionally, differences and similarities between pure elastin and collagen bundles (CB) were investigated by Fourier transform-infrared (FT-IR) microspectroscopy. Although elastin exhibited a faint birefringence, the elastin-collagen complex aggregates formed in solution exhibited a deep birefringence and formation of an ordered-supramolecular complex typical of collagen chiral structure. The FT-IR study revealed elastin and CB peptide NH groups involved in different types of H-bonding. More energy is absorbed in the vibrational transitions corresponding to CH, CH2 and CH3 groups (probably associated with the hydrophobicity demonstrated by 8-anilino-1-naphtalene sulfonic acid sodium salt [ANS] fluorescence), and to νCN, δNH and ωCH2 groups of elastin compared to CB. It is assumed that the α-helix contribution to the pure elastin amide I profile is 46.8%, whereas that of the B-sheet is 20% and that unordered structures contribute to the remaining percentage. An FT-IR profile library reveals that the elastin signature within the 1360-1189cm(-1) spectral range resembles that of Conex-Toray aramid fibers. Copyright © 2014 Elsevier GmbH. All rights reserved.

Matrix ageing and vascular impacts: focus on elastin fragmentation.

Science.gov (United States)

Duca, Laurent; Blaise, Sébastien; Romier, Béatrice; Laffargue, Muriel; Gayral, Stéphanie; El Btaouri, Hassan; Kawecki, Charlotte; Guillot, Alexandre; Martiny, Laurent; Debelle, Laurent; Maurice, Pascal

2016-06-01

Cardiovascular diseases (CVDs) are the leading cause of death worldwide and represent a major problem of public health. Over the years, life expectancy has considerably increased throughout the world, and the prevalence of CVD is inevitably rising with the growing ageing of the population. The normal process of ageing is associated with progressive deterioration in structure and function of the vasculature, commonly called vascular ageing. At the vascular level, extracellular matrix (ECM) ageing leads to molecular alterations in long half-life proteins, such as elastin and collagen, and have critical effects on vascular diseases. This review highlights ECM alterations occurring during vascular ageing with a specific focus on elastin fragmentation and also the contribution of elastin-derived peptides (EDP) in age-related vascular complications. Moreover, current and new pharmacological strategies aiming at minimizing elastin degradation, EDP generation, and associated biological effects are discussed. These strategies may be of major relevance for preventing and/or delaying vascular ageing and its complications. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.

A compensatory mutation provides resistance to disparate HIV fusion inhibitor peptides and enhances membrane fusion.

Directory of Open Access Journals (Sweden)

Matthew P Wood

Full Text Available Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide, are peptides designed to competitively inhibit the viral fusion protein gp41. With the emergence of drug resistance, there is an increased need for effective and unique alternatives within this class of antivirals. One such alternative is a class of cyclic, cationic, antimicrobial peptides known as θ-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the θ-defensin analog RC-101 is being developed as a microbicide due to its specific antiviral activity, lack of toxicity to cells and tissues, and safety in animals. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development. In previous studies, we identified a mutant, R5-tropic virus that had evolved partial resistance to RC-101 during in vitro selection. Here, we report that a secondary mutation in gp41 was found to restore replicative fitness, membrane fusion, and the rate of viral entry, which were compromised by an initial mutation providing partial RC-101 resistance. Interestingly, we show that RC-101 is effective against two enfuvirtide-resistant mutants, demonstrating the clinical importance of RC-101 as a unique fusion inhibitor. These findings both expand our understanding of HIV drug-resistance to diverse peptide fusion inhibitors and emphasize the significance of compensatory gp41 mutations.

Potent peptidic fusion inhibitors of influenza virus

Energy Technology Data Exchange (ETDEWEB)

Kadam, Rameshwar U.; Juraszek, Jarek; Brandenburg, Boerries; Buyck, Christophe; Schepens, Wim B. G.; Kesteleyn, Bart; Stoops, Bart; Vreeken, Rob J.; Vermond, Jan; Goutier, Wouter; Tang, Chan; Vogels, Ronald; Friesen, Robert H. E.; Goudsmit, Jaap; van Dongen, Maria J. P.; Wilson, Ian A.

2017-09-28

Influenza therapeutics with new targets and mechanisms of action are urgently needed to combat potential pandemics, emerging viruses, and constantly mutating strains in circulation. We report here on the design and structural characterization of potent peptidic inhibitors of influenza hemagglutinin. The peptide design was based on complementarity-determining region loops of human broadly neutralizing antibodies against the hemagglutinin (FI6v3 and CR9114). The optimized peptides exhibit nanomolar affinity and neutralization against influenza A group 1 viruses, including the 2009 H1N1 pandemic and avian H5N1 strains. The peptide inhibitors bind to the highly conserved stem epitope and block the low pH–induced conformational rearrangements associated with membrane fusion. These peptidic compounds and their advantageous biological properties should accelerate the development of new small molecule– and peptide-based therapeutics against influenza virus.

Lipid raft-like liposomes used for targeted delivery of a chimeric entry-inhibitor peptide with anti-HIV-1 activity.

Science.gov (United States)

Gómara, María José; Pérez-Pomeda, Ignacio; Gatell, José María; Sánchez-Merino, Victor; Yuste, Eloisa; Haro, Isabel

2017-02-01

The work reports the design and synthesis of a chimeric peptide that is composed of the peptide sequences of two entry inhibitors which target different sites of HIV-1 gp41. The chimeric peptide offers the advantage of targeting two gp41 regions simultaneously: the fusion peptide and the loop both of which are membrane active and participate in the membrane fusion process. We therefore use lipid raft-like liposomes as a tool to specifically direct the chimeric inhibitor peptide to the membrane domains where the HIV-1 envelope protein is located. Moreover, the liposomes that mimic the viral membrane composition protect the chimeric peptide against proteolytic digestion thereby increasing the stability of the peptide. The described liposome preparations are suitable nanosystems for managing hydrophobic entry-inhibitor peptides as putative therapeutics. Copyright © 2016 Elsevier Inc. All rights reserved.

Mechanistic Insights into Elastin Degradation by Pseudolysin, the Major Virulence Factor of the Opportunistic Pathogen Pseudomonas aeruginosa

Science.gov (United States)

Yang, Jie; Zhao, Hui-Lin; Ran, Li-Yuan; Li, Chun-Yang; Zhang, Xi-Ying; Su, Hai-Nan; Shi, Mei; Zhou, Bai-Cheng; Chen, Xiu-Lan; Zhang, Yu-Zhong

2015-01-01

Pseudolysin is the most abundant protease secreted by Pseudomonas aeruginosa and is the major extracellular virulence factor of this opportunistic human pathogen. Pseudolysin destroys human tissues by solubilizing elastin. However, the mechanisms by which pseudolysin binds to and degrades elastin remain elusive. In this study, we investigated the mechanism of action of pseudolysin on elastin binding and degradation by biochemical assay, microscopy and site-directed mutagenesis. Pseudolysin bound to bovine elastin fibers and preferred to attack peptide bonds with hydrophobic residues at the P1 and P1’ positions in the hydrophobic domains of elastin. The time-course degradation processes of both bovine elastin fibers and cross-linked human tropoelastin by pseudolysin were further investigated by microscopy. Altogether, the results indicate that elastin degradation by pseudolysin began with the hydrophobic domains on the fiber surface, followed by the progressive disassembly of macroscopic elastin fibers into primary structural elements. Moreover, our site-directed mutational results indicate that five hydrophobic residues in the S1-S1’ sub-sites played key roles in the binding of pseudolysin to elastin. This study sheds lights on the pathogenesis of P. aeruginosa infection. PMID:25905792

Fusion peptide of influenza hemagglutinin requires a fixed angle boomerang structure for activity.

Science.gov (United States)

Lai, Alex L; Park, Heather; White, Judith M; Tamm, Lukas K

2006-03-03

The fusion peptide of influenza hemagglutinin is crucial for cell entry of this virus. Previous studies showed that this peptide adopts a boomerang-shaped structure in lipid model membranes at the pH of membrane fusion. To examine the role of the boomerang in fusion, we changed several residues proposed to stabilize the kink in this structure and measured fusion. Among these, mutants E11A and W14A expressed hemagglutinins with hemifusion and no fusion activities, and F9A and N12A had no effect on fusion, respectively. Binding enthalpies and free energies of mutant peptides to model membranes and their ability to perturb lipid bilayer structures correlated well with the fusion activities of the parent full-length molecules. The structure of W14A determined by NMR and site-directed spin labeling features a flexible kink that points out of the membrane, in sharp contrast to the more ordered boomerang of the wild-type, which points into the membrane. A specific fixed angle boomerang structure is thus required to support membrane fusion.

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.

Sifuvirtide, a potent HIV fusion inhibitor peptide

International Nuclear Information System (INIS)

Wang, Rui-Rui; Yang, Liu-Meng; Wang, Yun-Hua; Pang, Wei; Tam, Siu-Cheung; Tien, Po; Zheng, Yong-Tang

2009-01-01

Enfuvirtide (ENF) is currently the only FDA approved HIV fusion inhibitor in clinical use. Searching for more drugs in this category with higher efficacy and lower toxicity seems to be a logical next step. In line with this objective, a synthetic peptide with 36 amino acid residues, called Sifuvirtide (SFT), was designed based on the crystal structure of gp41. In this study, we show that SFT is a potent anti-HIV agent with relatively low cytotoxicity. SFT was found to inhibit replication of all tested HIV strains. The effective concentrations that inhibited 50% viral replication (EC 50 ), as determined in all tested strains, were either comparable or lower than benchmark values derived from well-known anti-HIV drugs like ENF or AZT, while the cytotoxic concentrations causing 50% cell death (CC 50 ) were relatively high, rendering it an ideal anti-HIV agent. A GST-pull down assay was performed to confirm that SFT is a fusion inhibitor. Furthermore, the activity of SFT on other targets in the HIV life cycle was also investigated, and all assays showed negative results. To further understand the mechanism of action of HIV peptide inhibitors, resistant variants of HIV-1 IIIB were derived by serial virus passage in the presence of increasing doses of SFT or ENF. The results showed that there was cross-resistance between SFT and ENF. In conclusion, SFT is an ideal anti-HIV agent with high potency and low cytotoxicity, but may exhibit a certain extent of cross-resistance with ENF.

Construction of hybrid peptide synthetases by module and domain fusions.

Science.gov (United States)

Mootz, H D; Schwarzer, D; Marahiel, M A

2000-05-23

Nonribosomal peptide synthetases are modular enzymes that assemble peptides of diverse structures and important biological activities. Their modular organization provides a great potential for the rational design of novel compounds by recombination of the biosynthetic genes. Here we describe the extension of a dimodular system to trimodular ones based on whole-module fusion. The recombinant hybrid enzymes were purified to monitor product assembly in vitro. We started from the first two modules of tyrocidine synthetase, which catalyze the formation of the dipeptide dPhe-Pro, to construct such hybrid systems. Fusion of the second, proline-specific module with the ninth and tenth modules of the tyrocidine synthetases, specific for ornithine and leucine, respectively, resulted in dimodular hybrid enzymes exhibiting the combined substrate specificities. The thioesterase domain was fused to the terminal module. Upon incubation of these dimodular enzymes with the first tyrocidine module, TycA, incorporating dPhe, the predicted tripeptides dPhe-Pro-Orn and dPhe-Pro-Leu were obtained at rates of 0.15 min(-1) and 2.1 min(-1). The internal thioesterase domain was necessary and sufficient to release the products from the hybrid enzymes and thereby facilitate a catalytic turnover. Our approach of whole-module fusion is based on an improved definition of the fusion sites and overcomes the recently discovered editing function of the intrinsic condensation domains. The stepwise construction of hybrid peptide synthetases from catalytic subunits reinforces the inherent potential for the synthesis of novel, designed peptides.

A cytokine axis regulates elastin formation and degradation

Science.gov (United States)

Sproul, Erin P.; Argraves, W. Scott

2013-01-01

Underlying the dynamic regulation of tropoelastin expression and elastin formation in development and disease are transcriptional and post-transcriptional mechanisms that have been the focus of much research. Of particular importance is the cytokine–governed elastin regulatory axis in which the pro-elastogenic activities of transforming growth factor β-1 (TGFβ1) and insulin-like growth factor-I (IGF-I) are opposed by anti-elastogenic activities of basic fibroblast growth factor (bFGF/FGF-2), heparin-binding epidermal growth factor-like growth factor (HB-EGF), EGF, PDGF-BB, TGFα, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β and noncanonical TGFβ1 signaling. A key mechanistic feature of the regulatory axis is that cytokines influence elastin formation through effects on the cell cycle involving control of cyclin–cyclin dependent kinase complexes and activation of the Ras/MEK/ERK signaling pathway. In this article we provide an overview of the major cytokines/growth factors that modulate elastogenesis and describe the underlying molecular mechanisms for their action on elastin production. PMID:23160093

Elastin-derived peptides promote abdominal aortic aneurysm formation by modulating M1/M2 macrophage polarization1

Science.gov (United States)

Dale, Matthew A; Xiong, Wanfen; Carson, Jeffrey S; Suh, Melissa K; Karpisek, Andrew D.; Meisinger, Trevor M.; Casale, George P.; Baxter, B. Timothy

2016-01-01

Abdominal aortic aneurysm (AAA) is a dynamic vascular disease characterized by inflammatory cell invasion and extracellular matrix (ECM) degradation. Damage to elastin in the ECM results in release of elastin-derived peptides (EDPs), which are chemotactic for inflammatory cells such as monocytes. Their effect on macrophage polarization is less well known. Pro-inflammatory M1 macrophages initially are recruited to sites of injury but, if their effects are prolonged, they can lead to chronic inflammation that prevents normal tissue repair. Conversely, anti-inflammatory M2 macrophages reduce inflammation and aid in wound healing. Thus, a proper M1/M2 ratio is vital for tissue homeostasis. AAA tissue reveals a high M1/M2 ratio where pro-inflammatory cells and their associated markers dominate. In the present study, in vitro treatment of bone marrow-derived macrophages with EDPs induced M1 macrophage polarization. By using C57Bl/6 mice, antibody-mediated neutralization of EDPs reduced aortic dilation, matrix metalloproteinase activity, and pro-inflammatory cytokine expression at early and late time points after aneurysm induction. Furthermore, direct manipulation of the M1/M2 balance altered aortic dilation. Injection of M2 polarized macrophages reduced aortic dilation after aneurysm induction. EDPs promoted a pro-inflammatory environment in aortic tissue by inducing M1 polarization and neutralization of EDPs attenuated aortic dilation. The M1/M2 imbalance is vital to aneurysm formation. PMID:27183603

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.

Exploring the Properties of Genetically Engineered Silk-Elastin-Like Protein Films.

Science.gov (United States)

Machado, Raul; da Costa, André; Sencadas, Vitor; Pereira, Ana Margarida; Collins, Tony; Rodríguez-Cabello, José Carlos; Lanceros-Méndez, Senentxu; Casal, Margarida

2015-12-01

Free standing films of a genetically engineered silk-elastin-like protein (SELP) were prepared using water and formic acid as solvents. Exposure to methanol-saturated air promoted the formation of aggregated β-strands rendering aqueous insolubility and improved the mechanical properties leading to a 10-fold increase in strain-to-failure. The films were optically clear with resistivity values similar to natural rubber and thermally stable up to 180 °C. Addition of glycerol showed to enhance the flexibility of SELP/glycerol films by interacting with SELP molecules through hydrogen bonding, interpenetrating between the polymer chains and granting more conformational freedom. This detailed characterization provides cues for future and unique applications using SELP based biopolymers. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mechanical contribution of lamellar and interlamellar elastin along the mouse aorta.

Science.gov (United States)

Clark, T E; Lillie, M A; Vogl, A W; Gosline, J M; Shadwick, R E

2015-10-15

The mechanical properties of aortic elastin vary regionally, but the microstructural basis for this variation is unknown. This study was designed to identify the relative contributions of lamellar and interlamellar elastin to circumferential load bearing in the mouse thoracic and abdominal aortas. Forces developed in uniaxial tests of samples of fresh and autoclaved aorta were correlated with elastin content and morphology obtained from histology and multiphoton laser scanning microscopy. Autoclaving should render much of the interlamellar elastin mechanically incompetent. In autoclaved tissue force per unit sample width correlated with lamellar elastin content (P≪0.001) but not total elastin content. In fresh tissue at low strain where elastin dominates the mechanical response, forces were higher than in the autoclaved tissue, but force did not correlate with total elastin content. Therefore although interlamellar elastin likely contributed to the stiffness in the fresh aorta, its contribution appeared not in proportion to its quantity. In both fresh and autoclaved tissue, elastin stiffness consistently decreased along the abdominal aorta, a key area for aneurysm development, and this difference could not be fully accounted for on the basis of either lamellar or total elastin content. These findings are relevant to the development of mathematical models of arterial mechanics, particularly for mouse models of arterial diseases involving elastic tissue. In microstructural based models the quantity of each mural constituent determines its contribution to the total response. This study shows elastin's mechanical response cannot necessarily be accounted for on the basis of fibre quantity, orientation, and modulus. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fibril Formation by pH and Temperature Responsive Silk-Elastin Block Copolymers

NARCIS (Netherlands)

Golinska, M.D.; Pham, T.T.H.; Werten, M.W.T.; Wolf, de F.A.; Cohen Stuart, M.A.; Gucht, van der J.

2013-01-01

In this report, we study the self-assembly of two silk-elastin-like proteins: one is a diblock S24E40 composed of 24 silk-like (S) repeats and 40 elastin-like (E) repeats; the other is a triblock S12C4E40, in which the S and E blocks are separated by a random coil block (C4). Upon lowering the pH,

Remote Exosites of the Catalytic Domain of Matrix Metalloproteinase-12 Enhance Elastin Degradation┼

Science.gov (United States)

Fulcher, Yan G.; Van Doren, Steven R.

2011-01-01

How does matrix metalloproteinase-12 (MMP-12 or metalloelastase) degrade elastin with high specific activity? NMR suggested soluble elastin to cover surfaces of MMP-12 far from its active site. Two of these surfaces have been found, by mutagenesis guided by the BINDSIght approach, to affect degradation and affinity for elastin substrates but not a small peptide substrate. Main exosite 1 has been extended out to Asp124 that binds calcium. Novel exosite 2 comprises residues from the II–III loop and β-strand I near the back of the catalytic domain. The high exposure of these distal exosites may make them accessible to elastin made more flexible by partial hydrolysis. Importantly, combination of a lesion at each of exosites 1 and 2 and active site decreased catalytic competence towards soluble elastin by 13- to 18-fold to the level of MMP-3, homologue and poor elastase. Double mutant cycle analysis of conservative mutations of Met156 (exosite 2) and either Asp124 (exosite 1) or Ile180 (active site) had additive effects. Compared to polar substitutions observed in other MMPs, Met156 enhanced affinity and Ile180 kcat for soluble elastin. Both residues detracted from the higher folding stability with polar mutations. This resembles the trend in enzymes of an inverse relationship between folding stability and activity. Restoring Asp124 from combination mutants enhanced kcat for soluble elastin. In elastin degradation, exosites 1 and 2 contributed independently of each other and Ile180 at the active site, but with partial coupling to Ala182 near the active site. The concept of weak, separated interactions coalescing somewhat independently can be extended to this proteolytic digestion of a protein from fibrils. PMID:21967233

Dual stimuli-sensitive dendrimers: Photothermogenic gold nanoparticle-loaded thermo-responsive elastin-mimetic dendrimers.

Science.gov (United States)

Fukushima, Daichi; Sk, Ugir Hossain; Sakamoto, Yasuhiro; Nakase, Ikuhiko; Kojima, Chie

2015-08-01

Dendrimers are synthetic macromolecules with unique structures that can work as nanoplatforms for both photothermogenic gold nanoparticles (AuNPs) and thermosensitive elastin-like peptides (ELPs) with valine-proline-glycine-valine-glycine (VPGVG) repeats. In this study, photothermogenic AuNPs were loaded into thermo-responsive elastin-mimetic dendrimers (dendrimers conjugating ELPs at their periphery) to produce dual stimuli-sensitive nanoparticles. Polyamidoamine G4 dendrimers were modified with acetylated VPGVG and (VPGVG)2, and the resulting materials were named ELP1-den and ELP2-den, respectively. The AuNPs were prepared by the reduction of Au ions using a dendrimer-nanotemplated method. The AuNP-loaded elastin-mimetic dendrimers exhibited photothermal properties. ELP1-den and ELP2-den showed similar temperature-dependent changes in their conformations. Phase transitions were observed at around 55°C and 35°C for the AuNP-loaded ELP1-den and AuNP-loaded ELP2-den, respectively, but not for the corresponding PEGylated dendrimer. In contrast to the AuNP-loaded PEGylated dendrimer, AuNP-loaded ELP2-den readily associated with cells and induced efficient photocytotoxicity at 37°C. The cell association and the photocytotoxicity properties of AuNP-loaded ELP2-den could be controlled by temperature. These results therefore suggest that dual stimuli-sensitive dendrimer nanoparticles of this type could be used for photothermal therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Universal antibodies against the highly conserved influenza fusion peptide cross-neutralize several subtypes of influenza A virus

Energy Technology Data Exchange (ETDEWEB)

Hashem, Anwar M. [Centre for Vaccine Evaluation, Biologics and Genetic Therapies Directorate, HPFB, Health Canada, Ottawa, ON (Canada); Department of Microbiology, Faculty of Medicine, King Abdulaziz University, Jeddah (Saudi Arabia); Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON (Canada); Van Domselaar, Gary [National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB (Canada); Li, Changgui; Wang, Junzhi [National Institute for the Control of Pharmaceutical and Biological Products, Beijing (China); She, Yi-Min; Cyr, Terry D. [Centre for Vaccine Evaluation, Biologics and Genetic Therapies Directorate, HPFB, Health Canada, Ottawa, ON (Canada); Sui, Jianhua [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Department of Medicine, Harvard Medical School, 44 Binney Street, Boston, MA 02115 (United States); He, Runtao [National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB (Canada); Marasco, Wayne A. [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Department of Medicine, Harvard Medical School, 44 Binney Street, Boston, MA 02115 (United States); Li, Xuguang, E-mail: Sean.Li@hc-sc.gc.ca [Centre for Vaccine Evaluation, Biologics and Genetic Therapies Directorate, HPFB, Health Canada, Ottawa, ON (Canada); Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON (Canada)

2010-12-10

Research highlights: {yields} The fusion peptide is the only universally conserved epitope in all influenza viral hemagglutinins. {yields} Anti-fusion peptide antibodies are universal antibodies that cross-react with all influenza HA subtypes. {yields} The universal antibodies cross-neutralize different influenza A subtypes. {yields} The universal antibodies inhibit the fusion process between the viruses and the target cells. -- Abstract: The fusion peptide of influenza viral hemagglutinin plays a critical role in virus entry by facilitating membrane fusion between the virus and target cells. As the fusion peptide is the only universally conserved epitope in all influenza A and B viruses, it could be an attractive target for vaccine-induced immune responses. We previously reported that antibodies targeting the first 14 amino acids of the N-terminus of the fusion peptide could bind to virtually all influenza virus strains and quantify hemagglutinins in vaccines produced in embryonated eggs. Here we demonstrate that these universal antibodies bind to the viral hemagglutinins in native conformation presented in infected mammalian cell cultures and neutralize multiple subtypes of virus by inhibiting the pH-dependant fusion of viral and cellular membranes. These results suggest that this unique, highly-conserved linear sequence in viral hemagglutinin is exposed sufficiently to be attacked by the antibodies during the course of infection and merits further investigation because of potential importance in the protection against diverse strains of influenza viruses.

Universal antibodies against the highly conserved influenza fusion peptide cross-neutralize several subtypes of influenza A virus

International Nuclear Information System (INIS)

Hashem, Anwar M.; Van Domselaar, Gary; Li, Changgui; Wang, Junzhi; She, Yi-Min; Cyr, Terry D.; Sui, Jianhua; He, Runtao; Marasco, Wayne A.; Li, Xuguang

2010-01-01

Research highlights: → The fusion peptide is the only universally conserved epitope in all influenza viral hemagglutinins. → Anti-fusion peptide antibodies are universal antibodies that cross-react with all influenza HA subtypes. → The universal antibodies cross-neutralize different influenza A subtypes. → The universal antibodies inhibit the fusion process between the viruses and the target cells. -- Abstract: The fusion peptide of influenza viral hemagglutinin plays a critical role in virus entry by facilitating membrane fusion between the virus and target cells. As the fusion peptide is the only universally conserved epitope in all influenza A and B viruses, it could be an attractive target for vaccine-induced immune responses. We previously reported that antibodies targeting the first 14 amino acids of the N-terminus of the fusion peptide could bind to virtually all influenza virus strains and quantify hemagglutinins in vaccines produced in embryonated eggs. Here we demonstrate that these universal antibodies bind to the viral hemagglutinins in native conformation presented in infected mammalian cell cultures and neutralize multiple subtypes of virus by inhibiting the pH-dependant fusion of viral and cellular membranes. These results suggest that this unique, highly-conserved linear sequence in viral hemagglutinin is exposed sufficiently to be attacked by the antibodies during the course of infection and merits further investigation because of potential importance in the protection against diverse strains of influenza viruses.

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.

Small leucine-rich repeat proteoglycans associated with mature insoluble elastin serve as binding sites for galectins.

Science.gov (United States)

Itoh, Aiko; Nonaka, Yasuhiro; Ogawa, Takashi; Nakamura, Takanori; Nishi, Nozomu

2017-11-01

We previously reported that galectin-9 (Gal-9), an immunomodulatory animal lectin, could bind to insoluble collagen preparations and exerted direct cytocidal effects on immune cells. In the present study, we found that mature insoluble elastin is capable of binding Gal-9 and other members of the human galectin family. Lectin blot analysis of a series of commercial water-soluble elastin preparations, PES-(A) ~ PES-(E), revealed that only PES-(E) contained substances recognized by Gal-9. Gal-9-interacting substances in PES-(E) were affinity-purified, digested with trypsin and then analyzed by reversed-phase HPLC. Peptide fragments derived from five members of the small leucine-rich repeat proteoglycan family, versican, lumican, osteoglycin/mimecan, prolargin, and fibromodulin, were identified by N-terminal amino acid sequence analysis. The results indicate that Gal-9 and possibly other galectins recognize glycans attached to small leucine-rich repeat proteoglycans associated with insoluble elastin and also indicate the possibility that mature insoluble elastin serves as an extracellular reservoir for galectins.

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.

Antibodies to elastin peptides in sera of Belgian Draught horses with chronic progressive lymphoedema.

Science.gov (United States)

van Brantegem, L; de Cock, H E V; Affolter, V K; Duchateau, L; Hoogewijs, M K; Govaere, J; Ferraro, G L; Ducatelle, R

2007-09-01

Chronic progressive lymphoedema (CPL) is a recently recognised disease of the lymphatic system characterised by lesions in the skin of the lower legs in several draught horse breeds, including the Belgian Draught hourse. Clinical signs slowly progress and result in severe disfigurement of the limbs. Ideally, supportive treatment should be started early in the disease process. However early diagnosis and monitoring progression of CPL is still a challenge. Elastin changes, characterised by morphological alterations as well as increased desmosine levels, in the skin of the distal limbs of horses affected with CPL are probably associated with a marked release of elastin degradation products, which elicit production of circulating anti-elastin antibodies (AEAbs) in the serum. An enzyme-linked immunosorbent assay (ELISA) for detection of serum AEAbs may document elastin breakdown. An ELISA technique was used to evaluate levels of AEAbs in sera of 97 affected Belgian Draught horses that were clinically healthy except for possible skin lesions, associated with CPL in their distal limbs. The horses were divided into 5 groups according to the severity of these skin lesions: normal horses (Group 1, n = 36), horses with mild lesions (Group 2, n = 43), horses with moderate lesions (Group 3, n = 8), horses with severe lesions (Group 4, n = 10) and, as a control, healthy Warmblood horses, unaffected by the disease (Group 5, n = 83). Horses with clinical signs of CPL had significantly higher AEAb levels compared to clinically normal Belgian Draught horses and to healthy Warmblood horses. These levels correlated with severity of lesions. CPL in draught horses is associated with an increase of serum AEAbs. Evaluation of serum levels of AEAbs by ELISA might be a useful diagnostic aid for CPL. Pathological degradation of elastic fibres, resulting in deficient support of the distal lymphatics, is proposed as a contributing factor for CPL in Belgian Draught horses.

Coiled-coil formation of the membrane-fusion K/E peptides viewed by electron paramagnetic resonance.

Directory of Open Access Journals (Sweden)

Pravin Kumar

Full Text Available The interaction of the complementary K (Ac-(KIAALKE3-GW-NH2 and E (Ac-(EIAALEK3-GY-NH2 peptides, components of the zipper of an artificial membrane fusion system (Robson Marsden H. et al. Angew Chemie Int Ed. 2009 is investigated by electron paramagnetic resonance (EPR. By frozen solution continuous-wave EPR and double electron-electron resonance (DEER, the distance between spin labels attached to the K- and to the E-peptide is measured. Three constructs of spin-labelled K- and E-peptides are used in five combinations for low temperature investigations. The K/E heterodimers are found to be parallel, in agreement with previous studies. Also, K homodimers in parallel orientation were observed, a finding that was not reported before. Comparison to room-temperature, solution EPR shows that the latter method is less specific to detect this peptide-peptide interaction. Combining frozen solution cw-EPR for short distances (1.8 nm to 2.0 nm and DEER for longer distances thus proves versatile to detect the zipper interaction in membrane fusion. As the methodology can be applied to membrane samples, the approach presented suggests itself for in-situ studies of the complete membrane fusion process, opening up new avenues for the study of membrane fusion.

Aluminum Chloride Pretreatment of Elastin Inhibits Elastolysis by Matrix Metalloproteinases and Leads to Inhibition of Elastin-Oriented Calcification

OpenAIRE

Bailey, Michael; Xiao, Hui; Ogle, Matthew; Vyavahare, Naren

2001-01-01

Calcification of elastin occurs in many pathological cardiovascular diseases including atherosclerosis. We have previously shown that purified elastin when subdermally implanted in rats undergoes severe calcification and aluminum chloride (AlCl3) pretreatment of elastin inhibits calcification. In the present study we investigated whether matrix metalloproteinase (MMP) binding to elastin and elastin degradation is prevented by AlCl3 pretreatment. Subdermal implantation of AlCl3-pretreated elas...

Heterologous production of peptides in plants: fusion proteins and beyond.

Science.gov (United States)

Viana, Juliane Flávia Cançado; Dias, Simoni Campos; Franco, Octávio Luiz; Lacorte, Cristiano

2013-11-01

Recombinant DNA technology has allowed the ectopic production of proteins and peptides of different organisms leading to biopharmaceutical production in large cultures of bacterial, yeasts and mammalian cells. Otherwise, the expression of recombinant proteins and peptides in plants is an attractive alternative presenting several advantages over the commonly used expression systems including reduced production costs, easy scale-up and reduced risks of pathogen contamination. Different types of proteins and peptides have been expressed in plants, including antibodies, antigens, and proteins and peptides of medical, veterinary and industrial applications. However, apart from providing a proof of concept, the use of plants as platforms for heterologous protein and peptide production still depends on key steps towards optimization including the enhancement of expression levels, manipulation of post-transcriptional modifications and improvements in purification methods. In this review, strategies to increase heterologous protein and peptide stability and accumulation are discussed, focusing on the expression of peptides through the use of gene fusions.

A phage display selected 7-mer peptide inhibitor of the Tannerella forsythia metalloprotease-like enzyme Karilysin can be truncated to Ser-Trp-Phe-Pro.

Science.gov (United States)

Skottrup, Peter Durand; Sørensen, Grete; Ksiazek, Miroslaw; Potempa, Jan; Riise, Erik

2012-01-01

Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a likely drug target. In this study peptides were selected towards the catalytic domain from Karilysin (Kly18) by phage display. The peptides were linear with low micromolar binding affinities. The two best binders (peptide14 and peptide15), shared the consensus sequence XWFPXXXGGG. A peptide15 fusion with Maltose Binding protein (MBP) was produced with peptide15 fused to the N-terminus of MBP. The peptide15-MBP was expressed in E. coli and the purified fusion-protein was used to verify Kly18 specific binding. Chemically synthesised peptide15 (SWFPLRSGGG) could inhibit the enzymatic activity of both Kly18 and intact Karilysin (Kly48). Furthermore, peptide15 could slow down the autoprocessing of intact Kly48 to Kly18. The WFP motif was important for inhibition and a truncation study further demonstrated that the N-terminal serine was also essential for Kly18 inhibition. The SWFP peptide had a Ki value in the low micromolar range, which was similar to the intact peptide15. In conclusion SWFP is the first reported inhibitor of Karilysin and can be used as a valuable tool in structure-function studies of Karilysin.

Fingerprinting desmosine-containing elastin peptides

DEFF Research Database (Denmark)

Schräder, Christoph U; Heinz, Andrea; Majovsky, Petra

2015-01-01

, and DES-/IDES-containing peptides to determine characteristic product ions. It was found that all investigated compounds yielded the same product ion clusters at elevated collision energies. Elemental composition determination using the exact masses of these ions revealed molecular formulas of the type Cx...

Phase separation and mechanical properties of an elastomeric biomaterial from spider wrapping silk and elastin block copolymers.

Science.gov (United States)

Muiznieks, Lisa D; Keeley, Fred W

2016-10-01

Elastin and silk spidroins are fibrous, structural proteins with elastomeric properties of extension and recoil. While elastin is highly extensible and has excellent recovery of elastic energy, silks are particularly strong and tough. This study describes the biophysical characterization of recombinant polypeptides designed by combining spider wrapping silk and elastin-like sequences as a strategy to rationally increase the strength of elastin-based materials while maintaining extensibility. We demonstrate a thermo-responsive phase separation and spontaneous colloid-like droplet formation from silk-elastin block copolymers, and from a 34 residue disordered region of Argiope trifasciata wrapping silk alone, and measure a comprehensive suite of tensile mechanical properties from cross-linked materials. Silk-elastin materials exhibited significantly increased strength, toughness, and stiffness compared to an elastin-only material, while retaining high failure strains and low energy loss upon recoil. These data demonstrate the mechanical tunability of protein polymer biomaterials through modular, chimeric recombination, and provide structural insights into mechanical design. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 693-703, 2016. © 2016 Wiley Periodicals, Inc.

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.

Proline-poor hydrophobic domains modulate the assembly and material properties of polymeric elastin.

Science.gov (United States)

Muiznieks, Lisa D; Reichheld, Sean E; Sitarz, Eva E; Miao, Ming; Keeley, Fred W

2015-10-01

Elastin is a self-assembling extracellular matrix protein that provides elasticity to tissues. For entropic elastomers such as elastin, conformational disorder of the monomer building block, even in the polymeric form, is essential for elastomeric recoil. The highly hydrophobic monomer employs a range of strategies for maintaining disorder and flexibility within hydrophobic domains, particularly involving a minimum compositional threshold of proline and glycine residues. However, the native sequence of hydrophobic elastin domain 30 is uncharacteristically proline-poor and, as an isolated polypeptide, is susceptible to formation of amyloid-like structures comprised of stacked β-sheet. Here we investigated the biophysical and mechanical properties of multiple sets of elastin-like polypeptides designed with different numbers of proline-poor domain 30 from human or rat tropoelastins. We compared the contributions of these proline-poor hydrophobic sequences to self-assembly through characterization of phase separation, and to the tensile properties of cross-linked, polymeric materials. We demonstrate that length of hydrophobic domains and propensity to form β-structure, both affecting polypeptide chain flexibility and cross-link density, play key roles in modulating elastin mechanical properties. This study advances the understanding of elastin sequence-structure-function relationships, and provides new insights that will directly support rational approaches to the design of biomaterials with defined suites of mechanical properties. © 2015 Wiley Periodicals, Inc.

Micellar Self-Assembly of Recombinant Resilin-/Elastin-Like Block Copolypeptides.

Science.gov (United States)

Weitzhandler, Isaac; Dzuricky, Michael; Hoffmann, Ingo; Garcia Quiroz, Felipe; Gradzielski, Michael; Chilkoti, Ashutosh

2017-08-14

Reported here is the synthesis of perfectly sequence defined, monodisperse diblock copolypeptides of hydrophilic elastin-like and hydrophobic resilin-like polypeptide blocks and characterization of their self-assembly as a function of structural parameters by light scattering, cryo-TEM, and small-angle neutron scattering. A subset of these diblock copolypeptides exhibit lower critical solution temperature and upper critical solution temperature phase behavior and self-assemble into spherical or cylindrical micelles. Their morphologies are dictated by their chain length, degree of hydrophilicity, and hydrophilic weight fraction of the ELP block. We find that (1) independent of the length of the corona-forming ELP block there is a minimum threshold in the length of the RLP block below which self-assembly does not occur, but that once that threshold is crossed, (2) the RLP block length is a unique molecular parameter to independently tune self-assembly and (3) increasing the hydrophobicity of the corona-forming ELP drives a transition from spherical to cylindrical morphology. Unlike the self-assembly of purely ELP-based block copolymers, the self-assembly of RLP-ELPs can be understood by simple principles of polymer physics relating hydrophilic weight fraction and polymer-polymer and polymer-solvent interactions to micellar morphology, which is important as it provides a route for the de novo design of desired nanoscale morphologies from first principles.

A phage display selected 7-mer peptide inhibitor of the Tannerella forsythia metalloprotease-like enzyme Karilysin can be truncated to Ser-Trp-Phe-Pro.

Directory of Open Access Journals (Sweden)

Peter Durand Skottrup

Full Text Available Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a likely drug target. In this study peptides were selected towards the catalytic domain from Karilysin (Kly18 by phage display. The peptides were linear with low micromolar binding affinities. The two best binders (peptide14 and peptide15, shared the consensus sequence XWFPXXXGGG. A peptide15 fusion with Maltose Binding protein (MBP was produced with peptide15 fused to the N-terminus of MBP. The peptide15-MBP was expressed in E. coli and the purified fusion-protein was used to verify Kly18 specific binding. Chemically synthesised peptide15 (SWFPLRSGGG could inhibit the enzymatic activity of both Kly18 and intact Karilysin (Kly48. Furthermore, peptide15 could slow down the autoprocessing of intact Kly48 to Kly18. The WFP motif was important for inhibition and a truncation study further demonstrated that the N-terminal serine was also essential for Kly18 inhibition. The SWFP peptide had a Ki value in the low micromolar range, which was similar to the intact peptide15. In conclusion SWFP is the first reported inhibitor of Karilysin and can be used as a valuable tool in structure-function studies of Karilysin.

Modulated growth, stability and interactions of liquid-like coacervate assemblies of elastin

NARCIS (Netherlands)

Muiznieks, L.D.; Cirulis, J.T.; Reinhardt, D.P.; Wuite, G.J.L.; Pomes, R.; Keeley, F.W.

2014-01-01

Elastin self-assembles from monomers into polymer networks that display elasticity and resilience. The first major step in assembly is a liquid-liquid phase separation known as coacervation. This process represents a continuum of stages from initial phase separation to early growth of droplets by

Expression and purification of moricin CM4 and human β-defensins 4 in Escherichia coli using a new technology.

Science.gov (United States)

Shen, Yang; Ai, Hong-Xin; Song, Ren; Liang, Zhen-Ning; Li, Jian-Feng; Zhang, Shuang-Quan

2010-10-20

Different strategies have been developed to produce small antimicrobial peptides using recombinant techniques. Here we report a new technology of biosynthesis of moricin CM4 and human β-defensins 4 (HβD4) in the Escherichia coli. The CM4 and HβD4 gene were cloned into a vector containing the tags elastin-like peptide (ELP) and intein to construct the expression vector pET-EI-CM4 and pET-EI-HβD4. All the peptides, expressed as soluble fusions, were isolated from the protein debris by the method called inverse transition cycling (ITC) rather than traditional immobilized metal affinity chromatography (IMAC) and separated from the fusion leader by self-cleavage. Fully reduced peptides that were purified exhibited expected antimicrobial activity. The approach described here is a low-cost, convenient and potential way for generating small antimicrobial peptide. Copyright © 2010 Elsevier GmbH. All rights reserved.

Small Artery Elastin Distribution and Architecture-Focus on Three Dimensional Organization.

Science.gov (United States)

Hill, Michael A; Nourian, Zahra; Ho, I-Lin; Clifford, Philip S; Martinez-Lemus, Luis; Meininger, Gerald A

2016-11-01

The distribution of ECM proteins within the walls of resistance vessels is complex both in variety of proteins and structural arrangement. In particular, elastin exists as discrete fibers varying in orientation across the adventitia and media as well as often resembling a sheet-like structure in the case of the IEL. Adding to the complexity is the tissue heterogeneity that exists in these structural arrangements. For example, small intracranial cerebral arteries lack adventitial elastin while similar sized arteries from skeletal muscle and intestinal mesentery exhibit a complex adventitial network of elastin fibers. With regard to the IEL, several vascular beds exhibit an elastin sheet with punctate holes/fenestrae while in others the IEL is discontinuous and fibrous in appearance. Importantly, these structural patterns likely sub-serve specific functional properties, including mechanosensing, control of external forces, mechanical properties of the vascular wall, cellular positioning, and communication between cells. Of further significance, these processes are altered in vascular disorders such as hypertension and diabetes mellitus where there is modification of ECM. This brief report focuses on the three-dimensional wall structure of small arteries and considers possible implications with regard to mechanosensing under physiological and pathophysiological conditions. © 2016 John Wiley & Sons Ltd.

Impact of peptide clustering on unbinding forces in the context of fusion mimetics

International Nuclear Information System (INIS)

Pähler, Gesa; Lorenz, Bärbel; Janshoff, Andreas

2013-01-01

Highlights: ► Coiled-coil peptides as SNARE mimetics for membrane fusion. ► Interaction forces assessed by colloidal probe microscopy. ► Lateral organization of lipopeptides visualized by atomic force microscopy. -- Abstract: Coiled-coil zipping and unzipping is a pivotal process in SNARE-regulated membrane fusion. In this study we examine this process mediated by a minimal model for coiled-coil formation employing force spectroscopy in the context of membrane-coated surfaces and probes. The interaction forces of several hundred pN are surprisingly low considering the proposed amount of molecular bonds in the contact zone. However, by means of high-resolution imaging employing atomic force microscopy and studying the lateral mobility of lipids and peptides as a function of coiled-coil formation, we are able to supply a detailed view on processes occurring on the membrane surfaces during force measurements. The interaction forces determined here are not only dependent on the peptide concentration on the surface, but also on the regional organization of lateral peptide clusters found prior to coiled-coil formation

One-week glucose control via zero-order release kinetics from an injectable depot of glucagon-like peptide-1 fused to a thermosensitive biopolymer.

Science.gov (United States)

Luginbuhl, Kelli M; Schaal, Jeffrey L; Umstead, Bret; Mastria, Eric M; Li, Xinghai; Banskota, Samagya; Arnold, Susan; Feinglos, Mark; D'Alessio, David; Chilkoti, Ashutosh

2017-01-01

Stimulation of the glucagon-like peptide-1 (GLP1) receptor is a useful treatment strategy for type 2 diabetes because of pleiotropic effects, including the regulation of islet hormones and the induction of satiety. However, the native ligand for the GLP1 receptor has a short half-live owing to enzymatic inactivation and rapid clearance. Here, we show that a subcutaneous depot formed after a single injection of GLP1 recombinantly fused to a thermosensitive elastin-like polypeptide results in zero-order release kinetics and circulation times of up to 10 days in mice and 17 days in monkeys. The optimized pharmacokinetics leads to 10 days of glycemic control in three different mouse models of diabetes, as well as to the reduction of glycosylated hemoglobin levels and weight gain in ob/ob mice treated once weekly for 8 weeks. Our results suggest that the optimized GLP1 formulation could enhance therapeutic outcomes by eliminating peak-and-valley pharmacokinetics and improving overall safety and tolerability. The design principles that we established should be broadly applicable for improving the pharmacological performance of other peptide and protein therapeutics.

Mechanistic Insight into the Elastin Degradation Process by the Metalloprotease Myroilysin from the Deep-Sea Bacterium Myroides profundi D25

Science.gov (United States)

Yang, Jie; Zhao, Hui-Lin; Tang, Bai-Lu; Chen, Xiu-Lan; Su, Hai-Nan; Zhang, Xi-Ying; Song, Xiao-Yan; Zhou, Bai-Cheng; Xie, Bin-Bin; Weiss, Anthony S.; Zhang, Yu-Zhong

2015-01-01

Elastases have been widely studied because of their important uses as medicine and meat tenderizers. However, there are relatively few studies on marine elastases. Myroilysin, secreted by Myroides profundi D25 from deep-sea sediment, is a novel elastase. In this study, we examined the elastin degradation mechanism of myroilysin. When mixed with insoluble bovine elastin, myroilysin bound hydrophobically, suggesting that this elastase may interact with the hydrophobic domains of elastin. Consistent with this, analysis of the cleavage pattern of myroilysin on bovine elastin and recombinant tropoelastin revealed that myroilysin preferentially cleaves peptide bonds with hydrophobic residues at the P1 and/or P1′ positions. Scanning electron microscopy (SEM) of cross-linked recombinant tropoelastin degraded by myroilysin showed preferential damages of spherules over cross-links, as expected for a hydrophobic preference. The degradation process of myroilysin on bovine elastin fibres was followed by light microscopy and SEM, revealing that degradation begins with the formation of crevices and cavities at the fibre surface, with these openings increasing in number and size until the fibre breaks into small pieces, which are subsequently fragmented. Our results are helpful for developing biotechnological applications for myroilysin. PMID:25793427

Characterization of glucagon-like peptide-1 receptor beta-arrestin 2 interaction: a high-affinity receptor phenotype

DEFF Research Database (Denmark)

Jorgensen, Rasmus; Martini, Lene; Schwartz, Thue W

2005-01-01

To dissect the interaction between beta-arrestin ((beta)arr) and family B G protein-coupled receptors, we constructed fusion proteins between the glucagon-like peptide 1 receptor and (beta)arr2. The fusion constructs had an increase in apparent affinity selectively for glucagon, suggesting...... that (beta)arr2 interaction locks the receptor in a high-affinity conformation, which can be explored by some, but not all, ligands. The fusion constructs adopted a signaling phenotype governed by the tethered (beta)arr2 with an attenuated G protein-mediated cAMP signal and a higher maximal internalization...... of that which has previously been characterized for family A G protein-coupled receptors, suggesting similarities in the effect of (beta)arr interaction between family A and B receptors also at the molecular level....

Prolyl hydroxylation in elastin is not random.

Science.gov (United States)

Schmelzer, Christian E H; Nagel, Marcus B M; Dziomba, Szymon; Merkher, Yulia; Sivan, Sarit S; Heinz, Andrea

2016-10-01

This study aimed to investigate the prolyl and lysine hydroxylation in elastin from different species and tissues. Enzymatic digests of elastin samples from human, cattle, pig and chicken were analyzed using mass spectrometry and bioinformatics tools. It was confirmed at the protein level that elastin does not contain hydroxylated lysine residues regardless of the species. In contrast, prolyl hydroxylation sites were identified in all elastin samples. Moreover, the analysis of the residues adjacent to prolines allowed the determination of the substrate site preferences of prolyl 4-hydroxylase. It was found that elastins from all analyzed species contain hydroxyproline and that at least 20%-24% of all proline residues were partially hydroxylated. Determination of the hydroxylation degrees of specific proline residues revealed that prolyl hydroxylation depends on both the species and the tissue, however, is independent of age. The fact that the highest hydroxylation degrees of proline residues were found for elastin from the intervertebral disc and knowledge of elastin arrangement in this tissue suggest that hydroxylation plays a biomechanical role. Interestingly, a proline-rich domain of tropoelastin (domain 24), which contains several repeats of bioactive motifs, does not show any hydroxyproline residues in the mammals studied. The results show that prolyl hydroxylation is not a coincidental feature and may contribute to the adaptation of the properties of elastin to meet the functional requirements of different tissues. The study for the first time shows that prolyl hydroxylation is highly regulated in elastin. Copyright © 2016 Elsevier B.V. All rights reserved.

GABA promotes elastin synthesis and elastin fiber formation in normal human dermal fibroblasts (HDFs).

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Uehara, Eriko; Hokazono, Hideki; Hida, Mariko; Sasaki, Takako; Yoshioka, Hidekatsu; Matsuo, Noritaka

2017-06-01

The multiple physiological effects of γ-aminobutyric acid (GABA) as a functional food component have been recently reported. We previously reported that GABA upregulated the expression of type I collagen in human dermal fibroblasts (HDFs), and that oral administration of GABA significantly increased skin elasticity. However, details of the regulatory mechanism still remain unknown. In this study, we further examined the effects of GABA on elastin synthesis and elastin fiber formation in HDFs. Real-time PCR indicated that GABA significantly increased the expression of tropoelastin transcript in a dose-dependent manner. Additionally, the expression of fibrillin-1, fibrillin-2, and fibulin-5/DANCE, but not lysyl oxidase and latent transforming factor-β-binding protein 4, were also significantly increased in HDFs. Finally, immunohistochemical analysis confirmed that treatment with GABA dramatically increased the formation of elastic fibers in HDFs. Taken together, our results showed that GABA improves skin elasticity in HDFs by upregulating elastin synthesis and elastin fiber formation.

Structural Insights into the Mechanisms of Action of Short-Peptide HIV-1 Fusion Inhibitors Targeting the Gp41 Pocket

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Xiujuan Zhang

2018-02-01

Full Text Available The deep hydrophobic pocket of HIV-1 gp41 has been considered a drug target, but short-peptides targeting this site usually lack potent antiviral activity. By applying the M-T hook structure, we previously generated highly potent short-peptide fusion inhibitors that specifically targeted the pocket site, such as MT-SC22EK, HP23L, and LP-11. Here, the crystal structures of HP23L and LP-11 bound to the target mimic peptide N36 demonstrated the critical intrahelical and interhelical interactions, especially verifying that the hook-like conformation was finely adopted while the methionine residue was replaced by the oxidation-less prone residue leucine, and that addition of an extra glutamic acid significantly enhanced the binding and inhibitory activities. The structure of HP23L bound to N36 with two mutations (E49K and L57R revealed the critical residues and motifs mediating drug resistance and provided new insights into the mechanism of action of inhibitors. Therefore, the present data help our understanding for the structure-activity relationship (SAR of HIV-1 fusion inhibitors and facilitate the development of novel antiviral drugs.

Pulmonary alveolar macrophages (PAM) engulf and regain elastin particles and do not respond to some stimuli of neutrophil (PMN) elastinolysis

International Nuclear Information System (INIS)

Tricomi, S.M.; Hyers, T.M.; Yu, S.Y.; Liao, J.J.

1986-01-01

Elastin degradation by PMN and by PAM differs in the proteinases produced and in the method of cellular attack on the substrate. To further characterize the elastinolytic mechanisms of these two cells, 14 C-labelled bovine ligament elastin was dried onto 24-well culture plates and live cells were placed on the substrate in culture medium. Incubation times were 4 hours for PMN and 20 hours for PAM. Elastinolytic activity was determined by counting 14 C-elastin peptides in the supernatant. By lidocaine release of PAM from the surface, 14 C-elastin retained by the cell was measured. Studies on rabbit PAM showed that 40% of dpm remain associated with the cell at 20 hours. Transmission electron microscopy of human PAM confirmed that PAM can engulf and retain elastin particles at 4 and 24 hours of incubation when in close contact with the substrate. Of the number of dpm released by PMN in 4 hours, PAM in 20 hours released only 23% of that number into supernatant and retained 17% closely associated with the cell after lidocaine treatment. Platelet factor 4, a protein released by platelets upon aggregation which stimulates activity of PMN elastase on elastin, was shown to enhance elastinolysis by whole PMN by 57% at 10 μg/ml in this assay. Platelet factor 4 did not enhance elastinolysis by PAM at concentrations up to 100 μg/ml

Cues for cellular assembly of vascular elastin networks

Science.gov (United States)

Kothapalli, Chandrasekhar R.

Elastin, a structural protein distributed in the extracellular matrix of vascular tissues is critical to the maintenance of vascular mechanics, besides regulation of cell-signaling pathways involved in injury response and morphogenesis. Thus, congenital absence or disease-mediated degradation of vascular elastin and its malformation within native vessels due to innately poor elastin synthesis by adult vascular cells compromise vascular homeostasis. Current elastin regenerative strategies using tissue engineering principles are limited by the progressive destabilization of tropoelastin mRNA expression in adult vascular cells and the unavailability of scaffolds that can provide cellular cues necessary to up-regulate elastin synthesis and regenerate faithful mimics of native elastin. Since our earlier studies demonstrated the elastogenic utility of hyaluronan (HA)-based cues, we have currently sought to identify a unique set of culture conditions based on HA fragments (0.756-2000 kDa), growth factors (TGF-beta1, IGF-1) and other biomolecules (Cu2+ ions, LOX), which will together enhance synthesis, crosslinking, maturation and fibrous elastin matrix formation by adult SMCs, under both healthy and inflammatory conditions. It was observed that TGF-beta1 (1 ng/mL) together with HA oligomers (0.2 microg/mL) synergistically suppressed SMC proliferation, enhanced tropoelastin (8-fold) and matrix elastin synthesis (5.5-fold), besides improving matrix yield (4.5-fold), possibly by increasing production and activity of lysyl oxidase (LOX). Though addition of IGF-1 alone did not offer any advantage, HA fragments (20-200 kDa) in the presence of IGF-1 stimulated tropoelastin and soluble elastin synthesis more than 2.2-fold, with HMW HA contributing for ˜5-fold increase in crosslinked matrix elastin synthesis. Similarly, 0.1 M of Cu2+ ions, alone or together with HA fragments stimulated synthesis of tropoelastin (4-fold) and crosslinked matrix elastin (4.5-fold), via increases in

Preparation of Photocrosslinked Fish Elastin Polypeptide/Microfibrillated Cellulose Composite Gels with Elastic Properties for Biomaterial Applications

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Shinya Yano

2015-01-01

Full Text Available Photocrosslinked hydrogels reinforced by microfibrillated cellulose (MFC were prepared from a methacrylate-functionalized fish elastin polypeptide and MFC dispersed in dimethylsulfoxide (DMSO. First, a water-soluble elastin peptide with a molecular weight of ca. 500 g/mol from the fish bulbus arteriosus was polymerized by N,N′-dicyclohexylcarbodiimide (DCC, a condensation reagent, and then modified with 2-isocyanatoethyl methacrylate (MOI to yield a photocrosslinkable fish elastin polypeptide. The product was dissolved in DMSO and irradiated with UV light in the presence of a radical photoinitiator. We obtained hydrogels successfully by substitution of DMSO with water. The composite gel with MFC was prepared by UV irradiation of the photocrosslinkable elastin polypeptide mixed with dispersed MFC in DMSO, followed by substitution of DMSO with water. The tensile test of the composite gels revealed that the addition of MFC improved the tensile properties, and the shape of the stress–strain curve of the composite gel became more similar to the typical shape of an elastic material with an increase of MFC content. The rheology measurement showed that the elastic modulus of the composite gel increased with an increase of MFC content. The cell proliferation test on the composite gel showed no toxicity.

Expression and purification of chimeric peptide comprising EGFR B-cell epitope and measles virus fusion protein T-cell epitope in Escherichia coli.

Science.gov (United States)

Wu, Meizhi; Zhao, Lin; Zhu, Lei; Chen, Zhange; Li, Huangjin

2013-03-01

Chimeric peptide MVF-EGFR(237-267), comprising a B-cell epitope from the dimerization interface of human epidermal growth factor receptor (EGFR) and a promiscuous T-cell epitope from measles virus fusion protein (MVF), is a promising candidate antigen peptide for therapeutic vaccine. To establish a high-efficiency preparation process of this small peptide, the coding sequence was cloned into pET-21b and pET-32a respectively, to be expressed alone or in the form of fusion protein with thioredoxin (Trx) and His(6)-tag in Escherichia coli BL21 (DE3). The chimeric peptide failed to be expressed alone, but over-expressed in the fusion form, which presented as soluble protein and took up more than 30% of total proteins of host cells. The fusion protein was seriously degraded during the cell disruption, in which endogenous metalloproteinase played a key role. Degradation of target peptide was inhibited by combined application of EDTA in the cell disruption buffer and a step of Source 30Q anion exchange chromatography (AEC) before metal-chelating chromatography (MCAC) for purifying His(6)-tagged fusion protein. The chimeric peptide was recovered from the purified fusion protein by enterokinase digestion at a yield of 3.0 mg/L bacteria culture with a purity of more than 95%. Immunogenicity analysis showed that the recombinant chimeric peptide was able to arouse more than 1×10(4) titers of specific antibody in BALB/c mice. Present work laid a solid foundation for the development of therapeutic peptide vaccine targeting EGFR dimerization and provided a convenient and low-cost preparation method for small peptides. Copyright © 2012 Elsevier Inc. All rights reserved.

Prolyl hydroxylation in elastin is not random

DEFF Research Database (Denmark)

Schmelzer, Christian E H; Nagel, Marcus B M; Dziomba, Szymon

2016-01-01

BACKGROUND: This study aimed to investigate the prolyl and lysine hydroxylation in elastin from different species and tissues. METHODS: Enzymatic digests of elastin samples from human, cattle, pig and chicken were analyzed using mass spectrometry and bioinformatics tools. RESULTS: It was confirmed...... at the protein level that elastin does not contain hydroxylated lysine residues regardless of the species. In contrast, prolyl hydroxylation sites were identified in all elastin samples. Moreover, the analysis of the residues adjacent to prolines allowed the determination of the substrate site preferences...... of prolyl 4-hydroxylase. It was found that elastins from all analyzed species contain hydroxyproline and that at least 20%-24% of all proline residues were partially hydroxylated. Determination of the hydroxylation degrees of specific proline residues revealed that prolyl hydroxylation depends on both...

Alternative Splicing and Tissue-specific Elastin Misassembly Act as Biological Modifiers of Human Elastin Gene Frameshift Mutations Associated with Dominant Cutis Laxa*

Science.gov (United States)

Sugitani, Hideki; Hirano, Eiichi; Knutsen, Russell H.; Shifren, Adrian; Wagenseil, Jessica E.; Ciliberto, Christopher; Kozel, Beth A.; Urban, Zsolt; Davis, Elaine C.; Broekelmann, Thomas J.; Mecham, Robert P.

2012-01-01

Elastin is the extracellular matrix protein in vertebrates that provides elastic recoil to blood vessels, the lung, and skin. Because the elastin gene has undergone significant changes in the primate lineage, modeling elastin diseases in non-human animals can be problematic. To investigate the pathophysiology underlying a class of elastin gene mutations leading to autosomal dominant cutis laxa, we engineered a cutis laxa mutation (single base deletion) into the human elastin gene contained in a bacterial artificial chromosome. When expressed as a transgene in mice, mutant elastin was incorporated into elastic fibers in the skin and lung with adverse effects on tissue function. In contrast, only low levels of mutant protein incorporated into aortic elastin, which explains why the vasculature is relatively unaffected in this disease. RNA stability studies found that alternative exon splicing acts as a modifier of disease severity by influencing the spectrum of mutant transcripts that survive nonsense-mediated decay. Our results confirm the critical role of the C-terminal region of tropoelastin in elastic fiber assembly and suggest tissue-specific differences in the elastin assembly pathway. PMID:22573328

Gene design, fusion technology and TEV cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in Escherichia coli.

Science.gov (United States)

Sequeira, Ana Filipa; Turchetto, Jeremy; Saez, Natalie J; Peysson, Fanny; Ramond, Laurie; Duhoo, Yoan; Blémont, Marilyne; Fernandes, Vânia O; Gama, Luís T; Ferreira, Luís M A; Guerreiro, Catarina I P I; Gilles, Nicolas; Darbon, Hervé; Fontes, Carlos M G A; Vincentelli, Renaud

2017-01-17

Animal venoms are large, complex libraries of bioactive, disulphide-rich peptides. These peptides, and their novel biological activities, are of increasing pharmacological and therapeutic importance. However, recombinant expression of venom peptides in Escherichia coli remains difficult due to the significant number of cysteine residues requiring effective post-translational processing. There is also an urgent need to develop high-throughput recombinant protocols applicable to the production of reticulated peptides to enable efficient screening of their drug potential. Here, a comprehensive study was developed to investigate how synthetic gene design, choice of fusion tag, compartment of expression, tag removal conditions and protease recognition site affect levels of solubility of oxidized venom peptides produced in E. coli. The data revealed that expression of venom peptides imposes significant pressure on cysteine codon selection. DsbC was the best fusion tag for venom peptide expression, in particular when the fusion was directed to the bacterial periplasm. While the redox activity of DsbC was not essential to maximize expression of recombinant fusion proteins, redox activity did lead to higher levels of correctly folded target peptides. With the exception of proline, the canonical TEV protease recognition site tolerated all other residues at its C-terminus, confirming that no non-native residues, which might affect activity, need to be incorporated at the N-terminus of recombinant peptides for tag removal. This study reveals that E. coli is a convenient heterologous host for the expression of soluble and functional venom peptides. Using the optimal construct design, a large and diverse range of animal venom peptides were produced in the µM scale. These results open up new possibilities for the high-throughput production of recombinant disulphide-rich peptides in E. coli.

Ginkgotides: Proline-Rich Hevein-Like Peptides from Gymnosperm Ginkgo biloba.

Science.gov (United States)

Wong, Ka H; Tan, Wei Liang; Serra, Aida; Xiao, Tianshu; Sze, Siu Kwan; Yang, Daiwen; Tam, James P

2016-01-01

Hevein and hevein-like peptides belong to the family of chitin-binding cysteine-rich peptides. They are classified into three subfamilies, the prototypic 8C- and the 6C- and 10C-hevein-like peptides. Thus far, only five 8C-hevein-like peptides have been characterized from three angiosperms and none from gymnosperm. To determine their occurrence and distribution in the gymnosperm, Ginkgo biloba leaves were examined. Here, we report the discovery and characterization of 11 novel 8C-hevein-like peptides, namely ginkgotides gB1-gB11. Proteomic analysis showed that the ginkgotides contain 41-44 amino acids (aa), a chitin-binding domain and are Pro-rich, a distinguishing feature that differs from other hevein-like peptides. Solution NMR structure determination revealed that gB5 contains a three β-stranded structure shaped by a cystine knot with an additional disulfide bond at the C-terminus. Transcriptomic analysis showed that the ginkgotide precursors contain a three-domain architecture, comprised of a C-terminal tail (20 aa) that is significantly shorter than those of other 8C- and 10C-hevein-like peptides, which generally contain a protein cargo such as a Barwin-like protein (126 aa) or class I chitinase (254 aa). Transcriptomic data mining found an additional 48 ginkgotide homologs in 39 different gymnosperms. Phylogenetic analysis revealed that ginkgotides and their homologs belong to a new class of 8C-hevein-like peptides. Stability studies showed that ginkgotides are highly resistant to thermal, acidic and endopeptidase degradation. Ginkgotides flanked at both the N- and C-terminal ends by Pro were resistant to exopeptidase degradation by carboxypeptidase A and aminopeptidase. Antifungal assays showed that ginkgotides inhibit the hyphal growth of phyto-pathogenic fungi. Taken together, ginkgotides represent the first suite of hevein-like peptides isolated and characterized from gymnosperms. As a group, they represent a novel class of 8C-hevein-like peptides that

Multiple Factors Related to the Secretion of Glucagon-Like Peptide-1

Directory of Open Access Journals (Sweden)

XingChun Wang

2015-01-01

Full Text Available The glucagon-like peptide-1 is secreted by intestinal L cells in response to nutrient ingestion. It regulates the secretion and sensitivity of insulin while suppressing glucagon secretion and decreasing postprandial glucose levels. It also improves beta-cell proliferation and prevents beta-cell apoptosis induced by cytotoxic agents. Additionally, glucagon-like peptide-1 delays gastric emptying and suppresses appetite. The impaired secretion of glucagon-like peptide-1 has negative influence on diabetes, hyperlipidemia, and insulin resistance related diseases. Thus, glucagon-like peptide-1-based therapies (glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors are now well accepted in the management of type 2 diabetes. The levels of glucagon-like peptide-1 are influenced by multiple factors including a variety of nutrients. The component of a meal acts as potent stimulants of glucagon-like peptide-1 secretion. The levels of its secretion change with the intake of different nutrients. Some drugs also have influence on GLP-1 secretion. Bariatric surgery may improve metabolism through the action on GLP-1 levels. In recent years, there has been a great interest in developing effective methods to regulate glucagon-like peptide-1 secretion. This review summarizes the literature on glucagon-like peptide-1 and related factors affecting its levels.

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.

In Vivo Efficacy of Measles Virus Fusion Protein-Derived Peptides Is Modulated by the Properties of Self-Assembly and Membrane Residence

Science.gov (United States)

Figueira, T. N.; Palermo, L. M.; Veiga, A. S.; Huey, D.; Alabi, C. A.; Santos, N. C.; Welsch, J. C.; Mathieu, C.; Niewiesk, S.; Moscona, A.

2016-01-01

ABSTRACT Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV hemagglutinin (H) and fusion (F) envelope glycoproteins; upon receptor engagement by H, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad repeat (HR) regions of F can inhibit MV infection at the entry stage. In previous work, we have generated potent MV fusion inhibitors by dimerizing the F-derived peptides and conjugating them to cholesterol. We have shown that prophylactic intranasal administration of our lead fusion inhibitor efficiently protects from MV infection in vivo. We show here that peptides tagged with lipophilic moieties self-assemble into nanoparticles until they reach the target cells, where they are integrated into cell membranes. The self-assembly feature enhances biodistribution and the half-life of the peptides, while integration into the target cell membrane increases fusion inhibitor potency. These factors together modulate in vivo efficacy. The results suggest a new framework for developing effective fusion inhibitory peptides. IMPORTANCE Measles virus (MV) infection causes an acute illness that may be associated with infection of the central nervous system (CNS) and severe neurological disease. No specific treatment is available. We have shown that fusion-inhibitory peptides delivered intranasally provide effective prophylaxis against MV infection. We show here that specific biophysical properties regulate the in vivo efficacy of MV F-derived peptides. PMID:27733647

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.

Interaction between fatty acid and the elastin network

NARCIS (Netherlands)

Vreeswijk, van J.

1995-01-01

The aim of the present study was to investigate the interaction between salts of fatty acids (FAS) and elastin. Absorption of fatty acids in elastin may affect the elasticity of elastin-containing tissue. Such phenomena could, for instance, be of relevance for the understanding of the

Atrial natriuretic-like peptide and its prohormone within metasequoia.

Science.gov (United States)

Yang, Q; Gower, W R; Li, C; Chen, P; Vesely, D L

1999-07-01

Metasequoia glyptostroboides was one of the dominant conifers in North America, Asia, and Europe for more than 100 million years since the late Cretaceous Albian Age, but Quaternary glaciations drove the Metasequoia population to apparent extinction. A small pocket of Metasequoia, however, was found in central China in the 1940s representing the only surviving population of this "living fossil" species. Atrial natriuretic peptide, a 28-amino-acid peptide hormone that causes sodium and water excretion in animals, has been found to be part of the first peptide hormonal system in lower plants. The existence of this hormonal system has never been examined within trees of any genus. High-performance gel permeation chromatography of the leaves and stems (i.e., branches) of Metasequoia followed by atrial natriuretic peptide radioimmunoassay revealed an ANP-like peptide and its prohormone (i.e., approximately 13,000 mol wt) were present in both leaves and stems of this conifer. The elution profile of ANP-like peptide in stems of Metasequoia had a shoulder to the left of where pure synthetic ANP elutes suggesting the possibility of a slightly larger peptide eluting within this shoulder secondary to alternate processing of the ANP-like prohormone and similar to what occurs with the kidney of animals. The elution profile of ANP-like peptide in the leaves of Metasequoia revealed two peaks; one where ANP elutes and a second peak suggesting a smaller peptide that has been metabolically processed. The presence of the ANP-like prohormone strongly suggests that ANP-like gene expression is occurring in both leaves and stems of Metasequoia since this prohormone is the gene product of this hormonal system. The presence of the ANP-like hormonal system in trees implies that this hormonal system may have been present early in land plant evolution to allow trees to reach heights of greater than 30 feet where a water flow-enhancing substance is absolutely necessary for water flow to occur

Ginkgotides: Proline-rich Hevein-like Peptides from Gymnosperm Ginkgo biloba

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Ka Ho Wong

2016-11-01

Full Text Available Hevein and hevein-like peptides belong to the family of chitin-binding cysteine-rich peptides. They are classified into three subfamilies, the prototypic 8C- and the 6C- and 10C-hevein-like peptides. Thus far, only five 8C-hevein-like peptides have been characterized from three angiosperms and none from gymnosperm. To determine their occurrence and distribution in the gymnosperm, Ginkgo biloba leaves were examined. Here, we report the discovery and characterization of eleven novel 8C-hevein-like peptides, namely ginkgotides gB1–gB11. Proteomic analysis showed that the ginkgotides contain 41–44 amino acids (aa, a chitin-binding domain and are Pro-rich, a distinguishing feature that differs from other hevein-like peptides. Solution 1H-NMR structure determination revealed that gB5 contains a three β-stranded structure shaped by a cystine knot with an additional disulfide bond at the C-terminus. Transcriptomic analysis showed that the ginkgotide precursors contain a three-domain architecture, comprised of a C-terminal tail (20 aa that is significantly shorter than those of other 8C- and 10C-hevein-like peptides, which generally contain a protein cargo such as a Barwin-like protein (126 aa or class I chitinase (254 aa. Transcriptomic data mining found an additional 48 ginkgotide homologs in 39 different gymnosperms. Phylogenetic analysis revealed that ginkgotides and their homologs belong to a new class of 8C-hevein-like peptides. Stability studies showed that ginkgotides are highly resistant to thermal, acidic and endopeptidase degradation. Ginkgotides flanked at both the N- and C-terminal ends by Pro were resistant to exopeptidase degradation by carboxypeptidase A and aminopeptidase. Antifungal assays showed that ginkgotides inhibit the hyphal growth of phyto-pathogenic fungi. Taken together, ginkgotides represent the first suite of hevein-like peptides isolated and characterized from gymnosperms. As a group, they represent a novel class of 8C-hevein-like

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).

Identification of vimentin- and elastin-like transcripts specifically expressed in developing notochord of Atlantic salmon (Salmo salar L.).

Science.gov (United States)

Sagstad, Anita; Grotmol, Sindre; Kryvi, Harald; Krossøy, Christel; Totland, Geir K; Malde, Ketil; Wang, Shou; Hansen, Tom; Wargelius, Anna

2011-11-01

The notochord functions as the midline structural element of all vertebrate embryos, and allows movement and growth at early developmental stages. Moreover, during embryonic development, notochord cells produce secreted factors that provide positional and fate information to a broad variety of cells within adjacent tissues, for instance those of the vertebrae, central nervous system and somites. Due to the large size of the embryo, the salmon notochord is useful to study as a model for exploring notochord development. To investigate factors that might be involved in notochord development, a normalized cDNA library was constructed from a mix of notochords from ∼500 to ∼800 day°. From the 1968 Sanger-sequenced transcripts, 22 genes were identified to be predominantly expressed in the notochord compared to other organs of salmon. Twelve of these genes were found to show expressional regulation around mineralization of the notochord sheath; 11 genes were up-regulated and one gene was down-regulated. Two genes were found to be specifically expressed in the notochord; these genes showed similarity to vimentin (acc. no GT297094) and elastin (acc. no GT297478). In-situ results showed that the vimentin- like transcript was expressed in both chordocytes and chordoblasts, whereas the elastin- like transcript was uniquely expressed in the chordoblasts lining the notochordal sheath. In salmon aquaculture, vertebral deformities are a common problem, and some malformations have been linked to the notochord. The expression of identified transcripts provides further insight into processes taking place in the developing notochord, prior to and during the early mineralization period.

Vaccatides: Antifungal Glutamine-Rich Hevein-Like Peptides from Vaccaria hispanica

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Ka H. Wong

2017-06-01

Full Text Available Hevein and hevein-like peptides are disulfide-constrained chitin-binding cysteine-rich peptides. They are divided into three subfamilies, 6C-, 8C-, and 10C-hevein-like peptides, based on the number of cysteine residues. In addition, hevein-like peptides can exist in two forms, short and long. The long C-terminal form found in hevein and 10C-hevein-like peptides contain a C-terminal protein cargo. In contrast, the short form without a protein cargo is found in all three subfamilies. Here, we report the discovery and characterization of two novel glutamine-rich and protein cargo-free 8C-hevein-like peptides, vaccatides vH1 and vH2, from Vaccaria hispanica of the Caryophyllaceae family. Proteomic analyses showed that the vaccatides are 40–41 amino acids in length and contain a chitin-binding domain. NMR determination revealed that vaccatide vH2 displays a highly compact structure with a N-terminal cystine knot and an addition C-terminal disulfide bond. Stability studies showed that this compact structure renders vaccatide vH2 resistant to thermal, chemical and proteolytic degradation. The chitin-binding vH2 was shown to inhibit the mycelium growth of four phyto-pathogenic fungal strains with IC50 values in the micromolar range. Our findings show that vaccatides represent a new family of 8C-hevein-like peptides, which are protein cargo-free and glutamine-rich, characteristics that differentiate them from the prototypic hevein and the 10C-hevein-like peptides. In summary, this study enriches the existing library of hevein-like peptides and provides insight into their molecular diversity in sequence, structure and biosynthesis. Additionally, their highly disulfide-constrained structure could be used as a scaffold for developing metabolically and orally active peptidyl therapeutics.

Inhibition of the Hantavirus Fusion Process by Predicted Domain III and Stem Peptides from Glycoprotein Gc.

Science.gov (United States)

Barriga, Gonzalo P; Villalón-Letelier, Fernando; Márquez, Chantal L; Bignon, Eduardo A; Acuña, Rodrigo; Ross, Breyan H; Monasterio, Octavio; Mardones, Gonzalo A; Vidal, Simon E; Tischler, Nicole D

2016-07-01

Hantaviruses can cause hantavirus pulmonary syndrome or hemorrhagic fever with renal syndrome in humans. To enter cells, hantaviruses fuse their envelope membrane with host cell membranes. Previously, we have shown that the Gc envelope glycoprotein is the viral fusion protein sharing characteristics with class II fusion proteins. The ectodomain of class II fusion proteins is composed of three domains connected by a stem region to a transmembrane anchor in the viral envelope. These fusion proteins can be inhibited through exogenous fusion protein fragments spanning domain III (DIII) and the stem region. Such fragments are thought to interact with the core of the fusion protein trimer during the transition from its pre-fusion to its post-fusion conformation. Based on our previous homology model structure for Gc from Andes hantavirus (ANDV), here we predicted and generated recombinant DIII and stem peptides to test whether these fragments inhibit hantavirus membrane fusion and cell entry. Recombinant ANDV DIII was soluble, presented disulfide bridges and beta-sheet secondary structure, supporting the in silico model. Using DIII and the C-terminal part of the stem region, the infection of cells by ANDV was blocked up to 60% when fusion of ANDV occurred within the endosomal route, and up to 95% when fusion occurred with the plasma membrane. Furthermore, the fragments impaired ANDV glycoprotein-mediated cell-cell fusion, and cross-inhibited the fusion mediated by the glycoproteins from Puumala virus (PUUV). The Gc fragments interfered in ANDV cell entry by preventing membrane hemifusion and pore formation, retaining Gc in a non-resistant homotrimer stage, as described for DIII and stem peptide inhibitors of class II fusion proteins. Collectively, our results demonstrate that hantavirus Gc shares not only structural, but also mechanistic similarity with class II viral fusion proteins, and will hopefully help in developing novel therapeutic strategies against hantaviruses.

The Elastin Receptor Complex: a unique matricellular receptor with high anti-tumoral potential

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Amandine eScandolera

2016-03-01

Full Text Available Elastin, one of the longest-lived proteins, confers elasticity to tissues with high mechanical constraints. During aging or pathophysiological conditions such as cancer progression, this insoluble polymer of tropoelastin undergoes an important degradation leading to the release of bioactive elastin-derived peptides (EDP, named elastokines. EDP exhibit several biological functions able to drive tumor development by regulating cell proliferation, invasion, survival, angiogenesis, and matrix metalloproteinase expression in various tumor and stromal cells. Although several receptors have been suggested to bind elastokines (αvβ3 and αvβ5 integrins, galectin-3, their main receptor remains the Elastin Receptor Complex (ERC. This heterotrimer comprises a peripheral subunit, named Elastin Binding Protein (EBP, associated to the Protective Protein/Cathepsin A (PPCA. The latter is bound to a membrane-associated protein called Neuraminidase-1 (Neu-1. The pro-tumoral effects of elastokines have been linked to their binding onto EBP. Additionally, Neu-1 sialidase activity is essential for their signal transduction. Consistently, EDP-EBP interaction and Neu-1 activity emerge as original anti-tumoral targets. Interestingly, besides its direct involvement in cancer progression, the ERC also regulates diabetes outcome and thrombosis, an important risk factor for cancer development and a vascular process highly increased in patients suffering from cancer. In this review, we will describe ERC and elastokines involvement in cancer development suggesting that this unique receptor would be a promising therapeutic target. We will also discuss the pharmacological concepts aiming at blocking its pro-tumoral activities. Finally, its emerging role in cancer-associated complications and pathologies such as diabetes and thrombotic events will be also considered.

The Effect of Static Stretch on Elastin Degradation in Arteries

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Chow, Ming-Jay; Choi, Myunghwan; Yun, Seok Hyun; Zhang, Yanhang

2013-01-01

Previously we have shown that gradual changes in the structure of elastin during an elastase treatment can lead to important transition stages in the mechanical behavior of arteries [1]. However, in vivo arteries are constantly being loaded due to systolic and diastolic pressures and so understanding the effects of loading on the enzymatic degradation of elastin in arteries is important. With biaxial tensile testing, we measured the mechanical behavior of porcine thoracic aortas digested with a mild solution of purified elastase (5 U/mL) in the presence of a static stretch. Arterial mechanical properties and biochemical composition were analyzed to assess the effects of mechanical stretch on elastin degradation. As elastin is being removed, the dimensions of the artery increase by more than 20% in both the longitude and circumference directions. Elastin assays indicate a faster rate of degradation when stretch was present during the digestion. A simple exponential decay fitting confirms the time constant for digestion with stretch (0.11±0.04 h−1) is almost twice that of digestion without stretch (0.069±0.028 h−1). The transition from J-shaped to S-shaped stress vs. strain behavior in the longitudinal direction generally occurs when elastin content is reduced by about 60%. Multiphoton image analysis confirms the removal/fragmentation of elastin and also shows that the collagen fibers are closely intertwined with the elastin lamellae in the medial layer. After removal of elastin, the collagen fibers are no longer constrained and become disordered. Release of amorphous elastin during the fragmentation of the lamellae layers is observed and provides insights into the process of elastin degradation. Overall this study reveals several interesting microstructural changes in the extracellular matrix that could explain the resulting mechanical behavior of arteries with elastin degradation. PMID:24358135

Localization of receptors for bombesin-like peptides in the rat brain

International Nuclear Information System (INIS)

Moody, T.W.; Getz, R.; O'Donohue, T.L.; Rosenstein, J.M.

1988-01-01

BN-like peptides and receptors are present in discrete areas of the mammalian brain. By radioimmunoassay, endogenous BN/GRP, neuromedin B, and ranatensin-like peptides are present in the rat brain. High-to-moderate concentrations of BN/GRP are present in the rat hypothalamus and thalamus, whereas moderate-to-high densities of neuromedin B and ranatensin-like peptides are present in the olfactory bulb and hippocampus, as well as in the hypothalamus and thalamus. While the distribution of neuromedin B and ranatensin-like peptides appears similar, it is distinct from that of BN/GRP. When released from CNS neurons, these peptides may interact with receptors for BN-like peptides. BN, GRP, ranatensin, and neuromedin B inhibit specific [ 125 I-Tyr4]BN binding with high affinity. By use of in vitro autoradiographic techniques to detect binding of [ 125 I-Tyr4]BN to receptors for BN-like peptides, high grain densities were found in the olfactory bulb and tubercle, the nucleus accumbens, the suprachiasmatic and paraventricular nucleus of the hypothalamus, the central medial and paraventricular thalamic nuclei, the hippocampus, the dentate gyrus, and the amygdala of the rat brain. Some of these receptors may be biologically active and mediate the biological effects of BN-like peptides. For example, when BN is directly injected into the nucleus accumbens, pronounced grooming results and the effects caused by BN are reversed by spantide and [D-Phe12]BN. Thus, the putative BN receptor antagonists may serve as useful agents to investigate the biological significance of BN-like peptides in the CNS

Solid-state nuclear magnetic resonance measurements of HIV fusion peptide 13CO to lipid 31P proximities support similar partially inserted membrane locations of the α helical and β sheet peptide structures.

Science.gov (United States)

Gabrys, Charles M; Qiang, Wei; Sun, Yan; Xie, Li; Schmick, Scott D; Weliky, David P

2013-10-03

Fusion of the human immunodeficiency virus (HIV) membrane and the host cell membrane is an initial step of infection of the host cell. Fusion is catalyzed by gp41, which is an integral membrane protein of HIV. The fusion peptide (FP) is the ∼25 N-terminal residues of gp41 and is a domain of gp41 that plays a key role in fusion catalysis likely through interaction with the host cell membrane. Much of our understanding of the FP domain has been accomplished with studies of "HFP", i.e., a ∼25-residue peptide composed of the FP sequence but lacking the rest of gp41. HFP catalyzes fusion between membrane vesicles and serves as a model system to understand fusion catalysis. HFP binds to membranes and the membrane location of HFP is likely a significant determinant of fusion catalysis perhaps because the consequent membrane perturbation reduces the fusion activation energy. In the present study, many HFPs were synthesized and differed in the residue position that was (13)CO backbone labeled. Samples were then prepared that each contained a singly (13)CO labeled HFP incorporated into membranes that lacked cholesterol. HFP had distinct molecular populations with either α helical or oligomeric β sheet structure. Proximity between the HFP (13)CO nuclei and (31)P nuclei in the membrane headgroups was probed by solid-state NMR (SSNMR) rotational-echo double-resonance (REDOR) measurements. For many samples, there were distinct (13)CO shifts for the α helical and β sheet structures so that the proximities to (31)P nuclei could be determined for each structure. Data from several differently labeled HFPs were then incorporated into a membrane location model for the particular structure. In addition to the (13)CO labeled residue position, the HFPs also differed in sequence and/or chemical structure. "HFPmn" was a linear peptide that contained the 23 N-terminal residues of gp41. "HFPmn_V2E" contained the V2E mutation that for HIV leads to greatly reduced extent of fusion and

Serological assessment of neutrophil elastase activity on elastin during lung ECM remodeling.

Science.gov (United States)

Kristensen, Jacob H; Karsdal, Morten A; Sand, Jannie Mb; Willumsen, Nicholas; Diefenbach, Claudia; Svensson, Birte; Hägglund, Per; Oersnes-Leeming, Diana J

2015-05-03

During the pathological destruction of lung tissue, neutrophil elastase (NE) degrades elastin, one of the major constituents of lung parenchyma. However there are no non-invasive methods to quantify NE degradation of elastin. We selected specific elastin fragments generated by NE for antibody generation and developed an ELISA assay (EL-NE) for the quantification of NE-degraded elastin. Monoclonal antibodies were developed against 10 NE-specific cleavage sites on elastin. One EL-NE assay was tested for analyte stability, linearity and intra- and inter-assay variation. The NE specificity was demonstrated using elastin cleaved in vitro with matrix metalloproteinases (MMPs), cathepsin G (CatG), NE and intact elastin. Clinical relevance was assessed by measuring levels of NE-generated elastin fragments in serum of patients diagnosed with idiopathic pulmonary fibrosis (IPF, n = 10) or lung cancer (n = 40). Analyte recovery of EL-NE for human serum was between 85% and 104%, the analyte was stable for four freeze/thaw cycles and after 24 h storage at 4°C. EL-NE was specific for NE-degraded elastin. Levels of NE-generated elastin fragments for elastin incubated in the presence of NE were 900% to 4700% higher than those seen with CatG or MMP incubation or in intact elastin. Serum levels of NE-generated elastin fragments were significantly increased in patients with IPF (137%, p = 0.002) and in patients with lung cancer (510%, p elastin. The EL-NE assay was able to specifically quantify NE-degraded elastin in serum. Serum levels of NE-degraded elastin might be used to detect excessive lung tissue degradation in lung cancer and IPF.

Procyanidins-crosslinked aortic elastin scaffolds with distinctive anti-calcification and biological properties.

Science.gov (United States)

Wang, Xiaoya; Zhai, Wanyin; Wu, Chengtie; Ma, Bing; Zhang, Jiamin; Zhang, Hongfeng; Zhu, Ziyan; Chang, Jiang

2015-04-01

Elastin, a main component of decellularized extracellular matrices and elastin-containing materials, has been used for tissue engineering applications due to their excellent biocompatibility. However, elastin is easily calcified, leading to the decrease of life span for elastin-based substitutes. How to inhibit the calcification of elastin-based scaffolds, but maintain their good biocompatibility, still remains significantly challenging. Procyanidins (PC) are a type of natural polyphenols with crosslinking ability. To investigate whether pure elastin could be crosslinked by PC with anti-calcification effect, PC was first used to crosslink aortic elastin. Results show that PC can crosslink elastin and effectively inhibit elastin-initiated calcification. Further experiments reveal the possible mechanisms for the anti-calcification of PC crosslinking including (1) inhibiting inflammation cell attachment, and secretion of inflammatory factors such as MMPs and TNF-α, (2) preventing elastin degradation by elastase, and (3) direct inhibition of mineral nucleation in elastin. Moreover, the PC-crosslinked aortic elastin maintains natural structure with high pore volume (1111 μL/g), large pore size (10-300 μm) and high porosity (75.1%) which facilitates recellularization of scaffolds in vivo, and displays excellent hemocompatibility, anti-thrombus and anti-inflammatory potential. The advantages of PC-crosslinked porous aortic elastin suggested that it can serve as a promising scaffold for tissue engineering. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Ferroelectric switching of elastin

Science.gov (United States)

Liu, Yuanming; Cai, Hong-Ling; Zelisko, Matthew; Wang, Yunjie; Sun, Jinglan; Yan, Fei; Ma, Feiyue; Wang, Peiqi; Chen, Qian Nataly; Zheng, Hairong; Meng, Xiangjian; Sharma, Pradeep; Zhang, Yanhang; Li, Jiangyu

2014-01-01

Ferroelectricity has long been speculated to have important biological functions, although its very existence in biology has never been firmly established. Here, we present compelling evidence that elastin, the key ECM protein found in connective tissues, is ferroelectric, and we elucidate the molecular mechanism of its switching. Nanoscale piezoresponse force microscopy and macroscopic pyroelectric measurements both show that elastin retains ferroelectricity at 473 K, with polarization on the order of 1 μC/cm2, whereas coarse-grained molecular dynamics simulations predict similar polarization with a Curie temperature of 580 K, which is higher than most synthetic molecular ferroelectrics. The polarization of elastin is found to be intrinsic in tropoelastin at the monomer level, analogous to the unit cell level polarization in classical perovskite ferroelectrics, and it switches via thermally activated cooperative rotation of dipoles. Our study sheds light onto a long-standing question on ferroelectric switching in biology and establishes ferroelectricity as an important biophysical property of proteins. This is a critical first step toward resolving its physiological significance and pathological implications. PMID:24958890

Silk-elastin-like protein polymer matrix for intraoperative delivery of an oncolytic vaccinia virus.

Science.gov (United States)

Price, Daniel L; Li, Pingdong; Chen, Chun-Hao; Wong, Danni; Yu, Zhenkun; Chen, Nanhai G; Yu, Yong A; Szalay, Aladar A; Cappello, Joseph; Fong, Yuman; Wong, Richard J

2016-02-01

Oncolytic viral efficacy may be limited by the penetration of the virus into tumors. This may be enhanced by intraoperative application of virus immediately after surgical resection. Oncolytic vaccinia virus GLV-1h68 was delivered in silk-elastin-like protein polymer (SELP) in vitro and in vivo in anaplastic thyroid carcinoma cell line 8505c in nude mice. GLV-1h68 in SELP infected and lysed anaplastic thyroid cancer cells in vitro equally as effectively as in phosphate-buffered saline (PBS), and at 1 week retains a thousand fold greater infectious plaque-forming units. In surgical resection models of residual tumor, GLV-1h68 in SELP improves tumor control and shows increased viral β-galactosidase expression as compared to PBS. The use of SELP matrix for intraoperative oncolytic viral delivery protects infectious viral particles from degradation, facilitates sustained viral delivery and transgene expression, and improves tumor control. Such optimization of methods of oncolytic viral delivery may enhance therapeutic outcomes. © 2014 Wiley Periodicals, Inc.

Elastin as a biomaterial for tissue engineering.

NARCIS (Netherlands)

Daamen, W.F.; Veerkamp, J.H.; Hest, J.C.M. van; Kuppevelt, A.H.M.S.M. van

2007-01-01

Biomaterials based upon elastin and elastin-derived molecules are increasingly investigated for their application in tissue engineering. This interest is fuelled by the remarkable properties of this structural protein, such as elasticity, self-assembly, long-term stability, and biological activity.

Diversity in the organization of elastin bundles and intramembranous muscles in bat wings.

Science.gov (United States)

Cheney, Jorn A; Allen, Justine J; Swartz, Sharon M

2017-04-01

Unlike birds and insects, bats fly with wings composed of thin skin that envelops the bones of the forelimb and spans the area between the limbs, digits, and sometimes the tail. This skin is complex and unusual; it is thinner than typical mammalian skin and contains organized bundles of elastin and embedded skeletal muscles. These elements are likely responsible for controlling the shape of the wing during flight and contributing to the aerodynamic capabilities of bats. We examined the arrangement of two macroscopic architectural elements in bat wings, elastin bundles and wing membrane muscles, to assess the diversity in bat wing skin morphology. We characterized the plagiopatagium and dactylopatagium of 130 species from 17 families of bats using cross-polarized light imaging. This method revealed structures with distinctive relative birefringence, heterogeneity of birefringence, variation in size, and degree of branching. We used previously published anatomical studies and tissue histology to identify birefringent structures, and we analyzed their architecture across taxa. Elastin bundles, muscles, neurovasculature, and collagenous fibers are present in all species. Elastin bundles are oriented in a predominantly spanwise or proximodistal direction, and there are five characteristic muscle arrays that occur within the plagiopatagium, far more muscle than typically recognized. These results inform recent functional studies of wing membrane architecture, support the functional hypothesis that elastin bundles aid wing folding and unfolding, and further suggest that all bats may use these architectural elements for flight. All species also possess numerous muscles within the wing membrane, but the architecture of muscle arrays within the plagiopatagium varies among families. To facilitate present and future discussion of these muscle arrays, we refine wing membrane muscle nomenclature in a manner that reflects this morphological diversity. The architecture of the

Extraction and characterization of elastin from poultry skin

Energy Technology Data Exchange (ETDEWEB)

Nadalian, Mehdi; Yusop, Salma Mohamad; Mustapha, Wan Aida Wan; Babji, Abdul Salam [School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor (Malaysia); Azman, Mohd Azri [Strategic Livestock Research Centre, Malaysian Agricultural Research and Development Institute, MARDI Headquarters, 43400, Serdang, Selangor (Malaysia)

2013-11-27

Poultry by-products have a great economic potential that need to be exploited. Poultry skin could be utilized to produce elastin, which is often incorporated in the production of functional food or medicine due to its antioxidative properties. This study was conducted to determine the physicochemical and microstructural characteristics of elastins isolated from broiler and spent hen skin. Analyses including proximate and amino acid composition along with transmission electron microscopy (TEM) were carried out. In this study, elastin was successfully extracted from broiler and spent hen skin using three successive solvents extract of NaCl, acetone and NaOH respectively. It was apparent that the fat content of extracted elastin from broiler skin was higher (P < 0.05) than spent hen’s, with both samples recording less than 1% fat. Moreover, broiler skin elastin also had a higher protein content (68.3%) than spent hen’s (67.8%). Both skin sources contained glycine as the major amino acid (19–20%), followed by glutamic acid, proline, alanine and arginine. The results of TEM indicated that the use of collagenase enzyme or further purification efforts should be incorporated along with the extraction methods used because of the presence of collagen and other debris in the resultant elastin.

Extraction and characterization of elastin from poultry skin

International Nuclear Information System (INIS)

Nadalian, Mehdi; Yusop, Salma Mohamad; Mustapha, Wan Aida Wan; Babji, Abdul Salam; Azman, Mohd Azri

2013-01-01

Poultry by-products have a great economic potential that need to be exploited. Poultry skin could be utilized to produce elastin, which is often incorporated in the production of functional food or medicine due to its antioxidative properties. This study was conducted to determine the physicochemical and microstructural characteristics of elastins isolated from broiler and spent hen skin. Analyses including proximate and amino acid composition along with transmission electron microscopy (TEM) were carried out. In this study, elastin was successfully extracted from broiler and spent hen skin using three successive solvents extract of NaCl, acetone and NaOH respectively. It was apparent that the fat content of extracted elastin from broiler skin was higher (P < 0.05) than spent hen’s, with both samples recording less than 1% fat. Moreover, broiler skin elastin also had a higher protein content (68.3%) than spent hen’s (67.8%). Both skin sources contained glycine as the major amino acid (19–20%), followed by glutamic acid, proline, alanine and arginine. The results of TEM indicated that the use of collagenase enzyme or further purification efforts should be incorporated along with the extraction methods used because of the presence of collagen and other debris in the resultant elastin

High-contrast multimodel nonlinear optical imaging of collagen and elastin

Energy Technology Data Exchange (ETDEWEB)

Zhuo, S M [Key Laboratory of Optoelectronic Science and Technology for Medicine (Fujian Normal University), Ministry of Education, Fuzhou 350007 (China); Chen, J X [Key Laboratory of Optoelectronic Science and Technology for Medicine (Fujian Normal University), Ministry of Education, Fuzhou 350007 (China); Luo, T S [Key Laboratory of Optoelectronic Science and Technology for Medicine (Fujian Normal University), Ministry of Education, Fuzhou 350007 (China); Chen, H L [Key Laboratory of Optoelectronic Science and Technology for Medicine (Fujian Normal University), Ministry of Education, Fuzhou 350007 (China); Zhao, J J [Department of Skin, Affiliated Xiehe Hospital, Fujian Medical University, Fuzhou 350001 (China)

2007-07-15

Collagen and elastin, as the major components in the extracellular matrix (ECM), are intrinsic indicators of physiological and pathological states. Here, we have developed a high-contrast multimodel nonlinear optical imaging technique to imaging collagen and elastin by detecting simultaneously two photon-excited fluorescence (TPEF) from elastin and second-harmonic generation (SHG) from collagen. Our results show that this technique can obtain a high-contrast TPEF/SHG image in human dermis and permit direct visualization of collagen and elastin. It was shown that the technique can provide collagen and elastin structural information to determine collagen and elastin fibril orientation and distribution and acquire some morphometric properties. It was found that the in-depth TPEF/SHG imaging and 3-D reconstruction of TPEF/SHG images can extract more collagen and elastin structural and biochemical information. The study results suggest that the high-contrast multimodel nonlinear imaging provides a powerful tool to study ECM intrinsic components and has the potential to provide more important information for the diagnosis of tissue.

High-contrast multimodel nonlinear optical imaging of collagen and elastin

International Nuclear Information System (INIS)

Zhuo, S M; Chen, J X; Luo, T S; Chen, H L; Zhao, J J

2007-01-01

Collagen and elastin, as the major components in the extracellular matrix (ECM), are intrinsic indicators of physiological and pathological states. Here, we have developed a high-contrast multimodel nonlinear optical imaging technique to imaging collagen and elastin by detecting simultaneously two photon-excited fluorescence (TPEF) from elastin and second-harmonic generation (SHG) from collagen. Our results show that this technique can obtain a high-contrast TPEF/SHG image in human dermis and permit direct visualization of collagen and elastin. It was shown that the technique can provide collagen and elastin structural information to determine collagen and elastin fibril orientation and distribution and acquire some morphometric properties. It was found that the in-depth TPEF/SHG imaging and 3-D reconstruction of TPEF/SHG images can extract more collagen and elastin structural and biochemical information. The study results suggest that the high-contrast multimodel nonlinear imaging provides a powerful tool to study ECM intrinsic components and has the potential to provide more important information for the diagnosis of tissue

Transmural variation in elastin fiber orientation distribution in the arterial wall.

Science.gov (United States)

Yu, Xunjie; Wang, Yunjie; Zhang, Yanhang

2018-01-01

The complex three-dimensional elastin network is a major load-bearing extracellular matrix (ECM) component of an artery. Despite the reported anisotropic behavior of arterial elastin network, it is usually treated as an isotropic material in constitutive models. Our recent multiphoton microscopy study reported a relatively uniform elastin fiber orientation distribution in porcine thoracic aorta when imaging from the intima side (Chow et al., 2014). However it is questionable whether the fiber orientation distribution obtained from a small depth is representative of the elastin network structure in the arterial wall, especially when developing structure-based constitutive models. To date, the structural basis for the anisotropic mechanical behavior of elastin is still not fully understood. In this study, we examined the transmural variation in elastin fiber orientation distribution in porcine thoracic aorta and its association with elastin anisotropy. Using multi-photon microscopy, we observed that the elastin fibers orientation changes from a relatively uniform distribution in regions close to the luminal surface to a more circumferential distribution in regions that dominate the media, then to a longitudinal distribution in regions close to the outer media. Planar biaxial tensile test was performed to characterize the anisotropic behavior of elastin network. A new structure-based constitutive model of elastin network was developed to incorporate the transmural variation in fiber orientation distribution. The new model well captures the anisotropic mechanical behavior of elastin network under both equi- and nonequi-biaxial loading and showed improvements in both fitting and predicting capabilities when compared to a model that only considers the fiber orientation distribution from the intima side. We submit that the transmural variation in fiber orientation distribution is important in characterizing the anisotropic mechanical behavior of elastin network and

Acquired Localized Cutis Laxa due to Increased Elastin Turnover

DEFF Research Database (Denmark)

Nygaard, Rie Harboe; Maynard, Scott; Schjerling, Peter

2016-01-01

Cutis laxa is a rare disease characterized by abnormal skin wrinkling and laxity, due to decreased elastin synthesis or structural extracellular matrix defects. We have explored elastin metabolism in a case of adult onset cutis laxa localized to the upper body of a woman. For this purpose, we...... obtained skin biopsies from affected and unaffected skin areas of the patient and analyzed these with microscopy, polymerase chain reaction, western blotting and cell culture experiments. Skin from the affected area lacked elastin fibers in electron microscopy but had higher mRNA expression of elastin...... and total RNA. Levels of an apparent tropoelastin degradation product were higher in the affected area. Fibroblast cultures from the affected area were able to produce elastin and showed higher proliferation and survival after oxidative and UVB stress compared to fibroblasts from the unaffected area...

A general strategy to endow natural fusion-protein-derived peptides with potent antiviral activity.

Directory of Open Access Journals (Sweden)

Antonello Pessi

Full Text Available Fusion between the viral and target cell membranes is an obligatory step for the infectivity of all enveloped virus, and blocking this process is a clinically validated therapeutic strategy.Viral fusion is driven by specialized proteins which, although specific to each virus, act through a common mechanism, the formation of a complex between two heptad repeat (HR regions. The HR regions are initially separated in an intermediate termed "prehairpin", which bridges the viral and cell membranes, and then fold onto each other to form a 6-helical bundle (6HB, driving the two membranes to fuse. HR-derived peptides can inhibit viral infectivity by binding to the prehairpin intermediate and preventing its transition to the 6HB.The antiviral activity of HR-derived peptides differs considerably among enveloped viruses. For weak inhibitors, potency can be increased by peptide engineering strategies, but sequence-specific optimization is time-consuming. In seeking ways to increase potency without changing the native sequence, we previously reported that attachment to the HR peptide of a cholesterol group ("cholesterol-tagging" dramatically increases its antiviral potency, and simultaneously increases its half-life in vivo. We show here that antiviral potency may be increased by combining cholesterol-tagging with dimerization of the HR-derived sequence, using as examples human parainfluenza virus, Nipah virus, and HIV-1. Together, cholesterol-tagging and dimerization may represent strategies to boost HR peptide potency to levels that in some cases may be compatible with in vivo use, possibly contributing to emergency responses to outbreaks of existing or novel viruses.

Expression of the cationic antimicrobial peptide lactoferricin fused with the anionic peptide in Escherichia coli.

Science.gov (United States)

Kim, Ha-Kun; Chun, Dae-Sik; Kim, Joon-Sik; Yun, Cheol-Ho; Lee, Ju-Hoon; Hong, Soon-Kwang; Kang, Dae-Kyung

2006-09-01

Direct expression of lactoferricin, an antimicrobial peptide, is lethal to Escherichia coli. For the efficient production of lactoferricin in E. coli, we developed an expression system in which the gene for the lysine- and arginine-rich cationic lactoferricin was fused to an anionic peptide gene to neutralize the basic property of lactoferricin, and successfully overexpressed the concatemeric fusion gene in E. coli. The lactoferricin gene was linked to a modified magainin intervening sequence gene by a recombinational polymerase chain reaction, thus producing an acidic peptide-lactoferricin fusion gene. The monomeric acidic peptide-lactoferricin fusion gene was multimerized and expressed in E. coli BL21(DE3) upon induction with isopropyl-beta-D-thiogalactopyranoside. The expression levels of the fusion peptide reached the maximum at the tetramer, while further increases in the copy number of the fusion gene substantially reduced the peptide expression level. The fusion peptides were isolated and cleaved to generate the separate lactoferricin and acidic peptide. About 60 mg of pure recombinant lactoferricin was obtained from 1 L of E. coli culture. The purified recombinant lactoferricin was found to have a molecular weight similar to that of chemically synthesized lactoferricin. The recombinant lactoferricin showed antimicrobial activity and disrupted bacterial membrane permeability, as the native lactoferricin peptide does.

Effect of glucose on the biomechanical function of arterial elastin.

Science.gov (United States)

Wang, Yunjie; Zeinali-Davarani, Shahrokh; Davis, Elaine C; Zhang, Yanhang

2015-09-01

Elastin is essential to provide elastic support for blood vessels. As a remarkably long-lived protein, elastin can suffer from cumulative effects of exposure to biochemical damages, which can greatly compromise its biomechanical properties. Non-enzymatic glycation is one of the main mechanisms of aging and its effect is magnified in diabetic patients. The purpose of this study is to investigate the effects of glucose on mechanical properties of isolated porcine aortic elastin. Elastin samples were incubated in 2 M glucose solution and were allowed to equilibrate for 4, 7, 14, 21 or 28 days at 37 °C. Equibiaxial tensile tests were performed to study the changes of elastic properties of elastin due to glycation. Significant decreases in tissue dimension were observed after 7 days glucose incubation. Elastin samples treated for 14, 21 or 28 days demonstrate a significant increase in hysteresis in the stress-stretch curves, indicating a greater energy loss due to glucose treatment. Both the longitudinal and the circumferential directions show significant increases in tangent modulus with glucose treatment, however only significant increases are observed after 7 days for the circumferential direction. An eight-chain statistical mechanics based microstructural model was used to study the hyperelastic and orthotropic behavior of the glucose-treated elastin and the material parameters were estimated using a nonlinear least squares method. Material parameters in the model were related to elastin density and fiber orientation, and, hence, the possible microstructural changes in glucose-treated elastin. Estimated material parameters show a general increasing trend in elastin density per unit volume with glucose incubation. The simulation results also indicate that more elastic fibers are aligned in the longitudinal and circumferential directions, rather than in the radial direction. Copyright © 2015 Elsevier Ltd. All rights reserved.

Adherence of B16-F10 melanoma cells to elastin

International Nuclear Information System (INIS)

Zetter, B.R.; Netland, P.A.

1986-01-01

B16-F10 melanoma cells selectivity colonize lung tissue in vivo. The authors have previously shown that these cells adhere preferentially to lung tissue in vitro. To quantify the binding of B16-F10 cells to isolated components of lung tissue, the authors devised a dot-blot cell adhesion assay. Samples were absorbed to 4 mm dots of nylon based paper under non-denaturing conditions, blocked with albumin or hemoglobin, and incubated with radiolabelled cells for 30 min. at 4 0 C. 125 -I labelled B16-F10 cells demonstrated a dose dependent binding to mouse lung elastin. Autoradiography and scanning electron microscopy demonstrated that cells localized preferentially to the elastin dots. The melanoma cells bound more strongly to elastin relative to laminin, fibronectin, collagen types I and IV or heparan sulfate. Neither elastin-associated microfibrillar protein nor fragments of elastin produced by alkali or acid treatment demonstrated significant binding activity for these cells. The findings demonstrate that in addition to its unique mechanical properties that confer elasticity to tissues, elastin can also function as a cell adhesion molecule. The localization of elastin in the lung and its adhesive properties reported here suggest that elastin may facilitate the arrest and eventual colonization of circulating B16-F10 melanoma cells in the mouse lung

Intracellular protein delivery activity of peptides derived from insulin-like growth factor binding proteins 3 and 5

International Nuclear Information System (INIS)

Goda, Natsuko; Tenno, Takeshi; Inomata, Kosuke; Shirakawa, Masahiro; Tanaka, Toshiki; Hiroaki, Hidekazu

2008-01-01

Insulin-like growth factor binding proteins (IGFBPs) have various IGF-independent cellular activities, including receptor-independent cellular uptake followed by transcriptional regulation, although mechanisms of cellular entry remain unclear. Herein, we focused on their receptor-independent cellular entry mechanism in terms of protein transduction domain (PTD) activity, which is an emerging technique useful for clinical applications. The peptides of 18 amino acid residues derived from IGFBP-3 and IGFBP-5, which involve heparin-binding regions, mediated cellular delivery of an exogenous protein into NIH3T3 and HeLa cells. Relative protein delivery activities of IGFBP-3/5-derived peptides were approximately 20-150% compared to that of the HIV-Tat peptide, a potent PTD. Heparin inhibited the uptake of the fusion proteins with IGFBP-3 and IGFBP-5, indicating that the delivery pathway is heparin-dependent endocytosis, similar to that of HIV-Tat. The delivery of GST fused to HIV-Tat was competed by either IGFBP-3 or IGFBP-5-derived synthetic peptides. Therefore, the entry pathways of the three PTDs are shared. Our data has shown a new approach for designing protein delivery systems using IGFBP-3/5 derived peptides based on the molecular mechanisms of IGF-independent activities of IGFBPs

Solid-State Nuclear Magnetic Resonance Investigation of the Structural Topology and Lipid Interactions of a Viral Fusion Protein Chimera Containing the Fusion Peptide and Transmembrane Domain.

Science.gov (United States)

Yao, Hongwei; Lee, Myungwoon; Liao, Shu-Yu; Hong, Mei

2016-12-13

The fusion peptide (FP) and transmembrane domain (TMD) of viral fusion proteins play important roles during virus-cell membrane fusion, by inducing membrane curvature and transient dehydration. The structure of the water-soluble ectodomain of viral fusion proteins has been extensively studied crystallographically, but the structures of the FP and TMD bound to phospholipid membranes are not well understood. We recently investigated the conformations and lipid interactions of the separate FP and TMD peptides of parainfluenza virus 5 (PIV5) fusion protein F using solid-state nuclear magnetic resonance. These studies provide structural information about the two domains when they are spatially well separated in the fusion process. To investigate how these two domains are structured relative to each other in the postfusion state, when the ectodomain forms a six-helix bundle that is thought to force the FP and TMD together in the membrane, we have now expressed and purified a chimera of the FP and TMD, connected by a Gly-Lys linker, and measured the chemical shifts and interdomain contacts of the protein in several lipid membranes. The FP-TMD chimera exhibits α-helical chemical shifts in all the membranes examined and does not cause strong curvature of lamellar membranes or membranes with negative spontaneous curvature. These properties differ qualitatively from those of the separate peptides, indicating that the FP and TMD interact with each other in the lipid membrane. However, no 13 C- 13 C cross peaks are observed in two-dimensional correlation spectra, suggesting that the two helices are not tightly associated. These results suggest that the ectodomain six-helix bundle does not propagate into the membrane to the two hydrophobic termini. However, the loosely associated FP and TMD helices are found to generate significant negative Gaussian curvature to membranes that possess spontaneous positive curvature, consistent with the notion that the FP-TMD assembly may

Structural characterization of Mumps virus fusion protein core

International Nuclear Information System (INIS)

Liu Yueyong; Xu Yanhui; Lou Zhiyong; Zhu Jieqing; Hu Xuebo; Gao, George F.; Qiu Bingsheng; Rao Zihe; Tien, Po

2006-01-01

The fusion proteins of enveloped viruses mediating the fusion between the viral and cellular membranes comprise two discontinuous heptad repeat (HR) domains located at the ectodomain of the enveloped glycoproteins. The crystal structure of the fusion protein core of Mumps virus (MuV) was determined at 2.2 A resolution. The complex is a six-helix bundle in which three HR1 peptides form a central highly hydrophobic coiled-coil and three HR2 peptides pack against the hydrophobic grooves on the surface of central coiled-coil in an oblique antiparallel manner. Fusion core of MuV, like those of simian virus 5 and human respiratory syncytium virus, forms typical 3-4-4-4-3 spacing. The similar charecterization in HR1 regions, as well as the existence of O-X-O motif in extended regions of HR2 helix, suggests a basic rule for the formation of the fusion core of viral fusion proteins

Does human leukocyte elastase degrade intact skin elastin?

DEFF Research Database (Denmark)

Schmelzer, Christian E H; Jung, Michael C; Wohlrab, Johannes

2012-01-01

This study aimed to investigate the susceptibility of intact fibrillar human elastin to human leukocyte elastase and cathepsin G. Elastin is a vital protein of the extracellular matrix of vertebrates, and provides exceptional properties including elasticity and tensile strength to many tissues...... and organs, including the aorta, lung, cartilage, elastic ligaments and skin, and is thus critical for their long-term function. Mature elastin is an insoluble and extremely durable protein that undergoes very little turnover, but sustained exposure to proteases may lead to irreversible and severe damage......, and thus to functional loss of the elastic fiber network. Hence, it is a key issue to understand which enzymes actually initiate elastolysis under certain pathological conditions or during intrinsic aging. In this paper, we provide a complete workflow for isolation of pure and intact elastin from very...

Surface characterization of collagen/elastin based biomaterials for tissue regeneration

International Nuclear Information System (INIS)

Skopinska-Wisniewska, J.; Sionkowska, A.; Kaminska, A.; Kaznica, A.; Jachimiak, R.; Drewa, T.

2009-01-01

Collagen and elastin are the main proteins of extracellular matrix. Collagen plays a crucial role in tensile strength of tissues, whereas elastin provides resilience to many organs. Both biopolymers are readily available and biocompatible. These properties point out that collagen and elastin are good components of materials for many potential medical applications. The surface properties of biomaterials play an important role in biomedicine as the majority of biological reactions occur on the surface of implanted materials. One of the methods of surface modification is UV-irradiation. The exposition of the biomaterial on ultraviolet light can alterate surface properties of the materials, their chemical stability, swelling properties and mechanical properties as well. The aim of our work was to study the surface properties and biocompatibility of new collagen/elastin based biomaterials and consideration of the influence of ultraviolet light on these properties. The surface properties of collagen/elastin based biomaterials modified by UV-irradiation were studied using the technique of atomic force microscopy (AFM) and contact angle measurements. On the basis of the results the surface free energy and its polar component was calculated using Owens-Wendt method. To assess the biological performance of films based on collagen, elastin and their blends, the response of 3T3 cell was investigated. It was found that the surface of collagen/elastin film is enriched in less polar component - collagen. Exposition on UV light increases polarity of collagen/elastin based films, due to photooxidation process. The AFM images have shown that topography and roughness of the materials had been also affected by UV-irradiation. The changes in surface properties influence on interaction between the material's surface and cells. The investigation of 3T3 cells grown on films based on collagen, elastin and their blends, leads to the conclusion that higher content of elastin in biomaterial

Surface characterization of collagen/elastin based biomaterials for tissue regeneration

Energy Technology Data Exchange (ETDEWEB)

Skopinska-Wisniewska, J., E-mail: joanna@chem.uni.torun.pl [Faculty of Chemistry, Nicolaus Copernicus University, Gagarin 7, 87-100 Torun (Poland); Sionkowska, A.; Kaminska, A. [Faculty of Chemistry, Nicolaus Copernicus University, Gagarin 7, 87-100 Torun (Poland); Kaznica, A.; Jachimiak, R.; Drewa, T. [Collegium Medicum, Nicolaus Copernicus University, Karlowicz 24, 85-092 Bydgoszcz (Poland)

2009-07-15

Collagen and elastin are the main proteins of extracellular matrix. Collagen plays a crucial role in tensile strength of tissues, whereas elastin provides resilience to many organs. Both biopolymers are readily available and biocompatible. These properties point out that collagen and elastin are good components of materials for many potential medical applications. The surface properties of biomaterials play an important role in biomedicine as the majority of biological reactions occur on the surface of implanted materials. One of the methods of surface modification is UV-irradiation. The exposition of the biomaterial on ultraviolet light can alterate surface properties of the materials, their chemical stability, swelling properties and mechanical properties as well. The aim of our work was to study the surface properties and biocompatibility of new collagen/elastin based biomaterials and consideration of the influence of ultraviolet light on these properties. The surface properties of collagen/elastin based biomaterials modified by UV-irradiation were studied using the technique of atomic force microscopy (AFM) and contact angle measurements. On the basis of the results the surface free energy and its polar component was calculated using Owens-Wendt method. To assess the biological performance of films based on collagen, elastin and their blends, the response of 3T3 cell was investigated. It was found that the surface of collagen/elastin film is enriched in less polar component - collagen. Exposition on UV light increases polarity of collagen/elastin based films, due to photooxidation process. The AFM images have shown that topography and roughness of the materials had been also affected by UV-irradiation. The changes in surface properties influence on interaction between the material's surface and cells. The investigation of 3T3 cells grown on films based on collagen, elastin and their blends, leads to the conclusion that higher content of elastin in

Dual-affinity peptides to generate dense surface coverages of nanoparticles

International Nuclear Information System (INIS)

Del Re, Julia; Blum, Amy Szuchmacher

2014-01-01

Graphical abstract: - Highlights: • Stable nanoparticles were created with the Flg-A3 fusion peptide as a ligand. • Interactions of transition metal ions with Flg control aggregation of the nanoparticles in solution. • The QBP1-A3 fusion peptide improves surface attachment of gold nanoparticles. • Solution pre-aggregation of nanoparticles results in dense surface coverage. - Abstract: Depositing gold nanoparticles is of great interest because of the many potential applications of nanoparticle films; however, generating dense surface nanoparticle coverage remains a difficult challenge. Using dual-affinity peptides we have synthesized gold nanoparticles and then pre-aggregated the particles in solution via interactions with metal ions. These nanoparticle aggregates were then deposited onto silicon dioxide surfaces using another dual-affinity peptide to control binding to the substrate. The results demonstrate that when divalent ions like Zn 2+ or Ni 2+ are used, densely packed gold nanoparticle monolayers are formed on the silicon dioxide substrate, which may have applications in fields like molecular electronics

Elastin Fiber Accumulation in Liver Correlates with the Development of Hepatocellular Carcinoma.

Science.gov (United States)

Yasui, Yutaka; Abe, Tokiya; Kurosaki, Masayuki; Higuchi, Mayu; Komiyama, Yasuyuki; Yoshida, Tsubasa; Hayashi, Tsuguru; Kuwabara, Konomi; Takaura, Kenta; Nakakuki, Natsuko; Takada, Hitomi; Tamaki, Nobuharu; Suzuki, Shoko; Nakanishi, Hiroyuki; Tsuchiya, Kaoru; Itakura, Jun; Takahashi, Yuka; Hashiguchi, Akinori; Sakamoto, Michiie; Izumi, Namiki

2016-01-01

The fibrosis stage, which is evaluated by the distribution pattern of collagen fibers, is a major predictor for the development of hepatocellular carcinoma (HCC) for patients with hepatitis C. Meanwhile, the role of elastin fibers has not yet been elucidated. The present study was conducted to determine the significance of quantifying both collagen and elastin fibers. We enrolled 189 consecutive patients with hepatitis C and advanced fibrosis. Using Elastica van Gieson-stained whole-slide images of pretreatment liver biopsies, collagen and elastin fibers were evaluated pixel by pixel (0.46 μm/pixel) using an automated computational method. Consequently, fiber amount and cumulative incidences of HCC within 3 years were analyzed. There was a significant correlation between collagen and elastin fibers, whereas variation in elastin fiber was greater than in collagen fiber. Both collagen fiber (p = 0.008) and elastin fiber (p elastin fiber (p = 0.002) in addition to higher collagen fiber (p = 0.05) showed significantly higher incidences of HCC. With regard to elastin fiber, this difference remained significant in F3 patients. Furthermore, for patients with a higher collagen fiber amount, higher elastin was a significant predictor for HCC development (p = 0.02). Computational analysis is a novel technique for quantification of fibers with the added value of conventional staging. Elastin fiber is a predictor for the development of HCC independently of collagen fiber and F stage.

Engineering a pharmacologically superior form of granulocyte-colony-stimulating factor by fusion with gelatin-like-protein polymer.

Science.gov (United States)

Huang, Yan-Shan; Wen, Xiao-Fang; Wu, Yi-Liang; Wang, Ye-Fei; Fan, Min; Yang, Zhi-Yu; Liu, Wei; Zhou, Lin-Fu

2010-03-01

The plasma half-life of therapeutic proteins is a critical factor in many clinical applications. Therefore, new strategies to prolong plasma half-life of long-acting peptides and protein drugs are in high demand. Here, we designed an artificial gelatin-like protein (GLK) and fused this hydrophilic GLK polymer to granulocyte-colony-stimulating factor (G-CSF) to generate a chimeric GLK/G-CSF fusion protein. The genetically engineered recombinant GLK/G-CSF (rGLK/G-CSF) fusion protein was purified from Pichia pastoris. In vitro studies demonstrated that rGLK/G-CSF possessed an enlarged hydrodynamic radius, improved thermal stability and retained full bioactivity compared to unfused G-CSF. Following a single subcutaneous administration to rats, the rGLK/G-CSF fusion protein displayed a slower plasma clearance rate and stimulated greater and longer lasting increases in circulating white blood cells than G-CSF. Our findings indicate that fusion with this artificial, hydrophilic, GLK polymer provides many advantages in the construction of a potent hematopoietic factor with extended plasma half-life. This approach could be easily applied to other therapeutic proteins and have important clinical applications. (c) 2009 Elsevier B.V. All rights reserved.

Elastin in the human intervertebral disk. A histological and biochemical study comparing it with elastin in the human yellow ligament.

Science.gov (United States)

Mikawa, Y; Hamagami, H; Shikata, J; Yamamuro, T

1986-01-01

The elastic fiber and elastin in the human yellow ligament and intervertebral disk were studied histologically and biochemically. The elastic fiber in the human intervertebral disk, which until now had not been clearly identified microscopically, was observed clearly. We found the distribution of the elastic fiber in the intervertebral disk to be very sparse and irregular, and its diameter was small, being about one-tenth of that found in the yellow ligament. The elastin contents of the yellow ligament and intervertebral disk were 46.7% +/- 0.9% and 1.7% +/- 0.2% respectively (mean +/- SE) of the total dry weight. The amino acid composition of elastin in the yellow ligament is similar to that of other tissue, as reported in the literature; however, that found in the intervertebral disk is significantly different. It would appear, therefore, that the elastin in the intervertebral disk is of a different type from that found elsewhere.

Purification of Microbially Expressed Recombinant Proteins via a Dual ELP Split Intein System.

Science.gov (United States)

Shi, Changhua; Han, Tzu-Chiang; Wood, David W

2017-01-01

Fusions of elastin-like peptide (ELP) purification tags and self-cleaving inteins provide a powerful platform for purifying tagless recombinant proteins without the need for conventional packed-bed columns. A drawback to this method has been premature cleaving of the ELP tag during expression, before the purification procedure can take place. Here we demonstrate a split-intein method, where the self-cleaving intein is divided into two inactive segments during expression and purification. Spontaneous assembly of the purified intein segments then restores self-cleaving activity to deliver the tagless target protein.

Detection of closed influenza virus hemagglutinin fusion peptide structures in membranes by backbone {sup 13}CO-{sup 15}N rotational-echo double-resonance solid-state NMR

Energy Technology Data Exchange (ETDEWEB)

Ghosh, Ujjayini; Xie Li; Weliky, David P., E-mail: weliky@chemistry.msu.edu [Michigan State University, Department of Chemistry (United States)

2013-02-15

The influenza virus fusion peptide is the N-terminal {approx}20 residues of the HA2 subunit of the hemagglutinin protein and this peptide plays a key role in the fusion of the viral and endosomal membranes during initial infection of a cell. The fusion peptide adopts N-helix/turn/C-helix structure in both detergent and membranes with reports of both open and closed interhelical topologies. In the present study, backbone {sup 13}CO-{sup 15}N REDOR solid-state NMR was applied to the membrane-associated fusion peptide to detect the distribution of interhelical distances. The data clearly showed a large fraction of closed and semi-closed topologies and were best-fitted to a mixture of two structures that do not exchange. One of the earlier open structural models may have incorrect G13 dihedral angles derived from TALOS analysis of experimentally correct {sup 13}C shifts.

Structural Study of a New HIV-1 Entry Inhibitor and Interaction with the HIV-1 Fusion Peptide in Dodecylphosphocholine Micelles.

Science.gov (United States)

Pérez, Yolanda; Gómara, Maria José; Yuste, Eloísa; Gómez-Gutierrez, Patricia; Pérez, Juan Jesús; Haro, Isabel

2017-08-25

Previous studies support the hypothesis that the envelope GB virus C (GBV-C) E1 protein interferes the HIV-1 entry and that a peptide, derived from the region 139-156 of this protein, has been defined as a novel HIV-1 entry inhibitor. In this work, we firstly focus on the characterization of the structural features of this peptide, which are determinant for its anti-HIV-1 activity and secondly, on the study of its interaction with the proposed viral target (i.e., the HIV-1 fusion peptide). We report the structure of the peptide determined by NMR spectroscopy in dodecylphosphocholine (DPC) micelles solved by using restrained molecular dynamics calculations. The acquisition of different NMR experiments in DPC micelles (i.e., peptide-peptide titration, diffusion NMR spectroscopy, and addition of paramagnetic relaxation agents) allows a proposal of an inhibition mechanism. We conclude that a 18-mer peptide from the non-pathogenic E1 GBV-C protein, with a helix-turn-helix structure inhibits HIV-1 by binding to the HIV-1 fusion peptide at the membrane level, thereby interfering with those domains in the HIV-1, which are critical for stabilizing the six-helix bundle formation in a membranous environment. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Specific expression of GFPuv-β1,3-N-acetylglucosaminyltransferase 2 fusion protein in fat body of Bombyx mori silkworm larvae using signal peptide

International Nuclear Information System (INIS)

Kato, Tatsuya; Park, Enoch Y.

2007-01-01

Bombyxin (bx) and prophenoloxidase-activating enzyme (ppae) signal peptides from Bombyx mori, their modified signal peptides, and synthetic signal peptides were investigated for the secretion of GFP uv -β1,3-N-acetylglucosaminyltransferase 2 (GGT2) fusion protein in B. mori Bm5 cells and silkworm larvae using cysteine protease deficient B. mori multiple nucleopolyhedrovirus (BmMNPV-CP - ) and its bacmid. The secretion efficiencies of all signal peptides were 15-30% in Bm5 cells and 24-30% in silkworm larvae, while that of the +16 signal peptide was 0% in Bm5 cells and 1% in silkworm larvae. The fusion protein that contained the +16 signal peptide was expressed specifically in the endoplasmic reticulum (ER) and in the fractions of cell precipitations. Ninety-four percent of total intracellular β1,3-N-acetylglucosaminyltransferase (β3GnT) activity was detected in cell precipitations following the 600, 8000, and 114,000g centrifugations. In the case of the +38 signal peptide, 60% of total intracellular activity was detected in the supernatant from the 114,000g spin, and only 1% was found in the precipitate. Our results suggest that the +16 signal peptide might be situated in the transmembrane region and not cleaved by signal peptidase in silkworm or B. mori cells. Therefore, the fusion protein connected to the +16 signal peptide stayed in the fat body of silkworm larvae with biological function, and was not secreted extracellularly

Broadband diffuse optical characterization of elastin for biomedical applications.

Science.gov (United States)

Konugolu Venkata Sekar, Sanathana; Beh, Joo Sin; Farina, Andrea; Dalla Mora, Alberto; Pifferi, Antonio; Taroni, Paola

2017-10-01

Elastin is a key structural protein of dynamic connective tissues widely found in the extracellular matrix of skin, arteries, lungs and ligaments. It is responsible for a range of diseases related to aging of biological tissues. The optical characterization of elastin can open new opportunities for its investigation in biomedical studies. In this work, we present the absorption spectra of elastin using a broadband (550-1350nm) diffuse optical spectrometer. Distortions caused by fluorescence and finite bandwidth of the laser source on estimated absorption were effectively accounted for in measurements and data analysis and compensated. A comprehensive summary and comparison between collagen and elastin is presented, highlighting distinct features for its accurate quantification in biological applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Elastin density: Link between histological and biomechanical properties of vaginal tissue in women with pelvic organ prolapse?

Science.gov (United States)

de Landsheere, Laurent; Brieu, Mathias; Blacher, Silvia; Munaut, Carine; Nusgens, Betty; Rubod, Chrystèle; Noel, Agnès; Foidart, Jean-Michel; Nisolle, Michelle; Cosson, Michel

2016-04-01

The aim of the study was to correlate histological and biomechanical characteristics of the vaginal wall in women with pelvic organ prolapse (POP). Tissue samples were collected from the anterior [point Ba; POP Questionnaire (POP-Q)] and/or posterior (point Bp; POP-Q) vaginal wall of 15 women who underwent vaginal surgery for POP. Both histological and biomechanical assessments were performed from the same tissue samples in 14 of 15 patients. For histological assessment, the density of collagen and elastin fibers was determined by combining high-resolution virtual imaging and computer-assisted digital image analysis. For biomechanical testing, uniaxial tension tests were performed to evaluate vaginal tissue stiffness at low (C0) and high (C1) deformation rates. Biomechanical testing highlights the hyperelastic behavior of the vaginal wall. At low strains (C0), vaginal tissue appeared stiffer when elastin density was low. We found a statistically significant inverse relationship between C0 and the elastin/collagen ratio (p = 0.048) in the lamina propria. However, at large strain levels (C1), no clear relationship was observed between elastin density or elastin/collagen ratio and stiffness, likely reflecting the large dispersion of the mechanical behavior of the tissue samples. Histological and biomechanical properties of the vaginal wall vary from patient to patient. This study suggests that elastin density deserves consideration as a relevant factor of vaginal stiffness in women with POP.

Elastin distribution in the normal uterus, uterine leiomyomas, adenomyosis and adenomyomas: a comparison.

Science.gov (United States)

Zheng, Wei-Qiang; Ma, Rong; Zheng, Jian-Ming; Gong, Zhi-Jing

2006-04-01

To describe the histologic distribution of elastin in the nonpregnant human uterus, uterine leiomyomas, adenomyosis and adenomyomas. Uteri were obtained from women undergoing hysterectomy for benign conditions, including 26 cases of uterine leiomyomas, 24 cases of adenomyosis, 18 adenomyomas and 6 cases of autopsy specimens. Specific histochemical staining techniques were employed in order to demonstrate the distribution of elastin. The distribution of elastin components in the uterus was markedly uneven and showed a decreasing gradient from outer to inner myometrium. No elastin was present within leiomyomas, adenomyomas or adenomyosis. The distribution of elastin may help explain the normal function of the myometrium in labor. It implies that the uneven distribution of elastin components and absence of elastin within leiomyomas, adenomyomas and adenomyosis could be of some clinical significance. The altered elastin distribution in disease states may help explain such symptoms as dysmenorrhea in uterine endometriosis.

Liberation of Desmosine and Isodesmosine as Amino Acids from Insoluble Elastin by Elastolytic Proteases

Science.gov (United States)

Umeda, Hideyuki; Aikawa, Masanori; Libby, Peter

2011-01-01

The development of atherosclerotic lesions and abdominal aortic aneurysms involves degradation and loss of extracellular matrix components, such as collagen and elastin. Releases of the elastin cross-links desmosine (DES) and isodesmosine (IDE) may reflect elastin degradation in cardiovascular diseases. This study investigated the production of soluble elastin cross-linking structures by proteinases implicated in arterial diseases. Recombinant MMP-12 and neutrophil elastase liberated DES and IDE as amino acids from insoluble elastin. DES and IDE were also released from insoluble elastin exposed to monocyte/macrophage cell lines or human primary macrophages derived from peripheral blood monocytes. Elastin oxidized by reactive oxygen species (ROS) liberated more unconjugated DES and IDE than did non-oxidized elastin when incubated with MMP-12 or neutrophil elastase. These results support the exploration of free DES and IDE as biomarkers of elastin degradation. PMID:21726534

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.

Electrospun polydioxanone-elastin blends: potential for bioresorbable vascular grafts

Energy Technology Data Exchange (ETDEWEB)

Sell, S A [Virginia Commonwealth University, Richmond, VA 23298 (United States); McClure, M J [Virginia Commonwealth University, Richmond, VA 23298 (United States); Barnes, C P [Virginia Commonwealth University, Richmond, VA 23298 (United States); Knapp, D C [Virginia Commonwealth University, Richmond, VA 23298 (United States); Walpoth, B H [University Hospital, 1211 Geneva 14 (Switzerland); Simpson, D G [Virginia Commonwealth University, Richmond, VA 23298 (United States); Bowlin, G L [Virginia Commonwealth University, Richmond, VA 23298 (United States)

2006-06-15

An electrospun cardiovascular graft composed of polydioxanone (PDO) and elastin has been designed and fabricated with mechanical properties to more closely match those of native arterial tissue, while remaining conducive to tissue regeneration. PDO was chosen to provide mechanical integrity to the prosthetic, while elastin provides elasticity and bioactivity (to promote regeneration in vitro/in situ). It is the elastic nature of elastin that dominates the low-strain mechanical response of the vessel to blood flow and prevents pulsatile energy from being dissipated as heat. Uniaxial tensile and suture retention tests were performed on the electrospun grafts to demonstrate the similarities of the mechanical properties between the grafts and native vessel. Dynamic compliance measurements produced values that ranged from 1.2 to 5.6%/100 mmHg for a set of three different mean arterial pressures. Results showed the 50:50 ratio to closely mimic the compliance of native femoral artery, while grafts that contained less elastin exceeded the suture retention strength of native vessel. Preliminary cell culture studies showed the elastin-containing grafts to be bioactive as cells migrated through their full thickness within 7 days, but failed to migrate into pure PDO scaffolds. Electrospinning of the PDO and elastin-blended composite into a conduit for use as a small diameter vascular graft has extreme potential and warrants further investigation as it thus far compares favorably to native vessel.

Tyrosine kinase fusion genes in pediatric BCR-ABL1-like acute lymphoblastic leukemia

Science.gov (United States)

Boer, Judith M.; Steeghs, Elisabeth M.P.; Marchante, João R.M.; Boeree, Aurélie; Beaudoin, James J.; Berna Beverloo, H.; Kuiper, Roland P.; Escherich, Gabriele; van der Velden, Vincent H.J.; van der Schoot, C. Ellen; de Groot-Kruseman, Hester A.; Pieters, Rob; den Boer, Monique L.

2017-01-01

Approximately 15% of pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL) is characterized by gene expression similar to that of BCR-ABL1-positive disease and unfavorable prognosis. This BCR-ABL1-like subtype shows a high frequency of B-cell development gene aberrations and tyrosine kinase-activating lesions. To evaluate the clinical significance of tyrosine kinase gene fusions in children with BCP-ALL, we studied the frequency of recently identified tyrosine kinase fusions, associated genetic features, and prognosis in a representative Dutch/German cohort. We identified 14 tyrosine kinase fusions among 77 BCR-ABL1-like cases (18%) and none among 76 non-BCR-ABL1-like B-other cases. Novel exon fusions were identified for RCSD1-ABL2 and TERF2-JAK2. JAK2 mutation was mutually exclusive with tyrosine kinase fusions and only occurred in cases with high CRLF2 expression. The non/late response rate and levels of minimal residual disease in the fusion-positive BCR-ABL1-like group were higher than in the non-BCR-ABL1-like B-others (p<0.01), and also higher, albeit not statistically significant, compared with the fusion-negative BCR-ABL1-like group. The 8-year cumulative incidence of relapse in the fusion-positive BCR-ABL1-like group (35%) was comparable with that in the fusion-negative BCR-ABL1-like group (35%), and worse than in the non-BCR-ABL1-like B-other group (17%, p=0.07). IKZF1 deletions, predominantly other than the dominant-negative isoform and full deletion, co-occurred with tyrosine kinase fusions. This study shows that tyrosine kinase fusion-positive cases are a high-risk subtype of BCP-ALL, which warrants further studies with specific kinase inhibitors to improve outcome. PMID:27894077

Measuring the strength of interaction between the Ebola fusion peptide and lipid rafts: implications for membrane fusion and virus infection.

Directory of Open Access Journals (Sweden)

Mônica S Freitas

Full Text Available The Ebola fusion peptide (EBO₁₆ is a hydrophobic domain that belongs to the GP2 membrane fusion protein of the Ebola virus. It adopts a helical structure in the presence of mimetic membranes that is stabilized by the presence of an aromatic-aromatic interaction established by Trp8 and Phe12. In spite of its infectious cycle becoming better understood recently, several steps still remain unclear, a lacuna that makes it difficult to develop strategies to block infection. In order to gain insight into the mechanism of membrane fusion, we probed the structure, function and energetics of EBO₁₆ and its mutant W8A, in the absence or presence of different lipid membranes, including isolated domain-resistant membranes (DRM, a good experimental model for lipid rafts. The depletion of cholesterol from living mammalian cells reduced the ability of EBO₁₆ to induce lipid mixing. On the other hand, EBO₁₆ was structurally sensitive to interaction with lipid rafts (DRMs, but the same was not observed for W8A mutant. In agreement with these data, W8A showed a poor ability to promote membrane aggregation in comparison to EBO₁₆. Single molecule AFM experiments showed a high affinity force pattern for the interaction of EBO₁₆ and DRM, which seems to be a complex energetic event as observed by the calorimetric profile. Our study is the first to show a strong correlation between the initial step of Ebola virus infection and cholesterol, thus providing a rationale for Ebola virus proteins being co-localized with lipid-raft domains. In all, the results show how small fusion peptide sequences have evolved to adopt highly specific and strong interactions with membrane domains. Such features suggest these processes are excellent targets for therapeutic and vaccine approaches to viral diseases.

Measuring the strength of interaction between the Ebola fusion peptide and lipid rafts: implications for membrane fusion and virus infection.

Science.gov (United States)

Freitas, Mônica S; Follmer, Cristian; Costa, Lilian T; Vilani, Cecília; Bianconi, M Lucia; Achete, Carlos Alberto; Silva, Jerson L

2011-01-13

The Ebola fusion peptide (EBO₁₆) is a hydrophobic domain that belongs to the GP2 membrane fusion protein of the Ebola virus. It adopts a helical structure in the presence of mimetic membranes that is stabilized by the presence of an aromatic-aromatic interaction established by Trp8 and Phe12. In spite of its infectious cycle becoming better understood recently, several steps still remain unclear, a lacuna that makes it difficult to develop strategies to block infection. In order to gain insight into the mechanism of membrane fusion, we probed the structure, function and energetics of EBO₁₆ and its mutant W8A, in the absence or presence of different lipid membranes, including isolated domain-resistant membranes (DRM), a good experimental model for lipid rafts. The depletion of cholesterol from living mammalian cells reduced the ability of EBO₁₆ to induce lipid mixing. On the other hand, EBO₁₆ was structurally sensitive to interaction with lipid rafts (DRMs), but the same was not observed for W8A mutant. In agreement with these data, W8A showed a poor ability to promote membrane aggregation in comparison to EBO₁₆. Single molecule AFM experiments showed a high affinity force pattern for the interaction of EBO₁₆ and DRM, which seems to be a complex energetic event as observed by the calorimetric profile. Our study is the first to show a strong correlation between the initial step of Ebola virus infection and cholesterol, thus providing a rationale for Ebola virus proteins being co-localized with lipid-raft domains. In all, the results show how small fusion peptide sequences have evolved to adopt highly specific and strong interactions with membrane domains. Such features suggest these processes are excellent targets for therapeutic and vaccine approaches to viral diseases.

Design of an elastin-layered dermal regeneration template.

Science.gov (United States)

Mithieux, Suzanne M; Weiss, Anthony S

2017-04-01

We demonstrate a novel approach for the production of tunable quantities of elastic fibers. We also show that exogenous tropoelastin is rate-limiting for elastin synthesis regardless of the age of the dermal fibroblast donor. Additionally, we provide a strategy to further enhance synthesis by older cells through the application of conditioned media. We show that this approach delivers an elastin layer on one side of the leading dermal repair template for contact with the deep dermis in order to deliver prefabricated elastic fibers to a physiologically appropriate site during subsequent surgery. This system is attractive because it provides for the first time a viable path for sufficient, histologically detectable levels of patient elastin into full-thickness wound sites that have until now lacked this elastic underlayer. The scars of full thickness wounds typically lack elasticity. Elastin is essential for skin elasticity and is enriched in the deep dermis. This paper is significant because it shows that: (1) we can generate elastic fibers in tunable quantities, (2) tropoelastin is the rate-limiting component in elastin synthesis in vitro, (3) we can generate elastin fibers regardless of donor age, (4) we describe a novel approach to further increase the numbers and thickness of elastic fibers for older donors, (5) we improve on Integra Dermal Regeneration Template and generate a new hybrid biomaterial intended to subsequently surgically deliver these elastic fibers, (6) the elastic fiber layer is presented on the side of Integra that is intended for delivery into its physiologically appropriate site i.e. the deep dermis. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Investigation of water-soluble elastin as a multifunctional cosmetic material: Moisturizing and whitening effects.

Science.gov (United States)

Inoue, Asako; Hikima, Tomohiro; Taniguchi, Suguru; Nose, Takeru; Maeda, Iori

Elastin and collagen are extracellular matrix proteins that are widely distributed in the body. Although elastin essentially functions as a skin moisturizer, there have been few reports on its other fundamental chemical and biological functions. In this study, we investigated the moisturizing and whitening (tyrosinase inhibition) effects of elastin to examine its usefulness as a cosmetic material. Water-soluble hot alkali pig aorta (HAPA)-elastin was prepared from pig aorta using the hot alkali method. HAPA-elastin showed a widely distributed molecular weight and had a coacervation property that mediated reversible self-assembly of its molecules with increasing temperature. Amino acid analysis of HAPA-elastin showed a high content (81.5%) of hydrophobic amino acids such as Gly, Ala, Val, and Pro. Des (desmosine) and Ide (isodesmosine), which are characteristic amino acids of elastin, accounted for more than 0.4% of the total amino acid content. HAPA-elastin showed a moisture-retaining property. The water content of skin samples treated with and without HAPA-elastin was 77.2% ± 7.8% and 49.4% ± 10.1%, respectively. HAPA-elastin also inhibited tyrosinase activity by 11.3% ± 3.9%. The results obtained indicate that elastin has a useful function as a cosmetic material.

Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

International Nuclear Information System (INIS)

Fernández-Sainz, I.J.; Largo, E.; Gladue, D.P.; Fletcher, P.; O’Donnell, V.; Holinka, L.G.; Carey, L.B.; Lu, X.; Nieva, J.L.; Borca, M.V.

2014-01-01

E2, along with E rns and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, 818 CPIGWTGVIEC 828 , containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a β-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP 818 CPIGWTGVIEC 828 indicates a membrane fusion activity and a critical role in virus replication. - Highlights: • A putative fusion peptide (FP) region in CSFV E2 protein was shown to be critical for virus growth. • Synthetic FPs were shown to efficiently penetrate into lipid membranes using an in vitro model. • Individual residues in the FP affecting virus replication were identified by reverse genetics. • The same FP residues are also responsible for mediating membrane fusion

Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

Energy Technology Data Exchange (ETDEWEB)

Fernández-Sainz, I.J. [Plum Island Animal Disease Center, ARS, USDA (United States); Largo, E. [Biophysics Unit (CSIC-UPV/EHU), Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), P.O. Box 644, 48080 Bilbao (Spain); Gladue, D.P.; Fletcher, P. [Plum Island Animal Disease Center, ARS, USDA (United States); O’Donnell, V. [Plum Island Animal Disease Center, ARS, USDA (United States); Plum Island Animal Disease Center, DHS, Greenport, NY 11944 (United States); Holinka, L.G. [Plum Island Animal Disease Center, ARS, USDA (United States); Carey, L.B. [Department of Experimental and Health Sciences, Universitat Pompeu Fabra (UPF), E-08003 Barcelona (Spain); Lu, X. [Plum Island Animal Disease Center, DHS, Greenport, NY 11944 (United States); Nieva, J.L. [Biophysics Unit (CSIC-UPV/EHU), Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), P.O. Box 644, 48080 Bilbao (Spain); Borca, M.V., E-mail: manuel.borca@ars.usda.gov [Plum Island Animal Disease Center, ARS, USDA (United States)

2014-05-15

E2, along with E{sup rns} and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, {sup 818}CPIGWTGVIEC{sup 828}, containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a β-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP {sup 818}CPIGWTGVIEC{sup 828} indicates a membrane fusion activity and a critical role in virus replication. - Highlights: • A putative fusion peptide (FP) region in CSFV E2 protein was shown to be critical for virus growth. • Synthetic FPs were shown to efficiently penetrate into lipid membranes using an in vitro model. • Individual residues in the FP affecting virus replication were identified by reverse genetics. • The same FP residues are also responsible for mediating membrane fusion.

Structural and functional characterization of EIAV gp45 fusion peptide proximal region and asparagine-rich layer

Energy Technology Data Exchange (ETDEWEB)

Duan, Liangwei; Du, Jiansen [State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071 (China); Wang, Xuefeng; Zhou, Jianhua; Wang, Xiaojun [State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001 (China); Liu, Xinqi, E-mail: liu2008@nankai.edu.cn [State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071 (China)

2016-04-15

Equine infectious anaemia virus (EIAV) and human immunodeficiency virus (HIV) are members of the lentiviral genus. Similar to HIV gp41, EIAV gp45 is a fusogenic protein that mediates fusion between the viral particle and the host cell membrane. The crystal structure of gp45 reported reveals a different conformation in the here that includes the fusion peptide proximal region (FPPR) and neighboring asparagine-rich layer compared with previous HIV-1 gp41 structures. A complicated hydrogen-bond network containing a cluster of solvent molecules appears to be critical for the stability of the gp45 helical bundle. Interestingly, viral replication was relatively unaffected by site-directed mutagenesis of EIAV, in striking contrast to that of HIV-1. Based on these observations, we speculate that EIAV is more adaptable to emergent mutations, which might be important for the evolution of EIAV as a quasi-species, and could potentially contribute to the success of the EIAV vaccine. - Highlights: • The crystal structure of EIAV gp45 was determined. • The fusion peptide proximal region adopts a novel conformation different to HIV-1. • The asparagine-rich layer includes an extensive hydrogen-bond network. • These regions of EIAV are highly tolerant to mutations. • The results provide insight into the mechanism of gp41/gp45-mediated membrane fusion.

Serological assessment of neutrophil elastase activity on elastin during lung ECM remodeling

DEFF Research Database (Denmark)

Kristensen, Jacob Hull; Karsdal, Morten A.; Sand, Jannie M. B.

2015-01-01

Background: During the pathological destruction of lung tissue, neutrophil elastase (NE) degrades elastin, one of the major constituents of lung parenchyma. However there are no non-invasive methods to quantify NE degradation of elastin. We selected specific elastin fragments generated by NE for ...

Line-Tension Controlled Mechanism for Influenza Fusion

NARCIS (Netherlands)

Risselada, Herre Jelger; Marelli, Giovanni; Fuhrmans, Marc; Smirnova, Yuliya G.; Grubmueller, Helmut; Marrink, Siewert Jan; Mueller, Marcus

2012-01-01

Our molecular simulations reveal that wild-type influenza fusion peptides are able to stabilize a highly fusogenic pre-fusion structure, i.e. a peptide bundle formed by four or more trans-membrane arranged fusion peptides. We rationalize that the lipid rim around such bundle has a non-vanishing rim

Levels of circulating MMP-7 degraded elastin are elevated in pulmonary disorders.

Science.gov (United States)

Kristensen, J H; Larsen, L; Dasgupta, B; Brodmerkel, C; Curran, M; Karsdal, M A; Sand, J M B; Willumsen, N; Knox, A J; Bolton, C E; Johnson, S R; Hägglund, P; Svensson, B; Leeming, D J

2015-11-01

Elastin is a signature protein of the lungs. Matrix metalloproteinase-7 (MMP-7) is important in lung defence mechanisms and degrades elastin. However, MMP-7 activity in regard to elastin degradation has never been quantified serologically in patients with lung diseases. An assay for the quantification of MMP-7 generated elastin fragments (ELM7) was therefore developed to investigate MMP-7 derived elastin degradation in pulmonary disorders such as idiopathic pulmonary fibrosis (IPF) and lung cancer. Monoclonal antibodies (mABs) were raised against eight carefully selected MMP-7 cleavage sites on elastin. After characterisation and validation of the mABs, one mAB targeting the ELM7 fragment was chosen. ELM7 fragment levels were assessed in serum samples from patients diagnosed with IPF (n=123, baseline samples, CTgov reg. NCT00786201), and lung cancer (n=40) and compared with age- and sex-matched controls. The ELM7 assay was specific towards in vitro MMP-7 degraded elastin and the ELM7 neoepitope but not towards other MMP-7 derived elastin fragments. Serum ELM7 levels were significantly increased in IPF (113%, pelastin fragments can be quantified in serum and may reflect pathological lung tissue turnover in several important lung diseases. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Flunarizine Prevents Hepatitis C Virus Membrane Fusion in a Genotype-dependent Manner by Targeting the Potential Fusion Peptide within E1

Science.gov (United States)

Perin, Paula M.; Haid, Sibylle; Brown, Richard J. P.; Doerrbecker, Juliane; Schulze, Kai; Zeilinger, Carsten; von Schaewen, Markus; Heller, Brigitte; Vercauteren, Koen; Luxenburger, Eva; Baktash, Yasmine M.; Vondran, Florian W. R.; Speerstra, Sietkse; Awadh, Abdullah; Mukhtarov, Furkat; Schang, Luis M; Kirschning, Andreas; Müller, Rolf; Guzman, Carlos A.; Kaderali, Lars; Randall, Glenn; Meuleman, Philip; Ploss, Alexander; Pietschmann, Thomas

2015-01-01

To explore mechanisms of hepatitis C virus (HCV) replication we screened a compound library including licensed drugs. Flunarizine, a diphenylmethylpiperazine used to treat migraine, inhibited HCV cell entry in vitro and in vivo in a genotype-dependent fashion. Analysis of mosaic viruses between susceptible and resistant strains revealed that E1 and E2 glycoproteins confer susceptibility to flunarizine. Time of addition experiments and single particle tracking of HCV demonstrated that flunarizine specifically prevents membrane fusion. Related phenothiazines and pimozide also inhibited HCV infection and preferentially targeted HCV genotype 2 viruses. However, phenothiazines and pimozide exhibited improved genotype coverage including the difficult to treat genotype 3. Flunarizine-resistant HCV carried mutations within the alleged fusion peptide and displayed cross-resistance to these compounds, indicating that these drugs have a common mode of action. Conclusion: These observations reveal novel details about HCV membrane fusion. Moreover, flunarizine and related compounds represent first-in-class HCV fusion inhibitors that merit consideration for repurposing as cost-effective component of HCV combination therapies. PMID:26248546

Effects of brachytherapy on gene expressions of elastin and elastase

International Nuclear Information System (INIS)

Li Junming; Zhou Jingqun; Hu Bin; Li Shuguo

2004-01-01

Objective: To study the effects of brachytherapy on the gene expressions of elastin and elastase in cultured rat vascular smooth muscle cells (VSMCs). Methods: Rat VSMCs cultured in DMEM containing 10% FBS were irradiated by 60 Co γ-rays at 0, 7, 14, 28 Gy respectively. Then mRNA levels of elastin and elastase were determined by reverse transcription competitive PCR(RT-PCR). Results: Brachytherapy inhibited the expressions of elastase. Elastase mRNA decreased 25.3% and 50.1% in VSMC irradiated with 14, 28 Gy, respectively (P<0.05). The elastin mRNA level increased 80.7% and 102.3% in VSMC irradiated with 14, 25 Gy, respectively (P<0.05). Conclusion: Brachytherapy inhabits the expressions of elastase and increased elastin in VSMC cells

Evaluation of the amyloid beta-GFP fusion protein as a model of amyloid beta peptides-mediated aggregation: A study of DNAJB6 chaperone

Directory of Open Access Journals (Sweden)

Rasha Mohamed Hussein

2015-07-01

Full Text Available Alzheimer’s disease is a progressive neurodegenerative disease characterized by the accumulation and aggregation of extracellular amyloid β (Aβ peptides and intracellular aggregation of hyper-phosphorylated tau protein. Recent evidence indicates that accumulation and aggregation of intracellular amyloid β peptides may also play a role in disease pathogenesis. This would suggest that intracellular Heat Shock Proteins (HSP that maintain cellular protein homeostasis might be candidates for disease amelioration. We recently found that DNAJB6, a member of DNAJ family of heat shock proteins, effectively prevented the aggregation of short aggregation-prone peptides containing large poly glutamines (associated with CAG repeat diseases both in vitro and in cells. Moreover, recent in vitro data showed that DNAJB6 can delay the aggregation of Aβ42 peptides. In this study, we investigated the ability of DNAJB6 to prevent the aggregation of extracellular and intracellular Aβ peptides using transfection of HEK293 cells with Aβ-GFP fusion construct and performing western blotting and immunofluorescence techniques. We found that DNAJB6 indeed suppresses Aβ-GFP aggregation, but not seeded aggregation initiated by extracellular Aβ peptides. Unexpectedly and unlike what we found for peptide-mediated aggregation, DNAJB6 required interaction with HSP70 to prevent the aggregation of the Aβ-GFP fusion protein and its J-domain was crucial for its anti-aggregation effect. In addition, other DNAJ proteins as well as HSPA1a overexpression also suppressed Aβ-GFP aggregation efficiently. Our findings suggest that Aβ aggregation differs from poly Q peptide induced aggregation in terms of chaperone handling and sheds doubt on the usage of Aβ-GFP fusion construct for studying Aβ peptide aggregation in cells.

Tyrosinase-Mediated Construction of a Silk Fibroin/Elastin Nanofiber Bioscaffold.

Science.gov (United States)

Hong, Yanqing; Zhu, Xueke; Wang, Ping; Fu, Haitian; Deng, Chao; Cui, Li; Wang, Qiang; Fan, Xuerong

2016-04-01

Elastin has characteristics of elasticity, biological activity, and mechanical stability. In the present work, tyrosinase-mediated construction of a bioscaffold with silk fibroin and elastin was carried out, aiming at developing a novel medical biomaterial. The efficiency of enzymatic oxidation of silk fibroin and the covalent reaction between fibroin and elastin were examined by spectrophotometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and size exclusion chromatography (SEC). The properties of composite air-dried and nanofiber scaffolds were investigated. The results reveal that elastin was successfully bonded to silk fibroins, resulting in an increase in molecular weight of fibroin proteins. ATR-FTIR spectra indicated that tyrosinase treatment impacted the conformational structure of fibroin-based membrane. The thermal behaviors and mechanical properties of the tyrosinase-treated scaffolds were also improved compared with the untreated group. NIH/3T3 cells exhibited optimum densities when grown on the nanofiber scaffold, implying that the nanofiber scaffold has enhanced biocompatibility compared to the air-dried scaffold. A biological nanofiber scaffold constructed from tyrosinase-treated fibroin and elastin could potentially be utilized in biomedical applications.

Hormone-like peptides in the venoms of marine cone snails

Science.gov (United States)

Robinson, Samuel D.; Li, Qing; Bandyopadhyay, Pradip K.; Gajewiak, Joanna; Yandell, Mark; Papenfuss, Anthony T.; Purcell, Anthony W.; Norton, Raymond S.; Safavi-Hemami, Helena

2015-01-01

The venoms of cone snails (genus Conus) are remarkably complex, consisting of hundreds of typically short, disulfide-rich peptides termed conotoxins. These peptides have diverse pharmacological targets, with injection of venom eliciting a range of physiological responses, including sedation, paralysis and sensory overload. Most conotoxins target the prey’s nervous system but evidence of venom peptides targeting neuroendocrine processes is emerging. Examples include vasopressin, RFamide neuropeptides and recently also insulin. To investigate the diversity of hormone/neuropeptide-like molecules in the venoms of cone snails we systematically mined the venom gland transcriptomes of several cone snail species and examined secreted venom peptides in dissected and injected venom of the Australian cone snail Conus victoriae. Using this approach we identified several novel hormone/neuropeptide-like toxins, including peptides similar to the bee brain hormone prohormone-4, the mollusc ganglia neuropeptide elevenin, and thyrostimulin, a member of the glycoprotein hormone family, and confirmed the presence of insulin. We confirmed that at least two of these peptides are not only expressed in the venom gland but also form part of the injected venom cocktail, unambiguously demonstrating their role in envenomation. Our findings suggest that hormone/neuropeptide-like toxins are a diverse and integral part of the complex envenomation strategy of Conus. Exploration of this group of venom components offers an exciting new avenue for the discovery of novel pharmacological tools and drug candidates, complementary to conotoxins. PMID:26301480

Hormone-like peptides in the venoms of marine cone snails.

Science.gov (United States)

Robinson, Samuel D; Li, Qing; Bandyopadhyay, Pradip K; Gajewiak, Joanna; Yandell, Mark; Papenfuss, Anthony T; Purcell, Anthony W; Norton, Raymond S; Safavi-Hemami, Helena

2017-04-01

The venoms of cone snails (genus Conus) are remarkably complex, consisting of hundreds of typically short, disulfide-rich peptides termed conotoxins. These peptides have diverse pharmacological targets, with injection of venom eliciting a range of physiological responses, including sedation, paralysis and sensory overload. Most conotoxins target the prey's nervous system but evidence of venom peptides targeting neuroendocrine processes is emerging. Examples include vasopressin, RFamide neuropeptides and recently also insulin. To investigate the diversity of hormone/neuropeptide-like molecules in the venoms of cone snails we systematically mined the venom gland transcriptomes of several cone snail species and examined secreted venom peptides in dissected and injected venom of the Australian cone snail Conus victoriae. Using this approach we identified several novel hormone/neuropeptide-like toxins, including peptides similar to the bee brain hormone prohormone-4, the mollusc ganglia neuropeptide elevenin, and thyrostimulin, a member of the glycoprotein hormone family, and confirmed the presence of insulin. We confirmed that at least two of these peptides are not only expressed in the venom gland but also form part of the injected venom cocktail, unambiguously demonstrating their role in envenomation. Our findings suggest that hormone/neuropeptide-like toxins are a diverse and integral part of the complex envenomation strategy of Conus. Exploration of this group of venom components offers an exciting new avenue for the discovery of novel pharmacological tools and drug candidates, complementary to conotoxins. Copyright © 2015 Elsevier Inc. All rights reserved.

The FMRFamide-like peptide family in nematodes

Directory of Open Access Journals (Sweden)

Katleen ePeymen

2014-06-01

Full Text Available In the three decades since the FMRFamide peptide was isolated from the mollusk Macrocallista nimbosa, structurally similar peptides sharing a C-terminal RFamide motif have been identified across the animal kingdom. FMRFamide-like peptides (FLPs represent the largest known family of neuropeptides in invertebrates. In the phylum Nematoda, at least 32 flp genes are classified, making the FLP system of nematodes unusually complex. The diversity of the nematode FLP complement is most extensively mapped in Caenorhabditis elegans, where over 70 FLPs have been predicted. FLPs have shown to be expressed in the majority of the 302 C. elegans neurons including interneurons, sensory and motor neurons. The vast expression of FLPs is reflected in the broad functional repertoire of nematode FLP signaling, including neuroendocrine and neuromodulatory effects on locomotory activity, reproduction, feeding, and behavior. In contrast to the many identified nematode FLPs, only few peptides have been assigned a receptor and there is the need to clarify the pathway components and working mechanisms of the FLP signaling network. Here, we review the diversity, distribution, and functions of FLPs in nematodes.

Binding of cholesterol and inhibitory peptide derivatives with the fusogenic hydrophobic sequence of F-glycoprotein of HVJ (Sendai virus): possible implication in the fusion reaction

International Nuclear Information System (INIS)

Asano, K.; Asano, A.

1988-01-01

Specificity of the binding of sterols and related compounds with purified F-protein (fusion protein) of the HVJ (Sendai virus) was studied by binding competition with [ 3 H] cholesterol. Requirement for cholesterol or the A/B ring trans structure and nonrequirement for the 3-hydroxyl group were found in this binding. Binding of 125 I-labeled Z-Phe-Tyr, an inhibitory peptide of viral membrane-cell membrane fusion, was studied by using purified proteins and virions. F-Protein and virions showed a specific binding with the peptide, whereas the result was negative with hemagglutinin and neuraminidase protein. Thermolysin-truncated F-protein (an F-protein derivative deprived of a 2.5-kDa fragment from the N-terminal of the F 1 subunit and without fusogenic activity) exhibited a considerably diminished binding ability both to cholesterol and to inhibitory peptides. Therefore, the N-terminal hydrophobic sequence that was previously assigned as fusogenic seems to be the binding site of these molecules. In support of this, the binding of cholesterol with F-protein was inhibited by Z-Phe-Tyr and other fusion inhibitory peptides, whereas it was not affected with non-fusion-inhibitory Z-Gly-Phe. These results are discussed in relation to the notion that the binding of the N-terminal portion of the F 1 subunit of F-protein with cholesterol in the target cell membranes facilitiates the fusion reaction

Elastin is a key regulator of outward remodeling in arteriovenous fistulas.

Science.gov (United States)

Wong, C Y; Rothuizen, T C; de Vries, M R; Rabelink, T J; Hamming, J F; van Zonneveld, A J; Quax, P H A; Rotmans, J I

2015-04-01

Maturation failure is the major limitation of arteriovenous fistulas (AVFs) as hemodialysis access conduits. Indeed, 30-50% of AVFs fail to mature due to intimal hyperplasia and insufficient outward remodeling. Elastin has emerged as an important determinant of vascular remodeling. Here the role of elastin in AVF remodeling in elastin haplodeficient (eln(+/-)) mice undergoing AVF surgery has been studied. Unilateral AVFs between the branch of the jugular vein and carotid artery in an end to side manner were created in wild-type (WT) C57BL/6 (n = 11) and in eln(+/-) mice (n = 9). Animals were killed at day 21 and the AVFs were analyzed histologically and at an mRNA level using real-time quantitative polymerase chain reaction. Before AVF surgery, a marked reduction in elastin density in the internal elastic lamina (IEL) of eln(+/-) mice was observed. AVF surgery resulted in fragmentation of the venous internal elastic lamina in both groups while the expression of the tropoelastin mRNA was 53% lower in the eln(+/-) mice than in WT mice (p elastin has an important role in vascular remodeling following AVF creation, in which a lower amount of elastin results in enhanced outward remodeling. Interventions targeting elastin degradation might be a viable option in order to improve AVF maturation. Copyright © 2015 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.

Insights into the role of elastin in vocal fold health and disease

Science.gov (United States)

Moore, Jaime

2011-01-01

Elastic fibers are large, complex and surprisingly poorly understood extracellular matrix (ECM) macromolecules. The elastin fiber, generated from a single human gene - elastin (ELN), is a self assembling integral protein that endows critical mechanic proprieties to elastic tissues and organs such as the skin, lungs, and arteries. The biology of elastic fibers is complex because they have multiple components, a tightly regulated developmental deposition, a multi-step hierarchical assembly and unique biomechanical functions. Elastin is present in vocal folds, where it plays a pivotal role in the quality of phonation. This review article provides an overview of the genesis of elastin and its wide- ranging structure and function. Specific distribution within the vocal fold lamina propria across the lifespan in normal and pathological states and its contribution to vocal fold biomechanics will be examined. Elastin and elastin-derived molecules are increasingly investigated for their application in tissue engineering. The properties of various elastin– based materials will be discussed and their current and future applications evaluated. A new level of understanding of the biomechanical properties of vocal fold elastin composites and their molecular basis should lead to new strategies for elastic fiber repair and regeneration in aging and disease. PMID:21708449

Processing and characterization of α-elastin electrospun membranes

Science.gov (United States)

Araujo, J.; Padrão, J.; Silva, J. P.; Dourado, F.; Correia, D. M.; Botelho, G.; Gomez Ribelles, J. L.; Lanceros-Méndez, S.; Sencadas, V.

2014-06-01

Elastin isolated from fresh bovine ligaments was dissolved in a mixture of 1,1,1,3,3,3-Hexafluoro-2-propanol and water were electrospun into fiber membranes under different processing conditions. Fiber mats of randomly and aligned fibers were obtained with fixed and rotating ground collectors and fibrils were composed by thin ribbons whose width depends on electrospinning conditions; fibrils with 721 nm up to 2.12 μm width were achieved. After cross-linking with glutaraldehyde, α-elastin can uptake as much as 1700 % of PBS solution and a slight increase on fiber thickness was observed. The glass transition temperature of electrospun fiber mats was found to occur at ˜80 °C. Moreover, α-Elastin showed to be a perfect elastomeric material, and no mechanical hysteresis was found in cycle mechanical measurements. The elastic modulus obtained for random and aligned fibers mats in a PBS solution was 330±10 kPa and 732±165 kPa, respectively. Finally, the electrospinning and cross-linking process does not inhibit MC-3T3-E1 cell adhesion. Cell culture results showed good cell adhesion and proliferation in the cross-linked elastin fiber mats.

Photoreactive elastin-like proteins for use as versatile bioactive materials and surface coatings.

Science.gov (United States)

Raphel, Jordan; Parisi-Amon, Andreina; Heilshorn, Sarah

2012-10-07

Photocrosslinkable, protein-engineered biomaterials combine a rapid, controllable, cytocompatible crosslinking method with a modular design strategy to create a new family of bioactive materials. These materials have a wide range of biomedical applications, including the development of bioactive implant coatings, drug delivery vehicles, and tissue engineering scaffolds. We present the successful functionalization of a bioactive elastin-like protein with photoreactive diazirine moieties. Scalable synthesis is achieved using a standard recombinant protein expression host followed by site-specific modification of lysine residues with a heterobifunctional N-hydroxysuccinimide ester-diazirine crosslinker. The resulting biomaterial is demonstrated to be processable by spin coating, drop casting, soft lithographic patterning, and mold casting to fabricate a variety of two- and three-dimensional photocrosslinked biomaterials with length scales spanning the nanometer to millimeter range. Protein thin films proved to be highly stable over a three-week period. Cell-adhesive functional domains incorporated into the engineered protein materials were shown to remain active post-photo-processing. Human adipose-derived stem cells achieved faster rates of cell adhesion and larger spread areas on thin films of the engineered protein compared to control substrates. The ease and scalability of material production, processing versatility, and modular bioactive functionality make this recombinantly engineered protein an ideal candidate for the development of novel biomaterial coatings, films, and scaffolds.

Glucagon-like peptide 1--a cardiologic dimension

DEFF Research Database (Denmark)

Treiman, Marek; Elvekjaer, Mikkel; Engstrøm, Thomas

2010-01-01

Recent experimental data suggest glucagon-like peptide 1 (GLP-1) and its analogs to have direct effects on the cardiovascular system, in addition to their classic glucoregulatory actions. These direct effects may be cardioprotective, contractility augmenting, and vasorelaxant. A few preliminary c...

Monoclonal anti-elastin antibody labelled with technetium-99m

International Nuclear Information System (INIS)

Oliveira, Marcia B.N. de; Silva, Claudia R. da; Araujo, Adriano C. de; Bernardo Filho, Mario; Porto, Luis Cristovao M.S.; Gutfilen, Bianca; Souza, J.E.Q.; Frier, Malcolm

1999-01-01

Technetium-99m ( 99m Tc) is widely employed in nuclear medicine due to its desirable physical, chemical and biological properties. Moreover, it is easily available and normally is inexpensive. A reducing agent is necessary to label cells and molecules with 99m Tc and stannous chloride (Sn C L 2 ) is usually employed. Elastin is the functional protein component of the elastic fiber and it is related with some diseases such as arteriosclerosis, pulmonary emphysema and others. The present study refers to the preparation of the 99m Tc labeled monoclonal anti-elastin antibody. The monoclonal antibody was incubated with an excess of 2-iminothiolane. The free thiol groups created, were capable of binding with the reduced technetium. Labeling was an exchange reaction with 99m Tc-glucoheptonate. The labeled preparation was left at 4 deg C for one hour. Then, it was passed through a Sephadex G50 column. Various fractions were collected and counted. A peak corresponding to the radiolabeled antibody was obtained. Stability studies of the labelled anti-elastin were performed at 0,3 6, 24 hours, at both 4 deg C or room temperature. The biodistribution pattern of the 99m Tc-anti-elastin was studied in healthy male Swiss mice. The immunoreactivity was also determined. An useful labeled-anti-elastin was obtained to future immunoscintigraphic investigations. (author)

A Fusion-Inhibiting Peptide against Rift Valley Fever Virus Inhibits Multiple, Diverse Viruses

Science.gov (United States)

2013-09-12

performance liquid chroma- tography to greater than 95% purity (Bio-synthesis, Inc., Lewisville, TX). Lyophilized peptides were initially resuspended in...lesser extent RVFV-6sc, were found to precipitate Gc (Figure 5); however, in the presence of the non- ionic detergent Triton-X, which will solubilize...Viral membrane fusion. Nat Struct Mol Biol 15: 690–698. 12. Allison SL, Schalich J, Stiasny K, Mandl CW, Kunz C, et al. (1995) Oligomeric rearrangement

Membrane fusion is induced by a distinct peptide sequence of the sea urchin fertilization protein bindin

NARCIS (Netherlands)

Ulrich, AS; Glabe, CG; Hoekstra, D

1998-01-01

Fertilization in the sea urchin is mediated by the membrane-associated acrosomal protein bindin, which plays a key role in the adhesion and fusion between sperm and egg. We have investigated the structure/function relationship of an 18-amino acid peptide fragment "B18," which represents the minimal

Novel β-lactamase-random peptide fusion libraries for phage display selection of cancer cell-targeting agents suitable for enzyme prodrug therapy

Science.gov (United States)

Shukla, Girja S.; Krag, David N.

2010-01-01

Novel phage-displayed random linear dodecapeptide (X12) and cysteine-constrained decapeptide (CX10C) libraries constructed in fusion to the amino-terminus of P99 β-lactamase molecules were used for identifying β-lactamase-linked cancer cell-specific ligands. The size and quality of both libraries were comparable to the standards of other reported phage display systems. Using the single-round panning method based on phage DNA recovery, we identified severalβ-lactamase fusion peptides that specifically bind to live human breast cancer MDA-MB-361 cells. The β-lactamase fusion to the peptides helped in conducting the enzyme activity-based clone normalization and cell-binding screening in a very time- and cost-efficient manner. The methods were suitable for 96-well readout as well as microscopic imaging. The success of the biopanning was indicated by the presence of ~40% cancer cell-specific clones among recovered phages. One of the binding clones appeared multiple times. The cancer cell-binding fusion peptides also shared several significant motifs. This opens a new way of preparing and selecting phage display libraries. The cancer cell-specific β-lactamase-linked affinity reagents selected from these libraries can be used for any application that requires a reporter for tracking the ligand molecules. Furthermore, these affinity reagents have also a potential for their direct use in the targeted enzyme prodrug therapy of cancer. PMID:19751096

Transplantation of bone marrow-derived mesenchymal stem cells expressing elastin alleviates pelvic floor dysfunction.

Science.gov (United States)

Jin, Minfei; Chen, Ying; Zhou, Yun; Mei, Yan; Liu, Wei; Pan, Chenhao; Hua, Xiaolin

2016-04-05

Pelvic floor dysfunction (PFD) is a group of clinical conditions including stress urinary incontinence (SUI) and pelvic organ prolapse (POP). The abnormality of collagen and elastin metabolism in pelvic connective tissues is implicated in SUI and POP. To reconstitute the connective tissues with normal distribution of collagen and elastin, we transduced elastin to bone marrow-derived mesenchymal stem cells (BMSC). Elastin-expressing BMSCs were then differentiated to fibroblasts using bFGF, which produced collagen and elastin. To achieve the sustained release of bFGF, we formulated bFGF in poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NP). In an in vitro cell culture system of 7 days, when no additional bFGF was administrated, the initial PLGA-loaded bFGF NP induced prolonged production of collagen and elastin from elastin-expressing BMSCs. In vivo, co-injection of PLGA-loaded bFGF NP and elastin-expressing BMSCs into the PFD rats significantly improved the outcome of urodynamic tests. Together, these results provided an efficient model of connective tissue engineering using BMSC and injectable PLGA-loaded growth factors. Our results provided the first instance of a multidisciplinary approach, combining both stem cell and nanoparticle technologies, for the treatment of PFD.

Chromosomal mapping of quantitative trait loci controlling elastin content in rat aorta.

Science.gov (United States)

Gauguier, Dominique; Behmoaras, Jacques; Argoud, Karène; Wilder, Steven P; Pradines, Christelle; Bihoreau, Marie Thérèse; Osborne-Pellegrin, Mary; Jacob, Marie Paule

2005-03-01

Extracellular matrix molecules such as elastin and collagens provide mechanical support to the vessel wall. In addition to its structural role, elastin is a regulator that maintains homeostasis through biologic signaling. Genetically determined minor modifications in elastin and collagen in the aorta could influence the onset and evolution of arterial pathology, such as hypertension and its complications. We previously demonstrated that the inbred Brown Norway (BN) rat shows an aortic elastin deficit in both abdominal and thoracic segments, partly because of a decrease in tropoelastin synthesis when compared with the LOU rat, that elastin gene polymorphisms in these strains do not significantly account for. After a genome-wide search for quantitative trait loci (QTL) influencing the aortic elastin, collagen, and cell protein contents in an F2 population derived from BN and LOU rats, we identified on chromosomes 2 and 14, 3 QTL specifically controlling elastin levels, and a further highly significant QTL on chromosome 17 linked to the level of cell proteins. We also mapped 3 highly significant QTL linked to body weight (on chromosomes 1 and 3) and heart weight (on chromosome 1) in the cross. This study demonstrates the polygenic control of the content of key components of the arterial wall. Such information represents a first step in understanding possible mechanisms involved in dysregulation of these parameters in arterial pathology.

Multiscale mechanical integrity of human supraspinatus tendon in shear after elastin depletion.

Science.gov (United States)

Fang, Fei; Lake, Spencer P

2016-10-01

Human supraspinatus tendon (SST) exhibits region-specific nonlinear mechanical properties under tension, which have been attributed to its complex multiaxial physiological loading environment. However, the mechanical response and underlying multiscale mechanism regulating SST behavior under other loading scenarios are poorly understood. Furthermore, little is known about the contribution of elastin to tendon mechanics. We hypothesized that (1) SST exhibits region-specific shear mechanical properties, (2) fiber sliding is the predominant mode of local matrix deformation in SST in shear, and (3) elastin helps maintain SST mechanical integrity by facilitating force transfer among collagen fibers. Through the use of biomechanical testing and multiphoton microscopy, we measured the multiscale mechanical behavior of human SST in shear before and after elastase treatment. Three distinct SST regions showed similar stresses and microscale deformation. Collagen fiber reorganization and sliding were physical mechanisms observed as the SST response to shear loading. Measures of microscale deformation were highly variable, likely due to a high degree of extracellular matrix heterogeneity. After elastase treatment, tendon exhibited significantly decreased stresses under shear loading, particularly at low strains. These results show that elastin contributes to tendon mechanics in shear, further complementing our understanding of multiscale tendon structure-function relationships. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

Genetically Controlled Fusion, Exocytosis and Fission of Artificial Vesicles

DEFF Research Database (Denmark)

Bönzli, Eva; Hadorn, Maik; De Lucrezia, Davide

if a special class of viral proteins, termed fusogenic peptides, were added to the external medium. In the present work, we intend to develop genetically controlled fusion, fission and exocytosis of vesicles by the synthesis of peptides within vesicles. First, we enclosed synthesized peptides in vesicles...... to induce in a next step fusion of adjacent vesicles, fission and exocytosis of nested vesicles. Second, we will replace the peptides by an enclosed cell-free expression system to internally synthesize fusion peptides. To control the gene expression, different mechanisms are available, e.g. addition...... fusion, fission and exocytosis....

Designing of peptides with desired half-life in intestine-like environment

KAUST Repository

Sharma, Arun; Singla, Deepak; Rashid, Mamoon; Raghava, Gajendra Pal Singh

2014-01-01

hindered mainly because of their high susceptibility towards proteases degradation. We have developed an in silico method to predict the half-life of peptides in intestine-like environment and to design better peptides having optimized physicochemical

Molecular modeling of protein materials: case study of elastin

International Nuclear Information System (INIS)

Tarakanova, Anna; Buehler, Markus J

2013-01-01

Molecular modeling of protein materials is a quickly growing area of research that has produced numerous contributions in fields ranging from structural engineering to medicine and biology. We review here the history and methods commonly employed in molecular modeling of protein materials, emphasizing the advantages for using modeling as a complement to experimental work. We then consider a case study of the protein elastin, a critically important ‘mechanical protein’ to exemplify the approach in an area where molecular modeling has made a significant impact. We outline the progression of computational modeling studies that have considerably enhanced our understanding of this important protein which endows elasticity and recoil to the tissues it is found in, including the skin, lungs, arteries and the heart. A vast collection of literature has been directed at studying the structure and function of this protein for over half a century, the first molecular dynamics study of elastin being reported in the 1980s. We review the pivotal computational works that have considerably enhanced our fundamental understanding of elastin's atomistic structure and its extraordinary qualities—focusing on two in particular: elastin's superb elasticity and the inverse temperature transition—the remarkable ability of elastin to take on a more structured conformation at higher temperatures, suggesting its effectiveness as a biomolecular switch. Our hope is to showcase these methods as both complementary and enriching to experimental approaches that have thus far dominated the study of most protein-based materials. (topical review)

A biomaterial composed of collagen and solubilized elastin enhances angiogenesis and elastic fiber formation without calcification.

NARCIS (Netherlands)

Daamen, W.F.; Nillesen, S.T.M.; Wismans, P.G.P.; Reinhardt, D.; Hafmans, T.G.M.; Veerkamp, J.H.; Kuppevelt, A.H.M.S.M. van

2008-01-01

Elastin is the prime protein in elastic tissues that contributes to elasticity of, for example, lung, aorta, and skin. Upon injury, elastic fibers are not readily replaced, which hampers tissue regeneration. Incorporation of solubilized elastin (hydrolyzed insoluble elastin fibers or elastin

Intracellular delivery of cell-penetrating peptide-transcriptional factor fusion protein and its role in selective osteogenesis

Science.gov (United States)

Suh, Jin Sook; Lee, Jue Yeon; Choi, Yoon Jung; You, Hyung Keun; Hong, Seong-Doo; Chung, Chong Pyoung; Park, Yoon Jeong

2014-01-01

Protein-transduction technology has been attempted to deliver macromolecular materials, including protein, nucleic acids, and polymeric drugs, for either diagnosis or therapeutic purposes. Herein, fusion protein composed of an arginine-rich cell-penetrating peptide, termed low-molecular-weight protamine (LMWP), and a transcriptional coactivator with a PDZ-binding motif (TAZ) protein was prepared and applied in combination with biomaterials to increase bone-forming capacity. TAZ has been recently identified as a specific osteogenic stimulating transcriptional coactivator in human mesenchymal stem cell (hMSC) differentiation, while simultaneously blocking adipogenic differentiation. However, TAZ by itself cannot penetrate the cells, and thus needs a transfection tool for translocalization. The LMWP-TAZ fusion proteins were efficiently translocalized into the cytosol of hMSCs. The hMSCs treated with cell-penetrating LMWP-TAZ exhibited increased expression of osteoblastic genes and protein, producing significantly higher quantities of mineralized matrix compared to free TAZ. In contrast, adipogenic differentiation of the hMSCs was blocked by treatment of LMWP-TAZ fusion protein, as reflected by reduced marker-protein expression, adipocyte fatty acid-binding protein 2, and peroxisome proliferator-activated receptor-γ messenger ribonucleic acid levels. LMWP-TAZ was applied in alginate gel for the purpose of localization and controlled release. The LMWP-TAZ fusion protein-loaded alginate gel matrix significantly increased bone formation in rabbit calvarial defects compared with alginate gel matrix mixed with free TAZ protein. The protein transduction of TAZ fused with cell-penetrating LMWP peptide was able selectively to stimulate osteogenesis in vitro and in vivo. Taken together, this fusion protein-transduction technology for osteogenic protein can thus be applied in combination with biomaterials for tissue regeneration and controlled release for tissue

Elastin overexpression by cell-based gene therapy preserves matrix and prevents cardiac dilation

Science.gov (United States)

Li, Shu-Hong; Sun, Zhuo; Guo, Lily; Han, Mihan; Wood, Michael F G; Ghosh, Nirmalya; Alex Vitkin, I; Weisel, Richard D; Li, Ren-Ke

2012-01-01

After a myocardial infarction, thinning and expansion of the fibrotic scar contribute to progressive heart failure. The loss of elastin is a major contributor to adverse extracellular matrix remodelling of the infarcted heart, and restoration of the elastic properties of the infarct region can prevent ventricular dysfunction. We implanted cells genetically modified to overexpress elastin to re-establish the elastic properties of the infarcted myocardium and prevent cardiac failure. A full-length human elastin cDNA was cloned, subcloned into an adenoviral vector and then transduced into rat bone marrow stromal cells (BMSCs). In vitro studies showed that BMSCs expressed the elastin protein, which was deposited into the extracellular matrix. Transduced BMSCs were injected into the infarcted myocardium of adult rats. Control groups received either BMSCs transduced with the green fluorescent protein gene or medium alone. Elastin deposition in the infarcted myocardium was associated with preservation of myocardial tissue structural integrity (by birefringence of polarized light; P elastin showed the greatest functional improvement (P elastin in the infarcted heart preserved the elastic structure of the extracellular matrix, which, in turn, preserved diastolic function, prevented ventricular dilation and preserved cardiac function. This cell-based gene therapy provides a new approach to cardiac regeneration. PMID:22435995

Regeneration of hyaline cartilage promoted by xenogeneic mesenchymal stromal cells embedded within elastin-like recombinamer-based bioactive hydrogels.

Science.gov (United States)

Pescador, David; Ibáñez-Fonseca, Arturo; Sánchez-Guijo, Fermín; Briñón, Jesús G; Arias, Francisco Javier; Muntión, Sandra; Hernández, Cristina; Girotti, Alessandra; Alonso, Matilde; Del Cañizo, María Consuelo; Rodríguez-Cabello, José Carlos; Blanco, Juan Francisco

2017-08-01

Over the last decades, novel therapeutic tools for osteochondral regeneration have arisen from the combination of mesenchymal stromal cells (MSCs) and highly specialized smart biomaterials, such as hydrogel-forming elastin-like recombinamers (ELRs), which could serve as cell-carriers. Herein, we evaluate the delivery of xenogeneic human MSCs (hMSCs) within an injectable ELR-based hydrogel carrier for osteochondral regeneration in rabbits. First, a critical-size osteochondral defect was created in the femora of the animals and subsequently filled with the ELR-based hydrogel alone or with embedded hMSCs. Regeneration outcomes were evaluated after three months by gross assessment, magnetic resonance imaging and computed tomography, showing complete filling of the defect and the de novo formation of hyaline-like cartilage and subchondral bone in the hMSC-treated knees. Furthermore, histological sectioning and staining of every sample confirmed regeneration of the full cartilage thickness and early subchondral bone repair, which was more similar to the native cartilage in the case of the cell-loaded ELR-based hydrogel. Overall histological differences between the two groups were assessed semi-quantitatively using the Wakitani scale and found to be statistically significant (p hyaline cartilage in osteochondral lesions.

Elastin in large artery stiffness and hypertension

Science.gov (United States)

Wagenseil, Jessica E.; Mecham, Robert P.

2012-01-01

Large artery stiffness, as measured by pulse wave velocity (PWV), is correlated with high blood pressure and may be a causative factor in essential hypertension. The extracellular matrix components, specifically the mix of elastin and collagen in the vessel wall, determine the passive mechanical properties of the large arteries. Elastin is organized into elastic fibers in the wall during arterial development in a complex process that requires spatial and temporal coordination of numerous proteins. The elastic fibers last the lifetime of the organism, but are subject to proteolytic degradation and chemical alterations that change their mechanical properties. This review discusses how alterations in the amount, assembly, organization or chemical properties of the elastic fibers affect arterial stiffness and blood pressure. Strategies for encouraging or reversing alterations to the elastic fibers are addressed. Methods for determining the efficacy of these strategies, by measuring elastin amounts and arterial stiffness, are summarized. Therapies that have a direct effect on arterial stiffness through alterations to the elastic fibers in the wall may be an effective treatment for essential hypertension. PMID:22290157

Spike Protein Fusion Peptide and Feline Coronavirus Virulence

Science.gov (United States)

Chang, Hui-Wen; Egberink, Herman F.; Halpin, Rebecca; Spiro, David J.

2012-01-01

Coronaviruses are well known for their potential to change their host or tissue tropism, resulting in unpredictable new diseases and changes in pathogenicity; severe acute respiratory syndrome and feline coronaviruses, respectively, are the most recognized examples. Feline coronaviruses occur as 2 pathotypes: nonvirulent feline enteric coronaviruses (FECVs), which replicate in intestinal epithelium cells, and lethal feline infectious peritonitis viruses (FIPVs), which replicate in macrophages. Evidence indicates that FIPV originates from FECV by mutation, but consistent distinguishing differences have not been established. We sequenced the full genome of 11 viruses of each pathotype and then focused on the single most distinctive site by additionally sequencing hundreds of viruses in that region. As a result, we identified 2 alternative amino acid differences in the putative fusion peptide of the spike protein that together distinguish FIPV from FECV in >95% of cases. By these and perhaps other mutations, the virus apparently acquires its macrophage tropism and spreads systemically. PMID:22709821

Effect of Elastin Digestion on the Quasi-static Tensile Response of Medial Collateral Ligament

Science.gov (United States)

Henninger, Heath B.; Underwood, Clayton J.; Romney, Steven J.; Davis, Grant L.; Weiss, Jeffrey A.

2014-01-01

Elastin is a structural protein that provides resilience to biological tissues. We examined the contributions of elastin to the quasi-static tensile response of porcine medial collateral ligament through targeted disruption of the elastin network with pancreatic elastase. Elastase concentration and treatment time were varied to determine a dose response. Whereas elastin content decreased with increasing elastase concentration and treatment time, the change in peak stress after cyclic loading reached a plateau above 1 U/ml elastase and 6 hr treatment. For specimens treated with 2 U/ml elastase for 6 hr, elastin content decreased approximately 35%. Mean peak tissue strain after cyclic loading (4.8%, p≥0.300), modulus (275 MPa, p≥0.114) and hysteresis (20%, p≥0.553) were unaffected by elastase digestion, but stress decreased significantly after treatment (up to 2 MPa, p≤0.049). Elastin degradation had no effect on failure properties, but tissue lengthened under the same pre-stress. Stiffness in the linear region was unaffected by elastase digestion, suggesting that enzyme treatment did not disrupt collagen. These results demonstrate that elastin primarily functions in the toe region of the stress-strain curve, yet contributes load support in the linear region. The increase in length after elastase digestion suggests that elastin may pre-stress and stabilize collagen crimp in ligaments. PMID:23553827

Evaluation of cell binding peptide (p15) with silk fibre enhanced hydroxyappatite bone substitute for posterolateral spinal fusion in sheep

DEFF Research Database (Denmark)

Axelsen, M.; Jespersen, Stig; Overgaard, Søren

2015-01-01

Background: Spinal fusion is indicated in the surgical management of various spinal disorders. To ensure stabile fusion, bone graft materials are essential. Traditionally allo- or autograft has been used, but both are associated with limitations. Synthetic bone graft materials that reassemble today......: In this study, we compared fusion rates between silk fibre enhanced anorganic bovine derived hydroxyapatite matrix (ABM) with and without P15 peptide coating in uninstrumented PLF in a preclinical setting. Study design: Randomised prospective study in sheep. Method/materials: Twelve Tex/got sheep underwent open...

Improving the representation of peptide-like inhibitor and antibiotic molecules in the Protein Data Bank.

Science.gov (United States)

Dutta, Shuchismita; Dimitropoulos, Dimitris; Feng, Zukang; Persikova, Irina; Sen, Sanchayita; Shao, Chenghua; Westbrook, John; Young, Jasmine; Zhuravleva, Marina A; Kleywegt, Gerard J; Berman, Helen M

2014-06-01

With the accumulation of a large number and variety of molecules in the Protein Data Bank (PDB) comes the need on occasion to review and improve their representation. The Worldwide PDB (wwPDB) partners have periodically updated various aspects of structural data representation to improve the integrity and consistency of the archive. The remediation effort described here was focused on improving the representation of peptide-like inhibitor and antibiotic molecules so that they can be easily identified and analyzed. Peptide-like inhibitors or antibiotics were identified in over 1000 PDB entries, systematically reviewed and represented either as peptides with polymer sequence or as single components. For the majority of the single-component molecules, their peptide-like composition was captured in a new representation, called the subcomponent sequence. A novel concept called "group" was developed for representing complex peptide-like antibiotics and inhibitors that are composed of multiple polymer and nonpolymer components. In addition, a reference dictionary was developed with detailed information about these peptide-like molecules to aid in their annotation, identification and analysis. Based on the experience gained in this remediation, guidelines, procedures, and tools were developed to annotate new depositions containing peptide-like inhibitors and antibiotics accurately and consistently. © 2013 The Authors Biopolymers Published by Wiley Periodicals, Inc.

Development of Tissue-Engineered Ligaments: Elastin Promotes Regeneration of the Rabbit Medial Collateral Ligament.

Science.gov (United States)

Hirukawa, Masaki; Katayama, Shingo; Sato, Tatsuya; Yamada, Masayoshi; Kageyama, Satoshi; Unno, Hironori; Suzuki, Yoshiaki; Miura, Yoshihiro; Shiratsuchi, Eri; Hasegawa, Masahiro; Miyamoto, Keiichi; Horiuchi, Takashi

2017-12-21

When ligaments are injured, reconstructive surgery is sometimes required to restore function. Methods of reconstructive surgery include transplantation of an artificial ligament and autotransplantation of a tendon. However, these methods have limitations related to the strength of the bone-ligament insertion and biocompatibility of the transplanted tissue after surgery. Therefore, it is necessary to develop new reconstruction methods and pursue the development of artificial ligaments. Elastin is a major component of elastic fibers and ligaments. However, the role of elastin in ligament regeneration has not been described. Here, we developed a rabbit model of a medial collateral ligament (MCL) rupture and treated animal knees with exogenous elastin [100 µg/(0.5 mL·week)] for 6 or 12 weeks. Elastin treatment increased gene expression and protein content of collagen and elastin (gene expression, 6-fold and 42-fold, respectively; protein content, 1.6-fold and 1.9-fold, respectively), and also increased the elastic modulus of MCL increased with elastin treatment (2-fold) compared with the controls. Our data suggest that elastin is involved in the regeneration of damaged ligaments. © 2017 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Evaluation of beta-cell secretory capacity using glucagon-like peptide 1

DEFF Research Database (Denmark)

Vilsbøll, Tina; Nielsen, Mette Toft; Krarup, T

2000-01-01

Beta-cell secretory capacity is often evaluated with a glucagon test or a meal test. However, glucagon-like peptide 1 (GLP-1) is the most insulinotropic hormone known, and the effect is preserved in type 2 diabetic patients.......Beta-cell secretory capacity is often evaluated with a glucagon test or a meal test. However, glucagon-like peptide 1 (GLP-1) is the most insulinotropic hormone known, and the effect is preserved in type 2 diabetic patients....

Glucagon and glucagon-like peptides 1 and 2

DEFF Research Database (Denmark)

Holst, Jens Juul

2010-01-01

amino acid precursor, proglucagon, leaving behind proglucagon fragments (PG 1-30 and PG 72-158, the so-called major proglucagon fragment (MPGF)) that are probably inactive, the intestinal processing leads to the formation of glicentin (PG 1-69; action uncertain) and glucagon-like peptides 1 (PG 78....... After their release, the hormones are eliminated mainly in the kidneys, but both GLP-2 and in particular GLP-1, but not glucagon, are metabolized both locally and in the circulation and liver by dipeptidyl peptidase 4 (DPP-4) which inactivates the peptides, suggesting that GLP-1 acts locally rather than...

High-performance liquid chromatographic quantitation of desmosine plus isodesmosine in elastin and whole tissue hydrolysates

International Nuclear Information System (INIS)

Soskel, N.T.

1987-01-01

Quantitation of desmosine and isodesmosine, the major crosslinks in elastin, has been of interest because of their uniqueness and use as markers of that protein. Accurate measurement of these crosslinks may allow determination of elastin degradation in vivo and elastin content in tissues, obviating lengthy extraction procedures. We have developed a method of quantitating desmosine plus isodesmosine in hydrolysates of tissue and insoluble elastin using high-performance liquid chromatographic separation and absorbance detection that is rapid (21-35 min) and sensitive (accurate linearity from 100 pmol to 5 nmol). This method has been used to quantitate desmosines in elastin from bovine nuchal ligament and lung and in whole aorta from hamsters. The ability to completely separate [ 3 H]lysine from desmosine plus isodesmosine allows the method to be used to study incorporation of lysine into crosslinks in elastin

Determination of Glucagon-Like Peptide-1, Glucagon and Oxyntomodulin in Plasma

DEFF Research Database (Denmark)

Bak, Monika Judyta

Glucagon-like peptide-1, glucagon and oxyntomodulin are three peptide hormones which play a significant role in diabetes, however there is a major controversy regarding their exact roles due to difficulties in measuring of these peptides because of molecular heterogeneity, low circulating concent...... of the studies from the literature that have provided reliable measurements and thereby help resolve controversies regarding the metabolic roles of the peptides. The improved technology should also provide better reliability of future publications in the field......., the addition of aprotinin to plasma prior to glucagon sample analysis was investigated. Aprotinin addition has been recommended for many years to avoid peptide degradation during sampling and storage. To make sure that the analysed samples are handled correctly and that the peptides are not degraded, a study...

Preparation of alpha-elastin nanoparticles by gamma irradiation

International Nuclear Information System (INIS)

Fujimoto, Mari; Okamoto, Kouji; Furuta, Masakazu

2009-01-01

Nanoparticles were prepared by utilizing the thermosensitive aggregation of alpha-elastin and gamma ray crosslinking. Three different heating process, 'Slow heating', 'Fast heating', and 'Heat shock', were applied for the aggregation of the alpha-elastin and examined to yield nanoparticles by gamma rays crosslinking. As a result, only 'Slow heating' process yielded nanoparticles with diameters of about ca. 300 nm above cloud point (CP) and about ca. 100 nm below CP, and a narrow size distribution above 1.0 mg/ml concentration (exclude 1.0 mg/ml).

Effect of glucagon-like peptide-1 on alpha- and beta-cell function in C-peptide-negative type 1 diabetic patients

DEFF Research Database (Denmark)

Kielgast, Urd; Asmar, Meena; Madsbad, Sten

2010-01-01

The mechanism by which glucagon-like peptide-1 (GLP-1) suppresses glucagon secretion is uncertain, and it is not determined whether endogenous insulin is a necessary factor for this effect.......The mechanism by which glucagon-like peptide-1 (GLP-1) suppresses glucagon secretion is uncertain, and it is not determined whether endogenous insulin is a necessary factor for this effect....

Thermo-responsive human α-elastin self-assembled nanoparticles for protein delivery.

Science.gov (United States)

Kim, Jae Dong; Jung, Youn Jae; Woo, Chang Hee; Choi, Young Chan; Choi, Ji Suk; Cho, Yong Woo

2017-01-01

Self-assembled nanoparticles based on PEGylated human α-elastin were prepared as a potential vehicle for sustained protein delivery. The α-elastin was extracted from human adipose tissue and modified with methoxypolyethyleneglycol (mPEG) to control particle size and enhance the colloidal stability. The PEGylated human α-elastin showed sol-to-particle transition with a lower critical solution temperature (LCST) of 25°C-40°C in aqueous media. The PEGylated human α-elastin nanoparticles (PhENPs) showed a narrow size distribution with an average diameter of 330±33nm and were able to encapsulate significant amounts of insulin and bovine serum albumin (BSA) upon simple mixing at low temperature in water and subsequent heating to physiological temperature. The release profiles of insulin and BSA showed sustained release for 72h. Overall, the thermo-responsive self-assembled PhENPs provide a useful tool for a range of protein delivery and tissue engineering applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Elastin governs the mechanical response of medial collateral ligament under shear and transverse tensile loading.

Science.gov (United States)

Henninger, Heath B; Valdez, William R; Scott, Sara A; Weiss, Jeffrey A

2015-10-01

Elastin is a highly extensible structural protein network that provides near-elastic resistance to deformation in biological tissues. In ligament, elastin is localized between and along the collagen fibers and fascicles. When ligament is stretched along the primary collagen axis, elastin supports a relatively high percentage of load. We hypothesized that elastin may also provide significant load support under elongation transverse to the primary collagen axis and shear along the collagen axis. Quasi-static transverse tensile and shear material tests were performed to quantify the mechanical contributions of elastin during deformation of porcine medial collateral ligament. Dose response studies were conducted to determine the level of elastase enzymatic degradation required to produce a maximal change in the mechanical response. Maximal changes in peak stress occurred after 3h of treatment with 2U/ml porcine pancreatic elastase. Elastin degradation resulted in a 60-70% reduction in peak stress and a 2-3× reduction in modulus for both test protocols. These results demonstrate that elastin provides significant resistance to elongation transverse to the collagen axis and shear along the collagen axis while only constituting 4% of the tissue dry weight. The magnitudes of the elastin contribution to peak transverse and shear stress were approximately 0.03 MPa, as compared to 2 MPa for axial tensile tests, suggesting that elastin provides a highly anisotropic contribution to the mechanical response of ligament and is the dominant structural protein resisting transverse and shear deformation of the tissue. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Preparation of alpha-elastin nanoparticles by gamma irradiation

Energy Technology Data Exchange (ETDEWEB)

Fujimoto, Mari [Department of Biological Science, Graduate school of Science, Osaka Prefecture University, 1-2 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8570 (Japan); Okamoto, Kouji [Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka, Fukuoka 820-8502 (Japan); Furuta, Masakazu [Department of Biological Science, Graduate school of Science, Osaka Prefecture University, 1-2 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8570 (Japan)], E-mail: mfuruta@b.s.osakafu-u.ac.jp

2009-12-15

Nanoparticles were prepared by utilizing the thermosensitive aggregation of alpha-elastin and gamma ray crosslinking. Three different heating process, 'Slow heating', 'Fast heating', and 'Heat shock', were applied for the aggregation of the alpha-elastin and examined to yield nanoparticles by gamma rays crosslinking. As a result, only 'Slow heating' process yielded nanoparticles with diameters of about ca. 300 nm above cloud point (CP) and about ca. 100 nm below CP, and a narrow size distribution above 1.0 mg/ml concentration (exclude 1.0 mg/ml)

Pentagalloyl glucose increases elastin deposition, decreases reactive oxygen species and matrix metalloproteinase activity in pulmonary fibroblasts under inflammatory conditions.

Science.gov (United States)

Parasaram, Vaideesh; Nosoudi, Nasim; Chowdhury, Aniqa; Vyavahare, Naren

2018-04-30

Emphysema is characterized by degradation of lung alveoli that leads to poor airflow in lungs. Irreversible elastic fiber degradation by matrix metalloproteinases (MMPs) and reactive oxygen species (ROS) activity leads to loss of elasticity and drives the progression of this disease. We investigated if a polyphenol, pentagalloyl glucose (PGG) can increase elastin production in pulmonary fibroblasts. We also studied the effect of PGG treatment in reducing MMP activity and ROS levels in cells. We exposed rat pulmonary fibroblasts to two different types of inflammatory environments i.e., tumor necrosis factor-α (TNF-α) and cigarette smoke extract (CSE) to mimic the disease. Parameters like lysyl oxidase (LOX) and elastin gene expression, MMP-9 activity in the medium, lysyl oxidase (LOX) activity and ROS levels were studied to assess the effect of PGG on pulmonary fibroblasts. CSE inhibited lysyl oxidase (LOX) enzyme activity that resulted in a decreased elastin formation. Similarly, TNF-α treated cells showed less elastin in the cell layers. Both these agents caused increase in MMP activity and ROS levels in cells. However, when supplemented with PGG treatment along with these two inflammatory agents, we saw a significant increase in elastin deposition, reduction in both MMP activity and ROS levels. Thus PGG, which has anti-inflammatory, anti-oxidant properties coupled with its ability to aid in elastic fiber formation, can be a multifunctional drug to potentially arrest the progression of emphysema. Copyright © 2018 Elsevier Inc. All rights reserved.

Designing of peptides with desired half-life in intestine-like environment

KAUST Repository

Sharma, Arun

2014-08-20

Background: In past, a number of peptides have been reported to possess highly diverse properties ranging from cell penetrating, tumor homing, anticancer, anti-hypertensive, antiviral to antimicrobials. Owing to their excellent specificity, low-toxicity, rich chemical diversity and availability from natural sources, FDA has successfully approved a number of peptide-based drugs and several are in various stages of drug development. Though peptides are proven good drug candidates, their usage is still hindered mainly because of their high susceptibility towards proteases degradation. We have developed an in silico method to predict the half-life of peptides in intestine-like environment and to design better peptides having optimized physicochemical properties and half-life.Results: In this study, we have used 10mer (HL10) and 16mer (HL16) peptides dataset to develop prediction models for peptide half-life in intestine-like environment. First, SVM based models were developed on HL10 dataset which achieved maximum correlation R/R2 of 0.57/0.32, 0.68/0.46, and 0.69/0.47 using amino acid, dipeptide and tripeptide composition, respectively. Secondly, models developed on HL16 dataset showed maximum R/R2 of 0.91/0.82, 0.90/0.39, and 0.90/0.31 using amino acid, dipeptide and tripeptide composition, respectively. Furthermore, models that were developed on selected features, achieved a correlation (R) of 0.70 and 0.98 on HL10 and HL16 dataset, respectively. Preliminary analysis suggests the role of charged residue and amino acid size in peptide half-life/stability. Based on above models, we have developed a web server named HLP (Half Life Prediction), for predicting and designing peptides with desired half-life. The web server provides three facilities; i) half-life prediction, ii) physicochemical properties calculation and iii) designing mutant peptides.Conclusion: In summary, this study describes a web server \\'HLP\\' that has been developed for assisting scientific

Collagen and elastin cross-linking is altered during aberrant late lung development associated with hyperoxia.

Science.gov (United States)

Mižíková, Ivana; Ruiz-Camp, Jordi; Steenbock, Heiko; Madurga, Alicia; Vadász, István; Herold, Susanne; Mayer, Konstantin; Seeger, Werner; Brinckmann, Jürgen; Morty, Rory E

2015-06-01

Maturation of the lung extracellular matrix (ECM) plays an important role in the formation of alveolar gas exchange units. A key step in ECM maturation is cross-linking of collagen and elastin, which imparts stability and functionality to the ECM. During aberrant late lung development in bronchopulmonary dysplasia (BPD) patients and animal models of BPD, alveolarization is blocked, and the function of ECM cross-linking enzymes is deregulated, suggesting that perturbed ECM cross-linking may impact alveolarization. In a hyperoxia (85% O2)-based mouse model of BPD, blunted alveolarization was accompanied by alterations to lung collagen and elastin levels and cross-linking. Total collagen levels were increased (by 63%). The abundance of dihydroxylysinonorleucine collagen cross-links and the dihydroxylysinonorleucine-to-hydroxylysinonorleucine ratio were increased by 11 and 18%, respectively, suggestive of a profibrotic state. In contrast, insoluble elastin levels and the abundance of the elastin cross-links desmosine and isodesmosine in insoluble elastin were decreased by 35, 30, and 21%, respectively. The lung collagen-to-elastin ratio was threefold increased. Treatment of hyperoxia-exposed newborn mice with the lysyl oxidase inhibitor β-aminopropionitrile partially restored normal collagen levels, normalized the dihydroxylysinonorleucine-to-hydroxylysinonorleucine ratio, partially normalized desmosine and isodesmosine cross-links in insoluble elastin, and partially restored elastin foci structure in the developing septa. However, β-aminopropionitrile administration concomitant with hyperoxia exposure did not improve alveolarization, evident from unchanged alveolar surface area and alveoli number, and worsened septal thickening (increased by 12%). These data demonstrate that collagen and elastin cross-linking are perturbed during the arrested alveolarization of developing mouse lungs exposed to hyperoxia. Copyright © 2015 the American Physiological Society.

Oxygenation decreases elastin secretion from rat ductus arteriosus smooth muscle cells.

Science.gov (United States)

Kawakami, Shoji; Minamisawa, Susumu

2015-08-01

The ductus arteriosus (DA), a fetal arterial connection between the main pulmonary artery and the descending aorta, normally closes immediately after birth. The oxygen concentration in the blood rises after birth, and in the DA this increase in oxygen concentration causes functional closure, which is induced by smooth muscle contraction. Previous studies have demonstrated that hypoxia and/or oxygenation affect vascular remodeling of various vessels. Therefore, we hypothesized that the rise in oxygen concentration would affect the vascular structure of the DA due to production of proteins secreted from DA smooth muscle cells (SMC). Liquid chromatography-tandem mass spectrometry was used to comprehensively investigate the secreted proteins in the supernatant of rat DA SMC harvested under hypoxic conditions (1% oxygen) or under normoxic conditions (21% oxygen). We found that the rise in oxygen concentration reduced the secretion of elastin from DA SMC. On reverse transcription-polymerase chain reaction, the expression of elastin mRNA was not significantly changed in DA SMC from hypoxic to normoxic conditions. Given that elastin forms internal elastic lamina and elastic fibers in the vascular muscle layers, and that a rise in oxygen concentration reduced the secretion of elastin, this suggests that the rise in blood oxygen concentration after birth reduces the secretion of elastin, and therefore may play a role in DA structural remodeling after birth. © 2015 Japan Pediatric Society.

'Boomerang'-like insertion of a fusogenic peptide in a lipid membrane revealed by solid-state 19F NMR.

Science.gov (United States)

Afonin, Sergii; Dürr, Ulrich H N; Glaser, Ralf W; Ulrich, Anne S

2004-02-01

Solid state (19)F NMR revealed the conformation and alignment of the fusogenic peptide sequence B18 from the sea urchin fertilization protein bindin embedded in flat phospholipid bilayers. Single (19)F labels were introduced into nine distinct positions along the wild-type sequence by substituting each hydrophobic amino acid, one by one, with L-4-fluorophenylglycine. Their anisotropic chemical shifts were measured in uniaxially oriented membrane samples and used as orientational constraints to model the peptide structure in the membrane-bound state. Previous (1)H NMR studies of B18 in 30% TFE and in detergent micelles had shown that the peptide structure consists of two alpha-helical segments that are connected by a flexible hinge. This helix-break-helix motif was confirmed here by the solid-state (19)F NMR data, while no other secondary structure (beta-sheet, 3(10)-helix) was compatible with the set of orientational constraints. For both alpha-helical segments we found that the helical conformation extends all the way to the respective N- and C-termini of the peptide. Analysis of the corresponding tilt and azimuthal rotation angles showed that the N-terminal helix of B18 is immersed obliquely into the bilayer (at a tilt angle tau approximately 54 degrees), whereas the C-terminus is peripherally aligned (tau approximately 91 degrees). The azimuthal orientation of the two segments is consistent with the amphiphilic distribution of side-chains. The observed 'boomerang'-like mode of insertion into the membrane may thus explain how peptide binding leads to lipid dehydration and acyl chain perturbation as a prerequisite for bilayer fusion to occur. Copyright 2004 John Wiley & Sons, Ltd.

Radio-immunoassays for glucagon-like peptides 1 and 2 (GLP-1 and GLP-2)

DEFF Research Database (Denmark)

Orskov, C; Holst, J J

1987-01-01

Gene-sequencing studies have shown that the glucagon precursor contains two additional glucagon-like sequences, the so-called glucagon-like peptides 1 and 2 (GLP-1 and GLP-2). We developed radio-immunoassays against synthetic peptides corresponding to these sequences. Antisera were raised in rabb...

Quantitative comparison of structure and dynamics of elastin following three isolation schemes by 13C solid state NMR and MALDI mass spectrometry.

Science.gov (United States)

Papaioannou, A; Louis, M; Dhital, B; Ho, H P; Chang, E J; Boutis, G S

2015-05-01

Methods for isolating elastin from fat, collagen, and muscle, commonly used in the design of artificial elastin based biomaterials, rely on exposing tissue to harsh pH levels and temperatures that usually denature many proteins. At present, a quantitative measurement of the modifications to elastin following isolation from other extracellular matrix constituents has not been reported. Using magic angle spinning (13)C NMR spectroscopy and relaxation methodologies, we have measured the modification in structure and dynamics following three known purification protocols. Our experimental data reveal that the (13)C spectra of the hydrated samples appear remarkably similar across the various purification methods. Subtle differences in the half maximum widths were observed in the backbone carbonyl suggesting possible structural heterogeneity across the different methods of purification. Additionally, small differences in the relative signal intensities were observed between purified samples. Lyophilizing the samples results in a reduction of backbone motion and reveals additional differences across the purification methods studied. These differences were most notable in the alanine motifs indicating possible changes in cross-linking or structural rigidity. The measured correlation times of glycine and proline moieties are observed to also vary considerably across the different purification methods, which may be related to peptide bond cleavage. Lastly, the relative concentration of desmosine cross-links in the samples quantified by MALDI mass spectrometry is reported. Copyright © 2015 Elsevier B.V. All rights reserved.

Chemical hydrogels based on a hyaluronic acid-graft-α-elastin derivative as potential scaffolds for tissue engineering

International Nuclear Information System (INIS)

Palumbo, Fabio Salvatore; Pitarresi, Giovanna; Fiorica, Calogero; Rigogliuso, Salvatrice; Ghersi, Giulio; Giammona, Gaetano

2013-01-01

In this work hyaluronic acid (HA) functionalized with ethylenediamine (EDA) has been employed to graft α-elastin. In particular a HA-EDA derivative bearing 50 mol% of pendant amino groups has been successfully employed to produce the copolymer HA-EDA-g-α-elastin containing 32% w/w of protein. After grafting with α-elastin, remaining free amino groups reacted with ethylene glycol diglycidyl ether (EGDGE) for producing chemical hydrogels, proposed as scaffolds for tissue engineering. Swelling degree, resistance to chemical and enzymatic hydrolysis, as well as preliminary biological properties of HA-EDA-g-α-elastin/EGDGE scaffold have been evaluated and compared with a HA-EDA/EGDGE scaffold. The presence of α-elastin grafted to HA-EDA improves attachment, viability and proliferation of primary rat dermal fibroblasts and human umbilical artery smooth muscle cells. Biological performance of HA-EDA-g-α-elastin/EGDGE scaffold resulted comparable to that of a commercial collagen type I sponge (Antema®), chosen as a positive control. - Highlights: ► Hyaluronic acid (HA) has been functionalized with ethylenediamine (EDA). ► Amino groups of HA-EDA allow the reaction with α-elastin and ethylene glycol diglycidyl ether (EGDGE). ► Chemical scaffolds of HA-EDA-graft-α-elastin/EGDGE have been characterized. ► The presence of α-elastin affects porosity, swelling and enzymatic degradation of scaffolds. ► The presence of α-elastin improves attachment, viability and proliferation of fibroblasts and smooth muscle cells

Chemical hydrogels based on a hyaluronic acid-graft-α-elastin derivative as potential scaffolds for tissue engineering

Energy Technology Data Exchange (ETDEWEB)

Palumbo, Fabio Salvatore [Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari, Sezione di Chimica e Tecnologie Farmaceutiche, Università degli Studi di Palermo, Via Archirafi 32, 90123, Palermo (Italy); Pitarresi, Giovanna, E-mail: giovanna.pitarresi@unipa.it [Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari, Sezione di Chimica e Tecnologie Farmaceutiche, Università degli Studi di Palermo, Via Archirafi 32, 90123, Palermo (Italy); Institute of Biophysics at Palermo, Italian National Research Council, Via Ugo La Malfa 153, 90146 Palermo (Italy); Fiorica, Calogero [Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari, Sezione di Chimica e Tecnologie Farmaceutiche, Università degli Studi di Palermo, Via Archirafi 32, 90123, Palermo (Italy); Rigogliuso, Salvatrice; Ghersi, Giulio [Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari, Sezione di Biologia Cellulare, Università degli Studi di Palermo, Viale delle Scienze ed. 16, 90128, Palermo (Italy); Giammona, Gaetano [Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari, Sezione di Chimica e Tecnologie Farmaceutiche, Università degli Studi di Palermo, Via Archirafi 32, 90123, Palermo (Italy); IBIM-CNR, Via Ugo La Malfa 153, 90146 Palermo (Italy)

2013-07-01

In this work hyaluronic acid (HA) functionalized with ethylenediamine (EDA) has been employed to graft α-elastin. In particular a HA-EDA derivative bearing 50 mol% of pendant amino groups has been successfully employed to produce the copolymer HA-EDA-g-α-elastin containing 32% w/w of protein. After grafting with α-elastin, remaining free amino groups reacted with ethylene glycol diglycidyl ether (EGDGE) for producing chemical hydrogels, proposed as scaffolds for tissue engineering. Swelling degree, resistance to chemical and enzymatic hydrolysis, as well as preliminary biological properties of HA-EDA-g-α-elastin/EGDGE scaffold have been evaluated and compared with a HA-EDA/EGDGE scaffold. The presence of α-elastin grafted to HA-EDA improves attachment, viability and proliferation of primary rat dermal fibroblasts and human umbilical artery smooth muscle cells. Biological performance of HA-EDA-g-α-elastin/EGDGE scaffold resulted comparable to that of a commercial collagen type I sponge (Antema®), chosen as a positive control. - Highlights: ► Hyaluronic acid (HA) has been functionalized with ethylenediamine (EDA). ► Amino groups of HA-EDA allow the reaction with α-elastin and ethylene glycol diglycidyl ether (EGDGE). ► Chemical scaffolds of HA-EDA-graft-α-elastin/EGDGE have been characterized. ► The presence of α-elastin affects porosity, swelling and enzymatic degradation of scaffolds. ► The presence of α-elastin improves attachment, viability and proliferation of fibroblasts and smooth muscle cells.

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

Science.gov (United States)

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.

Glucagon-like peptide 1 receptor agonists: a new approach to type 2 ...

African Journals Online (AJOL)

Hypoglycaemia and weight gain that are associated with many antidiabetic medications may interfere with the implementation and long-term application of treatment strategies. Glucose homeostasis is dependent on a complex interplay of multiple hormones and gastrointestinal peptides, including glucagon-like peptide 1 ...

Characterisation and evaluation of antiviral recombinant peptides based on the heptad repeat regions of NDV and IBV fusion glycoproteins

International Nuclear Information System (INIS)

Wang Xiaojia; Li Chuangen; Chi Xiaojing; Wang Ming

2011-01-01

Mixed virus infections can cause livestock losses that are more devastating than those caused by single virus infections. Newcastle disease virus (NDV) and infectious bronchitis virus (IBV), serious threats to the poultry industry, can give rise to complex mixed infections that hinder diagnosis and prevention. In this study, we show that newly designed peptides, which are based on the heptad repeat (HR) region of the fusion glycoproteins from NDV and IBV, have more potent antiviral activity than the mother HR peptides. Plaque formation and chicken embryo infectivity assays confirmed these results. The novel peptides completely inhibited single virus infections and mixed infections caused by NDV and IBV. Furthermore, we assessed cell toxicity and possible targets for the peptides, thereby strengthening the notion that HR2 is an attractive site for therapeutic intervention. These results suggest the possibility of designing a relatively broad-spectrum class of antiviral peptides that can reduce the effects of mixed-infections.

The physiology of glucagon-like peptide 1

DEFF Research Database (Denmark)

Holst, Jens Juul

2007-01-01

Glucagon-like peptide 1 (GLP-1) is a 30-amino acid peptide hormone produced in the intestinal epithelial endocrine L-cells by differential processing of proglucagon, the gene which is expressed in these cells. The current knowledge regarding regulation of proglucagon gene expression in the gut...... and in the brain and mechanisms responsible for the posttranslational processing are reviewed. GLP-1 is released in response to meal intake, and the stimuli and molecular mechanisms involved are discussed. GLP-1 is extremely rapidly metabolized and inactivated by the enzyme dipeptidyl peptidase IV even before...... postprandial glucose excursions. It also inhibits gastrointestinal motility and secretion and thus acts as an enterogastrone and part of the "ileal brake" mechanism. GLP-1 also appears to be a physiological regulator of appetite and food intake. Because of these actions, GLP-1 or GLP-1 receptor agonists...

Elastins from patients with Williams-Beuren syndrome and healthy individuals differ on the molecular level

DEFF Research Database (Denmark)

Heinz, Andrea; Huertas, Angela C Mora; Schräder, Christoph U

2016-01-01

Williams-Beuren syndrome (WBS) is a congenital disorder, which involves the heterozygous deletion of the elastin gene and other genes on chromosome 7. Clinical symptoms that are associated with hemizygosity of the essential extracellular matrix protein elastin include premature aging of the skin...... and supravalvular aortic stenosis. However, only little is known about the molecular basis of structural abnormalities in the connective tissue of WBS patients. Therefore, for the first time this study aimed to systematically characterize and compare the structure and amount of elastin present in skin and aortic...... tissue from WBS patients and healthy individuals. Elastin fibers were isolated from tissue biopsies, and it was found that skin of WBS patients contains significantly less elastin compared to skin of healthy individuals. Scanning electron microscopy and mass spectrometric measurements combined...

Renal clearance of the thyrotropin-releasing hormone-like peptide pyroglutamyl-glutamyl-prolineamide in humans

NARCIS (Netherlands)

W. Klootwijk (Willem); E. Sleddens-Linkels (Esther); R. de Boer (Renske); C.A. Jansen; R. Autar; W.W. de Herder (Wouter); E.R. Boeve; T.J. Visser (Theo); W.J. de Greef (W.)

1997-01-01

textabstractTRH-like peptides have been identified that differ from TRH (pGlu-His- ProNH2) in the middle aminoacid. We have estimated TRH-like immunoreactivity (TRH-LI) in human serum and urine by RIA with TRH-specific antiserum 8880 or with antiserum 4319, which binds most peptides with the

Levels of circulating MMP-7 degraded elastin are elevated in pulmonary disorders

DEFF Research Database (Denmark)

Kristensen, J.H.; Larsen, L.; Dasgupta, B.

2015-01-01

for the quantification of MMP-7 generated elastin fragments (ELM7) was therefore developed to investigate MMP-7 derived elastin degradation in pulmonary disorders such as idiopathic pulmonary fibrosis (IPF) and lung cancer. Design and methods:  Monoclonal antibodies (mABs) were raised against eight carefully selected...

The alpha cell expresses glucagon-like peptide-2 receptors and glucagon-like peptide-2 stimulates glucagon secretion from the rat pancreas

DEFF Research Database (Denmark)

de Heer, J; Pedersen, J; Orskov, C

2007-01-01

AIMS/HYPOTHESIS: Glucagon-like peptide-2 (GLP-2) is a gut hormone regulating intestinal growth and nutrient absorption. Recently, GLP-2 has been reported to stimulate glucagon secretion in healthy humans. We sought to clarify the mechanism and physiological significance of this endocrine effect. ...... of the glucagonotropic action of GLP-2 in humans....

The coupled bio-chemo-electro-mechanical behavior of glucose exposed arterial elastin

International Nuclear Information System (INIS)

Zhang, Yanhang; Li, Jiangyu; Boutis, Gregory S

2017-01-01

Elastin, the principle protein component of the elastic fiber, is a critical extracellular matrix (ECM) component of the arterial wall providing structural resilience and biological signaling essential in vascular morphogenesis and maintenance of mechanical homeostasis. Pathogenesis of many cardiovascular diseases have been associated with alterations of elastin. As a long-lived ECM protein that is deposited and organized before adulthood, elastic fibers can suffer from cumulative effects of biochemical exposure encountered during aging and/or disease, which greatly compromise their mechanical function. This review article covers findings from recent studies of the mechanical and structural contribution of elastin to vascular function, and the effects of biochemical degradation. Results from diverse experimental methods including tissue-level mechanical characterization, fiber-level nonlinear optical imaging, piezoelectric force microscopy, and nuclear magnetic resonance are reviewed. The intriguing coupled bio-chemo-electro-mechanical behavior of elastin calls for a multi-scale and multi-physical understanding of ECM mechanics and mechanobiology in vascular remodeling. (topical review)

The coupled bio-chemo-electro-mechanical behavior of glucose exposed arterial elastin

Science.gov (United States)

Zhang, Yanhang; Li, Jiangyu; Boutis, Gregory S.

2017-04-01

Elastin, the principle protein component of the elastic fiber, is a critical extracellular matrix (ECM) component of the arterial wall providing structural resilience and biological signaling essential in vascular morphogenesis and maintenance of mechanical homeostasis. Pathogenesis of many cardiovascular diseases have been associated with alterations of elastin. As a long-lived ECM protein that is deposited and organized before adulthood, elastic fibers can suffer from cumulative effects of biochemical exposure encountered during aging and/or disease, which greatly compromise their mechanical function. This review article covers findings from recent studies of the mechanical and structural contribution of elastin to vascular function, and the effects of biochemical degradation. Results from diverse experimental methods including tissue-level mechanical characterization, fiber-level nonlinear optical imaging, piezoelectric force microscopy, and nuclear magnetic resonance are reviewed. The intriguing coupled bio-chemo-electro-mechanical behavior of elastin calls for a multi-scale and multi-physical understanding of ECM mechanics and mechanobiology in vascular remodeling.

Genetically controlled fusion, exocytosis and fission of artificial vesicles-a roadmap

DEFF Research Database (Denmark)

Bönzli, Eva; Hadorn, Maik; de Lucrezia, Davide

2011-01-01

were shown to fuse if a special class of viral proteins, termed fusogenic peptides, were added to the external medium (Nomura et al. 2004). In the present work, we intend to develop genetically controlled fusion, fission and exocytosis of vesicles by the synthesis of peptides within vesicles. First, we...... enclosed synthesized peptides in vesicles to induce in a next step fusion of adjacent vesicles, fission and exocytosis of nested vesicles. Second, we will replace the peptides by an enclosed cell-free expression system to internally synthesize fusion peptides. To control the gene expression, different...

Elastin and Mechanics of Pig Pericardial Resistance Arteries (pPRA)

DEFF Research Database (Denmark)

Bloksgaard, Maria; Leurgans, Thomas; Rosenstand, Kristoffer

Resistance arteries are remodeled in hypertension and diabetes. Elastin was reported to play a role herein. The parietal pericardium is opened during cardio-thoracic surgeries and might be a valuable biopsy for research in cardio-vascular diseases. We tested the hypothesis that resistance arteries...... can be isolated from the pericardium to study the micro-architecture of elastin and vascular wall mechanics. The pericardium of pigs served to test the hypothesis. pPRAs were microdissected. Their structure was examined using multiphoton excitation fluorescence microscopy. Diameter......-tension and pressure-diameter-length relationships were recorded in myographs. Findings are compared to rodent mesenteric resistance arteries and –basilar arteries (rMRA, rBA) with comparable lumen diameter (±300µm at 100mmHg). pPRA have no clear external elastic lamina (present in rMRA, but not rBA), scant elastin...

MiR-29-mediated elastin down-regulation contributes to inorganic phosphorus-induced osteoblastic differentiation in vascular smooth muscle cells.

Science.gov (United States)

Sudo, Ryo; Sato, Fumiaki; Azechi, Takuya; Wachi, Hiroshi

2015-12-01

Vascular calcification increases the risk of cardiovascular mortality. We previously reported that expression of elastin decreases with progression of inorganic phosphorus (Pi)-induced vascular smooth muscle cell (VSMC) calcification. However, the regulatory mechanisms of elastin mRNA expression during vascular calcification remain unclear. MicroRNA-29 family members (miR-29a, b and c) are reported to mediate elastin mRNA expression. Therefore, we aimed to determine the effect of miR-29 on elastin expression and Pi-induced vascular calcification. Calcification of human VSMCs was induced by Pi and evaluated measuring calcium deposition. Pi stimulation promoted Ca deposition and suppressed elastin expression in VSMCs. Knockdown of elastin expression by shRNA also promoted Pi-induced VSMC calcification. Elastin pre-mRNA measurements indicated that Pi stimulation suppressed elastin expression without changing transcriptional activity. Conversely, Pi stimulation increased miR-29a and miR-29b expression. Inhibition of miR-29 recovered elastin expression and suppressed calcification in Pi-treated VSMCs. Furthermore, over-expression of miR-29b promoted Pi-induced VSMC calcification. RT-qPCR analysis showed knockdown of elastin expression in VSMCs induced expression of osteoblast-related genes, similar to Pi stimulation, and recovery of elastin expression by miR-29 inhibition reduced their expression. Our study shows that miR-29-mediated suppression of elastin expression in VSMCs plays a pivotal role in osteoblastic differentiation leading to vascular calcification. © 2015 The Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

Elastin aging and lipid oxidation products in human aorta

Directory of Open Access Journals (Sweden)

Kamelija Zarkovic

2015-04-01

Full Text Available Vascular aging is associated with structural and functional modifications of the arteries, and by an increase in arterial wall thickening in the intima and the media, mainly resulting from structural modifications of the extracellular matrix (ECM components. Among the factors known to accumulate with aging, advanced lipid peroxidation end products (ALEs are a hallmark of oxidative stress-associated diseases such as atherosclerosis. Aldehydes generated from the peroxidation of polyunsaturated fatty acids (PUFA, (4-hydroxynonenal, malondialdehyde, acrolein, form adducts on cellular proteins, leading to a progressive protein dysfunction with consequences in the pathophysiology of vascular aging. The contribution of these aldehydes to ECM modification is not known. This study was carried out to investigate whether aldehyde-adducts are detected in the intima and media in human aorta, whether their level is increased in vascular aging, and whether elastin fibers are a target of aldehyde-adduct formation. Immunohistological and confocal immunofluorescence studies indicate that 4-HNE-histidine-adducts accumulate in an age-related manner in the intima, media and adventitia layers of human aortas, and are mainly expressed in smooth muscle cells. In contrast, even if the structure of elastin fiber is strongly altered in the aged vessels, our results show that elastin is not or very poorly modified by 4-HNE. These data indicate a complex role for lipid peroxidation and in particular for 4-HNE in elastin homeostasis, in the vascular wall remodeling during aging and atherosclerosis development.

Elastin cross-linking in the skin from patients with amyotrophic lateral sclerosis

Science.gov (United States)

Ono, S.; Yamauchi, M.

1994-01-01

Two cross-links unique to elastin, desmosine and isodesmosine were measured and compared in skin tissue (left upper arm) from 10 patients with amyotrophic lateral sclerosis (ALS) and from seven age-matched controls. The contents of desmosine and isodesmosine were significantly decreased (p elastin is affected in ALS.

Elastin Is Differentially Regulated by Pressure Therapy in a Porcine Model of Hypertrophic Scar.

Science.gov (United States)

Carney, Bonnie C; Liu, Zekun; Alkhalil, Abdulnaser; Travis, Taryn E; Ramella-Roman, Jessica; Moffatt, Lauren T; Shupp, Jeffrey W

Beneficial effects of pressure therapy for hypertrophic scars have been reported, but the mechanisms of action are not fully understood. This study evaluated elastin and its contribution to scar pliability. The relationship between changes in Vancouver Scar Scale (VSS) scores of pressure-treated scars and differential regulation of elastin was assessed. Hypertrophic scars were created and assessed weekly using VSS and biopsy procurement. Pressure treatment began on day 70 postinjury. Treated scars were compared with untreated shams. Treatment lasted 2 weeks, through day 84, and scars were assessed weekly through day 126. Transcript and protein levels of elastin were quantified. Pressure treatment resulted in lower VSS scores compared with sham-treated scars. Pliability (VSSP) was a key contributor to this difference. At day 70 pretreatment, VSSP = 2. Without treatment, sham-treated scars became less pliable, while pressure-treated scars became more pliable. The percentage of elastin in scars at day 70 was higher than in uninjured skin. Following treatment, the percentage of elastin increased and continued to increase through day 126. Untreated sham scars did not show a similar increase. Quantification of Verhoeff-Van Gieson staining corroborated the findings and immunofluorescence revealed the alignment of elastin fibers. Pressure treatment results in increased protein level expression of elastin compared with sham-untreated scars. These findings further characterize the extracellular matrix's response to the application of pressure as a scar treatment, which will contribute to the refinement of rehabilitation practices and ultimately improvements in functional and psychosocial outcomes for patients.

Elastin structure and its involvement in skin photoageing.

Science.gov (United States)

Weihermann, A C; Lorencini, M; Brohem, C A; de Carvalho, C M

2017-06-01

Skin aging is a complex process that may be caused by factors that are intrinsic and extrinsic to the body. Ultraviolet (UV) radiation represents one of the main sources of skin damage over the years and characterizes a process known as photoaging. Among the changes that affect cutaneous tissue with age, the loss of elastic properties caused by changes in elastin production, increased degradation and/or processing produces a substantial impact on tissue esthetics and health. The occurrence of solar elastosis is one of the main markers of cutaneous photoaging and is characterized by disorganized and non-functional deposition of elastic fibers. The occurrence of UV radiation-induced alternative splicing of the elastin gene, which leads to inadequate synthesis of the proteins required for the correct assembly of elastic fibers, is a potential explanation for this phenomenon. Innovative studies have been fundamental for the elucidation of rarely explored photoaging mechanisms and have enabled the identification of effective therapeutic alternatives such as cosmetic products. This review addresses cutaneous photoaging and the changes that affect elastin in this process. © 2016 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Glucagon-like peptide-1 analogues: An overview

Directory of Open Access Journals (Sweden)

Vishal Gupta

2013-01-01

Full Text Available Abnormalities of the incretin axis have been implicated in the pathogenesis of type 2 diabetes mellitus. Glucagon-like peptide-1 (GLP-1 and gastroinhibitory intestinal peptide constitutes >90% of all the incretin function. Augmentation of GLP-1 results in improvement of beta cell health in a glucose-dependant manner (post-prandial hyperglycemia and suppression of glucagon (fasting hyperglycemia, amongst other beneficial pleiotropic effects. Native GLP-1 has a very short plasma half-life and novel methods have been developed to augment its half life, such that its anti-hyperglycemic effects can be exploited. They can be broadly classified as exendin-based therapies (exenatide, exenatide once weekly, DPP-4-resistant analogues (lixisenatide, albiglutide, and analogues of human GLP-1 (liraglutide, taspoglutide. Currently, commercially available analogues are exenatide, exenatide once weekly, and liraglutide. This review aims to provide an overview of most GLP-1 analogues.

Controlled assembly of multi-segment nanowires by histidine-tagged peptides

Energy Technology Data Exchange (ETDEWEB)

Wang, Aijun A; Lee, Joun; Jenikova, Gabriela; Mulchandani, Ashok; Myung, Nosang V; Chen, Wilfred [Department of Chemical and Environmental Engineering, University of California, Riverside, CA 92521 (United States)

2006-07-28

A facile technique was demonstrated for the controlled assembly and alignment of multi-segment nanowires using bioengineered polypeptides. An elastin-like-polypeptide (ELP)-based biopolymer consisting of a hexahistine cluster at each end (His{sub 6}-ELP-His{sub 6}) was generated and purified by taking advantage of the reversible phase transition property of ELP. The affinity between the His{sub 6} domain of biopolymers and the nickel segment of multi-segment nickel/gold/nickel nanowires was exploited for the directed assembly of nanowires onto peptide-functionalized electrode surfaces. The presence of the ferromagnetic nickel segments on the nanowires allowed the control of directionality by an external magnetic field. Using this method, the directed assembly and positioning of multi-segment nanowires across two microfabricated nickel electrodes in a controlled manner was accomplished with the expected ohmic contact.

Characterization of a structural intermediate of flavivirus membrane fusion.

Directory of Open Access Journals (Sweden)

Karin Stiasny

2007-02-01

Full Text Available Viral membrane fusion proceeds through a sequence of steps that are driven by triggered conformational changes of viral envelope glycoproteins, so-called fusion proteins. Although high-resolution structural snapshots of viral fusion proteins in their prefusion and postfusion conformations are available, it has been difficult to define intermediate structures of the fusion pathway because of their transient nature. Flaviviruses possess a class II viral fusion protein (E mediating fusion at acidic pH that is converted from a dimer to a trimer with a hairpin-like structure during the fusion process. Here we show for tick-borne encephalitis virus that exposure of virions to alkaline instead of acidic pH traps the particles in an intermediate conformation in which the E dimers dissociate and interact with target membranes via the fusion peptide without proceeding to the merger of the membranes. Further treatment to low pH, however, leads to fusion, suggesting that these monomers correspond to an as-yet-elusive intermediate required to convert the prefusion dimer into the postfusion trimer. Thus, the use of nonphysiological conditions allows a dissection of the flavivirus fusion process and the identification of two separate steps, in which membrane insertion of multiple copies of E monomers precedes the formation of hairpin-like trimers. This sequence of events provides important new insights for understanding the dynamic process of viral membrane fusion.

Virus-cell fusion inhibitory activity of novel analogue peptides based on the HP (2-20) derived from N-terminus of Helicobacter pylori Ribosomal Protein L1.

Science.gov (United States)

Woo, Eun-Rhan; Lee, Dong Gun; Chang, Young-Su; Park, Yoonkyung; Hahm, Kyung-Soo

2002-12-01

HP (2-20) (AKKVFKRLEKLFSKIQNDK) is the antibacterial sequence derived from N-terminus of Helicobacter pylori Ribosomal Protein L1 (RPL1). It has a broad-spectrum microbicidal activity in vitro that is thought to be related to the membrane-disruptive properties of the peptide. Based on the putative membrane-targeted mode of action, we postulated that HP (2-20) might be possessed virus-cell fusion inhibitory activity. To develop the novel virus-cell fusion inhibitory peptides, several analogues with amino acid substitution were designed to increase or decrease only net hydrophobic region. In particular, substitution of Gln and Asp for hydrophobic amino acid, Trp at position 17 and 19 of HP (2-20) (Anal 3) caused a dramatic increase in virus-cell fusion inhibitory activity without hemolytic effect.

The role of blood cell membrane lipids on the mode of action of HIV-1 fusion inhibitor sifuvirtide

International Nuclear Information System (INIS)

Matos, Pedro M.; Freitas, Teresa; Castanho, Miguel A.R.B.; Santos, Nuno C.

2010-01-01

Research highlights: → Sifuvirtide interacts with erythrocyte and lymphocyte membrane in a concentration dependent manner by decreasing its dipole potential. → Dipole potential variations in lipid vesicles show sifuvirtide's lipid selectivity towards saturated phosphatidylcholines. → This peptide-membrane interaction may direct the drug towards raft-like membrane domains where the receptors used by HIV are located, facilitating its inhibitory action. -- Abstract: Sifuvirtide is a gp41 based peptide that inhibits HIV-1 fusion with the host cells and is currently under clinical trials. Previous studies showed that sifuvirtide partitions preferably to saturated phosphatidylcholine lipid membranes, instead of fluid-phase lipid vesicles. We extended the study to the interaction of the peptide with circulating blood cells, by using the dipole potential sensitive probe di-8-ANEPPS. Sifuvirtide decreased the dipole potential of erythrocyte and lymphocyte membranes in a concentration dependent manner, demonstrating its interaction. Also, the lipid selectivity of the peptide towards more rigid phosphatidylcholines was confirmed based on the dipole potential variations. Overall, the interaction of the peptide with the cell membranes is a contribution of different lipid preferences that presumably directs the peptide towards raft-like domains where the receptors are located, facilitating the reach of the peptide to its molecular target, the gp41 in its pre-fusion conformation.

Why bacteria derived R-M nucleic enzymatic peptides are likely ...

African Journals Online (AJOL)

STORAGESEVER

2008-06-17

Jun 17, 2008 ... (3) Human genome contains several palindromes recognizable by R-M peptides, making ..... mutation and likely has affected genome evolution. .... Microbicide to prevent sexual transmission of HIV among high risk.

Steviol Glycoside Rebaudioside A Induces Glucagon-like Peptide-1 and Peptide YY Release in a Porcine ex Vivo Intestinal Model

NARCIS (Netherlands)

Ripken, D.; Wielen, N. van der; Wortelboer, H.M.; Meijerink, J.; Witkamp, R.F.; Hendriks, H.F.J.

2014-01-01

Glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are hormones important for satiation and are involved in the process called “ileal brake”. The aim of this study was to investigate the GLP-1- and PYY-stimulating efficacy of rebaudioside A, casein, and sucrose. This was studied using tissue

Steviol glycoside rebaudioside A induces glucagon-like peptide-1 and peptide YY release in a porcine ex vivo intestinal model

NARCIS (Netherlands)

Ripken, D.; Wielen, van der N.; Wortelboer, H.M.; Meijerink, J.; Witkamp, R.F.; Hendriks, H.F.

2014-01-01

Glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are hormones important for satiation and are involved in the process called "ileal brake". The aim of this study was to investigate the GLP-1- and PYY-stimulating efficacy of rebaudioside A, casein, and sucrose. This was studied using tissue

Elastin aging and lipid oxidation products in human aorta.

Science.gov (United States)

Zarkovic, Kamelija; Larroque-Cardoso, Pauline; Pucelle, Mélanie; Salvayre, Robert; Waeg, Georg; Nègre-Salvayre, Anne; Zarkovic, Neven

2015-01-01

Vascular aging is associated with structural and functional modifications of the arteries, and by an increase in arterial wall thickening in the intima and the media, mainly resulting from structural modifications of the extracellular matrix (ECM) components. Among the factors known to accumulate with aging, advanced lipid peroxidation end products (ALEs) are a hallmark of oxidative stress-associated diseases such as atherosclerosis. Aldehydes generated from the peroxidation of polyunsaturated fatty acids (PUFA), (4-hydroxynonenal, malondialdehyde, acrolein), form adducts on cellular proteins, leading to a progressive protein dysfunction with consequences in the pathophysiology of vascular aging. The contribution of these aldehydes to ECM modification is not known. This study was carried out to investigate whether aldehyde-adducts are detected in the intima and media in human aorta, whether their level is increased in vascular aging, and whether elastin fibers are a target of aldehyde-adduct formation. Immunohistological and confocal immunofluorescence studies indicate that 4-HNE-histidine-adducts accumulate in an age-related manner in the intima, media and adventitia layers of human aortas, and are mainly expressed in smooth muscle cells. In contrast, even if the structure of elastin fiber is strongly altered in the aged vessels, our results show that elastin is not or very poorly modified by 4-HNE. These data indicate a complex role for lipid peroxidation and in particular for 4-HNE in elastin homeostasis, in the vascular wall remodeling during aging and atherosclerosis development. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Fusing simulation and experiment: The effect of mutations on the structure and activity of the influenza fusion peptide

Science.gov (United States)

Lousa, Diana; Pinto, Antónia R. T.; Victor, Bruno L.; Laio, Alessandro; Veiga, Ana S.; Castanho, Miguel A. R. B.; Soares, Cláudio M.

2016-01-01

During the infection process, the influenza fusion peptide (FP) inserts into the host membrane, playing a crucial role in the fusion process between the viral and host membranes. In this work we used a combination of simulation and experimental techniques to analyse the molecular details of this process, which are largely unknown. Although the FP structure has been obtained by NMR in detergent micelles, there is no atomic structure information in membranes. To answer this question, we performed bias-exchange metadynamics (BE-META) simulations, which showed that the lowest energy states of the membrane-inserted FP correspond to helical-hairpin conformations similar to that observed in micelles. BE-META simulations of the G1V, W14A, G12A/G13A and G4A/G8A/G16A/G20A mutants revealed that all the mutations affect the peptide’s free energy landscape. A FRET-based analysis showed that all the mutants had a reduced fusogenic activity relative to the WT, in particular the mutants G12A/G13A and G4A/G8A/G16A/G20A. According to our results, one of the major causes of the lower activity of these mutants is their lower membrane affinity, which results in a lower concentration of peptide in the bilayer. These findings contribute to a better understanding of the influenza fusion process and open new routes for future studies. PMID:27302370

Measurement of MMP-9 and -12 degraded elastin (ELM) provides unique information on lung tissue degradation

Science.gov (United States)

2012-01-01

Background Elastin is an essential component of selected connective tissues that provides a unique physiological elasticity. Elastin may be considered a signature protein of lungs where matrix metalloprotease (MMP) -9-and -12, may be considered the signature proteases of the macrophages, which in part are responsible for tissue damage during disease progression. Thus, we hypothesized that a MMP-9/-12 generated fragment of elastin may be a relevant biochemical maker for lung diseases. Methods Elastin fragments were identified by mass-spectrometry and one sequence, generated by MMP-9 and -12 (ELN-441), was selected for monoclonal antibody generation and used in the development of an ELISA. Soluble and insoluble elastin from lung was cleaved in vitro and the time-dependent release of fragments was assessed in the ELN-441 assay. The release of ELN-441 in human serum from patients with chronic obstructive pulmonary disease (COPD) (n = 10) and idiopathic pulmonary fibrosis (IPF) (n = 29) were compared to healthy matched controls (n = 11). Results The sequence ELN-441 was exclusively generated by MMP-9 and -12 and was time-dependently released from soluble lung elastin. ELN-441 levels were 287% higher in patients diagnosed with COPD (p elastin. This fragment was elevated in serum from patients with the lung diseases IPF and COPD, however these data needs to be validated in larger clinical settings. PMID:22818364

Effect of gamma radiation on tissue elastin content and serum elastolytic activity in rats

International Nuclear Information System (INIS)

Drozdz, M.; Olczyk, K.; Piwowarczyk, B.; Stawiarska, B.

1981-01-01

The elastin content of aorta, heart, skin and lungs as well as the serum elastolytic activity were determined in rats exposed to radiation. It was found that a single irradiation of rats with gamma rays (500 r) caused a decrease of the elastin content in all examined tissues. The serum elastolytic activity in the irradiated rats was increased. It is suggested that elastin degradation following radiation may be caused by changes in its molecular structure and possibly, due to increased serum elastolytic activity. (author)

Deficient Circumferential Growth Is the Primary Determinant of Aortic Obstruction Attributable to Partial Elastin Deficiency.

Science.gov (United States)

Jiao, Yang; Li, Guangxin; Korneva, Arina; Caulk, Alexander W; Qin, Lingfeng; Bersi, Matthew R; Li, Qingle; Li, Wei; Mecham, Robert P; Humphrey, Jay D; Tellides, George

2017-05-01

Williams syndrome is characterized by obstructive aortopathy attributable to heterozygous loss of ELN , the gene encoding elastin. Lesions are thought to result primarily from excessive smooth muscle cell (SMC) proliferation and consequent medial expansion, although an initially smaller caliber and increased stiffness of the aorta may contribute to luminal narrowing. The relative contributions of such abnormalities to the obstructive phenotype had not been defined. We quantified determinants of luminal stenosis in thoracic aortas of Eln -/- mice incompletely rescued by human ELN . Moderate obstruction was largely because of deficient circumferential growth, most prominently of ascending segments, despite increased axial growth. Medial thickening was evident in these smaller diameter elastin-deficient aortas, with medial area similar to that of larger diameter control aortas. There was no difference in cross-sectional SMC number between mutant and wild-type genotypes at multiple stages of postnatal development. Decreased elastin content was associated with medial fibrosis and reduced aortic distensibility because of increased structural stiffness but preserved material stiffness. Elastin-deficient SMCs exhibited greater contractile-to-proliferative phenotypic modulation in vitro than in vivo. We confirmed increased medial collagen without evidence of increased medial area or SMC number in a small ascending aorta with thickened media of a Williams syndrome subject. Deficient circumferential growth is the predominant mechanism for moderate obstructive aortic disease resulting from partial elastin deficiency. Our findings suggest that diverse aortic manifestations in Williams syndrome result from graded elastin content, and SMC hyperplasia causing medial expansion requires additional elastin loss superimposed on ELN haploinsufficiency. © 2017 American Heart Association, Inc.

Glucagon-like peptide-1, glucose homeostasis and diabetes

DEFF Research Database (Denmark)

Holst, Jens Juul; Deacon, Carolyn F; Vilsbøll, Tina

2008-01-01

Incretins, enhancers of insulin secretion, are essential for glucose tolerance, and a reduction in their function might contribute to poor beta-cell function in patients with type-2 diabetes mellitus. However, at supraphysiological doses, the incretin glucagon-like peptide-1 (GLP-1) protects...... as therapies for type-2 diabetes and have recently reached the market. The pathophysiological basis the clinical use of these therapeutics is reviewed here....

Loss of alpha-like MeV fusion products from TFTR

International Nuclear Information System (INIS)

Zweben, S.J.; Boivin, R.L.; Diesso, M.; Hayes, S.E.; Hendel, H.W.; Park, H.; Strachan, J.D.

1990-03-01

A detailed comparison between the observed and expected loss of alpha-like MeV fusion products in TFTR is presented. The D-D fusion products (mainly the 1 MeV triton) were measured with an 2-D imaging scintillation detector. The expected first-orbit loss was calculated with a simple Lorentz orbit code. In almost all cases the measured loss was consistent with the expected first-orbit loss model. Exceptions are noted for small major radius plasmas and during strong MHD activity. 37 refs., 28 figs

Exaggerated secretion of glucagon-like peptide-1 (GLP-1) could cause reactive hypoglycaemia

DEFF Research Database (Denmark)

Toft-Nielsen, M; Madsbad, Sten; Holst, Jens Juul

1998-01-01

The plasma concentrations of the insulinotropic incretin hormone, glucagon-like peptide-1 (GLP-1) are abnormally high after oral glucose in partially gastrectomised subjects with reactive hypoglycaemia, suggesting a causal relationship. Because of the glucose-dependency of its effects, it is impo......The plasma concentrations of the insulinotropic incretin hormone, glucagon-like peptide-1 (GLP-1) are abnormally high after oral glucose in partially gastrectomised subjects with reactive hypoglycaemia, suggesting a causal relationship. Because of the glucose-dependency of its effects...

A wrinkle in flight: the role of elastin fibres in the mechanical behaviour of bat wing membranes.

Science.gov (United States)

Cheney, Jorn A; Konow, Nicolai; Bearnot, Andrew; Swartz, Sharon M

2015-05-06

Bats fly using a thin wing membrane composed of compliant, anisotropic skin. Wing membrane skin deforms dramatically as bats fly, and its three-dimensional configurations depend, in large part, on the mechanical behaviour of the tissue. Large, macroscopic elastin fibres are an unusual mechanical element found in the skin of bat wings. We characterize the fibre orientation and demonstrate that elastin fibres are responsible for the distinctive wrinkles in the surrounding membrane matrix. Uniaxial mechanical testing of the wing membrane, both parallel and perpendicular to elastin fibres, is used to distinguish the contribution of elastin and the surrounding matrix to the overall membrane mechanical behaviour. We find that the matrix is isotropic within the plane of the membrane and responsible for bearing load at high stress; elastin fibres are responsible for membrane anisotropy and only contribute substantially to load bearing at very low stress. The architecture of elastin fibres provides the extreme extensibility and self-folding/self-packing of the wing membrane skin. We relate these findings to flight with membrane wings and discuss the aeromechanical significance of elastin fibre pre-stress, membrane excess length, and how these parameters may aid bats in resisting gusts and preventing membrane flutter. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

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....

Induction of macrophage chemotaxis by aortic extracts from patients with Marfan syndrome is related to elastin binding protein.

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Gao Guo

Full Text Available Marfan syndrome is an autosomal dominantly inherited disorder of connective tissue with prominent skeletal, ocular, and cardiovascular manifestations. Aortic aneurysm and dissection are the major determinants of premature death in untreated patients. In previous work, we showed that extracts of aortic tissues from the mgR mouse model of Marfan syndrome showed increased chemotactic stimulatory activity related to the elastin-binding protein. Aortic samples were collected from 6 patients with Marfan syndrome and 8 with isolated aneurysms of the ascending aorta. Control samples were obtained from 11 organ donors without known vascular or connective tissue diseases. Soluble proteins extracted from the aortic samples of the two patient groups were compared against buffer controls and against the aortic samples from controls with respect to the ability to induce macrophage chemotaxis as measured using a modified Boyden chamber, as well as the reactivity to a monoclonal antibody BA4 against bioactive elastin peptides using ELISA. Samples from Marfan patients displayed a statistically significant increase in chemotactic inductive activity compared to control samples. Additionally, reactivity to BA4 was significantly increased. Similar statistically significant increases were identified for the samples from patients with idiopathic thoracic aortic aneurysm. There was a significant correlation between the chemotactic index and BA4 reactivity, and the increases in chemotactic activity of extracts from Marfan patients could be inhibited by pretreatment with lactose, VGVAPG peptides, or BA4, which indicates the involvement of EBP in mediating the effects. Our results demonstrate that aortic extracts of patients with Marfan syndrome can elicit macrophage chemotaxis, similar to our previous study on aortic extracts of the mgR mouse model of Marfan syndrome (Guo et al., Circulation 2006; 114:1855-62.

Induction of Macrophage Chemotaxis by Aortic Extracts from Patients with Marfan Syndrome Is Related to Elastin Binding Protein

Science.gov (United States)

Guo, Gao; Gehle, Petra; Doelken, Sandra; Martin-Ventura, José Luis; von Kodolitsch, Yskert; Hetzer, Roland; Robinson, Peter N.

2011-01-01

Marfan syndrome is an autosomal dominantly inherited disorder of connective tissue with prominent skeletal, ocular, and cardiovascular manifestations. Aortic aneurysm and dissection are the major determinants of premature death in untreated patients. In previous work, we showed that extracts of aortic tissues from the mgR mouse model of Marfan syndrome showed increased chemotactic stimulatory activity related to the elastin-binding protein. Aortic samples were collected from 6 patients with Marfan syndrome and 8 with isolated aneurysms of the ascending aorta. Control samples were obtained from 11 organ donors without known vascular or connective tissue diseases. Soluble proteins extracted from the aortic samples of the two patient groups were compared against buffer controls and against the aortic samples from controls with respect to the ability to induce macrophage chemotaxis as measured using a modified Boyden chamber, as well as the reactivity to a monoclonal antibody BA4 against bioactive elastin peptides using ELISA. Samples from Marfan patients displayed a statistically significant increase in chemotactic inductive activity compared to control samples. Additionally, reactivity to BA4 was significantly increased. Similar statistically significant increases were identified for the samples from patients with idiopathic thoracic aortic aneurysm. There was a significant correlation between the chemotactic index and BA4 reactivity, and the increases in chemotactic activity of extracts from Marfan patients could be inhibited by pretreatment with lactose, VGVAPG peptides, or BA4, which indicates the involvement of EBP in mediating the effects. Our results demonstrate that aortic extracts of patients with Marfan syndrome can elicit macrophage chemotaxis, similar to our previous study on aortic extracts of the mgR mouse model of Marfan syndrome (Guo et al., Circulation 2006; 114:1855-62). PMID:21647416

Identification of a human protein-derived HIV-1 fusion inhibitor targeting the gp41 fusion core structure.

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Lijun Chao

Full Text Available The HIV-1 envelope glycoprotein (Env gp41 plays a crucial role in the viral fusion process. The peptides derived from the C-terminal heptad repeat (CHR of gp41 are potent HIV fusion inhibitors. However, the activity of these anti-HIV-1 peptides in vivo may be attenuated by their induction of anti-gp41 antibodies. Thus, it is essential to identify antiviral peptides or proteins with low, or no, immunogenicity to humans. Here, we found that the C-terminal fragment (aa 462-521 of the human POB1 (the partner of RalBP1, designated C60, is an HIV-1 fusion inhibitor. It bound to N36, the peptide derived from the N-terminal heptad repeat (NHR of gp41, and to the six-helix bundle (6-HB formed by N36 and C34, a CHR-peptide, but it did not bind to C34. Unlike the CHR-peptides, C60 did not block gp41 6-HB formation. Rather, results suggest that C60 inhibits HIV-1 fusion by binding to the 6-HB, in particular, the residues in the gp41 NHR domain that are exposed on the surface of 6-HB. Since 6-HB plays a crucial role in the late stage of fusion between the viral envelope and endosomal membrane during the endocytic process of HIV-1, C60 may serve as a host restriction factor to suppress HIV-1 entry into CD4+ T lymphocytes. Taken together, it can be concluded from these results that C60 can be used as a lead for the development of anti-HIV-1 therapeutics or microbicides for the treatment and prevention of HIV-1 infection, as well as a molecular probe to study the fusogenic mechanism of HIV-1.

Plasma glucagon-like peptide 1 and peptide YY levels are not altered in symptomatic fructose-sorbitol malabsorption

DEFF Research Database (Denmark)

Valeur, Jørgen; Øines, Eliann; Morken, Mette Helvik

2008-01-01

consecutive patients with functional abdominal complaints, referred to our clinic for investigation of self-reported food hypersensitivity, were included in the study and compared with 15 healthy volunteers. All subjects ingested a mixture of 25 g fructose and 5 g sorbitol. Pulmonary hydrogen and methane...... excretion and plasma glucagon-like peptide 1 (GLP-1) and peptide YY (PYY) levels were measured during the next 3 h. Both habitual and post-test symptoms were assessed. RESULTS: Malabsorption of fructose and sorbitol was present in 61% of the patients and 73% of the controls. Nevertheless, the patients...

Neutrophil elastase-induced elastin degradation mediates macrophage influx and lung injury in 60% O2-exposed neonatal rats.

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Masood, Azhar; Yi, Man; Belcastro, Rosetta; Li, Jun; Lopez, Lianet; Kantores, Crystal; Jankov, Robert P; Tanswell, A Keith

2015-07-01

Neutrophil (PMNL) influx precedes lung macrophage (LM) influx into the lung following exposure of newborn pups to 60% O2. We hypothesized that PMNL were responsible for the signals leading to LM influx. This was confirmed when inhibition of PMNL influx with a CXC chemokine receptor-2 antagonist, SB-265610, also prevented the 60% O2-dependent LM influx, LM-derived nitrotyrosine formation, and pruning of small arterioles. Exposure to 60% O2 was associated with increased lung contents of neutrophil elastase and α-elastin, a marker of denatured elastin, and a decrease in elastin fiber density. This led us to speculate that neutrophil elastase-induced elastin fragments were the chemokines that led to a LM influx into the 60% O2-exposed lung. Inhibition of neutrophil elastase with sivelestat or elafin attenuated the LM influx. Sivelestat also attenuated the 60% O2-induced decrease in elastin fiber density. Daily injections of pups with an antibody to α-elastin prevented the 60% O2-dependent LM influx, impaired alveologenesis, and impaired small vessel formation. This suggests that neutrophil elastase inhibitors may protect against neonatal lung injury not only by preventing structural elastin degradation, but also by blocking elastin fragment-induced LM influx, thus preventing tissue injury from LM-derived peroxynitrite formation. Copyright © 2015 the American Physiological Society.

Chemical hydrogels based on a hyaluronic acid-graft-α-elastin derivative as potential scaffolds for tissue engineering.

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Palumbo, Fabio Salvatore; Pitarresi, Giovanna; Fiorica, Calogero; Rigogliuso, Salvatrice; Ghersi, Giulio; Giammona, Gaetano

2013-07-01

In this work hyaluronic acid (HA) functionalized with ethylenediamine (EDA) has been employed to graft α-elastin. In particular a HA-EDA derivative bearing 50 mol% of pendant amino groups has been successfully employed to produce the copolymer HA-EDA-g-α-elastin containing 32% w/w of protein. After grafting with α-elastin, remaining free amino groups reacted with ethylene glycol diglycidyl ether (EGDGE) for producing chemical hydrogels, proposed as scaffolds for tissue engineering. Swelling degree, resistance to chemical and enzymatic hydrolysis, as well as preliminary biological properties of HA-EDA-g-α-elastin/EGDGE scaffold have been evaluated and compared with a HA-EDA/EGDGE scaffold. The presence of α-elastin grafted to HA-EDA improves attachment, viability and proliferation of primary rat dermal fibroblasts and human umbilical artery smooth muscle cells. Biological performance of HA-EDA-g-α-elastin/EGDGE scaffold resulted comparable to that of a commercial collagen type I sponge (Antema®), chosen as a positive control. Copyright © 2013 Elsevier B.V. All rights reserved.

Aspartic acid racemisation in purified elastin from arteries as basis for age estimation.

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Dobberstein, R C; Tung, S-M; Ritz-Timme, S

2010-07-01

Aspartic acid racemisation (AAR) results in an age-dependent accumulation of D: -aspartic acid in durable human proteins and can be used as a basis for age estimation. Routinely, age estimation based on AAR is performed by analysis of dentine. However, in forensic practise, teeth are not always available. Non-dental tissues for age estimation may be suitable for age estimation based on AAR if they contain durable proteins that can be purified and analysed. Elastin is such a durable protein. To clarify if purified elastin from arteries is a suitable sample for biochemical age estimation, AAR was determined in purified elastin from arteries from individuals of known age (n = 68 individuals, including n = 15 putrefied corpses), considering the influence of different stages of atherosclerosis and putrefaction on the AAR values. AAR was found to increase with age. The relationship between AAR and age was good enough to serve as basis for age estimation, but worse than known from dentinal proteins. Intravital and post-mortem degradation of elastin may have a moderate effect on the AAR values. Age estimation based on AAR in purified elastin from arteries may be a valuable additional tool in the identification of unidentified cadavers, especially in cases where other methods cannot be applied (e.g., no available teeth and body parts).

Somatic Cell Fusions Reveal Extensive Heterogeneity in Basal-like Breast Cancer

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Ying Su

2015-06-01

Full Text Available Basal-like and luminal breast tumors have distinct clinical behavior and molecular profiles, yet the underlying mechanisms are poorly defined. To interrogate processes that determine these distinct phenotypes and their inheritance pattern, we generated somatic cell fusions and performed integrated genetic and epigenetic (DNA methylation and chromatin profiling. We found that the basal-like trait is generally dominant and is largely defined by epigenetic repression of luminal transcription factors. Definition of super-enhancers highlighted a core program common in luminal cells but a high degree of heterogeneity in basal-like breast cancers that correlates with clinical outcome. We also found that protein extracts of basal-like cells are sufficient to induce a luminal-to-basal phenotypic switch, implying a trigger of basal-like autoregulatory circuits. We determined that KDM6A might be required for luminal-basal fusions, and we identified EN1, TBX18, and TCF4 as candidate transcriptional regulators of the luminal-to-basal switch. Our findings highlight the remarkable epigenetic plasticity of breast cancer cells.

Streptavidin-binding peptides and uses thereof

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Szostak, Jack W. (Inventor); Wilson, David S. (Inventor); Keefe, Anthony D. (Inventor)

2006-01-01

The invention provides peptides with high affinity for streptavidin. These peptides may be expressed as part of fusion proteins to facilitate the detection, quantitation, and purification of proteins of interest.

On the mechanical role of de novo synthesized elastin in the urinary bladder wall

NARCIS (Netherlands)

Wognum, Silvia; Schmidt, David E.; Sacks, Michael S.

2009-01-01

The urinary bladder wall (UBW), which is composed of smooth muscle, collagen, and elastin, undergoes profound remodeling in response to changes in mechanical loading resulting from various pathologies. In our laboratory, we have observed the production of fibrillar elastin in the extracellular

Multiphoton microscopy observations of 3D elastin and collagen fiber microstructure changes during pressurization in aortic media.

Science.gov (United States)

Sugita, Shukei; Matsumoto, Takeo

2017-06-01

Elastin and collagen fibers play important roles in the mechanical properties of aortic media. Because knowledge of local fiber structures is required for detailed analysis of blood vessel wall mechanics, we investigated 3D microstructures of elastin and collagen fibers in thoracic aortas and monitored changes during pressurization. Using multiphoton microscopy, autofluorescence images from elastin and second harmonic generation signals from collagen were acquired in media from rabbit thoracic aortas that were stretched biaxially to restore physiological dimensions. Both elastin and collagen fibers were observed in all longitudinal-circumferential plane images, whereas alternate bright and dark layers were observed along the radial direction and were recognized as elastic laminas (ELs) and smooth muscle-rich layers (SMLs), respectively. Elastin and collagen fibers are mainly oriented in the circumferential direction, and waviness of collagen fibers was significantly higher than that of elastin fibers. Collagen fibers were more undulated in longitudinal than in radial direction, whereas undulation of elastin fibers was equibiaxial. Changes in waviness of collagen fibers during pressurization were then evaluated using 2-dimensional fast Fourier transform in mouse aortas, and indices of waviness of collagen fibers decreased with increases in intraluminal pressure. These indices also showed that collagen fibers in SMLs became straight at lower intraluminal pressures than those in EL, indicating that SMLs stretched more than ELs. These results indicate that deformation of the aorta due to pressurization is complicated because of the heterogeneity of tissue layers and differences in elastic properties of ELs, SMLs, and surrounding collagen and elastin.

Neural regulation of glucagon-like peptide-1 secretion in pigs

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Hansen, Lene; Lampert, Sarah; Mineo, Hitoshi

2004-01-01

Glucagon-like peptide (GLP)-1 is secreted rapidly from the intestine postprandially. We therefore investigated its possible neural regulation. With the use of isolated perfused porcine ileum, GLP-1 secretion was measured in response to electrical stimulation of the mixed, perivascular nerve supply...

Intracellular delivery of cell-penetrating peptide-transcriptional factor fusion protein and its role in selective osteogenesis

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Suh JS

2014-03-01

Full Text Available Jin Sook Suh,1,* Jue Yeon Lee,2,* Yoon Jung Choi,1 Hyung Keun You,3 Seong-Doo Hong,4 Chong Pyoung Chung,2 Yoon Jeong Park1,2 1Dental Regenerative Biotechnology, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, 2Central Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC, Seoul, 3Department of Periodontology, College of Dentistry, Wonkwang University, Iksan, 4Department of Oral Pathology, School of Dentistry, Seoul National University, Seoul, Republic of Korea *These authors contributed equally to this work Abstract: Protein-transduction technology has been attempted to deliver macromolecular materials, including protein, nucleic acids, and polymeric drugs, for either diagnosis or therapeutic purposes. Herein, fusion protein composed of an arginine-rich cell-penetrating peptide, termed low-molecular-weight protamine (LMWP, and a transcriptional coactivator with a PDZ-binding motif (TAZ protein was prepared and applied in combination with biomaterials to increase bone-forming capacity. TAZ has been recently identified as a specific osteogenic stimulating transcriptional coactivator in human mesenchymal stem cell (hMSC differentiation, while simultaneously blocking adipogenic differentiation. However, TAZ by itself cannot penetrate the cells, and thus needs a transfection tool for translocalization. The LMWP-TAZ fusion proteins were efficiently translocalized into the cytosol of hMSCs. The hMSCs treated with cell-penetrating LMWP-TAZ exhibited increased expression of osteoblastic genes and protein, producing significantly higher quantities of mineralized matrix compared to free TAZ. In contrast, adipogenic differentiation of the hMSCs was blocked by treatment of LMWP-TAZ fusion protein, as reflected by reduced marker-protein expression, adipocyte fatty acid-binding protein 2, and peroxisome proliferator-activated receptor-γ messenger ribonucleic acid levels. LMWP-TAZ was applied in

Measurement of MMP-9 and -12 degraded elastin (ELM) provides unique information on lung tissue degradation

DEFF Research Database (Denmark)

Skjøt-Arkil, Helene; Clausen, Rikke E; Nguyen, Quoc Hai Trieu

2012-01-01

Elastin is an essential component of selected connective tissues that provides a unique physiological elasticity. Elastin may be considered a signature protein of lungs where matrix metalloprotease (MMP) -9-and -12, may be considered the signature proteases of the macrophages, which in part...... are responsible for tissue damage during disease progression. Thus, we hypothesized that a MMP-9/-12 generated fragment of elastin may be a relevant biochemical maker for lung diseases....

A mucin-like peptide from Fasciola hepatica induces parasite-specific Th1-type cell immunity.

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Noya, Verónica; Brossard, Natalie; Berasaín, Patricia; Rodríguez, Ernesto; Chiale, Carolina; Mazal, Daniel; Carmona, Carlos; Freire, Teresa

2016-03-01

Fasciolosis, caused by the liver fluke Fasciola hepatica, is a major parasitic disease of livestock that causes significant economic losses worldwide. Although drugs are effective against liver flukes, they do not prevent reinfection, and continuous treatment is costly. Moreover, resistant fluke strains are emerging. In this context, vaccination is a good alternative since it provides a cost-effective long-term prevention strategy to control fasciolosis. In this paper, we evaluate the Fhmuc peptide as a potential vaccine against fasciolosis. This peptide derives from a mucin-like protein highly expressed in the infective stage of Fasciola hepatica. Mucin-like molecules expressed by parasites can contribute to several infection processes by protecting the parasite from host proteases and recognition by the immune system. We show that the Fhmuc peptide induces Th1-like immune responses specific for F. hepatica excretion-secretion products (FhESP) with a high production of IFNγ. We also investigated whether this peptide could protect animals from infection, and present preliminary data indicating that animals treated with Fhmuc exhibited reduced liver damage compared to non-immunised animals and that this protection was associated with a recruitment of B and T lymphocytes in the peritoneum, as well as eosinophils and mature dendritic cells. These results suggest that the mucin-like peptide Fhmuc could constitute a potential vaccine candidate against fasciolosis and pave the way towards the development of vaccines against parasites.

Formation of active inclusion bodies induced by hydrophobic self-assembling peptide GFIL8.

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Wang, Xu; Zhou, Bihong; Hu, Weike; Zhao, Qing; Lin, Zhanglin

2015-06-16

In the last few decades, several groups have observed that proteins expressed as inclusion bodies (IBs) in bacteria could still be biologically active when terminally fused to an appropriate aggregation-prone partner such as pyruvate oxidase from Paenibacillus polymyxa (PoxB). More recently, we have demonstrated that three amphipathic self-assembling peptides, an alpha helical peptide 18A, a beta-strand peptide ELK16, and a surfactant-like peptide L6KD, have properties that induce target proteins into active IBs. We have developed an efficient protein expression and purification approach for these active IBs by introducing a self-cleavable intein molecule. In this study, the self-assembling peptide GFIL8 (GFILGFIL) with only hydrophobic residues was analyzed, and this peptide effectively induced the formation of cytoplasmic IBs in Escherichia coli when terminally attached to lipase A and amadoriase II. The protein aggregates in cells were confirmed by transmission electron microscopy analysis and retained ~50% of their specific activities relative to the native counterparts. We constructed an expression and separation coupled tag (ESCT) by incorporating an intein molecule, the Mxe GyrA intein. Soluble target proteins were successfully released from active IBs upon cleavage of the intein between the GFIL8 tag and the target protein, which was mediated by dithiothreitol. A variant of GFIL8, GFIL16 (GFILGFILGFILGFIL), improved the ESCT scheme by efficiently eliminating interference from the soluble intein-GFIL8 molecule. The yields of target proteins at the laboratory scale were 3.0-7.5 μg/mg wet cell pellet, which is comparable to the yields from similar ESCT constructs using 18A, ELK16, or the elastin-like peptide tag scheme. The all-hydrophobic self-assembling peptide GFIL8 induced the formation of active IBs in E. coli when terminally attached to target proteins. GFIL8 and its variant GFIL16 can act as a "pull-down" tag to produce purified soluble proteins with

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.

Elastin Based Cell-laden Injectable Hydrogels with Tunable Gelation, Mechanical and Biodegradation Properties

Science.gov (United States)

Fathi, Ali; Mithieux, Suzanne M.; Wei, Hua; Chrzanowski, Wojciech; Valtchev, Peter; Weiss, Anthony S.; Dehghani, Fariba

2015-01-01

Injectable hydrogels made from extracellular matrix proteins such as elastin show great promise for various biomedical applications. Use of cytotoxic reagents, fixed gelling behavior, and lack of mechanical strength in these hydrogels are the main associated drawbacks. The aim of this study was to develop highly cytocompatible and injectable elastin-based hydrogels with alterable gelation characteristics, favorable mechanical properties and structural stability for load bearing applications. A thermoresponsive copolymer, poly(N-isopropylacrylamide-co-polylactide-2-hydroxyethyl methacrylate-co-oligo(ethylene glycol)monomethyl ether methacrylate, was functionalized with succinimide ester groups by incorporating N-acryloxysuccinimide monomer. These ester groups were exploited to covalently bond this polymer, denoted as PNPHO, to different proteins with primary amine groups such as α-elastin in aqueous media. The incorporation of elastin through covalent bond formation with PNPHO promotes the structural stability, mechanical properties and live cell proliferation within the structure of hydrogels. Our results demonstrated that elastin-co-PNPHO solutions were injectable through fine gauge needles and converted to hydrogels in situ at 37 °C in the absence of any crosslinking reagent. By altering PNPHO content, the gelling time of these hydrogels can be finely tuned within the range of 2 to 15 min to ensure compatibility with surgical requirements. In addition, these hydrogels exhibited compression moduli in the range of 40 to 145 kPa, which are substantially higher than those of previously developed elastin-based hydrogels. These hydrogels were highly stable in the physiological environment with the evidence of 10 wt% mass loss in 30 days of incubation in a simulated environment. This class of hydrogels is in vivo bioabsorbable due to the gradual increase of the lower critical solution temperature of the copolymer to above 37 °C due to the cleavage of polylactide from

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.

Distribution and activity of a Dippu DH31-like peptide in the large milkweed bug Oncopeltus fasciatus.

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Te Brugge, V A; Orchard, I

2008-02-01

The milkweed bug, Oncopeltus fasciatus, is a plant feeding hemipteran. While there has been much research done on the neurohormonal control of the post-feeding diuresis in the blood-feeding hemipteran, Rhodnius prolixus, little is known about the control of the post-feeding diuresis in O. fasciatus. One of the neurohormones that may play a role in this rapid diuresis belongs to the calcitonin-like diuretic hormone (DH31) family of insect peptides. In this study we demonstrate the presence of DH31-like immunoreactivity in the central nervous system (CNS) and gut of O. fasciatus 5th instars. As well, DH31-like material was quantified and partially purified from the CNS of 5th instar O. fasciatus using reversed-phase liquid chromatography (RPLC) and monitored with an enzyme-linked immunosorbent assay (ELISA). When tested on O. fasciatus 5th instar Malpighian tubules, DH31-like peptides significantly increased the rate of secretion over saline controls. The results suggest that there is a DH31-like peptide(s) present in the CNS of O. fasciatus and that this peptide may play a role in the control of Malpighian tubule secretion.

Unprecedented high insulin secretion in a healthy human subject after intravenous glucagon-like peptide-1

DEFF Research Database (Denmark)

Knop, Filip K; Lund, Asger; Madsbad, Sten

2014-01-01

BACKGROUND: The gut-derived incretin hormones, glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1, are released in response to ingestion of nutrients. Both hormones are highly insulinotropic in strictly glucose-dependent fashions and glucagon-like peptide-1 is often referred...... to as one of the most insulinotropic substances known. CASE PRESENTATION: Plasma insulin and C-peptide concentrations were measured in a healthy Caucasian male (age: 53 years; body mass index: 28.6 kg/m2; fasting plasma glucose: 5.7 mM; 2 h plasma glucose value following 75 g-oral glucose tolerance test: 3...

Assessment of Myocardial Remodeling Using an Elastin/Tropoelastin Specific Agent with High Field Magnetic Resonance Imaging (MRI)

OpenAIRE

Protti, Andrea; Lavin, Begoña; Dong, Xuebin; Lorrio, Silvia; Robinson, Simon; Onthank, David; Shah, Ajay M; Botnar, Rene M

2015-01-01

BACKGROUND: Well-defined inflammation, proliferation, and maturation phases orchestrate the remodeling of the injured myocardium after myocardial infarction (MI) by controlling the formation of new extracellular matrix. The extracellular matrix consists mainly of collagen but also fractions of elastin. It is thought that elastin is responsible for maintaining elastic properties of the myocardium, thus reducing the risk of premature rupture. An elastin/tropoelastin-specific contrast agent (Gd-...

Lung matrix and vascular remodeling in mechanically ventilated elastin haploinsufficient newborn mice

Science.gov (United States)

Hilgendorff, Anne; Parai, Kakoli; Ertsey, Robert; Navarro, Edwin; Jain, Noopur; Carandang, Francis; Peterson, Joanna; Mokres, Lucia; Milla, Carlos; Preuss, Stefanie; Alcazar, Miguel Alejandre; Khan, Suleman; Masumi, Juliet; Ferreira-Tojais, Nancy; Mujahid, Sana; Starcher, Barry; Rabinovitch, Marlene

2014-01-01

Elastin plays a pivotal role in lung development. We therefore queried if elastin haploinsufficient newborn mice (Eln+/−) would exhibit abnormal lung structure and function related to modified extracellular matrix (ECM) composition. Because mechanical ventilation (MV) has been linked to dysregulated elastic fiber formation in the newborn lung, we also asked if elastin haploinsufficiency would accentuate lung growth arrest seen after prolonged MV of neonatal mice. We studied 5-day-old wild-type (Eln+/+) and Eln+/− littermates at baseline and after MV with air for 8–24 h. Lungs of unventilated Eln+/− mice contained ∼50% less elastin and ∼100% more collagen-1 and lysyl oxidase compared with Eln+/+ pups. Eln+/− lungs contained fewer capillaries than Eln+/+ lungs, without discernible differences in alveolar structure. In response to MV, lung tropoelastin and elastase activity increased in Eln+/+ neonates, whereas tropoelastin decreased and elastase activity was unchanged in Eln+/− mice. Fibrillin-1 protein increased in lungs of both groups during MV, more in Eln+/− than in Eln+/+ pups. In both groups, MV caused capillary loss, with larger and fewer alveoli compared with unventilated controls. Respiratory system elastance, which was less in unventilated Eln+/− compared with Eln+/+ mice, was similar in both groups after MV. These results suggest that elastin haploinsufficiency adversely impacts pulmonary angiogenesis and that MV dysregulates elastic fiber integrity, with further loss of lung capillaries, lung growth arrest, and impaired respiratory function in both Eln+/+ and Eln+/− mice. Paucity of lung capillaries in Eln+/− newborns might help explain subsequent development of pulmonary hypertension previously reported in adult Eln+/− mice. PMID:25539853

HIV-1 gp41 Fusion Intermediate: A Target for HIV Therapeutics

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Chungen Pan

2010-02-01

Full Text Available Human immunodeficiency virus (HIV-1 infection is initiated by the binding of gp120 envelope glyco-protein to its cell receptor (CD4 and a coreceptor (CXCR4 or CCR5, followed by a series of conformational changes in the gp41 transmembrane subunit. These changes include insertion of fusion peptide into the target cell membrane and association of C-heptad repeat (CHR peptide with the N-heptad repeat (NHR trimer, a pre-hairpin fusion intermediate. A stable six-helix bundle core is then formed, bringing the viral envelope and target cell membrane into close proximity for fusion. Peptides derived from the CHR region, such as T20 and C34, inhibit HIV-1 fusion by interacting with the gp41 fusion intermediate. A number of anti-HIV-1 peptides and small molecule compounds targeting the gp41 NHR-trimer have been identified. By combining HIV fusion/entry inhibitors targeting different sites in the gp41 fusion intermediate, a potent synergistic effect takes place, resulting in a potential new therapeutic strategy for the HIV infection/AIDS. Here, we present an overview of the current development of anti-HIV drugs, particularly those targeting the gp41 fusion intermediate.

Impaired elastin deposition in Fstl1-/- lung allograft under the renal capsule.

Directory of Open Access Journals (Sweden)

Yan Geng

Full Text Available Lung alveolar development in late gestation is a process important to postnatal survival. Follistatin-like 1 (Fstl1 is a matricellular protein of the Bmp antagonist class, which is involved in the differentiation/maturation of alveolar epithelial cells during saccular stage of lung development. This study investigates the role of Fstl1 on elastin deposition in mesenchyme and subsequent secondary septation in the late gestation stage of terminal saccular formation. To this aim, we modified the renal capsule allograft model for lung organ culture by grafting diced E15.5 distal lung underneath the renal capsule of syngeneic host and cultured up to 7 days. The saccular development of the diced lung allografts, as indicated by the morphology, epithelial and vascular developments, occurred in a manner similar to that in utero. Fstl1 deficiency caused atelectatic phenotype companied by impaired epithelial differentiation in D3 Fstl1(-/- lung allografts, which is similar to that of E18.5 Fstl1(-/- lungs, supporting the role of Fstl1 during saccular stage. Inhibition of Bmp signaling by intraperitoneal injection of dorsomorphin in the host mice rescued the pulmonary atelectasis of D3 Fstl1(-/- allografts. Furthermore, a marked reduction in elastin expression and deposition was observed in walls of air sacs of E18.5 Fstl1(-/- lungs and at the tips of the developing alveolar septae of D7 Fstl1(-/- allografts. Thus, in addition to its role on alveolar epithelium, Fstl1 is crucial for elastin expression and deposition in mesenchyme during lung alveologenesis. Our data demonstrates that the modified renal capsule allograft model for lung organ culture is a robust and efficient technique to increase our understanding of saccular stage of lung development.

Complete sequences of glucagon-like peptide-1 from human and pig small intestine

DEFF Research Database (Denmark)

Orskov, C; Bersani, M; Johnsen, A H

1989-01-01

intestine of the proglucagon precursor were determined by pairs of basic amino acid residues flanking the two peptides. Earlier studies have shown that synthetic glucagon-like peptide-1 (GLP-1) synthesized according to the proposed structure (proglucagon 71-108 or because residue 108 is Gly, 72-107 amide......) had no physiological effects, whereas a truncated from of GLP-1, corresponding to proglucagon 78-107 amide, strongly stimulated insulin secretion and depressed glucagon secretion. To determine the amino acid sequence of the naturally occurring peptide we isolated GLP-1 from human small intestine...

Design, construction, and characterization of high-performance membrane fusion devices with target-selectivity.

Science.gov (United States)

Kashiwada, Ayumi; Yamane, Iori; Tsuboi, Mana; Ando, Shun; Matsuda, Kiyomi

2012-01-31

Membrane fusion proteins such as the hemagglutinin glycoprotein have target recognition and fusion accelerative domains, where some synergistically working elements are essential for target-selective and highly effective native membrane fusion systems. In this work, novel membrane fusion devices bearing such domains were designed and constructed. We selected a phenylboronic acid derivative as a recognition domain for a sugar-like target and a transmembrane-peptide (Leu-Ala sequence) domain interacting with the target membrane, forming a stable hydrophobic α-helix and accelerating the fusion process. Artificial membrane fusion behavior between the synthetic devices in which pilot and target liposomes were incorporated was characterized by lipid-mixing and inner-leaflet lipid-mixing assays. Consequently, the devices bearing both the recognition and transmembrane domains brought about a remarkable increase in the initial rate for the membrane fusion compared with the devices containing the recognition domain alone. In addition, a weakly acidic pH-responsive device was also constructed by replacing three Leu residues in the transmembrane-peptide domain by Glu residues. The presence of Glu residues made the acidic pH-dependent hydrophobic α-helix formation possible as expected. The target-selective liposome-liposome fusion was accelerated in a weakly acidic pH range when the Glu-substituted device was incorporated in pilot liposomes. The use of this pH-responsive device seems to be a potential strategy for novel applications in a liposome-based delivery system. © 2011 American Chemical Society

Moyamoya disease and artery tortuosity as rare phenotypes in a patient with an elastin mutation.

Science.gov (United States)

Ishiwata, Tsukasa; Tanabe, Nobuhiro; Shigeta, Ayako; Yokota, Hajime; Tsushima, Kenji; Terada, Jiro; Sakao, Seiichiro; Morisaki, Hiroko; Morisaki, Takayuki; Tatsumi, Koichiro

2016-07-01

Sporadic and familial elastin mutations can occur in large vessel stenosis such as supravalvular aortic stenosis and narrowing of the descending aorta. However, there are very few reports regarding the arteriopathy of cerebral, pulmonary or abdominal arteries in elastin mutations. We herein report the case of a Japanese female patient presenting with multiple arteriopathy including moyamoya disease, a tortuosity of abdominal arteries and pulmonary hypertension due to peripheral pulmonary artery stenosis. This case suggests the possible progression of cerebral arteriopathy including moyamoya disease in patients with elastin mutations. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Ultrasonic delineation of aortic microstructure: The relative contribution of elastin and collagen to aortic elasticity

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Marsh, Jon N.; Takiuchi, Shin; Lin, Shiow Jiuan; Lanza, Gregory M.; Wickline, Samuel A.

2004-05-01

Aortic elasticity is an important factor in hemodynamic health, and compromised aortic compliance affects not only arterial dynamics but also myocardial function. A variety of pathologic processes (e.g., diabetes, Marfan's syndrome, hypertension) can affect aortic elasticity by altering the microstructure and composition of the elastin and collagen fiber networks within the tunica media. Ultrasound tissue characterization techniques can be used to obtain direct measurements of the stiffness coefficients of aorta by measurement of the speed of sound in specific directions. In this study we sought to define the contributions of elastin and collagen to the mechanical properties of aortic media by measuring the magnitude and directional dependence of the speed of sound before and after selective isolation of either the collagen or elastin fiber matrix. Formalin-fixed porcine aortas were sectioned for insonification in the circumferential, longitudinal, or radial direction and examined using high-frequency (50 MHz) ultrasound microscopy. Isolation of the collagen or elastin fiber matrices was accomplished through treatment with NaOH or formic acid, respectively. The results suggest that elastin is the primary contributor to aortic medial stiffness in the unloaded state, and that there is relatively little anisotropy in the speed of sound or stiffness in the aortic wall.

Impact of elastin incorporation into electrochemically aligned collagen fibers on mechanical properties and smooth muscle cell phenotype.

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Nguyen, Thuy-Uyen; Bashur, Chris A; Kishore, Vipuil

2016-03-17

Application of tissue-engineered vascular grafts (TEVGs) for the replacement of small-diameter arteries is limited due to thrombosis and intimal hyperplasia. Previous studies have attempted to address the limitations of TEVGs by developing scaffolds that mimic the composition (collagen and elastin) of native arteries to better match the mechanical properties of the graft with the native tissue. However, most existing scaffolds do not recapitulate the aligned topography of the collagen fibers found in native vessels. In the current study, based on the principles of isoelectric focusing, two different types of elastin (soluble and insoluble) were incorporated into highly oriented electrochemically aligned collagen (ELAC) fibers and the effect of elastin incorporation on the mechanical properties of the ELAC fibers and smooth muscle cell (SMC) phenotype was investigated. The results indicate that elastin incorporation significantly decreased the modulus of ELAC fibers to converge upon that of native vessels. Further, a significant increase in yield strain and decrease in Young's modulus was observed on all fibers post SMC culture compared with before the culture. Real-time polymerase chain reaction results showed a significant increase in the expression of α-smooth muscle actin and calponin on ELAC fibers with insoluble elastin, suggesting that incorporation of insoluble elastin induces a contractile phenotype in SMCs after two weeks of culture on ELAC fibers. Immunofluorescence results showed that calponin expression increased with time on all fibers. In conclusion, insoluble elastin incorporated ELAC fibers have the potential to be used for the development of functional TEVGs for the repair and replacement of small-diameter arteries.

Receptors for GRP/bombesin-like peptides in the rat forebrain

International Nuclear Information System (INIS)

Wolf, S.S.; Moody, T.W.

1985-01-01

Binding sites in the rat forebrain were characterized using ( 125 I-Tyr4)bombesin as a receptor probe. Pharmacology experiments indicate that gastrin releasing peptide (GRP) and the GRP fragments GRP as well as Ac-GRP inhibited radiolabeled (Tyr4)bombesin binding with high affinity. Biochemistry experiments indicated that heat, N-ethyl maleimide or trypsin greatly reduced radiolabeled (Tyr4)bombesin binding. Also, autoradiographic studies indicated that highest grain densities were present in the stria terminalis, periventricular and suprachiasmatic nucleus of the hypothalamus, dorsomedial and rhomboid thalamus, dentate gyrus, hippocampus and medial amygdaloid nucleus. The data suggest that CNS protein receptors, which are discretely distributed in the rat forebrain, may mediate the action of endogenous GRP/bombesin-like peptides

Hormone-like peptides in the venoms of marine cone snails

DEFF Research Database (Denmark)

Robinson, Samuel D.; Li, Qing; Bandyopadhyay, Pradip K.

2017-01-01

, paralysis and sensory overload. Most conotoxins target the prey's nervous system but evidence of venom peptides targeting neuroendocrine processes is emerging. Examples include vasopressin, RFamide neuropeptides and recently also insulin. To investigate the diversity of hormone/neuropeptide-like molecules...... but also form part of the injected venom cocktail, unambiguously demonstrating their role in envenomation. Our findings suggest that hormone/neuropeptide-like toxins are a diverse and integral part of the complex envenomation strategy of Conus. Exploration of this group of venom components offers...

Polymorphism of glucagon-like peptide-1 receptor gene (rs1042044 ...

African Journals Online (AJOL)

Previous investigations indicated that glucagon-like peptide-1 (GLP-1) played important roles in bone turnover via GLP-1 receptors (GLP1Rs) in postmenopausal state. Furthermore, polymorphisms in GLP1R gene were suggested to affect the function of GLP1Rs and be associated with many diseases. However, the ...

Design, Recombinant Fusion Expression and Biological Evaluation of Vasoactive Intestinal Peptide Analogue as Novel Antimicrobial Agent

Directory of Open Access Journals (Sweden)

Chunlan Xu

2017-11-01

Full Text Available Antimicrobial peptides represent an emerging category of therapeutic agents with remarkable structural and functional diversity. Modified vasoactive intestinal peptide (VIP (VIP analogue 8 with amino acid sequence “FTANYTRLRRQLAVRRYLAAILGRR” without haemolytic activity and cytotoxicity displayed enhanced antimicrobial activities against Staphylococcus aureus (S. aureus ATCC 25923 and Escherichia coli (E. coli ATCC 25922 than parent VIP even in the presence of 180 mM NaCl or 50 mM MgCl2, or in the range of pH 4–10. VIP analogue 8 was expressed as fusion protein thioredoxin (Trx-VIP8 in E. coli BL21(DE at a yield of 45.67 mg/L. The minimum inhibitory concentration (MIC of the recombinant VIP analogue 8 against S. aureus ATCC 25923 and E. coli ATCC 25922 were 2 μM. These findings suggest that VIP analogue 8 is a promising candidate for application as a new and safe antimicrobial agent.

Arterial extracellular matrix: a mechanobiological study of the contributions and interactions of elastin and collagen.

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Chow, Ming-Jay; Turcotte, Raphaël; Lin, Charles P; Zhang, Yanhang

2014-06-17

The complex network structure of elastin and collagen extracellular matrix (ECM) forms the primary load bearing components in the arterial wall. The structural and mechanobiological interactions between elastin and collagen are important for properly functioning arteries. Here, we examined the elastin and collagen organization, realignment, and recruitment by coupling mechanical loading and multiphoton imaging. Two-photon excitation fluorescence and second harmonic generation methods were performed with a multiphoton video-rate microscope to capture real time changes to the elastin and collagen structure during biaxial deformation. Enzymatic removal of elastin was performed to assess the structural changes of the remaining collagen structure. Quantitative analysis of the structural changes to elastin and collagen was made using a combination of two-dimensional fast Fourier transform and fractal analysis, which allows for a more complete understanding of structural changes. Our study provides new quantitative evidence, to our knowledge on the sequential engagement of different arterial ECM components in response to mechanical loading. The adventitial collagen exists as large wavy bundles of fibers that exhibit fiber engagement after 20% strain. The medial collagen is engaged throughout the stretching process, and prominent elastic fiber engagement is observed up to 20% strain after which the engagement plateaus. The fiber orientation distribution functions show remarkably different changes in the ECM structure in response to mechanical loading. The medial collagen shows an evident preferred circumferential distribution, however the fiber families of adventitial collagen are obscured by their waviness at no or low mechanical strains. Collagen fibers in both layers exhibit significant realignment in response to unequal biaxial loading. The elastic fibers are much more uniformly distributed and remained relatively unchanged due to loading. Removal of elastin produces

Isolation and characterization of the human parathyroid hormone-like peptide gene

International Nuclear Information System (INIS)

Mangin, M.; Ikeda, K.; Dreyer, B.E.; Broadus, A.E.

1989-01-01

A parathyroid hormone-like peptide (PTH-LP) has recently been identified in human tumors associated with the syndrome of humoral hypercalcemia of malignancy. The peptide appears to be encoded by a single-copy gene that gives rise to multiple mRNAs that are heterogeneous at both their 5' and their 3' ends. Alternative RNA splicing is responsible for the 3' heterogeneity and results in mRNAs encoding three different peptides, each with a unique C terminus. The authors have isolated and characterized the human PTHLP gene. The gene is a complex transcriptional unit spanning more than 12 kilobases of DNA and containing six exons. Two 5' exons encode distinct 5' untranslated regions and are separated by a putative promoter element, indicating that the gene either has two promoters or is alternatively spliced from a single promoter upstream of the first exon. The middle portion of the PTHLP gene, comprising exons 2-4, has an organizational pattern of introns and exons identical to that of the parathyroid hormone gene, consistent with a common ancestral origin of these two genes. Exon 4 of the PTHLP gene encodes the region common to all three peptides and the C terminus of the shortest peptide, and exons 5 and 6 encode the unique C termini of the other two peptides. Northern analysis of mRNAs from four human tumors of different histological types reveals the preferential use of 3' splicing patterns of individual tumors

The interaction with gold suppresses fiber-like conformations of the amyloid β (16-22) peptide

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Bellucci, Luca; Ardèvol, Albert; Parrinello, Michele; Lutz, Helmut; Lu, Hao; Weidner, Tobias; Corni, Stefano

2016-04-01

Inorganic surfaces and nanoparticles can accelerate or inhibit the fibrillation process of proteins and peptides, including the biomedically relevant amyloid β peptide. However, the microscopic mechanisms that determine such an effect are still poorly understood. By means of large-scale, state-of-the-art enhanced sampling molecular dynamics simulations, here we identify an interaction mechanism between the segments 16-22 of the amyloid β peptide, known to be fibrillogenic by itself, and the Au(111) surface in water that leads to the suppression of fiber-like conformations from the peptide conformational ensemble. Moreover, thanks to advanced simulation analysis techniques, we characterize the conformational selection vs. induced fit nature of the gold effect. Our results disclose an inhibition mechanism that is rooted in the details of the microscopic peptide-surface interaction rather than in general phenomena such as peptide sequestration from the solution.Inorganic surfaces and nanoparticles can accelerate or inhibit the fibrillation process of proteins and peptides, including the biomedically relevant amyloid β peptide. However, the microscopic mechanisms that determine such an effect are still poorly understood. By means of large-scale, state-of-the-art enhanced sampling molecular dynamics simulations, here we identify an interaction mechanism between the segments 16-22 of the amyloid β peptide, known to be fibrillogenic by itself, and the Au(111) surface in water that leads to the suppression of fiber-like conformations from the peptide conformational ensemble. Moreover, thanks to advanced simulation analysis techniques, we characterize the conformational selection vs. induced fit nature of the gold effect. Our results disclose an inhibition mechanism that is rooted in the details of the microscopic peptide-surface interaction rather than in general phenomena such as peptide sequestration from the solution. Electronic supplementary information (ESI

Nutrient-induced glucagon like peptide-1 release is modulated by serotonin

NARCIS (Netherlands)

Ripken, D.; Wielen, N. van der; Wortelboer, H.M.; Meijerink, J.; Witkamp, R.F.; Hendriks, H.F.J.

2016-01-01

Glucagon like peptide-1 (GLP-1) and serotonin are both involved in food intake regulation. GLP-1 release is stimulated upon nutrient interaction with G-protein coupled receptors by enteroendocrine cells (EEC), whereas serotonin is released from enterochromaffin cells (ECC). The central hypothesis

Agatoxin-like peptides in the neuroendocrine system of the honey bee and other insects.

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Sturm, Sebastian; Ramesh, Divya; Brockmann, Axel; Neupert, Susanne; Predel, Reinhard

2016-01-30

We investigated the peptide inventory of the corpora cardiaca (CC) of the honey bee, Apis mellifera, by direct tissue profiling using MALDI-TOF MS combined with proteomic approaches focusing on cysteine-containing peptides. An agatoxin-like peptide (ALP) was identified as a component of the glandular part of the CC and was associated with the presence of the adipokinetic hormone in mass spectra. Although abundant in the CC, ALP does not belong to the toxins observed in the venom gland of A. mellifera. Homologs of ALP are highly conserved in major groups of arthropods and in line with this we detected ALP in the CC of non-venomous insects such as cockroaches and silverfish. In the American cockroach, Periplaneta americana, ALP was also identified in the CNS and stomatogastric nervous system. This is the first report that establishes the presence of ALPs in the neuroendocrine tissues of insects and further studies are necessary to reveal common functions of these peptides, e.g. as antimicrobial agents, ion channel modulators or classical neuropeptides. Among the messenger molecules of the nervous system, neuropeptides represent the structurally most diverse class and basically participate in the regulation of all physiological processes. The set of neuropeptides, their functions and spatial distribution are particularly well-studied in insects. Until now, however, several potential neuropeptide receptors remained orphan, which indicates the existence of so far unknown ligands. In our study, we used proteomic methods such as cysteine modification, enzymatic digestion and peptide derivatization, combined with direct tissue profiling by MALDI-TOF mass spectrometry, for the discovery of novel putative messenger molecules in the neuroendocrine system. The described presence of agatoxin-like peptides in the nervous system of the honey bee and other insects was overseen so far and is thus a remarkable addition to the very well studied neuropeptidome of insects. It is not

Somatic Cell Fusions Reveal Extensive Heterogeneity in Basal-like Breast Cancer

DEFF Research Database (Denmark)

Su, Ying; Subedee, Ashim; Bloushtain-Qimron, Noga

2015-01-01

Basal-like and luminal breast tumors have distinct clinical behavior and molecular profiles, yet the underlying mechanisms are poorly defined. To interrogate processes that determine these distinct phenotypes and their inheritance pattern, we generated somatic cell fusions and performed integrate...

Does bombesin-like peptide mediate radiation-induced anorexia and satiety?

Energy Technology Data Exchange (ETDEWEB)

Aalto, Y.; Franzen, L.; Henriksson, R. [Umeaa Univ. (Sweden). Dept. of Oncology; Forsgren, S.; Kjoerell, U. [Umeaa Univ. (Sweden). Dept. of Anatomy; Funegaard, U. [Umeaa Univ. (Sweden). Dept. of Cardiology

1999-07-01

Bombesin (BN) and its mammalian counterpart gastrin-releasing peptide (GRP) act as neuroregulatory hormones and peripheral and central satiety-inducing agents. Previously, we demonstrated that irradiation induces an increase in the expression of BN/GRP in the innervation of the salivary glands in rats. We therefore carried out a study using radioimmunoassay (RIA) analysis and immunohistochemistry to examine whether saliva contains BN and whether irradiation affects the BN release to saliva in rats. Immunoreactivity for BN was detected not only in the innervation of the parenchyma but also in the duct cells and in the lumina of the ducts, suggesting entrance of BN into saliva. The RIA analysis confirmed that rat saliva contains a BN-like peptide. The observation shows that saliva contains this peptide but that there is no significant increase following the radiation schedule used. Nevertheless, the occurrence of an enhanced expression of BN in different peripheral tissues such as the salivary and laryngeal glands should be taken into consideration when discussing the clinically important problem of reduced food intake and anorexia in cancer patients. (orig.)

Does bombesin-like peptide mediate radiation-induced anorexia and satiety?

International Nuclear Information System (INIS)

Aalto, Y.; Franzen, L.; Henriksson, R.; Forsgren, S.; Kjoerell, U.; Funegaard, U.

1999-01-01

Bombesin (BN) and its mammalian counterpart gastrin-releasing peptide (GRP) act as neuroregulatory hormones and peripheral and central satiety-inducing agents. Previously, we demonstrated that irradiation induces an increase in the expression of BN/GRP in the innervation of the salivary glands in rats. We therefore carried out a study using radioimmunoassay (RIA) analysis and immunohistochemistry to examine whether saliva contains BN and whether irradiation affects the BN release to saliva in rats. Immunoreactivity for BN was detected not only in the innervation of the parenchyma but also in the duct cells and in the lumina of the ducts, suggesting entrance of BN into saliva. The RIA analysis confirmed that rat saliva contains a BN-like peptide. The observation shows that saliva contains this peptide but that there is no significant increase following the radiation schedule used. Nevertheless, the occurrence of an enhanced expression of BN in different peripheral tissues such as the salivary and laryngeal glands should be taken into consideration when discussing the clinically important problem of reduced food intake and anorexia in cancer patients. (orig.)

Nutrient-induced glucagon like peptide-1 release is modulated by serotonin

NARCIS (Netherlands)

Ripken, Dina; Wielen, van der Nikkie; Wortelboer, Heleen M.; Meijerink, Jocelijn; Witkamp, Renger F.; Hendriks, Henk F.J.

2016-01-01

Glucagon like peptide-1 (GLP-1) and serotonin are both involved in food intake regulation. GLP-1 release is stimulated upon nutrient interaction with G-protein coupled receptors by enteroendocrine cells (EEC), whereas serotonin is released from enterochromaffin cells (ECC). The central hypothesis

Study of human lung elastin degradation by different elastases using high-performance liquid chromatography/mass spectrometry

NARCIS (Netherlands)

Barroso, Begona; Abello, Nicolas; Bischoff, Rainer

2006-01-01

Elastin is a structural insoluble protein which gives elasticity to tissues and organs. Although its hydrophobic and highly cross-linked nature makes it a very durable polymer, degradation of elastin in relation with several pathological conditions, such as pulmonary emphysema, has been documented.

HIPdb: a database of experimentally validated HIV inhibiting peptides.

Science.gov (United States)

Qureshi, Abid; Thakur, Nishant; Kumar, Manoj

2013-01-01

Besides antiretroviral drugs, peptides have also demonstrated potential to inhibit the Human immunodeficiency virus (HIV). For example, T20 has been discovered to effectively block the HIV entry and was approved by the FDA as a novel anti-HIV peptide (AHP). We have collated all experimental information on AHPs at a single platform. HIPdb is a manually curated database of experimentally verified HIV inhibiting peptides targeting various steps or proteins involved in the life cycle of HIV e.g. fusion, integration, reverse transcription etc. This database provides experimental information of 981 peptides. These are of varying length obtained from natural as well as synthetic sources and tested on different cell lines. Important fields included are peptide sequence, length, source, target, cell line, inhibition/IC(50), assay and reference. The database provides user friendly browse, search, sort and filter options. It also contains useful services like BLAST and 'Map' for alignment with user provided sequences. In addition, predicted structure and physicochemical properties of the peptides are also included. HIPdb database is freely available at http://crdd.osdd.net/servers/hipdb. Comprehensive information of this database will be helpful in selecting/designing effective anti-HIV peptides. Thus it may prove a useful resource to researchers for peptide based therapeutics development.

Membrane fusion-competent virus-like proteoliposomes and proteinaceous supported bilayers made directly from cell plasma membranes.

Science.gov (United States)

Costello, Deirdre A; Hsia, Chih-Yun; Millet, Jean K; Porri, Teresa; Daniel, Susan

2013-05-28

Virus-like particles are useful materials for studying virus-host interactions in a safe manner. However, the standard production of pseudovirus based on the vesicular stomatitis virus (VSV) backbone is an intricate procedure that requires trained laboratory personnel. In this work, a new strategy for creating virus-like proteoliposomes (VLPLs) and virus-like supported bilayers (VLSBs) is presented. This strategy uses a cell blebbing technique to induce the formation of nanoscale vesicles from the plasma membrane of BHK cells expressing the hemagglutinin (HA) fusion protein of influenza X-31. These vesicles and supported bilayers contain HA and are used to carry out single particle membrane fusion events, monitored using total internal reflection fluorescence microscopy. The results of these studies show that the VLPLs and VLSBs contain HA proteins that are fully competent to carry out membrane fusion, including the formation of a fusion pore and the release of fluorophores loaded into vesicles. This new strategy for creating spherical and planar geometry virus-like membranes has many potential applications. VLPLs could be used to study fusion proteins of virulent viruses in a safe manner, or they could be used as therapeutic delivery particles to transport beneficial proteins coexpressed in the cells to a target cell. VLSBs could facilitate high throughput screening of antiviral drugs or pathogen-host cell interactions.

Bioinformatic prediction of arthropod/nematode-like peptides in non-arthropod, non-nematode members of the Ecdysozoa.

Science.gov (United States)

Christie, Andrew E; Nolan, Daniel H; Garcia, Zachery A; McCoole, Matthew D; Harmon, Sarah M; Congdon-Jones, Benjamin; Ohno, Paul; Hartline, Niko; Congdon, Clare Bates; Baer, Kevin N; Lenz, Petra H

2011-02-01

The Onychophora, Priapulida and Tardigrada, along with the Arthropoda, Nematoda and several other small phyla, form the superphylum Ecdysozoa. Numerous peptidomic studies have been undertaken for both the arthropods and nematodes, resulting in the identification of many peptides from each group. In contrast, little is known about the peptides used as paracrines/hormones by species from the other ecdysozoan taxa. Here, transcriptome mining and bioinformatic peptide prediction were used to identify peptides in members of the Onychophora, Priapulida and Tardigrada, the only non-arthropod, non-nematode members of the Ecdysozoa for which there are publicly accessible expressed sequence tags (ESTs). The extant ESTs for each phylum were queried using 106 arthropod/nematode peptide precursors. Transcripts encoding calcitonin-like diuretic hormone and pigment-dispersing hormone (PDH) were identified for the onychophoran Peripatopsis sedgwicki, with transcripts encoding C-type allatostatin (C-AST) and FMRFamide-like peptide identified for the priapulid Priapulus caudatus. For the Tardigrada, transcripts encoding members of the A-type allatostatin, C-AST, insect kinin, orcokinin, PDH and tachykinin-related peptide families were identified, all but one from Hypsibius dujardini (the exception being a Milnesium tardigradum orcokinin-encoding transcript). The proteins deduced from these ESTs resulted in the prediction of 48 novel peptides, six onychophoran, eight priapulid and 34 tardigrade, which are the first described from these phyla. Copyright © 2010 Elsevier Inc. All rights reserved.

Elastin-like protein-hyaluronic acid (ELP-HA) hydrogels with decoupled mechanical and biochemical cues for cartilage regeneration.

Science.gov (United States)

Zhu, Danqing; Wang, Huiyuan; Trinh, Pavin; Heilshorn, Sarah C; Yang, Fan

2017-05-01

Hyaluronic acid (HA) is a major component of cartilage extracellular matrix and is an attractive material for use as 3D injectable matrices for cartilage regeneration. While previous studies have shown the promise of HA-based hydrogels to support cell-based cartilage formation, varying HA concentration generally led to simultaneous changes in both biochemical cues and stiffness. How cells respond to the change of biochemical content of HA remains largely unknown. Here we report an adaptable elastin-like protein-hyaluronic acid (ELP-HA) hydrogel platform using dynamic covalent chemistry, which allows variation of HA concentration without affecting matrix stiffness. ELP-HA hydrogels were created through dynamic hydrazone bonds via the reaction between hydrazine-modified ELP (ELP-HYD) and aldehyde-modified HA (HA-ALD). By tuning the stoichiometric ratio of aldehyde groups to hydrazine groups while maintaining ELP-HYD concentration constant, hydrogels with variable HA concentration (1.5%, 3%, or 5%) (w/v) were fabricated with comparable stiffness. To evaluate the effects of HA concentration on cell-based cartilage regeneration, chondrocytes were encapsulated within ELP-HA hydrogels with varying HA concentration. Increasing HA concentration led to a dose-dependent increase in cartilage-marker gene expression and enhanced sGAG deposition while minimizing undesirable fibrocartilage phenotype. The use of adaptable protein hydrogels formed via dynamic covalent chemistry may be broadly applicable as 3D scaffolds with decoupled niche properties to guide other desirable cell fates and tissue repair. Copyright © 2017 Elsevier Ltd. All rights reserved.

Insoluble elastin reduces collagen scaffold stiffness, improves viscoelastic properties, and induces a contractile phenotype in smooth muscle cells.

Science.gov (United States)

Ryan, Alan J; O'Brien, Fergal J

2015-12-01

Biomaterials with the capacity to innately guide cell behaviour while also displaying suitable mechanical properties remain a challenge in tissue engineering. Our approach to this has been to utilise insoluble elastin in combination with collagen as the basis of a biomimetic scaffold for cardiovascular tissue engineering. Elastin was found to markedly alter the mechanical and biological response of these collagen-based scaffolds. Specifically, during extensive mechanical assessment elastin was found to reduce the specific tensile and compressive moduli of the scaffolds in a concentration dependant manner while having minimal effect on scaffold microarchitecture with both scaffold porosity and pore size still within the ideal ranges for tissue engineering applications. However, the viscoelastic properties were significantly improved with elastin addition with a 3.5-fold decrease in induced creep strain, a 6-fold increase in cyclical strain recovery, and with a four-parameter viscoelastic model confirming the ability of elastin to confer resistance to long term deformation/creep. Furthermore, elastin was found to result in the modulation of SMC phenotype towards a contractile state which was determined via reduced proliferation and significantly enhanced expression of early (α-SMA), mid (calponin), and late stage (SM-MHC) contractile proteins. This allows the ability to utilise extracellular matrix proteins alone to modulate SMC phenotype without any exogenous factors added. Taken together, the ability of elastin to alter the mechanical and biological response of collagen scaffolds has led to the development of a biomimetic biomaterial highly suitable for cardiovascular tissue engineering. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effect of gamma irradiation dose on the fabrication of α-elastin nanoparticles by gamma-ray crosslinking

International Nuclear Information System (INIS)

Fujimoto, Mari; Takeda, Mayuko; Okamoto, Kouji; Furuta, Masakazu

2011-01-01

Nanoparticles were prepared utilizing the thermosensitive aggregation of α-elastin and gamma-ray crosslinking. We investigated the effect of the α-elastin irradiation doses to verify the yield of crosslinked nanoparticles. Aqueous solution of α-elastin (10 mg/ml) was used for the aggregation on raising temperature above its cloudy point (CP), followed by gamma-ray crosslinking. A slow heating process (1.9 o C/min) effectively led to aggregation of polypeptide and irradiation with more than 15 kGy yielded stable crosslinked nanoparticles with diameters less than ca. 200 nm and a narrow size distribution.

Effect of gamma irradiation dose on the fabrication of {alpha}-elastin nanoparticles by gamma-ray crosslinking

Energy Technology Data Exchange (ETDEWEB)

Fujimoto, Mari; Takeda, Mayuko [Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-2 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8570 (Japan); Okamoto, Kouji [Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka, Fukuoka 820-8502 (Japan); Furuta, Masakazu, E-mail: mfuruta@b.s.osakafu-u.ac.j [Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-2 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8570 (Japan)

2011-02-15

Nanoparticles were prepared utilizing the thermosensitive aggregation of {alpha}-elastin and gamma-ray crosslinking. We investigated the effect of the {alpha}-elastin irradiation doses to verify the yield of crosslinked nanoparticles. Aqueous solution of {alpha}-elastin (10 mg/ml) was used for the aggregation on raising temperature above its cloudy point (CP), followed by gamma-ray crosslinking. A slow heating process (1.9 {sup o}C/min) effectively led to aggregation of polypeptide and irradiation with more than 15 kGy yielded stable crosslinked nanoparticles with diameters less than ca. 200 nm and a narrow size distribution.

Fusion of raft-like lipid bilayers operated by a membranotropic domain of the HSV-type I glycoprotein gH occurs through a cholesterol-dependent mechanism.

Science.gov (United States)

Vitiello, Giuseppe; Falanga, Annarita; Petruk, Ariel Alcides; Merlino, Antonello; Fragneto, Giovanna; Paduano, Luigi; Galdiero, Stefania; D'Errico, Gerardino

2015-04-21

A wealth of evidence indicates that lipid rafts are involved in the fusion of the viral lipid envelope with the target cell membrane. However, the interplay between these sterol- and sphingolipid-enriched ordered domains and viral fusion glycoproteins has not yet been clarified. In this work we investigate the molecular mechanism by which a membranotropic fragment of the glycoprotein gH of the Herpes Simplex Virus (HSV) type I (gH625) drives fusion of lipid bilayers formed by palmitoyl oleoyl phosphatidylcholine (POPC)-sphingomyelin (SM)-cholesterol (CHOL) (1 : 1 : 1 wt/wt/wt), focusing on the role played by each component. The comparative analysis of the liposome fusion assays, Dynamic Light Scattering (DLS), spectrofluorimetry, Neutron Reflectivity (NR) and Electron Spin Resonance (ESR) experiments, and Molecular Dynamics (MD) simulations shows that CHOL is fundamental for liposome fusion to occur. In detail, CHOL stabilizes the gH625-bilayer association by specific interactions with the peptide Trp residue. The interaction with gH625 causes an increased order of the lipid acyl chains, whose local rotational motion is significantly hampered. SM plays only a minor role in the process, favoring the propagation of lipid perturbation to the bilayer inner core. The stiffening of the peptide-interacting bilayer leaflet results in an asymmetric perturbation of the membrane, which is locally destabilized thus favoring fusion events. Our results show that viral fusion glycoproteins are optimally suited to exert a high fusogenic activity on lipid rafts and support the relevance of cholesterol as a key player of membrane-related processes.

Can Natural Proteins Designed with ‘Inverted’ Peptide Sequences Adopt Native-Like Protein Folds?

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Sridhar, Settu; Guruprasad, Kunchur

2014-01-01

We have carried out a systematic computational analysis on a representative dataset of proteins of known three-dimensional structure, in order to evaluate whether it would possible to ‘swap’ certain short peptide sequences in naturally occurring proteins with their corresponding ‘inverted’ peptides and generate ‘artificial’ proteins that are predicted to retain native-like protein fold. The analysis of 3,967 representative proteins from the Protein Data Bank revealed 102,677 unique identical inverted peptide sequence pairs that vary in sequence length between 5–12 and 18 amino acid residues. Our analysis illustrates with examples that such ‘artificial’ proteins may be generated by identifying peptides with ‘similar structural environment’ and by using comparative protein modeling and validation studies. Our analysis suggests that natural proteins may be tolerant to accommodating such peptides. PMID:25210740

Biochemistry and biophysics of HIV-1 gp41 - membrane interactions and implications for HIV-1 envelope protein mediated viral-cell fusion and fusion inhibitor design.

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Cai, Lifeng; Gochin, Miriam; Liu, Keliang

2011-12-01

Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes ~2 millions death every year and still defies an effective vaccine. HIV-1 infects host cells through envelope protein - mediated virus-cell fusion. The transmembrane subunit of envelope protein, gp41, is the molecular machinery which facilitates fusion. Its ectodomain contains several distinguishing functional domains, fusion peptide (FP), Nterminal heptad repeat (NHR), C-terminal heptad repeat (CHR) and membrane proximal extracellular region (MPER). During the fusion process, FP inserts into the host cell membrane, and an extended gp41 prehairpin conformation bridges the viral and cell membranes through MPER and FP respectively. Subsequent conformational change of the unstable prehairpin results in a coiled-coil 6-helix bundle (6HB) structure formed between NHR and CHR. The energetics of 6HB formation drives membrane apposition and fusion. Drugs targeting gp41 functional domains to prevent 6HB formation inhibit HIV-1 infection. T20 (enfuvirtide, Fuzeon) was approved by the US FDA in 2003 as the first fusion inhibitor. It is a 36-residue peptide from the gp41 CHR, and it inhibits 6HB formation by targeting NHR and lipids. Development of new fusion inhibitors, especially small molecule drugs, is encouraged to overcome the shortcomings of T20 as a peptide drug. Hydrophobic characteristics and membrane association are critical for gp41 function and mechanism of action. Research in gp41-membrane interactions, using peptides corresponding to specific functional domains, or constructs including several interactive domains, are reviewed here to get a better understanding of gp41 mediated virus-cell fusion that can inform or guide the design of new HIV-1 fusion inhibitors.

Hydrogels for lung tissue engineering: Biomechanical properties of thin collagen-elastin constructs.

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Dunphy, Siobhán E; Bratt, Jessica A J; Akram, Khondoker M; Forsyth, Nicholas R; El Haj, Alicia J

2014-10-01

In this study, collagen-elastin constructs were prepared with the aim of producing a material capable of mimicking the mechanical properties of a single alveolar wall. Collagen has been used in a wide range of tissue engineering applications; however, due to its low mechanical properties its use is limited to non load-bearing applications without further manipulation using methods such as cross-linking or mechanical compression. Here, it was hypothesised that the addition of soluble elastin to a collagen hydrogel could improve its mechanical properties. Hydrogels made from collagen only and collagen plus varying amounts elastin were prepared. Young׳s modulus of each membrane was measured using the combination of a non-destructive indentation and a theoretical model previously described. An increase in Young׳s modulus was observed with increasing concentration of elastin. The use of non-destructive indentation allowed for online monitoring of the elastic moduli of cell-seeded constructs over 8 days. The addition of lung fibroblasts into the membrane increased the stiffness of the hydrogels further and cell-seeded collagen hydrogels were found to have a stiffness equal to the theoretical value for a single alveolar wall (≈5kPa). Through provision of some of the native extracellular matrix components of the lung parenchyma these scaffolds may be able to provide an initial building block toward the regeneration of new functional lung tissue. Copyright © 2014 Elsevier Ltd. All rights reserved.

Assessment of Myocardial Remodeling Using an Elastin/Tropoelastin Specific Agent with High Field Magnetic Resonance Imaging (MRI).

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Protti, Andrea; Lavin, Begoña; Dong, Xuebin; Lorrio, Silvia; Robinson, Simon; Onthank, David; Shah, Ajay M; Botnar, Rene M

2015-08-13

Well-defined inflammation, proliferation, and maturation phases orchestrate the remodeling of the injured myocardium after myocardial infarction (MI) by controlling the formation of new extracellular matrix. The extracellular matrix consists mainly of collagen but also fractions of elastin. It is thought that elastin is responsible for maintaining elastic properties of the myocardium, thus reducing the risk of premature rupture. An elastin/tropoelastin-specific contrast agent (Gd-ESMA) was used to image tropoelastin and mature elastin fibers for in vivo assessment of extracellular matrix remodeling post-MI. Gd-ESMA enhancement was studied in a mouse model of myocardial infarction using a 7 T MRI scanner and results were compared to those achieved after injection of a nonspecific control contrast agent, gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA). In the infarcted tissue, Gd-ESMA uptake (measured as R1 relaxation rate) steadily increased from day 3 to day 21 as a result of the synthesis of elastin/tropoelastin. R1 values were in good agreement with histological findings. A similar R1 behavior was observed in the remote myocardium. No mature cross-linked elastin was found at any time point. In contrast, Gd-DTPA uptake was only observed in the infarct with no changes in R1 values between 3 and 21 days post-MI. We demonstrate the feasibility of in vivo imaging of extracellular matrix remodeling post-MI using a tropoelastin/elastin binding MR contrast agent, Gd-ESMA. We found that tropoelastin is the main contributor to the increased MRI signal at late stages of MI where its augmentation in areas of infarction was in good agreement with the R1 increase. © 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

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

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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

Glucagon-like peptide 2 treatment may improve intestinal adaptation during weaning

DEFF Research Database (Denmark)

Thymann, Thomas; Le Huërou-Luron, I; Petersen, Y M

2014-01-01

Transition from sow’s milk to solid feed is associated with intestinal atrophy and diarrhea. We hypothesized that the intestinotrophic hormone glucagon-like peptide 2 (GLP-2) would induce a dose- and health status-dependent effect on gut adaptation. In Exp. 1, weaned pigs (average BW at weaning 4...

Genetic Modifiers of Cardiovascular Phenotype Caused by Elastin Haploinsufficiency Act by Extrinsic Noncomplementation*

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Kozel, Beth A.; Knutsen, Russell H.; Ye, Li; Ciliberto, Christopher H.; Broekelmann, Thomas J.; Mecham, Robert P.

2011-01-01

Elastin haploinsufficiency causes the cardiovascular complications associated with Williams-Beuren syndrome and isolated supravalvular aortic stenosis. Significant variability exists in the vascular pathology in these individuals. Using the Eln+/− mouse, we sought to identify the source of this variability. Following outcrossing of C57Bl/6J Eln+/−, two backgrounds were identified whose cardiovascular parameters deviated significantly from the parental strain. F1 progeny of the C57Bl/6J; Eln+/−x129X1/SvJ were more hypertensive and their arteries less compliant. In contrast, Eln+/− animals crossed to DBA/2J were protected from the pathologic changes associated with elastin insufficiency. Among the crosses, aortic elastin and collagen content did not correlate with quantitative vasculopathy traits. Quantitative trait locus analysis performed on F2 C57; Eln+/−x129 intercrosses identified highly significant peaks on chromosome 1 (LOD 9.7) for systolic blood pressure and on chromosome 9 (LOD 8.7) for aortic diameter. Additional peaks were identified that affect only Eln+/−, including a region upstream of Eln on chromosome 5 (LOD 4.5). Bioinformatic analysis of the quantitative trait locus peaks revealed several interesting candidates, including Ren1, Ncf1, and Nos1; genes whose functions are unrelated to elastic fiber assembly, but whose effects may synergize with elastin insufficiency to predispose to hypertension and stiffer blood vessels. Real time RT-PCR studies show background-specific increased expression of Ncf1 (a subunit of the NOX2 NAPDH oxidase) that parallel the presence of increased oxidative stress in Eln+/− aortas. This finding raises the possibility that polymorphisms in genes affecting the generation of reactive oxygen species alter cardiovascular function in individuals with elastin haploinsufficiency through extrinsic noncomplementation. PMID:22049077

Effect of a Fusion Peptide by Covalent Conjugation of a Mitochondrial Cell-Penetrating Peptide and a Glutathione Analog Peptide

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Carmine Pasquale Cerrato

2017-06-01

Full Text Available Previously, we designed and synthesized a library of mitochondrial antioxidative cell-penetrating peptides (mtCPPs superior to the parent peptide, SS31, to protect mitochondria from oxidative damage. A library of antioxidative glutathione analogs called glutathione peptides (UPFs, exceptional in hydroxyl radical elimination compared with glutathione, were also designed and synthesized. Here, a follow-up study is described, investigating the effects of the most promising members from both libraries on reactive oxidative species scavenging ability. None of the peptides influenced cell viability at the concentrations used. Fluorescence microscopy studies showed that the fluorescein-mtCPP1-UPF25 (mtgCPP internalized into cells, and spectrofluorometric analysis determined the presence and extent of peptide into different cell compartments. mtgCPP has superior antioxidative activity compared with mtCPP1 and UPF25 against H2O2 insult, preventing ROS formation by 2- and 3-fold, respectively. Moreover, we neither observed effects on mitochondrial membrane potential nor production of ATP. These data indicate that mtgCPP is targeting mitochondria, protecting them from oxidative damage, while also being present in the cytosol. Our hypothesis is based on a synergistic effect resulting from the fused peptide. The mitochondrial peptide segment is targeting mitochondria, whereas the glutathione analog peptide segment is active in the cytosol, resulting in increased scavenging ability.

Lysyl Oxidase Induces Vascular Oxidative Stress and Contributes to Arterial Stiffness and Abnormal Elastin Structure in Hypertension: Role of p38MAPK.

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Martínez-Revelles, Sonia; García-Redondo, Ana B; Avendaño, María S; Varona, Saray; Palao, Teresa; Orriols, Mar; Roque, Fernanda R; Fortuño, Ana; Touyz, Rhian M; Martínez-González, Jose; Salaices, Mercedes; Rodríguez, Cristina; Briones, Ana M

2017-09-01

Vascular stiffness, structural elastin abnormalities, and increased oxidative stress are hallmarks of hypertension. Lysyl oxidase (LOX) is an elastin crosslinking enzyme that produces H 2 O 2 as a by-product. We addressed the interplay between LOX, oxidative stress, vessel stiffness, and elastin. Angiotensin II (Ang II)-infused hypertensive mice and spontaneously hypertensive rats (SHR) showed increased vascular LOX expression and stiffness and an abnormal elastin structure. Mice over-expressing LOX in vascular smooth muscle cells (TgLOX) exhibited similar mechanical and elastin alterations to those of hypertensive models. LOX inhibition with β-aminopropionitrile (BAPN) attenuated mechanical and elastin alterations in TgLOX mice, Ang II-infused mice, and SHR. Arteries from TgLOX mice, Ang II-infused mice, and/or SHR exhibited increased vascular H 2 O 2 and O 2 .- levels, NADPH oxidase activity, and/or mitochondrial dysfunction. BAPN prevented the higher oxidative stress in hypertensive models. Treatment of TgLOX and Ang II-infused mice and SHR with the mitochondrial-targeted superoxide dismutase mimetic mito-TEMPO, the antioxidant apocynin, or the H 2 O 2 scavenger polyethylene glycol-conjugated catalase (PEG-catalase) reduced oxidative stress, vascular stiffness, and elastin alterations. Vascular p38 mitogen-activated protein kinase (p38MAPK) activation was increased in Ang II-infused and TgLOX mice and this effect was prevented by BAPN, mito-TEMPO, or PEG-catalase. SB203580, the p38MAPK inhibitor, normalized vessel stiffness and elastin structure in TgLOX mice. We identify LOX as a novel source of vascular reactive oxygen species and a new pathway involved in vascular stiffness and elastin remodeling in hypertension. LOX up-regulation is associated with enhanced oxidative stress that promotes p38MAPK activation, elastin structural alterations, and vascular stiffness. This pathway contributes to vascular abnormalities in hypertension. Antioxid. Redox Signal. 27

Thermal Stability of RNA Phage Virus-Like Particles Displaying Foreign Peptides

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Peabody David S

2011-05-01

Full Text Available Abstract Background To be useful for genetic display of foreign peptides a viral coat protein must tolerate peptide insertions without major disruption of subunit folding and capsid assembly. The folding of the coat protein of RNA phage MS2 does not normally tolerate insertions in its AB-loop, but an engineered single-chain dimer readily accepts them as long as they are restricted to one of its two halves. Results Here we characterize the effects of peptide insertions on the thermal stabilities of MS2 virus-like particles (VLPs displaying a variety of different peptides in one AB-loop of the coat protein single-chain dimer. These particles typically denature at temperatures around 5-10°C lower than unmodified VLPs. Even so, they are generally stable up to about 50°C. VLPs of the related RNA phage PP7 are cross-linked with intersubunit disulfide bonds and are therefore significantly more stable. An AB-loop insertion also reduces the stability of PP7 VLPs, but they only begin to denature above about 70°C. Conclusions VLPs assembled from MS2 single-chain dimer coat proteins with peptide insertions in one of their AB-loops are somewhat less stable than the wild-type particle, but still resist heating up to about 50°C. Because they possess disulfide cross-links, PP7-derived VLPs provide an alternate platform with even higher stability.

Toll-like receptor and antimicrobial peptide expression in the bovine endometrium

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Conlan R Steven

2008-11-01

Full Text Available Abstract Background The endometrium is commonly infected with bacteria leading to severe disease of the uterus in cattle and humans. The endometrial epithelium is the first line of defence for this mucosal surface against bacteria and Toll-like receptors (TLRs are a critical component of the innate immune system for detection of pathogen associated molecular patterns (PAMPs. Antimicrobial peptides, acute phase proteins and Mucin-1 (MUC-1 also provide non-specific defences against microbes on mucosal surfaces. The present study examined the expression of innate immune defences in the bovine endometrium and tested the hypothesis that endometrial epithelial cells express functional receptors of the TLR family and the non-specific effector molecules for defence against bacteria. Methods Bovine endometrial tissue and purified populations of primary epithelial and stromal cells were examined using RT-PCR for gene expression of TLRs, antimicrobial peptides and MUC-1. Functional responses were tested by evaluating the secretion of prostaglandin E2 and acute phase proteins when cells were treated with bacterial PAMPs such as bacterial lipopolysaccharide (LPS and lipoproteins. Results The endometrium expressed TLRs 1 to 10, whilst purified populations of epithelial cells expressed TLRs 1 to 7 and 9, and stromal cells expressed TLRs 1 to 4, 6, 7, 9 and 10. The TLRs appear to be functional as epithelial cells secreted prostaglandin E2 in response to bacterial PAMPs. In addition, the epithelial cells expressed antimicrobial peptides, such as Tracheal and Lingual Antimicrobial Peptides (TAP and LAP and MUC-1, which were upregulated when the cells were treated with LPS. However, the epithelial cells did not express appreciable amounts of the acute phase proteins haptoglobin or serum amyloid A. Conclusion Epithelial cells have an essential role in the orchestration of innate immune defence of the bovine endometrium and are likely to be the key to prevention of

Exaggerated glucagon-like peptide-1 and blunted glucose-dependent insulinotropic peptide secretion are associated with Roux-en-Y gastric bypass but not adjustable gastric banding

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Korner, Judith; Bessler, Marc; Inabnet, William

2007-01-01

BACKGROUND: The aim of this study was to measure the circulating levels of glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), and glucagon in patients who had undergone adjustable gastric banding (BND) or Roux-en-Y gastric bypass (RYGB) to understand the differences...

A GALA lipopeptide mediates pH- and membrane charge dependent fusion with stable giant unilamellar vesicles

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Etzerodt, Thomas P.; Trier, Sofie; Henriksen, Jonas R.

2012-01-01

sporadic and there is a strong need to characterize and increase our understanding of the membrane fusion properties of these peptides. Many fusion studies have focused on the ability of free peptides in solution that mediate fusion between liposomes. For drug delivery purposes it is a necessity to attach......,2-diamino propanoic acid (Dap) moiety, yielding the lipopeptide dimyristoyl-Dap-GALA (DMDGALA). We have investigated DMDGALA as a component in large unilamellar vesicles (LUVs) and demonstrate pH-triggered fusion of peptide containing LUVs with stable target giant unilamellar vesicles (GUVs), which were...

Analysis of the thermodynamics of binding of an SH3 domain to proline-rich peptides using a chimeric fusion protein.

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Candel, Adela M; van Nuland, Nico A J; Martin-Sierra, Francisco M; Martinez, Jose C; Conejero-Lara, Francisco

2008-03-14

A complete understanding of the thermodynamic determinants of binding between SH3 domains and proline-rich peptides is crucial to the development of rational strategies for designing ligands for these important domains. Recently we engineered a single-chain chimeric protein by fusing the alpha-spectrin Src homology region 3 (SH3) domain to the decapeptide APSYSPPPPP (p41). This chimera mimics the structural and energetic features of the interaction between SH3 domains and proline-rich peptides. Here we show that analysing the unfolding thermodynamics of single-point mutants of this chimeric fusion protein constitutes a very useful approach to deciphering the thermodynamics of SH3-ligand interactions. To this end, we investigated the contribution of each proline residue of the ligand sequence to the SH3-peptide interaction by producing six single Pro-Ala mutants of the chimeric protein and analysing their unfolding thermodynamics by differential scanning calorimetry (DSC). Structural analyses of the mutant chimeras by circular dichroism, fluorescence and NMR together with NMR-relaxation measurements indicate conformational flexibility at the binding interface, which is strongly affected by the different Pro-Ala mutations. An analysis of the DSC thermograms on the basis of a three-state unfolding model has allowed us to distinguish and separate the thermodynamic magnitudes of the interaction at the binding interface. The model assumes equilibrium between the "unbound" and "bound" states at the SH3-peptide binding interface. The resulting thermodynamic magnitudes classify the different proline residues according to their importance in the interaction as P2 approximately P7 approximately P10>P9 approximately P6>P8, which agrees well with Lim's model for the interaction between SH3 domains and proline-rich peptides. In addition, the thermodynamic signature of the interaction is the same as that usually found for this type of binding, with a strong enthalpy

Measurement of cross-linked elastin synthesis in bleomycin-induced pulmonary fibrosis using a highly sensitive assay for desmosine and isodesmosine

International Nuclear Information System (INIS)

Cantor, J.O.; Osman, M.; Keller, S.; Cerreta, J.M.; Mandl, I.; Turino, G.M.

1984-01-01

Cross-linked elastin synthesis was measured in the intratracheal bleomycin model of interstitial pulmonary fibrosis by incorporation of 14C-lysine into the elastin-specific crosslinks, desmosine and isodesmosine. Detection of the labeled crosslinks was facilitated by development of a highly sensitive assay utilizing thin-layer electrophoresis. The results indicate that crosslinked elastin synthesis is significantly elevated from controls (p less than 0.05) at 1 to 3 weeks after exposure to bleomycin and returns to normal by 5 weeks. The increases in labeled elastin synthesis are not directly related to changes in either total lung protein synthesis or the pool size of the 14C-lysine. In comparison with collagen and glycosaminoglycan synthesis in this model of lung injury, maximal increases in cross-linked elastin formation occur later, but overlap with the elevated synthesis of these other connective tissue components. The marked increase from normal in cross-linked elastin synthesis in this model suggests that this tissue component is an important part of the fibrotic response of the pulmonary parenchyma and may play a role in the observed alterations in lung structure and function

Inhibition of Nipah virus infection in vivo: targeting an early stage of paramyxovirus fusion activation during viral entry.

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Matteo Porotto

2010-10-01

Full Text Available In the paramyxovirus cell entry process, receptor binding triggers conformational changes in the fusion protein (F leading to viral and cellular membrane fusion. Peptides derived from C-terminal heptad repeat (HRC regions in F have been shown to inhibit fusion by preventing formation of the fusogenic six-helix bundle. We recently showed that the addition of a cholesterol group to HRC peptides active against Nipah virus targets these peptides to the membrane where fusion occurs, dramatically increasing their antiviral effect. In this work, we report that unlike the untagged HRC peptides, which bind to the postulated extended intermediate state bridging the viral and cell membranes, the cholesterol tagged HRC-derived peptides interact with F before the fusion peptide inserts into the target cell membrane, thus capturing an earlier stage in the F-activation process. Furthermore, we show that cholesterol tagging renders these peptides active in vivo: the cholesterol-tagged peptides cross the blood brain barrier, and effectively prevent and treat in an established animal model what would otherwise be fatal Nipah virus encephalitis. The in vivo efficacy of cholesterol-tagged peptides, and in particular their ability to penetrate the CNS, suggests that they are promising candidates for the prevention or therapy of infection by Nipah and other lethal paramyxoviruses.

Influences of conformations of peptides on stereoinversions and/or isomerizations of aspartic acid residues.

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Oda, Akifumi; Nakayoshi, Tomoki; Fukuyoshi, Shuichi; Kurimoto, Eiji; Takahashi, Ohgi

2018-07-01

Recently, non-enzymatic stereoinversions of aspartic acid (Asp) residues in proteins and peptides have been reported. Here, we performed replica exchange molecular dynamics (REMD) simulations of model peptides (exon 6, 26A-1, and 26A-2) extracted from elastin to investigate their structural features, thereby revealing the factor that influences stereoinversions. For REMD trajectories, we calculated distances between carboxyl carbon in Asp and amide nitrogen in the (n + 1) residue (CN distances). Because bond formation between carbon and nitrogen is indispensable to the formation of a succinimide intermediate the distance between them seems to play an important role in stereoinversion. Moreover, we calculated polar surface areas (PSAs) for the trajectories, finding that CN distances and PSA were different for each peptide, with the longest CN distance and smallest PSA observed for exon 6 peptide, where stereoinversion of Asp is the slowest. Although the average CN distance was shorter for exon 26A-1 peptide than for exon 26A-2 peptide, the number of conformations with CN distances acids: biology in the mirror, edited by Dr. Loredano Pollegioni, Dr. Jean-Pierre Mothet and Dr. Molla Gianluca. Copyright © 2018 Elsevier B.V. All rights reserved.

Glucagon-like peptide-1, a new hormone of the entero-insular axis

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Orskov, C

1992-01-01

reported in Type 2 (noninsulin-dependent) diabetic patients, however, the consequences of the elevation are not yet known. However, elevated levels of glucagon-like peptide-1 in patients with increased gastric emptying rate (post-gastrectomy syndromes) may be responsible for the exaggerated insulin...

Knockdown of versican 1 blocks cigarette-induced loss of insoluble elastin in human lung fibroblasts.

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Xu, Lu-lu; Lu, Yun-tao; Zhang, Jing; Wu, Lian; Merrilees, Mervyn J; Qu, Jie-ming

2015-08-15

COPD lung is characterized by loss of alveolar elastic fibers and an increase in the chondroitin sulfate (CS) matrix proteoglycan versican V1 (V1). V1 is a known inhibitor of elastic fiber deposition and this study investigates the effects of knockdown of V1, and add-back of CS, on CCL-210 lung fibroblasts treated with cigarette smoke extract (CSE) as a model for COPD. CSE inhibited fibroblast proliferation, viability, tropoelastin synthesis, and elastin deposition, and increased V1 synthesis and secretion. V1 siRNA decreased V1 and constituent CS, did not affect tropoelastin production, but blocked the CSE-induced loss in insoluble elastin. Exogenous CS reduced insoluble elastin, even in the presence of V1 siRNA. These findings confirm that V1 and CS impair the assembly of tropoelastin monomers into insoluble fibers, and further demonstrate that specific knockdown of V1 alleviates the impaired assembly of elastin seen in cultures of pulmonary fibroblasts exposed to CSE, indicating a regulatory role for this protein in the pathophysiology of COPD. Copyright © 2015 Elsevier B.V. All rights reserved.

Fourier transform infrared spectroscopy to quantify collagen and elastin in an in vitro model of extracellular matrix degradation in aorta.

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Cheheltani, Rabee; McGoverin, Cushla M; Rao, Jayashree; Vorp, David A; Kiani, Mohammad F; Pleshko, Nancy

2014-06-21

Extracellular matrix (ECM) is a key component and regulator of many biological tissues including aorta. Several aortic pathologies are associated with significant changes in the composition of the matrix, especially in the content, quality and type of aortic structural proteins, collagen and elastin. The purpose of this study was to develop an infrared spectroscopic methodology that is comparable to biochemical assays to quantify collagen and elastin in aorta. Enzymatically degraded porcine aorta samples were used as a model of ECM degradation in abdominal aortic aneurysm (AAA). After enzymatic treatment, Fourier transform infrared (FTIR) spectra of the aortic tissue were acquired by an infrared fiber optic probe (IFOP) and FTIR imaging spectroscopy (FT-IRIS). Collagen and elastin content were quantified biochemically and partial least squares (PLS) models were developed to predict collagen and elastin content in aorta based on FTIR spectra. PLS models developed from FT-IRIS spectra were able to predict elastin and collagen content of the samples with strong correlations (RMSE of validation = 8.4% and 11.1% of the range respectively), and IFOP spectra were successfully used to predict elastin content (RMSE = 11.3% of the range). The PLS regression coefficients from the FT-IRIS models were used to map collagen and elastin in tissue sections of degraded porcine aortic tissue as well as a human AAA biopsy tissue, creating a similar map of each component compared to histology. These results support further application of FTIR spectroscopic techniques for evaluation of AAA tissues.

Fourier Transform Infrared Spectroscopy to Quantify Collagen and Elastin in an In Vitro Model of Extracellular Matrix Degradation in Aorta

Science.gov (United States)

Cheheltani, Rabee; McGoverin, Cushla M.; Rao, Jayashree; Vorp, David A.; Kiani, Mohammad F.; Pleshko, N.

2014-01-01

Extracellular matrix (ECM) is a key component and regulator of many biological tissues including aorta. Several aortic pathologies are associated with significant changes in the composition of the matrix, especially in the content, quality and type of aortic structural proteins, collagen and elastin. The purpose of this study was to develop an infrared spectroscopic methodology that is comparable to biochemical assays to quantify collagen and elastin in aorta. Enzymatically degraded porcine aorta samples were used as a model of ECM degradation in abdominal aortic aneurysm (AAA). After enzymatic treatment, Fourier transform infrared (FTIR) spectra of the aortic tissue were acquired by an infrared fiber optic probe (IFOP) and FTIR imaging spectroscopy (FT-IRIS). Collagen and elastin content were quantified biochemically and partial least squares (PLS) models were developed to predict collagen and elastin content in aorta based on FTIR spectra. PLS models developed from FT-IRIS spectra were able to predict elastin and collagen content of the samples with strong correlations (RMSE of validation = 8.4% and 11.1% of the range respectively), and IFOP spectra were successfully used to predict elastin content (RMSE = 11.3% of the range). The PLS regression coefficients from the FT-IRIS models were used to map collagen and elastin in tissue sections of degraded porcine aortic tissue as well as a human AAA biopsy tissue, creating a similar map of each component compared to histology. These results support further application of FTIR spectroscopic techniques for evaluation of AAA tissues. PMID:24761431

Formulation optimization and in vivo proof-of-concept study of thermosensitive liposomes balanced by phospholipid, elastin-like polypeptide, and cholesterol.

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Sun Min Park

Full Text Available One application of nanotechnology in medicine that is presently being developed involves a drug delivery system (DDS employing nanoparticles to deliver drugs to diseased sites in the body avoiding damage of healthy tissue. Recently, the mild hyperthermia-triggered drug delivery combined with anticancer agent-loaded thermosensitive liposomes was widely investigated. In this study, thermosensitive liposomes (TSLs, composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol-2000] (DSPE-PEG, cholesterol, and a fatty acid conjugated elastin-like polypeptide (ELP, were developed and optimized for triggered drug release, controlled by external heat stimuli. We introduced modified ELP, tunable for various biomedical purposes, to our thermosensitive liposome (e-TSL to convey a high thermoresponsive property. We modulated thermosensitivity and stability by varying the ratios of e-TSL components, such as phospholipid, ELP, and cholesterol. Experimental data obtained in this study corresponded to results from a simulation study that demonstrated, through the calculation of the lateral diffusion coefficient, increased permeation of the lipid bilayer with higher ELP concentrations, and decreased permeation in the presence of cholesterol. Finally, we identified effective drug accumulation in tumor tissues and antitumor efficacy with our optimized e-TSL, while adjusting lag-times for systemic accumulation.

Engineering D-Amino Acid Containing Collagen Like Peptide at the Cleavage Site of Clostridium histolyticum Collagenase for Its Inhibition.

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Punitha Velmurugan

Full Text Available Collagenase is an important enzyme which plays an important role in degradation of collagen in wound healing, cancer metastasis and even in embryonic development. However, the mechanism of this degradation has not yet been completely understood. In the field of biomedical and protein engineering, the design and development of new peptide based materials is of main concern. In the present work an attempt has been made to study the effect of DAla in collagen like peptide (imino-poor region of type I collagen on the structure and stability of peptide against enzyme hydrolysis. Effect of replacement of DAla in the collagen like peptide has been studied using circular dichroic spectroscopy (CD. Our findings suggest that, DAla substitution leads to conformational changes in the secondary structure and favours the formation of polyproline II conformation than its L-counterpart in the imino-poor region of collagen like peptides. Change in the chirality of alanine at the cleavage site of collagenase in the imino-poor region inhibits collagenolytic activity. This may find application in design of peptides and peptidomimics for enzyme-substrate interaction, specifically with reference to collagen and other extra cellular matrix proteins.

Selection of side-chain carbons in a high-molecular-weight, hydrophobic peptide using solid-state spectral editing methods

International Nuclear Information System (INIS)

Kumashiro, Kristin K.; Niemczura, Walter P.; Kim, Minna S.; Sandberg, Lawrence B.

2000-01-01

Solid-state spectral editing techniques have been used by others to simplify 13 C CPMAS spectra of small organic molecules, synthetic organic polymers, and coals. One approach utilizes experiments such as cross-polarization-with-polarization-inversion and cross-polarization-with-depolarization to generate subspectra. This work shows that this particular methodology is also applicable to natural-abundance 13 C CPMAS NMR studies of high-molecular-weight biopolymers. The editing experiments are demonstrated first with model peptides and then with α-elastin, a high-molecular-weight peptidyl preparation obtained from the elastic fibers in mammalian tissue. The latter has a predominance of small, nonpolar residues, which is evident in the crowded aliphatic region of typical 13 C CPMAS spectra. Spectral editing is particularly useful for simplifying the aliphatic region of the NMR spectrum of this elastin preparation

Renal clearance of the thyrotropin-releasing hormone-like peptide pyroglutamyl-glutamyl-prolineamide in humans

NARCIS (Netherlands)

W. Klootwijk (Willem); E. Sleddens-Linkels (Esther); R.D.H. de Boer (Remco); C.A. Jansen; R. Autar; W.W. de Herder (Wouter); E.R. Boeve; T.J. Visser (Theo); W.J. de Greef

1997-01-01

textabstractTRH-like peptides have been identified that differ from TRH (pGlu-His-ProNH2) in the middle amino acid. We have estimated TRH-like immunoreactivity (TRH-LI) in human serum and urine by RIA with TRH-specific antiserum 8880 or with antiserum 4319, which binds

Development of a Tissue-Engineered Artificial Ligament: Reconstruction of Injured Rabbit Medial Collateral Ligament With Elastin-Collagen and Ligament Cell Composite Artificial Ligament.

Science.gov (United States)

Hirukawa, Masaki; Katayama, Shingo; Sato, Tatsuya; Inoue, Kota; Niwa, Kosuke; Ito, Naoya; Hattori, Tetsuya; Hosoi, Takashi; Unno, Hironori; Suzuki, Yoshiaki; Hasegawa, Masahiro; Miyamoto, Keiichi; Horiuchi, Takashi

2018-04-16

Ligament reconstruction using a tissue-engineered artificial ligament (TEAL) requires regeneration of the ligament-bone junction such that fixation devices such as screws and end buttons do not have to be used. The objective of this study was to develop a TEAL consisting of elastin-coated polydioxanone (PDS) sutures covered with elastin and collagen fibers preseeded with ligament cells. In a pilot study, a ring-type PDS suture with a 2.5 mm (width) bone insertion was constructed with/without elastin coating (Ela-coat and Non-coat) and implanted into two bone tunnels, diameter 2.4 mm, in the rabbit tibia (6 cases each) to access the effect of elastin on the bond strength. PDS specimens taken together with the tibia at 6 weeks after implantation indicated growth of bone-like hard tissues around bone tunnels accompanied with narrowing of the tunnels in the Ela-coat group and not in the Non-coat group. The drawout load of the Ela-coat group was significantly higher (28.0 ± 15.1 N, n = 4) than that of the Non-coat group (7.6 ± 4.6 N, n = 5). These data can improve the mechanical bulk property of TEAL through extracellular matrix formation. To achieve this TEAL model, 4.5 × 10 6 ligament cells were seeded on elastin and collagen fibers (2.5 cm × 2.5 cm × 80 µm) prior to coil formation around the elastin-coated PDS core sutures having ball-shape ends with a diameter of 2.5 mm. Cell-seeded and cell-free TEALs were implanted across the femur and the tibia through bone tunnels with a diameter of 2.4 mm (6 cases each). There was no incidence of TEAL being pulled in 6 weeks. Regardless of the remarkable degradation of PDS observed in the cell-seeded group, both the elastic modulus and breaking load of the cell-seeded group (n = 3) were comparable to those of the sham-operation group (n = 8) (elastic modulus: 15.4 ± 1.3 MPa and 18.5 ± 5.7 MPa; breaking load: 73.0 ± 23.4 N and 104.8 ± 21.8 N, respectively) and higher than those

Electrospinning of collagen and elastin for tissue engineering applications

NARCIS (Netherlands)

Buttafoco, L.; Kolkman, N.G.; Engbers-Buijtenhuijs, P.; Poot, Andreas A.; Dijkstra, Pieter J.; Vermes, I.; Feijen, Jan

2006-01-01

Meshes of collagen and/or elastin were successfully prepared by means of electrospinning from aqueous solutions. Flow rate, applied electric field, collecting distance and composition of the starting solutions determined the morphology of the obtained fibres. Addition of PEO (Mw=8×106) and NaCl was

Fusion of protegrin-1 and plectasin to MAP30 shows significant inhibition activity against dengue virus replication.

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Hussin A Rothan

Full Text Available Dengue virus (DENV broadly disseminates in tropical and sub-tropical countries and there are no vaccine or anti-dengue drugs available. DENV outbreaks cause serious economic burden due to infection complications that requires special medical care and hospitalization. This study presents a new strategy for inexpensive production of anti-DENV peptide-fusion protein to prevent and/or treat DENV infection. Antiviral cationic peptides protegrin-1 (PG1 and plectasin (PLSN were fused with MAP30 protein to produce recombinant antiviral peptide-fusion protein (PG1-MAP30-PLSN as inclusion bodies in E. coli. High yield production of PG1-MAP30-PLSN protein was achieved by solubilization of inclusion bodies in alkaline buffer followed by the application of appropriate refolding techniques. Antiviral PG1-MAP30-PLSN protein considerably inhibited DENV protease (NS2B-NS3pro with half-maximal inhibitory concentration (IC50 0.5±0.1 μM. The real-time proliferation assay (RTCA and the end-point proliferation assay (MTT assay showed that the maximal-nontoxic dose of the peptide-fusion protein against Vero cells is approximately 0.67±0.2 μM. The cell-based assays showed considerable inhibition of the peptide-fusion protein against binding and proliferating stages of DENV2 into the target cells. The peptide-fusion protein protected DENV2-challeged mice with 100% of survival at the dose of 50 mg/kg. In conclusion, producing recombinant antiviral peptide-fusion protein by combining short antiviral peptide with a central protein owning similar activity could be useful to minimize the overall cost of short peptide production and take advantage of its synergistic antiviral activities.

Characterization of BIV Env core: Implication for mechanism of BIV-mediated cell fusion

International Nuclear Information System (INIS)

Li Shu; Zhu Jieqing; Peng Yu; Cui Shanshan; Wang Chunping; Gao, George F.; Tien Po

2005-01-01

Entry of lentiviruses, such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), requires folding of two heptad repeat regions (HR1 and HR2) of gp41 into a trimer-of-hairpins, which subsequently brings virus and cell membrane into fusion. This motif is a generalized feature of viral fusion proteins and has been exploited in generating antiviral fusion agents. In the present paper, we report structural characters of Env protein from another lentivirus, bovine immunodeficiency virus (BIV), which contributes to a good animal model of HIV. BIV HR1 and HR2 regions are predicted by two different programs and expressed separately or conjointly in Escherichia coli. Biochemical and biophysical analyses show that the predicted HRs of BIV Env can form a stable trimer-of-hairpins or six-helix bundle just like that formed by feline immunodeficiency virus Env. Cell fusion assay demonstrates that the HR2 peptide of BIV can efficiently inhibit the virus-mediated cell fusion

Oligomerization of a Glucagon-like Peptide 1 Analog: Bridging Experiment and Simulations

DEFF Research Database (Denmark)

Frederiksen, Tine Maja; Sønderby, Pernille; Ryberg, Line A.

2015-01-01

The glucagon-like peptide 1 (GLP-1) analog, liraglutide, is a GLP-1 agonist and is used in the treatment of type-2 diabetes mellitus and obesity. From a pharmaceutical perspective, it is important to know the oligomerization state of liraglutide with respect to stability. Compared to GLP-1...

Facilitating protein solubility by use of peptide extensions

Science.gov (United States)

Freimuth, Paul I; Zhang, Yian-Biao; Howitt, Jason

2013-09-17

Expression vectors for expression of a protein or polypeptide of interest as a fusion product composed of the protein or polypeptide of interest fused at one terminus to a solubility enhancing peptide extension are provided. Sequences encoding the peptide extensions are provided. The invention further comprises antibodies which bind specifically to one or more of the solubility enhancing peptide extensions.

Peptide-Based Membrane Fusion Inhibitors Targeting HCoV-229E Spike Protein HR1 and HR2 Domains

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Shuai Xia

2018-02-01

Full Text Available Human coronavirus 229E (HCoV-229E infection in infants, elderly people, and immunocompromised patients can cause severe disease, thus calling for the development of effective and safe therapeutics to treat it. Here we reported the design, synthesis and characterization of two peptide-based membrane fusion inhibitors targeting HCoV-229E spike protein heptad repeat 1 (HR1 and heptad repeat 2 (HR2 domains, 229E-HR1P and 229E-HR2P, respectively. We found that 229E-HR1P and 229E-HR2P could interact to form a stable six-helix bundle and inhibit HCoV-229E spike protein-mediated cell-cell fusion with IC50 of 5.7 and 0.3 µM, respectively. 229E-HR2P effectively inhibited pseudotyped and live HCoV-229E infection with IC50 of 0.5 and 1.7 µM, respectively. In a mouse model, 229E-HR2P administered intranasally could widely distribute in the upper and lower respiratory tracts and maintain its fusion-inhibitory activity. Therefore, 229E-HR2P is a promising candidate for further development as an antiviral agent for the treatment and prevention of HCoV-229E infection.

Escape from R-peptide deletion in a γ-retrovirus

International Nuclear Information System (INIS)

Schneider, Irene C.; Eckhardt, Manon; Brynza, Julia; Collins, Mary K.; Cichutek, Klaus; Buchholz, Christian J.

2011-01-01

The R peptide in the cytoplasmic tail (C-tail) of γ-retroviral envelope proteins (Env) prevents membrane fusion before budding. To analyse its role in the formation of replication competent, infectious particles, we developed chimeric murine leukaemia viruses (MLV) with unmodified or R-peptide deleted Env proteins of the gibbon ape leukaemia virus (GaLV). While titres of these viruses were unaffected, R-peptide deficiency led to strongly impaired spreading. Most remarkably, we isolated an escape mutant which had restored an open reading frame for a C-terminal extension of the truncated C-tail. A reconstituted virus encoding this escape C-tail replicated in cell culture. In contrast to R-peptide deficient Env, particle incorporation of the escape Env was effective due to an enhanced protein expression and restored intracellular co-localisation with Gag proteins. Our data demonstrate that the R peptide not only regulates membrane fusion but also mediates efficient Env protein particle incorporation in γ-retrovirus infected cells.

Physiological regulation of extracellular matrix collagen and elastin in the arterial wall of rats by noradrenergic tone and angiotensin II.

Science.gov (United States)

Dab, Houcine; Kacem, Kamel; Hachani, Rafik; Dhaouadi, Nadra; Hodroj, Wassim; Sakly, Mohsen; Randon, Jacques; Bricca, Giampiero

2012-03-01

The interactions between the effects of the sympathetic nervous system (SNS) and angiotensin II (ANG II) on vascular extracellular matrix (ECM) synthesis were determined in rats. The mRNA and protein content of collagen I, collagen III and elastin in the abdominal aorta (AA) and femoral artery (FA) was investigated in Wistar-Kyoto rats treated for 5 weeks with guanethidine, a sympathoplegic, losartan, an ANG II AT1 receptor (AT1R) blocker, or both. The effects of noradrenaline (NE) and ANG II on collagen III and elastin mRNA, and the receptor involved, were tested in cultured vascular smooth muscle cells (VSMCs) in vitro. Guanethidine increased collagen types I and III and decreased elastin, while losartan had an opposite effect, although without effect on collagen III. The combination of treatments abrogated changes induced by simple treatment with collagen I and elastin, but increased collagen III mRNA in AA and not in FA. NE stimulated collagen III mRNA via β receptors and elastin via α1 and α2 receptors. ANG II stimulated collagen III but inhibited elastin mRNA via AT1R. Overall, SNS and ANG II exert opposite and antagonistic effects on major components of ECM in the vascular wall. This may be of relevance for the choice of a therapeutic strategy in vascular diseases.

BmK-YA, an enkephalin-like peptide in scorpion venom.

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Yan Zhang

Full Text Available By screening extracts of venom from the Asian scorpion Buthus martensii Karsch (BmK for their abilities to activate opioid receptors, we have identified BmK-YA, an amidated peptide containing an enkephalin-like sequence. BmK-YA is encoded by a precursor that displays a signal sequence and contains four copies of BmK-YA sequences and four of His(4-BmK-YA, all flanked by single amino acid residues. BmK-YA and His(4-BmK-YA are amidated and thus fulfill the characteristics expected of bioactive peptides. BmK-YA can activate mammalian opioid receptors with selectivity for the δ subtype while His(4-BmK-YA is inactive at opioid receptors. The discovery of BmK-YA suggests that scorpion venom may represent a novel source of bioactive molecules targeting G protein-coupled receptors (GPCRs and reveal additional insights on the evolution of the opioid precursors.

Labeling bombesin-like peptide with 99mTc via hydrazinonicotinamide. Description of optimized radiolabeling conditions

International Nuclear Information System (INIS)

Yurt Lambrecht, F.; Durkan, K.; Bayrak, E.

2010-01-01

Bombesin (BNN)-like peptides have very high binding affinity for the gastrin-releasing peptide (GRP) receptor. The goal of the current study was to optimize the labeling conditions of a new 99m Tc-radiolabeled BNN-like peptide based on the bifunctional chelating ligand HYNIC using different co-ligands (EDDA and tricine). The radiolabeling conditions (pH, amount of co-ligand, amount of stannous chloride, temperature and reaction time) for newly-formed 99m Tc-tricine-HYNIC-Q-Litorin and 99m Tc-EDDA-HYNIC-Q-Litorin were optimized and evaluated by RHPLC and RTLC. Radiochemical yields for 99m Tc-tricine-HYNIC-Q-Litorin and 99m Tc-EDDA-HYNIC-Q-Litorin were 98.0 ± 1.7 and 97.5 ± 2.5%, respectively. When EDDA was used as co-ligand, the labeling of 99m Tc-EDDA-HYNIC-Q-Litorin was optimal in the following reaction mixture: HYNIC-peptide: EDDA: 10 μg/5 mg, pH 3, SnCl 2 concentration: 12 μg/0.1 mL, reaction temperature: 100 deg C, reaction time: 15 min. Besides, the optimum conditions were HYNIC-peptide:tricine: 10 μg/50 mg, pH 5, SnCl 2 concentration: 12 μg/0.1 mL, reaction temperature: 100 deg C, reaction time: 15 min for preparing 99m Tc-tricine-HYNIC-Q-Litorin. The manufactured 99m Tc-HYNIC-Q-Litorin conjugates may offer new possibilities for imaging cancer cells expressing bombesin receptors. (author)

Phosphorylated form of adrenocorticotropin and corticotropin-like intermediary lobe peptide in human tumors

International Nuclear Information System (INIS)

Massias, J.F.; Hardouin, S.; Vieau, D.; Lenne, F.; Bertagna, X.

1994-01-01

Many peptides contribute to the heterogeneity of immunoreactive adrenocorticotropin (ACTH) in man. The use of a radioimmunoassay (RIA) specifically directed against the C-terminal end of ACTH allowed the precise study of the following four peptides: ACTH itself, corticotropin-like intermediary lobe peptide (CLIP) or ACTH and their phosphorylated forms on SeR 31 . The authors have set up a high-performance liquid chromatography system that separates these four molecules in a single run, to establish their relative distributions in tumors responsible for Cushing's disease or for the ectopic ACTH syndrome, and to evaluate the possible interference of phospho-Ser 31 on various RIA or immuno-radiometric assay (IRMA) recognition systems for ACTH. In this system, alkaline phosphatase treatment shifted the retention time of the phosphorylated peptides to that of their non-phosphorylated counterparts. In three tumors responsible for the ectopic ACTH syndrome, CLIP peptides were predominant in two and phosphorylated molecules represented between 22% and 50% of immuno-reactive materials. In five pituitary tumors responsible for Cushing's disease, ACTH peptides were predominant and the phosphorylated molecules varied between 35% and 75% in four of them. In the same tumor the ratios of phosphorylated to non-phosphorylated CLIP or ACTH were identical. The presence of phospho-Ser 31 did not affect the recognition ability of two mid-ACTH and two C-terminal ACTH RIA's, nor of the ACTH IRMA. 15 refs., 5 figs., 2 tabs

Plasma levels of glucagon like peptide-1 associate with diastolic function in elderly men

DEFF Research Database (Denmark)

Nathanson, D; Zethelius, B; Berne, C

2011-01-01

Congestive heart failure is a major cause of morbidity and mortality in diabetes. Besides the glycaemic effects of glucagon-like peptide 1 (GLP-1) mimetics, their effects on the heart are of interest....

Gene expression levels of elastin and fibulin-5 according to differences between carotid plaque regions.

Science.gov (United States)

Sivrikoz, Emre; Timirci-Kahraman, Özlem; Ergen, Arzu; Zeybek, Ümit; Aksoy, Murat; Yanar, Fatih; İsbir, Turgay; Kurtoğlu, Mehmet

2015-01-01

The purpose of this study was to investigate the gene expression levels of elastin and fibulin-5 according to differences between carotid plaque regions and to correlate it with clinical features of plaque destabilization. The study included 44 endarterectomy specimens available from operated symptomatic carotid artery stenoses. The specimens were separated according to anatomic location: internal carotid artery (ICA), external carotid artery (ECA) and common carotid artery (CCA), and then stored in liquid nitrogen. The amounts of cDNA for elastin and fibulin-5 were determined by Quantitative real-time PCR (Q-RT-PCR). Target gene copy numbers were normalized using hypoxanthine-guanine phosphoribosyltransferase (HPRT1) gene. The delta-delta CT method was applied for relative quantification. Q-RT-PCR data showed that relative fibulin-5 gene expression was increased in ICA plaque regions when compared to CCA regions but not reaching significance (p=0.061). At the same time, no differences were observed in elastin mRNA level between different anatomic plaque regions (p>0.05). Moreover, elastin and fibulin-5 mRNA expression and clinical parameters were compared in ICA plaques versus CCA and ECA regions, respectively. Up-regulation of elastin and fibulin-5 mRNA levels in ICA were strongly correlated with family history of cardiovascular disease when compared to CCA (p<0.05). Up-regulation of fibulin-5 in ICA was significantly associated with diabetes, and elevated triglycerides and very low density lipoprotein (VLDL) when compared to ECA (p<0.05). The clinical significance is the differences between the proximal and distal regions of the lesion, associated with the ICA, CCA and ECA respectively, with increased fibulin-5 in the ICA region. Copyright © 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Identification of snake bradykinin-potentiating peptides (BPPs)-simile sequences in rat brain--Potential BPP-like precursor protein?

Science.gov (United States)

Campeiro, Joana D'Arc; Neshich, Izabella P; Sant'Anna, Osvaldo A; Lopes, Robson; Ianzer, Danielle; Assakura, Marina T; Neshich, Goran; Hayashi, Mirian A F

2015-08-01

Bradykinin-potentiating peptides (BPPs) from the South American pit viper snake venom were the first natural inhibitors of the human angiotensin I-converting enzyme (ACE) described. The pioneer characterization of the BPPs precursor from the snake venom glands by our group showed for the first time the presence of the C-type natriuretic peptide (CNP) in this same viper precursor protein. The confirmation of the BPP/CNP expression in snake brain regions correlated with neuroendocrine functions stimulated us to pursue the physiological correlates of these vasoactive peptides in mammals. Notably, several snake toxins were shown to have endogenous physiological correlates in mammals. In the present work, we expressed in bacteria the BPPs domain of the snake venom gland precursor protein, and this purified recombinant protein was used to raise specific polyclonal anti-BPPs antibodies. The correspondent single protein band immune-recognized in adult rat brain cytosol was isolated by 2D-SDS/PAGE and/or HPLC, before characterization by MS fingerprint analysis, which identified this protein as superoxide dismutase (SOD, EC 1.15.1.1), a classically known enzyme with antioxidant activity and important roles in the blood pressure modulation. In silico analysis showed the exposition of the BPP-like peptide sequences on the surface of the 3D structure of rat SOD. These peptides were chemically synthesized to show the BPP-like biological activities in ex vivo and in vivo pharmacological bioassays. Taken together, our data suggest that SOD protein have the potential to be a source for putative BPP-like bioactive peptides, which once released may contribute to the blood pressure control in mammals. Copyright © 2015 Elsevier Inc. All rights reserved.

Ligand-regulated peptides: a general approach for modulating protein-peptide interactions with small molecules.

Science.gov (United States)

Binkowski, Brock F; Miller, Russell A; Belshaw, Peter J

2005-07-01

We engineered a novel ligand-regulated peptide (LiRP) system where the binding activity of intracellular peptides is controlled by a cell-permeable small molecule. In the absence of ligand, peptides expressed as fusions in an FKBP-peptide-FRB-GST LiRP scaffold protein are free to interact with target proteins. In the presence of the ligand rapamycin, or the nonimmunosuppressive rapamycin derivative AP23102, the scaffold protein undergoes a conformational change that prevents the interaction of the peptide with the target protein. The modular design of the scaffold enables the creation of LiRPs through rational design or selection from combinatorial peptide libraries. Using these methods, we identified LiRPs that interact with three independent targets: retinoblastoma protein, c-Src, and the AMP-activated protein kinase. The LiRP system should provide a general method to temporally and spatially regulate protein function in cells and organisms.

In planta production of ELPylated spidroin-based proteins results in non-cytotoxic biopolymers.

Science.gov (United States)

Hauptmann, Valeska; Menzel, Matthias; Weichert, Nicola; Reimers, Kerstin; Spohn, Uwe; Conrad, Udo

2015-02-19

Spider silk is a tear-resistant and elastic biopolymer that has outstanding mechanical properties. Additionally, exiguous immunogenicity is anticipated for spider silks. Therefore, spider silk represents a potential ideal biomaterial for medical applications. All known spider silk proteins, so-called spidroins, reveal a composite nature of silk-specific units, allowing the recombinant production of individual and combined segments. In this report, a miniaturized spidroin gene, named VSO1 that contains repetitive motifs of MaSp1 has been synthesized and combined to form multimers of distinct lengths, which were heterologously expressed as elastin-like peptide (ELP) fusion proteins in tobacco. The elastic penetration moduli of layered proteins were analyzed for different spidroin-based biopolymers. Moreover, we present the first immunological analysis of synthetic spidroin-based biopolymers. Characterization of the binding behavior of the sera after immunization by competitive ELISA suggested that the humoral immune response is mainly directed against the fusion partner ELP. In addition, cytocompatibility studies with murine embryonic fibroblasts indicated that recombinant spidroin-based biopolymers, in solution or as coated proteins, are well tolerated. The results show that spidroin-based biopolymers can induce humoral immune responses that are dependent on the fusion partner and the overall protein structure. Furthermore, cytocompatibility assays gave no indication of spidroin-derived cytotoxicity, suggesting that recombinant produced biopolymers composed of spider silk-like repetitive elements are suitable for biomedical applications.

A highly scalable peptide-based assay system for proteomics.

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Igor A Kozlov

Full Text Available We report a scalable and cost-effective technology for generating and screening high-complexity customizable peptide sets. The peptides are made as peptide-cDNA fusions by in vitro transcription/translation from pools of DNA templates generated by microarray-based synthesis. This approach enables large custom sets of peptides to be designed in silico, manufactured cost-effectively in parallel, and assayed efficiently in a multiplexed fashion. The utility of our peptide-cDNA fusion pools was demonstrated in two activity-based assays designed to discover protease and kinase substrates. In the protease assay, cleaved peptide substrates were separated from uncleaved and identified by digital sequencing of their cognate cDNAs. We screened the 3,011 amino acid HCV proteome for susceptibility to cleavage by the HCV NS3/4A protease and identified all 3 known trans cleavage sites with high specificity. In the kinase assay, peptide substrates phosphorylated by tyrosine kinases were captured and identified by sequencing of their cDNAs. We screened a pool of 3,243 peptides against Abl kinase and showed that phosphorylation events detected were specific and consistent with the known substrate preferences of Abl kinase. Our approach is scalable and adaptable to other protein-based assays.

Elastin Degradation by Cathepsin V Requires Two Exosites*

Science.gov (United States)

Du, Xin; Chen, Nelson L. H.; Wong, Andre; Craik, Charles S.; Brömme, Dieter

2013-01-01

Cathepsin V is a highly effective elastase and has been implicated in physiological and pathological extracellular matrix degradation. However, its mechanism of action remains elusive. Whereas human cathepsin V exhibits a potent elastolytic activity, the structurally homologous cathepsin L, which shares a 78% amino acid sequence, has only a minimal proteolytic activity toward insoluble elastin. This suggests that there are distinct structural domains that play an important role in elastinolysis. In this study, a total of 11 chimeras of cathepsins V and L were generated to identify elastin-binding domains in cathepsin V. Evaluation of these chimeras revealed two exosites contributing to the elastolytic activity of cathepsin V that are distant from the active cleft of the protease and are located in surface loop regions. Replacement of exosite 1 or 2 with analogous residues from cathepsin L led to a 75 and 43% loss in the elastolytic activity, respectively. Replacement of both exosites yielded a non-elastase variant similar to that of cathepsin L. Identification of these exosites may contribute to the design of inhibitors that will only affect the elastolytic activity of cysteine cathepsins without interfering with other physiological protease functions. PMID:24121514

Individually timing high-protein preloads has no effect on daily energy intake, peptide YY and glucagon-like peptide-1.

Science.gov (United States)

Willbond, S M; Doucet, É

2011-01-01

Gut hormones have been shown to influence energy intake (EI). To our knowledge, no study has investigated the effects of dietary patterns aimed at optimizing fullness on EI, appetite and gut hormones. To determine whether individually timing high-protein preloads would impact EI, appetite, and peptide YY and glucagon-like peptide-1 (GLP-1) levels. Ten men (body mass index = 25.5 ± 2.6 kg/m(2)) participated in a randomized crossover trial. The three conditions consisted of the self-selection of snacks (condition 1), or the consumption of a preload (300 kcal: 40% protein, 40% carbohydrates and 20% fat) at either 15 min (condition 2) or ∼ 50 min (individually set) (condition 3) before lunch and dinner. During each condition, a standardized breakfast was served, whereas lunch and dinner were self-selected from a five-item menu, and eaten ad libitum. Mealtime and daily EI were measured. Appetite, peptide YY and GLP-1 were sampled over 9 h. No differences in daily EI were noted across conditions (1 = 3078 ± 720 kcal; 2 = 2929 ± 264 kcal; 3 = 2998 ± 437 kcal; not significant). For the most part, daily profiles as well as premeal levels of peptide YY and GLP-1 were not different between conditions. Desire to eat, hunger and prospective food consumption were found to be lowest during condition 1 (P daily EI in healthy human subjects.

Details of the Collagen and Elastin Architecture in the Human Limbal Conjunctiva, Tenon's Capsule and Sclera Revealed by Two-Photon Excited Fluorescence Microscopy.

Science.gov (United States)

Park, Choul Yong; Marando, Catherine M; Liao, Jason A; Lee, Jimmy K; Kwon, Jiwon; Chuck, Roy S

2016-10-01

To investigate the architecture and distribution of collagen and elastin in human limbal conjunctiva, Tenon's capsule, and sclera. The limbal conjunctiva, Tenon's capsule, and sclera of human donor corneal buttons were imaged with an inverted two-photon excited fluorescence microscope. No fixation process was necessary. The laser (Ti:sapphire) was tuned at 850 nm for two-photon excitation. Backscatter signals of second harmonic generation (SHG) and autofluorescence (AF) were collected through a 425/30-nm and a 525/45-nm emission filter, respectively. Multiple, consecutive, and overlapping (z-stack) images were acquired. Collagen signals were collected with SHG, whereas elastin signals were collected with AF. The size and density of collagen bundles varied widely depending on depth: increasing from conjunctiva to sclera. In superficial image planes, collagen bundles were image planes (episclera and superficial sclera), collagen bundles were thicker (near 100 μm in width) and densely packed. Comparatively, elastin fibers were thinner and sparse. The orientation of elastin fibers was independent of collagen fibers in superficial layers; but in deep sclera, elastin fibers wove through collagen interbundle gaps. At the limbus, both collagen and elastin fibers were relatively compact and were distributed perpendicular to the limbal annulus. Two-photon excited fluorescence microscopy has enabled us to understand in greater detail the collagen and elastin architecture of the human limbal conjunctiva, Tenon's capsule, and sclera.

Glucagon-like peptide-1 receptor ligand interactions: structural cross talk between ligands and the extracellular domain.

Directory of Open Access Journals (Sweden)

Graham M West

Full Text Available Activation of the glucagon-like peptide-1 receptor (GLP-1R in pancreatic β-cells potentiates insulin production and is a current therapeutic target for the treatment of type 2 diabetes mellitus (T2DM. Like other class B G protein-coupled receptors (GPCRs, the GLP-1R contains an N-terminal extracellular ligand binding domain. N-terminal truncations on the peptide agonist generate antagonists capable of binding to the extracellular domain, but not capable of activating full length receptor. The main objective of this study was to use Hydrogen/deuterium exchange (HDX to identify how the amide hydrogen bonding network of peptide ligands and the extracellular domain of GLP-1R (nGLP-1R were altered by binding interactions and to then use this platform to validate direct binding events for putative GLP-1R small molecule ligands. The HDX studies presented here for two glucagon-like peptide-1 receptor (GLP-1R peptide ligands indicates that the antagonist exendin-4[9-39] is significantly destabilized in the presence of nonionic detergents as compared to the agonist exendin-4. Furthermore, HDX can detect stabilization of exendin-4 and exendin-4[9-39] hydrogen bonding networks at the N-terminal helix [Val19 to Lys27] upon binding to the N-terminal extracellular domain of GLP-1R (nGLP-1R. In addition we show hydrogen bonding network stabilization on nGLP-1R in response to ligand binding, and validate direct binding events with the extracellular domain of the receptor for putative GLP-1R small molecule ligands.

Secretion of glucagon-like peptide-1 in patients with type 2 diabetes mellitus

DEFF Research Database (Denmark)

Calanna, S; Christensen, M; Holst, Jens Juul

2013-01-01

We carried out a systematic review of clinical studies investigating glucagon-like peptide-1 (GLP-1) secretion in patients with type 2 diabetes and non-diabetic controls and performed meta-analyses of plasma total GLP-1 concentrations during an OGTT and/or meal test....

Glucagon-like peptide-1 (GLP-1) receptor agonism or DPP-4 inhibition does not accelerate neoplasia in carcinogen treated mice

DEFF Research Database (Denmark)

Kissow, Hannelouise; Hartmann, Bolette; Holst, Jens Juul

2012-01-01

Glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2) are secreted in parallel from the intestinal endocrine cells after nutrient intake. GLP-1 is an incretin hormone and analogues are available for the treatment of type 2 diabetes mellitus (T2DM). GLP-2 is an intestinal growth horm...

Quantitative and qualitative evaluation of dermal elastin of draught horses with chronic progressive lymphoedema.

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De Cock, H E V; Van Brantegem, L; Affolter, V K; Oosterlinck, M; Ferraro, G L; Ducatelle, R

2009-01-01

Chronic progressive lymphoedema (CPL) in horses, a disease of certain draught breeds, is associated with altered elastin metabolism. The characteristic lesions are seen in the skin of the lower (distal) limbs. This study was based on horses of susceptible breeds, with and without CPL, and on horses of a non-susceptible breed. Skin samples were obtained for examination from the neck (considered a non-affected region) and from the distal limb. The skin lesions were characterized histologically and the dermal elastic fibres were evaluated morphologically and quantitatively. In all horses the mean elastin concentrations were highest in the superficial dermis, gradually decreasing in the mid-dermis and deep dermis. As compared with horses of a non-susceptible breed, affected horses had increased amounts of dermal elastin in both the distal limb and neck, while non-affected horses of a susceptible breed had decreased amounts. The findings support an earlier hypothesis that CPL of horses is a generalized disease. Reduced efficiency of the elastic network in supporting the dermal lymphatics may explain the development of CPL.

Effects of chronic glucagon-like peptide-2 therapy during weaning in neonatal pigs

DEFF Research Database (Denmark)

Sigalet, David L; de Heuvel, Elaine; Wallace, Laurie

2014-01-01

BACKGROUND: The enteroendocrine hormone glucagon like peptide-2 (GLP-2) and its ligands are under development as therapeutic agents for a variety of intestinal pathologies. A number of these conditions occur in neonates and infants, and thus a detailed understanding of the effects of GLP-2 during...

Glucagon like peptide-1-induced glucose metabolism in differentiated human muscle satellite cells is attenuated by hyperglycemia

DEFF Research Database (Denmark)

Green, Charlotte J; Henriksen, Tora I; Pedersen, Bente K

2012-01-01

Glucagon like peptide-1 (GLP-1) stimulates insulin secretion from the pancreas but also has extra-pancreatic effects. GLP-1 may stimulate glucose uptake in cultured muscle cells but the mechanism is not clearly defined. Furthermore, while the pancreatic effects of GLP-1 are glucose-dependent, the......Glucagon like peptide-1 (GLP-1) stimulates insulin secretion from the pancreas but also has extra-pancreatic effects. GLP-1 may stimulate glucose uptake in cultured muscle cells but the mechanism is not clearly defined. Furthermore, while the pancreatic effects of GLP-1 are glucose...

Mitochondrial N-formyl peptides induce cardiovascular collapse and sepsis-like syndrome

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McCarthy, Cameron G.; Szasz, Theodora; Goulopoulou, Styliani; Webb, R. Clinton

2015-01-01

Fifty percent of trauma patients who present sepsis-like syndrome do not have bacterial infections. This condition is known as systemic inflammatory response syndrome (SIRS). A unifying factor of SIRS and sepsis is cardiovascular collapse. Trauma and severe blood loss cause the release of endogenous molecules known as damage-associated molecular patterns. Mitochondrial N-formyl peptides (F-MIT) are damage-associated molecular patterns that share similarities with bacterial N-formylated peptides and are potent immune system activators. The goal of this study was to investigate whether F-MIT trigger SIRS, including hypotension and vascular collapse via formyl peptide receptor (FPR) activation. We evaluated cardiovascular parameters in Wistar rats treated with FPR or histamine receptor antagonists and inhibitors of the nitric oxide pathway before and after F-MIT infusion. F-MIT, but not nonformylated peptides or mitochondrial DNA, induced severe hypotension via FPR activation and nitric oxide and histamine release. Moreover, F-MIT infusion induced hyperthermia, blood clotting, and increased vascular permeability. To evaluate the role of leukocytes in F-MIT-induced hypotension, neutrophil, basophil, or mast cells were depleted. Depletion of basophils, but not neutrophils or mast cells, abolished F-MIT-induced hypotension. Rats that underwent hemorrhagic shock increased plasma levels of mitochondrial formylated proteins associated with lung damage and antagonism of FPR ameliorated hemorrhagic shock-induced lung injury. Finally, F-MIT induced vasodilatation in isolated resistance arteries via FPR activation; however, F-MIT impaired endothelium-dependent relaxation in the presence of blood. These data suggest that F-MIT may be the link among trauma, SIRS, and cardiovascular collapse. PMID:25637548

Small Molecule Drug Discovery at the Glucagon-Like Peptide-1 Receptor

Directory of Open Access Journals (Sweden)

Francis S. Willard

2012-01-01

Full Text Available The therapeutic success of peptide glucagon-like peptide-1 (GLP-1 receptor agonists for the treatment of type 2 diabetes mellitus has inspired discovery efforts aimed at developing orally available small molecule GLP-1 receptor agonists. Although the GLP-1 receptor is a member of the structurally complex class B1 family of GPCRs, in recent years, a diverse array of orthosteric and allosteric nonpeptide ligands has been reported. These compounds include antagonists, agonists, and positive allosteric modulators with intrinsic efficacy. In this paper, a comprehensive review of currently disclosed small molecule GLP-1 receptor ligands is presented. In addition, examples of “ligand bias” and “probe dependency” for the GLP-1 receptor are discussed; these emerging concepts may influence further optimization of known molecules or persuade designs of expanded screening strategies to identify novel chemical starting points for GLP-1 receptor drug discovery.

Peptide YY3-36 and glucagon-like peptide-1 in functional dyspepsia. Secretion and role in symptom generation

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Witte, Anne-Barbara; Hilsted, Linda; Holst, Jens Juul

2016-01-01

method. Secondly, participants drank 75 mL (90 kcal) per five min until maximal satiety. PYY3-36, GLP-1, glucose, and insulin concentrations were assessed. Satiety measures and dyspeptic symptoms were registered using visual analogue scales. RESULTS: Gastric emptying, glucose, PYY3-36, and GLP-1......OBJECTIVE: The role of peptide YY3-36 (PYY3-36), glucagon-like peptide-1 (GLP-1), and glucose homoeostasis in symptom development in functional dyspepsia (FD) is unclear. The aim was to investigate postprandial changes in plasma PYY3-36, GLP-1, glucose and insulin, and the relationship between PYY3......-36, GLP-1, dyspeptic symptoms, and satiety measurements. MATERIALS AND METHODS: Thirty-six patients with functional dyspepsia and 18 healthy controls consumed a liquid meal at two occasions. Firstly, a fixed amount of 250 mL (300 kcal) was consumed and gastric emptying was assessed using the paracetamol...

Impact of exogenous hyperglucagonemia on postprandial concentrations of gastric inhibitory polypeptide and glucagon-like peptide-1 in humans

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Meier, Juris J; Ritter, Peter R; Jacob, Alexandra

2010-01-01

Postprandial secretion of glucagon-like peptide 1 (GLP-1) has been found diminished in some patients with type 2 diabetes mellitus (T2DM) and high glucagon concentrations. We examined the effects of exogenous glucagon on the release of incretin hormones.......Postprandial secretion of glucagon-like peptide 1 (GLP-1) has been found diminished in some patients with type 2 diabetes mellitus (T2DM) and high glucagon concentrations. We examined the effects of exogenous glucagon on the release of incretin hormones....

The association between newborn regional body composition and cord blood concentrations of C-peptide and insulin-like growth factor I

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Carlsen, Emma M; Renault, Kristina M; Jensen, Rikke B

2015-01-01

BACKGROUND: Third trimester fetal growth is partially regulated by C-peptide and insulin-like growth factor I (IGF-I). Prenatal exposures including maternal obesity and high gestational weight gain as well as high birth weight have been linked to subsequent metabolic disease. We evaluated...... with both C-peptide (p tissue accumulation was associated with cord blood C-peptide and IGF-I. Total and abdominal fat masses were related to C-peptide but not to IGF-I. Thus, newborn adiposity is partially mediated through C-peptide and early...

Quantification of aortic and cutaneous elastin and collagen morphology in Marfan syndrome by multiphoton microscopy.

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Cui, Jason Z; Tehrani, Arash Y; Jett, Kimberly A; Bernatchez, Pascal; van Breemen, Cornelis; Esfandiarei, Mitra

2014-09-01

In a mouse model of Marfan syndrome, conventional Verhoeff-Van Gieson staining displays severe fragmentation, disorganization and loss of the aortic elastic fiber integrity. However, this method involves chemical fixatives and staining, which may alter the native morphology of elastin and collagen. Thus far, quantitative analysis of fiber damage in aorta and skin in Marfan syndrome has not yet been explored. In this study, we have used an advanced noninvasive and label-free imaging technique, multiphoton microscopy to quantify fiber fragmentation, disorganization, and total volumetric density of aortic and cutaneous elastin and collagen in a mouse model of Marfan syndrome. Aorta and skin samples were harvested from Marfan and control mice aged 3-, 6- and 9-month. Elastin and collagen were identified based on two-photon excitation fluorescence and second-harmonic-generation signals, respectively, without exogenous label. Measurement of fiber length indicated significant fragmentation in Marfan vs. control. Fast Fourier transform algorithm analysis demonstrated markedly lower fiber organization in Marfan mice. Significantly reduced volumetric density of elastin and collagen and thinner skin dermis were observed in Marfan mice. Cutaneous content of elastic fibers and thickness of dermis in 3-month Marfan resembled those in the oldest control mice. Our findings of early signs of fiber degradation and thinning of skin dermis support the potential development of a novel non-invasive approach for early diagnosis of Marfan syndrome. Copyright © 2014 Elsevier Inc. All rights reserved.

Remodeling of the notochord during development of vertebral fusions in Atlantic salmon (Salmo salar).

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Ytteborg, Elisabeth; Torgersen, Jacob Seilø; Pedersen, Mona E; Baeverfjord, Grete; Hannesson, Kirsten O; Takle, Harald

2010-12-01

Histological characterization of spinal fusions in Atlantic salmon (Salmo salar) has demonstrated shape alterations of vertebral body endplates, a reduced intervertebral space, and replacement of intervertebral cells by ectopic bone. However, the significance of the notochord during the fusion process has not been addressed. We have therefore investigated structural and cellular events in the notochord during the development of vertebral fusions. In order to induce vertebral fusions, Atlantic salmon were exposed to elevated temperatures from fertilization until they attained a size of 15g. Based on results from radiography, intermediate and terminal stages of the fusion process were investigated by immunohistochemistry and real-time quantitative polymerase chain reaction. Examination of structural extracellular matrix proteins such as Perlecan, Aggrecan, Elastin, and Laminin revealed reduced activity and reorganization at early stages in the pathology. Staining for elastic fibers visualized a thinner elastic membrane surrounding the notochord of developing fusions, and immunohistochemistry for Perlecan showed that the notochordal sheath was stretched during fusion. These findings in the outer notochord correlated with the loss of Aggrecan- and Substance-P-positive signals and the further loss of vacuoles from the chordocytes in the central notochord. At more progressed stages of fusion, chordocytes condensed, and the expression of Aggrecan and Substance P reappeared. The hyperdense regions seem to be of importance for the formation of notochordal tissue into bone. Thus, the remodeling of notochord integrity by reduced elasticity, structural alterations, and cellular changes is probably involved in the development of vertebral fusions.

Extracellular secretion of a recombinant therapeutic peptide by Bacillus halodurans utilizing a modified flagellin type III secretion system

CSIR Research Space (South Africa)

Berger, E

2011-08-01

Full Text Available further 3.5-fold increase in the secretion of recombinant peptide fusions. Conclusions: The type III flagellar secretion system of B. halodurans has been shown to successfully secrete a therapeutic peptide as a heterologous flagellin fusion. Improvements...

Peroxisome proliferator-activated receptor δ modulates MMP-2 secretion and elastin expression in human dermal fibroblasts exposed to ultraviolet B radiation.

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Ham, Sun Ah; Yoo, Taesik; Hwang, Jung Seok; Kang, Eun Sil; Paek, Kyung Shin; Park, Chankyu; Kim, Jin-Hoi; Do, Jeong Tae; Seo, Han Geuk

2014-10-01

Changes in skin connective tissues mediated by ultraviolet (UV) radiation have been suggested to cause the skin wrinkling normally associated with premature aging of the skin. Recent investigations have shown that peroxisome proliferator-activated receptor (PPAR) δ plays multiple biological roles in skin homeostasis. We attempted to investigate whether PPARδ modulates elastin protein levels and secretion of matrix metalloproteinase (MMP)-2 in UVB-irradiated human dermal fibroblasts (HDFs) and mouse skin. These studies were undertaken in primary HDFs or HR-1 hairless mice using Western blot analyses, small interfering (si)RNA-mediated gene silencing, and Fluorescence microscopy. In HDFs, UVB irradiation induced increased secretion of MMP-2 and reduced levels of elastin. Activation of PPARδ by GW501516, a ligand specific for PPARδ, markedly attenuated UVB-induced MMP-2 secretion with a concomitant increase in the level of elastin. These effects were reduced by the presence of siRNAs against PPARδ or treatment with GSK0660, a specific inhibitor of PPARδ. Furthermore, GW501516 elicited a dose- and time-dependent increase in the expression of elastin. Modulation of MMP-2 secretion and elastin levels by GW501516 was associated with a reduction in reactive oxygen species (ROS) production in HDFs exposed to UVB. Finally, in HR-1 hairless mice, administration of GW501516 significantly reduced UVB-induced MMP-2 expression with a concomitant increase in elastin levels, and these effects were significantly reduced by the presence of GSK0660. Our results suggest that PPARδ-mediated modulation of MMP-2 secretion and elastin expression may contribute to the maintenance of skin integrity by inhibiting ROS generation. Copyright © 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Effects of solvent concentration and composition on protein dynamics: 13C MAS NMR studies of elastin in glycerol-water mixtures.

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Demuth, Dominik; Haase, Nils; Malzacher, Daniel; Vogel, Michael

2015-08-01

We use (13)C CP MAS NMR to investigate the dependence of elastin dynamics on the concentration and composition of the solvent at various temperatures. For elastin in pure glycerol, line-shape analysis shows that larger-scale fluctuations of the protein backbone require a minimum glycerol concentration of ~0.6 g/g at ambient temperature, while smaller-scale fluctuations are activated at lower solvation levels of ~0.2 g/g. Immersing elastin in various glycerol-water mixtures, we observe at room temperature that the protein mobility is higher for lower glycerol fractions in the solvent and, thus, lower solvent viscosity. When decreasing the temperature, the elastin spectra approach the line shape for the rigid protein at 245 K for all studied samples, indicating that the protein ceases to be mobile on the experimental time scale of ~10(-5) s. Our findings yield evidence for a strong coupling between elastin fluctuations and solvent dynamics and, hence, such interaction is not restricted to the case of protein-water mixtures. Spectral resolution of different carbon species reveals that the protein-solvent couplings can, however, be different for side chain and backbone units. We discuss these results against the background of the slaving model for protein dynamics. Copyright © 2015 Elsevier B.V. All rights reserved.

Elastin Cables Define the Axial Connective Tissue System in the Murine Lung.

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Wagner, Willi; Bennett, Robert D; Ackermann, Maximilian; Ysasi, Alexandra B; Belle, Janeil; Valenzuela, Cristian D; Pabst, Andreas; Tsuda, Akira; Konerding, Moritz A; Mentzer, Steven J

2015-11-01

The axial connective tissue system is a fiber continuum of the lung that maintains alveolar surface area during changes in lung volume. Although the molecular anatomy of the axial system remains undefined, the fiber continuum of the lung is central to contemporary models of lung micromechanics and alveolar regeneration. To provide a detailed molecular structure of the axial connective tissue system, we examined the extracellular matrix of murine lungs. The lungs were decellularized using a 24 hr detergent treatment protocol. Systematic evaluation of the decellularized lungs demonstrated no residual cellular debris; morphometry demonstrated a mean 39 ± 7% reduction in lung dimensions. Scanning electron microscopy (SEM) demonstrated an intact structural hierarchy within the decellularized lung. Light, fluorescence, and SEM of precision-cut lung slices demonstrated that alveolar duct structure was defined by a cable line element encased in basement membrane. The cable line element arose in the distal airways, passed through septal tips and inserted into neighboring blood vessels and visceral pleura. The ropelike appearance, collagenase resistance and anti-elastin immunostaining indicated that the cable was an elastin macromolecule. Our results indicate that the helical line element of the axial connective tissue system is composed of an elastin cable that not only defines the structure of the alveolar duct, but also integrates the axial connective tissue system into visceral pleura and peripheral blood vessels. © 2015 Wiley Periodicals, Inc.

Intracranial arteries in individuals with the elastin gene hemideletion of Williams syndrome.

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Wint, D P; Butman, J A; Masdeu, J C; Meyer-Lindenberg, A; Mervis, C B; Sarpal, D; Morris, C A; Berman, K F

2014-01-01

Williams syndrome, a rare genetic disorder with a striking neurobehavioral profile characterized by extreme sociability and impaired visuospatial construction abilities, is caused by a hemideletion that includes the elastin gene, resulting in frequent supravavular aortic stenosis and other stenotic arterial lesions. Strokes have been reported in Williams syndrome. Although the extracranial carotid artery has been studied in a sample of patients with Williams syndrome, proximal intracranial arteries have not. Using MRA, we studied the intracranial vessels in 27 participants: 14 patients with Williams syndrome (age range, 18-44 years; mean age, 27.3 ± 9.1; 43% women) and 13 healthy control participants with similar age and sex distribution (age range, 22-52 years; mean age, 33.4 ± 7.6; 46% women). All participants with Williams syndrome had hemideletions of the elastin gene. Blinded to group allocation or to any other clinical data, a neuroradiologist determined the presence of intracranial vascular changes in the 2 groups. The Williams syndrome group and the healthy control group had similar patency of the proximal intracranial arteries, including the internal carotid and vertebral arteries; basilar artery; and stem and proximal branches of the anterior cerebral artery, MCA, and posterior cerebral arteries. The postcommunicating segment of the anterior cerebral artery was longer in the Williams syndrome group. Despite the elastin haploinsufficiency, the proximal intracranial arteries in Williams syndrome preserve normal patency.

Three dimensional microstructural network of elastin, collagen, and cells in Achilles tendons.

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Pang, Xin; Wu, Jian-Ping; Allison, Garry T; Xu, Jiake; Rubenson, Jonas; Zheng, Ming-Hao; Lloyd, David G; Gardiner, Bruce; Wang, Allan; Kirk, Thomas Brett

2017-06-01

Similar to most biological tissues, the biomechanical, and functional characteristics of the Achilles tendon are closely related to its composition and microstructure. It is commonly reported that type I collagen is the predominant component of tendons and is mainly responsible for the tissue's function. Although elastin has been found in varying proportions in other connective tissues, previous studies report that tendons contain very small quantities of elastin. However, the morphology and the microstructural relationship among the elastic fibres, collagen, and cells in tendon tissue have not been well examined. We hypothesize the elastic fibres, as another fibrillar component in the extracellular matrix, have a unique role in mechanical function and microstructural arrangement in Achilles tendons. It has been shown that elastic fibres present a close connection with the tenocytes. The close relationship of the three components has been revealed as a distinct, integrated and complex microstructural network. Notably, a "spiral" structure within fibril bundles in Achilles tendons was observed in some samples in specialized regions. This study substantiates the hierarchical system of the spatial microstructure of tendon, including the mapping of collagen, elastin and tenocytes, with 3-dimensional confocal images. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1203-1214, 2017. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Glucagon-like Peptide-1 and the Central/Peripheral Nervous System

DEFF Research Database (Denmark)

Muscogiuri, Giovanna; DeFronzo, Ralph A; Gastaldelli, Amalia

2017-01-01

Glucagon-like peptide-1 (GLP-1) is released in response to meals and exerts important roles in the maintenance of normal glucose homeostasis. GLP-1 is also important in the regulation of neurologic and cognitive functions. These actions are mediated via neurons in the nucleus of the solitary trac...... human trials report a neuroprotective effect of GLP-1-RAs in Alzheimer's and Parkinson's disease. In this review, we discuss the role of GLP-1 and GLP-1-RAs in the nervous system with focus on GLP-1 actions on appetite regulation, glucose homeostasis, and neuroprotection....

Does Glucagon-like Peptide-1 Ameliorate Oxidative Stress in Diabetes?

DEFF Research Database (Denmark)

Petersen, Karen Ekkelund; Rakipovski, Günaj; Raun, Kirsten

2016-01-01

Glucagon-like peptide-1 (GLP-1) has shown to influence the oxidative stress status in a number of in vitro, in vivo and clinical studies. Well-known effects of GLP-1 including better glycemic control, decreased food intake, increased insulin release and increased insulin sensitivity may indirectly...... a controversial topic but could hold a therapeutic potential against micro- and macrovascular diabetic complications. This review discusses the presently available knowledge from experimental and clinical studies on the effects of GLP-1 on oxidative stress in diabetes and diabetes-related complications....

Crystal structure of the ligand-bound glucagon-like peptide-1 receptor extracellular domain.

Science.gov (United States)

Runge, Steffen; Thøgersen, Henning; Madsen, Kjeld; Lau, Jesper; Rudolph, Rainer

2008-04-25

The glucagon-like peptide-1 receptor (GLP-1R) belongs to Family B1 of the seven-transmembrane G protein-coupled receptors, and its natural agonist ligand is the peptide hormone glucagon-like peptide-1 (GLP-1). GLP-1 is involved in glucose homeostasis, and activation of GLP-1R in the plasma membrane of pancreatic beta-cells potentiates glucose-dependent insulin secretion. The N-terminal extracellular domain (nGLP-1R) is an important ligand binding domain that binds GLP-1 and the homologous peptide Exendin-4 with differential affinity. Exendin-4 has a C-terminal extension of nine amino acid residues known as the "Trp cage", which is absent in GLP-1. The Trp cage was believed to interact with nGLP-1R and thereby explain the superior affinity of Exendin-4. However, the molecular details that govern ligand binding and specificity of nGLP-1R remain undefined. Here we report the crystal structure of human nGLP-1R in complex with the antagonist Exendin-4(9-39) solved by the multiwavelength anomalous dispersion method to 2.2A resolution. The structure reveals that Exendin-4(9-39) is an amphipathic alpha-helix forming both hydrophobic and hydrophilic interactions with nGLP-1R. The Trp cage of Exendin-4 is not involved in binding to nGLP-1R. The hydrophobic binding site of nGLP-1R is defined by discontinuous segments including primarily a well defined alpha-helix in the N terminus of nGLP-1R and a loop between two antiparallel beta-strands. The structure provides for the first time detailed molecular insight into ligand binding of the human GLP-1 receptor, an established target for treatment of type 2 diabetes.

Gold nanoparticles-based electrochemical method for the detection of protein kinase with a peptide-like inhibitor as the bioreceptor

Directory of Open Access Journals (Sweden)

Sun K

2017-03-01

Full Text Available Kai Sun, Yong Chang, Binbin Zhou, Xiaojin Wang, Lin Liu Henan Province of Key Laboratory of New Optoelectronic Functional Materials, College of Chemistry and Chemical Engineering, Anyang Normal University, Anyang, Henan, People’s Republic of China Abstract: This article presents a general method for the detection of protein kinase with a peptide-like kinase inhibitor as the bioreceptor, and it was done by converting gold nanoparticles (AuNPs-based colorimetric assay into sensitive electrochemical analysis. In the colorimetric assay, the kinase-specific aptameric peptide triggered the aggregation of AuNPs in solution. However, the specific binding of peptide to the target protein (kinase inhibited its ability to trigger the assembly of AuNPs. In the electrochemical analysis, peptides immobilized on a gold electrode and presented as solution triggered together the in situ formation of AuNPs-based network architecture on the electrode surface. Nevertheless, the formation of peptide–kinase complex on the electrode surface made the peptide-triggered AuNPs assembly difficult. Electrochemical impedance spectroscopy was used to measure the change in surface property in the binding events. When a ferrocene-labeled peptide (Fc-peptide was used in this design, the network of AuNPs/Fc-peptide produced a good voltammetric signal. The competitive assay allowed for the detection of protein kinase A with a detection limit of 20 mU/mL. This work should be valuable for designing novel optical or electronic biosensors and likely lead to many detection applications. Keywords: electrochemical biosensor, colorimetric assay, gold nanoparticle, aptameric peptide, protein kinase A, signal amplification 

Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor-Derived Peptides for Regulation of Mast Cell Degranulation.

Science.gov (United States)

Yang, Yoosoo; Kong, Byoungjae; Jung, Younghoon; Park, Joon-Bum; Oh, Jung-Mi; Hwang, Jaesung; Cho, Jae Youl; Kweon, Dae-Hyuk

2018-01-01

Vesicle-associated V-soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and target membrane-associated T-SNAREs (syntaxin 4 and SNAP-23) assemble into a core trans -SNARE complex that mediates membrane fusion during mast cell degranulation. This complex plays pivotal roles at various stages of exocytosis from the initial priming step to fusion pore opening and expansion, finally resulting in the release of the vesicle contents. In this study, peptides with the sequences of various SNARE motifs were investigated for their potential inhibitory effects against SNARE complex formation and mast cell degranulation. The peptides with the sequences of the N-terminal regions of vesicle-associated membrane protein 2 (VAMP2) and VAMP8 were found to reduce mast cell degranulation by inhibiting SNARE complex formation. The fusion of protein transduction domains to the N-terminal of each peptide enabled the internalization of the fusion peptides into the cells equally as efficiently as cell permeabilization by streptolysin-O without any loss of their inhibitory activities. Distinct subsets of mast cell granules could be selectively regulated by the N-terminal-mimicking peptides derived from VAMP2 and VAMP8, and they effectively decreased the symptoms of atopic dermatitis in mouse models. These results suggest that the cell membrane fusion machinery may represent a therapeutic target for atopic dermatitis.

Glucagon-like peptide-2 induces rapid digestive adaptation following intestinal resection in preterm neonates

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Short bowel syndrome (SBS) is a frequent complication after intestinal resection in infants suffering from intestinal disease. We tested whether treatment with the intestinotrophic hormone glucagon-like peptide-2 (GLP-2) increases intestinal volume and function in the period immediately following in...

Controlling the porosity of collagen, gelatin and elastin biomaterials by ultrashort laser pulses

International Nuclear Information System (INIS)

Daskalova, A.; Nathala, Chandra S.R.; Bliznakova, I.; Stoyanova, E.; Zhelyazkova, A.; Ganz, T.; Lueftenegger, S.; Husinsky, W.

2014-01-01

We report on the structural investigation of self-organized micropores generated in thin gelatin, collagen, and collagen–elastin films after single and multishot irradiation with pulse durations ranging from 30–100 fs at 800 nm. We systematically studied the effect of laser parameters: laser energy, number of pulses, and pulse duration on the development of the micropores. This work showed that applying laser pulses at different rates significantly modified the thin film surface. The results clearly revealed that femtosecond laser treatment of thin films of biomaterials: gelatin, collagen and collagen–elastin, results in creation of micro/nanopores with different size of cavity formations. Experimentally, it is demonstrated that it is possible to influence the dimensions of the pore sizes, ranging from 100 nm to 2 μm by tuning the laser parameters. We are currently further exploring the possibility of structuring these biomaterials by applying a time delay between separate pulses. First results from cell culture experiments on laser created surface foam of collagen–elastin were successfully obtained, showing the potential of the method to cultivate cells on superficial porous substrates and the preferable selectivity of the cells to proliferate on the laser modified parts of the biopolymer substrate.

Controlling the porosity of collagen, gelatin and elastin biomaterials by ultrashort laser pulses

Energy Technology Data Exchange (ETDEWEB)

Daskalova, A., E-mail: a_daskalova@code.bg [Institute of Electronics, Bulgarian Academy of Sciences, 72, Tsarigradsko Chaussee blvd., 1784 Sofia (Bulgaria); Nathala, Chandra S.R. [IAP, Vienna University of Technology, Wiedner Hauptstrasse 8-10, 1040 Vienna (Austria); Femtolasers Productions GmbH, Fernkorngasse10, 1100 Vienna (Austria); Bliznakova, I. [Institute of Electronics, Bulgarian Academy of Sciences, 72, Tsarigradsko Chaussee blvd., 1784 Sofia (Bulgaria); Stoyanova, E. [IBIR, Department of Molecular Immunology, Bulgarian Academy of Sciences, 73, Tzarigradsko Chaussee blvd., 1113 Sofia (Bulgaria); Zhelyazkova, A. [Institute of Electronics, Bulgarian Academy of Sciences, 72, Tsarigradsko Chaussee blvd., 1784 Sofia (Bulgaria); Ganz, T. [Femtolasers Productions GmbH, Fernkorngasse10, 1100 Vienna (Austria); Lueftenegger, S.; Husinsky, W. [IAP, Vienna University of Technology, Wiedner Hauptstrasse 8-10, 1040 Vienna (Austria)

2014-02-15

We report on the structural investigation of self-organized micropores generated in thin gelatin, collagen, and collagen–elastin films after single and multishot irradiation with pulse durations ranging from 30–100 fs at 800 nm. We systematically studied the effect of laser parameters: laser energy, number of pulses, and pulse duration on the development of the micropores. This work showed that applying laser pulses at different rates significantly modified the thin film surface. The results clearly revealed that femtosecond laser treatment of thin films of biomaterials: gelatin, collagen and collagen–elastin, results in creation of micro/nanopores with different size of cavity formations. Experimentally, it is demonstrated that it is possible to influence the dimensions of the pore sizes, ranging from 100 nm to 2 μm by tuning the laser parameters. We are currently further exploring the possibility of structuring these biomaterials by applying a time delay between separate pulses. First results from cell culture experiments on laser created surface foam of collagen–elastin were successfully obtained, showing the potential of the method to cultivate cells on superficial porous substrates and the preferable selectivity of the cells to proliferate on the laser modified parts of the biopolymer substrate.

Deposition of insoluble elastin by pulmonary fibroblasts from patients with COPD is increased by treatment with versican siRNA.

Science.gov (United States)

Wu, Lian; Zhang, Jing; Qu, Jie Ming; Bai, Chun-Xue; Merrilees, Mervyn J

2017-01-01

A reduced content of alveolar elastic fibers is a key feature of COPD lung. Despite continued elastogenic potential by alveolar fibroblasts in the lung affected by COPD, repair of elastic fibers does not take place, which is due to increased levels of the chondroitin sulfate proteoglycan versican that inhibits the assembly of tropoelastin into fibers. In this study, primary pulmonary fibroblast cell lines from COPD and non-COPD patients were treated with a small interfering RNA (siRNA) against versican to determine if knockdown of versican could restore the deposition of insoluble elastin. Versican siRNA treatment reduced versican expression and secretion by pulmonary fibroblasts from both COPD and non-COPD patients ( P elastin in the COPD cell cultures ( P elastin (tropoelastin) in either the COPD or non-COPD cell cultures, supporting a role for versican in inhibiting assembly but not synthesis of tropoelastin. These results suggest that removal or knockdown of versican may be a possible therapeutic strategy for increasing deposition of insoluble elastin and stimulating repair of elastic fibers in COPD lung.

Effect of glycosylation on biodistribution of radiolabeled glucagon-like peptide 1

International Nuclear Information System (INIS)

Watanabe, Ayahisa; Nishijima, Ken-ichi; Zhao, Songji; Tamaki, Nagara; Kuge, Yuji; Tanaka, Yoshikazu; Itoh, Takeshi; Takemoto, Hiroshi

2012-01-01

Glycosylation is generally applicable as a strategy for increasing the activity of bioactive proteins. In this study, we examined the effect of glycosylation on biodistribution of radiolabeled glucagon-like peptide 1 (GLP-1) as a bioactive peptide for type 2 diabetes. Noninvasive imaging studies were performed using a gamma camera after the intravenous administration of 123 I-GLP-1 or 123 I-α2, 6-sialyl N-acetyllactosamine (glycosylated) GLP-1 in rats. In ex vivo biodistribution studies using 125 I-GLP-1 or 125 I-glycosylated GLP-1, organ samples were measured for radioactivity. Plasma samples were added to 15% trichloroacetic acid (TCA) to obtain TCA-insoluble and TCA-soluble fractions. The radioactivity in the TCA-insoluble and TCA-soluble fractions was measured. In the noninvasive imaging studies, a relatively high accumulation level of 123 I-GLP-1 was found in the liver, which is the major organ to eliminate exogenous GLP-1. The area under the time-activity curve (AUC) of 123 I-glycosylated GLP-1 in the liver was significantly lower (89%) than that of 123 I-GLP-1. These results were consistent with those of ex vivo biodistribution studies using 125 I-labeled peptides. The AUC of 125 I-glycosylated GLP-1 in the TCA-insoluble fraction was significantly higher (1.7-fold) than that of GLP-1. This study demonstrated that glycosylation significantly decreased the distribution of radiolabeled GLP-1 into the liver and increased the concentration of radiolabeled GLP-1 in plasma. These results suggested that glycosylation is a useful strategy for decreasing the distribution into the liver of bioactive peptides as desirable pharmaceuticals. (author)

Incretin-based treatment of type 2 diabetes: glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors

DEFF Research Database (Denmark)

Deacon, Carolyn F

2007-01-01

Incretins are gut peptides that potentiate nutrient-stimulated insulin secretion following meal ingestion. Activities of the dominant incretins, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide, include glucose-dependent stimulation of insulin secretion and, in preclin...... and liraglutide) and DPP-4 inhibitors that act to increase concentrations of endogenous intact incretins (e.g. sitagliptin and vildagliptin). Clinical trials of these incretin-based therapies have shown them to be effective in improving glycaemic control in patients with T2DM....

The secreted peptide PIP1 amplifies immunity through receptor-like kinase 7.

Directory of Open Access Journals (Sweden)

Shuguo Hou

2014-09-01

Full Text Available In plants, innate immune responses are initiated by plasma membrane-located pattern recognition receptors (PRRs upon recognition of elicitors, including exogenous pathogen-associated molecular patterns (PAMPs and endogenous damage-associated molecular patterns (DAMPs. Arabidopsis thaliana produces more than 1000 secreted peptide candidates, but it has yet to be established whether any of these act as elicitors. Here we identified an A. thaliana gene family encoding precursors of PAMP-induced secreted peptides (prePIPs through an in-silico approach. The expression of some members of the family, including prePIP1 and prePIP2, is induced by a variety of pathogens and elicitors. Subcellular localization and proteolytic processing analyses demonstrated that the prePIP1 product is secreted into extracellular spaces where it is cleaved at the C-terminus. Overexpression of prePIP1 and prePIP2, or exogenous application of PIP1 and PIP2 synthetic peptides corresponding to the C-terminal conserved regions in prePIP1 and prePIP2, enhanced immune responses and pathogen resistance in A. thaliana. Genetic and biochemical analyses suggested that the receptor-like kinase 7 (RLK7 functions as a receptor of PIP1. Once perceived by RLK7, PIP1 initiates overlapping and distinct immune signaling responses together with the DAMP PEP1. PIP1 and PEP1 cooperate in amplifying the immune responses triggered by the PAMP flagellin. Collectively, these studies provide significant insights into immune modulation by Arabidopsis endogenous secreted peptides.

Programmable fusion of liposomes mediated by lipidated PNA

DEFF Research Database (Denmark)

Rabe, A; Löffler, P M G; Ries, O

2017-01-01

We recently reported a DNA-programmed fusion cascade enabling the use of liposomes as nanoreactors for compartmentalized chemical reactions. This communication reports an alternative and robust strategy based on lipidated peptide nucleic acids (LiPs). LiPs enabled fusion of liposomes with remarka...... with remarkable 31% efficiency at 50 °C with low leakage (5%)....

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

DEFF Research Database (Denmark)

Ipharraguerre, Ignacio R; Tedó, Gemma; Menoyo, David

2013-01-01

Early weaning is a stressful event characterized by a transient period of intestinal atrophy that may be mediated by reduced secretion of glucagon-like peptide (GLP) 2. We tested whether enterally fed bile acids or plant sterols could increase nutrient-dependent GLP-2 secretion and improve.......05) but did not affect plasma GLP-1 and feed intake. The intestinal expression of glucagon-like peptide 2 receptor, sodium-dependent bile acid transporter, farnesoid X receptor, and guanosine protein-coupled bile acid receptor genes were not affected by CDC treatment. The intragastric administration of CDC...

Fixed ratio combinations of glucagon like peptide 1 receptor agonists with basal insulin: a systematic review and meta-analysis.

Science.gov (United States)

Liakopoulou, Paraskevi; Liakos, Aris; Vasilakou, Despoina; Athanasiadou, Eleni; Bekiari, Eleni; Kazakos, Kyriakos; Tsapas, Apostolos

2017-06-01

Basal insulin controls primarily fasting plasma glucose but causes hypoglycaemia and weight gain, whilst glucagon like peptide 1 receptor agonists induce weight loss without increasing risk for hypoglycaemia. We conducted a systematic review and meta-analysis of randomised controlled trials to investigate the efficacy and safety of fixed ratio combinations of basal insulin with glucagon like peptide 1 receptor agonists. We searched Medline, Embase, and the Cochrane Library as well as conference abstracts up to December 2016. We assessed change in haemoglobin A 1c , body weight, and incidence of hypoglycaemia and gastrointestinal adverse events. We included eight studies with 5732 participants in the systematic review. Switch from basal insulin to fixed ratio combinations with a glucagon like peptide 1 receptor agonist was associated with 0.72% reduction in haemoglobin A 1c [95% confidence interval -1.03 to -0.41; I 2  = 93%] and 2.35 kg reduction in body weight (95% confidence interval -3.52 to -1.19; I 2  = 93%), reducing also risk for hypoglycaemia [odds ratio 0.70; 95% confidence interval 0.57 to 0.86; I 2  = 85%] but increasing incidence of nausea (odds ratio 6.89; 95% confidence interval 3.73-12.74; I 2  = 79%). Similarly, switching patients from treatment with a glucagon like peptide 1 receptor agonist to a fixed ratio combination with basal insulin was associated with 0.94% reduction in haemoglobin A 1c (95% confidence interval -1.11 to -0.77) and an increase in body weight by 2.89 kg (95% confidence interval 2.17-3.61). Fixed ratio combinations of basal insulin with glucagon like peptide 1 receptor agonists improve glycaemic control whilst balancing out risk for hypoglycaemia and gastrointestinal side effects.

The Relationship between Glucagon-Like Peptide 2 and Feeding Intolerance in Preterm Infants

DEFF Research Database (Denmark)

Ozer, Esra Arun; Holst, Jens Juul; Duman, Nuray

2008-01-01

Glucagon-like peptide 2 (GLP-2) is a hormone produced primarily in the distal intestine, stimulated by enteral nutrients, and playing diverse roles in the intestinal adaptation and growth. We aimed to investigate whether GLP-2 may play a role in the development of feeding intolerance which is a c...... with low levels of GLP-2 carry a risk for development of feeding intolerance. It may, therefore, be of relevance to investigate the therapeutic and prophylactic effects of GLP-2 administration in the preterm babies.......Glucagon-like peptide 2 (GLP-2) is a hormone produced primarily in the distal intestine, stimulated by enteral nutrients, and playing diverse roles in the intestinal adaptation and growth. We aimed to investigate whether GLP-2 may play a role in the development of feeding intolerance which...... is a common problem in preterm newborns resulting from the intestinal immaturity. The study included 20 term and 28 preterm neonates. Of preterm babies, 13 showed feeding intolerance fulfilling at least one of the following criteria: abdominal distension, increased gastric residual volume and presence of bile...

Glucagon-like peptide-1 7-36 amide and peptide YY from the L-cell of the ileal mucosa are potent inhibitors of vagally induced gastric acid secretion in man

DEFF Research Database (Denmark)

Wettergren, A; Petersen, H; Orskov, C

1994-01-01

BACKGROUND: Glucagon-like peptide (GLP-1) 7-36 amide and peptide YY (PYY) from the L-cell of the ileal mucosa are potent inhibitors of gastric acid secretion in man. It is not clear, however, by which mechanism(s) they inhibit acid secretion. In dogs the inhibitory effect of PYY on acid secretion...

The contribution of vascular smooth muscle, elastin and collagen on the passive mechanics of porcine carotid arteries

International Nuclear Information System (INIS)

Kochová, P; Cimrman, R; Kuncová, J; Švíglerová, J; Miklíková, M; Liška, V; Tonar, Z

2012-01-01

The main components responsible for the mechanical behavior of the arterial wall are collagen, elastin, and smooth muscle cells (SMCs) in the medial layer. We determined the structural and mechanical changes in porcine carotid arteries after administration of Triton® X-100, elastase, and collagenase using the inflation–deflation test. The arteries were intraluminarly pressurized from 0 to 200 mmHg, and the outer diameter of the artery was measured. The pressure–strain elastic modulus was determined based on the pressure/diameter ratio. The intima–media thickness, wall thickness, thickness of the tunica adventitia layer, and the area fractions of SMCs, elastin, and collagen within the arterial wall (A A (SMC/elastin/collagen, wall)) were measured using stereological methods. The relative changes in the relevant components of the treated samples were as follows: the decrease in A A (SMC, wall) after administration of Triton® X-100 was 11% ± 7%, the decrease in A A (elastin, wall) after administration of elastase was 40% ± 22%, and the decrease in A A (collagen, wall) after the application of collagenase was 51% ± 22%. The Triton® X-100 treatment led to a decrease in the SMC content that was associated with enlargement of the arterial wall (outer diameter) for pressures up to 120 mmHg, and with mechanical stiffening of the arterial wall at higher pressures. Elastase led to a decrease in the elastin content that was associated with enlargement of the arterial wall, but not with stiffening or softening. Collagenase led to a decrease in collagen content that was associated with a change in the stiffness of the arterial wall, although the exact contribution of mechanical loading and the duration of treatment (enlargement) could not be quantified. (paper)

Cathepsin-Mediated Cleavage of Peptides from Peptide Amphiphiles Leads to Enhanced Intracellular Peptide Accumulation

Energy Technology Data Exchange (ETDEWEB)

Acar, Handan [Institute; Department; Samaeekia, Ravand [Institute; Department; Schnorenberg, Mathew R. [Institute; Department; Medical; Sasmal, Dibyendu K. [Institute; Huang, Jun [Institute; Tirrell, Matthew V. [Institute; Institute; LaBelle, James L. [Department

2017-08-24

Peptides synthesized in the likeness of their native interaction domain(s) are natural choices to target protein protein interactions (PPIs) due to their fidelity of orthostatic contact points between binding partners. Despite therapeutic promise, intracellular delivery of biofunctional peptides at concentrations necessary for efficacy remains a formidable challenge. Peptide amphiphiles (PAs) provide a facile method of intracellular delivery and stabilization of bioactive peptides. PAs consisting of biofunctional peptide headgroups linked to hydrophobic alkyl lipid-like tails prevent peptide hydrolysis and proteolysis in circulation, and PA monomers are internalized via endocytosis. However, endocytotic sequestration and steric hindrance from the lipid tail are two major mechanisms that limit PA efficacy to target intracellular PPIs. To address these problems, we have constructed a PA platform consisting of cathepsin-B cleavable PAs in which a selective p53-based inhibitory peptide is cleaved from its lipid tail within endosomes, allowing for intracellular peptide accumulation and extracellular recycling of the lipid moiety. We monitor for cleavage and follow individual PA components in real time using a resonance energy transfer (FRET)-based tracking system. Using this platform, components in real time using a Forster we provide a better understanding and quantification of cellular internalization, trafficking, and endosomal cleavage of PAs and of the ultimate fates of each component.

Effects of prepartum fat supplementation on plasma concentrations of glucagon-like peptide-1, peptide YY, adropin, insulin, and leptin in periparturient dairy cows.

Science.gov (United States)

Zapata, Rizaldy C; Salehi, Reza; Ambrose, Divakar J; Chelikani, Prasanth K

2015-10-01

Dietary fat supplementation during the periparturient period is one strategy to increase energy intake and attenuate the degree of negative energy balance during early lactation; however, little is known of the underlying hormonal and metabolic adaptations. We evaluated the effects of prepartum fat supplementation on energy-balance parameters and plasma concentrations of glucagon-like peptide-1, peptide tyrosine-tyrosine (PYY), adropin, insulin, leptin, glucose, nonesterified fatty acid, and β-hydroxybutyric acid in dairy cows. Twenty-four pregnant dairy cows were randomized to diets containing either rolled canola or sunflower seed at 8% of dry matter, or no oilseed supplementation, during the last 5 wk of gestation and then assigned to a common lactation diet postpartum. Blood samples were collected at -2, +2, and +14 h relative to feeding, at 2 wk after the initiation of the diets, and at 2 wk postpartum. Dietary canola and sunflower supplementation alone did not affect energy balance, body weight, and plasma concentrations of glucagon-like peptide-1, PYY, adropin, insulin, leptin, nonesterified fatty acid, and β-hydroxybutyric acid; however, canola decreased and sunflower tended to decrease dry matter intake. We also observed that the physiological stage had a significant, but divergent, effect on circulating hormones and metabolite concentrations. Plasma glucagon-like peptide-1, PYY, adropin, nonesterified fatty acid, and β-hydroxybutyric acid concentrations were greater postpartum than prepartum, whereas glucose, insulin, leptin, body weight, and energy balance were greater prepartum than postpartum. Furthermore, the interaction of treatment and stage was significant for leptin and adropin, and tended toward significance for PYY and insulin; only insulin exhibited an apparent postprandial increase. Postpartum PYY concentrations exhibited a strong negative correlation with body weight, suggesting that PYY may be associated with body weight regulation during

Targeted recombinant fusion proteins of IFNγ and mimetic IFNγ with PDGFβR bicyclic peptide inhibits liver fibrogenesis in vivo.

Directory of Open Access Journals (Sweden)

Ruchi Bansal

Full Text Available Hepatic stellate cells (HSCs, following transdifferentiation to myofibroblasts plays a key role in liver fibrosis. Therefore, attempts to attenuate this myofibroblastic phenotype would be a promising therapeutic approach. Interferon gamma (IFNγ is a potent anti-fibrotic cytokine, but its pleiotropic receptor expression leading to severe adverse effects has limited its clinical application. Since, activated HSC express high-level of platelet derived growth factor beta receptor (PDGFβR, we investigated the potential of PDGFβR-specific targeting of IFNγ and its signaling peptide that lacks IFNγR binding site (mimetic IFNγ or mimIFNγ in liver fibrosis. We prepared DNA constructs expressing IFNγ, mimIFNγ or BiPPB (PDGFβR-specific bicyclic peptide-IFNγ, BiPPB-mimIFNγ fusion proteins. Both chimeric proteins alongwith IFNγ and mimIFNγ were produced in E.coli. The expressed proteins were purified and analyzed for PDGFβR-specific binding and in vitro effects. Subsequently, these recombinant proteins were investigated for the liver uptake (pSTAT1α signaling pathway, for anti-fibrotic effects and adverse effects (platelet counts in CCl4-induced liver fibrogenesis in mice. The purified HSC-targeted IFNγ and mimIFNγ fusion proteins showed PDGFβR-specific binding and significantly reduced TGFβ-induced collagen-I expression in human HSC (LX2 cells, while mouse IFNγ and mimIFNγ did not show any effect. Conversely, mouse IFNγ and BiPPB-IFNγ induced activation and dose-dependent nitric oxide release in mouse macrophages (express IFNγR while lack PDGFβR, which was not observed with mimIFNγ and BiPPB-mimIFNγ, due to the lack of IFNγR binding sites. In vivo, targeted BiPPB-IFNγ and BiPPB-mimIFNγ significantly activated intrahepatic IFNγ-signaling pathway compared to IFNγ and mimIFNγ suggesting increased liver accumulation. Furthermore, the targeted fusion proteins ameliorated liver fibrogenesis in mice by significantly reducing

Design and expression of a short peptide as an HIV detection probe

Energy Technology Data Exchange (ETDEWEB)

Lines, Jamie A.; Yu, Zhiqiang; Dedkova, Larisa M.; Chen, Shengxi, E-mail: shengxi.chen.1@asu.edu

2014-01-03

Highlights: •We designed a short fusion peptide (FP-50) for in vivo expression. •This peptide is a very promising component for detection of gp120 protein. •The detectable level is about 20–200 times lower than previously published methods. •It is a novel probe to detect HIV-1 gp120 during early stages of HIV infection. -- Abstract: To explore a low-cost novel probe for HIV detection, we designed and prepared a 50-amino acid-length short fusion peptide (FP-50) via Escherichia coli in vivo expression. It was employed as a novel probe to detect HIV-1 gp120 protein. The detectable level of gp120 protein using the FP-50 peptide was approximately 20–200 times lower than previously published methods that used a pair of monoclonal antibodies. Thus, this short peptide is a very promising component for detection of gp120 protein during early stages of HIV infection.

Adding an Artificial Tail—Anchor to a Peptide-Based HIV-1 Fusion Inhibitor for Improvement of Its Potency and Resistance Profile

Directory of Open Access Journals (Sweden)

Shan Su

2017-11-01

Full Text Available Peptides derived from the C-terminal heptad repeat (CHR of human immunodeficiency virus type 1 (HIV-1 envelope protein transmembrane subunit gp41, such as T20 (enfuvirtide, can bind to the N-terminal heptad repeat (NHR of gp41 and block six-helix bundle (6-HB formation, thus inhibiting HIV-1 fusion with the target cell. However, clinical application of T20 is limited because of its low potency and genetic barrier to resistance. HP23, the shortest CHR peptide, exhibits better anti-HIV-1 activity than T20, but the HIV-1 strains with E49K mutations in gp41 will become resistant to it. Here, we modified HP23 by extending its C-terminal sequence using six amino acid residues (E6 and adding IDL (Ile-Asp-Leu to the C-terminus of E6, which is expected to bind to the shallow pocket in the gp41 NHR N-terminal region. The newly designed peptide, designated HP23-E6-IDL, was about 2- to 16-fold more potent than HP23 against a broad spectrum of HIV-1 strains and more than 12-fold more effective against HIV-1 mutants resistant to HP23. These findings suggest that addition of an anchor–tail to the C-terminus of a CHR peptide will allow binding with the pocket in the gp41 NHR that may increase the peptide’s antiviral efficacy and its genetic barrier to resistance.

Material Binding Peptides for Nanotechnology