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Sample records for hepatocytes polypeptide assembly

  1. Principles Governing the Self Assembly of Polypeptide Nanoparticles

    Science.gov (United States)

    Wahome, Newton

    Self assembling systems on the nanometer scale afford the advantage of being able to control submicron level events. In this study, we focus on the self-assembling polypeptide nanoparticles (SAPN). The SAPN scaffold is made up of oligomerizing domains that align along the principle rotational axes of icosahedral symmetry. By aligning them along these axes, a particle with spherical geometry can be achieved. This particle can be utilized as a vaccine, as a drug delivery vehicle, or as a biomedical imaging device. This research will try to answer why the SAPN self-assembles into distinct molecular weight ranges while mostly maintaining a spherical morphology. The first means will be theoretical and computational, where we will utilize a mathematical formalism to find out how the packing of SAPN's monomeric units can occur within symmetric space. Then molecular dynamics will be run within this symmetric space to test the per amino acid residue susceptibility of SAPN towards becoming polymorphic in nature. Means for examining the aggregation propensity of SAPN will be also be tested. Specifically, the relationship of different sequences of SAPN with pH will be elucidated. Co-assembly of SAPN to reduce the surface density of an aggregation prone epitope will be tested. Also, aggregation reduction consisting of the exchange of an anionic denaturant with a positively charged suppressor in order to mitigate a priori peptide association and misfolding, will also be attempted. SAPN has been shown to be an immunogenic platform for the presentation of pathogen derived antigens. We will attempt to show the efficacy of presenting an antigen from HIV-1 which is structurally restrained to best match the native conformation on the virus. Immunological studies will be performed to test the effect of this approach, as well testing the antigenicity of the nanoparticle in the absence of adjuvant. Finally, the antigen presenting nanoparticles will undergo formulation testing, to measure

  2. Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments.

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    Gradišar, Helena; Božič, Sabina; Doles, Tibor; Vengust, Damjan; Hafner-Bratkovič, Iva; Mertelj, Alenka; Webb, Ben; Šali, Andrej; Klavžar, Sandi; Jerala, Roman

    2013-06-01

    Protein structures evolved through a complex interplay of cooperative interactions, and it is still very challenging to design new protein folds de novo. Here we present a strategy to design self-assembling polypeptide nanostructured polyhedra based on modularization using orthogonal dimerizing segments. We designed and experimentally demonstrated the formation of the tetrahedron that self-assembles from a single polypeptide chain comprising 12 concatenated coiled coil-forming segments separated by flexible peptide hinges. The path of the polypeptide chain is guided by a defined order of segments that traverse each of the six edges of the tetrahedron exactly twice, forming coiled-coil dimers with their corresponding partners. The coincidence of the polypeptide termini in the same vertex is demonstrated by reconstituting a split fluorescent protein in the polypeptide with the correct tetrahedral topology. Polypeptides with a deleted or scrambled segment order fail to self-assemble correctly. This design platform provides a foundation for constructing new topological polypeptide folds based on the set of orthogonal interacting polypeptide segments.

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

    KAUST Repository

    Junnila, Susanna

    2012-03-27

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

  4. Beaded nanofibers assembled from double-hydrophobic elastin-like block polypeptides: Effects of trifluoroethanol.

    Science.gov (United States)

    Le, Duc H T; Okubo, Tatsuya; Sugawara-Narutaki, Ayae

    2015-03-01

    A "double-hydrophobic" elastin-like triblock polypeptide GPG has been constructed by mimicking the localization of proline- and glycine-rich hydrophobic domains of native elastin, a protein that provides elasticity and resilience to connective tissues. In this study, the effects of trifluoroethanol (TFE), an organic solvent that strongly affects secondary structures of polypeptides on self-assembly of GPG in aqueous solutions were systematically studied. Beaded nanofiber formation of GPG, where nanoparticles are initially formed by coacervation of the polypeptides followed by their connection into one-dimensional nanostructures, is accelerated by the addition of TFE at the concentrations up to 30% (v/v), whereas aggregates of nanoparticles are formed at 60% TFE. The concentration-dependent assembly pattern discussed is based on the influence of TFE on the secondary structures of GPG. Well-defined nanofibers whose diameter and secondary structures are controlled by TFE concentration may be ideal building blocks for constructing bioelastic materials in tissue engineering.

  5. Molecular Self-Assembly Strategy for Generating Catalytic Hybrid Polypeptides

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    Ikezoe, Yasuhiro; Pike, Douglas H.; Nanda, Vikas; Matsui, Hiroshi

    2016-01-01

    Recently, catalytic peptides were introduced that mimicked protease activities and showed promising selectivity of products even in organic solvents where protease cannot perform well. However, their catalytic efficiency was extremely low compared to natural enzyme counterparts presumably due to the lack of stable tertiary fold. We hypothesized that assembling these peptides along with simple hydrophobic pockets, mimicking enzyme active sites, could enhance the catalytic activity. Here we fused the sequence of catalytic peptide CP4, capable of protease and esterase-like activities, into a short amyloidogenic peptide fragment of Aβ. When the fused CP4-Aβ construct assembled into antiparallel β-sheets and amyloid fibrils, a 4.0-fold increase in the hydrolysis rate of p-nitrophenyl acetate (p-NPA) compared to neat CP4 peptide was observed. The enhanced catalytic activity of CP4-Aβ assembly could be explained both by pre-organization of a catalytically competent Ser-His-acid triad and hydrophobic stabilization of a bound substrate between the triad and p-NPA, indicating that a design strategy for self-assembled peptides is important to accomplish the desired functionality. PMID:27116246

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

    Science.gov (United States)

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

    2009-12-01

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

  7. Cellular Interactions and Biocompatibility of Self-Assembling Diblock Polypeptide Hydrogels

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    Pakstis, Lisa; Ozbas, Bulent; Pochan, Darrin; Robinson, Clifford; Nowak, Andrew; Deming, Timothy

    2002-03-01

    Self-assembling peptide based hydrogels having a unique nano- and microscopic morphology are being studied for potential use as tissue engineering scaffolds. Low molecular weight ( ~20 kg/mol), amphiphilic, diblock polypeptides of hydrophilic lysine (K) or glutamic acid (E) and hydrophobic leucine (L) or valine (V) form hydrogels in aqueous solution at neutral pH and at very low volume fraction of polymer (vol. fraction polypeptide >=0.5 wt%). The morphology of these hydrogels has been characterized using laser confocal microscopy (LCM), small angle neutron scattering (SANS), and cryogenic transmission electron microscopy (cryoTEM) imaging. Studies of the interactions of the hydrogels with bacterial and mammalian cells reveal that these materials are non-cytotoxic and biocompatible. Hence, the chemistry of the assembled diblock polypeptides allows for cellular proliferation whereas the same chemistry in the homopolyeric form is cytotoxic. Current research is directed at the design and incorporation of binding sites within the polypeptide to specifically target interactions of the hydrogel with desired cells types.

  8. Self-assemble nanoparticles based on polypeptides containing C-terminal luminescent Pt-cysteine complex

    Science.gov (United States)

    Vlakh, E. G.; Grachova, E. V.; Zhukovsky, D. D.; Hubina, A. V.; Mikhailova, A. S.; Shakirova, J. R.; Sharoyko, V. V.; Tunik, S. P.; Tennikova, T. B.

    2017-01-01

    The growing attention to the luminescent nanocarriers is strongly stimulated by their potential application as drug delivery systems and by the necessity to monitor their distribution in cells and tissues. In this communication we report on the synthesis of amphiphilic polypeptides bearing C-terminal phosphorescent label together with preparation of nanoparticles using the polypeptides obtained. The approach suggested is based on a unique and highly technological process where the new phosphorescent Pt-cysteine complex serves as initiator of the ring-opening polymerization of α-amino acid N-carboxyanhydrides to obtain the polypeptides bearing intact the platinum chromophore covalently bound to the polymer chain. It was established that the luminescent label retains unchanged its emission characteristics not only in the polypeptides but also in more complicated nanoaggregates such as the polymer derived amphiphilic block-copolymers and self-assembled nanoparticles. The phosphorescent nanoparticles display no cytotoxicity and hemolytic activity in the tested range of concentrations and easily internalize into living cells that makes possible in vivo cell visualization, including prospective application in time resolved imaging and drug delivery monitoring. PMID:28155880

  9. Self-assemble nanoparticles based on polypeptides containing C-terminal luminescent Pt-cysteine complex

    Science.gov (United States)

    Vlakh, E. G.; Grachova, E. V.; Zhukovsky, D. D.; Hubina, A. V.; Mikhailova, A. S.; Shakirova, J. R.; Sharoyko, V. V.; Tunik, S. P.; Tennikova, T. B.

    2017-02-01

    The growing attention to the luminescent nanocarriers is strongly stimulated by their potential application as drug delivery systems and by the necessity to monitor their distribution in cells and tissues. In this communication we report on the synthesis of amphiphilic polypeptides bearing C-terminal phosphorescent label together with preparation of nanoparticles using the polypeptides obtained. The approach suggested is based on a unique and highly technological process where the new phosphorescent Pt-cysteine complex serves as initiator of the ring-opening polymerization of α-amino acid N-carboxyanhydrides to obtain the polypeptides bearing intact the platinum chromophore covalently bound to the polymer chain. It was established that the luminescent label retains unchanged its emission characteristics not only in the polypeptides but also in more complicated nanoaggregates such as the polymer derived amphiphilic block-copolymers and self-assembled nanoparticles. The phosphorescent nanoparticles display no cytotoxicity and hemolytic activity in the tested range of concentrations and easily internalize into living cells that makes possible in vivo cell visualization, including prospective application in time resolved imaging and drug delivery monitoring.

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

    Science.gov (United States)

    Fluitt, Aaron Michael

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

  11. Assembly of Hepatocyte Spheroids Using Magnetic 3D Cell Culture for CYP450 Inhibition/Induction

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    Pujan K. Desai

    2017-05-01

    Full Text Available There is a significant need for in vitro methods to study drug-induced liver injury that are rapid, reproducible, and scalable for existing high-throughput systems. However, traditional monolayer and suspension cultures of hepatocytes are difficult to handle and risk the loss of phenotype. Generally, three-dimensional (3D cell culture platforms help recapitulate native liver tissue phenotype, but suffer from technical limitations for high-throughput screening, including scalability, speed, and handling. Here, we developed a novel assay for cytochrome P450 (CYP450 induction/inhibition using magnetic 3D cell culture that overcomes the limitations of other platforms by aggregating magnetized cells with magnetic forces. With this platform, spheroids can be rapidly assembled and easily handled, while replicating native liver function. We assembled spheroids of primary human hepatocytes in a 384-well format and maintained this culture over five days, including a 72 h induction period with known CYP450 inducers/inhibitors. CYP450 activity and viability in the spheroids were assessed and compared in parallel with monolayers. CYP450 activity was induced/inhibited in spheroids as expected, separate from any toxic response. Spheroids showed a significantly higher baseline level of CYP450 activity and induction over monolayers. Positive staining in spheroids for albumin and multidrug resistance-associated protein (MRP2 indicates the preservation of hepatocyte function within spheroids. The study presents a proof-of-concept for the use of magnetic 3D cell culture for the assembly and handling of novel hepatic tissue models.

  12. Characterization of liver-specific structure and function during hepatocyte spheroid self-assembly: Implications for a bioartificial liver device

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    Friend, Julie Renee

    A hollow fiber bioreactor containing collagen-entrapped hepatocytes has been developed as a bioartificial liver device. For clinical application, further scale-up of the device is desirable. This may be achieved through the use of hepatocyte spheroids, which are compacted aggregates that exhibit prolonged viability, higher liver-specific function and a more tissue-like ultrastructure compared to hepatocytes cultured as monolayers. In order to gain a better understanding of structural changes in spheroids over the course of their self-assembly, confocal microscopy was used to optically section spheroids and monitor changes in situ. Channels within spheroids hypothesized to be bile canaliculi were first evaluated by monitoring the diffusion of a fluorescent tracer, FITC-dextran, into spheroids. Three-dimensional reconstruction of spheroids showed that a continuous network of channels was forming within spheroids. Functionality of these channels as bile canaliculi was demonstrated by monitoring secretion of a fluorescently tagged bile acid, FITC-glycocholate, by hepatocytes in spheroids. Secretion of FITC-glycocholate could be seen in both rat and porcine hepatocyte spheroids. To elucidate changes in metabolism occurring during spheroid self-assembly, metabolic flux analysis was applied to hepatocyte spinner cultures. Glucose, lactate, amino acid, albumin and urea concentration in culture medium were measured and used to estimate intracellular fluxes within hepatocytes. Metabolism before and after spheroid formation was compared. Overall, little difference was seen in metabolism before and after spheroid self-assembly. As the BAL approaches clinical trials, methods of bioreactor storage for shipping and inventory purposed need to be developed. Storage conditions were tested in various hepatocyte culture systems. A protocol for storing reactors for 24 hours without significant loss in function was developed. Further optimization will be necessary for storage for longer

  13. Self-assembly model, hepatocytes attachment and inflammatory response for silk fibroin/chitosan scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    She Zhending; Feng Qingling [State Key Laboratory of New Ceramics and Fine Processing, Department of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China); Liu Weiqiang, E-mail: biomater@mail.tsinghua.edu.c [Center for Advanced Materials and Biotechnology, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057 (China)

    2009-08-15

    Silk fibroin is an attractive natural fibrous protein for biomedical application due to its good biocompatibility and high tensile strength. Silk fibroin is apt to form a sheet-like structure during the freeze-drying process, which is not suitable for the scaffold of tissue engineering. In our former study, the adding of chitosan promoted the self-assembly of silk fibroin/chitosan (SFCS) into a three-dimensional (3D) homogeneous porous structure. In this study, a model of the self-assembly is proposed; furthermore, hepatocytes attachment and inflammatory response for the SFCS scaffold were examined. The rigid chain of chitosan may be used as a template for beta-sheet formation of silk fibroin, and this may break the sheet structure of the silk fibroin scaffold and promote the formation of a 3D porous structure of the SFCS scaffold. Compared with the polylactic glycolic acid scaffold, the SFCS scaffold further facilitates the attachment of hepatocytes. To investigate the inflammatory response, SFCS scaffolds were implanted into the greater omentum of rats. From the results of implantation, we could demonstrate in vivo that the implantation of SFCS scaffolds resulted in only slight inflammation. Keeping the good histocompatibility and combining the advantages of both fibroin and chitosan, the SFCS scaffold could be a prominent candidate for soft tissue engineering, for example, in the liver.

  14. Regulating Cell Apoptosis on Layer-by-Layer Assembled Multilayers of Photosensitizer-Coupled Polypeptides and Gold Nanoparticles

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    Xing, Ruirui; Jiao, Tifeng; Ma, Kai; Ma, Guanghui; Möhwald, Helmuth; Yan, Xuehai

    2016-05-01

    The design of advanced, nanostructured materials by layer-by-layer (LbL) assembly at the molecular level is of great interest because of the broad application of these materials in the biomedical field especially in regulating cell growth, adhesion, movement, differentiation and detachment. Here, we fabricated functional hybrid multilayer films by LbL assembly of biocompatible photosensitizer-coupled polypeptides and collagen-capped gold nanoparticles. The resulting multilayer film can well accommodate cells for adhesion, growth and proliferation. Most significantly, controlled cell apoptosis (detachment) and patterning of the multilayer film is achieved by a photochemical process yielding reactive oxygen species (ROS). Moreover, the site and shape of apoptotic cells can be controlled easily by adjusting the location and shape of the laser beam. The LbL assembled multilayer film with integration of functions provides an efficient platform for regulating cell growth and apoptosis (detachment).

  15. Direct Assembly of Mesoporous Silica Functionalized with Polypeptides for Efficient Dye Adsorption.

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    Lu, Yi-Syuan; Bastakoti, Bishnu Prasad; Pramanik, Malay; Malgras, Victor; Yamauchi, Yusuke; Kuo, Shiao-Wei

    2016-01-18

    Herein, we introduce a new polypeptide-functionalized mesoporous silica template fabricated from a biodegradable poly(ethylene oxide-b-ɛ-caprolactone) (PEO-b-PCL) diblock copolymer and a poly(tyrosine) (PTyr) biopolymer. The crystallization behavior of the PEO-b-PCL diblock copolymer changes after blending, but the secondary structure of PTry remains stable. After selective solvent extraction in THF, the PEO-b-PCL is removed, but PTyr remains within the silica matrix due to its different solubility. Fourier-transform IR spectroscopic analysis (FTIR), thermal gravitometry analysis (TGA), small-angle X-ray scattering (SAXS), and X-ray diffraction (XRD) studies confirm the retention of PTyr to form a polypeptide-functionalized mesoporous material. The adsorption of methylene blue hydrate (MB) from aqueous solution into the polypeptide-functionalized mesoporous silica is investigated, thus revealing that the nanocomposite exhibits a high adsorption capacity relative to pure silica due to hydrogen-bonding interactions between the hydroxy phenolic group of PTyr and the N-containing aromatic ring from MB.

  16. Self-assembled micelles of amphiphilic poly(L-phenylalanine-b-poly(L-serine polypeptides for tumor-targeted delivery

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    Zhao ZM

    2014-12-01

    Full Text Available Ziming Zhao,1,2,* Yu Wang,1,2,* Jin Han,1,2 Keli Wang,1 Dan Yang,1,2 Yihua Yang,1,2 Qian Du,1,2 Yuanjian Song,3 Xiaoxing Yin1,2 1Department of Pharmacy, 2Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, 3Department of Basic Medical Sciences, Xuzhou Medical College, Xuzhou, People’s Republic of China *These authors contributed equally to this work Abstract: The aim of this work was to design, synthesize, and characterize self-assembled micelles based on polypeptides as a potential antitumor drug carrier. Amphiphilic poly(L-phenylalanine-b-poly(L-serine (PFS polypeptides were obtained through the polymerization of N-carboxyanhydride. As a novel hydrophilic segment, poly(L-serine was utilized to enhance tumor targeting due to a large demand of tumors for serine. PFS could self-assemble into micelles with an average diameter of 110–240 nm and a slightly negative charge. PFS polypeptides adopted random coil in pH 7.4 phosphate-buffered saline and could partly transform to a-helix induced by trifluoroethanol. PFS micelles with a low critical micelle concentration of 4.0 µg mL-1 were stable in pH 5–9 buffers and serum albumin solution. PFS micelles had a loading capacity of 3.8% for coumarin-6 and exhibited a sustained drug release. Coumarin-6 loaded rhodamine B isothiocyanate-labeled PFS micelles were incubated with Huh-7 tumor cells to study the correlation between drugs and carriers during endocytosis. The uptake of drugs was consistent with the micelles, illustrating that the intracellular transport of drugs highly depended on the micelles. PFS micelles diffused in whole cytoplasm while coumarin-6 assumed localized distribution, suggesting that the micelles could release the loaded drugs in particular areas. The internalization mechanism of PFS micelles was involved with clathrin-mediated endocytosis and macropinocytosis. Excess serine inhibited the uptake of PFS micelles, which demonstrated that serine receptors played

  17. Incorporation of a polypeptide segment into the beta-domain pore during the assembly of a bacterial autotransporter.

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    Ieva, Raffaele; Skillman, Kristen M; Bernstein, Harris D

    2008-01-01

    Bacterial autotransporters consist of an N-terminal 'passenger domain' that is transported into the extracellular space by an unknown mechanism and a C-terminal 'beta-domain' that forms a beta-barrel in the outer membrane. Recent studies have revealed that fully assembled autotransporters have an unusual architecture in which a small passenger domain segment traverses the pore formed by the beta-domain. It is unclear, however, whether this configuration forms prior to passenger domain translocation or results from the translocation of the passenger domain through the beta-domain pore. By examining the accessibility of tobacco etch virus protease sites and single-cysteine residues in the passenger domain of the Escherichia coli O157:H7 autotransporter EspP at different stages of protein biogenesis, we identified a novel pre-translocation intermediate whose topology resembles that of the fully assembled protein. This intermediate was isolated in the periplasm in cell fractionation experiments. The data strongly suggest that the EspP beta-domain and an embedded polypeptide segment are integrated into the outer membrane as a single pre-formed unit. The data also provide indirect evidence that at least some outer membrane proteins acquire considerable tertiary structure prior to their membrane integration.

  18. Optimizing the refolding conditions of self-assembling polypeptide nanoparticles that serve as repetitive antigen display systems.

    Science.gov (United States)

    Yang, Yongkun; Ringler, Philippe; Müller, Shirley A; Burkhard, Peter

    2012-01-01

    Nanoparticles show great promise as potent vaccine candidates since they are readily taken up by the antigen presenting cells of the immune system. The particle size and the density of the B cell epitopes on the surface of the particles greatly influences the strength of the humoral immune response. We have developed a novel type of nanoparticle composed of peptide building blocks (Raman et al., 2006) and have used such particles to design vaccines against malaria and SARS (Kaba et al., 2009; Pimentel et al., 2009). Here we investigate the biophysical properties and the refolding conditions of a prototype of these self-assembling polypeptide nanoparticles (SAPNs). SAPNs are formed from a peptide containing a pentameric and a trimeric coiled-coil domain. At near physiological conditions the peptide self-assembles into about 27 nm, roughly spherical SAPNs. The average size of the SAPNs increases with the salt concentration. The optimal pH for their formation is between 7.5 and 8.5, while aggregation occurs at lower and higher values. A glycerol concentration of about 5% v/v is required for the formation of SAPNs with regular spherical shapes. These studies will help to optimize the immunological properties of SAPNs.

  19. Residue specific effects of human islet polypeptide amyloid on self-assembly and on cell toxicity.

    Science.gov (United States)

    Khemtemourian, Lucie; Guillemain, Ghislaine; Foufelle, Fabienne; Killian, J Antoinette

    2017-08-01

    Type 2 diabetes mellitus is characterized histopathologically by the presence of fibrillary amyloid deposits in the pancreatic islets of Langerhans. Human islet amyloid polypeptide (hIAPP), the 37-residue pancreatic hormone, is the major constituent of these amyloid deposits. The propensity of IAPP to form amyloid fibrils is strongly dependent on its primary sequence. An intriguing example is His at residue 18. Although H18 is located outside the amyloidogenic region, it has been suggested that this residue and its charge state play an important role in the kinetics of conformational changes and fibril formation as well as in mediating cell toxicity. To gain more insight into the importance of this residue, we have synthesized four analogues (H18R-IAPP, H18K-IAPP, H18A-IAPP and H18E-IAPP) and we performed a full biophysical study on the properties of these peptides. Kinetic experiments as monitored by thioflavin-T fluorescence, transmission electron microscopy, circular dichroism and cell toxicity assays revealed that all variants are less fibrillogenic and less toxic than native hIAPP both at neutral pH and at low pH. This demonstrates that the effect of H18 in native IAPP is not simply determined by its charge state, but rather that residue 18 is important for specific intra- and intermolecular interactions that occur during fibril formation and that may involve charge, size and hydrophobicity. Furthermore, our results indicate that H18R-IAPP has a strong inhibiting effect on native hIAPP fibril formation. Together these results highlight the large impact of modifying a single residue outside the amyloidogenic domain on fibril formation and cell toxicity induced by IAPP, opening up new avenues for design of inhibitors or modulators of IAPP aggregation. Copyright © 2017. Published by Elsevier B.V.

  20. Capturing "Extraordinary" Soft-Assembled Charge-Like Polypeptides as a Strategy for Nanocarrier Design.

    Science.gov (United States)

    Duro-Castano, Aroa; Nebot, Vicent J; Niño-Pariente, Amaya; Armiñán, Ana; Arroyo-Crespo, Juan J; Paul, Alison; Feiner-Gracia, Natalia; Albertazzi, Lorenzo; Vicent, María J

    2017-08-23

    The rational design of nanomedicines is a challenging task given the complex architectures required for the construction of nanosized carriers with embedded therapeutic properties and the complex interface of these materials with the biological environment. Herein, an unexpected charge-like attraction mechanism of self-assembly for star-shaped polyglutamates in nonsalty aqueous solutions is identified, which matches the ubiquitous "ordinary-extraordinary" phenomenon previously described by physicists. For the first time, a bottom-up methodology for the stabilization of these nanosized soft-assembled star-shaped polyglutamates is also described, enabling the translation of theoretical research into nanomaterials with applicability within the drug-delivery field. Covalent capture of these labile assemblies provides access to unprecedented architectures to be used as nanocarriers. The enhanced in vitro and in vivo properties of these novel nanoconstructs as drug-delivery systems highlight the potential of this approach for tumor-localized as well as lymphotropic delivery. © 2017 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. 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.

  2. Human Islet Amyloid Polypeptide N-Terminus Fragment Self-Assembly: Effect of Conserved Disulfide Bond on Aggregation Propensity

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    Ilitchev, Alexandre I.; Giammona, Maxwell J.; Do, Thanh D.; Wong, Amy G.; Buratto, Steven K.; Shea, Joan-Emma; Raleigh, Daniel P.; Bowers, Michael T.

    2016-06-01

    Amyloid formation by human islet amyloid polypeptide (hIAPP) has long been implicated in the pathogeny of type 2 diabetes mellitus (T2DM) and failure of islet transplants, but the mechanism of IAPP self-assembly is still unclear. Numerous fragments of hIAPP are capable of self-association into oligomeric aggregates, both amyloid and non-amyloid in structure. The N-terminal region of IAPP contains a conserved disulfide bond between cysteines at position 2 and 7, which is important to hIAPP's in vivo function and may play a role in in vitro aggregation. The importance of the disulfide bond in this region was probed using a combination of ion mobility-based mass spectrometry experiments, molecular dynamics simulations, and high-resolution atomic force microscopy imaging on the wildtype 1-8 hIAPP fragment, a reduced fragment with no disulfide bond, and a fragment with both cysteines at positions 2 and 7 mutated to serine. The results indicate the wildtype fragment aggregates by a different pathway than either comparison peptide and that the intact disulfide bond may be protective against aggregation due to a reduction of inter-peptide hydrogen bonding.

  3. Self-assembled drug delivery systems. Part 7: hepatocyte-targeted nanoassemblies of an adefovir lipid derivative with cytochrome P450-triggered drug release.

    Science.gov (United States)

    Du, Lina; Wu, Lailong; Jin, Yiguang; Jia, Junwei; Li, Miao; Wang, Yu

    2014-09-10

    A novel strategy was used in the design of self-assembled drug delivery systems (SADDSs) in this study. The nanoassemblies of an amphiphilic adefovir lipid derivative were prepared and demonstrated to have the functions of hepatocyte targeting, enzyme-triggered drug release and high anti-hepatitis effect. An amphiphilic adefovir lipid derivative, N-lauroyl-1-(3-chlorophenyl)-1,3-propanyl phosphonyl adefovir (LCPA) was prepared and formed the nanoassemblies by injecting the mixture of LCPA and another amphiphilic polymer, d-galactide polyoxyethylene (20) cetyl ether (GPCE) (ca. 20:1, mol/mol) into water. The nanoassemblies were very stable and showed negative charge. LCPA was sensitive to the cytochrome P450 isozymes that were expressed predominantly in the hepatocytes to produce adefovir. GPCE contained a long hydrophilic chain and a galactose ligand targeting the asialoglycoprotein receptors overexpressed on the surface of hepatocytes. The nanoassemblies showed the long-circulating and liver targeting effects according to the results of pharmacokinetics, tissue distribution and fluorescence imagination after bolus intravenous administration of the nanoassemblies to the mice. The highly efficient hepatitis B treatment was achieved by 10 day continuous administration of the nanoassemblies to the HBV-infected mice. Many functions were combined in the nanoassemblies, including prodrug, molecular self-assembly, nanotechnology, long-circulating, hepatocyte targeting and hepatocyte over expressing enzyme-triggered drug release.

  4. Intracellular maturation of apolipoprotein[a] and assembly of lipoprotein[a] in primary baboon hepatocytes.

    Science.gov (United States)

    White, A L; Rainwater, D L; Lanford, R E

    1993-03-01

    The glycoprotein apolipoprotein[a] (apo[a]) is present in plasma at highly variable concentrations and appears as a number of genetically determined size isoforms (400-800 kDa), disulfide linked to apoB-100 in low density lipoprotein to produce lipoprotein [a](Lp[a]). Apo[a] is synthesized by the liver, but the site of association of apo[a] and apoB and factors that regulate its production are unknown. To examine the morphogenesis of the Lp[a] particle, baboon hepatocytes expressing a single, low molecular weight isoform of apo[a] were labeled with [35S]cysteine and methionine, and apo[a] was analyzed by immunoprecipitation and SDS-PAGE. Steady-state labeling revealed two molecular weight forms of apo[a] inside the cell. Only the large form was recovered from the culture medium. Pulse-chase studies and endoglycosidase treatment revealed that the lower molecular weight form of apo[a] represented a precursor with a prolonged residence time in the endoplasmic reticulum or an early Golgi compartment, after which it was processed to the mature form. A proportion of the mature form of apo[a] was rapidly secreted after synthesis, whereas the remainder had a prolonged residence time in a late Golgi compartment. In all experiments, apoB co-precipitated with apo[a] from the culture medium, but not from cell lysates. Density gradient ultracentrifugation and immunoblot analysis revealed that the majority of apo[a] was secreted into the medium in a free form, suggesting that the association between apo[a] and apoB occurred after secretion. Regulation of the movement of apo[a] between intracellular compartments may be one mechanism by which the plasma levels of Lp[a] are influenced.

  5. Synthesis and studies of polypeptide materials: Self-assembled block copolypeptide amphiphiles, DNA-condensing block copolypeptides and membrane-interactive random copolypeptides

    Science.gov (United States)

    Wyrsta, Michael Dmytro

    A new class of transition metal initiators for the controlled polymerization of alpha-aminoacid-N-carboxyanhydrides (alpha-NCAs), has been developed by Deming et al. This discovery has allowed for the synthesis of well-defined "protein-like" polymers. Using this chemistry we have made distinct block/random copolypeptides for biomedical applications. Drug delivery, gene delivery, and antimicrobial polymers were the focus of our research efforts. The motivation for the synthesis and study of synthetic polypeptide based materials comes from proteins. Natural proteins are able to adopt a staggeringly large amount of uniquely well-defined folded structures. These structures account for the diversity in properties of proteins. As catalysts (enzymes) natural proteins perform some of the most difficult chemistry with ease and precision at ambient pressures and temperatures. They also exhibit incredible structural properties that directly result from formation of complex hierarchical assemblies. Self-assembling block copolymers were synthesized with various compositions and architectures. In general, di- and tri-block amphiphiles were studied for their self-assembling properties. Both spherical and tubular vesicles were found to assemble from di- and tri-block amphiphiles, respectively. In addition to self-assembly, pH responsiveness was engineered into these amphiphiles by the incorporation of basic residues (lysine) into the hydrophobic block. Another form of self-assembly studied was the condensation of DNA using cationic block copolymers. It was found that cationic block copolymers could condense DNA into compact, ordered, water-soluble aggregates on the nanoscale. These aggregates sufficiently protected DNA from nucleases and yet were susceptible to proteases. These studies form the basis of a gene delivery platform. The ease with which NCAs are polymerized renders them completely amenable to parallel synthetic methods. We have employed this technique to discover new

  6. Self-assembled cationic micelles based on PEG-PLL-PLLeu hybrid polypeptides as highly effective gene vectors.

    Science.gov (United States)

    Deng, Jizhe; Gao, Ningning; Wang, Yanan; Yi, Huqiang; Fang, Shengtao; Ma, Yifan; Cai, Lintao

    2012-11-12

    Developing safe and effective nonviral gene vector is highly crucial for successful gene therapy. In the present study, we designed a series of biodegradable micelles based on hybrid polypeptide copolymers of poly(ethylene glycol)-b-poly(l-lysine)-b-poly(l-leucine) (PEG-PLL-PLLeu) for efficient gene delivery. A group of amphiphilic PEG-PLL-PLLeu hybrid polypeptide copolymers were synthesized by ring-opening polymerization of N-carboxyanhydride, and the chemical structure of each copolymer was characterized by (1)H NMR and FT-IR spectroscopy measurement. The PEG-PLL-PLLeu micelles were positively charged with tunable sizes ranging from 40 to 90 nm depending on the length of PLL and PLLeu segment. Compared with PEG-PLL copolymers, PEG-PLL-PLLeu micelles demonstrated significantly higher transfection efficiency and less cytotoxicity. Furthermore, the transfection efficiency and biocompatibility of the micelles can be simultaneously improved by tuning the length of PLL and PLLeu segments. The transfection efficiency of PEG-PLL-PLLeu micelles in vivo was two to three times higher than that of PEI(25k), which was attributable to their capability of promoting DNA condensation and cell internalization as well as successful lysosome escape. Hence well-defined PEG-PLL-PLLeu micelles would serve as highly effective nonviral vectors for in vivo gene delivery.

  7. Evidence of PPII-like helical conformation and glass transition in a self-assembled solid-state polypeptide-surfactant complex: poly(L-histidine)/docylbenzenesulfonic acid.

    Science.gov (United States)

    Ramani, Ramasubbu; Hanski, Sirkku; Laiho, Ari; Tuma, Roman; Kilpeläinen, Simo; Tuomisto, Filip; Ruokolainen, Janne; Ikkala, Olli

    2008-05-01

    We present lamellar self-assembly of cationic poly(L-histidine) (PLH) stoichiometrically complexed with an anionic surfactant, dodecyl benzenesulfonic acid (DBSA), which allows a stabilized conformation reminiscent of polyproline type II (PPII) left-handed helices. Such a conformation has no intrapeptide hydrogen bonds, and it has previously been found to be one source of flexibility, e.g., in collagen and elastin, as well as an intermediate in silk processing. PLH(DBSA)1.0 complexes were characterized by Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), and differential scanning calorimetry (DSC). The PPII-like conformation in PLH(DBSA)1.0 is revealed by characteristic CD and FTIR spectra, where the latter indicates absence of intrachain peptide hydrogen bonds. In addition, a glass transition was directly verified by DSC at ca. 135 degrees C for PLH(DBSA)1.0 and indirectly by SAXS and TEM in comparison to pure PLH at 165 degrees C, thus indicating plasticization. Glass transitions have not been observed before in polypeptide-surfactant complexes. The present results show that surfactant binding can be a simple scheme to provide steric crowding to stabilize PPII conformation to tune the polypeptide properties, plasticization and flexibility.

  8. Nanostructured self-assembling peptides as a defined extracellular matrix for long-term functional maintenance of primary hepatocytes in a bioartificial liver modular device

    Directory of Open Access Journals (Sweden)

    Giri S

    2013-04-01

    Full Text Available Shibashish Giri,1 Ulf-Dietrich Braumann,2,3 Priya Giri,1,3 Ali Acikgöz,1,4 Patrick Scheibe,3,5 Karen Nieber,6 Augustinus Bader1 1Department of Cell Techniques and Applied Stem Cell Biology, Center for Biotechnology and Biomedicine (BBZ, 2Institute for Medical Informatics, Statistics, and Epidemiology (IMISE, University of Leipzig, Leipzig, Germany; 3Interdisciplinary Center for Bioinformatics (IZBI, University of Leipzig, Leipzig, Germany; 4Klinikum St Georg, Leipzig, Germany; 5Translational Center for Regenerative Medicine (TRM Leipzig, 6Department of Pharmacology for Natural Sciences, Institute of Pharmacy, University of Leipzig, Leipzig, Germany Abstract: Much effort has been directed towards the optimization of the capture of in vivo hepatocytes from their microenvironment. Some methods of capture include an ex vivo cellular model in a bioreactor based liver module, a micropatterned module, a microfluidic 3D chip, coated plates, and other innovative approaches for the functional maintenance of primary hepatocytes. However, none of the above methods meet US Food and Drug Administration (FDA guidelines, which recommend and encourage that the duration of a toxicity assay of a drug should be a minimum of 14 days, to a maximum of 90 days for a general toxicity assay. Existing innovative reports have used undefined extracellular matrices like matrigel, rigid collagen, or serum supplementations, which are often problematic, unacceptable in preclinical and clinical applications, and can even interfere with experimental outcomes. We have overcome these challenges by using integrated nanostructured self-assembling peptides and a special combination of growth factors and cytokines to establish a proof of concept to mimic the in vivo hepatocyte microenvironment pattern in vitro for predicting the in vivo drug hepatotoxicity in a scalable bioartificial liver module. Hepatocyte functionality (albumin, urea was measured at days 10, 30, 60, and 90 and we

  9. Strip-Pattern-Spheres Self-Assembled from Polypeptide-Based Polymer Mixtures: Structure and Defect Features

    Science.gov (United States)

    Zhu, Xingyu; Guan, Zhou; Lin, Jiaping; Cai, Chunhua

    2016-07-01

    We found that poly(γ-benzyl-L-glutamate)-block-poly(ethylene glycol) (PBLG-b-PEG) rod-coil block copolymers and polystyrene (PS) homopolymers can cooperatively self-assemble into nano-spheres with striped patterns on their surfaces (strip-pattern-spheres) in aqueous solution. With assistance of dissipative particle dynamics simulation, it is discovered that the PS homopolymers form a spherical template core and the PBLG-b-PEG block copolymers assemble into striped patterns on the spherical surface. The hydrophobic PBLG rods are packed orderly in the strips, while the hydrophilic PEG blocks stabilize the strip-pattern-spheres in solution. Defects such as dislocations and disclinations can be observed in the striped patterns. Self-assembling temperature and sphere radius are found to affect defect densities in the striped patterns. A possible mechanism is proposed to illustrate how PBLG-b-PEG and PS cooperatively self-assemble into hierarchical spheres with striped patterns on surfaces.

  10. Dual-layered nanogel-coated hollow lipid/polypeptide conjugate assemblies for potential pH-triggered intracellular drug release.

    Directory of Open Access Journals (Sweden)

    Wen-Hsuan Chiang

    Full Text Available To achieve effective intracellular anticancer drug delivery, the polymeric vesicles supplemented with the pH-responsive outlayered gels as a delivery system of doxorubicin (DOX were developed from self-assembly of the lipid/polypeptide adduct, distearin grafted poly(γ-glutamic acid (poly(γ-GA, followed by sequential deposition of chitosan and poly(γ-GA-co-γ-glutamyl oxysuccinimide-g-monomethoxy poly(ethylene glycol in combination with in situ covalent cross-linking on assembly surfaces. The resultant gel-caged polymeric vesicles (GCPVs showed superior performance in regulating drug release in response to the external pH change. Under typical physiological conditions (pH 7.4 and 37 °C at which the γ-GA/DOX ionic pairings remained mostly undisturbed, the dense outlayered gels of GCPVs significantly reduced the premature leakage of the uncomplexed payload. With the environmental pH being reduced from pH 7.4 to 4.7, the drug liberation was appreciably promoted by the massive disruption of the ionic γ-GA/DOX complexes along with the significant swelling of nanogel layers upon the increased protonation of chitosan chain segments. After being internalized by HeLa cells via endocytosis, GCPVs exhibited cytotoxic effect comparable to free DOX achieved by rapidly releasing the payload in intracellular acidic endosomes and lysosomes. This strongly implies the great promise of such unique GCPVs as an intracellular drug delivery carrier for potential anticancer treatment.

  11. Conformation and assembly of polypeptide scaffolds in templating the synthesis of silica: an example of a polylysine macromolecular "switch".

    Science.gov (United States)

    Patwardhan, Siddharth V; Maheshwari, Ronak; Mukherjee, Niloy; Kiick, Kristi L; Clarson, Stephen J

    2006-02-01

    Although the role of polycationic macromolecules in catalyzing the synthesis of silica structures is well established, detailed understanding of the mechanisms behind the production of silica structures of controlled morphologies remains unclear. In this study, we have used both poly-L-lysine (PLL) and/or poly-D-lysine (PDL) for silica synthesis to investigate mechanisms controlling inorganic morphologies. The formation of both spherical silica particles and hexagonal plates was observed. The formation of hexagonal plates was suggested, via circular dichroic spectroscopy (CD), to result from the assembly of helical polylysine molecules. We confirm that the formation of PLL helices is a prerequisite to the hexagonal silica synthesis. In addition, we present for the first time that the handedness of the helicity of the macromolecule does not affect the formation of hexagonal silica. We also show, by using two different silica precursors, that the precursor does not have a direct effect on the formation of hexagonal silica plates. Furthermore, when polylysine helices were converted to beta-sheet structure, only silica particles were obtained, thus suggesting that the adoption of a helical conformation by PLL is required for the formation of hexagonally organized silica. These results demonstrate that the change in polylysine conformation can act as a "switch" in silica structure formation and suggest the potential for controlling morphologies and structures of inorganic materials via control of the conformation of soft macromolecular templates.

  12. Sustained release of hepatocyte growth factor by cationic self-assembling peptide/heparin hybrid hydrogel improves β-cell survival and function through modulating inflammatory response

    Science.gov (United States)

    Liu, Shuyun; Zhang, Lanlan; Cheng, Jingqiu; Lu, Yanrong; Liu, Jingping

    2016-01-01

    Inflammatory response is a major cause of grafts dysfunction in islet transplantation. Hepatocyte growth factor (HGF) had shown anti-inflammatory activity in multiple diseases. In this study, we aim to deliver HGF by self-assembling peptide/heparin (SAP/Hep) hybrid gel to protect β-cell from inflammatory injury. The morphological and slow release properties of SAPs were analyzed. Rat INS-1 β-cell line was treated with tumor necrosis factor α in vitro and transplanted into rat kidney capsule in vivo, and the viability, apoptosis, function, and inflammation of β-cells were evaluated. Cationic KLD1R and KLD2R self-assembled to nanofiber hydrogel, which showed higher binding affinity for Hep and HGF because of electrostatic interaction. Slow release of HGF from cationic SAP/Hep gel is a two-step mechanism involving binding affinity with Hep and molecular diffusion. In vitro and in vivo results showed that HGF-loaded KLD2R/Hep gel promoted β-cell survival and insulin secretion, and inhibited cell apoptosis, cytokine release, T-cell infiltration, and activation of NFκB/p38 MAPK pathways in β-cells. This study suggested that SAP/Hep gel is a promising carrier for local delivery of bioactive proteins in islet transplantation. PMID:27729786

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    Full Text Available NoD.Liv.05.AllAg.Hepatocytes mm9 No description Liver Hepatocytes ERX008721,ERX1129...,ERX008753,ERX008747,ERX008746,ERX113010 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Liv.05.AllAg.Hepatocytes.bed ...

  5. File list: NoD.Liv.05.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  6. File list: ALL.Liv.20.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. File list: InP.Liv.10.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Liv.10.AllAg.Hepatocytes mm9 Input control Liver Hepatocytes SRX019015,SRX55553...3,SRX1334843 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Liv.10.AllAg.Hepatocytes.bed ...

  8. File list: Oth.Liv.10.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Liv.10.AllAg.Hepatocytes mm9 TFs and others Liver Hepatocytes SRX019007,SRX1169...9006,SRX116906,SRX1334844,SRX555532,ERX204067,ERX204061,ERX204065,ERX204058 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Liv.10.AllAg.Hepatocytes.bed ...

  9. File list: InP.Liv.50.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Liv.50.AllAg.Hepatocytes hg19 Input control Liver Hepatocytes SRX530185,SRX5301...83 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/InP.Liv.50.AllAg.Hepatocytes.bed ...

  10. File list: InP.Liv.20.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Liv.20.AllAg.Hepatocytes hg19 Input control Liver Hepatocytes SRX530185,SRX5301...83 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/InP.Liv.20.AllAg.Hepatocytes.bed ...

  11. File list: InP.Liv.50.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Liv.50.AllAg.Hepatocytes mm9 Input control Liver Hepatocytes SRX019015,SRX55553...3,SRX1334843 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Liv.50.AllAg.Hepatocytes.bed ...

  12. Recombinant fibronectin polypeptide antagonizes hepatic failure induced by endotoxin in mice

    Institute of Scientific and Technical Information of China (English)

    Yong WU; Yuan-zhong CHEN; Hui-fang HUANG; Ping CHEN

    2004-01-01

    AIM: To study the preventive effect of recombinant human fibronectin (rhFN) polypeptide on hepatic failure induced by endotoxin in mice. METHODS: A cDNA fragment coding for Ile1363-Tyr1725 of human FN was inserted into the PinPoint Xa-3 plasmid, and the constructed plasmid was transformed into E coli BL21 (DE3) cells,and then the expression of rhFN polypeptide in DE3 cells was identified through SDS-PAGE. The target protein from the supernatant of bacteria lysate was purified through biotin-affinity chromatography. The bioactivity of the purified rhFN polypeptide was determined with cell adhesive activity. The survival rate was observed in endotoxemia mice injected with rhFN polypeptide. The tissue damage in hepatocyte was detected using histology, ultrastructure,and DNA fragmentation assay. RESULTS: The expression of rhFN polypeptide reached approximately 20 % of the total cellular protein. The adhesion ability of rhFN polypeptide was concentration-dependent. The value of EC50 was 0.8 nmol/L. The survival rate of endotoxemia mice sensitized by D-galactosamine (D-GalN) was 60 % in rhFN polypeptide treated group, while that of endotoxemia mice sensitized by D-GalN was 20 % in the control group (P<0.01). Histopathology showed that less necrosis occurred on the hepatocyte of endotoxemia mice injected with rhFN polypeptide compared with saline control. Ultrastructure and DNA fragmentation assay showed that no apoptotic hepatocyte was observed in the liver of rhFN-treated endotoxemia mice. CONCLUSION: Recombinant fibronectin polypeptide antagonizes hepatic failure induced by endotoxin in mice.

  13. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes.

    Science.gov (United States)

    Matsuda, Shigeo; Keiser, Kristofer; Nair, Jayaprakash K; Charisse, Klaus; Manoharan, Rajar M; Kretschmer, Philip; Peng, Chang G; V Kel'in, Alexander; Kandasamy, Pachamuthu; Willoughby, Jennifer L S; Liebow, Abigail; Querbes, William; Yucius, Kristina; Nguyen, Tuyen; Milstein, Stuart; Maier, Martin A; Rajeev, Kallanthottathil G; Manoharan, Muthiah

    2015-05-15

    Asialoglycoprotein receptor (ASGPR) mediated delivery of triantennary N-acetylgalactosamine (GalNAc) conjugated short interfering RNAs (siRNAs) to hepatocytes is a promising paradigm for RNAi therapeutics. Robust and durable gene silencing upon subcutaneous administration at therapeutically acceptable dose levels resulted in the advancement of GalNAc-conjugated oligonucleotide-based drugs into preclinical and clinical developments. To systematically evaluate the effect of display and positioning of the GalNAc moiety within the siRNA duplex on ASGPR binding and RNAi activity, nucleotides carrying monovalent GalNAc were designed. Evaluation of clustered and dispersed incorporation of GalNAc units to the sense (S) strand indicated that sugar proximity is critical for ASGPR recognition, and location of the clustered ligand impacts the intrinsic potency of the siRNA. An array of nucleosidic GalNAc monomers resembling a trivalent ligand at or near the 3' end of the S strand retained in vitro and in vivo siRNA activity, similar to the parent conjugate design. This work demonstrates the utility of simple, nucleotide-based, cost-effective siRNA-GalNAc conjugation strategies.

  14. Tunable drug loading and release from polypeptide multilayer nanofilms

    Directory of Open Access Journals (Sweden)

    Bingbing Jiang

    2009-03-01

    Full Text Available Bingbing Jiang1, Bingyun Li1,2,31Biomaterials, Bioengineering and Nanotechnology Laboratory, Department of Orthopaedics, School of Medicine, West Virginia University, Morgantown, WV, USA; 2WVNano Initiative, WV, USA; 3Department of Chemical Engineering, College of Engineering and Mineral Resources, West Virginia University, Morgantown, WV, USA Abstract: Polypeptide multilayer nanofilms were prepared using electrostatic layer-by-layer self-assembly nanotechnology. Small charged drug molecules (eg, cefazolin, gentamicin, and methylene blue were loaded in polypeptide multilayer nanofilms. Their loading and release were found to be pH-dependent and could also be controlled by changing the number of film layers and drug incubation time, and applying heat-treatment after film formation. Antibiotic-loaded polypeptide multilayer nanofilms showed controllable antibacterial properties against Staphylococcus aureus. The developed biodegradable polypeptide multilayer nanofilms are capable of loading both positively- and negatively-charged drug molecules and promise to serve as drug delivery systems on biomedical devices for preventing biomedical device-associated infection, which is a significant clinical complication for both civilian and military patients.Keywords: polypeptide, self-assembly, polyelectrolyte multilayer, nanofilm, charged molecule, tunable release

  15. Smart systems related to polypeptide sequences

    Directory of Open Access Journals (Sweden)

    Lourdes Franco

    2016-03-01

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

  16. Hydrogenase polypeptide and methods of use

    Energy Technology Data Exchange (ETDEWEB)

    Adams, Michael W.W.; Hopkins, Robert C.; Jenney, JR, Francis E.; Sun, Junsong

    2016-02-02

    Provided herein are polypeptides having hydrogenase activity. The polypeptide may be multimeric, and may have hydrogenase activity of at least 0.05 micromoles H.sub.2 produced min.sup.-1 mg protein.sup.-1. Also provided herein are polynucleotides encoding the polypeptides, genetically modified microbes that include polynucleotides encoding one or more subunits of the multimeric polypeptide, and methods for making and using the polypeptides.

  17. Hepatocyte differentiation of mesenchymalstemcells

    Institute of Scientific and Technical Information of China (English)

    Xu-Bo Wu; Ran Tao

    2012-01-01

    BACKGROUND: Liver cell transplantation and bioartiifcial liver may provide metabolic support of liver function temporary and are prospective treatments for patients with liver failure. Mesenchymal stem cells (MSCs) are expected to be an ideal cell source for transplantation or liver tissue engineering, however the hepatic differentiation of MSCs is still insufifcient for clinical application. DATA  SOURCES: A PubMed search on "mesenchymal stem cells","liver cell"and"hepatocyte differentiation"was performed on the topic, and the relevant articles published in the past ten years were reviewed. RESULTS:Hepatocyte-like cells differentiated from MSCs are a promising cell source for liver regeneration or tissue engineering. Although it is still a matter of debate as to whether MSC-derived hepatocytes may efifciently repopulate a host liver to provide adequate functional substitution, the majority of animal studies support that MSCs can become key players in liver-directed regenerative medicine. However the clinical application of human stem cells in the treatment of liver diseases is still in its infancy. CONCLUSIONS: Future studies are required to improve the efifcacy and consistency of hepatic differentiation from MSCs. It is necessary to better understand the mechanism to achieve transdifferentiation with high efifciency. More clinical trials are warranted to prove their efifcacy in the management of patients with liver failure.

  18. Glucose-dependent Insulinotropic Polypeptide

    DEFF Research Database (Denmark)

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

    2014-01-01

    CONTEXT: Patients with type 2 diabetes mellitus (T2DM) have clinically relevant disturbances in the effects of the hormone glucose-dependent insulinotropic polypeptide (GIP). OBJECTIVE: We aimed to evaluate the importance of the prevailing plasma glucose levels for the effect of GIP on responses...

  19. Methods for using polypeptides having cellobiohydrolase activity

    Energy Technology Data Exchange (ETDEWEB)

    Morant, Marc D; Harris, Paul

    2016-08-23

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

  20. A Novel Polypeptide from Cervus elaphus Linnaeus

    Institute of Scientific and Technical Information of China (English)

    LiangWENG; QiuLiZHOU; 等

    2002-01-01

    A novel polypeptide having stimulant effect on some cell proliferation was isolated from the velvet antler (Cervus elaphus Linnaeus). The velvet antler polypeptide consists of a single chain of 32 amino acid residues. Amino acid sequence of the polypeptide was identified as:VLSAADKSNVKAAWGKVGGNAPAFGAEALLRM.

  1. Nano polypeptide particles reinforced polymer composite fibers.

    Science.gov (United States)

    Li, Jiashen; Li, Yi; Zhang, Jing; Li, Gang; Liu, Xuan; Li, Zhi; Liu, Xuqing; Han, Yanxia; Zhao, Zheng

    2015-02-25

    Because of the intensified competition of land resources for growing food and natural textile fibers, there is an urgent need to reuse and recycle the consumed/wasted natural fibers as regenerated green materials. Although polypeptide was extracted from wool by alkaline hydrolysis, the size of the polypeptide fragments could be reduced to nanoscale. The wool polypeptide particles were fragile and could be crushed down to nano size again and dispersed evenly among polymer matrix under melt extrusion condition. The nano polypeptide particles could reinforce antiultraviolet capability, moisture regain, and mechanical properties of the polymer-polypeptide composite fibers.

  2. Culture of cryopreserved rat hepatocyte

    Institute of Scientific and Technical Information of China (English)

    Haitao Yin; Gaojun Teng; Lifeng Wang; Baorui Liu; Xiaoping Qian

    2006-01-01

    Objective: To study the method of cryopreserving rat hepatocytes and double collagen gel culture measurement after its cryopreservation. Methods: Rat hepatocytes, isolated by two-step perfusion with collagenase using an extra corporeal perfusion apparatus, were cryopreserved in double collagen gel with culture medium added by epidermal growth factor(EGF).The expression of cell function and cellular morphology were examined during culture. Results: The hepatocytes cryopreserved in double collagen gel concluding EGF showed good morphology and biological characteristics. After thawing, the MTT metabolism and protein synthesis of hepatocytes in sandwich ± EGF groups were better than those in control group. And the morphology and function of hepatocytes in sandwich group was better than that in EGF group(P < 0.05). Conclusion: Double collagen gel culture can keep hepatocyte's activities. Thawed hepatocytes can be cultivated with collagenous matrix, which provides an environment that more closely resembles that in vivo and maintain the expression of certain liver-specific function of hepatocytes.

  3. Glucose-dependent insulinotropic polypeptide

    DEFF Research Database (Denmark)

    Christensen, Mikkel; Vedtofte, Louise; Holst, Jens Juul

    2011-01-01

    OBJECTIVE To evaluate the glucose dependency of glucose-dependent insulinotropic polypeptide (GIP) effects on insulin and glucagon release in 10 healthy male subjects ([means ± SEM] aged 23 ± 1 years, BMI 23 ± 1 kg/m2, and HbA1c 5.5 ± 0.1%). RESEARCH DESIGN AND METHODS Saline or physiological doses....... In contrast, GIP increases glucagon levels during fasting and hypoglycemic conditions, where it has little or no effect on insulin secretion. Thus, GIP seems to be a physiological bifunctional blood glucose stabilizer with diverging glucose-dependent effects on the two main pancreatic glucoregulatory hormones....

  4. Current status of hepatocyte xenotransplantation.

    Science.gov (United States)

    Meier, Raphael P H; Navarro-Alvarez, Nalu; Morel, Philippe; Schuurman, Henk-Jan; Strom, Stephen; Bühler, Leo H

    2015-11-01

    The treatment of acute liver failure, a condition with high mortality, comprises optimal clinical care, and in severe cases liver transplantation. However, there are limitations in availability of organ donors. Hepatocyte transplantation is a promising alternative that could fill the medical need, in particular as the bridge to liver transplantation. Encapsulated porcine hepatocytes represent an unlimited source that could function as a bioreactor requiring minimal immunosuppression. Besides patients with acute liver failure, patients with alcoholic hepatitis who are unresponsive to a short course of corticosteroids are a target for hepatocyte transplantation. In this review we present an overview of the innate immune barriers in hepatocyte xenotransplantation, including the role of complement and natural antibodies; the role of phagocytic cells and ligands like CD47 in the regulation of phagocytic cells; and the role of Natural Killer cells. We present also some illustrations of physiological species incompatibilities in hepatocyte xenotransplantation, such as incompatibilities in the coagulation system. An overview of the methodology for cell microencapsulation is presented, followed by proof-of-concept studies in rodent and nonhuman primate models of fulminant liver failure: these studies document the efficacy of microencapsulated porcine hepatocytes which warrants progress towards clinical application. Lastly, we present an outline of a provisional clinical trial, that upon completion of preclinical work could start within the upcoming 2-3 years.

  5. Methods for engineering polypeptide variants via somatic hypermutation and polypeptide made thereby

    Science.gov (United States)

    Tsien, Roger Y; Wang, Lei

    2015-01-13

    Methods using somatic hypermutation (SHM) for producing polypeptide and nucleic acid variants, and nucleic acids encoding such polypeptide variants are disclosed. Such variants may have desired properties. Also disclosed are novel polypeptides, such as improved fluorescent proteins, produced by the novel methods, and nucleic acids, vectors, and host cells comprising such vectors.

  6. Polypeptide Chirality Influences Multilayer Thin Film Growth and Structure

    Science.gov (United States)

    Bell, Zephra; Khadka, Dhan; Haynie, Donald

    2011-03-01

    Polypeptide multilayer thin films are being developed for a variety of applications.These include coatings for implant devices and systems for drug delivery in thebiomedical sciences, and optical coatings. Subsequent polymer adsorption steps involve polymers of opposite polarity. Here, the polymers were polypeptides. This project compared the consequences of changing polypeptide chirality on film growth and structure. The peptides were poly(L-glutamic acid), its right-handed counterpart, poly(D-glutamic acid), and poly(lysine-tyrosine). The first two are negatively charged at neutral pH, the third one is positively charged. Poly(lysine-tyrosine)/poly(L-glutamic acid) films and poly(lysine-tyrosine)/poly(D-glutamic acid) films werefabricated on 1 mm-thick quartz plates. In one experiment, films were grown to 34layers. The UV absorption spectrum was taken after each layer deposited to determinethe rate of polymer self-assembly. Separately, UV or visible wavelength spectra wereobtained for films stained with a dye cooled/heated in the range 4-65 °C. In anotherexperiment, a mixture of poly-L-glutamic acid and poly-D-glutamic acid was used as thepolyanion for film buildup. The data show that poly(lysine-tyrosine)/poly(L-glutamicacid) films built up at a higher rate than the corresponding right-handed films.

  7. Chirality-mediated polypeptide micelles for regulated drug delivery.

    Science.gov (United States)

    Ding, Jianxun; Li, Chen; Zhang, Ying; Xu, Weiguo; Wang, Jincheng; Chen, Xuesi

    2015-01-01

    Two kinds of triblock poly(ethylene glycol)-polyleucine (PEG-PLeu) copolymers were synthesized through the ring-opening polymerization of L-Leu N-carboxyanhydride (NCA), or equivalent D-Leu NCA and L-Leu NCA with amino-terminated PEG as a macroinitiator. The amphiphilic copolymers spontaneously self-assembled into spherical micellar aggregations in an aqueous environment. The micelle with a racemic polypeptide core exhibited smaller critical micelle concentration and diameter compared to those with a levorotatory polypeptide core. A model anthracycline antineoplastic agent, i.e., doxorubicin (DOX), was loaded into micelles through nanoprecipitation, and the PEG-P(D,L-Leu) micelle exhibited higher drug-loading efficacy than that with a P(L-Leu) core-this difference was attributed to the flexible and compact P(L-Leu) core. Sustained in vitro DOX release from micelles with both levorotatory and racemic polypeptide cores was observed, and the DOX-loaded PEG-P(D,L-Leu) micelle exhibited a slower release rate. More interestingly, DOX-loaded micelles exhibited chirality-mediated antitumor efficacy in vitro and in vivo, which are all better than that of free DOX. Furthermore, both enhanced tumor inhibition and excellent security in vivo were confirmed by histopathological or in situ cell apoptosis analyses. Therefore, DOX-loaded PEG-PLeu micelles appear to be an interesting nanoscale polymeric formulation for promising malignancy chemotherapy.

  8. Ordered biological nanostructures formed from chaperonin polypeptides

    Science.gov (United States)

    Trent, Jonathan D. (Inventor); McMillan, R. Andrew (Inventor); Kagawa, Hiromi (Inventor); Paavola, Chad D. (Inventor)

    2010-01-01

    The following application relates to nanotemplates, nanostructures, nanoarrays and nanodevices formed from wild-type and mutated chaperonin polypeptides, methods of producing such compositions, methods of using such compositions and particular chaperonin polypeptides that can be utilized in producing such compositions.

  9. Glucose-dependent insulinotropic polypeptide

    DEFF Research Database (Denmark)

    Christensen, Mikkel Bring

    2016-01-01

    The hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are secreted by enteroendocrine cells in the intestinal mucosa in response to nutrient ingestion. They are called incretin hormones because of their ability to enhance insulin secretion. However...... the blood glucose levels. In Study 3, we also used stable glucose isotopes to estimate the endogenous glucose production and assessed symptoms and cognitive function during hypoglycaemia. The results from the three studies indicate that GIP has effects on insulin and glucagon responses highly dependent upon...... glucose to prevent hypoglycaemia. In conclusion, the studies position GIP as a bifunctional blood glucose stabilising hormone that glucose-dependently regulates insulin and glucagon responses in humans....

  10. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yu; Tang, Lan; Henriksen, Svend Hostgaard Bang

    2016-05-17

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

  11. Polypeptides having cellobiohydrolase activitiy and polynucleotides encoding same

    Science.gov (United States)

    Liu, Ye; Tang, Lan; Duan, Junxin

    2015-12-15

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

  12. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Spodsberg, Nikolaj

    2016-12-13

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

  13. Polypeptides having endoglucanase activity and polynucleotides encoding same

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-07-18

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

  14. Hybrid polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    Science.gov (United States)

    Liu, Ye; Shaghasi, Tarana

    2016-11-01

    The present invention provides hybrid polypeptides having cellobiohydrolase activity. The present invention also provides polynucleotides encoding the hybrid polypeptides; nucleic acid constructs, vectors and host cells comprising the polynucleotides; and processes of using the hybrid polypeptides.

  15. Protective effect of Cordyceps militaris polypeptide against acute alcoholic liver injury in rats

    Directory of Open Access Journals (Sweden)

    CAI Qi

    2016-04-01

    Full Text Available ObjectiveTo investigate the protective effect of Cordyceps militaris polypeptide against acute alcoholic liver injury in rats and related mechanism. MethodsA total of 60 Wistar rats were randomly divided into blank control group, model group, and low-, medium-, and high-dose Cordyceps militaris polypeptide groups. All rats except those in the blank control group were given 10 ml/kg 56° liquor by gavage once a day; the rats in the blank control group were given distilled water of the same dose by gavage once a day. At 1 hour after gavage with liquor, the rats in the model group and low-, medium-, and high-dose Cordyceps militaris polypeptide groups were given distilled water or Cordyceps militaris polypeptide solution (6 ml/kg by gavage. Blood samples were collected from the orbit 4 weeks later. The serum levels of alanine aminotransferase (ALT and aspartate aminotransferase (AST and the activity of superoxide dismutase (SOD and level of malondialdehyde (MDA in the liver were measured for each group, and the pathological changes in the liver were observed under a light microscope. Analysis of variance was applied for comparison between multiple groups, and the SNK-q test was applied for comparison between any two groups. ResultsCompared with the model group, the low-, medium-, and high-dose Cordyceps militaris polypeptide groups showed significant reductions in the serum levels of ALT and AST and the level of MDA in the liver (all P<0.05, as well as a significant increase in the activity of SOD in the liver (all P<0.05, while these indices showed significant differences between the low-, medium-, and high-dose Cordyceps militaris polypeptide groups (all P<0.05. The liver pathological sections from the low-, medium-, and high-dose Cordyceps militaris polypeptide groups showed alleviated hepatocyte fatty degeneration and necrosis induced by alcohol under a light microscope. ConclusionCordyceps militaris polypeptide has a protective effect

  16. Biolubricant Polypeptides and Therapeutic Uses Thereof

    NARCIS (Netherlands)

    Sharma, Prashant; Herrmann, Andreas; Kolbe, Anke; Veeregowda, Deepak

    2016-01-01

    The invention relates to the field of medicine. In particular, it relates to recombinant cationic polypeptides and their use as biolubricant. Provided is a biolubricant substance comprising the amino acid sequence[(GKGVP)9]n, wherein n is ≧5.

  17. Restriction/modification polypeptides, polynucleotides, and methods

    Energy Technology Data Exchange (ETDEWEB)

    Westpheling, Janet; Chung, DaeHwan; Huddleston, Jennifer; Farkas, Joel A

    2015-02-24

    The present invention relates to the discovery of a novel restriction/modification system in Caldicellulosiruptor bescii. The discovered restriction enzyme is a HaeIII-like restriction enzyme that possesses a thermophilic activity profile. The restriction/modification system also includes a methyltransferase, M.CbeI, that methylates at least one cytosine residue in the CbeI recognition sequence to m.sup.4C. Thus, the invention provides, in various aspects, isolated CbeI or M.CbeI polypeptides, or biologically active fragments thereof; isolated polynucleotides that encode the CbeI or M.CbeI polypeptides or biologically active fragments thereof, including expression vectors that include such polynucleotide sequences; methods of digesting DNA using a CbeI polypeptide; methods of treating a DNA molecule using a M.CbeI polypeptide; and methods of transforming a Caldicellulosiruptor cell.

  18. Artificial Polypeptide Scaffold for Protein Immobilization

    OpenAIRE

    Zhang, Kechun; Diehl, Michael R.; Tirrell, David A.

    2005-01-01

    An artificial polypeptide scaffold composed of surface anchor and protein capture domains was designed and expressed in vivo. By using a mutant E. coli phenylalanyl−tRNA synthetase, the photoreactive amino acid para-azidophenylalanine was incorporated into the surface anchor domain. Octyltrichlorosilane-treated surfaces were functionalized with this polypeptide by spin coating and photocrosslinking. The resulting protein films were shown to immobilize recombinant proteins through association ...

  19. Polypeptides having laccase activity and polynucleotides encoding same

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-08-22

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

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

    Science.gov (United States)

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

    1987-02-01

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

  1. Competition between surface adsorption and folding of fibril-forming polypeptides

    Science.gov (United States)

    Ni, Ran; Kleijn, J. Mieke; Abeln, Sanne; Cohen Stuart, Martien A.; Bolhuis, Peter G.

    2015-02-01

    Self-assembly of polypeptides into fibrillar structures can be initiated by planar surfaces that interact favorably with certain residues. Using a coarse-grained model, we systematically studied the folding and adsorption behavior of a β -roll forming polypeptide. We find that there are two different folding pathways depending on the temperature: (i) at low temperature, the polypeptide folds in solution into a β -roll before adsorbing onto the attractive surface; (ii) at higher temperature, the polypeptide first adsorbs in a disordered state and folds while on the surface. The folding temperature increases with increasing attraction as the folded β -roll is stabilized by the surface. Surprisingly, further increasing the attraction lowers the folding temperature again, as strong attraction also stabilizes the adsorbed disordered state, which competes with folding of the polypeptide. Our results suggest that to enhance the folding, one should use a weakly attractive surface. They also explain the recent experimental observation of the nonmonotonic effect of charge on the fibril formation on an oppositely charged surface [C. Charbonneau et al., ACS Nano 8, 2328 (2014), 10.1021/nn405799t].

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

    Science.gov (United States)

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

    2017-08-01

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

  3. Hepatocytes: critical for glucose homeostasis.

    Science.gov (United States)

    Klover, Peter J; Mooney, Robert A

    2004-05-01

    Maintaining blood glucose levels within a narrow range is a critical physiological function requiring multiple metabolic pathways and involving several cell types, including a prominent role for hepatocytes. Under hormonal control, hepatocytes can respond to either feeding or fasting conditions by storing or producing glucose as necessary. In the fasting state, the effects of glucagon avoid hypoglycemia by stimulating glucogenesis and glycogenolysis and initiating hepatic glucose release. Postprandially, insulin prevents hyperglycemia, in part, by suppressing hepatic gluconeogenesis and glycogenolysis and facilitating hepatic glycogen synthesis. Both transcriptional regulation of rate limiting enzymes and modulation of enzyme activity through phosphorylation and allosteric regulation are involved. Type 2 diabetes mellitus is the most common serious metabolic condition in the world, and results from a subnormal response of tissues to insulin (insulin resistance) and a failure of the insulin-secreting beta cells to compensate. In type 2 diabetes, glucose is overproduced by the hepatocyte and is ineffectively metabolized by other organs. Impairments in the insulin signal transduction pathway appear to be critical lesions contributing to insulin resistance and type 2 diabetes.

  4. Strategies for immortalization of primary hepatocytes

    Science.gov (United States)

    Eva, Ramboer; Bram, De Craene; Joery, De Kock; Tamara, Vanhaecke; Geert, Berx; Vera, Rogiers; Mathieu, Vinken

    2014-01-01

    The liver has the unique capacity to regenerate in response to a damaging event. Liver regeneration is hereby largely driven by hepatocyte proliferation, which in turn relies on cell cycling. The hepatocyte cell cycle is a complex process that is tightly regulated by several well-established mechanisms. In vitro, isolated hepatocytes do not longer retain this proliferative capacity. However, in vitro cell growth can be boosted by immortalization of hepatocytes. Well-defined immortalization genes can be artificially overexpressed in hepatocytes or the cells can be conditionally immortalized leading to controlled cell proliferation. This paper discusses the current immortalization techniques and provides a state-of-the-art overview of the actually available immortalized hepatocyte-derived cell lines and their applications. PMID:24911463

  5. Tuning Ice Nucleation with Supercharged Polypeptides

    NARCIS (Netherlands)

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

    2016-01-01

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

  6. Endogenous pancreatic polypeptide in different vascular beds

    DEFF Research Database (Denmark)

    Henriksen, J H; Schwartz, Tania; Bülow, J B

    1986-01-01

    The plasma concentration of pancreatic polypeptide (PP-like immunoreactivity) was measured in different vascular beds in order to determine regional kinetics of endogenous PP in fasting, supine subjects with normal or moderately decreased kidney function. Patients with kidney disease (n = 10) had...

  7. Recombinant Supercharged Polypeptides Restore and Improve Biolubrication

    NARCIS (Netherlands)

    Veeregowda, Deepak H.; Kolbe, Anke; van der Mei, Henny C.; Busscher, Henk J.; Herrmann, Andreas; Sharma, Prashant K.

    2013-01-01

    Recombinant supercharged polypeptides (SUPs) with low cytotoxicity are developed and applied to rejuvenate the lubrication of naturally occurring salivary conditioning films (SCFs). SUPs with 72 positive charges adsorbed and rigidified the SCFs and recruited mucins to form a hydrated layer. These SC

  8. Investigation of nonfouling polypeptides of poly(glutamic acid) with lysine side chains synthesized by EDC·HCl/HOBt chemistry.

    Science.gov (United States)

    Yang, Qinghua; Li, Wenchen; Wang, Longgang; Wang, Guangzhi; Wang, Zhen; Liu, Lingyun; Chen, Shengfu

    2014-01-01

    Nonfouling polypeptides with homogenous alternating charges draw peoples' attentions for their potential capability in biodegradation. Homogenous glutamic acid (E) and lysine (K) polypeptides were proposed and synthesized before. In this work, a new polypeptide formed by poly(glutamic acid) with lysine side chains (poly(E)-K) was synthesized by facile EDC·HCl/HOBt chemistry and investigated. Results show that these polypeptides also have good nonspecific protein resistance determined by enzyme-linked immunosorbent assay. The lowest nonspecific adsorption of the model proteins, anti-IgG and fibrinogen (Fg), on the self-assembling monolayers (SAMs) surface of poly(E)-K was only 3.3 ± 1.8 and 4.4 ± 1.6%, respectively, when protein adsorption on tissue culture polystyrene surface was set as 100%. And, the relative nonspecific protein adsorption increases when the polypeptide molecular weight increases due to the repression of low density polymer brushes. Moreover, almost no obvious cytotoxicity and hemolytic activity in vitro were detected. This work suggests that polypeptides with various formats of homogenous balanced charges could achieve excellent nonspecific protein resistance, which might be the intrinsic reason for the coexistence of high concentration serum proteins in blood.

  9. Mechanisms and prevention of hepatocyte cell death

    NARCIS (Netherlands)

    Vrenken, Titia Eveline

    2008-01-01

    In many liver diseases, hepatocyte damage occurs upon exposure to toxic bile acids, inflammatory cytokines and increased levels of reactive oxygen species (ROS). Detailed information of the signaling pathways involved in hepatocyte damage will facilitate the discovery of new targets for intervention

  10. Chemical synthesis of a polypeptide backbone derived from the primary sequence of the cancer protein NY-ESO-1 enabled by kinetically controlled ligation and pseudoprolines.

    Science.gov (United States)

    Harris, Paul W R; Brimble, Margaret A

    2015-03-01

    The cancer protein NY-ESO-1 has been shown to be one of the most promising vaccine candidates although little is known about its cellular function. Using a chemical protein strategy, the 180 amino acid polypeptide, tagged with an arginine solubilizing tail, was assembled in a convergent manner from four unprotected peptide α-thioester peptide building blocks and one cysteinyl polypeptide, which were in turn prepared by Boc and Fmoc solid phase peptide synthesis (SPPS) respectively. To facilitate the assembly by ligation chemistries, non-native cysteines were introduced as chemical handles into the polypeptide fragments; pseudoproline dipeptides and microwave assisted Fmoc SPPS were crucial techniques to prepare the challenging hydrophobic C-terminal fragment. Three sequential kinetically controlled ligations, which exploited the reactivity between peptide arylthioesters and peptide alkylthioesters, were then used in order to assemble the more tractable N-terminal region of NY-ESO-1. The ensuing 147 residue polypeptide thioester then underwent successful final native chemical ligation with the very hydrophobic C-terminal polypeptide bearing an N-terminal cysteine affording the 186 residue polypeptide as an advanced intermediate en route to the native NY-ESO-1 protein. © 2015 Wiley Periodicals, Inc.

  11. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yu; Duan, Junxin; Tang, Lan; Wu, Wenping

    2016-06-14

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

  12. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Schnorr, Kirk; Kramer, Randall

    2017-08-08

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

  13. Polypeptides having beta-xylosidase activity and polynucleotides encoding same

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-07-04

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

  14. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Lan; Liu, Ye; Duan, Junxin; Wu, Wenping; Kramer, Randall

    2017-09-19

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

  15. Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

    2017-09-05

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

  16. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ye; Tang, Lan; Duan, Junxin

    2017-02-07

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

  17. Polypeptides having endoglucanase activity and polynucleotides encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Harris, Paul [Carnation, WA; Lopez de Leon, Alfredo [Davis, CA; Rey, Micheal [Davis, CA; Ding, Hanshu [Davis, CA; Vlasenko, Elena [Davis, CA

    2012-02-21

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

  18. Polypeptide from a cellulolytic fungus having cellulolytic enhancing activity

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Kimberly (Elk Grove, CA); Harris, Paul (Carnation, WA); Zaretsky, Elizabeth (Reno, NV); Re, Edward (Davis, CA); Vlasenko, Elena (Davis, CA); McFarland, Keith (Davis, CA); Lopez de Leon, Alfredo (Davis, CA)

    2008-04-22

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

  19. Polypeptides having beta-glucosidase activity and polynucleotides encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Harris, Paul (Carnation, WA); Golightly, Elizabeth (Reno, NV)

    2011-06-14

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

  20. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Kimberly (Elk Grove, CA); Harris, Paul (Carnation, WA); Lopez De Leon, Alfredo (Davis, CA); Merino, Sandra (West Sacremento, CA)

    2007-05-22

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

  1. Polypeptides having beta-glucosidase activity and polynucleotides encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Harris, Paul (Carnation, WA); Golightly, Elizabeth (Reno, NV)

    2007-07-17

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

  2. Polypeptides having endoglucanase activity and polynucleotides encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Harris, Paul (Carnation, WA); Lopez de Leon, Alfredo (Davis, CA); Rey, Michael (Davis, CA); Ding, Hanshu (Davis, CA); Vlasenko, Elena (Davis, CA)

    2010-11-02

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

  3. Polynucleotides encoding polypeptides having beta-glucosidase activity

    Energy Technology Data Exchange (ETDEWEB)

    Harris, Paul (Carnation, WA); Golightly, Elizabeth (Reno, NV)

    2010-03-02

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

  4. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Stringer, Mary Ann; McBrayer, Brett

    2016-11-29

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

  5. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Schnorr, Kirk; Kramer, Randall

    2016-08-09

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

  6. Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

    2016-08-09

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

  7. Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

    2014-09-30

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

  8. Organic Anion Transporting Polypeptides Contribute to the Disposition of Perfluoroalkyl Acids in Humans and Rats.

    Science.gov (United States)

    Zhao, Wen; Zitzow, Jeremiah D; Weaver, Yi; Ehresman, David J; Chang, Shu-Ching; Butenhoff, John L; Hagenbuch, Bruno

    2016-12-24

    Perfluoroalkyl sulfonates (PFSAs) such as perfluorohexane sulfonate (PFHxS) and perfluorooctane sulfonate (PFOS) have very long serum elimination half-lives in humans, and preferentially distribute to serum and liver. The enterohepatic circulation of PFHxS and PFOS likely contributes to their extended elimination half-lives. We previously demonstrated that perfluorobutane sulfonate (PFBS), PFHxS, and PFOS are transported into hepatocytes both in a sodium-dependent and a sodium-independent manner. We identified Na(+)/taurocholate cotransporting polypeptide (NTCP) as the responsible sodium-dependent transporter. Furthermore, we demonstrated that the human apical sodium-dependent bile salt transporter (ASBT) contributes to the intestinal reabsorption of PFOS. However, so far no sodium-independent uptake transporters for PFSAs have been identified in human hepatocytes or enterocytes. In addition, perfluoroalkyl carboxylates (PFCAs) with 8 and 9 carbons were shown to preferentially distribute to the liver of rodents; however, no rat or human liver uptake transporters are known to transport these PFCAs. Therefore, we tested whether PFBS, PFHxS, PFOS, and PFCAs with 7-10 carbons are substrates of organic anion transporting polypeptides (OATPs). We used CHO and HEK293 cells to demonstrate that human OATP1B1, OATP1B3, and OATP2B1 can transport PFBS, PFHxS, PFOS, and the 2 PFCAs (C8 and C9). In addition, we show that rat OATP1A1, OATP1A5, OATP1B2, and OATP2B1 transport all 3 PFSAs. In conclusion, our results suggest that besides NTCP and ASBT, OATPs also are capable of contributing to the enterohepatic circulation and extended human serum elimination half-lives of the tested perfluoroalkyl acids.

  9. Biosynthesis and characterization of typical fibroin crystalline polypeptides of silkworm Bombyx mori

    Energy Technology Data Exchange (ETDEWEB)

    Wang Jiannan, E-mail: wangjn@suda.edu.cn [College of Material Engineering, Soochow University, Suzhou 215021 (China); Yan Shuqin [College of Material Engineering, Soochow University, Suzhou 215021 (China); Lu Changde [Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Bai Lun [College of Material Engineering, Soochow University, Suzhou 215021 (China)

    2009-05-05

    We aimed to investigate the self-organization/self-assembly mechanisms of silkworm fibroin-based material. In the present study, for the first time, we designed and multimerized four DNA 'monomer' sequences from structurally simple fibroin crystalline peptides or analog, [GAGAGX] (X = A, S, Y and V) to encode polypeptides [GAGAGX]{sub 16} (eGA, eGS, eGY and eGV) using a 'head-to-tail' construction strategy. Multimers were cloned into pGEX-KG and fusion proteins GST-[GAGAGX]{sub 16} (KGA, KGS, KGY and KGV) were efficiently expressed in Escherichia coli. These fusion proteins were isolated and purified by GST affinity chromatography and confirmed by SDS-PAGE and Western blot analysis using antibody reactive to GST. The polypeptides were cleavaged from GST fusion proteins by digesting with thrombin enzyme. The composition of the four polypeptides was confirmed by composition analysis of amino acids, and their abilities to form {beta}-sheet structure were determined by ThT fluorescence spectral analysis. The content of {beta}-sheet among the four polypeptides followed the order: eGS > eGV > eGY > eGA.

  10. POLYPEPTIDE AND POLYSACCHARIDE PROCESSING IN HYPERTHERMOPHILIC MICROORGANISMS

    Energy Technology Data Exchange (ETDEWEB)

    KELLY, ROBERT M.

    2008-12-22

    This project focused on the microbial physiology and biochemistry of heterotrophic hyperthermophiles with respect to mechanisms by which these organisms process polypeptides and polysaccharides under normal and stressed conditions. Emphasis is on two model organisms, for which completed genome sequences are available: Pyrococcus furiosus (growth Topt of 98°C), an archaeon, and Thermotoga maritima (growth Topt of 80°C), a bacterium. Both organisms are obligately anaerobic heterotrophs that reduce sulfur facultatively. Whole genome cDNA spotted microarrays were used to follow transcriptional response to a variety of environmental conditions in order to identify genes encoding proteins involved in the acquisition, synthesis, processing and utilization of polypeptides and polysaccharides. This project provided new insights into the physiological aspects of hyperthermophiles as these relate to microbial biochemistry and biological function in high temperature habitats. The capacity of these microorganisms to produce biohydrogen from renewable feedstocks makes them important for future efforts to develop biofuels.

  11. Is there proofreading during polypeptide synthesis?

    OpenAIRE

    Ruusala, T; Ehrenberg, M.; Kurland, C. G.

    1982-01-01

    The stoichiometric efficiency with which ternary complexes containing Phe-tRNAphe and Leu-tRNAleu support polypeptide synthesis has been compared in a poly(U)-directed, steady-state translation system. When unfractionated tRNA is used to support synthesis, the number of discharged ternary complexes per peptide bond formed is an average of 48 times greater for leucine than for phenylalanine. When three purified leucine isoacceptor species are tested, they each show a characteristic ratio of te...

  12. Biosynthetic Polypeptides as Templates in Materials Design

    Science.gov (United States)

    Kiick, Kristi

    2007-03-01

    Biosynthetic routes to protein-based polymeric materials offer important opportunities for the production of well-defined macromolecular templates, owing to the control of sequence and molecular weight inherent in the biosynthesis of proteins. In particular, the biosynthesis of polypeptides with controlled presentation of functional groups in multiple positions, coupled with their subsequent chemical modification with biologically relevant ligands, will permit the production of well-defined, bioactive macromolecules that may provide insight into biological binding events in which multivalent binding is important. Modification of the well-defined macromolecules with ligands such as saccharides has application in the study of events such as toxin neutralization and mediation of the immune and inflammatory responses. In this work, alanine-rich polypeptides of both random coil and helical conformations, equipped with glutamic acid residues to impart chemical versatility, have been produced via biosynthetic strategies. Analysis via spectroscopic and calorimetric methods indicates that the polypeptides adopt helical, beta-sheet, or random-coil conformations that can be controlled with variations in temperature, pH, and salt concentration; the conformational behavior of the polypeptides is not compromised upon chemical modification with saccharides. The binding of these macromolecules to bacterial toxins has been characterized via immunochemical and spectroscopic methods; results indicate that specific architectural features of the glycopolymer scaffold cause changes in the binding of these molecules to multivalent receptors. Given the chemical flexibility in the design of such scaffolds, they can be modified with many different moieties in addition to saccharides, so multiple opportunities exist for their application in areas where control of active side chains is important, such as in biomaterials, electronic devices, and bioinorganic structures.

  13. Protein kinase C induces endocytosis of the sodium taurocholate cotransporting polypeptide.

    Science.gov (United States)

    Stross, Claudia; Helmer, Angelika; Weissenberger, Katrin; Görg, Boris; Keitel, Verena; Häussinger, Dieter; Kubitz, Ralf

    2010-08-01

    Bile salts influence signaling and metabolic pathways. In hepatocytes, the sodium taurocholate cotransporting polypeptide (Ntcp) is a major determinant of intracellular bile salt levels. Short-term downregulation of Ntcp is not well characterized to date. FLAG and enhanced green fluorescent protein (EGFP) tags were cloned to the extra- and intracellular termini of Ntcp. Endocytosis of Ntcp in transfected HepG2 cells was visualized by fluorescence of EGFP, and membrane surface expression of Ntcp was quantified by flow cytometry with fluorochrome-labeled FLAG antibodies. Activation of protein kinase C (PKC) by phorbolester or thymeleatoxin an activator of Ca(2+)-dependent conventional PKCs (cPKCs), induced endocytosis of Ntcp, whereas the Na(+)-K(+)-ATPase remained in the plasma membrane. The PKC inhibitor BIM I and the cPKC-selective inhibitor Gö6976 abolished PMA-induced endocytosis. Because of this internalization, cell surface expression of Ntcp was reduced by 36 +/- 7%, bile salt uptake was decreased by 25%, and taurolithocholate sulfate-induced cell toxicity was prevented. In conclusion, Ca(2+)-dependent PKCs induce vesicular retrieval of Ntcp, thereby reducing bile salt uptake. This mechanism may protect hepatocytes from toxic intracellular bile salt concentrations.

  14. Clinical applications of hepatocyte transplantation

    Institute of Scientific and Technical Information of China (English)

    Giada Pietrosi; Giovanni Battista Vizzini; Salvatore Gruttadauria; Bruno Gridelli

    2009-01-01

    The shortage of organ donors is a problem worldwide, with approximately 15% of adult patients with lifethreatening liver diseases dying while on the waiting list. The use of cell transplantation for liver disease is an attempt to correct metabolic defects, or to support liver function as a bridge to liver transplantation and, as such, has raised a number of expectations. Most of the available studies briefly reported here focus on adult hepatocyte transplantation (HT), and the results are neither reproducible nor comparable, because the means of infusion, amount of injected cells and clinical variability differ among the studies. To better understand the specific role of HT in the management of end-stage liver disease, it is important that controlled studies, designed on the principles of evidence-based medicine, be done in order to guarantee the reproducibility of results.

  15. Primary 3-dimensional culture of mouse hepatocytes

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Complex 3-dimensional structures with good functions have been obtained under the primary mixcoculture of mouse hepatocytes with mouse liver fibroblasts without serum. Albumin secretion is kept above 10 μg/106 cells and urea synthesis reaches 25 μg/106 on the 7th day of culture. Avoiding serum affection, liver fibroblasts' effects on hepatocytes' viability, functions and 3-dimensional structure forming in primary serum-free culture have been studied. Important effects of the mesenchyma, especially the direct adherence of fibroblasts to hepatocytes, are shown.

  16. Domed Silica Microcylinders Coated with Oleophilic Polypeptides and Their Behavior in Lyotropic Cholesteric Liquid Crystals of the Same Polypeptide.

    Science.gov (United States)

    Rosu, Cornelia; Jacobeen, Shane; Park, Katherine; Reichmanis, Elsa; Yunker, Peter; Russo, Paul S

    2016-12-13

    Liquid crystals can organize dispersed particles into useful and exotic structures. In the case of lyotropic cholesteric polypeptide liquid crystals, polypeptide-coated particles are appealing because the surface chemistry matches that of the polymeric mesogen, which permits a tighter focus on factors such as extended particle shape. The colloidal particles developed here consist of a magnetic and fluorescent cylindrically symmetric silica core with one rounded, almost hemispherical end. Functionalized with helical poly(γ-stearyl-l-glutamate) (PSLG), the particles were dispersed at different concentrations in cholesteric liquid crystals (ChLC) of the same polymer in tetrahydrofuran (THF). Defects introduced by the particles to the director field of the bulk PSLG/THF host led to a variety of phases. In fresh mixtures, the cholesteric mesophase of the PSLG matrix was distorted, as reflected in the absence of the characteristic fingerprint pattern. Over time, the fingerprint pattern returned, more quickly when the concentration of the PSLG-coated particles was low. At low particle concentration the particles were "guided" by the PSLG liquid crystal to organize into patterns similar to that of the re-formed bulk chiral nematic phase. When their concentration increased, the well-dispersed PSLG-coated particles seemed to map onto the distortions in the bulk host's local director field. The particles located near the glass vial-ChLC interfaces were stacked lengthwise into architectures with apparent two-dimensional hexagonal symmetry. The size of these "crystalline" structures increased with particle concentration. They displayed remarkable stability toward an external magnetic field; hydrophobic interactions between the PSLG polymers in the shell and those in the bulk LC matrix may be responsible. The results show that bio-inspired LCs can assemble suitable colloidal particles into soft crystalline structures.

  17. Polymer Directed Protein Assemblies

    Directory of Open Access Journals (Sweden)

    Patrick van Rijn

    2013-05-01

    Full Text Available Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e.g., virus particles. Viruses are a multi-protein assembly of which the morphology is dictated by poly-nucleotides namely RNA or DNA. This “biopolymer” directs the proteins and imposes limitations on the structure like the length or diameter of the particle. Not only do these bionanoparticles use polymer-directed self-assembly, also processes like amyloid formation are in a way a result of directed protein assembly by partial unfolded/misfolded biopolymers namely, polypeptides. The combination of proteins and synthetic polymers, inspired by the natural processes, are therefore regarded as a highly promising area of research. Directed protein assembly is versatile with respect to the possible interactions which brings together the protein and polymer, e.g., electrostatic, v.d. Waals forces or covalent conjugation, and possible combinations are numerous due to the large amounts of different polymers and proteins available. The protein-polymer interacting behavior and overall morphology is envisioned to aid in clarifying protein-protein interactions and are thought to entail some interesting new functions and properties which will ultimately lead to novel bio-hybrid materials.

  18. Differential disposition of chenodeoxycholic acid versus taurocholic acid in response to acute troglitazone exposure in rat hepatocytes.

    Science.gov (United States)

    Marion, Tracy L; Perry, Cassandra H; St Claire, Robert L; Yue, Wei; Brouwer, Kim L R

    2011-04-01

    Inhibition of bile acid (BA) transport may contribute to the hepatotoxicity of troglitazone (TRO), a peroxisome proliferator-activated receptor gamma agonist. Typically, studies use taurocholic acid (TCA) as a model substrate to investigate effects of xenobiotics on BA disposition. However, TRO may differentially affect the transport of individual BAs, potentially causing hepatocyte accumulation of more cytotoxic BAs. The effects of TRO on the disposition of [(14)C]-labeled chenodeoxycholic acid ([(14)C]CDCA), an unconjugated cytotoxic BA, were determined in suspended hepatocytes and sandwich-cultured hepatocytes (SCH) from rats. (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK571), a multidrug resistance-associated protein (MRP) inhibitor, was included to evaluate involvement of MRPs in CDCA disposition. Accumulation in cells + bile of total [(14)C]CDCA species in SCH was sixfold greater than [(3)H]TCA and unaffected by 1 and 10μM TRO; 100μM TRO and 50μM MK571 ablated biliary excretion and significantly increased intracellular accumulation of total [(14)C]CDCA species. Results were similar in Mrp2-deficient TR(-) rat hepatocytes. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that taurine- and glycine-conjugated CDCA, in addition to unconjugated CDCA, accumulated in hepatocytes during the 10-min incubation. In suspended rat hepatocytes, initial [(14)C]CDCA uptake was primarily Na(+)-independent, whereas initial [(3)H]TCA uptake was primarily Na(+)-dependent; TRO and MK571 decreased [(14)C]CDCA uptake to a lesser extent than [(3)H]TCA. Unexpectedly, MK571 inhibited Na(+)-taurocholate cotransporting polypeptide and bile salt export pump. Differential effects on uptake and efflux of individual BAs may contribute to TRO hepatotoxicity. Although TCA is the prototypic BA used to investigate the effects of xenobiotics on BA transport, it may not be reflective of other

  19. Experimental Milestones in the Discovery of Molecular Chaperones as Polypeptide Unfolding Enzymes.

    Science.gov (United States)

    Finka, Andrija; Mattoo, Rayees U H; Goloubinoff, Pierre

    2016-06-01

    Molecular chaperones control the cellular folding, assembly, unfolding, disassembly, translocation, activation, inactivation, disaggregation, and degradation of proteins. In 1989, groundbreaking experiments demonstrated that a purified chaperone can bind and prevent the aggregation of artificially unfolded polypeptides and use ATP to dissociate and convert them into native proteins. A decade later, other chaperones were shown to use ATP hydrolysis to unfold and solubilize stable protein aggregates, leading to their native refolding. Presently, the main conserved chaperone families Hsp70, Hsp104, Hsp90, Hsp60, and small heat-shock proteins (sHsps) apparently act as unfolding nanomachines capable of converting functional alternatively folded or toxic misfolded polypeptides into harmless protease-degradable or biologically active native proteins. Being unfoldases, the chaperones can proofread three-dimensional protein structures and thus control protein quality in the cell. Understanding the mechanisms of the cellular unfoldases is central to the design of new therapies against aging, degenerative protein conformational diseases, and specific cancers.

  20. Genetic tracing of hepatocytes in liver homeostasis, injury, and regeneration.

    Science.gov (United States)

    Wang, Yue; Huang, XiuZhen; He, Lingjuan; Pu, Wenjuan; Li, Yan; Liu, Qiaozhen; Li, Yi; Zhang, Libo; Yu, Wei; Zhao, Huan; Zhou, Yingqun; Zhou, Bin

    2017-05-26

    The liver possesses a remarkable capacity to regenerate after damage. There is a heated debate on the origin of new hepatocytes after injuries in adult liver. Hepatic stem/progenitor cells have been proposed to produce functional hepatocytes after injury. Recent studies have argued against this model and suggested that pre-existing hepatocytes, rather than stem cells, contribute new hepatocytes. This hepatocyte-to-hepatocyte model is mainly based on labeling of hepatocytes with Cre-recombinase delivered by the adeno-associated virus. However, the impact of virus infection on cell fate determination, consistency of infection efficiency, and duration of Cre-virus in hepatocytes remain confounding factors that interfere with the data interpretation. Here, we generated a new genetic tool Alb-DreER to label almost all hepatocytes (>99.5%) and track their contribution to different cell lineages in the liver. By "pulse-and-chase" strategy, we found that pre-existing hepatocytes labeled by Alb-DreER contribute to almost all hepatocytes during normal homeostasis and after liver injury. Virtually all hepatocytes in the injured liver are descendants of pre-existing hepatocytes through self-expansion. We concluded that stem cell differentiation is unlikely to be responsible for the generation of a substantial number of new hepatocytes in adult liver. Our study also provides a new mouse tool for more precise in vivo genetic study of hepatocytes in the field. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Selective insulin resistance in hepatocyte senescence

    Energy Technology Data Exchange (ETDEWEB)

    Aravinthan, Aloysious [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Challis, Benjamin [Institute of Metabolic Sciences, University of Cambridge, Cambridge (United Kingdom); Shannon, Nicholas [Cancer Research UK Cambridge Institute, Cambridge (United Kingdom); Hoare, Matthew [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Cancer Research UK Cambridge Institute, Cambridge (United Kingdom); Heaney, Judith [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Foundation for Liver Research, Institute of Hepatology, London (United Kingdom); Alexander, Graeme J.M., E-mail: gja1000@doctors.org.uk [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom)

    2015-02-01

    Insulin resistance has been described in association with chronic liver disease for decades. Hepatocyte senescence has been demonstrated in chronic liver disease and as many as 80% of hepatocytes show a senescent phenotype in advanced liver disease. The aim of this study was to understand the role of hepatocyte senescence in the development of insulin resistance. Senescence was induced in HepG2 cells via oxidative stress. The insulin metabolic pathway was studied in control and senescent cells following insulin stimulation. GLUT2 and GLUT4 expressions were studied in HepG2 cells and human liver tissue. Further, GLUT2 and GLUT4 expressions were studied in three independent chronic liver disease cohorts. Signalling impairment distal to Akt in phosphorylation of AS160 and FoxO1 was evident in senescent HepG2 cells. Persistent nuclear localisation of FoxO1 was demonstrated in senescent cells despite insulin stimulation. Increased GLUT4 and decreased GLUT2 expressions were evident in senescent cells, human cirrhotic liver tissue and publically available liver disease datasets. Changes in GLUT expressions were associated with a poor clinical prognosis. In conclusion, selective insulin resistance is evident in senescent HepG2 cells and changes in GLUT expressions can be used as surrogate markers of hepatocyte senescence. - Highlights: • Senescent hepatocytes demonstrate selective insulin resistance. • GLUT changes act as markers of hepatocyte senescence and have prognostic value. • Study offers insight into long noticed intimacy of cirrhosis and insulin resistance.

  2. Tissue polypeptide antigen activity in cerebrospinal fluid

    DEFF Research Database (Denmark)

    Bach, F; Söletormos, Georg; Dombernowsky, P

    1991-01-01

    in the CSF and neurological clinical function. TPpA concentrations decreased in parallel with the clinical response and increased prior to CNS disease progression. As a marker for CNS metastases, the level of TPpA in the CSF in breast cancer patients appears to be superior to the level of protein, lactate......Tissue polypeptide antigen (TPpA) in the cerebrospinal fluid (CSF) was measured in 59 consecutive breast cancer patients with suspected central nervous system (CNS) metastases. Subsequently, we determined that 13 patients had parenchymal brain metastases, 10 had leptomeningeal carcinomatosis...

  3. Pulsed ELDOR in spin-labeled polypeptides

    Science.gov (United States)

    Milov, Alexander D.; Maryasov, Alexander G.; Tsvetkov, Yuri D.; Raap, Jan

    1999-04-01

    The pulsed electron-electron double-resonance (PELDOR) technique was applied to obtain information about the structure of the synthetic polypeptide-biradical in a frozen glassy solution. From the concentration dependence of the PELDOR signal, the effects of intermolecular and intramolecular interactions were separated. It was found that the intramolecular dipole-dipole interactions in the biradical peptide led to the modulation effects in the PELDOR signal decay. This may be attributed to the existence of a conformational population having a distance between the two unpaired electrons of ˜20 Å with a distribution of (˜2 Å). Its fraction is estimated as about 25%.

  4. Polypeptide having beta-glucosidase activity and uses thereof

    Energy Technology Data Exchange (ETDEWEB)

    Schoonneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; De Jong, Rene Marcel; Damveld, Robbertus Antonius

    2016-09-13

    The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

  5. Phase transitions in polypeptides: analysis of energy fluctuations

    DEFF Research Database (Denmark)

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

    2009-01-01

    The helix random coil transition in alanine, valine, and leucine polypeptides consisting of 30 amino acids is studied in vacuo using the Langevin molecular dynamics approach. The influence of side chain radicals on internal energy and heat capacity of the polypeptides is discussed. The heat...... capacity of these polypeptides is calculated as a function of temperature using two different methods, namely, as the derivative of the energy with respect to temperature, and on the basis of energy fluctuations in the system. The convergence of the fluctuations based approach is analyzed as a function...... of simulation time. This study provides a comparison of methods for the description of structural transitions in polypeptides....

  6. Computer simulation of polypeptides in a confinement.

    Science.gov (United States)

    Sikorski, Andrzej; Romiszowski, Piotr

    2007-02-01

    A coarse-grained model of polypeptide chains confined in a slit formed by two parallel impenetrable surfaces was studied. The chains were flexible heteropolymers (polypeptides) built of two kinds of united atoms-hydrophobic and hydrophilic. The positions of the united atoms were restricted to the vertices of a [310] lattice. The force field consisted of a rigorous excluded volume, a long-distance potential between a pair of amino-acid residues and a local preference for forming secondary structure (helices). The properties of the chains were studied at a wide range of temperatures from good to bad solvent conditions. Monte-Carlo simulations were carried out using the algorithm based on the chain's local changes of conformation and employing the Replica Exchange technique. The influence of the chain length, the distances between the confining surfaces, the temperature and the force field on the dimension and the structure of chains were studied. It was shown that the presence of the confinement chain complicates the process of the chain collapse to low-temperature structures. For some conditions, one can find a rapid decrease of chain size and a second transition indicated by the rapid decrease of the total energy of the system.

  7. Extracellular calmodulin: A polypeptide signal in plants?

    Institute of Scientific and Technical Information of China (English)

    孙大业; 唐文强; 马力耕

    2001-01-01

    Traditionally, calmodulin (CaM) was thought to be a multi-functional receptor for intracellular Ca2+ signals. But in the last ten years, it was found that CaM also exists and acts extracellularly in animal and plant cells to regulate many important physiological functions. Laboratory studies by the authors showed that extracellular CaM in plant cells can stimulate the proliferation of suspension cultured cell and protoplast; regulate pollen germination and pollen tube elongation,and stimulate the light-independent gene expression of Rubisco small subunit (rbcS). Furthermore,we defined the trans-membrane and intracellular signal transduction pathways for extracellular CaM by using a pollen system. The components in this pathway include heterotrimeric G-protein,phospholipase C, IP3, calcium signal and protein phosphorylation etc. Based on our findings, we suggest that extracellular CaM is a polypeptide signal in plants. This idea strongly argues against the traditional concept that there is no intercellular polypeptide signal in plants.

  8. Conserved balance of hepatocyte nuclear DNA content in mononuclear and binuclear hepatocyte populations during the course of chronic viral hepatitis

    Institute of Scientific and Technical Information of China (English)

    Hidenori Toyoda; Takashi Kumada; Olivier Bregerie; Christian Brechot; Chantal Desdouets

    2006-01-01

    AIM: To analyze the percentages of hepatocytes with increased nuclear DNA content, i.e., tetraploid (4n) and octoploid (8n) nuclei, and then compared mononuclear and binuclear hepatocyte populations:METHODS: The percentages of mononuclear diploid(2n), 4n, and 8n hepatocytes and those of binuclear 2× 2n, 2 × 4n, and 2 × 8n hepatocytes were determined with a method that can simultaneously measure hepatocyte nuclear DNA content and binuclearity in 62patients with chronic hepatitis B or C. The percentage of 4n and 8n hepatocytes in the mononuclear hepatocyte population was compared with the percentage of 2 ×4n and 2 × 8n hepatocytes in the binuclear hepatocyte population.RESULTS: The percentages of 4n and 8n hepatocytes in mononuclear hepatocytes and 2 × 4n and 2 × 8n hepatocytes in binuclear hepatocytes were similar,regardless of the activity or fibrosis grade of chronic hepatitis and regardless of the infecting virus.CONCLUSION: The distribution of nuclear DNA content within mononuclear and binuclear hepatocyte populations was conserved during the course of chronic viral hepatitis.

  9. Transcriptome of extracellular vesicles released by hepatocytes.

    Directory of Open Access Journals (Sweden)

    Felix Royo

    Full Text Available The discovery that the cells communicate through emission of vesicles has opened new opportunities for better understanding of physiological and pathological mechanisms. This discovery also provides a novel source for non-invasive disease biomarker research. Our group has previously reported that hepatocytes release extracellular vesicles with protein content reflecting the cell-type of origin. Here, we show that the extracellular vesicles released by hepatocytes also carry RNA. We report the messenger RNA composition of extracellular vesicles released in two non-tumoral hepatic models: primary culture of rat hepatocytes and a progenitor cell line obtained from a mouse foetal liver. We describe different subpopulations of extracellular vesicles with different densities and protein and RNA content. We also show that the RNA cargo of extracellular vesicles released by primary hepatocytes can be transferred to rat liver stellate-like cells and promote their activation. Finally, we provide in vitro and in vivo evidence that liver-damaging drugs galactosamine, acetaminophen, and diclofenac modify the RNA content of these vesicles. To summarize, we show that the extracellular vesicles secreted by hepatocytes contain various RNAs. These vesicles, likely to be involved in the activation of stellate cells, might become a new source for non-invasive identification of the liver toxicity markers.

  10. Induced pluripotent stem cell-derived hepatocytes and endothelial cells in multi-component hydrogel fibers for liver tissue engineering.

    Science.gov (United States)

    Du, Chan; Narayanan, Karthikeyan; Leong, Meng Fatt; Wan, Andrew C A

    2014-07-01

    Liver tissue engineering requires a suitable cell source, methodologies to assemble the cells within their niche microenvironments in a spatially defined manner, and vascularization of the construct in vivo for maintenance of hepatocyte viability and function. Recently, we have developed methods of encapsulating cells within separate domains in multi-component hydrogel fibers and methods of assembling fibers to form 3D-patterned tissue constructs. In the present work, we have combined these approaches to encapsulate hepatocytes and endothelial cells within their specific niches, and to assemble them into endothelialized liver tissue constructs. The hepatocytes and endothelial cells were obtained in parallel by differentiating human recombinant protein-induced human pluripotent stem cells, resulting in a construct which contained genetically identical endothelial and parenchymal elements. We were able to demonstrate that the presence of endothelial cells in the scaffold significantly improved hepatocyte function in vitro and facilitated vascularization of the scaffold when implanted in a mouse partial hepatectomy model. The in vivo studies further asserted that integration of the scaffold with host vasculature had occurred, as demonstrated by the presence of human albumin in the mouse serum. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Characterization, structure and function of linker polypeptides in phycobilisomes of cyanobacteria and red algae: an overview.

    Science.gov (United States)

    Liu, Lu-Ning; Chen, Xiu-Lan; Zhang, Yu-Zhong; Zhou, Bai-Cheng

    2005-06-30

    Cyanobacteria and red algae have intricate light-harvesting systems comprised of phycobilisomes that are attached to the outer side of the thylakoid membrane. The phycobilisomes absorb light in the wavelength range of 500-650 nm and transfer energy to the chlorophyll for photosynthesis. Phycobilisomes, which biochemically consist of phycobiliproteins and linker polypeptides, are particularly wonderful subjects for the detailed analysis of structure and function due to their spectral properties and their various components affected by growth conditions. The linker polypeptides are believed to mediate both the assembly of phycobiliproteins into the highly ordered arrays in the phycobilisomes and the interactions between the phycobilisomes and the thylakoid membrane. Functionally, they have been reported to improve energy migration by regulating the spectral characteristics of colored phycobiliproteins. In this review, the progress regarding linker polypeptides research, including separation approaches, structures and interactions with phycobiliproteins, as well as their functions in the phycobilisomes, is presented. In addition, some problems with previous work on linkers are also discussed.

  12. Doxorubicin-loaded polypeptide nanorods based on electrostatic interactions for cancer therapy.

    Science.gov (United States)

    Zhang, Longlong; Zhang, Pei; Zhao, Qingyun; Zhang, Yongchun; Cao, Longqiao; Luan, Yuxia

    2016-02-15

    An amphiphilic anionic polypeptide, methoxypolyethylene glycol-poly (glutamic acid) (mPEG-PGA), was synthesized, characterized and evaluated as a nanocarrier for the cationic anticancer drug doxorubicin hydrochloride (DOX·HCl). The complex self-assembled into nanorods in aqueous solutions via electrostatic interactions and exhibited a superior drug loading content (50.8%) and drug loading efficiency (90.2%). The average major axis of the drug-loaded nanorods was approximately 300nm, as determined by transmission electron microscopy. An in vitro release assay showed that drug-loaded nanorods exhibited pH-sensitivity and sustained release. Haemolysis assays demonstrated that the polypeptide was haemocompatible, and the polypeptide drug carrier significantly reduced the haemolysis ratio of DOX·HCl. The pharmacokinetics study showed that DOX-loaded nanorods significantly prolonged the resident time in blood. An in vitro cytotoxicity study and cellular uptake assays demonstrated that the DOX-loaded nanorods resulted in higher cell proliferation inhibition and a higher level of tumour cell uptake in A549 cells than with free DOX·HCl. The prolonged circulation and enhanced antitumor efficacy of DOX-loaded nanorods shows promise for efficient cancer chemotherapy.

  13. Cholangiocarcinomas can originate from hepatocytes in mice.

    Science.gov (United States)

    Fan, Biao; Malato, Yann; Calvisi, Diego F; Naqvi, Syed; Razumilava, Nataliya; Ribback, Silvia; Gores, Gregory J; Dombrowski, Frank; Evert, Matthias; Chen, Xin; Willenbring, Holger

    2012-08-01

    Intrahepatic cholangiocarcinomas (ICCs) are primary liver tumors with a poor prognosis. The development of effective therapies has been hampered by a limited understanding of the biology of ICCs. Although ICCs exhibit heterogeneity in location, histology, and marker expression, they are currently thought to derive invariably from the cells lining the bile ducts, biliary epithelial cells (BECs), or liver progenitor cells (LPCs). Despite lack of experimental evidence establishing BECs or LPCs as the origin of ICCs, other liver cell types have not been considered. Here we show that ICCs can originate from fully differentiated hepatocytes. Using a mouse model of hepatocyte fate tracing, we found that activated NOTCH and AKT signaling cooperate to convert normal hepatocytes into biliary cells that act as precursors of rapidly progressing, lethal ICCs. Our findings suggest a previously overlooked mechanism of human ICC formation that may be targetable for anti-ICC therapy.

  14. Block co-polypeptide and hydrogels made thereof

    NARCIS (Netherlands)

    Wolf, de F.A.; Werten, M.W.T.; Moers, A.P.H.A.; Wolbert, E.J.H.; Eggink, G.

    2009-01-01

    Disclosed is a block co-polypeptide comprising at least two blocks T and at least one block S, with blocks T and S alternating, wherein each T independently denotes a Trimerizing Block being a polypeptide block capable of forming a thermoreversible collagen-like triple helix structure, and

  15. Fibrillar dimer formation of islet amyloid polypeptides

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-05-08

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

  16. Successful mouse hepatocyte culture with sandwich collagen gel formation

    National Research Council Canada - National Science Library

    Choi, Hyun Jung; Choi, Dongho

    2013-01-01

    .... However, long-term cultures of functional hepatocytes are difficult to establish. To increase the longevity and maintain the differentiated functions of hepatocytes in primary culture, cells can be cultured in a sandwich configuration of collagen...

  17. Nucleic acids encoding antifungal polypeptides and uses thereof

    Science.gov (United States)

    Altier, Daniel J.; Ellanskaya, I. A.; Gilliam, Jacob T.; Hunter-Cevera, Jennie; Presnail, James K; Schepers, Eric; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser

    2010-11-02

    Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include an amino acid sequence, and variants and fragments thereof, for an antipathogenic polypeptide that was isolated from a fungal fermentation broth. Nucleic acid molecules that encode the antipathogenic polypeptides of the invention, and antipathogenic domains thereof, are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention are also disclosed.

  18. Long-term culture and expansion of primary human hepatocytes

    NARCIS (Netherlands)

    Levy, G.; Bomze, D.; Heinz, S.; Ramachandran, S.D.; Noerenberg, A.; Cohen, M.; Shibolet, O.; Sklan, E.; Braspenning, J.C.; Nahmias, Y.

    2015-01-01

    Hepatocytes have a critical role in metabolism, but their study is limited by the inability to expand primary hepatocytes in vitro while maintaining proliferative capacity and metabolic function. Here we describe the oncostatin M (OSM)-dependent expansion of primary human hepatocytes by low

  19. Long-term culture and expansion of primary human hepatocytes

    NARCIS (Netherlands)

    Levy, G.; Bomze, D.; Heinz, S.; Ramachandran, S.D.; Noerenberg, A.; Cohen, M.; Shibolet, O.; Sklan, E.; Braspenning, J.C.; Nahmias, Y.

    2015-01-01

    Hepatocytes have a critical role in metabolism, but their study is limited by the inability to expand primary hepatocytes in vitro while maintaining proliferative capacity and metabolic function. Here we describe the oncostatin M (OSM)-dependent expansion of primary human hepatocytes by low expressi

  20. Hepatocyte Growth Factor Reduces Free Cholesterol-Mediated Lipotoxicity in Primary Hepatocytes by Countering Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Mayra Domínguez-Pérez

    2016-01-01

    Full Text Available Cholesterol overload in the liver has shown toxic effects by inducing the aggravation of nonalcoholic fatty liver disease to steatohepatitis and sensitizing to damage. Although the mechanism of damage is complex, it has been demonstrated that oxidative stress plays a prominent role in the process. In addition, we have proved that hepatocyte growth factor induces an antioxidant response in hepatic cells; in the present work we aimed to figure out the protective effect of this growth factor in hepatocytes overloaded with free cholesterol. Hepatocytes from mice fed with a high-cholesterol diet were treated or not with HGF, reactive oxygen species present in cholesterol overloaded hepatocytes significantly decreased, and this effect was particularly associated with the increase in glutathione and related enzymes, such as γ-gamma glutamyl cysteine synthetase, GSH peroxidase, and GSH-S-transferase. Our data clearly indicate that HGF displays an antioxidant response by inducing the glutathione-related protection system.

  1. Hepatocyte Growth Factor Reduces Free Cholesterol-Mediated Lipotoxicity in Primary Hepatocytes by Countering Oxidative Stress

    Science.gov (United States)

    Domínguez-Pérez, Mayra; Nuño-Lámbarri, Natalia; Clavijo-Cornejo, Denise; Luna-López, Armando; Souza, Verónica; Bucio, Leticia; Miranda, Roxana U.; Muñoz, Linda; Gomez-Quiroz, Luis Enrique; Uribe-Carvajal, Salvador; Gutiérrez-Ruiz, María Concepción

    2016-01-01

    Cholesterol overload in the liver has shown toxic effects by inducing the aggravation of nonalcoholic fatty liver disease to steatohepatitis and sensitizing to damage. Although the mechanism of damage is complex, it has been demonstrated that oxidative stress plays a prominent role in the process. In addition, we have proved that hepatocyte growth factor induces an antioxidant response in hepatic cells; in the present work we aimed to figure out the protective effect of this growth factor in hepatocytes overloaded with free cholesterol. Hepatocytes from mice fed with a high-cholesterol diet were treated or not with HGF, reactive oxygen species present in cholesterol overloaded hepatocytes significantly decreased, and this effect was particularly associated with the increase in glutathione and related enzymes, such as γ-gamma glutamyl cysteine synthetase, GSH peroxidase, and GSH-S-transferase. Our data clearly indicate that HGF displays an antioxidant response by inducing the glutathione-related protection system. PMID:27143995

  2. Constrained spheroids for prolonged hepatocyte culture.

    Science.gov (United States)

    Tong, Wen Hao; Fang, Yu; Yan, Jie; Hong, Xin; Hari Singh, Nisha; Wang, Shu Rui; Nugraha, Bramasta; Xia, Lei; Fong, Eliza Li Shan; Iliescu, Ciprian; Yu, Hanry

    2016-02-01

    Liver-specific functions in primary hepatocytes can be maintained over extended duration in vitro using spheroid culture. However, the undesired loss of cells over time is still a major unaddressed problem, which consequently generates large variations in downstream assays such as drug screening. In static culture, the turbulence generated by medium change can cause spheroids to detach from the culture substrate. Under perfusion, the momentum generated by Stokes force similarly results in spheroid detachment. To overcome this problem, we developed a Constrained Spheroids (CS) culture system that immobilizes spheroids between a glass coverslip and an ultra-thin porous Parylene C membrane, both surface-modified with poly(ethylene glycol) and galactose ligands for optimum spheroid formation and maintenance. In this configuration, cell loss was minimized even when perfusion was introduced. When compared to the standard collagen sandwich model, hepatocytes cultured as CS under perfusion exhibited significantly enhanced hepatocyte functions such as urea secretion, and CYP1A1 and CYP3A2 metabolic activity. We propose the use of the CS culture as an improved culture platform to current hepatocyte spheroid-based culture systems.

  3. Laser enhanced hydrolysis of selected polypeptides

    Science.gov (United States)

    Ouzts, Mary Paige

    This project serves as a preliminary examination of selectively enhancing bond cleavage during chemical reactions in biological molecules by using continuous wave infrared lasers. To analyze protein content, polypeptides are broken into their constituent amino acids through hydrolysis. The cleaving of the peptide bond has traditionally been accomplished under harsh conditions, 110°C in 6 N hydrochloric acid for 24 hours. In this project hydrolysis was strongly enhanced by irradiating the dipeptides, threonyl-aspartate and alanyl-alanine, for 30 minutes with coherent infrared radiation from a tunable carbon dioxide laser. The dipeptide tyrosyl-tyrosine, the chemical N- methylacetimide, and the protein BSA were successfully hydrolyzed with the laser. The effect of reaction parameters such as laser power and HCl concentration were studied, as well as the effect of the primary parameter, the beam wavelength. The samples were analyzed using standard biological methods for determining the amino acid concentration, thin layer chromatography and ion exchange chromatography. These methods gave consistent results for the irradiated samples as well as for standard amino acids and polypeptide samples. The results from these methods were used to create the hydrolysis spectra. The catalytic action of the laser was strongly wavelength dependent. The hydrolysis spectra of the molecules were compared to the absorption spectra of the samples. Laser enhanced hydrolysis occurred when the laser wavelength coincided with a line in the dipeptide spectra. This weak line in each of the dipeptide spectra is consistent both in position and strength with a line in NMA, which has been identified as a fundamental mode associated with the peptide bond. From the experimental results, the enhanced process appears to occur in the vapor phase. The initially liquid sample was progressively evaporated, and fully hydrolyzed material was carried to a collection trap by the vapor. It can, in principle

  4. Binary polypeptide system for permanent and oriented protein immobilization

    Directory of Open Access Journals (Sweden)

    Bailes Julian

    2010-05-01

    Full Text Available Abstract Background Many techniques in molecular biology, clinical diagnostics and biotechnology rely on binary affinity tags. The existing tags are based on either small molecules (e.g., biotin/streptavidin or glutathione/GST or peptide tags (FLAG, Myc, HA, Strep-tag and His-tag. Among these, the biotin-streptavidin system is most popular due to the nearly irreversible interaction of biotin with the tetrameric protein, streptavidin. The major drawback of the stable biotin-streptavidin system, however, is that neither of the two tags can be added to a protein of interest via recombinant means (except for the Strep-tag case leading to the requirement for chemical coupling. Results Here we report a new immobilization system which utilizes two monomeric polypeptides which self-assemble to produce non-covalent yet nearly irreversible complex which is stable in strong detergents, chaotropic agents, as well as in acids and alkali. Our system is based on the core region of the tetra-helical bundle known as the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex. This irreversible protein attachment system (IPAS uses either a shortened syntaxin helix and fused SNAP25-synaptobrevin or a fused syntaxin-synaptobrevin and SNAP25 allowing a two-component system suitable for recombinant protein tagging, capture and immobilization. We also show that IPAS is suitable for use with traditional beads and chromatography, planar surfaces and Biacore, gold nanoparticles and for protein-protein interaction in solution. Conclusions IPAS offers an alternative to chemical cross-linking, streptavidin-biotin system and to traditional peptide affinity tags and can be used for a wide range of applications in nanotechnology and molecular sciences.

  5. Role of apoptotic hepatocytes in HCV dissemination: regulation by acetaldehyde.

    Science.gov (United States)

    Ganesan, Murali; Natarajan, Sathish Kumar; Zhang, Jinjin; Mott, Justin L; Poluektova, Larisa I; McVicker, Benita L; Kharbanda, Kusum K; Tuma, Dean J; Osna, Natalia A

    2016-06-01

    Alcohol consumption exacerbates hepatitis C virus (HCV) pathogenesis and promotes disease progression, although the mechanisms are not quite clear. We have previously observed that acetaldehyde (Ach) continuously produced by the acetaldehyde-generating system (AGS), temporarily enhanced HCV RNA levels, followed by a decrease to normal or lower levels, which corresponded to apoptosis induction. Here, we studied whether Ach-induced apoptosis caused depletion of HCV-infected cells and what role apoptotic bodies (AB) play in HCV-alcohol crosstalk. In liver cells exposed to AGS, we observed the induction of miR-122 and miR-34a. As miR-34a has been associated with apoptotic signaling and miR-122 with HCV replication, these findings may suggest that cells with intensive viral replication undergo apoptosis. Furthermore, when AGS-induced apoptosis was blocked by a pan-caspase inhibitor, the expression of HCV RNA was not changed. AB from HCV-infected cells contained HCV core protein and the assembled HCV particle that infect intact hepatocytes, thereby promoting the spread of infection. In addition, AB are captured by macrophages to switch their cytokine profile to the proinflammatory one. Macrophages exposed to HCV(+) AB expressed more IL-1β, IL-18, IL-6, and IL-10 mRNAs compared with those exposed to HCV(-) AB. The generation of AB from AGS-treated HCV-infected cells even enhanced the induction of aforementioned cytokines. We conclude that HCV and alcohol metabolites trigger the formation of AB containing HCV particles. The consequent spread of HCV to neighboring hepatocytes via infected AB, as well as the induction of liver inflammation by AB-mediated macrophage activation potentially exacerbate the HCV infection course by alcohol and worsen disease progression.

  6. Translation regulation via nascent polypeptide-mediated ribosome stalling.

    Science.gov (United States)

    Wilson, Daniel N; Arenz, Stefan; Beckmann, Roland

    2016-04-01

    As the nascent polypeptide chain is being synthesized, it passes through a tunnel within the large ribosomal subunit. Interaction between the nascent polypeptide chain and the ribosomal tunnel can modulate the translation rate and induce translational stalling to regulate gene expression. In this article, we highlight recent structural insights into how the nascent polypeptide chain, either alone or in cooperation with co-factors, can interact with components of the ribosomal tunnel to regulate translation via inactivating the peptidyltransferase center of the ribosome and inducing ribosome stalling.

  7. Selective posttranslational modification of phage-displayed polypeptides

    Energy Technology Data Exchange (ETDEWEB)

    Tsao, Meng-Lin; Tian, Feng; Schultz, Peter

    2013-02-05

    The invention relates to posttranslational modification of phage-displayed polypeptides. These displayed polypeptides comprise at least one unnatural amino acid, e.g., an aryl-azide amino acid such as p-azido-L-phenylalanine, or an alkynyl-amino acid such as para-propargyloxyphenylalanine, which are incorporated into the phage-displayed fusion polypeptide at a selected position by using an in vivo orthogonal translation system comprising a suitable orthogonal aminoacyl-tRNA synthetase and a suitable orthogonal tRNA species. These unnatural amino acids advantageously provide targets for posttranslational modifications such as azide-alkyne [3+2]cycloaddition reactions and Staudinger modifications.

  8. Selective posttranslational modification of phage-displayed polypeptides

    Energy Technology Data Exchange (ETDEWEB)

    Tsao, Meng-Lin; Tian, Feng; Schultz, Peter

    2013-11-19

    The invention relates to posttranslational modification of phage-displayed polypeptides. These displayed polypeptides comprise at least one unnatural amino acid, e.g., an aryl-azide amino acid such as p-azido-L-phenylalanine, or an alkynyl-amino acid such as para-propargyloxyphenylalanine, which are incorporated into the phage-displayed fusion polypeptide at a selected position by using an in vivo orthogonal translation system comprising a suitable orthogonal aminoacyl-tRNA synthetase and a suitable orthogonal tRNA species. These unnatural amino acids advantageously provide targets for posttranslational modifications such as azide-alkyne [3+2] cycloaddition reactions and Staudinger modifications.

  9. Mechanisms of stability of electrospun polypeptide fibers

    Science.gov (United States)

    Gitnik, Alina; Khadka, Dhan; Cross, Michael; Le, Nicole; Haynie, Donald

    2013-03-01

    Electrospun nano- and microfibers made of biodegradable and absorbable polymers are of great interest in biomedical engineering for tissue engineering, wound healing and other purposes. We have investigated physical properties of fibers made of the synthetic organic polymer co-poly(L-glutamic acid4, L-tyrosine1) (PLEY). This water-soluble polypeptide has a net negative charge at neutral pH. Dehydrated fibers are crosslinked with a diimide reagent dissolved in ethanol, giving a maximum average number of crosslinks of 1 per polymer molecule. Fiber integrity has been assessed in an aqueous medium at pH 2, 7 and 12, before and after crosslinking. Non-crosslinked fibers dissolved rapidly at all pH values, on a timescale of seconds to minutes. Crosslinked fibers dissolved completely at pH 12, but not at pH 2 or pH 7, the rate depending on the concentration of crosslinking reagent and therefore the density of crosslinks. Dissolution at pH 12 is attributable to ionization of the tyrosine side chain, which has a nominal pKa of 10.4, an increase in electrostatic repulsion between side chains and the migration of counterions into the fiber. Fibers crosslinked in 50 mM EDC buckled on a timescale of minutes at pH 12 and dissolved shortly thereafter. Funding provided by the National Science Foundation

  10. Islet Amyloid Polypeptide: Structure, Function, and Pathophysiology

    Directory of Open Access Journals (Sweden)

    Rehana Akter

    2016-01-01

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

  11. Reduction of Gap Junctional Conductance by Microinjection of Antibodies against the 27-kDa Liver Gap Junction Polypeptide

    Science.gov (United States)

    Hertzberg, E. L.; Spray, D. C.; Bennett, M. V. L.

    1985-04-01

    Antibody raised against isolated rat liver gap junctions was microinjected into coupled cells in culture to assess its influence on gap junctional conductance. A rapid inhibition of fluorescent dye transfer and electrical coupling was produced in pairs of freshly dissociated adult rat hepatocytes and myocardial cells as well as in pairs of superior cervical ganglion neurons from neonatal rats cultured under conditions in which electrotonic synapses form. The antibodies have been shown by indirect immunofluorescence to bind to punctate regions of the plasma membrane in liver. By immunoreplica analysis of rat liver homogenates, plasma membranes, and isolated gap junctions resolved on NaDodSO4/polyacrylamide gels, binding was shown to be specific for the 27-kDa major polypeptide of gap junctions. This and similar antibodies should provide a tool for further investigation of the role of cell-cell communication mediated by gap junctions and indicate that immunologically similar polypeptides comprise gap junctions in adult mammalian cells derived from all three germ layers.

  12. Synthesis and Cleavage Activity of Artifical Minic Polypeptides

    Institute of Scientific and Technical Information of China (English)

    Yong YE; Xiao Lian HU; Ping LI; Ming Yu NIU; Li Feng CAO; Yu Fen ZHAO

    2006-01-01

    Two artificial minic polypeptides which are synthetic analogues of natural products with DNA affinity were synthesized, and theirs cleavage activity with DNA were examined. The structures of these compounds was confirmed by 1H NMR, MS and IR.

  13. Biomedical applications of polypeptide multilayer nanofilms and microcapsules

    Science.gov (United States)

    Rudra, Jai Simha S.

    The past few years have witnessed considerable growth in synthetic polymer chemistry and physics, biomaterials science, and nano-scale engineering. Research on polypeptide multilayer films, coatings, and microcapsules is located at the intersection of these areas and are promising materials for applications in medicine, biotechnology, environmental science. Most envisioned applications of polypeptide multilayers have a biomedical bent. This dissertation on polypeptide multilayer film applications covers key points of polypeptides as materials, means of polymer production, film preparation, film characterization methods, and key points of current research in basic science. Both commercial and designed peptides have been used to fabricate films for in-vitro applications such as antimicrobial coatings and cell culture coatings and also microcapsules for drug delivery applications. Other areas of product development include artificial red blood cells, anisotropic coatings, enantioselective membranes, and artificial viruses.

  14. Isolation and characterization of lipid-associated and neurosecretory polypeptides

    OpenAIRE

    Stark, Margareta

    2000-01-01

    Lipid-interacting proteins play important roles in all living organisms. This thesis focuses on isolation and characterization of an enzyme in the triacylglycerol biosynthesis (phosphatidic acid phosphatase, PAP), hydrophobic polypeptides in bile, and polypeptides in cerebrospinal fluid. These fields constitute methodological challenges and mean development of suitable tools in between lipid and protein biochemistry. Two methods used to measure PAP activity were compared. I...

  15. Fruit juice, organic anion transporting polypeptides, and drug interactions in psychiatry.

    Science.gov (United States)

    Andrade, Chittaranjan

    2014-11-01

    Organic anion transporting polypeptides (OATPs) are a group of membrane transport proteins that facilitate the influx of endogenous and exogenous substances across biological membranes. OATPs are found in enterocytes and hepatocytes and in brain, kidney, and other tissues. In enterocytes, OATPs facilitate the gastrointestinal absorption of certain orally administered drugs. Fruit juices such as grapefruit juice, orange juice, and apple juice contain substances that are OATP inhibitors. These fruit juices diminish the gastrointestinal absorption of certain antiallergen, antibiotic, antihypertensive, and β-blocker drugs. While there is no evidence, so far, that OATP inhibition affects the absorption of psychotropic medications, there is no room for complacency because the field is still nascent and because the necessary studies have not been conducted. Patients should therefore err on the side of caution, taking their medications at least 4 hours distant from fruit juice intake. Doing so is especially desirable with grapefruit juice, orange juice, and apple juice; with commercial fruit juices in which OATP-inhibiting substances are likely to be present in higher concentrations; with calcium-fortified fruit juices; and with medications such as atenolol and fexofenadine, the absorption of which is substantially diminished by concurrent fruit juice intake.

  16. Study on Hydrolysis Conditions of Flavourzyme in Soybean Polypeptide Alcalase Hydrolysate and Soybean Polypeptide Refining Process

    Directory of Open Access Journals (Sweden)

    Yongsheng Ma

    2014-10-01

    Full Text Available Soybean protein Alcalase hydrolysate was further hydrolyzed by adopting Flavourzyme as hydrolytic enzyme. The optimal hydrolysis conditions of Flavourzyme was that pH was 7.0 at temperature 50°C and E/S(ratio of enzyme and substrate was 20LAPU/g. Bitterness value was reduced to 2 after Flavourzyme hydrolysis reaction in optimal hydrolysis conditions. The change of molecular weight distribution range from Alcalase hydrolysate to Flavourzyme hydrolysate was not obvious. DH (Degree of hydrolysis of soybean protein hydrolysate was increased to 24.2% which was improved 3.5% than Alcalase hydrolysate. Protein recovery proportion was increased to 73.2% which was improved 0.8% than Alcalase hydrolysate. Soybean polypeptide Flavourzyme hydrolysate was decolorized with activated carbon which optimal dosage was 1.2% solution amount (w/w. Anion/cation exchange process was used in the desalination processing of soybean polypeptide. Ratio of anion resin and cation resin was 2:3(V/V. The volume of hydrolysate processed was 5 times as the volume of anion resin. Ash content of soybean peptide solution reduced to 2.11% (dry basis, salinity decreased by 86% after desalination processing.

  17. EXPERIMENTAL HEPATOCYTE XENOTRANSPLANTATION – A COMPREHENSIVE REVIEW OF THE LITERATURE

    Science.gov (United States)

    Zhou, Huidong; Liu, Hong; Ezzelarab, Mohamed; Schmelzer, Eva; Wang, Yi; Gerlach, Jörg; Gridelli, Bruno; Cooper, David K. C.

    2015-01-01

    Background Hepatocyte transplantation is a potential therapy for certain diseases of the liver, including hepatic failure. However, there is a limited supply of human livers as a source of cells and, after isolation, human hepatocytes can be difficult to expand in culture, limiting the number available for transplantation. Hepatocytes from other species, e.g., the pig, have therefore emerged as a potential alternative source. We searched the literature through the end of 2014 to assess the current status of experimental research into hepatocyte xenotransplantation. Literature search and results The literature search identified 51 reports of in vivo cross-species transplantation of hepatocytes in a variety of experimental models. Most studies investigated the transplantation of human (n=23) or pig (n=19) hepatocytes. No studies explored hepatocytes from genetically-engineered pigs. The spleen was the most common site of transplantation (n=23), followed by the liver (through the portal vein [n=6]) and peritoneal cavity (n=19). In 47 studies (92%), there was evidence of hepatocyte engraftment and function across a species barrier. Conclusions The data provided by this literature search strengthen the hypothesis that xenotransplantation of hepatocytes is feasible and potentially successful as a clinical therapy for certain liver diseases, including hepatic failure. By excluding vascular structures, hepatocytes isolated from genetically-engineered pig livers may address some of the immunological problems of xenotransplantation. PMID:25950141

  18. Experimental hepatocyte xenotransplantation--a comprehensive review of the literature.

    Science.gov (United States)

    Zhou, Huidong; Liu, Hong; Ezzelarab, Mohamed; Schmelzer, Eva; Wang, Yi; Gerlach, Jörg; Gridelli, Bruno; Cooper, David K C

    2015-01-01

    Hepatocyte transplantation (Tx) is a potential therapy for certain diseases of the liver, including hepatic failure. However, there is a limited supply of human livers as a source of cells and, after isolation, human hepatocytes can be difficult to expand in culture, limiting the number available for Tx. Hepatocytes from other species, for example, the pig, have therefore emerged as a potential alternative source. We searched the literature through the end of 2014 to assess the current status of experimental research into hepatocyte xenoTx. The literature search identified 51 reports of in vivo cross-species Tx of hepatocytes in a variety of experimental models. Most studies investigated the Tx of human (n = 23) or pig (n = 19) hepatocytes. No studies explored hepatocytes from genetically engineered pigs. The spleen was the most common site of Tx (n = 23), followed by the liver (through the portal vein [n = 6]) and peritoneal cavity (n = 19). In 47 studies (92%), there was evidence of hepatocyte engraftment and function across a species barrier. The data provided by this literature search strengthen the hypothesis that xenoTx of hepatocytes is feasible and potentially successful as a clinical therapy for certain liver diseases, including hepatic failure. By excluding vascular structures, hepatocytes isolated from genetically engineered pig livers may address some of the immunological problems of xenoTx.

  19. Bile acid formation in primary human hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Curt Einarsson; Ewa Ellis; Anna Abrahamsson; Bo-G6ran Ericzon; Ingemar Bj rkhem; Magnus Axelson

    2000-01-01

    AIM To evaluate a culture system for bile acid formation in primary human hepatocytes in comparison with HepG2 cells. METHODS Hepatocytes were isolated from normal human liver tissue and were cultured in serum-free William's E medium. The medium was collected and renewed every 24 h. Bile acids and their precursors in media were finally analysed by gas chromatography-mass spectrometry. RESULTS Cholic acid ( CA ) andchenodeoxycholic acid (CDCA) conjugated with glycine or taurine accounted for 70% and 25% of total steroids. A third of CDCA was also conjugated with sulphuric acid. Dexamathasone and thyroid hormorm alone or in combination did not significantly effect bile acid formation. The addition of cyclosporin A (10 μmol/L) inhibited the synthesis of CA and CDCA by about 13% and 30%, respectively. CONCLUSION Isolated human hepatocytes in primary culture behave as in the intact liver by converting cholesterol to conjugated CA and CDCA. This is in contrast to cultured HepG2 cells, which release large amounts of bile acid precursors and unconjugated bile acids into the medium.

  20. Bile acid formation in primary human hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Curt Einarsson; Ewa Ellis; Anna Abrahamsson; Bo-G ran Ericzon; Ingemar Bj rkhem; Magnus Axelson

    2000-01-01

    AIM To evaluate a system for bile acid formation in human hepatocytes in comparison with HepG2 cells.METHODS Hepatocytes were isolated from normal human liver tissue and were cultured in serum-freeWilliam's E medium. The medium was collected and renewed every 24 h. Bile acids and their precursors inmedia were finally analysed by gas chromatography-mass spectrometry.RESULTS Cholic acid (CA) and chenodeoxycholic acid (CDCA) conjugated with glycine or taurineaccounted for 70% and 25% of total steroids. One third of CDCA was also conjugated with sulphuric acid.Dexamethasone and thyroid hormone alone or in combination did not significantly affect bile acid formation.The addition of cyclosporin A (10 tm) inhibited the synthesis of CA and CDCA by about 13% and 30%,respectively.CONCLUSION Isolated human hepatocytes in primary culture behave as in the intact liver by convertingalmost quantitatively cholesterol to conjugated CA and CDCA. This is in contrast to cultured HepG2 cells,which release large amounts of bile acid precursors and unconjugated bile acids into the medium.

  1. Hepatocyte xenotransplantation for treating liver disease.

    Science.gov (United States)

    Bonavita, André Gustavo; Quaresma, Kátia; Cotta-de-Almeida, Vinícius; Pinto, Marcelo Alves; Saraiva, Roberto Magalhães; Alves, Luiz Anastácio

    2010-01-01

    The treatment of acute and chronic liver failure is still a challenge despite modern therapeutic innovations. While liver transplantation can restore liver function and improve patient survival, donor shortages limit this treatment to a small number of patients. Cellular xenotransplantation has emerged as an alternative for treating liver failure. Xenohepatocytes could be readily available in sufficient quantities to treat patients in critical condition and thereby reduce the donor shortage. The use of isolated encapsulated or non-encapsulated cells can reduce the immunorejection response. Several studies using animal models of acute or chronic liver failure have demonstrated improved survival and recovery of liver function after xenotransplantation of adult hepatocytes. Porcine liver cells are a potential source of xenohepatocytes due to similarities with human physiology and the great number of hepatocytes that can be obtained. The recent development of less immunogenic transgenic pigs, new immunosuppressive drugs, and cellular encapsulation systems represents important advances in the field of cellular xenotransplantation. In this study, we review the work carried out in animal models that deals with the advantages and limitations of hepatocyte xenotransplantation, and we propose new studies needed in this field.

  2. Biosynthesis of the neural cell adhesion molecule: characterization of polypeptide C

    DEFF Research Database (Denmark)

    Nybroe, O; Albrechtsen, M; Dahlin, J;

    1985-01-01

    and a 115,000 Mr polypeptide C, whereas neurons expressed a 200,000 Mr polypeptide A as well as polypeptide B. Skeletal muscle cells produced polypeptide B. The polypeptides synthesized by the three cell types were immunochemically identical. The membrane association of polypeptide C was investigated......The biosynthesis of the neural cell adhesion molecule (N-CAM) was studied in primary cultures of rat cerebral glial cells, cerebellar granule neurons, and skeletal muscle cells. The three cell types produced different N-CAM polypeptide patterns. Glial cells synthesized a 135,000 Mr polypeptide B...... with methods that distinguish peripheral and integral membrane proteins. Polypeptide C was found to be a peripheral membrane protein, whereas polypeptides A and B were integral membrane proteins with cytoplasmic domains of approximately 50,000 and approximately 25,000 Mr, respectively. The affinity...

  3. Sulindac and its metabolites inhibit multiple transport proteins in rat and human hepatocytes.

    Science.gov (United States)

    Lee, Jin Kyung; Paine, Mary F; Brouwer, Kim L R

    2010-08-01

    Sulindac is a commonly used nonsteroidal anti-inflammatory drug. This study tested the hypothesis that sulindac-mediated drug-drug interactions and/or hepatotoxicity may be caused, in part, by inhibition of proteins responsible for the hepatic transport of drugs and/or bile acids by sulindac and/or sulindac metabolites [sulindac sulfone (S-sulfone) and sulindac sulfide (S-sulfide)]. The uptake and excretion of model substrates, [(3)H]taurocholate (TC), [(3)H]estradiol 17-beta-glucuronide (E217G), and nitrofurantoin (NF), were investigated in rat and human suspended and sandwich-cultured hepatocytes (SCH). In suspended rat hepatocytes, S-sulfone and S-sulfide inhibited Na(+)-dependent TC initial uptake (IC(50) of 24.9 +/- 6.4 and 12.5 +/- 1.8 microM, respectively) and Na(+)-independent E217G initial uptake (IC(50) of 12.1 +/- 1.6 and 6.3 +/- 0.3 microM, respectively). In rat SCH, sulindac metabolites (100 microM) decreased the in vitro biliary clearance (Cl(biliary)) of TC, E217G, and NF by 38 to 83%, 81 to 97%, and 33 to 57%, respectively; S-sulfone and S-sulfide also decreased the TC and NF biliary excretion index by 39 to 55%. In suspended human hepatocytes, S-sulfone and S-sulfide inhibited Na(+)-dependent TC initial uptake (IC(50) of 42.2 and 3.1 microM, respectively); S-sulfide also inhibited the TC Cl(biliary) in human SCH. Sulindac/metabolites markedly inhibited hepatic uptake and biliary excretion of E217G by 51 to 100% in human SCH. In conclusion, sulindac and metabolites are potent inhibitors of the uptake and biliary clearance of bile acids in rat and human hepatocytes and also inhibit substrates of rat breast cancer resistance protein, rat and human organic anion-transporting polypeptides, and human multidrug resistance-associated protein 2. Inhibition of multiple hepatic transport proteins by sulindac/metabolites may play an important role in clinically significant sulindac-mediated drug-drug interactions and/or liver injury.

  4. CKAP2 is necessary to ensure the faithful spindle bipolarity in a dividing diploid hepatocyte.

    Science.gov (United States)

    Yoo, Bum Ho; Park, Chi-Hu; Kim, Hyun-Jun; Kang, Du-Seock; Bae, Chang-Dae

    2016-05-13

    Spindle bipolarity is crucial for segregating chromosome during somatic cell division. Previous studies have suggested that cytoskeleton associated protein 2 (CKAP2) is involved in spindle assembly and chromosome segregation. In this study, we show that CKAP2-depleted primary hepatocytes exhibit over-duplicated centrosomes with disjoined chromosomes from metaphase plate. These cells proceed to apoptosis or multipolar cell division and subsequent apoptotic cell death. In addition, a mouse liver regeneration experiment showed a marked decrease in efficiency of hepatic regeneration in CKAP2-depleted liver. These data suggest a physiological role of CKAP2 in the formation of spindle bipolarity, which is necessary for maintaining chromosomal stability.

  5. Antiangiogenic Effect of Oyster Polypeptide (OPP

    Directory of Open Access Journals (Sweden)

    Zhenhua WANG

    2009-08-01

    Full Text Available Background and objective Drugs which block tumor angiogenesis will be likely effective towards inhibiting tumor growth for angiogenesis being a prerequisite for tumor growth and metastasis. Therefore, antiangiogenesis has become a promising strategy for the treatment of cancer. Investigation on both antiangiogenic effect and mechanism(s of oyster polypeptide (OPP were performed via experiments of chicken embryos model in vivo and human umbilical vein endothelial cells (HUVECs in vitro. Methods The methods employed in experiment were chorioallantoic membrane (CAM angiogenesis in chicken embryos in vivo, MTT cell survival assay, flat plate scarification, transwell plates assay, matrigel-induced tube formation assay and transmission electron microscope et al. and the OPP’s effects on angiogenesis was observed. Results Study showed that treatment with OPP resulted in significant inhibition of chorioallantoic membrane (CAM angiogenesis in chicken embryos. MTT cell survival assay showed that treatment with OPP resulted in strong inhibition of HUVECs growth, with an IC50 of 400 μg/mL. Flat plate scarification suggested that OPP (200 μg/mL, 400 μg/mL and 800 μg/mL distinctly inhibited HUVECs’ migration (18.75%, 37.93%, 74.07% respectively, treatment for 12 h. Treatment with OPP of different concentrations (200 μg/mL, 400 μg/mL and 800 μg/mL significantly reduced the density of the migration cells by 15.5%, 37.2% and 67.24% (P<0.05 respectively. Matrigel-induced tube formation assay showed that OPP resulted in striking inhibition of tube formation of 52.43%, 84.47% and 96.12% (P<0.01 at 200 μg/mL, 400 μg/mL and 800 μg/mL (treatment for 10 h respectively. In addition, the apoptotic analysis by transmission electron microscope showed that OPP (400 μg/mL, treatment for 48 h distinctly induced HUVECs’ apoptosis. Conclusion This study strikingly showed that OPP could inhibit angiogenesis through its effects on vascular endothelial cells

  6. Organization of the lamin scaffold in the internal nuclear matrix of normal and transformed hepatocytes.

    Science.gov (United States)

    Barboro, Paola; D'Arrigo, Cristina; Repaci, Erica; Patrone, Eligio; Balbi, Cecilia

    2010-04-01

    Nuclear lamins are among the more abundant proteins making up the internal nuclear matrix, but very little is known about their structure in the nucleoplasm. Using immunoelectron microscopy, we demonstrate the organization of lamins in the nuclear matrix isolated from rat hepatocytes for the first time. Lamin epitopes are arrayed both in locally ordered clusters and in quasi-regular rows. Fourier filtering of the images demonstrates that the epitopes are placed at the nodes and halfway between the nodes of square or rhombic lattices that are about 50 nm on each side, as well as along rows at regular approximately 25-nm intervals. In addition, we have compared this structure with that of the internal nuclear matrix isolated from persistent hepatocyte nodules. In transformed hepatocytes, the islands of lamin lattice are lost, and only a partial regularity in the rows of gold particles remains. We suggest that orthogonal lattice assembly might be an intrinsic property of lamin molecules, and that the disassembly may be triggered by simple molecular events such as phosphorylation. Copyright (c) 2009 Elsevier Inc. All rights reserved.

  7. Qualitative Mapping of Structural Different Polypeptide Nanotubes

    DEFF Research Database (Denmark)

    Clausen, Casper Hyttel; Jensen, Jason; Castillo, Jaime

    2008-01-01

    that it is possible to distinguish between these three types of structures using this method. Further, an agreement between the detected signal and the structure of the hollow peptide was demonstrated; however only qualitative agreement with the mathematical expressing of the tubes is shown.......Biological self-assembled structures are receiving increasing focus within micro- and nanotechnology, for example, as sensing devices, due to the fact that they are cheap to produce and easy to functionalize. Therefore, methods for the characterization of these structures are much needed...

  8. Surface active complexes formed between keratin polypeptides and ionic surfactants.

    Science.gov (United States)

    Pan, Fang; Lu, Zhiming; Tucker, Ian; Hosking, Sarah; Petkov, Jordan; Lu, Jian R

    2016-12-15

    Keratins are a group of important proteins in skin and hair and as biomaterials they can provide desirable properties such as strength, biocompatibility, and moisture regaining and retaining. The aim of this work is to develop water-soluble keratin polypeptides from sheep wool and then explore how their surface adsorption behaves with and without surfactants. Successful preparation of keratin samples was demonstrated by identification of the key components from gel electrophoresis and the reproducible production of gram scale samples with and without SDS (sodium dodecylsulphate) during wool fibre dissolution. SDS micelles could reduce the formation of disulphide bonds between keratins during extraction, reducing inter-molecular crosslinking and improving keratin polypeptide solubility. However, Zeta potential measurements of the two polypeptide batches demonstrated almost identical pH dependent surface charge distributions with isoelectric points around pH 3.5, showing complete removal of SDS during purification by dialysis. In spite of different solubility from the two batches of keratin samples prepared, very similar adsorption and aggregation behavior was revealed from surface tension measurements and dynamic light scattering. Mixing of keratin polypeptides with SDS and C12TAB (dodecyltrimethylammonium bromide) led to the formation of keratin-surfactant complexes that were substantially more effective at reducing surface tension than the polypeptides alone, showing great promise in the delivery of keratin polypeptides via the surface active complexes. Neutron reflection measurements revealed the coexistence of surfactant and keratin polypeptides at the interface, thus providing the structural support to the observed surface tension changes associated with the formation of the surface active complexes.

  9. Primary hepatocyte culture in collagen gel mixture and collagen sandwich

    Institute of Scientific and Technical Information of China (English)

    Ying-Jie Wang; Hong-Ling Liu; Hai-Tao Guo; Hong-Wei Wen; Jun Liu

    2004-01-01

    AIM: To explore the methods of hepatocytes culture in a collagen gel mixture or between double layers of collagen sandwich configuration and to examine the functional and cytomorphological characteristics of cultured hepatocytes.METHODS: A two-step collagenase perfusion technique was used to isolate the hepatocytes from Wistar rats or newborn Chinese experimental piglets. The isolated hepatocytes were cultured in a collagen gel mixture or between double layers of collagen sandwich configuration respectively. The former was that rat hepatocytes were mixed with type I rat tail collagen solution till gelled, and the medium was added onto the gel. The latter was that swine hepatocytes were seeded on a plate precoated with collagen gel for 24 h, then another layer of collagen gel was overlaid, resulting in a sandwich configuration. The cytomorphological characteristics, albumin secretion, and LDH-release of the hepatocytes cultured in these two models were examined.RESULTS: Freshly isolated rat hepatocytes were successfully mixed and fixed in collagen gel, and cultured in the gel condition. During the culture period, the urea synthesized and secreted by rat hepatocytes was detected throughout the period. Likewise, newborn experimental piglet hepatocytes were successfully fixed between the double layers of collagen gel, forming a sandwich configuration.Within a week of culture, the albumin secreted by swine hepatocytes was detected by SDS/PAGE analysis. The typical cytomorphological characteristics of the hepatocytes cultured by the above two culture models were found under a phasecontrast microscope. There was little LDH-release during the culture period.CONCLUSION: Both collagen gel mixture and double layers of collagen sandwich configuration can provide cultural conditions much closer to in vivoenvironment, and are helpful for maintaining specific hepatic fiJnctions and cytomorphological characteristics. A collagen gel mixture culture may be more eligible for the

  10. Stem cell-derived hepatocytes for functional liver replacement

    Directory of Open Access Journals (Sweden)

    Bruno eChrist

    2012-06-01

    Full Text Available Mesenchymal stem cells (MSC represent an alternate cell source to substitute for primary hepatocytes in hepatocyte transplantation because of their multiple differentiation potential and nearly unlimited availability. They may differentiate into hepatocyte-like cells in vitro and maintain specific hepatocyte functions also after transplantation into the regenerating livers of mice or rats both under injury and non-injury conditions. Depending on the underlying liver disease their mode of action is either to replace the diseased liver tissue or to support liver regeneration through their anti-inflammatory and anti-apoptotic as well as their pro-proliferative action.

  11. SURFACE MODIFICATION OF POLYPROPYLENE MICROPOROUS MEMBRANE BY TETHERING POLYPEPTIDES

    Institute of Scientific and Technical Information of China (English)

    Zhen-mei Liu; Zhi-kang Xu; Mathias Ulbricht

    2006-01-01

    Two kinds of polypeptides were tethered onto the surface of polypropylene microporous membrane (PPMM)through a ring opening polymerization of L-glutamate N-carboxyanhydride initiated by amino groups which were introduced by ammonia plasma and γ-aminopropyl triethanoxysilane treatments. X-ray photoelectron spectroscopy (XPS), attenuated total reflectance Fourier transform infrared spectroscopy (FT-IR/ATR), scanning electron microscopy (SEM), together with water contact angle measurements were used to characterize the modified membranes. XPS analyses and FT-IR/ATR spectra demonstrated that polypeptides are actually grafted onto the membrane surface. The wettability of the membrane surface increases at first and then decreases with the increase in grafting degrees of polypeptide. Platelet adhesion and murine macrophage attachment experiments reveal an enhanced hemocompatibility for the polypeptide modified PPMMs. All these results give evidence that polypeptide grafting can simultaneously improve the hemocompatibility as well as reserve the hydrophobicity for the membrane, which will provide a potential approach to improve the performance of polypropylene hollow fiber microporous membrane used in artificial oxygenator.

  12. Assembly of lamins in vitro

    Institute of Scientific and Technical Information of China (English)

    MINGUNGWEI; XIANGJUNTONG; 等

    1996-01-01

    After lamins A,B and C were isolated and purified from rat liver,their assembly properties were examined by electron microscopy and scanning tunneling microscopy by electron microscopy and scanning tunneling microscopy using negative staining and the glycerol coating method,respectively.By varying the assembly time or the ionic conditions under which polymerization takes place,we have observed different stages of lamin assembly,which may provide clues on the structure of the 10 nm lamin filaments.At the first level of structural organization,two lamin polypeptides associate laterally into dimers with the two domains being parallel and in register.At the second level of structural organization,two dimers associate in a half-staggered and antiparallel fashion to form a tetramer 75 nm in length.At the third level of structural organization,4-10 lamin tetramers associate laterally in register to form 75 nm long 10nm filaments,which in turn combine head to head into long,fully assembled lamin filaments.The assembled lamin filaments are nonpolar.

  13. Immortalized human hepatocytes as a tool for the study of hepatocytic (de-)differentiation

    NARCIS (Netherlands)

    Schippers, IJ; Moshage, H; Roelofsen, H; Muller, M; Heymans, HSA; Ruiters, M; Kuipers, F

    Primary human hepatocytes were immortalized by stable transfection with a recombinant plasmid containing the early region of simian virus (SV) 40. The cells were cultured in serum-free, hormonally defined medium during the immortalization procedure. Foci of dividing cells were seen after 3 months.

  14. Cryopreservation and gel collagen culture of porcine hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Hong-Ling Liu; Ying-Jie Wang; Hai-Tao Guo; Yu-Ming Wang; Jun Liu; Yue-Cheng Yu

    2004-01-01

    AIM: To study the method of cryopreserving porcine hepatocytes and gel collagen culture measure after its cryopreservation.METHODS: Hepatocytes, isolated from Chinese experimental suckling mini-pigs by two-step perfusion with collagenase using an extra corporeal perfusion apparatus, were cryopreserved with 50 mL/L to 200 mL/L DMSO in liquid nitrogen for 4 mo, then thawed and seeded in 1 or between 2 layers of gel collagen. The expression of porcine albumin message RNA, cellular morphology and content of aspartate aminotransferase (AST) and urea nitrogen (UN) were examined during culture in gel.RESULTS: Viability of 150 mL/L DMSO group thawed hepatocytes was (83±4)%, but after purification, its viability was (90±5)%, attachment efficiency was (86±7)%, the viability of thawed hepatocytes was near to fresh cells. When the thawed hepatocytes were cultivated in gel collagen with culture medium adding epidermal growth factor, the hepatocytes grew in various administrative levels in mixed collagen gel, and bunchy in the sandwich configuration cultures. For up to 10 days' culture, the typical cellular morphological characteristics of cultivated hepatocytes could be observed. The leakage of AST was lower during culture in gel than that in common culture. At the same time, the UN synthesized by cells cultivated in mixed gel collagen was higher than that in other groups.CONCLUSION: Storage in liquid nitrogen can long keep hepatocytes' activities, the concentration of 150 mL/L DMSO is fit for porcine hepatocytes' cryopreservation. Thawed hepatocytes can be cultivated with coliagenous matrix, which provides an environment that more closely resembles that in vivo and maintain the expression of certain liver-specific function of hepatocytes.

  15. Grid polymeric scaffolds with polypeptide gel filling as patches for infarcted tissue regeneration.

    Science.gov (United States)

    Vallés-Lluch, A; Arnal-Pastor, M; Martínez-Ramos, C; Vilariéño-Feltrer, G; Vikingsson, L; Monleón Pradas, M

    2013-01-01

    Scaffolds of poly(ethyl acrylate) (PEA) with interconnected cylindrical orthogonal pores filled with a self-assembling peptide (SAP) gel are here proposed as patches for infarcted tissue regeneration. These combined systems aim to support cell therapy and meet further requirements posed by the application: the three-dimensional architecture of the elastomeric scaffold is expected to lodge the cells of interest in the damaged zone avoiding their death or migration, and at the same time conduct cell behavior and give mechanical support if necessary; the ECM-like polypeptide gel provides a cell-friendly aqueous microenvironment, facilitates diffusion of nutrients and cell wastes and is expected to improve the distribution and viability of the seeded cells within the pores and stimulate angiogenesis.

  16. Optimal screening of surface-displayed polypeptide libraries.

    Science.gov (United States)

    Boder, E T; Wittrup, K D

    1998-01-01

    Cell surface display of polypeptide libraries combined with flow cytometric cell sorting presents remarkable potential for enhancement of protein-ligand recognition properties. To maximize the utility of this approach, screening and purification conditions must be optimized to take full advantage of the quantitative feature of this technique. In particular, discrimination of improved library mutants from an excess of wild-type polypeptides is dependent upon an effective screening methodology. Fluorescence discrimination profiles for improved library mutants were derived from a mathematical model of expected cell fluorescence intensities for polypeptide libraries screened with fluorescent ligand. Profiles for surface-displayed libraries under equilibrium or kinetic screening conditions demonstrate distinct discrimination optima from which optimal equilibrium and kinetic screening parameters were derived. In addition, a statistical model of low cytometrically analyzed cell populations indicates the importance of low-stringency sorting followed by amplification through regrowth and resorting at increased stringency. This analysis further yields quantitative recommendations for cell-sorting stringency.

  17. Elastin-like polypeptides: biomedical applications of tunable biopolymers.

    Science.gov (United States)

    MacEwan, Sarah R; Chilkoti, Ashutosh

    2010-01-01

    Artificial repetitive polypeptides have grown in popularity as a bioinspired alternative to synthetic polymers. The genetically encoded synthesis, monodispersity, potential lack of toxicity, and biocompatibility are attractive features of these biopolymers for biological applications. Elastin-like polypeptides (ELPs) are one such class of biopolymers that are of particular interest because of their "smart"-stimuli responsive-properties. Herein, we discuss the genetically encoded design and recombinant synthesis of ELPs that enable precise control of their physicochemical properties and which have led to a wide range of biomedical applications of these biopolymers in the last decade.

  18. On the theory of phase transitions in polypeptides

    DEFF Research Database (Denmark)

    Yakubovich, Alexander V.; Solov'yov, Ilia; Greiner, Walter

    2008-01-01

    We suggest a theoretical method based on the statistical mechanics for treating the alpha-helix random coil transition in polypeptides. This process is considered as a first-order-like phase transition. The developed theory is free of model parameters and is based solely on fundamental physical...... principles. We apply the developed formalism for the description of thermodynamical properties of alanine polypeptides of different length. We analyze the essential thermodynamical properties of the system such as heat capacity, phase transition temperature and latent heat of the phase transition...

  19. Scavenger receptor BI boosts hepatocyte permissiveness to Plasmodium infection.

    NARCIS (Netherlands)

    Yalaoui, S.; Huby, T.; Franetich, J.F.; Gego, A.; Rametti, A.; Moreau, M.; Collet, X.; Siau, A.; Gemert, G.J.A. van; Sauerwein, R.W.; Luty, A.J.F.; Vaillant, J.C.; Hannoun, L.; Chapman, J.; Mazier, D.; Froissard, P.

    2008-01-01

    Infection of hepatocytes by Plasmodium falciparum sporozoites requires the host tetraspanin CD81. CD81 is also predicted to be a coreceptor, along with scavenger receptor BI (SR-BI), for hepatitis C virus. Using SR-BI-knockout, SR-BI-hypomorphic and SR-BI-transgenic primary hepatocytes, as well as s

  20. Potency of individual bile acids to regulate bile acid synthesis and transport genes in primary human hepatocyte cultures.

    Science.gov (United States)

    Liu, Jie; Lu, Hong; Lu, Yuan-Fu; Lei, Xiaohong; Cui, Julia Yue; Ellis, Ewa; Strom, Stephen C; Klaassen, Curtis D

    2014-10-01

    Bile acids (BAs) are known to regulate their own homeostasis, but the potency of individual bile acids is not known. This study examined the effects of cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA) and ursodeoxycholic acid (UDCA) on expression of BA synthesis and transport genes in human primary hepatocyte cultures. Hepatocytes were treated with the individual BAs at 10, 30, and 100μM for 48 h, and RNA was extracted for real-time PCR analysis. For the classic pathway of BA synthesis, BAs except for UDCA markedly suppressed CYP7A1 (70-95%), the rate-limiting enzyme of bile acid synthesis, but only moderately (35%) down-regulated CYP8B1 at a high concentration of 100μM. BAs had minimal effects on mRNA of two enzymes of the alternative pathway of BA synthesis, namely CYP27A1 and CYP7B1. BAs increased the two major target genes of the farnesoid X receptor (FXR), namely the small heterodimer partner (SHP) by fourfold, and markedly induced fibroblast growth factor 19 (FGF19) over 100-fold. The BA uptake transporter Na(+)-taurocholate co-transporting polypeptide was unaffected, whereas the efflux transporter bile salt export pump was increased 15-fold and OSTα/β were increased 10-100-fold by BAs. The expression of the organic anion transporting polypeptide 1B3 (OATP1B3; sixfold), ATP-binding cassette (ABC) transporter G5 (ABCG5; sixfold), multidrug associated protein-2 (MRP2; twofold), and MRP3 (threefold) were also increased, albeit to lesser degrees. In general, CDCA was the most potent and effective BA in regulating these genes important for BA homeostasis, whereas DCA and CA were intermediate, LCA the least, and UDCA ineffective.

  1. Pluripotent stem cell-derived hepatocyte-like cells.

    Science.gov (United States)

    Schwartz, R E; Fleming, H E; Khetani, S R; Bhatia, S N

    2014-01-01

    Liver disease is an important clinical problem, impacting over 30 million Americans and over 600 million people worldwide. It is the 12th leading cause of death in the United States and the 16th worldwide. Due to a paucity of donor organs, several thousand Americans die yearly while waiting for liver transplantation. Unfortunately, alternative tissue sources such as fetal hepatocytes and hepatic cell lines are unreliable, difficult to reproduce, and do not fully recapitulate hepatocyte phenotype and functions. As a consequence, alternative cell sources that do not have these limitations have been sought. Human embryonic stem (hES) cell- and induced pluripotent stem (iPS) cell-derived hepatocyte-like cells may enable cell based therapeutics, the study of the mechanisms of human disease and human development, and provide a platform for screening the efficacy and toxicity of pharmaceuticals. iPS cells can be differentiated in a step-wise fashion with high efficiency and reproducibility into hepatocyte-like cells that exhibit morphologic and phenotypic characteristics of hepatocytes. In addition, iPS-derived hepatocyte-like cells (iHLCs) possess some functional hepatic activity as they secrete urea, alpha-1-antitrypsin, and albumin. However, the combined phenotypic and functional traits exhibited by iHLCs resemble a relatively immature hepatic phenotype that more closely resembles that of fetal hepatocytes rather than adult hepatocytes. Specifically, iHLCs express fetal markers such as alpha-fetoprotein and lack key mature hepatocyte functions, as reflected by drastically reduced activity (~0.1%) of important detoxification enzymes (i.e. CYP2A6, CYP3A4). These key differences between iHLCs and primary adult human hepatocytes have limited the use of stem cells as a renewable source of functional adult hepatocytes for in vitro and in vivo applications. Unfortunately, the developmental pathways that control hepatocyte maturation from a fetal into an adult hepatocyte are

  2. Metabolism of dicarboxylic acids in rat hepatocytes.

    Science.gov (United States)

    Bergseth, S; Poisson, J P; Bremer, J

    1990-02-06

    [carboxyl-14C]Dodecanedioic acid (DC12) is metabolized in hepatocytes at a rate about two thirds that of [1-14C]palmitate. Shorter dicarboxylates (sebacic (DC10), suberic (DC8), and adipic (DC6) acid) are formed, mainly DC6, less DC8 and only a little DC10. In hepatocytes from clofibrate-treated rats, more polar products account for most of the breakdown products, presumably because the beta-oxidation proceeds all the way to succinate and acetyl-CoA. [carboxyl-14C]Suberic acid (DC8) is oxidized at a rate only one fifth that of dodecanedioic acid. (+)-Decanoylcarnitine inhibits palmitate oxidation but not the oxidation of dodecanedioic acid. At low concentrations of [carboxyl-14C]dodecanedioic acid or of [1-14C]palmitate, acetylsulfanilamide is more efficiently labeled by the former. High concentrations of dodecanedioic acid inhibit palmitate oxidation and the acetylation of sulfanilamide, presumably because their CoA-esters accumulate in the cytosol. These results indicate that medium-chain dicarboxylic acids are beta-oxidized mainly in the peroxisomes.

  3. Mechanisms of the statins cytotoxicity in freshly isolated rat hepatocytes.

    Science.gov (United States)

    Abdoli, Narges; Heidari, Reza; Azarmi, Yadollah; Eghbal, Mohammad Ali

    2013-06-01

    Statins are potent drugs, used as lipid-lowering agents in cardiovascular diseases. Hepatotoxicity is one of the serious adverse effects of statins, and the exact mechanism of hepatotoxicity is not yet clear. In this study, the cytotoxic effects of the most commonly used statins, that is, atorvastatin, lovastatin, and simvastatin toward isolated rat hepatocytes, were evaluated. Markers, such as cell death, reactive oxygen species (ROS) formation, lipid peroxidation, mitochondrial membrane potential, and the amount of reduced and oxidized glutathione in the statin-treated hepatocytes, were investigated. It was found that the statins caused cytotoxicity toward rat hepatocytes dose dependently. An elevation in ROS formation, accompanied by a significant amount of lipid peroxidation and mitochondrial depolarization, was observed. Cellular glutathione reservoirs were decreased, and a significant amount of oxidized glutathione was formed. This study suggests that the adverse effect of statins toward hepatocytes is mediated through oxidative stress and the hepatocytes mitochondria play an important role in the statin-induced toxicity.

  4. Property of hepatitis B virus replication in Tupaia belangeri hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Sanada, Takahiro [Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506 (Japan); Tsukiyama-Kohara, Kyoko, E-mail: kkohara@vet.kagoshima-u.ac.jp [Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24, Korimoto, Kagoshima-city, Kagoshima 890-0065 (Japan); Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24, Korimoto, Kagoshima, Kagoshima 890-0065 (Japan); Yamamoto, Naoki [Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506 (Japan); Ezzikouri, Sayeh; Benjelloun, Soumaya [Viral Hepatitis Laboratory, Virology Unit, Institut Pasteur du Maroc, 1, Louis Pasteur, Casablanca 20360 (Morocco); Murakami, Shuko; Tanaka, Yasuhito [Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Kawasumi 1, Mizuho-ku, Nagoya, Aichi 467-8601 (Japan); Tateno, Chise [PhoenixBio Co. Ltd., 3-4-1, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046 (Japan); Kohara, Michinori, E-mail: kohara-mc@igakuken.or.jp [Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506 (Japan)

    2016-01-08

    The northern treeshrew (Tupaia belangeri) has been reported to be an effective candidate for animal infection model with hepatitis B virus (HBV). The objective of our study was to analyze the growth characteristics of HBV in tupaia hepatocytes and the host response to HBV infection. We established primary tupaia hepatocytes (3–6-week old tupaia) and infected them with HBV genotypes A, B and C, and all the genotypes proliferated as well as those in human primary hepatocytes (>10{sup 5} copies/ml in culture supernatant). We next generated a chimeric mouse with tupaia liver by transplantation of tupaia primary hepatocytes to urokinase-type plasminogen activator cDNA (cDNA-uPA)/severe combined immunodeficient (SCID) mice and the replacement ratio with tupaia hepatocytes was found to be more than 95%. Infection of chimeric mice with HBV (genotypes B, C, and D) resulted in HBV-DNA level of 10{sup 4}-10{sup 6} copies/ml after 8 weeks of infection, which were almost similar to that in humanized chimeric mouse. In contrast, serum HBV level in adult tupaia (1-year-old tupaia) was quite low (<10{sup 3} copies/ml). Understanding the differences in the response to HBV infection in primary tupaia hepatocytes, chimeric mouse, and adult tupaia will contribute to elucidating the mechanism of persistent HBV infection and viral eradication. Thus, T. belangeri was found to be efficient for studying the host response to HBV infection, thereby providing novel insight into the pathogenesis of HBV. - Highlights: • Primary hepatocytes were established from tupaia that is a novel HBV infection model. • Tupaia primary hepatocytes were susceptible for HBV infection. • The immunodeficient chimeric mice with tupaia hepatocytes were established. • The chimeric mice with tupaia hepatocytes were susceptible for HBV infection.

  5. Polypeptide Modulators of TRPV1 Produce Analgesia without Hyperthermia

    Directory of Open Access Journals (Sweden)

    Yaroslav A. Andreev

    2013-12-01

    Full Text Available Transient receptor potential vanilloid 1 receptors (TRPV1 play a significant physiological role. The study of novel TRPV1 agonists and antagonists is essential. Here, we report on the characterization of polypeptide antagonists of TRPV1 based on in vitro and in vivo experiments. We evaluated the ability of APHC1 and APHC3 to inhibit TRPV1 using the whole-cell patch clamp approach and single cell Ca2+ imaging. In vivo tests were performed to assess the biological effects of APHC1 and APHC3 on temperature sensation, inflammation and core body temperature. In the electrophysiological study, both polypeptides partially blocked the capsaicin-induced response of TRPV1, but only APHC3 inhibited acid-induced (pH 5.5 activation of the receptor. APHC1 and APHC3 showed significant antinociceptive and analgesic activity in vivo at reasonable doses (0.01–0.1 mg/kg and did not cause hyperthermia. Intravenous administration of these polypeptides prolonged hot-plate latency, blocked capsaicin- and formalin-induced behavior, reversed CFA-induced hyperalgesia and produced hypothermia. Notably, APHC3’s ability to inhibit the low pH-induced activation of TRPV1 resulted in a reduced behavioural response in the acetic acid-induced writhing test, whereas APHC1 was much less effective. The polypeptides APHC1 and APHC3 could be referred to as a new class of TRPV1 modulators that produce a significant analgesic effect without hyperthermia.

  6. Research progress of active polypeptides of Cordyceps militaris

    Directory of Open Access Journals (Sweden)

    Xu Guangyu

    2017-01-01

    Full Text Available It is recognized at home and abroad that Cordyceps militaris is a fungus that can be edible and used for medicinal application, and also a common cordyceps taishanensis that is widely distributed and with very high medicinal value in China. Active Cordyceps militaris polypeptide has shown several highlights in its absorption mechanism, such as directly absorbed without the need for digestion, and absorbed quickly and completely, and more and more international researchers are attaching great importance to it. Physiological and pharmacological activities of Cordyceps militaris polypeptide are mainly to activate related enzyme systems, promote intermediary metabolisms or control the DNA transcription and translation, simultaneously activate the reticuloendothelial system and macrophage, promote the transformation of lymphocytes, and as a nonspecific immune enhancer and regulator, activate the immune competent cells, especially lymphocytes, lymphokines, mononuclear macrophage system and NK cells, thereby attacking the target cells to play an antitumor role, and also exerting its anti-aging, anticoagulant, hypolipidemic to enhance the immunity, improve the liver function and delay the aging. In this paper, the immunity improvement, anticancer, antioxidation and antimicrobial activities of active polypeptides from Cordyceps militaris are reviewed, in order to provide a solid theoretical help for the further development and application of active polypeptides of Cordyceps militaris.

  7. Kinetics of network formation by telechelic polypeptides with trimeric nodes

    NARCIS (Netherlands)

    Skrzeszewska, P.J.; Wolf, de F.A.; Cohen Stuart, M.A.; Gucht, van der J.

    2010-01-01

    We study the kinetics of transient network formation by monodisperse telechelic polypeptides with collagen-like end blocks and a random coil middle block. Upon cooling, the end blocks associate reversibly into triple helices, leading to gels with well-defined, trimeric crosslinks. Formation of tripl

  8. In vitro assembly of catalase.

    Science.gov (United States)

    Baureder, Michael; Barane, Elisabeth; Hederstedt, Lars

    2014-10-10

    Most aerobic organisms contain catalase, which functions to decompose hydrogen peroxide. Typical catalases are structurally complex homo-tetrameric enzymes with one heme prosthetic group buried in each subunit. It is not known how catalase in the cell is assembled from its constituents. The bacterium Enterococcus faecalis cannot synthesize heme but can acquire it from the environment to form a cytoplasmic catalase. We have in E. faecalis monitored production of the enzyme polypeptide (KatA) depending on the availability of heme and used our findings to devise a procedure for the purification of preparative amounts of in vivo-synthesized apocatalase. We show that fully active catalase can be obtained in vitro by incubating isolated apoprotein with hemin. We have characterized features of the assembly process and describe a temperature-trapped hemylated intermediate of the enzyme maturation process. Hemylation of apocatalase does not require auxiliary cell components, but rapid assembly of active enzyme seemingly is assisted in the cell. Our findings provide insight about catalase assembly and offer new experimental possibilities for detailed studies of this process.

  9. Organic anion transporting polypeptides in the hepatic uptake of PBDE congeners in mice

    Energy Technology Data Exchange (ETDEWEB)

    Pacyniak, Erik [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Hagenbuch, Bruno [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); The University of Kansas Cancer Center, Kansas City, KS (United States); Klaassen, Curtis D. [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Lehman-McKeeman, Lois [Bristol Myers Squibb Co., Princeton, NJ (United States); Guo, Grace L., E-mail: lguo@kumc.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States)

    2011-11-15

    BDE47, BDE99 and BDE153 are the predominant polybrominated diphenyl ether (PBDE) congeners detected in humans and can induce drug metabolizing enzymes in the liver. We have previously demonstrated that several human liver organic anion transporting polypeptides (humans: OATPs; rodents: Oatps) can transport PBDE congeners. Mice are commonly used to study the toxicity of chemicals like the PBDE congeners. However, the mechanism of the hepatic PBDE uptake in mice is not known. Therefore, the purpose of the current study was to test the hypothesis that BDE47, BDE99, and BDE153 are substrates of mouse hepatic Oatps (Oatp1a1, Oatp1a4, Oatp1b2, and Oatp2b1). We used Human Embryonic Kidney 293 (HEK293) cells transiently expressing individual Oatps and quantified the uptake of BDE47, BDE99, and BDE153. Oatp1a4, Oatp1b2, and Oatp2b1 transported all three PBDE congeners, whereas Oatp1a1 did transport none. Kinetic studies demonstrated that Oatp1a4 and Oatp1b2 transported BDE47 with the greatest affinity, followed by BDE99 and BDE153. In contrast, Oatp2b1 transported all three PBDE congeners with similar affinities. The importance of hepatic Oatps for the liver accumulation of BDE47 was confirmed using Oatp1a4-, and Oatp1b2-null mice. -- Highlights: Black-Right-Pointing-Pointer PBDE congeners are substrates of OATPs expressed in human hepatocytes. Black-Right-Pointing-Pointer Mice are commonly used to study the toxicity of chemicals like the PBDE congeners. Black-Right-Pointing-Pointer Oatp1a4, Oatp1b2, and Oatp2b1 transported all three PBDE congeners in vitro. Black-Right-Pointing-Pointer In vivo Oatp1a4 plays a minor and Oatp1b2 a major role in BDE47 liver accumulation.

  10. Polypeptide models to understand misfolding and amyloidogenesis and their relevance in protein design and therapeutics.

    Science.gov (United States)

    Zurdo, Jesús

    2005-02-01

    The study of amyloid polypeptide models (polypeptides able to generate amyloid structures not necessarily connected with any pathology) provides an excellent tool to increase the understanding of the generic aspects of misfolding and aggregation as well as the details of the mechanism of polypeptide deposition in disease. This knowledge can be integrated and applied to different problems in therapy and biotechnology, and in particular to re-designing bio-active polypeptides (biopharmaceuticals) with improved properties.

  11. Hepatic maturation of human iPS cell-derived hepatocyte-like cells by ATF5, c/EBPα, and PROX1 transduction.

    Science.gov (United States)

    Nakamori, Daiki; Takayama, Kazuo; Nagamoto, Yasuhito; Mitani, Seiji; Sakurai, Fuminori; Tachibana, Masashi; Mizuguchi, Hiroyuki

    2016-01-15

    Hepatocyte-like cells differentiated from human iPS cells (human iPS-HLCs) are expected to be utilized in drug development and research. However, recent hepatic characterization of human iPS-HLCs showed that these cells resemble fetal hepatocytes rather than adult hepatocytes. Therefore, in this study, we aimed to develop a method to enhance the hepatic function of human iPS-HLCs. Because the gene expression levels of the hepatic transcription factors (activating transcription factor 5 (ATF5), CCAAT/enhancer-binding protein alpha (c/EBPα), and prospero homeobox protein 1 (PROX1)) in adult liver were significantly higher than those in human iPS-HLCs and fetal liver, we expected that the hepatic functions of human iPS-HLCs could be enhanced by adenovirus (Ad) vector-mediated ATF5, c/EBPα, and PROX1 transduction. The gene expression levels of cytochrome P450 (CYP) 2C9, 2E1, alpha-1 antitrypsin, transthyretin, Na+/taurocholate cotransporting polypeptide, and uridine diphosphate glucuronosyl transferase 1A1 and protein expression levels of CYP2C9 and CYP2E1 were upregulated by ATF5, c/EBPα, and PROX1 transduction. These results suggest that the hepatic functions of the human iPS-HLCs could be enhanced by ATF5, c/EBPα, and PROX1 transduction. Our findings would be useful for the hepatic maturation of human iPS-HLCs.

  12. Isolation and Primary Culture of Rat Hepatocytes Using Kiwifruit Actinidin

    Directory of Open Access Journals (Sweden)

    Z. Shirvani Farsani

    2007-07-01

    Full Text Available Introduction & Objective: Isolation of cells from different tissues rely on proteolytic enzymes mainly collagenases that selectively digest collagen fibers of extra-cellular matrix. It is important to find new and proper collagenases from plant sources. In the present research actinidin, a cysteine protease abundant in Kiwifruit, was used to isolate and culture of rat hepatocytes. Materials & Methods: Different concentrations of actinidin was used to isolate rat hepatocytes by one or two-step perfusion method. The viability of the separated cells was examined by the trypan blue test. The isolated rat hepatocytes were cultured on collagen coated plates in William´s E medium. The morphology of hepatocytes was examined microscopically after staining with the Papanicolaou method.Results: Actinidin in the concentration of 0.4 mg/ml in two-step perfusion method properly isolated hepatocytes from rat liver. The viability of isolated hepatocytes that successfully cultured in collagen coated plates was 90-95 percent.Conclusion: These results showed that actinidin is a proper protease for isolation of hepatocytes. In addition, purification of this enzyme is simpler than the collagenases.

  13. Effect of dithiotheritol on viability of cryopreserved rat hepatocytes

    Directory of Open Access Journals (Sweden)

    A Jamshidzadeh

    2009-10-01

    Full Text Available Background: Prolonged storage of cryopreserved isolated hepatocytes is now possible and such cells have been used with success for drug metabolism and toxicity studies. Quality of the cells before cryopreservation is important for viability and function after freezing. Methods: In this study, fresh rat hepatocytes were incubated with the thiol-containing compounds N-acetylcysteine (NAC, dithiotheritol (DTT and fructose as ATP supplier, at incubation times 1 and 3 hrs. The preincubated hepatocytes cryopreserved for 24 hour, and 1 and 3 months. Hepatocytes, viability were determined immediately postthaw by Trypan Blue exclusion. Results: Fructose preincubation improved the viability of hepatocytes at a concentration of 300 mM. Preincubation with DTT (50, 100 and 200 μM prior to cryopreservation had beneficial effects on viability of hepatocytes. The postthaw viability of hepatocytes preincubated with NAC was uniformly poor. Conclusion: Preincubation with DTT, improved GSH levels before freezing which could be responsible for the reduction in membrane damage during cryopreservation. It may be concluded that the intracellular

  14. Sequence assembly

    DEFF Research Database (Denmark)

    Scheibye-Alsing, Karsten; Hoffmann, S.; Frankel, Annett Maria

    2009-01-01

    Despite the rapidly increasing number of sequenced and re-sequenced genomes, many issues regarding the computational assembly of large-scale sequencing data have remain unresolved. Computational assembly is crucial in large genome projects as well for the evolving high-throughput technologies...

  15. Recombinant host cells and nucleic acid constructs encoding polypeptides having cellulolytic enhancing activity

    Energy Technology Data Exchange (ETDEWEB)

    Schnorr, Kirk; Kramer, Randall

    2017-03-28

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

  16. Alginate microencapsulated hepatocytes optimised for transplantation in acute liver failure.

    Directory of Open Access Journals (Sweden)

    Suttiruk Jitraruch

    Full Text Available BACKGROUND AND AIM: Intraperitoneal transplantation of alginate-microencapsulated human hepatocytes is an attractive option for the management of acute liver failure (ALF providing short-term support to allow native liver regeneration. The main aim of this study was to establish an optimised protocol for production of alginate-encapsulated human hepatocytes and evaluate their suitability for clinical use. METHODS: Human hepatocyte microbeads (HMBs were prepared using sterile GMP grade materials. We determined physical stability, cell viability, and hepatocyte metabolic function of HMBs using different polymerisation times and cell densities. The immune activation of peripheral blood mononuclear cells (PBMCs after co-culture with HMBs was studied. Rats with ALF induced by galactosamine were transplanted intraperitoneally with rat hepatocyte microbeads (RMBs produced using a similar optimised protocol. Survival rate and biochemical profiles were determined. Retrieved microbeads were evaluated for morphology and functionality. RESULTS: The optimised HMBs were of uniform size (583.5±3.3 µm and mechanically stable using 15 min polymerisation time compared to 10 min and 20 min (p<0.001. 3D confocal microscopy images demonstrated that hepatocytes with similar cell viability were evenly distributed within HMBs. Cell density of 3.5×10(6 cells/ml provided the highest viability. HMBs incubated in human ascitic fluid showed better cell viability and function than controls. There was no significant activation of PBMCs co-cultured with empty or hepatocyte microbeads, compared to PBMCs alone. Intraperitoneal transplantation of RMBs was safe and significantly improved the severity of liver damage compared to control groups (empty microbeads and medium alone; p<0.01. Retrieved RMBs were intact and free of immune cell adherence and contained viable hepatocytes with preserved function. CONCLUSION: An optimised protocol to produce GMP grade alginate

  17. Ploidy reductions in murine fusion-derived hepatocytes.

    Directory of Open Access Journals (Sweden)

    Andrew W Duncan

    2009-02-01

    Full Text Available We previously showed that fusion between hepatocytes lacking a crucial liver enzyme, fumarylacetoacetate hydrolase (FAH, and wild-type blood cells resulted in hepatocyte reprogramming. FAH expression was restored in hybrid hepatocytes and, upon in vivo expansion, ameliorated the effects of FAH deficiency. Here, we show that fusion-derived polyploid hepatocytes can undergo ploidy reductions to generate daughter cells with one-half chromosomal content. Fusion hybrids are, by definition, at least tetraploid. We demonstrate reduction to diploid chromosome content by multiple methods. First, cytogenetic analysis of fusion-derived hepatocytes reveals a population of diploid cells. Secondly, we demonstrate marker segregation using ss-galactosidase and the Y-chromosome. Approximately 2-5% of fusion-derived FAH-positive nodules were negative for one or more markers, as expected during ploidy reduction. Next, using a reporter system in which ss-galactosidase is expressed exclusively in fusion-derived hepatocytes, we identify a subpopulation of diploid cells expressing ss-galactosidase and FAH. Finally, we track marker segregation specifically in fusion-derived hepatocytes with diploid DNA content. Hemizygous markers were lost by >or=50% of Fah-positive cells. Since fusion-derived hepatocytes are minimally tetraploid, the existence of diploid hepatocytes demonstrates that fusion-derived cells can undergo ploidy reduction. Moreover, the high degree of marker loss in diploid daughter cells suggests that chromosomes/markers are lost in a non-random fashion. Thus, we propose that ploidy reductions lead to the generation of genetically diverse daughter cells with about 50% reduction in nuclear content. The generation of such daughter cells increases liver diversity, which may increase the likelihood of oncogenesis.

  18. Raman spectroscopic identification of normal and malignant hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Jianyu Guo; Bing Du; Min Qian; Weiying Cai; Zugeng Wang; Zhenrong Sun

    2009-01-01

    Raman spectroscopy has strong potential for providing non-invasion diagnosis of cancers. In this paper, micro-Raman spectroscopy is used to diagnose one most common liver cancer, hepatocellular carcinoma (HCC). The statistical analyzes, including i-test, principal component analysis (PCA), and linear discrim inant analysis (LDA), are performed on the Raman spectra of malignant and normal hepatocytes. The t-test-LDA results show that the 786- and 1004-cm-1 bands of the malignant and normal hepatocytes are significantly different, and PCA-LDA results show an overall accuracy of 100% for the Raman spectro scopic identification of normal and malignant hepatocytes in our experiment.

  19. Interaction between amyloid beta peptide and an aggregation blocker peptide mimicking islet amyloid polypeptide.

    Directory of Open Access Journals (Sweden)

    Nasrollah Rezaei-Ghaleh

    Full Text Available Assembly of amyloid-beta peptide (Aβ into cytotoxic oligomeric and fibrillar aggregates is believed to be a major pathologic event in Alzheimer's disease (AD and interfering with Aβ aggregation is an important strategy in the development of novel therapeutic approaches. Prior studies have shown that the double N-methylated analogue of islet amyloid polypeptide (IAPP IAPP-GI, which is a conformationally constrained IAPP analogue mimicking a non-amyloidogenic IAPP conformation, is capable of blocking cytotoxic self-assembly of Aβ. Here we investigate the interaction of IAPP-GI with Aβ40 and Aβ42 using NMR spectroscopy. The most pronounced NMR chemical shift changes were observed for residues 13-20, while residues 7-9, 15-16 as well as the C-terminal half of Aβ--that is both regions of the Aβ sequence that are converted into β-strands in amyloid fibrils--were less accessible to solvent in the presence of IAPP-GI. At the same time, interaction of IAPP-GI with Aβ resulted in a concentration-dependent co-aggregation of Aβ and IAPP-GI that was enhanced for the more aggregation prone Aβ42 peptide. On the basis of the reduced toxicity of the Aβ peptide in the presence of IAPP-GI, our data are consistent with the suggestion that IAPP-GI redirects Aβ into nontoxic "off-pathway" aggregates.

  20. Ghrelin Inhibition Restores Glucose Homeostasis in Hepatocyte Nuclear Factor-1α (MODY3)-Deficient Mice.

    Science.gov (United States)

    Brial, François; Lussier, Carine R; Belleville, Karine; Sarret, Philippe; Boudreau, François

    2015-09-01

    Hepatocyte nuclear factor-1α (HNF1α) is a transcription factor expressed in tissues of endoderm origin. Mutations in HNF1A are associated with maturity-onset diabetes of the young 3 (MODY3). Mice deficient for Hnf1α are hyperglycemic, with their pancreatic β-cells being defective in glucose-sensing insulin secretion. The specific mechanisms involved in this defect are unclear. Gut hormones control glucose homeostasis. Our objective was to explore whether changes in these hormones play a role in glucose homeostasis in the absence of Hnf1α. An increase in ghrelin gene transcript and a decrease in glucose-dependent insulinotropic polypeptide (GIP) gene transcripts were observed in the gut of Hnf1α-null mice. These changes correlated with an increase of ghrelin and a decrease of GIP-labeled cells. Ghrelin serological levels were significantly induced in Hnf1α-null mice. Paradoxically, GIP levels were also induced in these mice. Treatment of Hnf1α-null mice with a ghrelin antagonist led to a recovery of the diabetic symptoms. We conclude that upregulation of ghrelin in the absence of Hnf1α impairs insulin secretion and can be reversed by pharmacological inhibition of ghrelin/GHS-R interaction. These observations open up on future strategies to counteract ghrelin action in a program that could become beneficial in controlling non-insulin-dependent diabetes.

  1. Self-assembly of polypeptides into left-handedly twisted fibril-like structures

    Science.gov (United States)

    Mu, Yan; Gao, Yi Qin

    2009-10-01

    In this paper, we investigated the spontaneous formation of aggregation structures of amyloid-forming peptide (GGVVIA) using a coarse-grained model and Monte Carlo simulations. The effects of concentration and temperature on the formation of different aggregation structures were studied. Three types of aggregation structures, single-layer β sheet, amorphous β -sheet aggregate, and fibril-like structures, were observed in our simulations. The fibril-like structures obtained in simulations have a common cross- β spine structure in which β sheets twist in a left-handed fashion. The averaged twisting angle of the β sheet in the fibril-like structures is 12°±2° . Moreover, it was found that the peptides in the same β sheets prefer to arrange in a parallel way, which is consistent with the corresponding GGVVIA crystalline structure. On the other hand, it was found that there is a rich family of β -sheet stacking patterns in the fibril-like structures suggesting that the fibril structures are more complex than the corresponding crystalline structure and there exist many local free-energy minima rather than a distinct global minimum.

  2. Dose-dependent effect of N′-Nitrosodiethylamine on hepatic architecture, RBC rheology and polypeptide repertoire in Wistar rats

    Directory of Open Access Journals (Sweden)

    Mukherjee Devoshree

    2015-03-01

    Full Text Available N′-Nitrosodiethylamine (NDEA is an effective hepatotoxicant, carcinogen and mutagen. NDEA-induced hepatic necrosis, through metabolic activation by CYP2E1, is an extensively used experimental model. In the present study, we analysed the dose- and time-dependent effect of NDEA on hepatic damage, RBC rheology and proteomic profile in male Wistar rats. The rats, 5–6 weeks old, were divided into four groups: Group-1 served as control and received normal saline, Group-2 received a single dose of 200 mg/kg body weight NDEA intraperitoneally (i.p. and the animals were sacrificed after one week; the rats of Group-3 received a single dose of 100 mg/kg body weight NDEA and were sacrificed after one week; Group-4 received 100 mg/kg body weight/wk NDEA for two weeks and were then sacrificed. Various biochemical parameters such as ALT, AST, ALP and bilirubin were determined. Further, RBC rheology, histopathology (H&E staining of liver biopsies and polypeptide profiling (SDS-PAGE in sera and liver sections were also carried out both in control and NDEA treated groups. Our results showed a significant increase in all the biochemical parameters of the liver function test (p<0.05. In NDEA treated categories dacryocytes (tear drop cells, schistocytes (fragmented cells, codocytes (target cells, acanthocytes (spur cells and ovalocytes (oval cells were observed. H & E stained liver biopsies treated with NDEA showed abnormal liver architecture with severe haemorrhage, neutrophilic infiltration and dysplastic hepatocytes manifested in a dose-dependent manner. Software analysis of SDS-PAGE of control and NDEA treated rat sera and liver revealed qualitative and quantitative differences in polypeptide composition. Based on the presence/absence, polypeptides were classified in three different categories: (1 house-keeping, present in all the groups investigated; (2 novel, present in either control or NDEA treated group at any given time; (3 differential expression

  3. Compositions and methods for making selenocysteine containing polypeptides

    Energy Technology Data Exchange (ETDEWEB)

    Soll, Dieter; Aldag, Caroline; Hohn, Michael

    2016-10-11

    Non-naturally occurring tRNA.sup.Sec and methods of using them for recombinant expression of proteins engineered to include one or more selenocysteine residues are disclosed. The non-naturally occurring tRNA.sup.Sec can be used for recombinant manufacture of selenocysteine containing polypeptides encoded by mRNA without the requirement of an SECIS element. In some embodiments, selenocysteine containing polypeptides are manufactured by co-expressing a non-naturally occurring tRNA.sup.Sec a recombinant expression system, such as E. coli, with SerRS, EF-Tu, SelA, or PSTK and SepSecS, and an mRNA with at least one codon that recognizes the anticodon of the non-naturally occurring tRNA.sup.Sec.

  4. Separation and nanoencapsulation of antitumor polypeptide from Spirulina platensis.

    Science.gov (United States)

    Zhang, Bochao; Zhang, Xuewu

    2013-01-01

    Spirulina platensis is a multicellular edible blue-green alga with abundant proteins (∼ 60%). No report is available on the antitumor polypeptides from the whole proteins of S. platensis. In this study, for the first time, an antitumor polypeptide Y2 from trypsin digest of S. platensis proteins was obtained by using freeze-thawing plus ultrasonication extraction, hydrolysis with four enzymes (trypsin, alcalase, papain, and pepsin), and gel filtration chromatography. The results showed that the degree of hydrolysis can be ordered as: trypsin (38.5%) > alcalase (31.2%) > papain (27.8%) > pepsin (7.1%). For MCF-7 and HepG2 cells, at 250 µg/mL, the maximum inhibitory rate of Y2 was 97%, while standard drug 5-FU was 55 and 97%, respectively. Furthermore, the nanoencapsulation of Y2 with chitosan (CS) was also investigated. After nanoencapsulation, the maximum encapsulation efficiency and polypeptides contents are 49 and 15%, respectively; and the antitumor activity is basically not lost. These data demonstrated the potential of nanopolypeptides (Y2-CS) in food and pharmaceutical applications.

  5. Polypeptide synthesis induced in Nicotiana clevelandii protoplasts by infection with raspberry ringspot nepovirus.

    Science.gov (United States)

    Acosta, O; Mayo, M A

    1993-01-01

    Infection of Nicotiana clevelandii protoplasts by raspberry ringspot nepovirus resulted in the accumulation of about 24 polypeptides that differed in M(r) and pI from polypeptides accumulating in mock-inoculated protoplasts. Similar polypeptides accumulated in protoplasts infected with the S and E strains of RRV but different infection-specific polypeptides were detected in protoplasts infected with tobacco ringspot nepovirus. The M(r) of RRV-specific polypeptides ranged from 210,000 to 18,000 and most are presumed to be derived from others by proteolytic cleavage. No evidence was found for marked changes in polypeptide abundance with time after inoculation or for any virus-specific polypeptide becoming disproportionately abundant in the medium during culture.

  6. The Signature Sequence Region of the Human Drug Transporter Organic Anion Transporting Polypeptide 1B1 Is Important for Protein Surface Expression

    Directory of Open Access Journals (Sweden)

    Jennina Taylor-Wells

    2014-01-01

    Full Text Available The organic anion transporting polypeptides (OATPs encompass a family of membrane transport proteins responsible for the uptake of xenobiotic compounds. Human organic anion transporting polypeptide 1B1 (OATP1B1 mediates the uptake of clinically relevant compounds such as statins and chemotherapeutic agents into hepatocytes, playing an important role in drug delivery and detoxification. The OATPs have a putative 12-transmembrane domain topology and a highly conserved signature sequence (human OATP1B1: DSRWVGAWWLNFL, spanning the extracellular loop 3/TM6 boundary. The presence of three conserved tryptophan residues at the TM interface suggests a structural role for the sequence. This was investigated by site-directed mutagenesis of selected amino acids within the sequence D251E, W254F, W258/259F, and N261A. Transport was measured using the substrate estrone-3-sulfate and surface expression detected by luminometry and confocal microscopy, facilitated by an extracellular FLAG epitope. Uptake of estrone-3-sulfate and the surface expression of D251E, W254F, and W258/259F were both significantly reduced from the wild type OATP1B1-FLAG in transfected HEK293T cells. Confocal microscopy revealed that protein was produced but was retained intracellularly. The uptake and expression of N261A were not significantly different. The reduction in surface expression and intracellular protein retention indicates a structural and/or membrane localization role for these signature sequence residues in the human drug transporter OATP1B1.

  7. The Signature Sequence Region of the Human Drug Transporter Organic Anion Transporting Polypeptide 1B1 Is Important for Protein Surface Expression.

    Science.gov (United States)

    Taylor-Wells, Jennina; Meredith, David

    2014-01-01

    The organic anion transporting polypeptides (OATPs) encompass a family of membrane transport proteins responsible for the uptake of xenobiotic compounds. Human organic anion transporting polypeptide 1B1 (OATP1B1) mediates the uptake of clinically relevant compounds such as statins and chemotherapeutic agents into hepatocytes, playing an important role in drug delivery and detoxification. The OATPs have a putative 12-transmembrane domain topology and a highly conserved signature sequence (human OATP1B1: DSRWVGAWWLNFL), spanning the extracellular loop 3/TM6 boundary. The presence of three conserved tryptophan residues at the TM interface suggests a structural role for the sequence. This was investigated by site-directed mutagenesis of selected amino acids within the sequence D251E, W254F, W258/259F, and N261A. Transport was measured using the substrate estrone-3-sulfate and surface expression detected by luminometry and confocal microscopy, facilitated by an extracellular FLAG epitope. Uptake of estrone-3-sulfate and the surface expression of D251E, W254F, and W258/259F were both significantly reduced from the wild type OATP1B1-FLAG in transfected HEK293T cells. Confocal microscopy revealed that protein was produced but was retained intracellularly. The uptake and expression of N261A were not significantly different. The reduction in surface expression and intracellular protein retention indicates a structural and/or membrane localization role for these signature sequence residues in the human drug transporter OATP1B1.

  8. General review on in vitro hepatocyte models and their applications.

    Science.gov (United States)

    Guguen-Guillouzo, Christiane; Guillouzo, Andre

    2010-01-01

    In vitro hepatocyte models represent very useful systems in both fundamental research and various application areas. Primary hepatocytes appear as the closest model for the liver in vivo. However, they are phenotypically unstable, have a limited life span and in addition, exhibit large interdonor variability when of human origin. Hepatoma cell lines appear as an alternative but only the HepaRG cell line exhibits various functions, including major cytochrome P450 activities, at levels close to those found in primary hepatocytes. In vitro hepatocyte models have brought a substantial contribution to the understanding of the biochemistry, physiology, and cell biology of the normal and diseased liver and in various application domains such as xenobiotic metabolism and toxicity, virology, parasitology, and more generally cell therapies. In the future, new well-differentiated hepatocyte cell lines derived from tumors or from either embryonic or adult stem cells might be expected and although hepatocytes will continue to be used in various fields, these in vitro liver models should allow marked advances, especially in cell-based therapies and predictive and mechanistic hepatotoxicity of new drugs and other chemicals. All models will benefit from new developments in throughput screening based on cell chips coupled with high-content imaging and in toxicogenomics technologies.

  9. Biomechanics and functionality of hepatocytes in liver cirrhosis.

    Science.gov (United States)

    Sun, Shan; Song, Zhenyuan; Cotler, Scott J; Cho, Michael

    2014-06-27

    Cirrhosis is a life-threatening condition that is generally attributed to overproduction of collagen fibers in the extracellular matrix that mechanically stiffens the liver. Chronic liver injury due to causes including viral hepatitis, inherited and metabolic liver diseases and external factors such as alcohol abuse can result in the development of cirrhosis. Progression of cirrhosis leads to hepatocellular dysfunction. While extensive studies to understand the complexity underlying liver fibrosis have led to potential application of anti-fibrotic drugs, no such FDA-approved drugs are currently available. Additional studies of hepatic fibrogenesis and cirrhosis primarily have focused on the extracellular matrix, while hepatocyte biomechanics has received limited attention. The role of hepatocyte biomechanics in liver cirrhosis remains elusive, and how the cell stiffness is correlated with biological functions of hepatocytes is also unknown. In this study, we demonstrate that the biomechanical properties of hepatocytes are correlated with their functions (e.g., glucose metabolism), and that hepatic dysfunction can be restored through modulation of the cellular biomechanics. Furthermore, our results indicate the hepatocyte functionality appears to be regulated through a crosstalk between the Rho and Akt signaling. These novel findings may lead to biomechanical intervention of hepatocytes and the development of innovative tissue engineering for clinical treatment to target liver cells rather than exclusively focusing on the extracellular matrix alone in liver cirrhosis.

  10. alpha-Amanitin induced apoptosis in primary cultured dog hepatocytes.

    Directory of Open Access Journals (Sweden)

    Adam Szelag

    2010-06-01

    Full Text Available Amatoxin poisoning is caused by mushroom species belonging to the genera Amanita, Galerina and Lepiota with the majority of lethal mushroom exposures attributable to Amanita phalloides. High mortality rate in intoxications with these mushrooms is principally a result of the acute liver failure following significant hepatocyte damage due to hepatocellular uptake of amatoxins. A wide variety of amatoxins have been isolated; however, alpha-amanitin (alpha-AMA appears to be the primary toxin. Studies in vitro and in vivo suggest that alpha-AMA does not only cause hepatocyte necrosis, but also may lead to apoptotic cell death. The objective of this study was to evaluate the complex hepatocyte apoptosis in alpha-AMA cytotoxicity. All experiments were performed on primary cultured canine hepatocytes. The cells were incubated for 12 h with alpha-AMA at a final concentration of 1, 5, 10 and 20 microM. Viability test (MTT assay, apoptosis evaluation (TUNEL reaction, detection of DNA laddering and electron microscopy were performed at 6 and 12 h of exposure to alpha-AMA. There was a clear correlation between hepatocyte viability, concentration of alpha-AMA and time of exposure to this toxin. The decline in cultured dog hepatocyte viability during the exposure to alpha-AMA is most likely preceded by enhanced cellular apoptosis. Our results demonstrate that apoptosis might contribute to pathogenesis of the severe liver injury in the course of amanitin intoxication, particularly during the early phase of poisoning.

  11. A dileucine motif is involved in plasma membrane expression and endocytosis of rat sodium taurocholate cotransporting polypeptide (Ntcp).

    Science.gov (United States)

    Stross, Claudia; Kluge, Stefanie; Weissenberger, Katrin; Winands, Elisabeth; Häussinger, Dieter; Kubitz, Ralf

    2013-11-15

    The sodium taurocholate cotransporting polypeptide (Ntcp) is the major uptake transporter for bile salts into liver parenchymal cells, and PKC-mediated endocytosis was shown to regulate the number of Ntcp molecules at the plasma membrane. In this study, mechanisms of Ntcp internalization were analyzed by flow cytometry, immunofluorescence, and Western blot analyses in HepG2 cells. PKC activation induced endocytosis of Ntcp from the plasma membrane by ~30%. Endocytosis of Ntcp was clathrin dependent and was followed by lysosomal degradation. A dileucine motif located in the third intracellular loop of Ntcp was essential for endocytosis but also for processing and plasma membrane targeting, suggesting a dual function of this motif for intracellular trafficking of Ntcp. Mutation of two of five potential phosphorylation sites surrounding the dileucine motif (Thr225 and Ser226) inhibited PKC-mediated endocytosis. In conclusion, we could identify a motif, which is critical for Ntcp plasma membrane localization. Endocytic retrieval protects hepatocytes from elevated bile salt concentrations and is of special interest, because NTCP has been identified as a receptor for the hepatitis B and D virus.

  12. Linker polypeptides of the phycobilisome from the cyanobacterium Mastigocladus laminosus. I. Isolation and characterization of phycobiliprotein-linker-polypeptide complexes.

    Science.gov (United States)

    Füglistaller, P; Suter, F; Zuber, H

    1986-07-01

    Phycobilisomes from the cyanobacterium Mastigocladus laminosus cultured in white and red light were isolated and compared with respect to the phycoerythrocyanin (PEC) and linker polypeptide contents. It was verified that the production of PEC is induced by low light intensities. A PEC complex, (alpha PEC beta PEC)6LR34.5,PEC, and a phycocyanin (PC) complex, (alpha PC beta PC)6LR34.5,PC, were isolated from phycobilisomes by Cellex-D anion exchange chromatography and sucrose density gradient centrifugation. The absorption and fluorescence emission maxima of the PEC complex are at 575 and 620 nm and those of the PC complex are at 631 and 647 nm, respectively. The extinction coefficients of the two complexes were determined. From different experiments it was concluded that PEC is present as a hexameric complex, (alpha PEC beta PEC)6LR34.5,PEC, in the phycobilisome. The two linker polypeptides LR34.5,PEC and LR34.5,PC were isolated from their phycobiliprotein complexes by gel filtration on Bio-Gel P-100 in 50% formic acid. A 5-kDa terminal segment of both linker polypeptides was found to influence the hexamer formation of the phycobiliproteins. The same segments have been described to be responsible for the hexamer-hexamer linkage (Yu, M.-H. & Glazer, A.N. (1982) J. Biol. Chem. 257, 3429-3433). A 8.9-kDa linker polypeptide, LR(C)8.9, was isolated from a PEC fraction of the Cellex-D column by Bio-Gel P-100 gel filtration in 50% formic acid. Localisation of this protein within the phycobilisome was attempted. Its most probable function is to terminate the phycobilisomal rods at the end distal to the allophycocyanin core.

  13. Force spectroscopy of hepatocytic extracellular matrix components

    Energy Technology Data Exchange (ETDEWEB)

    Yongsunthon, R., E-mail: YongsuntR@Corning.com [Corning Incorporated, SP-FR-01, R1S32D, Corning, NY 14831 (United States); Baker, W.A.; Bryhan, M.D.; Baker, D.E.; Chang, T.; Petzold, O.N.; Walczak, W.J.; Liu, J.; Faris, R.A.; Senaratne, W.; Seeley, L.A.; Youngman, R.E. [Corning Incorporated, SP-FR-01, R1S32D, Corning, NY 14831 (United States)

    2009-07-15

    We present atomic force microscopy and force spectroscopy data of live hepatocytes (HEPG2/C3A liver cell line) grown in Eagle's Minimum Essential Medium, a complex solution of salts and amino acids commonly used for cell culture. Contact-mode imaging and force spectroscopy of this system allowed correlation of cell morphology and extracellular matrix (ECM) properties with substrate properties. Force spectroscopy analysis of cellular 'footprints' indicated that the cells secrete large polymers (e.g., 3.5 {mu}m contour length and estimated MW 1000 kDa) onto their substrate surface. Although definitive identification of the polymers has not yet been achieved, fluorescent-labeled antibody staining has specified the presence of ECM proteins such as collagen and laminin in the cellular footprints. The stretched polymers appear to be much larger than single molecules of known ECM components, such as collagen and heparan sulfate proteoglycan, thus suggesting that the cells create larger entangled, macromolecular structures from smaller components. There is strong evidence which suggests that the composition of the ECM is greatly influenced by the hydrophobicity of the substrate surface, with preferential production and/or adsorption of larger macromolecules on hydrophobic surfaces.

  14. Inhibitors and pathways of hepatocytic protein degradation.

    Science.gov (United States)

    Seglen, P O; Gordon, P B; Grinde, B; Solheim, A; Kovács, A L; Poli, A

    1981-01-01

    On the basis of experiments using amino acids and various inhibitors (lysosomotropic amines, leupeptin, chymostatin, vanadate, vinblastine, anoxia, methylaminopurines), five different modes of endogenous protein degradation in isolated rat hepatocytes can be distinguished. The two non-lysosomal (amine-resistant) mechanisms preferentially degrade relatively labile (short-lived) proteins: one of these mechanisms is energy-dependent and chymostatin-sensitive, the other is not. Of the three lysosomal (amine-sensitive) mechanisms, one--quantitatively minor--is amino acid-resistant and preferentially degrades labile proteins. The two amino acid-sensitive mechanisms each seen account for about one-half of the degradation of relatively stable (long-lived) proteins; one of them is suppressed by leucine and apparently corresponds to the formation of electron microscopically visible autophagosomes; the other may represent a different type of autophagy, inhibited by asparagine and glutamine. A new class of inhibitors, the purine derivatives (methylated 6-aminopurines, and 6-mercaptopurines) appear to specifically suppress autophagic/lysosomal protein degradation, and may help to further elucidate the mechanisms of autophagy.

  15. HEMETβ: improvement of hepatocyte metabolism mathematical model.

    Science.gov (United States)

    Orsi, G; De Maria, C; Guzzardi, M; Vozzi, F; Vozzi, G

    2011-10-01

    This article describes hepatocyte metabolism mathematical model (HEMETβ), which is an improved version of HEMET, an effective and versatile virtual cell model based on hepatic cell metabolism. HEMET is based on a set of non-linear differential equations, implemented in Simulink®, which describes the biochemical reactions and energetic cell state, and completely mimics the principal metabolic pathways in hepatic cells. The cell energy function and modular structure are the core of this model. HEMETβ as HEMET model describes hepatic cellular metabolism in standard conditions (cell culture in a plastic multi-well placed in an incubator at 37° C with 5% of CO2) and with excess substrates concentration. The main improvements in HEMETβ are the introductions of Michaelis-Menten models for reversible reactions and enzymatic inhibition. In addition, we eliminated hard non-linearities and modelled cell proliferation and every single aminoacid degradation pathway. All these innovations, combined with a user-friendly aspect, allow researchers to create new cell types and validate new experimental protocols just varying 'peripheral' pathways or model inputs.

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

    DEFF Research Database (Denmark)

    Ottesen, B; Fahrenkrug, J

    1995-01-01

    in the control of erection. Vasoactive intestinal polypeptide has been suggested as a causative factor in some diseases of the genital organs (e.g., it may play a pathophysiologic role in male impotence and the peptide is currently used in the treatment of this condition). Vasoactive intestinal polypeptide may...

  17. In vitro culture of isolated primary hepatocytes and stem cell-derived hepatocyte-like cells for liver regeneration.

    Science.gov (United States)

    Hu, Chenxia; Li, Lanjuan

    2015-08-01

    Various liver diseases result in terminal hepatic failure, and liver transplantation, cell transplantation and artificial liver support systems are emerging as effective therapies for severe hepatic disease. However, all of these treatments are limited by organ or cell resources, so developing a sufficient number of functional hepatocytes for liver regeneration is a priority. Liver regeneration is a complex process regulated by growth factors (GFs), cytokines, transcription factors (TFs), hormones, oxidative stress products, metabolic networks, and microRNA. It is well-known that the function of isolated primary hepatocytes is hard to maintain; when cultured in vitro, these cells readily undergo dedifferentiation, causing them to lose hepatocyte function. For this reason, most studies focus on inducing stem cells, such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), hepatic progenitor cells (HPCs), and mesenchymal stem cells (MSCs), to differentiate into hepatocyte-like cells (HLCs) in vitro. In this review, we mainly focus on the nature of the liver regeneration process and discuss how to maintain and enhance in vitro hepatic function of isolated primary hepatocytes or stem cell-derived HLCs for liver regeneration. In this way, hepatocytes or HLCs may be applied for clinical use for the treatment of terminal liver diseases and may prolong the survival time of patients in the near future.

  18. Conformational Study of Polypeptide Chains Grafted on the Surface of Polylactide Latex Particle

    Directory of Open Access Journals (Sweden)

    Satoshi Tanimoto

    2009-01-01

    Full Text Available Polylactide (PLA latex particle covered with polypeptide chains were prepared by means of solvent exchange method from PLA and PLA-block-polypeptide block copolymer solutions. PLA segment of the block copolymer and PLA homopolymer formed a core of the particle, and the polypeptide segment of the block copolymer, which is designed as tightly fixed biodegradable emulsifier, formed corona around the particle surface. This picture was supported by the fact that zeta-potential of PLA latex particle covered with polypeptide segment was different from that of bare PLA particle because of the presence of the ionizable group in the polypeptide chains. To clarify the effect of the ionizable group on conformation of the polypeptide chain, the relation between the polypeptide chain length and the area occupied by the single block chain was evaluated. The result that the occupied area per a polypeptide chain was linearly increased with the increase in the polypeptide chain length indicates that the polypeptide chains trail on the particle surface and did not take helical structures.

  19. Bile canaliculi formation and biliary transport in 3D sandwich-cultured hepatocytes in dependence of the extracellular matrix composition.

    Science.gov (United States)

    Deharde, Daniela; Schneider, Christin; Hiller, Thomas; Fischer, Nicolas; Kegel, Victoria; Lübberstedt, Marc; Freyer, Nora; Hengstler, Jan G; Andersson, Tommy B; Seehofer, Daniel; Pratschke, Johann; Zeilinger, Katrin; Damm, Georg

    2016-10-01

    CDF from the bile canaliculi into the culture supernatant with variations in dependence on the used matrix combination. In conclusion, the results of this study show that the choice of ECM has an impact on the morphology, cell assembly and bile canaliculi formation in PHH sandwich cultures. The morphology and the multicellular arrangement were essentially influenced by the underlaying matrix, while bile excretion and leakage of sandwich-cultured hepatocytes were mainly influenced by the overlay matrix. Leaking and damaged bile canaliculi could be a limitation of the investigated sandwich culture models in long-term excretion studies.

  20. Caffeine induces CYP1A2 expression in rat hepatocytes but not in human hepatocytes.

    Science.gov (United States)

    Vaynshteyn, David; Jeong, Hyunyoung

    2012-06-01

    Caffeine is the active constituent in coffee. Continual consumption of caffeine can lead to an attenuated response also known as tolerance. Results from rat studies have shown that caffeine is an inducer of CYP1A2, the enzyme responsible for caffeine's metabolism. This suggests that CYP1A2 induction by caffeine may be in part responsible for caffeine tolerance. However, whether caffeine induces CYP1A2 expression in humans remains unknown. Our results from luciferase assays performed in HepG2 cells showed that caffeine is not an activator of the aromatic hydrocarbon receptor (AhR), a major transcription factor involved in upregulation of CYP1A2. Furthermore, caffeine did not induce CYP1A2 expression in primary human hepatocytes at a concentration attained by ordinary coffee drinking. On the other hand, caffeine enhanced CYP1A2 expression by 9-fold in rat hepatocytes. Our results suggest that caffeine from ordinary coffee drinking does not induce CYP1A2 expression in humans and that factors other than CYP1A2 induction by caffeine likely contribute to development of caffeine tolerance in humans.

  1. Keratin 8 modulation of desmoplakin deposition at desmosomes in hepatocytes.

    Science.gov (United States)

    Loranger, Anne; Gilbert, Stéphane; Brouard, Jean-Simon; Magin, Thomas M; Marceau, Normand

    2006-12-10

    Keratins, the intermediate filament proteins of epithelial cells, connect to desmosomes, the cell-cell adhesion structures at the surface membrane. The building elements of desmosomes include desmoglein and desmocollin, which provide the actual cell adhesive properties, and desmoplakins, which anchor the keratin intermediate filaments to desmosomes. In the work reported here, we address the role of keratin 8 in modulating desmoplakin deposition at surface membrane in mouse hepatocytes. The experimental approach is based on the use of keratin 8- and keratin 18-null mouse hepatocytes as cell models. In wild-type mouse hepatocytes, desmoplakin is aligned with desmoglein and keratin 8 at the surface membrane. In keratin 8-null hepatocytes, the intermediate filament loss leads to alterations in desmoplakin distribution at the surface membrane, but not of desmoglein. Intriguingly, a significant proportion of keratin 18-null hepatocytes express keratin 8 at the surface membrane, associated with a proper desmoplakin alignment with desmoglein at desmosomes. A Triton treatment of the monolayer reveals that most of the desmoplakin present in either wild-type, keratin 8- or keratin 18-null hepatocytes is insoluble. Deletion analysis of keratin 8 further suggests that the recovery of desmoplakin alignment requires the keratin 8 rod domain. In addition, similarly to other works revealing a key role of desmoplakin phosphorylation on its interaction with intermediate filaments, we find that the phosphorylation status of the keratin 8 head domain affects desmoplakin distribution at desmosomes. Together, the data indicate that a proper alignment/deposition of desmoplakin with keratins and desmoglein in hepatocytes requires keratin 8, through a reciprocal phosphoserine-dependent process.

  2. Genetic abolishment of hepatocyte proliferation activates hepatic stem cells.

    Directory of Open Access Journals (Sweden)

    Yoko Endo

    Full Text Available Quiescent hepatic stem cells (HSCs can be activated when hepatocyte proliferation is compromised. Chemical injury rodent models have been widely used to study the localization, biomarkers, and signaling pathways in HSCs, but these models usually exhibit severe promiscuous toxicity and fail to distinguish damaged and non-damaged cells. Our goal is to establish new animal models to overcome these limitations, thereby providing new insights into HSC biology and application. We generated mutant mice with constitutive or inducible deletion of Damaged DNA Binding protein 1 (DDB1, an E3 ubiquitin ligase, in hepatocytes. We characterized the molecular mechanism underlying the compensatory activation and the properties of oval cells (OCs by methods of mouse genetics, immuno-staining, cell transplantation and gene expression profiling. We show that deletion of DDB1 abolishes self-renewal capacity of mouse hepatocytes in vivo, leading to compensatory activation and proliferation of DDB1-expressing OCs. Partially restoring proliferation of DDB1-deficient hepatocytes by ablation of p21, a substrate of DDB1 E3 ligase, alleviates OC proliferation. Purified OCs express both hepatocyte and cholangiocyte markers, form colonies in vitro, and differentiate to hepatocytes after transplantation. Importantly, the DDB1 mutant mice exhibit very minor liver damage, compared to a chemical injury model. Microarray analysis reveals several previously unrecognized markers, including Reelin, enriched in oval cells. Here we report a genetic model in which irreversible inhibition of hepatocyte duplication results in HSC-driven liver regeneration. The DDB1 mutant mice can be broadly applied to studies of HSC differentiation, HSC niche and HSCs as origin of liver cancer.

  3. Sabot assembly

    Energy Technology Data Exchange (ETDEWEB)

    Bzorgi, Fariborz

    2016-11-08

    A sabot assembly includes a projectile and a housing dimensioned and configured for receiving the projectile. An air pressure cavity having a cavity diameter is disposed between a front end and a rear end of the housing. Air intake nozzles are in fluid communication with the air pressure cavity and each has a nozzle diameter less than the cavity diameter. In operation, air flows through the plurality of air intake nozzles and into the air pressure cavity upon firing of the projectile from a gun barrel to pressurize the air pressure cavity for assisting in separation of the housing from the projectile upon the sabot assembly exiting the gun barrel.

  4. Single-particle cryo-electron microscopy of macromolecular assemblies

    OpenAIRE

    Cheng, Kimberley

    2009-01-01

    In this thesis, single-particle cryo-electron microscopy (cryo-EM) was used to study the structure of three macromolecular assemblies: the two hemocyanin isoforms from Rapana thomasiana, the Pyrococcus furiosus chaperonin, and the ribosome from Escherichia coli. Hemocyanins are large respiratory proteins in arthropods and molluscs. Most molluscan hemocyanins exist as two distinct isoforms composed of related polypeptides. In most species the two isoforms differ in terms of their oligomeric st...

  5. Multi-layered molecular mechanisms of polypeptide holding, unfolding and disaggregation by HSP70/HSP110 chaperones

    Science.gov (United States)

    Finka, Andrija; Sharma, Sandeep K.; Goloubinoff, Pierre

    2015-01-01

    Members of the HSP70/HSP110 family (HSP70s) form a central hub of the chaperone network controlling all aspects of proteostasis in bacteria and the ATP-containing compartments of eukaryotic cells. The heat-inducible form HSP70 (HSPA1A) and its major cognates, cytosolic HSC70 (HSPA8), endoplasmic reticulum BIP (HSPA5), mitochondrial mHSP70 (HSPA9) and related HSP110s (HSPHs), contribute about 3% of the total protein mass of human cells. The HSP70s carry out a plethora of housekeeping cellular functions, such as assisting proper de novo folding, assembly and disassembly of protein complexes, pulling polypeptides out of the ribosome and across membrane pores, activating and inactivating signaling proteins and controlling their degradation. The HSP70s can induce structural changes in alternatively folded protein conformers, such as clathrin cages, hormone receptors and transcription factors, thereby regulating vesicular trafficking, hormone signaling and cell differentiation in development and cancer. To carry so diverse cellular housekeeping and stress-related functions, the HSP70s act as ATP-fuelled unfolding nanomachines capable of switching polypeptides between different folded states. During stress, the HSP70s can bind (hold) and prevent the aggregation of misfolding proteins and thereafter act alone or in collaboration with other unfolding chaperones to solubilize protein aggregates. Here, we discuss the common ATP-dependent mechanisms of holding, unfolding-by-clamping and unfolding-by-entropic pulling, by which the HSP70s can apparently convert various alternatively folded and misfolded polypeptides into differently active conformers. Understanding how HSP70s can prevent the formation of cytotoxic protein aggregates, pull, unfold, and solubilize them into harmless species is central to the design of therapies against protein conformational diseases. PMID:26097841

  6. Do the ependymal cells contain vasoactive intestinal polypeptide?

    Science.gov (United States)

    Kawano, H; Masuko, S

    1989-12-04

    Immunoreaction for vasoactive intestinal polypeptide (VIP) in the ependymal cells was investigated using two different commercially available polyclonal antisera for VIP. Immunostaining with an anti-VIP serum showed strong reaction products in the ependymal cells of the central canal and the third ventricle, in addition to immunoreactive neuronal elements in the spinal cord and in the suprachiasmatic nucleus. Staining of the ependymal cells was not reduced by preabsorption of the antiserum with synthetic VIP, while the immunoreactive neuronal elements disappeared. Such staining of the ependymal cells was not found using other antiserum.

  7. Topology-dependent entropic differences for chiral polypeptides in solvents

    Energy Technology Data Exchange (ETDEWEB)

    Bernido, Christopher C., E-mail: cbernido@mozcom.co [Research Center for Theoretical Physics, Central Visayan Institute Foundation, Jagna, Bohol 6308 (Philippines); Carpio-Bernido, M. Victoria [Research Center for Theoretical Physics, Central Visayan Institute Foundation, Jagna, Bohol 6308 (Philippines); Aringa, Henry P. [Department of Physics, Mindanao State University, Marawi City 9700 (Philippines)

    2011-02-28

    The entropy of topological conformations of helical biopolymers in a solvent is determined as a function of the chirality of winding. Probability distributions are obtained by taking all possible winding conformations of a polypeptide. The results show that between mirror image distributions, there is a difference in entropy arising from a net topological winding number and the interaction of a chiral solute with its environment. This interaction is reflected in a drift coefficient which varies from segment to segment along the length of a biopolymer.

  8. Well-defined (co)polypeptides bearing pendant alkyne groups

    KAUST Repository

    Zhao, Wei

    2016-03-18

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

  9. Reaction mechanisms in the radiolysis of peptides, polypeptides and proteins

    Energy Technology Data Exchange (ETDEWEB)

    Garrison, W.M.

    1985-01-01

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

  10. 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.

  11. Molecular cytotoxic mechanisms of chlorpromazine in isolated rat hepatocytes.

    Science.gov (United States)

    MacAllister, Stephanie L; Young, Cheryl; Guzdek, Anna; Zhidkov, Nickholas; O'Brien, Peter J

    2013-01-01

    Chlorpromazine (CPZ), a member of the largest class of first-generation antipsychotic agents, is known to cause hepatotoxicity in the form of cholestasis and hepatocellular necrosis in some patients. The mechanism of CPZ hepatotoxicity is unclear, but is thought to result from reactive metabolite formation. The goal of this research was to assess potential cytotoxic mechanisms of CPZ using the accelerated cytotoxicity mechanism screening (ACMS) technique with freshly isolated rat hepatocytes. This study identified CPZ cytotoxicity and inhibition of mitochondrial membrane potential (MMP) to be concentration-dependent. Furthermore, inhibition of cytochrome P450s (CYPs), including CYP2D1 and 1A2, delayed CPZ cytotoxicity, suggesting a role for CYP activation of CPZ to a toxic metabolite(s) in this model. Metabolism studies also demonstrated glucuronide and glutathione (GSH) requirement for CPZ detoxification in hepatocytes. Inactivating the 2-electron reduction pathway, NAD(P)H quinone oxidoreductase (NQO1), caused a significant increase in hepatocyte susceptibility to CPZ, indicating quinoneimine contribution to CPZ cytotoxicity. Nontoxic concentrations of peroxidase/H(2)O(2) (inflammatory model) increased cytotoxicity in CPZ-treated hepatocytes and caused additional mitochondrial toxicity. Inflammation further depleted GSH and increased oxidized glutathione (GSSG) levels. Results suggest activation of CPZ to reactive metabolites by 2 pathways in hepatocytes: (i) a CYP-catalyzed quinoneimine pathway, and (ii) a peroxidase-catalyzed oxidation of CPZ to CPZ radicals.

  12. Morphology and function of isolated hepatocytes transplanted into rat spleen.

    Science.gov (United States)

    Mito, M; Ebata, H; Kusano, M; Onishi, T; Saito, T; Sakamoto, S

    1979-12-01

    Hepatocytes isolated by the collagenase digestive method were transplanted into the spleens of syngeneic rats. Morphology and function of the hepatocytes in the spleen were investigated for 12 to 17 months after transplantation. The transplanted hepatocytes proliferated and reconfigured in the spleen without direct perfusion of portal venous blood and with the presence of an intact host liver. Fourteen to 17 months after transplantation, the hepatocytes which had formed a demarcated nodule occupied approximately 40% of the area of the splenic parenchyma without undifferentiation on microscopic examination. However, the weight of the hepatized spleen did not increase beyond the weight of a normal spleen and the weight of the host liver that had normal morphology also did not differ from a normal liver. Light and electron microscopic studies demonstrated differentiated cord structure and normal architecture for each heptocyte. Furthermore, the hepatized spleen synthesized albumin and glycogen as demonstrated by immunofluorescence and histochemical studies. Ammonia tolerance and indocyanine green clearance tests revealed functioning hepatocytes in the spleen proper. These results indicate that our experimental model lends itself well to investigations in cell growth mechanism and that hepatocellular transplantation has potential clinical application to compensate for impaired hepatic function.

  13. Hepatitis B antigen in hepatocytes of chronic active liver disease.

    Science.gov (United States)

    Kawanishi, H

    1979-04-01

    To study the morphologic interrelation of hepatocytes with the replication of hepatitis B vius (HBV) and immunocompetent cells in chronic active liver disease(CALD), organ cultures were prepared from liver biopsy specimens. Replication of hepatitis B core antigen (HBcAg) appears to occur in the nucleus of the hepatocyte in close association with intranuclear electron-dense strands and sometimes intranucleolar matrixes (likely HBcAg genomes), and cytoplasmic maturation of the HBcAg takes place in the preautolytic condition of host hepatocytes. Immunocompetent cells became progressively autolyzed in the early period of cultures. No difference in progression of hepatocyte injury in tissues from normal subjects and from hepatitis B surface antigen (HBsAg)-positive and HBsAg-negative patients with CALD may suggest that intracellular synthesis of HBV alone is not cytopathic to host hepatocytes. This model is promising for the study of HBV replication and development, and also for testing the efficacy of new antiviral agents against the virus.

  14. Polypeptide having or assisting in carbohydrate material degrading activity and uses thereof

    Energy Technology Data Exchange (ETDEWEB)

    Schooneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; Los, Alrik Pieter

    2016-02-16

    The invention relates to a polypeptide which comprises the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 76% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 76% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

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

    DEFF Research Database (Denmark)

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

    1997-01-01

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

  16. Assembling consumption

    DEFF Research Database (Denmark)

    Assembling Consumption marks a definitive step in the institutionalisation of qualitative business research. By gathering leading scholars and educators who study markets, marketing and consumption through the lenses of philosophy, sociology and anthropology, this book clarifies and applies...... societies. This is an essential reading for both seasoned scholars and advanced students of markets, economies and social forms of consumption....

  17. In vitro culture of functionally active buffalo hepatocytes isolated by using a simplified manual perfusion method.

    Directory of Open Access Journals (Sweden)

    Santanu Panda

    Full Text Available In farm animals, there is no suitable cell line available to understand liver-specific functions. This has limited our understanding of liver function and metabolism in farm animals. Culturing and maintenance of functionally active hepatocytes is difficult, since they survive no more than few days. Establishing primary culture of hepatocytes can help in studying cellular metabolism, drug toxicity, hepatocyte specific gene function and regulation. Here we provide a simple in vitro method for isolation and short-term culture of functionally active buffalo hepatocytes.Buffalo hepatocytes were isolated from caudate lobes by using manual enzymatic perfusion and mechanical disruption of liver tissue. Hepatocyte yield was (5.3 ± 0.66×107 cells per gram of liver tissue with a viability of 82.3 ± 3.5%. Freshly isolated hepatocytes were spherical with well contrasted border. After 24 hours of seeding onto fibroblast feeder layer and different extracellular matrices like dry collagen, matrigel and sandwich collagen coated plates, hepatocytes formed confluent monolayer with frequent clusters. Cultured hepatocytes exhibited typical cuboidal and polygonal shape with restored cellular polarity. Cells expressed hepatocyte-specific marker genes or proteins like albumin, hepatocyte nuclear factor 4α, glucose-6-phosphatase, tyrosine aminotransferase, cytochromes, cytokeratin and α1-antitrypsin. Hepatocytes could be immunostained with anti-cytokeratins, anti-albumin and anti α1-antitrypsin antibodies. Abundant lipid droplets were detected in the cytosol of hepatocytes using oil red stain. In vitro cultured hepatocytes could be grown for five days and maintained for up to nine days on buffalo skin fibroblast feeder layer. Cultured hepatocytes were viable for functional studies.We developed a convenient and cost effective technique for hepatocytes isolation for short-term culture that exhibited morphological and functional characteristics of active hepatocytes

  18. The effects of human umbilical cord perivascular cells on rat hepatocyte structure and functional polarity.

    Science.gov (United States)

    Gómez-Aristizábal, Alejandro; Davies, John Edward

    2013-06-01

    Hepatocyte culture is a useful tool for the study of their biology and the development of bioartificial livers. However, many challenges have to be overcome since hepatocytes rapidly lose their normal phenotype in vitro. We have recently demonstrated that human umbilical cord perivascular cells (HUCPVCs) are able to provide support to hepatocytes. In the present study we go further into exploring the effects that HUCPVCs have in the functional polarization, and both the internal and external organization, of hepatocytes. Also, we investigate HUCPVC-hepatocyte crosstalk by tracking both the effects of HUCPVCs on hepatocyte transcription factors and those of hepatocytes on the expression of hepatotrophic factors in HUCPVCs. Our results show that HUCPVCs maintain the functional polarity of hepatocytes ex vivo, as judged by the secretion of fluorescein into bile canaliculi, for at least 40 days. Transmission electron microscopy revealed that hepatocytes in coculture organize in an organoid-like structure embedded in extracellular matrix surrounded by HUCPVCs. In coculture, hepatocytes displayed a higher expression of C/EBPα, implicated in maintenance of the mature hepatocyte phenotype, and HUCPVCs upregulated hepatocyte growth factor and Jagged1 indicating that these genes may play important roles in HUCPVC-hepatocyte interactions.

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

    Science.gov (United States)

    Colaco, Martin Francis

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

  20. Trigger sequence can influence final morphology in the self-assembly of asymmetric telechelic polymers

    NARCIS (Netherlands)

    Kumar, Aatish; Lowe, C.P.; Cohen Stuart, M.A.; Bolhuis, P.G.

    2016-01-01

    We report on a numerical study of polymer network formation of asymmetric biomimetic telechelic polymers with two reactive ends based on a self-assembling collagen, elastin or silk-like polypeptide sequence. The two reactive ends of the polymer can be activated independently using physicochemical

  1. Endogenous bile acid disposition in rat and human sandwich-cultured hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Marion, Tracy L., E-mail: tracylmarion@qualyst.com [Curriculum in Toxicology, UNC School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7270 (United States); Perry, Cassandra H., E-mail: cassandraperry@qualyst.com [Qualyst, Inc., Durham, NC 27713 (United States); St Claire, Robert L., E-mail: bobstclaire@qualyst.com [Qualyst, Inc., Durham, NC 27713 (United States); Brouwer, Kim L.R., E-mail: kbrouwer@unc.edu [Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, CB 7569 Kerr Hall, Chapel Hill, NC 27599-7569 (United States)

    2012-05-15

    Sandwich-cultured hepatocytes (SCH) are used commonly to investigate hepatic transport protein-mediated uptake and biliary excretion of substrates. However, little is known about the disposition of endogenous bile acids (BAs) in SCH. In this study, four endogenous conjugated BAs common to rats and humans [taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), and glycochenodeoxycholic acid (GCDCA)], as well as two BA species specific to rodents (α- and β-tauromuricholic acid; α/β TMCA), were profiled in primary rat and human SCH. Using B-CLEAR{sup ®} technology, BAs were measured in cells + bile canaliculi, cells, and medium of SCH by LC-MS/MS. Results indicated that, just as in vivo, taurine-conjugated BA species were predominant in rat SCH, while glycine-conjugated BAs were predominant in human SCH. Total intracellular BAs remained relatively constant over days in culture in rat SCH. Total BAs in control (CTL) cells + bile, cells, and medium were approximately 3.4, 2.9, and 8.3-fold greater in human than in rat. The estimated intracellular concentrations of the measured total BAs were 64.3 ± 5.9 μM in CTL rat and 183 ± 56 μM in CTL human SCH, while medium concentrations of the total BAs measured were 1.16 ± 0.21 μM in CTL rat SCH and 9.61 ± 6.36 μM in CTL human SCH. Treatment of cells for 24 h with 10 μM troglitazone (TRO), an inhibitor of the bile salt export pump (BSEP) and the Na{sup +}-taurocholate cotransporting polypeptide (NTCP), had no significant effect on endogenous BAs measured at the end of the 24-h culture period, potentially due to compensatory mechanisms that maintain BA homeostasis. These data demonstrate that BAs in SCH are similar to in vivo, and that SCH may be a useful in vitro model to study alterations in BA disposition if species differences are taken into account. -- Highlights: ► Bile acids (BAs) were measured in rat and human sandwich-cultured hepatocytes (SCH). ► Cell and medium BA

  2. Self-assembled bionanostructures: proteins following the lead of DNA nanostructures

    OpenAIRE

    Gradišar, Helena; Jerala, Roman

    2014-01-01

    Natural polymers are able to self-assemble into versatile nanostructures based on the information encoded into their primary structure. The structural richness of biopolymer-based nanostructures depends on the information content of building blocks and the available biological machinery to assemble and decode polymers with a defined sequence. Natural polypeptides comprise 20 amino acids with very different properties in comparison to only 4 structurally similar nucleotides, building elements ...

  3. Compositions comprising a polypeptide having cellulolytic enhancing activity and a quinone compound and uses thereof

    Energy Technology Data Exchange (ETDEWEB)

    Quinlan, Jason; Xu, Feng; Sweeney, Matthew

    2017-09-05

    The present invention relates to compositions comprising: a polypeptide having cellulolytic enhancing activity and a quinone compound. The present invention also relates to methods of using the compositions.

  4. Compositions comprising a polypeptide having cellulolytic enhancing activity and a bicycle compound and uses thereof

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Feng; Sweeney, Matthew; Quinlan, Jason

    2015-06-16

    The present invention relates to compositions comprising: a polypeptide having cellulolytic enhancing activity and a bicyclic compound. The present invention also relates to methods of using the compositions.

  5. Compositions comprising a polypeptide having cellulolytic enhancing activity and a bicyclic compound and uses thereof

    Energy Technology Data Exchange (ETDEWEB)

    Quinlan, Jason; Xu, Feng; Sweeney, Matthew

    2016-10-04

    The present invention relates to compositions comprising: a polypeptide having cellulolytic enhancing activity and a bicyclic compound. The present invention also relates to methods of using the compositions.

  6. Compositions comprising a polypeptide having cellulolytic enhancing activity and a heterocyclic compound and uses thereof

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Feng; Sweeney, Matthew; Quinlan, Jason

    2016-08-02

    The present invention relates to compositions comprising: a polypeptide having cellulolytic enhancing activity and a heterocyclic compound. The present invention also relates to methods of using the compositions.

  7. A nonadjuvanted polypeptide nanoparticle vaccine confers long-lasting protection against rodent malaria.

    Science.gov (United States)

    Kaba, Stephen A; Brando, Clara; Guo, Qin; Mittelholzer, Christian; Raman, Senthilkumar; Tropel, David; Aebi, Ueli; Burkhard, Peter; Lanar, David E

    2009-12-01

    We have designed and produced a prototypic malaria vaccine based on a highly versatile self-assembling polypeptide nanoparticle (SAPN) platform that can repetitively display antigenic epitopes. We used this platform to display a tandem repeat of the B cell immunodominant repeat epitope (DPPPPNPN)(2)D of the malaria parasite Plasmodium berghei circumsporozoite protein. Administered in saline, without the need for a heterologous adjuvant, the SAPN construct P4c-Mal conferred a long-lived, protective immune response to mice with a broad range of genetically distinct immune backgrounds including the H-2(b), H-2(d), and H-2(k) alleles. Immunized mice produced a CD4(+) T cell-dependent, high-titer, long-lasting, high-avidity Ab response against the B cell epitope. Mice were protected against an initial challenge of parasites up to 6 mo after the last immunization or for up to 15 mo against a second challenge after an initial challenge of parasites had successfully been cleared. Furthermore, we demonstrate that the SAPN platform not only functions to deliver an ordered repetitive array of B cell peptide epitopes but operates as a classical immunological carrier to provide cognate help to the P4c-Mal-specific B cells.

  8. Measuring the rate of intramolecular contact formation in polypeptides

    Science.gov (United States)

    Lapidus, Lisa; Eaton, William; Hofrichter, James

    2001-03-01

    Formation of a specific contact between two residues of a polypeptide chain is an important elementary process in protein folding. Here we describe a method for studying contact formation between tryptophan and cysteine based on measurements of the lifetime of the tryptophan triplet state. With tryptophan at one end of a flexible peptide and cysteine at the other, the triplet decay rate is identical to the rate of quenching by cysteine and is also close to the diffusion-limited rate of contact formation. The length dependence of this end-to-end contact rate was studied in a series of Cys-(Ala-Gly-Gln)_k-Trp peptides, with k varying from 1 to 6. The rate decreases from -1/(40 ns) for k = 1 to -1/(140 ns) for k = 6, approaching the length-dependence expected for a random coil (n-3/2) for the longest peptides. Comparing the intramolecular and bimolecular quenching rates for cysteine with two disulfide molecules (cystine and lipoate) determines the ratio of the equilibrium constants for forming the intramolecular and bimolecular contact pairs. This ratio depends on the persistence length of the chain and is therefore a measure of the flexibility of the polypeptide.

  9. Polypeptide profiles of South Indian isolate of Trypanosoma evansi.

    Science.gov (United States)

    Sivajothi, S; Rayulu, V C; Bhaskar Reddy, B V; Malakondaiah, P; Sreenivasulu, D; Sudhakara Reddy, B

    2016-09-01

    The field isolates of Trypanosoma evansi was collected from the infected cattle and it was propagated in rats. Trypanosoma evansi parasites were separated from the blood of infected rats by using diethylaminoethyl cellulose column chromatography. Whole cell lysate antigen (WCL) was prepared from purified trypanosomes by ultrasonication and centrifugation. The prepared WCL antigen was further purified by 50 % ammonium sulphate precipitation. Protein concentration of WCL antigen of T. evansi was 60 mg/ml. Protein concentration was adjusted to 1.0 mg/ml in PBS, pH 8.0 and stored at -20(0) C.   Polypeptide profiles of WCL antigen of T. evansi was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. A total of eight polypeptide bands of the size ranging from 25 to 85 kDa in WCL antigen of T. evansi were obtained. Five prominent bands with molecular weight of 74, 60, 53, 42 and 37 kDa and three light bands with molecular weight of 85, 34 and 25 kDa were observed.

  10. Mechanically Controlled Electron Transfer in a Single-Polypeptide Transistor

    Science.gov (United States)

    Sheu, Sheh-Yi; Yang, Dah-Yen

    2017-01-01

    Proteins are of interest in nano-bio electronic devices due to their versatile structures, exquisite functionality and specificity. However, quantum transport measurements produce conflicting results due to technical limitations whereby it is difficult to precisely determine molecular orientation, the nature of the moieties, the presence of the surroundings and the temperature; in such circumstances a better understanding of the protein electron transfer (ET) pathway and the mechanism remains a considerable challenge. Here, we report an approach to mechanically drive polypeptide flip-flop motion to achieve a logic gate with ON and OFF states during protein ET. We have calculated the transmission spectra of the peptide-based molecular junctions and observed the hallmarks of electrical current and conductance. The results indicate that peptide ET follows an NC asymmetric process and depends on the amino acid chirality and α-helical handedness. Electron transmission decreases as the number of water molecules increases, and the ET efficiency and its pathway depend on the type of water-bridged H-bonds. Our results provide a rational mechanism for peptide ET and new perspectives on polypeptides as potential candidates in logic nano devices.

  11. Prediction of the absolute aggregation rates of amyloidogenic polypeptide chains.

    Science.gov (United States)

    DuBay, Kateri F; Pawar, Amol P; Chiti, Fabrizio; Zurdo, Jesús; Dobson, Christopher M; Vendruscolo, Michele

    2004-08-27

    Protein aggregation is associated with a variety of pathological conditions, including Alzheimer's and Creutzfeldt-Jakob diseases and type II diabetes. Such degenerative disorders result from the conversion of the normal soluble state of specific proteins into aggregated states that can ultimately form the characteristic amyloid fibrils found in diseased tissue. Under appropriate conditions it appears that many, perhaps all, proteins can be converted in vitro into amyloid fibrils. The aggregation propensities of different polypeptide chains have, however, been observed to vary substantially. Here, we describe an approach that uses the knowledge of the amino acid sequence and of the experimental conditions to reproduce, with a correlation coefficient of 0.92 and over five orders of magnitude, the in vitro aggregation rates of a wide range of unstructured peptides and proteins. These results indicate that the formation of protein aggregates can be rationalised to a considerable extent in terms of simple physico-chemical parameters that describe the properties of polypeptide chains and their environment.

  12. Uncharged Helical Modular Polypeptide Hydrogels for Cellular Scaffolds.

    Science.gov (United States)

    Ahrens, Caroline C; Welch, M Elizabeth; Griffith, Linda G; Hammond, Paula T

    2015-12-14

    Grafted synthetic polypeptides hold appeal for extending the range of biophysical properties achievable in synthetic extracellular matrix (ECM) hydrogels. Here, N-carboxyanhydride polypeptide, poly(γ-propargyl-l-glutamate) (PPLG) macromers were generated by fully grafting the "clickable" side chains with mixtures of short polyethylene glycol (PEG) chains terminated with inert (-OH) or reactive (maleimide and/or norbornene) groups, then reacting a fraction of these groups with an RGD cell attachment motif. A panel of synthetic hydrogels was then created by cross-linking the PPLG macromers with a 4-arm PEG star molecule. Compared to well-established PEG-only hydrogels, gels containing PPLG exhibited dramatically less dependence on swelling as a function of cross-link density. Further, PPLG-containing gels, which retain an α-helical chain conformation, were more effective than standard PEG gels in fostering attachment of a human mesenchymal stem cell (hMSC) line for a given concentration of RGD in the gel. These favorable properties of PPLG-containing PEG hydrogels suggest they may find broad use in synthetic ECM.

  13. CDNA encoding a polypeptide including a hevein sequence

    Science.gov (United States)

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

    1995-03-21

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

  14. Alternative Cell Sources to Adult Hepatocytes for Hepatic Cell Therapy.

    Science.gov (United States)

    Pareja, Eugenia; Gómez-Lechón, María José; Tolosa, Laia

    2017-01-01

    Adult hepatocyte transplantation is limited by scarce availability of suitable donor liver tissue for hepatocyte isolation. New cell-based therapies are being developed to supplement whole-organ liver transplantation, to reduce the waiting-list mortality rate, and to obtain more sustained and significant metabolic correction. Fetal livers and unsuitable neonatal livers for organ transplantation have been proposed as potential useful sources of hepatic cells for cell therapy. However, the major challenge is to use alternative cell sources for transplantation that can be derived from reproducible methods. Different types of stem cells with hepatic differentiation potential are eligible for generating large numbers of functional hepatocytes for liver cell therapy to treat degenerative disorders, inborn hepatic metabolic diseases, and organ failure. Clinical trials are designed to fully establish the safety profile of such therapies and to define target patient groups and standardized protocols.

  15. Protective effect of liver ischemic preconditioning on rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Ischemic preconditioning (IPC) protects liver graft function following ischemia in liver transplantation and liver resection. The aim of this study was to assess the advantages and any potential disadvantages of liver IPC prior to isolation for rat hepatocytes during isolation and cryopreservation. After isolating and thawing the cryopreserved hepatocytes after 14 and 28 d,cell viability,efficiency,and lactate dehydrogenase (LDH) levels in preserve solution were examined for every group. Groups treated with IPC had better cell viability determination,assessment of plating efficiency and lactate dehydrogenase (LDH) assay than Group without IPC,suggesting that IPC prior to isolation may have a significant protective effect on hepatocytes subjected to isolation and short period cryopreservation.

  16. Acetaminophen metabolism, cytotoxicity, and genotoxicity in rat primary hepatocyte cultures

    Energy Technology Data Exchange (ETDEWEB)

    Milam, K.M.; Byard, J.L.

    1985-06-30

    Acetaminophen (APAP) metabolism, cytotoxicity, and genotoxicity were measured in primary cultures of rat hepatocytes. Although 3 mM APAP caused a slight increase in cellular release of lactate dehydrogenase into the culture medium, cellular glutathione concentration (an index of APAP metabolism) was reduced by 50%. APAP at 7 mM was significantly more toxic to these hepatocytes and had a similar but more marked effect on glutathione concentrations. In spite of its cytotoxicity, neither dose of APAP stimulated DNA repair synthesis when monitored by the rate of incorporation of (/sup 3/H)thymidine into DNA following exposure to APAP. Thus, although APAP has been shown to be both hepato- and nephrotoxic in several in vivo and in vitro systems, the reactive toxic metabolite of APAP is not genotoxic in rat primary hepatocyte cultures.

  17. Early membrane depressions in hepatocytes cultured on Primaria supports.

    Science.gov (United States)

    Griffiths, B J; Evans, P J

    2001-01-01

    The topography of spreading hepatocytes on positively charged Primaria plates was examined using scanning electron microscopy. The cells acquired a uniform morphology within 2 h. The spreading was rapid and the surface of the cells showed early prominent depressions or dips. The hepatocytes had either one or two of these structures which corresponded to the frequency of mononuclear and binuclear cells, respectively. The nuclear origin of the dips was strengthened after 6 h. They contained solid structures, whose number, size and shape were the same as nucleoli. The membrane dipping was independent of cell density and took place under conditions where phenotypic changes can occur. Kidney proximal tubule cells had no dips. Co-cultures of hepatocytes and kidney proximal tubule cells showed that the cell types behave differently.

  18. Identification of a novel human islet amyloid polypeptide beta-sheet domain and factors influencing fibrillogenesis.

    Science.gov (United States)

    Jaikaran, E T; Higham, C E; Serpell, L C; Zurdo, J; Gross, M; Clark, A; Fraser, P E

    2001-05-04

    Human islet amyloid polypeptide (hIAPP) accumulates as pancreatic amyloid in type 2 diabetes and readily forms fibrils in vitro. Investigations into the mechanism of hIAPP fibril formation have focused largely on residues 20 to 29, which are considered to comprise a primary amyloidogenic domain. In rodents, proline substitutions within this region and the subsequent beta-sheet disruption, prevents fibril formation. An additional amyloidogenic fragment within the C-terminal sequence, residues 30 to 37, has been identified recently. We have extended these observations by examining a series of overlapping peptide fragments from the human and rodent sequences. Using protein spectroscopy (CD/FTIR), electron microscopy and X-ray diffraction, a previously unrecognised amyloidogenic domain was localised within residues 8 to 20. Synthetic peptides corresponding to this region exhibited a transition from random coil to beta-sheet conformation and assembled into fibrils having a typical amyloid-like morphology. The comparable rat 8-20 sequence, which contains a single His18Arg substitution, was also capable of assembling into amyloid-like fibrils. Examination of peptide fragments corresponding to residues 1 to 13 revealed that the immediate N-terminal region is likely to have only a modulating influence on fibril formation or conformational conversion. The contributions of charged residues as they relate to the amyloid-forming 8-20 sequence were also investigated using IAPP fragments and by assessing the effects of pH and counterions. The identification of these principal amyloidogenic sequences and the effects of associated factors provide details on the IAPP aggregation pathway and structure of the peptide in its fibrillar state. Copyright 2001 Academic Press.

  19. Morphological and functional behaviors of rat hepatocytes cultured on single-walled carbon nanotubes.

    Science.gov (United States)

    Koga, Haruka; Fujigaya, Tsuyohiko; Nakashima, Naotoshi; Nakazawa, Kohji

    2011-09-01

    This study describes the morphological and functional behaviors of rat hepatocytes on single-walled carbon nanotube (CNT)-coated surfaces. Although the hydrophobic characteristics of CNT-coated surfaces increased with increasing CNT density, hepatocyte adhesion decreased, indicating that the interaction between hepatocytes and CNTs is weak. We found that hepatocytes on a CNT-coated surface gradually gather together and form spheroids (spherical multicellular aggregates). These spheroids exhibit compact spherical morphology with a smooth surface and express connexin-32, an intracellular communication molecule. In contrast, collagen treatment in conjunction with the CNT-coated surface improved hepatocyte adhesion, and the cells maintained a monolayer configuration throughout the culture period. The albumin secretion and ammonia removal activities of hepatocyte spheroids were maintained at elevated levels for at least 15 days of culturing as compared with hepatocyte monolayers. These results indicate that CNTs can be used for the formation and long-term culture of hepatocyte spheroids.

  20. In vivo hepatocyte MR imaging using lactose functionalized magnetoliposomes.

    Science.gov (United States)

    Ketkar-Atre, Ashwini; Struys, Tom; Dresselaers, Tom; Hodenius, Michael; Mannaerts, Inge; Ni, Yicheng; Lambrichts, Ivo; Van Grunsven, Leo A; De Cuyper, Marcel; Himmelreich, Uwe

    2014-01-01

    The aim of this study was to assess a novel lactose functionalized magnetoliposomes (MLs) as an MR contrast agent to target hepatocytes as well as to evaluate the targeting ability of MLs for in vivo applications. In the present work, 17 nm sized iron oxide cores functionalized with anionic MLs bearing lactose moieties were used for targeting the asialoglycoprotein receptor (ASGP-r), which is highly expressed in hepatocytes. Non-functionalized anionic MLs were tested as negative controls. The size distribution of lactose and anionic MLs was determined by transmission electron microscopy (TEM) and dynamic light scattering (DLS). After intravenous administration of both MLs, contrast enhancement in the liver was observed by magnetic resonance imaging (MRI). Label retention was monitored non-invasively by MRI and validated with Prussian blue staining and TEM for up to eight days post MLs administration. Although the MRI signal intensity did not show significant differences between functionalized and non-functionalized particles, iron-specific Prussian blue staining and TEM analysis confirmed the uptake of lactose MLs mainly in hepatocytes. In contrast, non-functionalized anionic MLs were mainly taken up by Kupffer and sinusoidal cells. Target specificity was further confirmed by high-resolution MR imaging of phantoms containing isolated hepatocytes, Kupffer cell (KCs) and hepatic stellate cells (HSCs) fractions. Hypointense signal was observed for hepatocytes isolated from animals which received lactose MLs but not from animals which received anionic MLs. These data demonstrate that galactose-functionalized MLs can be used as a hepatocyte targeting MR contrast agent to potentially aid in the diagnosis of hepatic diseases if the non-specific uptake by KCs is taken into account.

  1. Intrasplenic transplantation of allogeneic hepatocytes prolongs survival in anhepatic rats.

    Science.gov (United States)

    Arkadopoulos, N; Lilja, H; Suh, K S; Demetriou, A A; Rozga, J

    1998-11-01

    To examine whether hepatocytes transplanted in the spleen can function as an ectopic liver, we performed hepatocyte transplantation in rats that were rendered anhepatic. Total hepatectomy was performed by using a novel single-stage technique. Following hepatectomy, Group 1 rats (n = 16) were monitored until death to determine survival time without prior intervention. Group 2 anhepatic rats (n = 20) were sacrificed at various times to measure blood hepatocyte growth factor (HGF) and transforming growth factor beta1 (TGF-beta1) levels. Group 3 (n = 16) rats received intrasplenic injection of isolated hepatocytes (2.5 x 10(7) cells/rat) followed by total hepatectomy after 3 days. Group 4 (n = 12) sham-transplanted rats received intrasplenic saline infusion, and after 3 days they were rendered anhepatic. Group 2, 3, and 4 rats were maintained on daily Cyclosporine A (10 mg/kg; intramuscularly). Group 1 anhepatic rats survived for 22.4 +/- 5.2 hours (standard deviation). The anhepatic state was associated with a progressive and statistically significant rise in blood HGF and TGF-beta1 levels. Rats that received hepatocyte transplantation before total hepatectomy had a significantly longer survival time than sham-transplanted anhepatic controls (34.1 +/- 8.5 vs. 15.5 +/- 4.8 hrs, P ammonia, prothrombin time, international normalized ratio, and TGF-beta1 levels when compared with sham-transplanted controls. In conclusion, intrasplenic transplantation of allogeneic hepatocytes prolonged survival, improved blood chemistry, and lowered blood TGF-beta1 levels in rats rendered anhepatic.

  2. Hepatocyte autophagy model established by physical method

    Directory of Open Access Journals (Sweden)

    ZHU Xuemin

    2016-08-01

    Full Text Available ObjectiveTo establish the autophagy model of normal human liver cell line 7702 induced by hypoxia and starvation, and to lay a foundation for further studies on the influence of autophagy on liver function. MethodsThe 7702 cells were selected and incubated with 95% air and 5% CO2 at a temperature of 37 ℃(normal control group. The Binder three-gas incubator was used, with a temperature of 37 ℃, a CO2 concentration of 5%, and an O2 concentration of 0.3% to provide a hypoxic environment, and the serum-free DMEM was used to induce starvation. These cells were divided into 6-, 12-, 18-, and 24-hour hypoxia-starvation groups. Western blot was used to measure the protein expression of Beclin 1, Atg5, and LC3 in the normal control group and experimental groups, RT-qPCR was used to measure the mRNA expression of Beclin 1 and Atg5 in each group, and after transfection of LC3 plasmid, immunofluorescence assay was used to observe autophagy in each group. An analysis of variance was used for comparison of continuous data between groups, and the least significant difference t-test was used for further comparison between any two groups; the chi-square test was used for comparison of categorical data between groups. ResultsThe 6-hour hypoxia-starvation groups had higher protein expression of Beclin 1, Atg5, and LC3 than the normal control group or other treated groups. Compared with all the other groups, the 6-hour hypoxia-starvation group showed significantly increased mRNA expression of Beclin 1 and Atg5, as well as significantly greater increases in the mRNA expression of Beclin 1 and Atg5 (all P<0.05. The hypoxia-starvation groups had significantly lower numbers of autophagosomes than the normal control group, and the 6-hour hypoxia-starvation group had the highest number of autophagosomes (all P<0.05. ConclusionHypoxia and starvation established by physical methods can successfully induce hepatocyte autophagy, which is the most remarkable at 6

  3. No evidence for protective erythropoietin alpha signalling in rat hepatocytes

    Directory of Open Access Journals (Sweden)

    Frede Stilla

    2009-04-01

    Full Text Available Abstract Background Recombinant human erythropoietin alpha (rHu-EPO has been reported to protect the liver of rats and mice from ischemia-reperfusion injury. However, direct protective effects of rHu-EPO on hepatocytes and the responsible signalling pathways have not yet been described. The aim of the present work was to study the protective effect of rHu-EPO on warm hypoxia-reoxygenation and cold-induced injury to hepatocytes and the rHu-EPO-dependent signalling involved. Methods Loss of viability of isolated rat hepatocytes subjected to hypoxia/reoxygenation or incubated at 4°C followed by rewarming was determined from released lactate dehydrogenase activity in the absence and presence of rHu-EPO (0.2–100 U/ml. Apoptotic nuclear morphology was assessed by fluorescence microscopy using the nuclear fluorophores H33342 and propidium iodide. Erythropoietin receptor (EPOR, EPO and Bcl-2 mRNAs were quantified by real time PCR. Activation of JAK-2, STAT-3 and STAT-5 in hepatocytes and rat livers perfused in situ was assessed by Western blotting. Results In contrast to previous in vivo studies on ischemia-reperfusion injury to the liver, rHu-EPO was without any protective effect on hypoxic injury, hypoxia-reoxygenation injury and cold-induced apoptosis to isolated cultured rat hepatocytes. EPOR mRNA was identified in these cells but specific detection of the EPO receptor protein was not possible due to the lack of antibody specificity. Both, in the cultured rat hepatocytes (10 U/ml for 15 minutes and in the rat liver perfused in situ with rHu-EPO (8.9 U/ml for 15 minutes no evidence for EPO-dependent signalling was found as indicated by missing effects of rHu-EPO on phosphorylation of JAK-2, STAT-3 and STAT-5 and on the induction of Bcl-2 mRNA. Conclusion Together, these results indicate the absence of any protective EPO signalling in rat hepatocytes. This implies that the protection provided by rHu-EPO in vivo against ischemia-reperfusion and

  4. Dump assembly

    Science.gov (United States)

    Goldmann, Louis H.

    1986-01-01

    A dump assembly having a fixed conduit and a rotatable conduit provided with overlapping plates, respectively, at their adjacent ends. The plates are formed with openings, respectively, normally offset from each other to block flow. The other end of the rotatable conduit is provided with means for securing the open end of a filled container thereto. Rotation of the rotatable conduit raises and inverts the container to empty the contents while concurrently aligning the conduit openings to permit flow of material therethrough.

  5. A new splice variant of the major subunit of human asialoglycoprotein receptor encodes a secreted form in hepatocytes.

    Directory of Open Access Journals (Sweden)

    Jia Liu

    Full Text Available BACKGROUND: The human asialoglycoprotein receptor (ASGPR is composed of two polypeptides, designated H1 and H2. While variants of H2 have been known for decades, the existence of H1 variants has never been reported. PRINCIPAL FINDINGS: We identified two splice variants of ASGPR H1 transcripts, designated H1a and H1b, in human liver tissues and hepatoma cells. Molecular cloning of ASGPR H1 variants revealed that they differ by a 117 nucleotide segment corresponding to exon 2 in the ASGPR genomic sequence. Thus, ASGPR variant H1b transcript encodes a protein lacking the transmembrane domain. Using an H1b-specific antibody, H1b protein and a functional soluble ASGPR (sASGPR composed of H1b and H2 in human sera and in hepatoma cell culture supernatant were identified. The expression of ASGPR H1a and H1b in Hela cells demonstrated the different cellular loctions of H1a and H1b proteins at cellular membranes and in intracellular compartments, respectively. In vitro binding assays using fluorescence-labeled sASGPR or the substract ASOR revealed that the presence of sASGPR reduced the binding of ASOR to cells. However, ASOR itself was able to enhance the binding of sASGPR to cells expressing membrane-bound ASGPR. Further, H1b expression is reduced in liver tissues from patients with viral hepatitis. CONCLUSIONS: We conclude that two naturally occurring ASGPR H1 splice variants are produced in human hepatocytes. A hetero-oligomeric complex sASGPR consists of the secreted form of H1 and H2 and may bind to free substrates in circulation and carry them to liver tissue for uptake by ASGPR-expressing hepatocytes.

  6. General Assembly

    CERN Multimedia

    Staff Association

    2016-01-01

    5th April, 2016 – Ordinary General Assembly of the Staff Association! In the first semester of each year, the Staff Association (SA) invites its members to attend and participate in the Ordinary General Assembly (OGA). This year the OGA will be held on Tuesday, April 5th 2016 from 11:00 to 12:00 in BE Auditorium, Meyrin (6-2-024). During the Ordinary General Assembly, the activity and financial reports of the SA are presented and submitted for approval to the members. This is the occasion to get a global view on the activities of the SA, its financial management, and an opportunity to express one’s opinion, including taking part in the votes. Other points are listed on the agenda, as proposed by the Staff Council. Who can vote? Only “ordinary” members (MPE) of the SA can vote. Associated members (MPA) of the SA and/or affiliated pensioners have a right to vote on those topics that are of direct interest to them. Who can give his/her opinion? The Ordinary General Asse...

  7. Non-viral FoxM1 gene delivery to hepatocytes enhances liver repopulation.

    Science.gov (United States)

    Xiang, D; Liu, C-C; Wang, M-J; Li, J-X; Chen, F; Yao, H; Yu, B; Lu, L; Borjigin, U; Chen, Y-X; Zhong, L; Wangensteen, K J; He, Z-Y; Wang, X; Hu, Y-P

    2014-05-22

    Hepatocyte transplantation as a substitute strategy of orthotopic liver transplantation is being studied for treating end-stage liver diseases. Several technical hurdles must be overcome in order to achieve the therapeutic liver repopulation, such as the problem of insufficient expansion of the transplanted hepatocytes in recipient livers. In this study, we analyzed the application of FoxM1, a cell-cycle regulator, to enhance the proliferation capacity of hepatocytes. The non-viral sleeping beauty (SB) transposon vector carrying FoxM1 gene was constructed for delivering FoxM1 into the hepatocytes. The proliferation capacities of hepatocytes with FoxM1 expression were examined both in vivo and in vitro. Results indicated that the hepatocytes with FoxM1 expression had a higher proliferation rate than wild-type (WT) hepatocytes in vitro. In comparison with WT hepatocytes, the hepatocytes with FoxM1 expression had an enhanced level of liver repopulation in the recipient livers at both sub-acute injury (fumaryl acetoacetate hydrolase (Fah)(-/-) mice model) and acute injury (2/3 partial hepatectomy mice model). Importantly, there was no increased risk of tumorigenicity with FoxM1 expression in recipients even after serial transplantation. In conclusion, expression of FoxM1 in hepatocytes enhanced the capacity of liver repopulation without inducing tumorigenesis. FoxM1 gene delivered by non-viral SB vector into hepatocytes may be a viable approach to promote therapeutic repopulation after hepatocyte transplantation.

  8. Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration.

    Science.gov (United States)

    Malato, Yann; Naqvi, Syed; Schürmann, Nina; Ng, Raymond; Wang, Bruce; Zape, Joan; Kay, Mark A; Grimm, Dirk; Willenbring, Holger

    2011-12-01

    Recent evidence has contradicted the prevailing view that homeostasis and regeneration of the adult liver are mediated by self duplication of lineage-restricted hepatocytes and biliary epithelial cells. These new data suggest that liver progenitor cells do not function solely as a backup system in chronic liver injury; rather, they also produce hepatocytes after acute injury and are in fact the main source of new hepatocytes during normal hepatocyte turnover. In addition, other evidence suggests that hepatocytes are capable of lineage conversion, acting as precursors of biliary epithelial cells during biliary injury. To test these concepts, we generated a hepatocyte fate-tracing model based on timed and specific Cre recombinase expression and marker gene activation in all hepatocytes of adult Rosa26 reporter mice with an adenoassociated viral vector. We found that newly formed hepatocytes derived from preexisting hepatocytes in the normal liver and that liver progenitor cells contributed minimally to acute hepatocyte regeneration. Further, we found no evidence that biliary injury induced conversion of hepatocytes into biliary epithelial cells. These results therefore restore the previously prevailing paradigms of liver homeostasis and regeneration. In addition, our new vector system will be a valuable tool for timed, efficient, and specific loop out of floxed sequences in hepatocytes.

  9. In vitro proliferation of primary rat hepatocytes expressing ureogenesis activity by coculture with STO cells.

    Science.gov (United States)

    Takagi, Mutsumi; Kondo, Hiroki; Yoshida, Toshiomi

    2002-01-01

    A coculture system for in vitro proliferation of rat primary hepatocytes with feeder cells was investigated with the view of constructing a hybrid artificial liver module using human hepatocytes. A mouse cell line, STO, was apparently effective compared with other kinds of feeder cells such as FLS5 and MRC5 in increasing the total cell number in the coculture of rat primary hepatocytes. The parenchymal hepatocyte, which are polygonal cells, spread well as the cultivation progressed. Monitoring of parenchymal hepatocyte colonies under a microscope showed that the area of each colony increased as the single culture and cocultures progressed. The adhesion area per hepatocyte increased little during the coculture with STO cells, while the adhesion area increased markedly in the single culture of hepatocytes. The density of hepatocytes increased more than two times during the coculture for 5 d, while it decreased during the single culture. The production rate of urea by hepatocytes markedly increased during the coculture, while the rate in the single culture was lower than the coculture and increased only slightly. The specific rate of urea production by hepatocytes in the coculture did not decrease even as the hepatocytes grew. Consequently, rat primary hepatocytes could proliferate in vitro by coculture with STO cells without a decrease in urea production activity.

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

    KAUST Repository

    Pahovnik, David

    2015-09-01

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

  11. Thymus Polypeptide Preparation Tactivin Restores Learning and Memory in Thymectomied Rats.

    Science.gov (United States)

    Novoseletskaya, A V; Kiseleva, N M; Zimina, I V; Bystrova, O V; Belova, O V; Inozemtsev, A N; Arion, V Ya; Sergienko, V I

    2015-09-01

    We studied the effects of tactivin and splenic polypeptides on learning and memory of thymectomized animals. In 3-week rats, thymectomy blocked active avoidance conditioning. Injections of tactivin (0.5 mg/kg) during 1 month after surgery restored learning capacity; splenic polypeptides were ineffective.

  12. Directed evolution methods for improving polypeptide folding and solubility and superfolder fluorescent proteins generated thereby

    Science.gov (United States)

    Waldo, Geoffrey S.

    2007-09-18

    The current invention provides methods of improving folding of polypeptides using a poorly folding domain as a component of a fusion protein comprising the poorly folding domain and a polypeptide of interest to be improved. The invention also provides novel green fluorescent proteins (GFPs) and red fluorescent proteins that have enhanced folding properties.

  13. Polypeptide synthesis in alphavirus-infected Aedes albopictus cells during the establishment of persistent infection.

    Science.gov (United States)

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

    1980-01-01

    Polypeptide synthesis was examined in mosquito cells during the establishment of a persistent infection with two alphaviruses, Ross River virus (RRV) and Semliki Forest virus (SFV), and in vertebrate cells cytopathically-infected with the same viruses. In Aedes albopictus cell, RRV reached peak titres at 34--48 hours p.i. At 12 hours 85 per cent of cells assayed as infected by infective centre assay; by 48 hours when persistence was established, virus production was reduced and less than 5 per cent of cells assayed as infected. There was no shut-down of host polypeptide synthesis during infection. Viral polypeptide synthesis was maximal between 10 and 24 hours p.i. The major viral polypeptides labelled were nucleocapsid protein and envelope protein(s). The precursor polypeptide p95 which was prominent in infected BHK cells was not detected in mosquito cells. Similar results were obtained on SFV infection. During the establishment of persistence there was a coordinate decline in the synthesis of RRV polypeptides, reaching undetectable levels by 72 hours p.i. Subculturing persitently-infected cells led to a small increase in viral polypeptide synthesis and virus titre. In contrast, during RRV growth in BHK celos host protein synthesis was severly inhibited and by 9--11 hours p.i. virus-specific polypeptide synthesis represented more than 90 per cent of total protein synthetic activity.

  14. Ab initio study of alanine polypeptide chains twisting

    CERN Document Server

    Solovyov, I A; Solovyov, A V; Yakubovitch, A V; Greiner, Walter; Solov'yov, Andrey V.; Solov'yov, Ilia A.; Yakubovitch, Alexander V.

    2005-01-01

    We have investigated the potential energy surfaces for alanine chains consisting of three and six amino acids. For these molecules we have calculated potential energy surfaces as a function of the Ramachandran angles Phi and Psi, which are widely used for the characterization of the polypeptide chains. These particular degrees of freedom are essential for the characterization of proteins folding process. Calculations have been carried out within ab initio theoretical framework based on the density functional theory and accounting for all the electrons in the system. We have determined stable conformations and calculated the energy barriers for transitions between them. Using a thermodynamic approach, we have estimated the times of characteristic transitions between these conformations. The results of our calculations have been compared with those obtained by other theoretical methods and with the available experimental data extracted from the Protein Data Base. This comparison demonstrates a reasonable corres...

  15. Vasoactive intestinal polypeptide (VIP) in the pig pancreas

    DEFF Research Database (Denmark)

    Poulsen, Steen Seier

    1984-01-01

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

  16. Asialoglycoprotein receptor mediated hepatocyte targeting - strategies and applications.

    Science.gov (United States)

    D'Souza, Anisha A; Devarajan, Padma V

    2015-04-10

    Hepatocyte resident afflictions continue to affect the human population unabated. The asialoglycoprotein receptor (ASGPR) is primarily expressed on hepatocytes and minimally on extra-hepatic cells. This makes it specifically attractive for receptor-mediated drug delivery with minimum concerns of toxicity. ASGPR facilitates internalization by clathrin-mediated endocytosis and exhibits high affinity for carbohydrates specifically galactose, N-acetylgalactosamine and glucose. Isomeric forms of sugar, galactose density and branching, spatial geometry and galactose linkages are key factors influencing ligand-receptor binding. Popular ligands for ASGPR mediated targeting are carbohydrate polymers, arabinogalactan and pullulan. Other ligands include galactose-bearing glycoproteins, glycopeptides and galactose modified polymers and lipids. Drug-ligand conjugates provide a viable strategy; nevertheless ligand-anchored nanocarriers provide an attractive option for ASGPR targeted delivery and are widely explored. The present review details various ligands and nanocarriers exploited for ASGPR mediated delivery of drugs to hepatocytes. Nanocarrier properties affecting ASGPR mediated uptake are discussed at length. The review also highlights the clinical relevance of ASGPR mediated targeting and applications in diagnostics. ASGPR mediated hepatocyte targeting provides great promise for improved therapy of hepatic afflictions.

  17. Stem cell-derived hepatocytes and their use in toxicology.

    Science.gov (United States)

    Guguen-Guillouzo, Christiane; Corlu, Anne; Guillouzo, Andre

    2010-03-30

    Better prediction of safety risk and understanding of mechanism of action of drug candidates remain a major challenge in order to prevent late stage attrition. Continuous efforts are made to improve and develop new models, especially in some areas such as hepatotoxicity. Besides primary hepatocytes and transformed liver cell lines, stem cells either isolated from embryos or adult tissues or obtained by reprogramming somatic cells are emerging as a new potential source of unlimited numbers of hepatocytes. Presently, only hepatocyte-like cells expressing low levels of liver-specific markers, especially drug metabolizing and detoxifying enzymes, are usually obtained, making them still unsuitable as metabolically competent cells for toxicity studies. The only exceptions are some hepatoma cell lines, particularly the HepaRG cell line that can differentiate from a bipotent progenitor stage to attain the functional capacity of normal adult hepatocytes in primary culture without losing the indefinite growth property of transformed cells. Since the research field on stem cells is growing fast marked advances might be expected in the next future.

  18. Metformin stimulates FGF21 expression in primary hepatocytes

    DEFF Research Database (Denmark)

    Nygaard, Eva B; Vienberg, Sara G; Ørskov, Cathrine

    2012-01-01

    incubated with Compound C (an AMPK inhibitor) in the presence of metformin. A strong dose-dependent increase in FGF21 expression was observed in both rat and human hepatocytes treated with metformin. This effect was blocked by addition of the AMPK-inhibitor Compound C. The study shows that metformin...

  19. The use of pig hepatocytes for biotransformation and toxicity studies

    NARCIS (Netherlands)

    Hoogenboom, L.A.P.

    1991-01-01

    The three main objectives of this study were, (1) to investigate the possibility to isolate viable hepatocytes from liver samples of pigs, (2) to study their use for biotransformation and toxicity studies, and (3) to demonstrate the value of this model, in particular in the field of residue

  20. The use of pig hepatocytes for biotransformation and toxicity studies.

    NARCIS (Netherlands)

    Hoogenboom, L.A.P.

    1991-01-01

    The three main objectives of this study were, (1) to investigate the possibility to isolate viable hepatocytes from liver samples of pigs, (2) to study their use for biotransformation and toxicity studies, and (3) to demonstrate the value of this model, in particular in the field of residue toxicolo

  1. 3D Cultivation Techniques for Primary Human Hepatocytes

    Directory of Open Access Journals (Sweden)

    Anastasia Bachmann

    2015-02-01

    Full Text Available One of the main challenges in drug development is the prediction of in vivo toxicity based on in vitro data. The standard cultivation system for primary human hepatocytes is based on monolayer cultures, even if it is known that these conditions result in a loss of hepatocyte morphology and of liver-specific functions, such as drug-metabolizing enzymes and transporters. As it has been demonstrated that hepatocytes embedded between two sheets of collagen maintain their function, various hydrogels and scaffolds for the 3D cultivation of hepatocytes have been developed. To further improve or maintain hepatic functions, 3D cultivation has been combined with perfusion. In this manuscript, we discuss the benefits and drawbacks of different 3D microfluidic devices. For most systems that are currently available, the main issues are the requirement of large cell numbers, the low throughput, and expensive equipment, which render these devices unattractive for research and the drug-developing industry. A higher acceptance of these devices could be achieved by their simplification and their compatibility with high-throughput, as both aspects are of major importance for a user-friendly device.

  2. Mesenchymal stem cells support hepatocyte function in engineered liver grafts.

    Science.gov (United States)

    Kadota, Yoshie; Yagi, Hiroshi; Inomata, Kenta; Matsubara, Kentaro; Hibi, Taizo; Abe, Yuta; Kitago, Minoru; Shinoda, Masahiro; Obara, Hideaki; Itano, Osamu; Kitagawa, Yuko

    2014-01-01

    Recent studies suggest that organ decellularization is a promising approach to facilitate the clinical application of regenerative therapy by providing a platform for organ engineering. This unique strategy uses native matrices to act as a reservoir for the functional cells which may show therapeutic potential when implanted into the body. Appropriate cell sources for artificial livers have been debated for some time. The desired cell type in artificial livers is primary hepatocytes, but in addition, other supportive cells may facilitate this stem cell technology. In this context, the use of mesenchymal stem cells (MSC) is an option meeting the criteria for therapeutic organ engineering. Ideally, supportive cells are required to (1) reduce the hepatic cell mass needed in an engineered liver by enhancing hepatocyte function, (2) modulate hepatic regeneration in a paracrine fashion or by direct contact, and (3) enhance the preservability of parenchymal cells during storage. Here, we describe enhanced hepatic function achieved using a strategy of sequential infusion of cells and illustrate the advantages of co-cultivating bone marrow-derived MSCs with primary hepatocytes in the engineered whole-liver scaffold. These co-recellularized liver scaffolds colonized by MSCs and hepatocytes were transplanted into live animals. After blood flow was established, we show that expression of adhesion molecules and proangiogenic factors was upregulated in the graft.

  3. Inhibitory effect of picroside Ⅱ on hepatocyte apoptosis

    Institute of Scientific and Technical Information of China (English)

    Hua GAO; Ya-wei ZHOU

    2005-01-01

    Aim: To investigate the influence of picroside Ⅱ on hepatocyte apoptosis and its mechanism. Methods: Morphological changes and quantification of apoptotic cells were determined under transmission electron microscopy and flow cytometry respectively. DNA fragmentation was visualized by agarose gel electrophoresis.Semi-quantitative reverse transcription-PCR (RT-PCR) was used to analyze the expression of bcl-2 and bax genes. The content of manganese-superoxide dismutase (SOD) in liver mitochondria was detected by the Marland method. The content of malonic aldehyde (MDA) and the protein level in liver tissue were determined by thiobarbituric acid colorimetry and Lowry method. Results:Picroside Ⅱ decreased the levels of alanine aminotransferase and aspartate aminotransferase in the serum resulting from acute-liver injured mice induced with D-GalN and LPS; it also reduced the content of MDA, and thus, enhanced the activity of SOD. Picroside Ⅱ 10 mg/kg was found to protect hepatocytes against apoptosis in a dose-dependent manner; it up-regulated the expression of bcl-2 genes,thus increased the bcl-2/bax ratio. Conclusion: Picroside Ⅱ can protect hepatocytes against injury and prevent hepatocytes from apoptosis. It might by upregulating the bcl-2 gene expression and antioxidation.

  4. Effect of matrine on primary human hepatocytes in vitro.

    Science.gov (United States)

    Gong, Xiaobing; Gao, Yuan; Guo, Guoqing; Vondran, Florian W R; Schwartlander, Ruth; Efimova, Ekaterina; Pless, Gesine; Sauera, Igor M; Neuhaus, Peter

    2015-03-01

    Matrine is a bioactive component of the traditional Chinese medical herb Sophora flavescens that has been used in China to treat various kinds of diseases including virus hepatitis. However, the molecular mechanisms underlying its hepatoprotective effects remains elusive. In the present study, primary human hepatocytes were employed to elucidate the protective effects and molecular mechanisms of matrine. We observed that low concentrations of matrine had no significant impact on albumin secretion, but high concentrations (>140 mg/L) of matrine decreased the albumin secretion in hepatocytes. Western blot data indicated that matrine at 140 mg/L at 72 h induced protein expression of CYP2A6, CYP2B6 and CYP3A4. Furthermore, high concentrations of matrine reduced LDH and AST levels and were cytotoxic to hepatocytes, leading to a decreased cell viability and total protein amount. Moreover, low concentrations of matrine, enhanced the ECOD activity and decreased the level of NO2 (-) induced by cytokines in human hepatocytes. Taken together, the present study sheds novel light on the molecular mechanisms of matrine and potential application of matrine in hepatic diseases.

  5. Transplantation of human hepatocytes into tolerized genetically immunocompetent rats

    Institute of Scientific and Technical Information of China (English)

    Edwin C. Ouyang; Catherine H. Wu; Cherie Walton; Kittichai Promrat; George Y. Wu

    2001-01-01

    AIM To determine whether normal geneticallyirnmunocornpetent rodent hosts could bemanipulated to accept human hepatocytetransplants with long term survival withoutirnrnunosuppression.METHODS Tolerance towards humanhepatocytes was established by injection ofprimary human hepatocytes or Huh7 humanhepatoma cells into the peritoneal cavities offetal rats. Corresponding cells weresubsequently transplanted into newborn rats viaintrasplenic injection within 24 h after birth.RESULTS Mixed lymphocyte assays showedthat spleen cells from non-tolerized rats werestimulated to proliferate when exposed to humanhepatocytes, while cells from tolerized ratswere not. Injections made between 15 d and 17 dof gestation produced optimal tolerizaton.Transplanted human hepatocytes in rat liverswere visualized by immunohistochemicalstaining of human albumin. By dot blotting ofgenomic DNA in livers of tolerized rats 16 weeksafter hepatocyte transplantation, it was foundthat approximately 2.5 × 105 human hepatocytessurvived per rat liver. Human albumin mRNA wasdetected in rat livers by RT-PCR for 15 wk, andhuman albumin protein was also detectable in ratserum.CONCLUSION Tolerization of an immuno-competent rat can permit transplantation, andsurvival of functional human hepatocytes.

  6. A Microfabricated Platform for Generating Physiologically-Relevant Hepatocyte Zonation

    Science.gov (United States)

    McCarty, William J.; Usta, O. Berk; Yarmush, Martin L.

    2016-05-01

    In vitro liver models have been important tools for more than 40 years for academic research and preclinical toxicity screening by the pharmaceutical industry. Hepatocytes, the highly metabolic parenchymal cells of the liver, are efficient at different metabolic chemistries depending on their relative spatial location along the sinusoid from the portal triad to the central vein. Although replicating hepatocyte metabolic zonation is vitally important for physiologically-relevant in vitro liver tissue and organ models, it is most often completely overlooked. Here, we demonstrate the creation of spatially-controlled zonation across multiple hepatocyte metabolism levels through the application of precise concentration gradients of exogenous hormone (insulin and glucagon) and chemical (3-methylcholanthrene) induction agents in a microfluidic device. Observed gradients in glycogen storage via periodic acid-Schiff staining, urea production via carbamoyl phosphatase synthetase I staining, and cell viability after exposure to allyl alcohol and acetaminophen demonstrated the in vitro creation of hepatocyte carbohydrate, nitrogen, alcohol degradation, and drug conjugation metabolic zonation. This type of advanced control system will be crucial for studies evaluating drug metabolism and toxicology using in vitro constructs.

  7. Aniline Induces Oxidative Stress and Apoptosis of Primary Cultured Hepatocytes

    Directory of Open Access Journals (Sweden)

    Yue Wang

    2016-11-01

    Full Text Available The toxicity and carcinogenicity of aniline in humans and animals have been well documented. However, the molecular mechanism involved in aniline-induced liver toxicity and carcinogenesis remains unclear. In our research, primary cultured hepatocytes were exposed to aniline (0, 1.25, 2.50, 5.0 and 10.0 μg/mL for 24 h in the presence or absence of N-acetyl-l-cysteine (NAC. Levels of reactive oxygen species (ROS, malondialdehyde (MDA, and glutathione (GSH, activities of superoxide dismutase (SOD and catalase (CAT, mitochondrial membrane potential, DNA damage, cell viability, and apoptosis were detected. Levels of ROS and MDA were significantly increased and levels of GSH and CAT, activity of SOD, and mitochondrial membrane potential in hepatocytes were significantly decreased by aniline compared with the negative control group. The tail moment and DNA content of the tail in exposed groups were significantly higher than those in the negative control group. Cell viability was reduced and apoptotic death was induced by aniline in a concentration-dependent manner. The phenomena of ROS generation, oxidative damage, loss of mitochondrial membrane potential, DNA damage and apoptosis could be prevented if ROS inhibitor NAC was added. ROS generation is involved in the loss of mitochondrial membrane potential and DNA injury, which may play a role in aniline-induced apoptosis in hepatocytes. Our study provides insight into the mechanism of aniline-induced toxicity and apoptosis of hepatocytes.

  8. Preservation of hepatocyte suspensions at 4 degrees C.

    Science.gov (United States)

    Lund, P; Wiggins, D

    1988-10-01

    Suspensions of rat hepatocytes are resistant to hypoxia for at least 24 hr at 4 degrees C. After storage for 24 hr with xylitol, sorbitol, or glycerol, their subsequent capacity for glucogenesis from these substrates is increased. Even storage under N2/CO2 as the gas phase has little deleterious effect.

  9. Structural features that distinguish kinetically distinct biomineralization polypeptides.

    Science.gov (United States)

    Collino, Sebastiano; Evans, John Spencer

    2007-05-01

    AP7 and AP24 are mollusk shell proteins which are responsible for aragonite polymorph formation and stabilization within the nacre layer of the Pacific red abalone, Haliotis rufescens. It is known that the 30-AA N-terminal mineral modification domains of both proteins (AP7N, AP24N) possess identical multifunctional mineralization capabilities within in vitro assays but differ in terms of rate kinetics, with AP24N > AP7N. In this report, we identify previously unreported molecular features of AP24N and contrast the lowest energy polypeptide backbone structures of AP24N (planar configuration) with that of AP7N ("bent paper clip" configuration) using NMR data and simulated annealing molecular dynamics structure refinement. Like AP7N, we find that AP24N possesses an unfolded conformation, can sequester Ca(II) and other multivalent metal ions, can adsorb onto or within calcite crystals, and possesses anionic and cationic electrostatic "pocket" regions on its molecular surfaces. However, AP24N has some unique features: greater conformational responsiveness to Ca(II), the tendency to form a more planar backbone configuration, and longer anionic and hydrogen-bonding donor/acceptor sequence blocks. We conclude that the presence of unfolded polypeptide conformation, electrostatic surface pockets, and interactive sequence clustering endow both AP7N and AP24N with similar features that lead to comparable effects on crystal morphology and nucleation. However, AP24N possesses longer anionic and hydrogen-bonding sequence clusters and exhibits a tendency to adopt a more planar backbone configuration than AP7N does. We believe that these features facilitate peptide-mineral, peptide-ion, or water cluster interactions, thereby enhancing the mineralization kinetics of AP24N over AP7N.

  10. Polypeptides of Treponema pallidum: progress toward understanding their structural, functional, and immunologic roles. Treponema Pallidum Polypeptide Research Group.

    Science.gov (United States)

    Norris, S J

    1993-09-01

    Treponema pallidum subsp. pallidum, the spirochete that causes syphilis, is unusual in a number of respects, including its small genome size, inability to grow under standard in vitro culture conditions, microaerophilism, apparent paucity of outer membrane proteins, structurally complex periplasmic flagella, and ability to evade the host immune responses and cause disease over a period of years to decades. Many of these attributes are related ultimately to its protein content. Our knowledge of the activities, structure, and immunogenicity of its proteins has been expanded by the application of recombinant DNA, hybridoma, and structural fractionation techniques. The purpose of this monograph is to summarize and correlate this new information by using two-dimensional gel electrophoresis, monoclonal antibody reactivity, sequence data, and other properties as the bases of polypeptide identification. The protein profiles of the T. pallidum subspecies causing syphilis, yaws, and endemic syphilis are virtually indistinguishable but differ considerably from those of other treponemal species. Among the most abundant polypeptides are a group of lipoproteins of unknown function that appear to be important in the immune response during syphilitic infection. The periplasmic flagella of T. pallidum and other spirochetes are unique with regard to their protein content and ultrastructure, as well as their periplasmic location. They are composed of three core proteins (homologous to the other members of the eubacterial flagellin family) and a single, unrelated sheath protein; the functional significance of this arrangement is not understood at present. Although the bacterium contains the chaperonins GroEL and DnaK, these proteins are not under the control of the heat shock regulon as they are in most organisms. Studies of the immunogenicity of T. pallidum proteins indicate that many may be useful for immunodiagnosis and immunoprotection. Future goals in T. pallidum polypeptide

  11. General Assembly

    CERN Multimedia

    Staff Association

    2015-01-01

    Mardi 5 mai à 11 h 00 Salle 13-2-005 Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : 1- Adoption de l’ordre du jour. 2- Approbation du procès-verbal de l’Assemblée générale ordinaire du 22 mai 2014. 3- Présentation et approbation du rapport d’activités 2014. 4- Présentation et approbation du rapport financier 2014. 5- Présentation et approbation du rapport des vérificateurs aux comptes pour 2014. 6- Programme 2015. 7- Présentation et approbation du projet de budget 2015 et taux de cotisation pour 2015. 8- Pas de modifications aux Statuts de l'Association du personnel proposée. 9- Élections des membres de la Commission é...

  12. General Assembly

    CERN Multimedia

    Staff Association

    2016-01-01

    Mardi 5 avril à 11 h 00 BE Auditorium Meyrin (6-2-024) Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : Adoption de l’ordre du jour. Approbation du procès-verbal de l’Assemblée générale ordinaire du 5 mai 2015. Présentation et approbation du rapport d’activités 2015. Présentation et approbation du rapport financier 2015. Présentation et approbation du rapport des vérificateurs aux comptes pour 2015. Programme de travail 2016. Présentation et approbation du projet de budget 2016 Approbation du taux de cotisation pour 2017. Modifications aux Statuts de l'Association du personnel proposée. Élections des membres de la Commissio...

  13. General assembly

    CERN Multimedia

    Staff Association

    2015-01-01

    Mardi 5 mai à 11 h 00 Salle 13-2-005 Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : Adoption de l’ordre du jour. Approbation du procès-verbal de l’Assemblée générale ordinaire du 22 mai 2014. Présentation et approbation du rapport d’activités 2014. Présentation et approbation du rapport financier 2014. Présentation et approbation du rapport des vérificateurs aux comptes pour 2014. Programme 2015. Présentation et approbation du projet de budget 2015 et taux de cotisation pour 2015. Pas de modifications aux Statuts de l'Association du personnel proposée. Élections des membres de la Commission électorale. &am...

  14. General Assembly

    CERN Multimedia

    Staff Association

    2017-01-01

    Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : Adoption de l’ordre du jour. Approbation du procès-verbal de l’Assemblée générale ordinaire du 5 avril 2016. Présentation et approbation du rapport d’activités 2016. Présentation et approbation du rapport financier 2016. Présentation et approbation du rapport des vérificateurs aux comptes pour 2016. Programme de travail 2017. Présentation et approbation du projet de budget 2017 Approbation du taux de cotisation pour 2018. Modifications aux Statuts de l'Association du personnel proposées. Élections des membres de la Commission électorale. Élections des vérifica...

  15. SREBP-1c Gene Silencing can Decrease Lipid Deposits in Bovine Hepatocytes Cultured in Vitro

    Directory of Open Access Journals (Sweden)

    Qinghua Deng

    2014-05-01

    Full Text Available Background: Fatty liver is a major metabolic disorder that occurs during early lactation in high-producing dairy cows. Sterol regulatory element-binding protein-1c (SREBP-1c is an important transcription factor that regulates lipid synthesis by regulating the expression of lipid metabolism genes. Methods: In this study, we reduced the expression of SREBP-1c by adenovirus-mediated SREBP-1c with a low expression vector (AD-GFP-SREBP-1c to study the effects of SREBP-1c on lipid deposits in bovine hepatocytes. The expression levels and enzyme activities of SERBP-1c and its target genes were determined by real-time PCR, western blot, and ELISA. Results: These results showed that Ad-GFP-SREBP-1c could inhibit SREBP-1c expression. The expression of the lipid synthesis enzyme acetyl-CoA carboxylase (ACC was down-regulated. The expression levels of the lipid oxidation enzymes long-chain fatty acyl-COA synthetase (ACSL-1, carnitine palmitoyltransferase І (CPT-І, carnitine palmitoyltransferase II (CPT- II, and β-hydroxyacyl-CoA-DH (HADH were significantly elevated. Furthermore, the expression levels of factors involved in the assembly and transport of very low-density lipoproteins (VLDLs, such as apolipoprotein B100 (ApoB, apolipoprotein E (ApoE, and microsomal triglyceride transfer protein (MTTP were decreased comparison with the negative control and the blank control groups, but the low-density lipoprotein receptor (LDLR was elevated. The concentrations of TG (triglyceride and VLDL were also reduced. Conclusion: These data suggest that low SREBP-1c expression can decrease lipid synthesis, increase lipid oxidation, and decrease the TG and VLDL content in bovine hepatocytes.

  16. Isolation of modulators of the liver-specific organic anion-transporting polypeptides (OATPs) 1B1 and 1B3 from Rollinia emarginata Schlecht (Annonaceae).

    Science.gov (United States)

    Roth, Megan; Araya, Juan J; Timmermann, Barbara N; Hagenbuch, Bruno

    2011-11-01

    Organic anion-transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3) are liver-specific transporters that mediate the uptake of a broad range of drugs into hepatocytes, including statins, antibiotics, and many anticancer drugs. Compounds that alter transport by one or both of these OATPs could potentially be used to target drugs to hepatocytes or improve the bioavailability of drugs that are cleared by the liver. In this study, we applied a bioassay-guided isolation approach to identify such compounds from the organic extract of Rollinia emarginata Schlecht (Annonaceae). Fractions of the plant extract were screened for effects on OATP1B1- and OATP1B3-mediated transport of the model substrates estradiol-17β-glucuronide and estrone-3-sulfate. We isolated three compounds, ursolic acid, oleanolic acid, and 8-trans-p-coumaroyloxy-α-terpineol, which inhibited estradiol-17β-glucuronide uptake by OATP1B1 but not OATP1B3. In addition, a rare compound, quercetin 3-O-α-l-arabinopyranosyl(1→2) α-L-rhamnopyranoside, was identified that had distinct effects on each OATP. OATP1B1 was strongly inhibited, as was OATP1B3-mediated transport of estradiol-17β-glucuronide. However, OATP1B3-mediated uptake of estrone-3-sulfate was stimulated 4- to 5-fold. Kinetic analysis of this stimulation revealed that the apparent affinity for estrone-3-sulfate was increased (decreased K(m)), whereas the maximal rate of transport (V(max)) was significantly reduced. These results demonstrate a mechanism through which the hepatic uptake of drug OATP substrates could be stimulated.

  17. Chemically induced hepatotoxicity in human stem cell-induced hepatocytes compared with primary hepatocytes and HepG2.

    Science.gov (United States)

    Kang, Seok-Jin; Lee, Hyuk-Mi; Park, Young-Il; Yi, Hee; Lee, Hunjoo; So, ByungJae; Song, Jae-Young; Kang, Hwan-Goo

    2016-10-01

    Stem cell-induced hepatocytes (SC-iHeps) have been suggested as a valuable model for evaluating drug toxicology. Here, human-induced pluripotent stem cells (QIA7) and embryonic stem cells (WA01) were differentiated into hepatocytes, and the hepatotoxic effects of acetaminophen (AAP) and aflatoxin B1 (AFB1) were compared with primary hepatocytes (p-Heps) and HepG2. In a cytotoxicity assay, the IC50 of SC-iHeps was similar to that in p-Heps and HepG2 in the AAP groups but different from that in p-Heps of the AFB1 groups. In a multi-parameter assay, phenotypic changes in mitochondrial membrane potential, calcium influx and oxidative stress were similar between QIA7-iHeps and p-Heps following AAP and AFB1 treatment but relatively low in WA01-iHeps and HepG2. Most hepatic functional markers (hepatocyte-specific genes, albumin/urea secretion, and the CYP450 enzyme activity) were decreased in a dose-dependent manner following AAP and AFB1 treatment in SC-iHeps and p-Heps but not in HepG2. Regarding CYP450 inhibition, the cell viability of SC-iHeps and p-Heps was increased by ketoconazole, a CYP3A4 inhibitor. Collectively, SC-iHeps and p-Heps showed similar cytotoxicity and hepatocyte functional effects for AAP and AFB1 compared with HepG2. Therefore, SC-iHeps have phenotypic characteristics and sensitivity to cytotoxic chemicals that are more similar to p-Heps than to HepG2 cells.

  18. Overexpression of hepatocyte growth factor in SBMA model mice has an additive effect on combination therapy with castration

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Ying [Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Adachi, Hiroaki, E-mail: hadachi-ns@umin.org [Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Department of Neurology, University of Occupational and Environmental Health School of Medicine, 1-1 Iseigaoka, Yahata-nishi-ku, Kitakyushu 807-8555 (Japan); Katsuno, Masahisa [Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Huang, Zhe [Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Department of Neurology, University of Occupational and Environmental Health School of Medicine, 1-1 Iseigaoka, Yahata-nishi-ku, Kitakyushu 807-8555 (Japan); Jiang, Yue-Mei; Kondo, Naohide; Iida, Madoka; Tohnai, Genki; Nakatsuji, Hideaki [Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Funakoshi, Hiroshi [Center for Advanced Research and Education, Asahikawa Medical University, 1-1-1- Higashinijo Midorigaoka, Asahikawa 078-8510 (Japan); Nakamura, Toshikazu [Neurogen Inc., 1-1-52-201 Nakahozumi, Ibaraki 567-0034 (Japan); Sobue, Gen, E-mail: sobueg@med.nagoya-u.ac.jp [Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Research Division of Dementia and Neurodegenerative Disease, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan)

    2015-12-25

    Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of a polyglutamine (polyQ)-encoding tract within the androgen receptor (AR) gene. The pathologic features of SBMA are motor neuron loss in the spinal cord and brainstem and diffuse nuclear accumulation and nuclear inclusions of mutant AR in residual motor neurons and certain visceral organs. Hepatocyte growth factor (HGF) is a polypeptide growth factor which has neuroprotective properties. To investigate whether HGF overexpression can affect disease progression in a mouse model of SBMA, we crossed SBMA transgenic model mice expressing an AR gene with an expanded CAG repeat with mice overexpressing HGF. Here, we report that high expression of HGF induces Akt phosphorylation and modestly ameliorated motor symptoms in an SBMA transgenic mouse model treated with or without castration. These findings suggest that HGF overexpression can provide a potential therapeutic avenue as a combination therapy with disease-modifying therapies in SBMA. - Highlights: • HGF overexpression ameliorates the motor phenotypes of the SBMA mouse model. • HGF overexpression induces Akt phosphorylation in the SBMA mouse model. • This is the first report of combination therapy in a mouse model of polyQ diseases.

  19. Overexpression of hepatocyte growth factor in SBMA model mice has an additive effect on combination therapy with castration.

    Science.gov (United States)

    Ding, Ying; Adachi, Hiroaki; Katsuno, Masahisa; Huang, Zhe; Jiang, Yue-Mei; Kondo, Naohide; Iida, Madoka; Tohnai, Genki; Nakatsuji, Hideaki; Funakoshi, Hiroshi; Nakamura, Toshikazu; Sobue, Gen

    2015-12-25

    Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of a polyglutamine (polyQ)-encoding tract within the androgen receptor (AR) gene. The pathologic features of SBMA are motor neuron loss in the spinal cord and brainstem and diffuse nuclear accumulation and nuclear inclusions of mutant AR in residual motor neurons and certain visceral organs. Hepatocyte growth factor (HGF) is a polypeptide growth factor which has neuroprotective properties. To investigate whether HGF overexpression can affect disease progression in a mouse model of SBMA, we crossed SBMA transgenic model mice expressing an AR gene with an expanded CAG repeat with mice overexpressing HGF. Here, we report that high expression of HGF induces Akt phosphorylation and modestly ameliorated motor symptoms in an SBMA transgenic mouse model treated with or without castration. These findings suggest that HGF overexpression can provide a potential therapeutic avenue as a combination therapy with disease-modifying therapies in SBMA. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Peptides and polypeptides as scaffolds for optoelectronics and biomaterials applications

    Science.gov (United States)

    Charati, Manoj B.

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

  1. Polypeptide stimulators of the Ms-Lon protease.

    Science.gov (United States)

    Rudyak, S G; Shrader, T E

    2000-09-01

    Both the peptidase activity against small fluorescent peptide substrates and the ATPase activity of Lon (La) proteases are stimulated by unstructured proteins such as alpha-casein. This stimulation reveals the simultaneous interaction of Lon with two proteolytic substrates--alpha-casein and the peptide substrate. To understand the cellular function of this stimulation, it is important to determine the physical properties of Lon stimulators. The abilities of compositionally simple random copolymers of amino acids (rcAAs) to stimulate the peptidase and ATPase activities of the Lon protease from Mycobacterium smegmatis (Ms-Lon) and its N-terminal truncation mutant (N-E226) were determined. We report that cationic but not anionic rcAAs stimulated Ms-Lon's peptidase activity but were themselves poor substrates for the enzyme. Peptidase stimulation by rcAAs correlated approximately with the degree of hydrophobicity of these polypeptides and reached levels >10-fold higher than observed previously for Ms-Lon stimulators such as alpha-casein. In contrast to alpha-casein, which stimulates Ms-Lon's peptidase activity by 40% and ATPase activity by 150%, rcAAs stimulated peptidase activity without concomitant stimulation of ATPase activity. Active site labeling experiments suggested that both rcAAs and ATP increased peptidase activity by increasing accessibility to the peptidase active site. Peptidase activity assays in the presence of both alpha-casein and rcAAs revealed that interactions of rcAAs and alpha-casein with Ms-Lon are extremely complex and not mutually exclusive. Specifically, (1) additions of low concentrations of alpha-casein (rcAA-stimulated peptidase activity; (2) additions of higher concentrations of alpha-casein inhibited Ms-Lon's rcAA-stimulated peptidase activity; (3) additions of all concentrations of alpha-casein inhibited N-E226's rcAA-stimulated peptidase activity. We conclude the Ms-Lon can interact with an rcAA, alpha-casein, and a substrate peptide

  2. Synthesis of hyperbranched polypeptide and PEO block copolymer by consecutive thiol-yne chemistry.

    Science.gov (United States)

    Chang, Xiao; Dong, Chang-Ming

    2013-09-09

    Hyperbranched poly(ε-benzyloxycarbonyl-L-lysine) (HPlys) with multiple alkyne peripheries was synthesized through the click polycondensation of an AB2 type Plys macromonomer with α-thiol and ω-alkyne terminal groups (thiol is the A unit, and each π bond in alkyne is the B unit), and the resulting HPlys was further conjugated with thiol-termined poly(ethylene oxide) (PEO) to generate HPlys-b-PEO block copolymer by consecutive thiol-yne chemistry. Their molecular structures and physical properties were characterized in detail by FT-IR, (1)H NMR, gel permeation chromatography, differential scanning calorimetry, wide-angle X-ray diffraction, and polarized optical microscopy. HPlys and HPlys-b-PEO mainly assumed an α-helix conformation similar to the linear precursors, while the liquid crystalline phase transition of Plys segment disappeared within HPlys and HPlys-b-PEO. HPlys-b-PEO self-assembled into nearly spherical micelles in aqueous solution, while it gave a 5-fold lower critical aggregation concentration (8.9 × 10(-3) mg/mL) than a linear counterpart (4.5 × 10(-2) mg/mL), demonstrating a dendritic topology effect. Compared with a linear counterpart, HPlys-b-PEO gave a higher drug-loading capacity and efficiency for the anticancer drug doxorubicin (DOX) and a slower drug-release rate with an improved burst-release profile, enabling them useful for drug delivery systems. Importantly, this work provides a versatile strategy for the synthesis of hyperbranched polypeptides and related block copolymers by utilizing thiol-yne chemistry.

  3. DIFFERENTIAL-EFFECTS OF EICOSAPENTAENOIC ACID ON GLYCEROLIPID AND APOLIPOPROTEIN-B METABOLISM IN PRIMARY HUMAN HEPATOCYTES COMPARED TO HEPG2 CELLS AND PRIMARY RAT HEPATOCYTES

    NARCIS (Netherlands)

    LIN, YG; SMIT, MJ; HAVINGA, R; VERKADE, HJ; VONK, RJ; KUIPERS, F

    1995-01-01

    We compared the effects of eicosapentaenoic acid (EPA) and oleic acid (OA) on glycerolipid and apolipoprotein B (apoB) metabolism in primary human hepatocytes, HepG2 cells and primary rat hepatocytes. Cells were incubated for 1 to 5 h with 0.25 mM bovine serum albumin in the absence (control) or

  4. Transplantation of human stem cell-derived hepatocytes in an animal model of acute liver failure.

    Science.gov (United States)

    Ramanathan, Rajesh; Pettinato, Giuseppe; Beeston, John T; Lee, David D; Wen, Xuejun; Mangino, Martin J; Fisher, Robert A

    2015-08-01

    Hepatocyte cell transplantation can be life-saving in patients with acute liver failure (ALF); however, primary human hepatocyte transplantation is limited by the scarcity of donor hepatocytes. We investigated the effect of stem cell-derived, hepatocyte-like cells in an animal xenotransplant model of ALF. Intraperitoneal d-galactosamine was used to develop a lethal model of ALF in the rat. Human induced pluripotent stem cells (iPSC), human mesenchymal stem cells, and human iPSC combined with human endothelial cells (iPSC + EC) were differentiated into hepatocyte-like cells and transplanted into the spleens of athymic nude rats with ALF. A reproducible lethal model of ALF was achieved with nearly 90% death within 3 days. Compared with negative controls, rats transplanted with stem cell-derived, hepatocyte-like cells were associated with increased survival. Human albumin was detected in the rat serum 3 days after transplantation in more than one-half the animals transplanted with hepatocyte-like cells. Only animals transplanted with iPSC + EC-derived hepatocytes had serum human albumin at 14 days posttransplant. Transplanted hepatocyte-like cells homed to the injured rat liver, whereas the ECs were only detected in the spleen. Transplantation of stem cell-derived, hepatocyte-like cells improved survival with evidence of in vivo human albumin production. Combining ECs may prolong cell function after transplantation. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. [Cytoskeletal reorganization in hepatocytes of the regenerating mouse liver].

    Science.gov (United States)

    Gleĭberman, A S; Troianovskiĭ, S M; Bannikov, G A

    1984-12-01

    The intracellular pattern of prekeratin and actin filaments has been studied on sections of mouse livers regenerating after CCl4 injury. Monoclonal antibodies against one of liver prekeratins and monospecific polyclonal actin antibodies were used in the indirect immunofluorescent test. The presence of alpha-fetoprotein and bile canaliculi antigen was also monitored during regeneration. In control livers, prekeratin and actin filaments formed thick bundles adjacent to plasma membranes. The cytoplasmic prekeratin network was unmarked. In contrast to the latter, the bright well developed intracytoplasmic prekeratin network and intracytoplasmic actin fibers were identified in the perinecrotic hepatocytes by the 3d-4th day of regeneration. This rearrangement of the cytoskeleton coincided in time with the appearance of alpha-fetoprotein and the loss of the bile canaliculi antigen in the perinecrotic hepatocytes.

  6. Effects of electroporation on primary rat hepatocytes in vitro

    Institute of Scientific and Technical Information of China (English)

    Yun-Qing Yao; Ding-Feng Zhang; Ai-Long Huang; Yun Luo; Da-Zhi Zhang; Bo Wang; Wei-Ping Zhou; Hong Ren; Shu-Hua Guo

    2002-01-01

    AIM: To investigate the effects of electroporation on primaryrat hepatocyte and to optimize the electroporation conditionsintroducing foreign genes into primary hepatocytes.METHODS: A single-pulse procedure was performed at Iowvoltage (220-400 V) but with high capacitance (500-950 μF).Hepatocytes were divided into 4 groups according to theelectroporation conditions: group Ⅰ, 220 V and 500 μF;group Ⅱ, 220 Vand 950 μF; group Ⅲ, 400 V and 950 μF,and group Ⅳ.The control group was freshly isolatedhepatocytes and directly cultured under the same conditionsas those of electroporation groups. The effects ofelectroporation on primary rat hepatocytes were detectedby trypan blue exclusion (TBE) and MTT analysis. Besides,albumin (AIb), alanine transaminase (ALT) and lactatedehydrogenase (LDH) in the supernatants of culturedhepatocytes were measured by biochemical assay.RESULTS: Between day 1 and day 15 after incubation,primary rat hepatocytes of each electroporation groupappeared normal, being the same with those of controlgroup. TBE staining showed that slight hepatocyte damageand high survival rate were found in the electroporationgroups and the control group. Cultured for 3, 7, 11 and 15days, hepatocyte viability was approximatly 92.6±2.5 %,89.5±3.3 %, 82.0±3.5 % and 74.3±1.2 %, respectively.MTT analysis indicated that the viabilities of hepatocyteshad no significant difference between each electroporationgroup, and those were similar to that of control group. Atthe 36th hour after electroporation, AIb, ALT and LDH in thesupernatants of control group were 5.3±0.1 g. L-1, 183.7±8.4 nkat. L-1 and 896.8±58.5 nkat. L-1; those of group Ⅱwere 5.7±0.1 g. L-1, 215.4±16.7 nkat. L-1 and 1063.8±51.8 nkat. L-1; and those of group Ⅲ were 5.80.2 g. L-1,217.1 ± 8.4 nkat. L-1 and 1063.8± 10.0 nkat. L-1 . Statistically,the proteins of group Ⅱ and group Ⅲ were significantlyhigher than those of control group (P<0.05), whereas theprotein production of group

  7. [Current status and future perspectives of hepatocyte transplantation].

    Science.gov (United States)

    Pareja, Eugenia; Cortés, Miriam; Gómez-Lechón, M José; Maupoey, Javier; San Juan, Fernando; López, Rafael; Mir, Jose

    2014-02-01

    The imbalance between the number of potential beneficiaries and available organs, originates the search for new therapeutic alternatives, such as Hepatocyte transplantation (HT).Even though this is a treatment option for these patients, the lack of unanimity of criteria regarding indications and technique, different cryopreservation protocols, as well as the different methodology to assess the response to this therapy, highlights the need of a Consensus Conference to standardize criteria and consider future strategies to improve the technique and optimize the results.Our aim is to review and update the current state of hepatocyte transplantation, emphasizing the future research attempting to solve the problems and improve the results of this treatment. Copyright © 2013 AEC. Published by Elsevier Espana. All rights reserved.

  8. The cytoskeleton of digitonin-treated rat hepatocytes.

    Science.gov (United States)

    Fiskum, G; Craig, S W; Decker, G L; Lehninger, A L

    1980-06-01

    Treatment of isolated rat hepatocptes with low concentrations of digitonin increases the permeability of the plsma membrane to cytosolic proteins without causing release of organelles such as mitochondria into the surrounding medium. Electron microscopy showed that treatment of the cells with increasing concentations of digitonin results in a progressive loss in the continuity of the plasma membrane, while most other aspects of cellular morphology remain normal. Depletion of background staining material from the cytosol by digitonin treatment of the cells greatly enhances the visualization of the cytoskeleton. The use of this technique, together with immunofluorescent light microscopy, has verified the presence of an actin-containing filamentous network at the hepatocyte cortex as well as intermediate filaments distributed throughout the cell. Digitonin is thus useful both for selectively permeabilizing the plasma membrane and for intensifying the appearance of intracellular structures such as microfilaments that are normally difficult to observe in cells such as hepatocytes.

  9. Polypeptide growth factors in the course of surgical periodontal treatment.

    Science.gov (United States)

    Pietruska, M D; Pietruski, J K; Stokowska, W

    2000-01-01

    The aim of the study was to evaluate the behaviour of certain polypeptide growth factors in patients with rapidly progressive periodontitis (RPP) during periodontal therapy using alloplastic grafts. Concentrations of epidermal growth factor (EGF), fibroblastic growth factor (FGF), platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF beta) were assayed in blood serum and saliva. Significant differences in the behaviour of growth factors in blood referred to EGF and PDGF. Their mean concentrations in serum of RPP patients were higher both in the preliminary examination and in the final investigation after 3 months compared with control group. However, mean FGF concentrations in serum were significantly higher only in the distant examination. In saliva, the concentrations of EGF, PDGF and FGF were not significantly different compared with control group. Salivary TGF beta in patients with RPP was significantly higher than in controls and increased in the final examination. The differences observed in the concentrations of growth factors in the serum and saliva of patients with RPP indicate that these factors can be involved in inflammation, destruction and regeneration of periodontal tissues.

  10. Atrial natriuretic polypeptide-like material in rat lung

    Energy Technology Data Exchange (ETDEWEB)

    Chang, J.K.; Chang, D.; Xie, C.W.; Song, D.L.; Li, X.R.; Zhang, S.X.; Wang, T.L.; Tang, J.

    1986-03-05

    Atrial natriuretic polypeptide-like immunoreactive material (ANP-IR) was found in rat lung by radioimmunoassay, with the concentration ranging from 0.6-1.2 pmol/g of tissue in each lobe. PAP-immunohistochemical study demonstrated that specific staining of granules for ..cap alpha..-human ANP are mainly located in the muscular layer of the pulmonary vein. Fractionation of lung extract by gel filtration and reserve phase HPLC revealed the presence of multiple forms of ANP-IR, which possibly possessed molecular structure partially different from rat ANP, atriopeptin I and III. Intravenous injection of lung extract induced potent diuresis and natriuresis in rats. These responses could be abolished when the lung extract was preincubated with antiserum for ..cap alpha..-human ANP. Specific binding sites for /sup 125/I-labeled rat ANP were also found in lung membrane preparation by radioreceptor assay. Incubation of synthetic atriopeptin III (10/sup -9/ to 10/sup -6/M) with lung tissue induced 1-28 fold increase in lung cGMP content. The results suggest that ANP-IR and its receptors existing in rat lung may be involved in the regulation of pulmonary function and have a synergic effect with ANP of cardiac origin in the control of water-electrolytes balance.

  11. Free radical scavenging abilities of polypeptide from Chlamys farreri

    Science.gov (United States)

    Han, Zhiwu; Chu, Xiao; Liu, Chengjuan; Wang, Yuejun; Mi, Sun; Wang, Chunbo

    2006-09-01

    We investigated the radical scavenging effect and antioxidation property of polypeptide extracted from Chlamys farreri (PCF) in vitro using chemiluminescence and electron spin resonance (ESR) methods. We examined the scavenging effects of PCF on superoxide anions (O{2/-}), hydroxyl radicals (OH·), peroxynitrite (ONOO-) and the inhibiting capacity of PCF on peroxidation of linoleic acid. Our experiment suggested that PCF could scavenge oxygen free radicals including superoxide anions (O{2/-}) (IC50=0.3 mg/ml), hydroxyl radicals (OH·) (IC50=0.2 μg/ml) generated from the reaction systems and effectively inhibit the oxidative activity of ONOO- (IC50=0.2 mg/ml). At 1.25 mg/ml of PCF, the inhibition ratio on lipid peroxidation of linoleic acid was 43%. The scavenging effect of PCF on O{2/-}, OH· and ONOO- free radicals were stronger than those of vitamin C but less on lipid peroxidation of linoleic acid. Thus PCF could scavenge free radicals and inhibit the peroxidation of linoleic acid in vitro. It is an antioxidant from marine products and potential for industrial production in future.

  12. Volumetric properties of human islet amyloid polypeptide in liquid water.

    Science.gov (United States)

    Brovchenko, I; Andrews, M N; Oleinikova, A

    2010-04-28

    The volumetric properties of human islet amyloid polypeptide (hIAPP) in water were studied in a wide temperature range by computer simulations. The intrinsic density rho(p) and the intrinsic thermal expansion coefficient alpha(p) of hIAPP were evaluated by taking into account the difference between the volumetric properties of hydration and bulk water. The density of hydration water rho(h) was found to decrease almost linearly with temperature upon heating and its thermal expansion coefficient was found to be notably higher than that of bulk water. The peptide surface exposed to water is more hydrophobic and its rho(h) is smaller in conformation with a larger number of intrapeptide hydrogen bonds. The two hIAPP peptides studied (with and without disulfide bridge) show negative alpha(p), which is close to zero at 250 K and decreases to approximately -1.5 x 10(-3) K(-1) upon heating to 450 K. The analysis of various structural properties of peptides shows a correlation between the intrinsic peptide volumes and the number of intrapeptide hydrogen bonds. The obtained negative values of alpha(p) can be attributed to the shrinkage of the inner voids of the peptides upon heating.

  13. Molecular Dynamics Simulation of Cholera Toxin A-1 Polypeptide

    Directory of Open Access Journals (Sweden)

    Badshah Syed Lal

    2016-01-01

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

  14. Stimulation of growth hormone by vasoactive intestinal polypeptide in acromegaly.

    Science.gov (United States)

    Chihara, K; Kaji, H; Minamitani, N; Kodama, H; Kita, T; Goto, B; Chiba, T; Coy, D H; Fujita, T

    1984-01-01

    Vasoactive intestinal polypeptide (VIP) was administered as an iv bolus of 1 micrograms/kg BW to 8 acromegalic patients and in doses of 0.5 and 1 microgram/kg BW to 15 normal volunteers. Both systolic and diastolic blood pressures decreased, and pulse rate increased transiently after VIP injection. VIP stimulated PRL release from the anterior pituitary in normal subjects. Plasma PRL responses to VIP in women were dose dependent and larger than those in men. On the other hand, plasma GH levels rose markedly after VIP injection in all 6 patients with untreated acromegaly. In 2 patients studied after transsphenoidal microadenomectomy, there was no plasma GH response to VIP. In 2 other patients with inactive acromegaly as well as in normal subjects, VIP failed to affect plasma GH levels. In all 6 patients with active acromegaly, LRH (1-2 micrograms/kg BW, iv) did not increase plasma GH levels, but TRH (5-10 micrograms/kg BW, iv) caused significant increases in plasma GH, the magnitude of which was not similar to that of increases seen after VIP injection. Paradoxical GH responses to TRH were not observed in patients in the inactive phase after transsphenoidal surgery. These findings suggest that VIP stimulates GH release in vivo in acromegalic patients. A VIP test as well as a TRH test offer promise as simple and reliable techniques to evaluate the activity of acromegaly, particularly after transsphenoidal surgery.

  15. Vasoactive intestinal polypeptide provokes acetylcholine release from the myenteric plexus

    Energy Technology Data Exchange (ETDEWEB)

    Kusunoki, M.; Tsai, L.H.; Taniyama, K.; Tanaka, C.

    1986-07-01

    Effects of vasoactive intestinal polypeptide (VIP) on the release of acetylcholine (ACh) from longitudinal muscle strips with myenteric plexus (LM) preparations were examined in the guinea pig small intestine. VIP (10 to 10 W M) induced a concentration-dependent contraction of LM preparation. The VIP-induced contractions seem to be related to three components, the scopolamine-sensitive, the scopolamine-insensitive, the tetrodotoxin-sensitive, and the tetrodotoxin-insensitive contractions. VIP (10 to 10 W M) induced a concentration-dependent increase in the release of (TH)ACh from LM preparations preloaded with (TH)choline. The VIP-evoked (TH)ACh release was inhibited by removal of CaS from the perfusion medium and by treatment with tetrodotoxin but not by scopolamine and hexamethonium. The spontaneous and VIP-evoked (TH)ACh release was not affected by phentolamine, propranolol, methysergide, diphenhydramine, cimetidine, bicuculline, or (D-ProS, D-Trp/sup 7,9/)substance P. The result demonstrates that VIP induces contractions of longitudinal smooth muscle directly and indirectly by the stimulation of both cholinergic neurons and noncholinergic excitatory neurons.

  16. Islet amyloid polypeptide, islet amyloid, and diabetes mellitus.

    Science.gov (United States)

    Westermark, Per; Andersson, Arne; Westermark, Gunilla T

    2011-07-01

    Islet amyloid polypeptide (IAPP, or amylin) is one of the major secretory products of β-cells of the pancreatic islets of Langerhans. It is a regulatory peptide with putative function both locally in the islets, where it inhibits insulin and glucagon secretion, and at distant targets. It has binding sites in the brain, possibly contributing also to satiety regulation and inhibits gastric emptying. Effects on several other organs have also been described. IAPP was discovered through its ability to aggregate into pancreatic islet amyloid deposits, which are seen particularly in association with type 2 diabetes in humans and with diabetes in a few other mammalian species, especially monkeys and cats. Aggregated IAPP has cytotoxic properties and is believed to be of critical importance for the loss of β-cells in type 2 diabetes and also in pancreatic islets transplanted into individuals with type 1 diabetes. This review deals both with physiological aspects of IAPP and with the pathophysiological role of aggregated forms of IAPP, including mechanisms whereby human IAPP forms toxic aggregates and amyloid fibrils.

  17. Aspects of structural landscape of human islet amyloid polypeptide

    Science.gov (United States)

    He, Jianfeng; Dai, Jin; Li, Jing; Peng, Xubiao; Niemi, Antti J.

    2015-01-01

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

  18. Free radical scavenging abilities of polypeptide from Chlamys farreri

    Institute of Scientific and Technical Information of China (English)

    HAN Zhiwu; CHU Xiao; LIU Chengjuan; WANG Yuejun; SUN Mi; WANG Chunbo

    2006-01-01

    We investigated the radical scavenging effect and antioxidation property of polypeptide extracted from Chlamys farreri (PCF) in vitro using chemiluminescence and electron spin resonance (ESR) methods. We examined the scavenging effects of PCF on superoxide anions (O-2), hydroxyl radicals (OH·), peroxynitrite (ONOO-) and the inhibiting capacity of PCF on peroxidation of linoleic acid. Our experiment suggested that PCF could scavenge oxygen free radicals including superoxide anions (O-2) (IC50 =0.3 mg/ml), hydroxyl radicals (OH·) (IC50 = 0.2 μg/ml) generated from the reaction systems and effectively inhibit the oxidative activity of ONOO- (IC50 = 0.2 mg/ml). At 1.25 mg/ml of PCF, the inhibition ratio on lipid peroxidation of linoleic acid was 43 %. The scavenging effect of PCF on (O-2), OH·and ONOO- free radicals were stronger than those of vitamin C but less on lipid peroxidation of linoleic acid. Thus PCF could scavenge free radicals and inhibit the peroxidation of linoleic acid in vitro. It is an antioxidant from marine products and potential for industrial production in future.

  19. Aspects of structural landscape of human islet amyloid polypeptide

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-01-28

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

  20. Inhibitory effects of berberine on ion channels of rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Fang Wang; Hong-Yi Zhou; Gang Zhao; Li-Ying Fu; Lan Cheng; Jian-Guo Chen; Wei-Xing Yao

    2004-01-01

    AIM: To examine the effects of berberine, an isoquinoline alkaloid with a long history used as a tonic remedy for liver and heart, on ion channels of isolated rat hepatocytes.METHODS: Tight-seal whole-cell patch-clamp techniques were performed to investigate the effects of berberine on the delayed outward potassium currents (IK), inward rectifier potassium currents (IK1) and Ca2+ release-activated Ca2+currents (ICRAC) in enzymatically isolated rat hepatocytes.RESULTS: Berberine 1-300 nmol/L reduced IK in a concentration dependent manner with EC50 of 38.86±5.37 μmol/L and nH of 0.82±0.05 (n = 8). When the bath solution was changed to tetraethylammonium (TEA) 8 mmol/L, IK was inhibited.Berberine 30 μmol/L reduced IK at all examined membrane potentials, especially at potentials positive to +60 mV (n = 8,P<0.05 or P<0.01 vs control). Berberine had mild inhibitory effects on IK1 in rat hepatocytes. Berberine 1-300 μmol/L also inhibited ICRAC in a concentration-dependent fashion.The fitting parameters were EC50 = 47.20±10.86 μmol/L,nH = 0.71±0.09 (n = 8). The peak value of ICRAC in the Ⅰ-Ⅴrelationship was decreased by berberine 30 μmol/L at potential negative to -80 mV (n = 8, P<0.05 vscontrol). But the reverse potential of ICRAC occurred at voltage 0 mV in all cells.CONCLUSION: Berberine has inhibitory effects on potassium and calcium currents in isolated rat hepatocytes, which may be involved in hepatoprotection.

  1. Detection of Dichlorvos Adducts in a Hepatocyte Cell Line

    Science.gov (United States)

    2014-06-30

    G6019) was obtained from Sigma (St. Louis, MS), human actin gamma recombinant protein (ACTG1, H00000071) and lactate dehydrogenase A recombi - nant...microRNA and mRNA expression profiling analysis of dichlorvos cytotoxicity in porcine kidney epithelial PK15 cells. DNA Cell Biol. 2011, 30 (12), 1073...Dissociation of DDVP-induced DNA strand breaks from oxidative damage in isolated rat hepatocytes. Toxicology 1996, 108 (1−2), 49−56. (40) Clapp, C.; Portt, L

  2. Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Yeom, Chul-gon; Kim, Dong-il; Park, Min-jung; Choi, Joo-hee [College of Veterinary Medicine, Chonnam National University, Gwangju 500-757 (Korea, Republic of); Jeong, Jieun; Wi, Anjin; Park, Whoashig [Jeollanamdo Forest Resources Research Institute, Naju 520-833 (Korea, Republic of); Han, Ho-jae [College of Veterinary Medicine, Seoul National University, Seoul 151-741 (Korea, Republic of); Park, Soo-hyun, E-mail: parksh@chonnam.ac.kr [College of Veterinary Medicine, Chonnam National University, Gwangju 500-757 (Korea, Republic of)

    2015-06-05

    Previously, we reported that CARM1 undergoes ubiquitination-dependent degradation in renal podocytes. It was also reported that CARM1 is necessary for fasting-induced hepatic gluconeogenesis. Based on these reports, we hypothesized that treatment with insulin, a hormone typically present under the ‘fed’ condition, would inhibit gluconeogenesis via CARM1 degradation. HepG2 cells, AML-12 cells, and rat primary hepatocytes were treated with insulin to confirm CARM1 downregulation. Surprisingly, insulin treatment increased CARM1 expression in all cell types examined. Furthermore, treatment with insulin increased histone 3 methylation at arginine 17 and 26 in HepG2 cells. To elucidate the role of insulin-induced CARM1 upregulation, the HA-CARM1 plasmid was transfected into HepG2 cells. CARM1 overexpression did not increase the expression of lipogenic proteins generally increased by insulin signaling. Moreover, CARM1 knockdown did not influence insulin sensitivity. Insulin is known to facilitate hepatic proliferation. Like insulin, CARM1 overexpression increased CDK2 and CDK4 expression. In addition, CARM1 knockdown reduced the number of insulin-induced G2/M phase cells. Moreover, GFP-CARM1 overexpression increased the number of G2/M phase cells. Based on these results, we concluded that insulin-induced CARM1 upregulation facilitates hepatocyte proliferation. These observations indicate that CARM1 plays an important role in liver pathophysiology. - Highlights: • Insulin treatment increases CARM1 expression in hepatocytes. • CARM1 overexpression does not increase the expression of lipogenic proteins. • CARM1 knockdown does not influence insulin sensitivity. • Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation.

  3. Hepatocyte and Sertoli Cell Aquaporins, Recent Advances and Research Trends

    Directory of Open Access Journals (Sweden)

    Raquel L. Bernardino

    2016-07-01

    Full Text Available Aquaporins (AQPs are proteinaceous channels widespread in nature where they allow facilitated permeation of water and uncharged through cellular membranes. AQPs play a number of important roles in both health and disease. This review focuses on the most recent advances and research trends regarding the expression and modulation, as well as physiological and pathophysiological functions of AQPs in hepatocytes and Sertoli cells (SCs. Besides their involvement in bile formation, hepatocyte AQPs are involved in maintaining energy balance acting in hepatic gluconeogenesis and lipid metabolism, and in critical processes such as ammonia detoxification and mitochondrial output of hydrogen peroxide. Roles are played in clinical disorders including fatty liver disease, diabetes, obesity, cholestasis, hepatic cirrhosis and hepatocarcinoma. In the seminiferous tubules, particularly in SCs, AQPs are also widely expressed and seem to be implicated in the various stages of spermatogenesis. Like in hepatocytes, AQPs may be involved in maintaining energy homeostasis in these cells and have a major role in the metabolic cooperation established in the testicular tissue. Altogether, this information represents the mainstay of current and future investigation in an expanding field.

  4. Hepatocyte and Sertoli Cell Aquaporins, Recent Advances and Research Trends.

    Science.gov (United States)

    Bernardino, Raquel L; Marinelli, Raul A; Maggio, Anna; Gena, Patrizia; Cataldo, Ilaria; Alves, Marco G; Svelto, Maria; Oliveira, Pedro F; Calamita, Giuseppe

    2016-07-09

    Aquaporins (AQPs) are proteinaceous channels widespread in nature where they allow facilitated permeation of water and uncharged through cellular membranes. AQPs play a number of important roles in both health and disease. This review focuses on the most recent advances and research trends regarding the expression and modulation, as well as physiological and pathophysiological functions of AQPs in hepatocytes and Sertoli cells (SCs). Besides their involvement in bile formation, hepatocyte AQPs are involved in maintaining energy balance acting in hepatic gluconeogenesis and lipid metabolism, and in critical processes such as ammonia detoxification and mitochondrial output of hydrogen peroxide. Roles are played in clinical disorders including fatty liver disease, diabetes, obesity, cholestasis, hepatic cirrhosis and hepatocarcinoma. In the seminiferous tubules, particularly in SCs, AQPs are also widely expressed and seem to be implicated in the various stages of spermatogenesis. Like in hepatocytes, AQPs may be involved in maintaining energy homeostasis in these cells and have a major role in the metabolic cooperation established in the testicular tissue. Altogether, this information represents the mainstay of current and future investigation in an expanding field.

  5. Hepatocyte ABCA1 Deletion Impairs Liver Insulin Signaling and Lipogenesis

    Directory of Open Access Journals (Sweden)

    Chia-Chi C. Key

    2017-06-01

    Full Text Available Plasma membrane (PM free cholesterol (FC is emerging as an important modulator of signal transduction. Here, we show that hepatocyte-specific knockout (HSKO of the cellular FC exporter, ATP-binding cassette transporter A1 (ABCA1, leads to decreased PM FC content and defective trafficking of lysosomal FC to the PM. Compared with controls, chow-fed HSKO mice had reduced hepatic (1 insulin-stimulated Akt phosphorylation, (2 activation of the lipogenic transcription factor Sterol Regulatory Element Binding Protein (SREBP-1c, and (3 lipogenic gene expression. Consequently, Western-type diet-fed HSKO mice were protected from steatosis. Surprisingly, HSKO mice had intact glucose metabolism; they showed normal gluconeogenic gene suppression in response to re-feeding and normal glucose and insulin tolerance. We conclude that: (1 ABCA1 maintains optimal hepatocyte PM FC, through intracellular FC trafficking, for efficient insulin signaling; and (2 hepatocyte ABCA1 deletion produces a form of selective insulin resistance so that lipogenesis is suppressed but glucose metabolism remains normal.

  6. Homocysteine inhibits hepatocyte proliferation via endoplasmic reticulum stress.

    Directory of Open Access Journals (Sweden)

    Xue Yu

    Full Text Available Homocysteine is an independent risk factor for coronary, cerebral, and peripheral vascular diseases. Recent studies have shown that levels of homocysteine are elevated in patients with impaired hepatic function, but the precise role of homocysteine in the development of hepatic dysfunction is unclear. In this study, we examined the effect of homocysteine on hepatocyte proliferation in vitro. Our results demonstrated that homocysteine inhibited hepatocyte proliferation by up-regulating protein levels of p53 as well as mRNA and protein levels of p21(Cip1 in primary cultured hepatocytes. Homocysteine induced cell growth arrest in p53-positive hepatocarcinoma cell line HepG2, but not in p53-null hepatocarcinoma cell line Hep3B. A p53 inhibitor pifithrin-α inhibited the expression of p21(Cip1 and attenuated homocysteine-induced cell growth arrest. Homocysteine induced TRB3 expression via endoplasmic reticulum stress pathway, resulting in Akt dephosphorylation. Knock-down of endogenous TRB3 significantly suppressed the inhibitory effect of homocysteine on cell proliferation and the phosphorylation of Akt. LiCl reversed homocysteine-mediated cell growth arrest by inhibiting TRB3-mediated Akt dephosphorylation. These results demonstrate that both TRB3 and p21(Cip1 are critical molecules in the homocysteine signaling cascade and provide a mechanistic explanation for impairment of liver regeneration in hyperhomocysteinemia.

  7. Biomaterial Scaffolds with Biomimetic Fluidic Channels for Hepatocyte Culture

    Institute of Scientific and Technical Information of China (English)

    Xiao Li; Jiankang He; Yaxiong Liu; Qian Zhao; Wanquan Wu; Dichen Li; Zhongmin Jin

    2013-01-01

    Biomaterial scaffolds play an important role in maintaining the viability and biological functions of highly metabolic hepatocytes in liver tissue engineering.One of the major challenges involves building a complex microchannel network inside three-dimensional (3D) scaffolds for efficient mass transportation.Here we presented a biomimetic strategy to generate a microchannel network within porous biomaterial scaffolds by mimicking the vascular tree of rat liver.The typical parameters of the blood vessels were incorporated into the biomimetic design of the microchannel network such as branching angle and diameter.Silk fibroin-gelatin scaffolds with biomimetic vascular tree were fabricated by combining micromolding,freeze drying and 3D rolling techniques.The relationship between the micro-channeled design and flow pattern was revealed by a flow experiment,which indicated that the scaffolds with biomimetic vascular tree exhibited unique capability in improving mass transportation inside the 3D scaffold.The 3D scaffolds,preseeded with primary hepatocytes,were dynamically cultured in a bioreactor system.The results confirmed that the pre-designed biomimetic microchannel network facilitated the generation and expansion of hepatocytes.

  8. A fast and robust hepatocyte quantification algorithm including vein processing

    Directory of Open Access Journals (Sweden)

    Homeyer André

    2010-03-01

    Full Text Available Abstract Background Quantification of different types of cells is often needed for analysis of histological images. In our project, we compute the relative number of proliferating hepatocytes for the evaluation of the regeneration process after partial hepatectomy in normal rat livers. Results Our presented automatic approach for hepatocyte (HC quantification is suitable for the analysis of an entire digitized histological section given in form of a series of images. It is the main part of an automatic hepatocyte quantification tool that allows for the computation of the ratio between the number of proliferating HC-nuclei and the total number of all HC-nuclei for a series of images in one processing run. The processing pipeline allows us to obtain desired and valuable results for a wide range of images with different properties without additional parameter adjustment. Comparing the obtained segmentation results with a manually retrieved segmentation mask which is considered to be the ground truth, we achieve results with sensitivity above 90% and false positive fraction below 15%. Conclusions The proposed automatic procedure gives results with high sensitivity and low false positive fraction and can be applied to process entire stained sections.

  9. Hepatocyte and Sertoli Cell Aquaporins, Recent Advances and Research Trends

    Science.gov (United States)

    Bernardino, Raquel L.; Marinelli, Raul A.; Maggio, Anna; Gena, Patrizia; Cataldo, Ilaria; Alves, Marco G.; Svelto, Maria; Oliveira, Pedro F.; Calamita, Giuseppe

    2016-01-01

    Aquaporins (AQPs) are proteinaceous channels widespread in nature where they allow facilitated permeation of water and uncharged through cellular membranes. AQPs play a number of important roles in both health and disease. This review focuses on the most recent advances and research trends regarding the expression and modulation, as well as physiological and pathophysiological functions of AQPs in hepatocytes and Sertoli cells (SCs). Besides their involvement in bile formation, hepatocyte AQPs are involved in maintaining energy balance acting in hepatic gluconeogenesis and lipid metabolism, and in critical processes such as ammonia detoxification and mitochondrial output of hydrogen peroxide. Roles are played in clinical disorders including fatty liver disease, diabetes, obesity, cholestasis, hepatic cirrhosis and hepatocarcinoma. In the seminiferous tubules, particularly in SCs, AQPs are also widely expressed and seem to be implicated in the various stages of spermatogenesis. Like in hepatocytes, AQPs may be involved in maintaining energy homeostasis in these cells and have a major role in the metabolic cooperation established in the testicular tissue. Altogether, this information represents the mainstay of current and future investigation in an expanding field. PMID:27409609

  10. Adropin induction of lipoprotein lipase expression in tilapia hepatocytes.

    Science.gov (United States)

    Lian, Anji; Wu, Keqiang; Liu, Tianqiang; Jiang, Nan; Jiang, Quan

    2016-01-01

    The peptide hormone adropin plays a role in energy homeostasis. However, biological actions of adropin in non-mammalian species are still lacking. Using tilapia as a model, we examined the role of adropin in lipoprotein lipase (LPL) regulation in hepatocytes. To this end, the structural identity of tilapia adropin was established by 5'/3'-rapid amplification of cDNA ends (RACE). The transcripts of tilapia adropin were ubiquitously expressed in various tissues with the highest levels in the liver and hypothalamus. The prolonged fasting could elevate tilapia hepatic adropin gene expression, whereas no effect of fasting was observed on hypothalamic adropin gene levels. In primary cultures of tilapia hepatocytes, synthetic adropin was effective in stimulating LPL release, cellular LPL content, and total LPL production. The increase in LPL production also occurred with parallel rises in LPL gene levels. In parallel experiments, adropin could elevate cAMP production and up-regulate protein kinase A (PKA) and PKC activities. Using a pharmacological approach, cAMP/PKA and PLC/inositol trisphosphate (IP3)/PKC cascades were shown to be involved in adropin-stimulated LPL gene expression. Parallel inhibition of p38MAPK and Erk1/2, however, were not effective in these regards. Our findings provide, for the first time, evidence that adropin could stimulate LPL gene expression via direct actions in tilapia hepatocytes through the activation of multiple signaling mechanisms.

  11. Gene expression changes after hypoxic preconditioning in rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Wei Chen; Jiang-Feng Qiu; Zhi-Qi Zhang; Hai-Feng Luo; Joan Rosello-Catafau; Zhi-Yong Wu

    2006-01-01

    BACKGROUND: Hypoxic preconditioning can protect hepatocytes against hypoxic injury, but its mechanism has not been elucidated. The aim of this study was to proifle gene expression patterns involved in hypoxic preconditioning and probable mechanism at the level of gene expression. METHODS: Hepatocytes were divided into 2 groups:control group and hypoxic preconditioning group. Biotin-labeled cRNA from the control group and the hypoxic preconditioning group was hybridized by oligonucleotide microarray. Genes that were signiifcantly associated with hypoxic preconditioning were ifltered, and validated at the level of transcript expression. RESULTS: Forty-three genes with signiifcantly altered expression patterns were discovered and most of them had not been previously reported. Among these genes, genes encoding superoxide dismutase 2 (SOD2)and interleukin 10 (IL-10) in the hypoxic preconditioning group were conifrmed to be up-regulated with real-time quantitative PCR. CONCLUSIONS:Many cytokines are involved in hypoxic preconditioning and protect hepatocytes from hypoxia-reoxygenation injury, and the increase of oxygen free-radical scavengers and anti-inlfammatory factors may play a key role in this phenomenon. Diverse signal pathways are probably involved.

  12. Silver nanoparticles stimulate glycogenolysis in rainbow trout (Oncorhynchus mykiss) hepatocytes.

    Science.gov (United States)

    Massarsky, Andrey; Labarre, Justine; Trudeau, Vance L; Moon, Thomas W

    2014-02-01

    Silver nanoparticles (AgNPs) are found in many consumer products yet their biological effects on non-target aquatic organisms are yet to be fully understood. This research aimed to investigate the effects of AgNPs on cell signaling in rainbow trout (Oncorhynchus mykiss) hepatocytes. We focused on the β-adrenoreceptor (AR), which mediates glycogenolysis, and the glucocorticoid receptor (GCR), which mediates gluconeogenesis. These two receptors have been extensively studied in trout hepatocytes due to their key roles during the stress response to increase glucose availability (among other things), allowing the organisms to cope with the stressor. We show for the first time that AgNPs at a concentration of 1 μg/mL did not interfere with the function of either the β-AR or the GCR systems in rainbow trout hepatocytes, but at the concentration of 10 μg/mL AgNPs stimulated glycogenolysis which was apparently receptor-independent. This study suggests that AgNPs could affect hormone-regulated cell signaling pathways at a concentration of 10 μg/mL.

  13. Purinergic receptor functionality is necessary for infection of human hepatocytes by hepatitis delta virus and hepatitis B virus.

    Directory of Open Access Journals (Sweden)

    John M Taylor

    Full Text Available Hepatitis B virus (HBV and hepatitis delta virus (HDV are major sources of acute and chronic hepatitis. HDV requires the envelope proteins of HBV for the processes of assembly and infection of new cells. Both viruses are able to infect hepatocytes though previous studies have failed to determine the mechanism of entry into such cells. This study began with evidence that suramin, a symmetrical hexasulfated napthylurea, could block HDV entry into primary human hepatocytes (PHH and was then extrapolated to incorporate findings of others that suramin is one of many compounds that can block activation of purinergic receptors. Thus other inhibitors, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS and brilliant blue G (BBG, both structurally unrelated to suramin, were tested and found to inhibit HDV and HBV infections of PHH. BBG, unlike suramin and PPADS, is known to be more specific for just one purinergic receptor, P2X7. These studies provide the first evidence that purinergic receptor functionality is necessary for virus entry. Furthermore, since P2X7 activation is known to be a major component of inflammatory responses, it is proposed that HDV and HBV attachment to susceptible cells, might also contribute to inflammation in the liver, that is, hepatitis.

  14. Catalytic diversity in self-propagating peptide assemblies

    Science.gov (United States)

    Omosun, Tolulope O.; Hsieh, Ming-Chien; Childers, W. Seth; Das, Dibyendu; Mehta, Anil K.; Anthony, Neil R.; Pan, Ting; Grover, Martha A.; Berland, Keith M.; Lynn, David G.

    2017-08-01

    The protein-only infectious agents known as prions exist within cellular matrices as populations of assembled polypeptide phases ranging from particles to amyloid fibres. These phases appear to undergo Darwinian-like selection and propagation, yet remarkably little is known about their accessible chemical and biological functions. Here we construct simple peptides that assemble into well-defined amyloid phases and define paracrystalline surfaces able to catalyse specific enantioselective chemical reactions. Structural adjustments of individual amino acid residues predictably control both the assembled crystalline order and their accessible catalytic repertoire. Notably, the density and proximity of the extended arrays of enantioselective catalytic sites achieve template-directed polymerization of new polymers. These diverse amyloid templates can now be extended as dynamic self-propagating templates for the construction of even more complex functional materials.

  15. Hepatitis B e antigen polypeptides isolated from sera of individuals infected with hepatitis B virus: comparison with HBeAg polypeptide derived from Dane particles.

    Science.gov (United States)

    Takahashi, K; Imai, M; Gotanda, T; Sano, T; Oinuma, A; Mishiro, S; Miyakawa, Y; Mayumi, M

    1980-09-01

    Hepatitis B e antigen (HBeAg) occurs in the serum of individuals infected with hepatitis B virus both free and in association with IgG. Utilizing a succession of steps involving salt precipitation, affinity chromatography, ion-exchange chromatography and isoelectrofocusing, we isolated free and IgG-bound forms of HBeAg from the sera of infected individuals with an overall gain in specific activity of 3000-fold and 540-fold, respectively. Polypeptide profiles of purified HBeAg preparations were studied by SDS-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol. Both free and IgG-bound preparations revealed polypeptides with mol. wt. of 15500 (P15.5) and 16 500 (P16.5), and HBeAg activity was detected corresponding to their positions. The HBeAg polypeptides (P15.5/16.5) derived from sera were physicochemically different from the two polypeptides with HBeAg activity (P19 and P45) liberated from Dane particle cores by the conventional method involving incubation with Nonidet P40 and 2-mercaptoethanol. However, when core particles were prepared in the presence of a proteolytic enzyme, in addition to Nonidet P40 and 2-mercaptoethanol, they gave rise to HBeAg polypeptides with mol. wt. of 31000 (P31) and 15 500. Furthermore, P31 split into P15.5 when heated at 100 degrees C for 2 min. On the basis of these results, P15.5 may be assumed to be the essential polypeptide bearing HBeAg activity in the serum and also in Dane particles.

  16. Pericarp polypeptides and SRAP markers associated with fruit quality traits in an interspecific tomato backcross.

    Science.gov (United States)

    Pereira da Costa, J H; Rodríguez, G R; Pratta, G R; Picardi, L A; Zorzoli, R

    2014-01-24

    The aim of this study was to detect polypeptides and genomic regions associated with fruit quality traits in a backcross generation using as parent the Argentinean cultivated tomato Caimanta of Solanum lycopersicum and the wild accession LA722 of S. pimpinellifolium. We tested two types of molecular marker: polypeptide profile (at two ripening stages, mature green and red ripe) and SRAP (sequence-related amplified polymorphism). A polypeptide of 45 kDa present in the wild parents at the mature green stage was associated with larger fruit and long shelf life. Some amplification fragments from SRAP markers were associated with more than one quality trait such as fruit color, firmness, titratable acidity, and fruit soluble solids content. This study demonstrated for the first time the usefulness of the polypeptide profiles of pericarp and SRAP markers in finding associations with quality fruit traits in a tomato backcross generation.

  17. Biosynthesis of metal-binding polypeptides and their precursors in response to cadmium in Datura innoxia

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, P.J.; Delhaize, E.; Kuske, C.R.

    1991-01-01

    Metal-tolerant Datura innoxia cells synthesize large amounts of a class of metal-binding polypeptides, poly({gamma}-glutamylcysteinyl) glycines (({gamma}-EC){sub n}G, n=2-5), when exposed to Cd. These polypeptides have a high affinity for Cd (2) and certain other metal ions and are thought to play a role in metal tolerance in higher plants. ({gamma}-EC){sub n}G is biosynthetically derived from glutathione. Therefore, the response of Datura cells to Cd must include an increase in production of glutathione and its precursors, since cells rapidly accumulate very high concentrations of these metal-binding polypeptides. The biosynthesis of ({gamma}-EC){sub n}Gs, glutathione, and cysteine in response to Cd exposure is described. The physiological significance of the synthesis of these polypeptides and their precursors and its relevance to Cd tolerance and metal homeostasis are discussed. 34 refs., 6 figs., 1 tab.

  18. Theoretical investigations on model ternary polypeptides using genetic algorithm—Some new results

    Science.gov (United States)

    Arora, Vinita; Bakhshi, A. K.

    2011-04-01

    Using genetic algorithm (GA) model ternary polypeptides containing glycine, alanine and serine in β-pleated conformation have been theoretically investigated. In designing, the criterion to attain the optimum solution at the end of GA run is minimum band gap and maximum delocalization in the polypeptide chain. Ab initio results obtained using Clementi's minimal basis set are used as input. Effects of (i) change of basis set from minimal to double zeta, (ii) change in secondary structure from β-pleated to α-helical, (iii) presence of solvation shell and (iv) binding of H + and Li + ions to peptide group on the resulting solution as well as on electronic structure and conduction properties of polypeptides are investigated. A comparison is drawn between results obtained for the two cationic adducts. The protonated adduct is expected to withdraw more negative charge from the polypeptide chain due to smaller size of H + and is found to have high electron affinity compared to Li + adduct.

  19. Freshly isolated hepatocyte transplantation in acetaminophen-induced hepatotoxicity model in rats

    Directory of Open Access Journals (Sweden)

    Daniela Rodrigues

    2012-12-01

    Full Text Available CONTEXT: Hepatocyte transplantation is an attractive therapeutic modality for liver disease as an alternative for orthotopic liver transplantation. OBJECTIVE: The aim of the current study was to investigate the feasibility of freshly isolated rat hepatocyte transplantation in acetaminophen-induced hepatotoxicity model. METHODS: Hepatocytes were isolated from male Wistar rats and transplanted 24 hours after acetaminophen administration in female recipients. Female rats received either 1x10(7 hepatocytes or phosphate buffered saline through the portal vein or into the spleen and were sacrificed after 48 hours. RESULTS: Alanine aminotransferase levels measured within the experiment did not differ between groups at any time point. Molecular analysis and histology showed presence of hepatocytes in liver of transplanted animals injected either through portal vein or spleen. CONCLUSION: These data demonstrate the feasibility and efficacy of hepatocyte transplantation in the liver or spleen in a mild acetaminophen-induced hepatotoxicity model.

  20. Effects of quercetin on hepatocyte stimulating factor production by mouse peritoneal macrophases

    Institute of Scientific and Technical Information of China (English)

    周斌; 张俊平; 刘宏; 殷明; 钱定华

    2003-01-01

    Objective: To study the effects of quercetin on hepatocyte stimulating factor production from mouse peritoneal macrophages. Methods: Hepatocyte stimulating factor was evaluated by the amount of fibrinogen synthesized in Hep3B cells. Interleukin-6 activity was measured by B9 cell proliferation methyl thiazolyl tetrazolium colorimetric method. Hep3B cell supernatant fibrinogen was quantitated with ELISA. Results: LPS induced the synthesis of hepatocyte stimulating factor in mouse peritoneal macrophages, and hepatocyte stimulating factor promotes the synthesis of fibrinogen from Hep3B cells. Quercetin(5 to 40 μmol/ L)inhibited the synthesis of hepatocyte stimulating factor stimulated by LPS. Quercetin(5 to 20 μmol/ L) inhibited release of interleukin-6 from mouse peritoneal macrophages induced by 0.5 g/ L fibrin fibrinogen degradation products. Conclusion: Quercetin inhibits the synthesis of hepatocyte stimulating factor in macrophages.

  1. Effect of Concentrated Fibroblast-Conditioned Media on In Vitro Maintenance of Rat Primary Hepatocyte.

    Directory of Open Access Journals (Sweden)

    Dayeong Jeong

    Full Text Available The effects of concentrated fibroblast-conditioned media were tested to determine whether hepatocyte function can be maintained without direct contact between hepatocytes and fibroblasts. Primary rat hepatocytes cultured with a concentrated conditioned media of NIH-3T3 J2 cell line (final concentration of 55 mg/ml showed significantly improved survival and functions (albumin and urea compared to those of control groups. They also showed higher expression levels of mRNA, albumin and tyrosine aminotransferase compared to hepatocyte monoculture. The results suggest that culture with concentrated fibroblast-conditioned media could be an easy method for in vitro maintenance of primary hepatocytes. They also could be contribute to understand and analyze co-culture condition of hepatocyte with stroma cells.

  2. Theoretical investigations on model ternary polypeptides using genetic algorithm-Some new results

    Energy Technology Data Exchange (ETDEWEB)

    Arora, Vinita [Department of Chemistry, University of Delhi, Delhi 110 007 (India); Bakhshi, A.K., E-mail: akbakhshi2000@yahoo.com [Department of Chemistry, University of Delhi, Delhi 110 007 (India)

    2011-04-28

    Graphical abstract: Model ternary polypeptide chains consisting of glycine, alanine and serine amino acids as repeat units in anti-parallel {beta}-pleated sheet conformation have been theoretically investigated and designed using the genetic algorithm. The optimum solution or the polypeptide chain being searched for using the algorithm is the one having minimum band gap and maximum electronic delocalization in the polypeptide chain. The effects of (i) change of basis set from minimal to double zeta, (ii) change in secondary structure from {beta}-pleated to {alpha}-helical, (iii) presence of solvation shell, and (iv) binding of ions such as H{sup +} and Li{sup +} to the peptide group on the resulting optimum solution as well as on electronic structure and conduction properties of polypeptides have been investigated taking the ab initio Hartree-Fock crystal orbital results as input. The band gap value was also found to decrease in presence of a solvation shell, in presence of cations in the vicinity of the polypeptide chain as well as with the use of an improved basis set. Highlights: {yields} GA has been used for theoretical tailoring of aperiodic ternary polypeptides. {yields} Band gap of polypeptide chain decreases in presence of solvation shell. {yields} Band gap decreases in presence of cations in the vicinity of the chain. {yields} H{sup +} ion acts as a strong electron acceptor than Li{sup +} ion due to smaller size. - Abstract: Using genetic algorithm (GA) model ternary polypeptides containing glycine, alanine and serine in {beta}-pleated conformation have been theoretically investigated. In designing, the criterion to attain the optimum solution at the end of GA run is minimum band gap and maximum delocalization in the polypeptide chain. Ab initio results obtained using Clementi's minimal basis set are used as input. Effects of (i) change of basis set from minimal to double zeta, (ii) change in secondary structure from {beta}-pleated to {alpha

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

    Directory of Open Access Journals (Sweden)

    Joensuu Jussi J

    2009-08-01

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

  4. Piceatannol increases the expression of hepatocyte growth factor and IL-10 thereby protecting hepatocytes in thioacetamide-induced liver fibrosis.

    Science.gov (United States)

    Abd-Elgawad, Hazem; Abu-Elsaad, Nashwa; El-Karef, Amr; Ibrahim, Tarek

    2016-07-01

    Piceatannol is a polyphenolic analog of resveratrol that selectively inhibits the non-receptor tyrosine kinase-Syk. This study investigates the potential ability of piceatannol to attenuate liver fibrosis and protect hepatocytes from injury. Thioacetamide was injected in adult male mice (100 mg/kg, i.p., 3 times/week) for 8 weeks. Piceatannol (1 or 5 mg/kg per day) was administered by oral gavage during the last 4 weeks. Liver function biomarkers, tissue malondialdehyde (MDA), cytokeratin-18 (CK18), hepatocyte growth factor (HGF), and interleukin-10 (IL-10) were measured. Necroinflammation, fibrosis, expression of transforming growth factor (TGF)-β1, and α-smooth muscle actin (SMA) were scored by histopathological examination and immunohistochemistry. Obtained results showed ability of piceatannol (1 mg/kg) to restore liver function and reduce inflammation. It significantly (p IL-10. It can be concluded that piceatannol at low dose can inhibit TGF-β1 induced hepatocytes apoptosis and exerts an anti-inflammatory effect attenuating fibrosis progression.

  5. Identification and characterization of salt-inducible polypeptide in Paenibacillus sp., a moderately halophilic bacterium.

    Science.gov (United States)

    Sokhansanj, Ashrafaddin; Karkhane, Ali Asghar; Jazii, Ferdous Rastgar

    2005-11-01

    In response to salt, Paenibacillus sp. strain XII expresses a 21.4 kDa polypeptide. N-terminal sequencing and sequence homology analysis indicate homology between the N-terminal sequence of the polypeptide and a segment of the N-terminus of the spore coat associated protein CotN of Oceanobacillus iheyensis, an extremely halotolerant bacteria of the deep-sea.

  6. Metal ion-dependent, reversible, protein filament formation by designed beta-roll polypeptides

    Directory of Open Access Journals (Sweden)

    Campbell Robert L

    2007-10-01

    Full Text Available Abstract Background A right-handed, calcium-dependent β-roll structure found in secreted proteases and repeat-in-toxin proteins was used as a template for the design of minimal, soluble, monomeric polypeptides that would fold in the presence of Ca2+. Two polypeptides were synthesised to contain two and four metal-binding sites, respectively, and exploit stacked tryptophan pairs to stabilise the fold and report on the conformational state of the polypeptide. Results Initial analysis of the two polypeptides in the presence of calcium suggested the polypeptides were disordered. The addition of lanthanum to these peptides caused aggregation. Upon further study by right angle light scattering and electron microscopy, the aggregates were identified as ordered protein filaments that required lanthanum to polymerize. These filaments could be disassembled by the addition of a chelating agent. A simple head-to-tail model is proposed for filament formation that explains the metal ion-dependency. The model is supported by the capping of one of the polypeptides with biotin, which disrupts filament formation and provides the ability to control the average length of the filaments. Conclusion Metal ion-dependent, reversible protein filament formation is demonstrated for two designed polypeptides. The polypeptides form filaments that are approximately 3 nm in diameter and several hundred nm in length. They are not amyloid-like in nature as demonstrated by their behaviour in the presence of congo red and thioflavin T. A capping strategy allows for the control of filament length and for potential applications including the "decoration" of a protein filament with various functional moieties.

  7. EXPRESSED PROTEIN LIGATION. A NEW TOOL FOR THE BIOSYNTHESIS OF CYCLIC POLYPEPTIDES

    Energy Technology Data Exchange (ETDEWEB)

    Kimura, R; Camarero, J A

    2004-11-11

    The present paper reviews the use of expressed protein ligation for the biosynthesis of backbone cyclic polypeptides. This general method allows the in vivo and in vitro biosynthesis of cyclic polypeptides using recombinant DNA expression techniques. Biosynthetic access to backbone cyclic peptides opens the possibility to generate cell-based combinatorial libraries that can be screened inside living cells for their ability to attenuate or inhibit cellular processes.

  8. Purification and Primary Structure Determination of a Novel Polypeptide Isolated from Mistletoe Viscum coloratum

    Institute of Scientific and Technical Information of China (English)

    Jing Lin KONG; Xiu Bao DU; Chong Xu FAN; Ying CAO; Hui JIANG; Jian Fu XU; Xiao Jun ZHENG

    2004-01-01

    A novel polypeptide was isolated from mistletoe Viscum coloratum. The primary structure of the polypeptide 'named viscotoxin B2' was determined to be KSCCKNTTGRNIYNT CRFAGGSRERCAKLSGCKIISASTCPSDYPK by Edman degradation. Viscotoxin B2 shared high sequence homology with viscotoxins isolated from Viscum album. Pharmacological experiments showed that viscotoxin B2 had distinct cytotoxic activity on tumor cells. Viscotoxin B2 could be used as a leading compound in cancer therapy.

  9. Glucagon-like peptide 1 and glucose-dependent insulinotropic polypeptide: new advances

    DEFF Research Database (Denmark)

    Asmar, Meena; Holst, Jens Juul

    2010-01-01

    This article highlights recent advances in our understanding of glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) physiology and their various sites of action beyond the incretin effect.......This article highlights recent advances in our understanding of glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) physiology and their various sites of action beyond the incretin effect....

  10. Species-specific toxicity of troglitazone on rats and human by gel entrapped hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Chong [College of Materials Science and Chemical Engineering, Zhejiang University, Zhejiang 310027 (China); Meng, Qin, E-mail: mengq@zju.edu.cn [College of Materials Science and Chemical Engineering, Zhejiang University, Zhejiang 310027 (China); Zhang, Guoliang [Institute of Biological and Environmental Engineering, Zhejiang University of Technology, Zhejiang 310012 (China)

    2012-01-01

    Troglitazone, despite passing preclinical trials on animals, was shortly withdrawn from market due to its severe hepatotoxicity in clinic. As rat hepatocyte monolayer consistently showed sensitive troglitazone toxicity as human hepatocyte monolayer in contrast to the species-specific toxicity in vivo, this paper utilized both hepatocytes in three-dimensional culture of gel entrapment to reflect the species difference on hepatotoxicity. Rat hepatocytes in gel entrapment did not show obvious cellular damage even under a long-term exposure for 21 days while gel entrapped human hepatocytes significantly displayed oxidative stress, steatosis, mitochondrial damage and cell death at a short exposure for 4 days. As a result, the detected species-specific toxicity of troglitazone between gel entrapped rat and human hepatocytes consisted well with the situation in vivo but was in a sharp contrast to the performance of two hepatocytes by monolayer culture. Such contradictory toxicity of rat hepatocytes between monolayer and gel entrapment culture could be explained by the fact that troglitazone was cleared more rapidly in gel entrapment than in monolayer culture. Similarly, the differential clearance of troglitazone in rat and human might also explain its species-specific toxicity. Therefore, gel entrapment of hepatocytes might serve as a platform for evaluation of drug toxicity at early stage of drug development by reducing costs, increasing the likelihood of clinical success and limiting human exposure to unsafe drugs. -- Highlights: ► Species-specific toxicity of troglitazone reflected by rat/human hepatocytes ► 3D hepatocytes in 21 days’ long-term culture used for drug hepatotoxicity ► Oversensitive toxicity in hepatocyte monolayer by slow troglitazone clearance.

  11. TRANSPLANTATION OF HEPATOCYTES AS THE METHOD OF TREATMENT OF LIVER FAILURE: EXPERIMENTAL AND CLINICAL EXPERIENCE

    Directory of Open Access Journals (Sweden)

    M. Y. Shagidulin

    2010-01-01

    Full Text Available For correction and treatment of liver failure before liver transplantation were proposed severe methods such as: extracorporal devices, transplantation of hepatocytes and implanted tissue-engineering units. The function of healthy hepatocytes presumes to stabilize the state of patients with chronic liver diseases and to wait a donor organ transplantation. In this review the results of experimental and clinical therapy of liver diseases by method of hepatocyte transplantation were summarized. 

  12. Hepatocytes isolated from neoplastic liver-immunomagnetic purging as a new source for transplantation

    Institute of Scientific and Technical Information of China (English)

    Aravin Gunasegaram; Javed Akhter; Peng Yao; Loreena A Johnson; Stephen M Riodan; David L Morris

    2008-01-01

    AIM: To investigate whether hepatocytes isolated from macroscopically normal liver during hepatic resection for neoplasia could provide a novel source of healthy hepatocytes, including the development of reliable protocols for malignant cells removal from the hepatocyte preparation.METHODS: Hepatocytes were procured from resected liver of 18 patients with liver tumors using optimised digestion and cell-enrichment protocols. Suspensions of various known quantities of the HT-29 tumor cell line and patient hepatocytes were treated or not with Ep-CAM-antibody-coated immunomagnetic beads in order to investigate the efficacy of tumor-purging by immunomagnetic depletion, using a semi-quantitative RT-PCR method developed to detect tumor cells.Immunomagnetic bead-treated or bead-untreated tumor cell-hepatocyte suspensions were transplanted intra-peritoneally in Balb/C nude mice to assess the rates of tumor development.RESULTS: Mean viable hepatocyte yield was 9.3 x 106 cells per gram of digested liver with mean viability of 70.5%. Immunomagnetic depletion removed tumor cells to below the RT-PCR detection-threshold of 1 tumor cell in 106 hepatocytes, representing a maximum tumor purging efficacy of greater than 400000-fold.Transplanted, immunomagnetic bead-purged tumor cell-hepatocyte suspensions did not form peritoneal tumors in Balb/C nude mice. Co-transplantation of hepatocytes with tumor cells did not increase tumorigenesis of the tumor cells.CONCLUSION: Immunomagnetic depletion appears to be an effective method of purging contaminating tumor cells to below threshold for likely tumorigenesis.Along with improved techniques for isolation of large numbers of viable hepatocytes, normal liver resected for neoplasia has potential as another clinically useful source of hepatocytes for transplantation.

  13. Heparanase enhances early hepatocyte inclusion in the recipient liver after transplantation in partially hepatectomized rats.

    Science.gov (United States)

    Tsiperson, Vladislav; Goldshmidt, Orit; Ilan, Neta; Shoshany, Gideon; Vlodavsky, Israel; Veitsman, Ella; Baruch, Yaacov

    2008-03-01

    Hepatocyte transplantation is an emerging approach for the treatment of liver diseases. However, broad clinical application of this method has been limited by restricted source of cells and low efficiency of cell integration within the recipient liver. Heparanase cleaves heparan sulfate proteoglycans in the extracellular matrix and basement membrane, activity that affects cellular invasion associated with cancer metastasis and inflammation. This activity has a multifunctional effect on cell-cell interaction, cell adhesion, and angiogenesis. All these factors are important for successful integration of transplanted hepatocytes. Male donor hepatocytes pretreated with heparanase or untreated were transplanted into recipient female rat spleen following partial hepatectomy. Engraftment efficacy was evaluated by PCR for Y chromosome, histology and PCNA, and heparanase immunohistochemistry. In addition, proliferative activity of hepatocytes in vitro was determined by bromodeoxyuridine immunostaining. The number of heparanase-treated cells detected in the recipient liver was significantly increased three- to fivefold within 24-48 h posttransplantation and twofold at 14 days compared with untreated cells. The transplanted hepatocytes treated with heparanase were clearly seen inside portal vein radicles as cell aggregates up to 72 h posttransplantation. The number of portal radicles filled with heparanase-treated hepatocytes was increased compared to control early after transplantation. Heparanase treatment enhanced hepatocyte and sinusoidal endothelial cell proliferation in the liver, and hepatocyte proliferation within the spleen tissue. Preliminary in vitro studies with isolated hepatocytes treated with heparanase showed increased proliferative activity within 24-48 h of cell culture. These results suggest that preincubation of hepatocytes with heparanase increases the presence of hepatocytes within the recipient liver early following cell transplantation and stimulates

  14. Freshly isolated hepatocyte transplantation in acetaminophen-induced hepatotoxicity model in rats

    OpenAIRE

    Daniela Rodrigues; Themis Reverbel Da Silveira; Ursula Matte

    2012-01-01

    CONTEXT: Hepatocyte transplantation is an attractive therapeutic modality for liver disease as an alternative for orthotopic liver transplantation. OBJECTIVE: The aim of the current study was to investigate the feasibility of freshly isolated rat hepatocyte transplantation in acetaminophen-induced hepatotoxicity model. METHODS: Hepatocytes were isolated from male Wistar rats and transplanted 24 hours after acetaminophen administration in female recipients. Female rats received either 1x10(7) ...

  15. FGF19-induced Hepatocyte Proliferation Is Mediated through FGFR4 Activation

    OpenAIRE

    Wu, Xinle; Ge, Hongfei; Lemon, Bryan; Vonderfecht, Steven; Weiszmann, Jennifer; Hecht, Randy; Gupte, Jamila; Hager, Todd; Wang, Zhulun; Lindberg, Richard; Li, Yang

    2009-01-01

    FGF19 and FGF21, unique members of the fibroblast growth factor (FGF) family, are hormones that regulate glucose, lipid, and energy homeostasis. Increased hepatocyte proliferation and liver tumor formation have also been observed in FGF19 transgenic mice. Here, we report that, in contrast to FGF19, FGF21 does not induce hepatocyte proliferation in vivo. To identify the mechanism for FGF19-induced hepatocyte proliferation, we explored similarities and differences in receptor specificity betwee...

  16. Influence of the synthetic polypeptide c25-mms6 on cobalt ferrite nanoparticle formation

    Energy Technology Data Exchange (ETDEWEB)

    Wolff, Annalena, E-mail: awolff@physik.uni-bielefeld.de [Bielefeld University, Department of Physics (Germany); Frese, Katrin; Wissbrock, Marco [Bielefeld University, Department of Chemistry (Germany); Eckstaedt, Katrin [Bielefeld University, Department of Physics (Germany); Ennen, Inga; Hetaba, Walid; Loeffler, Stefan [Technische Universitaet Wien, Institut fuer Festkoerperphysik (Austria); Regtmeier, Anna; Thomas, Patrick [Bielefeld University, Department of Physics (Germany); Sewald, Norbert [Bielefeld University, Department of Chemistry (Germany); Schattschneider, Peter [Technische Universitaet Wien, Service Center fuer Elektronenmikroskopie (Austria); Huetten, Andreas [Bielefeld University, Department of Physics (Germany)

    2012-10-15

    Nanoparticle syntheses utilizing biomimetic approaches have advanced in recent years. Polypeptides, with their ability to influence inorganic crystal growth, are a topic of great interest. Their effect on the particle formation has not been completely understood yet. Here we report a bioinspired synthesis of cobalt ferrite nanoparticles carried out in vitro under mild conditions using a short, synthetic polypeptide c25-mms6. The influence of c25-mms6 on the nanoparticle formation was investigated by comparing the particles synthesized with the polypeptide to particles synthesized under equivalent conditions without c25-mms6. A separation into D{sub small,av} = 10 nm small, superparamagnetic spheres and D{sub big,av} = 48 nm disc-like single-domain particles was observed. Non-stoichiometric cobalt ferrite particles with a shape-dependent stoichiometry were produced in the polypeptide-free synthesis. Stoichiometric D{sub small,av} = 10 nm CoFe{sub 2}O{sub 4} spheres and D{sub big,av} = 60-70 nm Co{sub 2}FeO{sub 4} ferromagnetic discs were obtained in the polypeptide-enhanced synthesis. The results indicate that the polypeptide acts as a catalyst during the multistep biomineralization process and allows the formation of stoichiometric phases which cannot be synthesized at room temperature using conventional bottom-up syntheses.

  17. New Kunitz-Type HCRG Polypeptides from the Sea Anemone Heteractis crispa

    Directory of Open Access Journals (Sweden)

    Irina Gladkikh

    2015-09-01

    Full Text Available Sea anemones are a rich source of Kunitz-type polypeptides that possess not only protease inhibitor activity, but also Kv channels toxicity, analgesic, antihistamine, and anti-inflammatory activities. Two Kunitz-type inhibitors belonging to a new Heteractis crispa RG (HCRG polypeptide subfamily have been isolated from the sea anemone Heteractis crispa. The amino acid sequences of HCRG1 and HCRG2 identified using the Edman degradation method share up to 95% of their identity with the representatives of the HCGS polypeptide multigene subfamily derived from H. crispa cDNA. Polypeptides are characterized by positively charged Arg at the N-terminus as well as P1 Lys residue at their canonical binding loop, identical to those of bovine pancreatic trypsin inhibitor (BPTI. These polypeptides are shown by our current evidence to be more potent inhibitors of trypsin than the known representatives of the HCGS subfamily with P1Thr. The kinetic and thermodynamic characteristics of the intermolecular interactions between inhibitors and serine proteases were determined by the surface plasmon resonance (SPR method. Residues functionally important for polypeptide binding to trypsin were revealed using molecular modeling methods. Furthermore, HCRG1 and HCRG2 possess anti-inflammatory activity, reducing tumor necrosis factor-α (TNF-α and interleukin 6 (IL-6 secretions, as well as proIL-1β expression in lipopolysaccharide (LPS-activated macrophages. However, there was no effect on nitric oxide (NO generation.

  18. The Mitochondrial Peptidase Pitrilysin Degrades Islet Amyloid Polypeptide in Beta-Cells.

    Directory of Open Access Journals (Sweden)

    Hanjun Guan

    Full Text Available Amyloid formation and mitochondrial dysfunction are characteristics of type 2 diabetes. The major peptide constituent of the amyloid deposits in type 2 diabetes is islet amyloid polypeptide (IAPP. In this study, we found that pitrilysin, a zinc metallopeptidase of the inverzincin family, degrades monomeric, but not oligomeric, islet amyloid polypeptide in vitro. In insulinoma cells when pitrilysin expression was decreased to 5% of normal levels, there was a 60% increase in islet amyloid polypeptide-induced apoptosis. In contrast, overexpression of pitrilysin protects insulinoma cells from human islet amyloid polypeptide-induced apoptosis. Since pitrilysin is a mitochondrial protein, we used immunofluorescence staining of pancreases from human IAPP transgenic mice and Western blot analysis of IAPP in isolated mitochondria from insulinoma cells to provide evidence for a putative intramitochondrial pool of IAPP. These results suggest that pitrilysin regulates islet amyloid polypeptide in beta cells and suggest the presence of an intramitochondrial pool of islet amyloid polypeptide involved in beta-cell apoptosis.

  19. Algorithm to design inhibitors using stereochemically mixed l,d polypeptides: Validation against HIV protease.

    Science.gov (United States)

    Gupta, Pooja; Durani, Susheel

    2015-11-01

    Polypeptides have potential to be designed as drugs or inhibitors against the desired targets. In polypeptides, every chiral α-amino acid has enantiomeric structural possibility to become l or d amino acids and can be used as design monomer. Among the various possibilities, use of stereochemistry as a design tool has potential to determine both functional specificity and metabolic stability of the designed polypeptides. The polypeptides with mixed l,d amino acids are a class of peptidomimitics, an attractive drug like molecules and also less susceptible to proteolytic activities. Therefore in this study, a three step algorithm is proposed to design the polypeptides against desired drug targets. For this, all possible configurational isomers of mixed l,d polyleucine (Ac-Leu8-NHMe) structure were randomly modeled with simulated annealing molecular dynamics and the resultant library of discrete folds were scored against HIV protease as a model target. The best scored folds of mixed l,d structures were inverse optimized for sequences in situ and the resultant sequences as inhibitors were validated for conformational integrity using molecular dynamics. This study presents and validates an algorithm to design polypeptides of mixed l,d structures as drugs/inhibitors by inverse fitting them as molecular ligands against desired target.

  20. Purification and Characterization of a Novel Tetradecapeptide from Ginseng Polypeptides with Enhancing Memory Activity for Mice

    Institute of Scientific and Technical Information of China (English)

    LUO Hao-ming; JIANG Rui-zhi; YANG Xiao-hong; CHEN Ying-hong; HONG Tie; WANG Ying

    2013-01-01

    Aiming at isolating and investigating the active ingredients of the aqueous extract from Panax ginseng which showed enhancing memory activity,the authors characterized one ingredient.To separate the oligosaccharides and polypeptides,a DEAE-Sephadex A-50 colum was used.The enhanced memory activity in mice was studied by Mirros water maze tesk in mice.The dose of oligosacchrides,polypeptides or Piracetam was 30 mg/kg per day with intraperitoneal administration.The oligosaccharides did not show enhancing memory effect,but polypeptides did show.This result demonstrates that the active ingredients of the aqueous extract from Panax ginseng which showed enhancing memory effect was polypeptides.The purification of the polypeptides was performed on a Sephadex G-25 column.A novel tetradecapeptide was purified from the polypeptides and its structure was determined by liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry(LC-ESI-Q-TOF-MS) with the amino acid sequence of Lys-Ser-Leu-Thr-Leu-Thr-Ser-Ser-Leu-Ser-Tyr-Thr-Asp-Ser.

  1. Synthesis of Tacaribe virus polypeptides in an in vitro coupled transcription and translation system.

    Science.gov (United States)

    Boersma, D P; Compans, R W

    1985-04-01

    We have analyzed polypeptides synthesized in a coupled in vitro transcription and translation system in response to detergent-disrupted Tacaribe virus. Analysis of the major Tacaribe virus-specified product by two-dimensional polyacrylamide gel electrophoresis indicated that it had an isoelectric point similar to that of the Tacaribe nucleocapsid polypeptide N; however, the in vitro product had an approximate mol. wt. of 73 000, compared to a mol. wt. of 68 000 for the N protein. The 73 000 dalton product was found to yield proteolytic cleavage products with similar electrophoretic mobilities to those obtained from the virion P and N proteins. These results, as well as pulse-chase experiments in Tacaribe virus-infected cells, suggest that a 73 000 dalton polypeptide may be processed to yield the N polypeptide. The polypeptides synthesized in the coupled system depended on the amount and type of virus added; addition of purified Shark River (SR) virus, a member of the Patois group of bunyaviruses, resulted in synthesis of a polypeptide of mol. wt. 22 000 which corresponds to the SR nucleocapsid protein.

  2. Cholesterol Enhances the Toxic Effect of Ethanol and Acetaldehyde in Primary Mouse Hepatocytes

    Directory of Open Access Journals (Sweden)

    Anayelly López-Islas

    2016-01-01

    Full Text Available Obesity and alcohol consumption are risk factors for hepatic steatosis, and both commonly coexist. Our objective was to evaluate the effect of ethanol and acetaldehyde on primary hepatocytes obtained from mice fed for two days with a high cholesterol (HC diet. HC hepatocytes increased lipid and cholesterol content. HC diet sensitized hepatocytes to the toxic effect of ethanol and acetaldehyde. Cyp2E1 content increased with HC diet, as well as in those treated with ethanol or acetaldehyde, while the activity of this enzyme determined in microsomes increased in the HC and in all ethanol treated hepatocytes, HC and CW. Oxidized proteins were increased in the HC cultures treated or not with the toxins. Transmission electron microscopy showed endoplasmic reticulum (ER stress and megamitochondria in hepatocytes treated with ethanol as in HC and the ethanol HC treated hepatocytes. ER stress determined by PERK content was increased in ethanol treated hepatocytes from HC mice and CW. Nuclear translocation of ATF6 was observed in HC hepatocytes treated with ethanol, results that indicate that lipids overload and ethanol treatment favor ER stress. Oxidative stress, ER stress, and mitochondrial damage underlie potential mechanisms for increased damage in steatotic hepatocyte treated with ethanol.

  3. Three-dimensional (3D) printing of mouse primary hepatocytes to generate 3D hepatic structure

    Science.gov (United States)

    Kim, Yohan; Kang, Kyojin; Jeong, Jaemin; Paik, Seung Sam; Kim, Ji Sook; Park, Su A; Kim, Wan Doo; Park, Jisun

    2017-01-01

    Purpose The major problem in producing artificial livers is that primary hepatocytes cannot be cultured for many days. Recently, 3-dimensional (3D) printing technology draws attention and this technology regarded as a useful tool for current cell biology. By using the 3D bio-printing, these problems can be resolved. Methods To generate 3D bio-printed structures (25 mm × 25 mm), cells-alginate constructs were fabricated by 3D bio-printing system. Mouse primary hepatocytes were isolated from the livers of 6–8 weeks old mice by a 2-step collagenase method. Samples of 4 × 107 hepatocytes with 80%–90% viability were printed with 3% alginate solution, and cultured with well-defined culture medium for primary hepatocytes. To confirm functional ability of hepatocytes cultured on 3D alginate scaffold, we conducted quantitative real-time polymerase chain reaction and immunofluorescence with hepatic marker genes. Results Isolated primary hepatocytes were printed with alginate. The 3D printed hepatocytes remained alive for 14 days. Gene expression levels of Albumin, HNF-4α and Foxa3 were gradually increased in the 3D structures. Immunofluorescence analysis showed that the primary hepatocytes produced hepatic-specific proteins over the same period of time. Conclusion Our research indicates that 3D bio-printing technique can be used for long-term culture of primary hepatocytes. It can therefore be used for drug screening and as a potential method of producing artificial livers. PMID:28203553

  4. Cholesterol Enhances the Toxic Effect of Ethanol and Acetaldehyde in Primary Mouse Hepatocytes.

    Science.gov (United States)

    López-Islas, Anayelly; Chagoya-Hazas, Victoria; Pérez-Aguilar, Benjamin; Palestino-Domínguez, Mayrel; Souza, Verónica; Miranda, Roxana U; Bucio, Leticia; Gómez-Quiroz, Luis Enrique; Gutiérrez-Ruiz, María-Concepción

    2016-01-01

    Obesity and alcohol consumption are risk factors for hepatic steatosis, and both commonly coexist. Our objective was to evaluate the effect of ethanol and acetaldehyde on primary hepatocytes obtained from mice fed for two days with a high cholesterol (HC) diet. HC hepatocytes increased lipid and cholesterol content. HC diet sensitized hepatocytes to the toxic effect of ethanol and acetaldehyde. Cyp2E1 content increased with HC diet, as well as in those treated with ethanol or acetaldehyde, while the activity of this enzyme determined in microsomes increased in the HC and in all ethanol treated hepatocytes, HC and CW. Oxidized proteins were increased in the HC cultures treated or not with the toxins. Transmission electron microscopy showed endoplasmic reticulum (ER) stress and megamitochondria in hepatocytes treated with ethanol as in HC and the ethanol HC treated hepatocytes. ER stress determined by PERK content was increased in ethanol treated hepatocytes from HC mice and CW. Nuclear translocation of ATF6 was observed in HC hepatocytes treated with ethanol, results that indicate that lipids overload and ethanol treatment favor ER stress. Oxidative stress, ER stress, and mitochondrial damage underlie potential mechanisms for increased damage in steatotic hepatocyte treated with ethanol.

  5. Cholesterol Enhances the Toxic Effect of Ethanol and Acetaldehyde in Primary Mouse Hepatocytes

    Science.gov (United States)

    López-Islas, Anayelly; Chagoya-Hazas, Victoria; Pérez-Aguilar, Benjamin; Palestino-Domínguez, Mayrel; Souza, Verónica; Miranda, Roxana U.; Bucio, Leticia; Gómez-Quiroz, Luis Enrique; Gutiérrez-Ruiz, María-Concepción

    2016-01-01

    Obesity and alcohol consumption are risk factors for hepatic steatosis, and both commonly coexist. Our objective was to evaluate the effect of ethanol and acetaldehyde on primary hepatocytes obtained from mice fed for two days with a high cholesterol (HC) diet. HC hepatocytes increased lipid and cholesterol content. HC diet sensitized hepatocytes to the toxic effect of ethanol and acetaldehyde. Cyp2E1 content increased with HC diet, as well as in those treated with ethanol or acetaldehyde, while the activity of this enzyme determined in microsomes increased in the HC and in all ethanol treated hepatocytes, HC and CW. Oxidized proteins were increased in the HC cultures treated or not with the toxins. Transmission electron microscopy showed endoplasmic reticulum (ER) stress and megamitochondria in hepatocytes treated with ethanol as in HC and the ethanol HC treated hepatocytes. ER stress determined by PERK content was increased in ethanol treated hepatocytes from HC mice and CW. Nuclear translocation of ATF6 was observed in HC hepatocytes treated with ethanol, results that indicate that lipids overload and ethanol treatment favor ER stress. Oxidative stress, ER stress, and mitochondrial damage underlie potential mechanisms for increased damage in steatotic hepatocyte treated with ethanol. PMID:26788255

  6. Hepatitis B virus infection and replication in primarily cultured human fetal hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Min Lin; Qun Chen; Li-Ye Yang; Wen-Yu Li; Xi-Biao Cao; Jiao-Ren Wu; You-Peng Peng; Mo-Rui Chen

    2007-01-01

    AIM:To investigate the infection and replication of hepatitis B virus(HBV)in primarily cultured human fetal hepatocytes(HFHs).METHODS:The human fetal hepatocytes were cultured in serum-free medium,HBV-positive serum was added into the medium to study the susceptibility of hepatocytes to HBV infection.The supernatant was collected for ELISA assay of HBsAg and HBeAg,and quantitative fluorescence PCR for HBV-DNA assay daily.Albumin and HBcAg,CK8 and CK18 expressions were detected by immunohistochemistry in cultured hepatocytes.Content of lactate dehydrogenate(LDH)was measured to find out the integrity of the cell membrane.RESULTS:A stable hepatocyte culture system was established.HBV could infect the hepatocytes and replicate,and HBcAg expression could be detected by immunohistochemistry in hepatocyte-like cells.HBV-DNA in the supernatant could be detected from d 2 to d 18 and HBsAg and HBeAg were positive on d 3-d 18 after HBV infection.HBV in medium increased from d 0 to d 6 and subsequently decreased as the cells were progressively loosing their hepatocyte phenotypes.CONCLUSION:HBV could infect human fetal hepatocytes and replicate.This in vitro model allowed a detailed Study on early events associated with human HBV entry into cells and subsequent replication.

  7. Three-dimensional (3D) printing of mouse primary hepatocytes to generate 3D hepatic structure.

    Science.gov (United States)

    Kim, Yohan; Kang, Kyojin; Jeong, Jaemin; Paik, Seung Sam; Kim, Ji Sook; Park, Su A; Kim, Wan Doo; Park, Jisun; Choi, Dongho

    2017-02-01

    The major problem in producing artificial livers is that primary hepatocytes cannot be cultured for many days. Recently, 3-dimensional (3D) printing technology draws attention and this technology regarded as a useful tool for current cell biology. By using the 3D bio-printing, these problems can be resolved. To generate 3D bio-printed structures (25 mm × 25 mm), cells-alginate constructs were fabricated by 3D bio-printing system. Mouse primary hepatocytes were isolated from the livers of 6-8 weeks old mice by a 2-step collagenase method. Samples of 4 × 10(7) hepatocytes with 80%-90% viability were printed with 3% alginate solution, and cultured with well-defined culture medium for primary hepatocytes. To confirm functional ability of hepatocytes cultured on 3D alginate scaffold, we conducted quantitative real-time polymerase chain reaction and immunofluorescence with hepatic marker genes. Isolated primary hepatocytes were printed with alginate. The 3D printed hepatocytes remained alive for 14 days. Gene expression levels of Albumin, HNF-4α and Foxa3 were gradually increased in the 3D structures. Immunofluorescence analysis showed that the primary hepatocytes produced hepatic-specific proteins over the same period of time. Our research indicates that 3D bio-printing technique can be used for long-term culture of primary hepatocytes. It can therefore be used for drug screening and as a potential method of producing artificial livers.

  8. Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes.

    Science.gov (United States)

    Mallanna, Sunil K; Cayo, Max A; Twaroski, Kirk; Gundry, Rebekah L; Duncan, Stephen A

    2016-09-13

    When comparing hepatic phenotypes between iPSC-derived hepatocyte-like cells from different liver disease patients, cell heterogeneity can confound interpretation. We proposed that homogeneous cell populations could be generated by fluorescence-activated cell sorting (FACS). Using cell-surface capture proteomics, we identified a total of 300 glycoproteins on hepatocytes. Analyses of the expression profiles during the differentiation of iPSCs revealed that SLC10A1, CLRN3, and AADAC were highly enriched during the final stages of hepatocyte differentiation. FACS purification of hepatocyte-like cells expressing SLC10A1, CLRN3, or AADAC demonstrated enrichment of cells with hepatocyte characteristics. Moreover, transcriptome analyses revealed that cells expressing the liver gene regulatory network were enriched while cells expressing a pluripotent stem cell network were depleted. In conclusion, we report an extensive catalog of cell-surface N-linked glycoproteins expressed in primary hepatocytes and identify cell-surface proteins that facilitate the purification of homogeneous populations of iPSC-derived hepatocyte-like cells.

  9. Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes

    Directory of Open Access Journals (Sweden)

    Sunil K. Mallanna

    2016-09-01

    Full Text Available When comparing hepatic phenotypes between iPSC-derived hepatocyte-like cells from different liver disease patients, cell heterogeneity can confound interpretation. We proposed that homogeneous cell populations could be generated by fluorescence-activated cell sorting (FACS. Using cell-surface capture proteomics, we identified a total of 300 glycoproteins on hepatocytes. Analyses of the expression profiles during the differentiation of iPSCs revealed that SLC10A1, CLRN3, and AADAC were highly enriched during the final stages of hepatocyte differentiation. FACS purification of hepatocyte-like cells expressing SLC10A1, CLRN3, or AADAC demonstrated enrichment of cells with hepatocyte characteristics. Moreover, transcriptome analyses revealed that cells expressing the liver gene regulatory network were enriched while cells expressing a pluripotent stem cell network were depleted. In conclusion, we report an extensive catalog of cell-surface N-linked glycoproteins expressed in primary hepatocytes and identify cell-surface proteins that facilitate the purification of homogeneous populations of iPSC-derived hepatocyte-like cells.

  10. Herpes simplex virus mutants defective in the virion-associated shutoff of host polypeptide synthesis and exhibiting abnormal synthesis of alpha (immediate early) viral polypeptides.

    Science.gov (United States)

    Read, G S; Frenkel, N

    1983-05-01

    Six mutants isolated from herpes simplex virus type 1 were judged to be defective with respect to the virion-associated function acting to rapidly shut off host polypeptide synthesis in herpes simplex virus-infected cells. The mutants were capable of proper entry into the cells, but, unlike the parent wild-type virus, they failed to shut off host polypeptide syntehsis in the presence of actinomycin D. They were consequently designated as virion-associated host shutoff (vhs) mutants. In the presence of actinomycin D, three of the mutants, vhs1, -2, and -3, failed to shut off the host at both 34 and 39 degrees C, whereas vhs4, -5, and -6 exhibited a temperature-dependent vhs phenotype. Since the mutants were capable of growth at 34 degrees C, it appeared that the vhs function was not essential for virus replication in cultured cells. Temperature-shift experiments performed with the vhs4 mutant showed that an active vhs function was required throughout the shutoff process and that, once established, the translational shutoff could not be reversed. In the absence of actinomycin D, the mutants induced a generalized, secondary shutoff of host translation, which required the synthesis of beta (early) or gamma (late) viral polypeptide(s). The vhs mutants appeared to be defective also with respect to post-transcriptional shutoff of alpha (immediate early) viral gene expression, since (i) cells infected with mutant viruses overproduced alpha viral polypeptides, (ii) there was an increased functional stability of alpha mRNA in the vhs1 mutant virus-infected cells, and (iii) superinfection of vhs1-infected cells with wild-type virus, in the presence of actinomycin D, resulted in a more pronounced shutoff of alpha polypeptide synthesis from preformed alpha mRNA than equivalent superinfection with vhs1 virus. The data suggest that the synthesis of alpha polypeptides in wild-type virus infections is subject to a negative post-transcriptional control involving viral gene product

  11. Probe tip heating assembly

    Science.gov (United States)

    Schmitz, Roger William; Oh, Yunje

    2016-10-25

    A heating assembly configured for use in mechanical testing at a scale of microns or less. The heating assembly includes a probe tip assembly configured for coupling with a transducer of the mechanical testing system. The probe tip assembly includes a probe tip heater system having a heating element, a probe tip coupled with the probe tip heater system, and a heater socket assembly. The heater socket assembly, in one example, includes a yoke and a heater interface that form a socket within the heater socket assembly. The probe tip heater system, coupled with the probe tip, is slidably received and clamped within the socket.

  12. Newnes electronics assembly handbook

    CERN Document Server

    Brindley, Keith

    2013-01-01

    Newnes Electronics Assembly Handbook: Techniques, Standards and Quality Assurance focuses on the aspects of electronic assembling. The handbook first looks at the printed circuit board (PCB). Base materials, basic mechanical properties, cleaning of assemblies, design, and PCB manufacturing processes are then explained. The text also discusses surface mounted assemblies and packaging of electromechanical assemblies, as well as the soldering process. Requirements for the soldering process; solderability and protective coatings; cleaning of PCBs; and mass solder/component reflow soldering are des

  13. Contextualizing Hepatocyte Functionality of Cryopreserved HepaRG Cell Cultures.

    Science.gov (United States)

    Jackson, Jonathan P; Li, Linhou; Chamberlain, Erica D; Wang, Hongbing; Ferguson, Stephen S

    2016-09-01

    Over the last decade HepaRG cells have emerged as a promising alternative to primary human hepatocytes (PHH) and have been featured in over 300 research publications. Most of these reports employed freshly differentiated HepaRG cells that require time-consuming culture (∼28 days) for full differentiation. Recently, a cryopreserved, predifferentiated format of HepaRG cells (termed here "cryo-HepaRG") has emerged as a new model that improves global availability and experimental flexibility; however, it is largely unknown whether HepaRG cells in this format fully retain their hepatic characteristics. Therefore, we systematically investigated the hepatocyte functionality of cryo-HepaRG cultures in context with the range of interindividual variation observed with PHH in both sandwich-culture and suspension formats. These evaluations uncovered a novel adaptation period for the cryo-HepaRG format and demonstrated the impact of extracellular matrix on cryo-HepaRG functionality. Pharmacologically important drug-metabolizing alleles were genotyped in HepaRG cells and poor metabolizer alleles for CYP2D6, CYP2C9, and CYP3A5 were identified and consistent with higher frequency alleles found in individuals of Caucasian decent. We observed liver enzyme inducibility with aryl hydrocarbon receptor, constitutive androstane receptor (CAR), and pregnane X receptor activators comparable to that of sandwich-cultured PHH. Finally, we show for the first time that cryo-HepaRG supports proper CAR cytosolic sequestration and translocation to hepatocyte nuclei in response to phenobarbital treatment. Taken together, these data reveal important considerations for the use of this cell model and demonstrate that cryo-HepaRG are suitable for metabolism and toxicology screening.

  14. Curcumin inhibits activation of TRPM2 channels in rat hepatocytes

    Directory of Open Access Journals (Sweden)

    E. Kheradpezhouh

    2016-04-01

    Full Text Available Oxidative stress is a hallmark of many liver diseases including viral and drug-induced hepatitis, ischemia-reperfusion injury, and non-alcoholic steatohepatitis. One of the consequences of oxidative stress in the liver is deregulation of Ca2+ homeostasis, resulting in a sustained elevation of the free cytosolic Ca2+ concentration ([Ca2+]c in hepatocytes, which leads to irreversible cellular damage. Recently it has been shown that liver damage induced by paracetamol and subsequent oxidative stress is, in large part, mediated by Ca2+ entry through Transient Receptor Potential Melastatin 2 (TRPM2 channels. Involvement of TRPM2 channels in hepatocellular damage induced by oxidative stress makes TRPM2 a potential therapeutic target for treatment of a range of oxidative stress-related liver diseases. We report here the identification of curcumin ((1E,6E-1,7-bis(4-hydroxy-3-methoxyphenyl-1,6-heptadiene-3,5-dione, a natural plant-derived polyphenol in turmeric spice, as a novel inhibitor of TRPM2 channel. Presence of 5 µM curcumin in the incubation medium prevented the H2O2- and paracetamol-induced [Ca2+]c rise in rat hepatocytes. Furthermore, in patch clamping experiments incubation of hepatocytes with curcumin inhibited activation of TRPM2 current by intracellular ADPR with IC50 of approximately 50 nM. These findings enhance understanding of the actions of curcumin and suggest that the known hepatoprotective properties of curcumin are, at least in part, mediated through inhibition of TRPM2 channels.

  15. Effects of ethanol on antioxidant capacity in isolated rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Sien-Sing Yang; Chi-Chang Huang; Jiun-Rong Chen; Che-Lin Chiu; Ming-Jer Shieh; Su-Jiun Lin; Suh-Ching Yang

    2005-01-01

    AIM: To investigate dose-response and time-course of the effects of ethanol on the cell viability and antioxidant capacity in isolated rat hepatocytes.METHODS: Hepatocytes were isolated from male adult Wistar rats and seeded into 100-mm dishes. Hepatocytes were treated with ethanol at concentrations between 0 (C), 10 (E10), 50 (E50), and 100 (E100) mmol/L (dose response) for 12, 24, and 36 h (time course). Then,lactate dehydrogenase (LDH) leakage, malondialdehyde (MDA) concentration, glutathione (GSH) level, and activities of glutathione peroxidase (GPX), glutathione reductase (GRD), superoxide dismutase (SOD), and catalase (CAT) were measured.RESULTS: Our data revealed that LDH leakage was significantly increased by about 30% in group E100 over those in groups C and E10 at 24 and 36 h, The MDA concentration in groups C, E10 and E50 were significantly lower than that in group E100 at 36 h. Furthermore,the concentration of MDA in group E100 at 36 h was significantly higher by 4.5- and 1.7-fold, respectively,than that at 12 and 24 h. On the other hand, the GSH level in group E100 at 24 and 36 h was significantly decreased, by 32% and 28%, respectively, compared to that at 12 h. The activities of GRD and CAT in group E100 at 36 h were significantly less than those in groups C and E10. However, The GPX and SOD activities showed no significant change in each group.CONCLUSION: These results suggest that longtime incubation with higher concentration of ethanol (100 mmol/L) decreased the cell viability by means of reducing GRD and CAT activities and increasing lipid peroxidation.

  16. Electrocatalytic water oxidation with a copper(II) polypeptide complex.

    Science.gov (United States)

    Zhang, Ming-Tian; Chen, Zuofeng; Kang, Peng; Meyer, Thomas J

    2013-02-13

    A self-assembly-formed triglycylglycine macrocyclic ligand (TGG(4-)) complex of Cu(II), [(TGG(4-))Cu(II)-OH(2)](2-), efficiently catalyzes water oxidation in a phosphate buffer at pH 11 at room temperature by a well-defined mechanism. In the mechanism, initial oxidation to Cu(III) is followed by further oxidation to a formal "Cu(IV)" with formation of a peroxide intermediate, which undergoes further oxidation to release oxygen and close the catalytic cycle. The catalyst exhibits high stability and activity toward water oxidation under these conditions with a high turnover frequency of 33 s(-1).

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

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    Grishin Eugene

    2011-01-01

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

  18. Insulin infusion reduces hepatocyte growth factor in lean humans

    DEFF Research Database (Denmark)

    de Courten, Barbora; de Courten, Maximilian; Dougherty, Sonia;

    2013-01-01

    OBJECTIVE: Plasma Hepatocyte Growth Factor (HGF) is significantly elevated in obesity and may contribute to vascular disease, metabolic syndrome or cancer in obese individuals. The current studies were done to determine if hyperinsulinemia increases plasma HGF. MATERIALS/METHODS: Twenty......-two participants (10 women/12 men, BMI 20.6-34.5 kg/m(2), age 18-49 years) underwent a hyperinsulinemic euglycemic clamp with measurement of HGF at baseline and steady state. Relationships between baseline HGF, anthropometrics, triglycerides, liver enzymes, c-reactive protein and adiponectin were also evaluated...

  19. Phosphatidylinositol 3-Kinase and Protein Kinase C Contribute to the Inhibition by Interleukin 6 of Phosphoenolpyruvate Carboxykinase Gene Expression in Cultured Rat Hepatocytes

    DEFF Research Database (Denmark)

    Christ, Bruno; Yazici, Emine; Nath, Annegret

    2000-01-01

    Gluconeogenesis, hepatocytes, interleukin 6, liver, phosphoinositide 3-kinase, phosphoenolpyruvate carboxykinase......Gluconeogenesis, hepatocytes, interleukin 6, liver, phosphoinositide 3-kinase, phosphoenolpyruvate carboxykinase...

  20. A sandwich-cultured rat hepatocyte system with increased metabolic competence evaluated by gene expression profiling

    NARCIS (Netherlands)

    Kienhuis, A.S.; Wortelboer, H.M.; Maas, W.J.; Herwijnen, M. van; Kleinjans, J.C.S.; Delft, J.H.M. van; Stierum, R.H.

    2007-01-01

    A rapid decline of cytochrome P450 (CYP450) enzyme activities remains a drawback of rat hepatocyte-based in vitro cultures. Consequently, judgment of the toxic potential of compounds that need bioactivation by CYP450s may not be adequate using this model. In the present study, an improved hepatocyte

  1. A dual-functional fibrous scaffold enhances P450 activity of cultured primary rat hepatocytes.

    Science.gov (United States)

    Chua, Kian-Ngiap; Tang, Yen-Ni; Quek, Chai-Hoon; Ramakrishna, Seeram; Leong, Kam W; Mao, Hai-Quan

    2007-09-01

    We have designed a novel dual-functional electrospun fibrous scaffold comprising two fiber mesh layers that were modified differently to induce two separate biological responses from hepatocytes. The first fiber layer was galactosylated on the surface to mediate hepatocyte attachment, while the second layer was loaded with 3-methylcholanthrene (3-Mc) to enhance cytochrome P450 activity of hepatocytes. Primary rat hepatocytes cultured on the galactosylated fibrous scaffolds loaded with different concentrations of 3-Mc were compared for their cell attachment efficiency, albumin secretion activity and cytochrome P450-dependent 7-ethoxycoumarin O-deethylase activity. This hybrid fibrous scaffold mediated hepatocyte attachment with slightly lower efficiency (76+/-2.3%) than a single-layer galactosylated fibrous scaffold (84+/-3.5%). More importantly, the cytochrome P450 activity of the hepatocytes cultured on the hybrid scaffold correlated well with the 3-Mc loading level. The results also showed that transfer of 3-Mc to hepatocytes through direct cell-fiber contact was the dominant transport route, with the induced cytochrome P450 activity being 1.9- to 4.8-fold higher than that of transfer of 3-Mc to hepatocytes via dissolution from fibers to medium. This study demonstrates the feasibility of creating multi-functional fibrous scaffolds that serve both as an adhesive substrate and as a delivery vehicle for bioactive molecules.

  2. Interactions between macrophage/Kupffer cells and hepatocytes in surgical sepsis

    Energy Technology Data Exchange (ETDEWEB)

    West, M.A.

    1988-01-01

    Experiments were performed to investigate the role of Kupffer cell/macrophage interactions with hepatocytes in modulating liver function during infections using direct in vitro cocultivation of rat macrophages or Kupffer cells with rat hepatocytes. Protein synthesis was assayed as a sensitive indicator of integrated hepatocellular function by measuring {sup 3}H-leucine incorporation into hepatocyte protein. Septic stimuli such as lipoploysaccharide and killed bacteria were added to cocultures of hepatocytes and macrophages or Kupffer cells and the responses compared to hepatocytes alone. Information about the types of proteins synthesized by hepatocytes under various culture conditions was determined using polyacrylamide gel electrophoresis and autoradiography. These experiments showed that septic stimuli alter the amount and type of protein synthesized by hepatocytes and had no direct effect on hepatocytes in the absence of macrophages or Kupffer cells. The mediator(s) appears to be a heat labile, soluble monokine(s) which is distinct from interleukin-1 or tumor necrosis factor. The important role of Kupffer cells/macrophages in mediating alterations in hepatocellular function in sepsis may ultimately improve patient care.

  3. Effects of nuclear translocation of tissue transglutaminase and the release of cytochrome C on hepatocyte apoptosis

    Institute of Scientific and Technical Information of China (English)

    宋良文; 马宪梅; 李扬; 崔雪梅; 王晓民

    2003-01-01

    Objective To assess the effects of nuclear translocation of tissue transglutaminase (TTG) and the release of cytochrome C on hepatocyte apoptosis and to reveal the mechanism of signal transduction of early apoptosis in injured hepatocytes. Methods Hepatocytes isolated from tissue transglutaminase gene knock-out rats and mice were stimulated with ethanol. Proteins from whole cell, cytoplasm and nuclei were extracted for determination of TTG activity by 14 C-putrescine incorporation. Distribution of TTG throughout the entire cell, as well as just nucleus was observed under a confocal scanning microscope. The amount of cytochrome C released from mitochondria was determined by ELISA. Cell apoptosis was observed by fluorescent cytochemistry.Results TTG activity in whole cells and nuclei was significantly increased after the hepatocytes were treated with ethanol. Cytochrome C release was remarkably increased in the cells isolated from rat and wild-type mouse after treatment with ethanol but not in TTG gene knock-out mice. Cellular apoptosis appeared in hepatocytes isolated from rats and wild-type mice but not in the hepatocytes from TTG gene knock-out mice after stimulation with ethanol.Conclusions Increased TTG in hepatocytes can be translocated into the nucleus and promote release of mitochondrial cytochrome C into the cytoplasm. Passing through a series of signal pathways, hepatocyte apoptosis is induced eventually.

  4. HepatoDyn: A Dynamic Model of Hepatocyte Metabolism That Integrates 13C Isotopomer Data.

    Directory of Open Access Journals (Sweden)

    Carles Foguet

    2016-04-01

    Full Text Available The liver performs many essential metabolic functions, which can be studied using computational models of hepatocytes. Here we present HepatoDyn, a highly detailed dynamic model of hepatocyte metabolism. HepatoDyn includes a large metabolic network, highly detailed kinetic laws, and is capable of dynamically simulating the redox and energy metabolism of hepatocytes. Furthermore, the model was coupled to the module for isotopic label propagation of the software package IsoDyn, allowing HepatoDyn to integrate data derived from 13C based experiments. As an example of dynamical simulations applied to hepatocytes, we studied the effects of high fructose concentrations on hepatocyte metabolism by integrating data from experiments in which rat hepatocytes were incubated with 20 mM glucose supplemented with either 3 mM or 20 mM fructose. These experiments showed that glycogen accumulation was significantly lower in hepatocytes incubated with medium supplemented with 20 mM fructose than in hepatocytes incubated with medium supplemented with 3 mM fructose. Through the integration of extracellular fluxes and 13C enrichment measurements, HepatoDyn predicted that this phenomenon can be attributed to a depletion of cytosolic ATP and phosphate induced by high fructose concentrations in the medium.

  5. Measurement of acetyl-CoA carboxylase activity in isolated hepatocytes

    NARCIS (Netherlands)

    Bijleveld, C.; Geelen, M.J.H.

    1987-01-01

    An assay is described for acetyl-CoA carboxylase activity in isolated hepatocytes. The assay is based on two principles: (a) The hepatocytes are made permeable by digitonin. 64 μg of digitonin per mg of cellular protein were most effective in exposing enzyme activity without a significant effect on

  6. Partial hepatectomy induces delayed hepatocyte proliferation and normal liver regeneration in ovariectomized mice

    Directory of Open Access Journals (Sweden)

    Umeda M

    2015-07-01

    Full Text Available Makoto Umeda,1 Masaki Hiramoto,1,2 Takeshi Imai1 1Department of Aging Intervention, National Center for Geriatrics and Gerontology, Obu, Aichi, Japan; 2Department of Biochemistry, Tokyo Medical University, Tokyo, Japan Abstract: Estrogens play central roles in sexual development, reproduction, and hepatocyte proliferation. The ovaries are one of the main organs for estradiol (E2 production. Ovariectomies (OVXs were performed on the female mice, and hepatocyte proliferation was analyzed. The ovariectomized mice exhibited delayed hepatocyte proliferation after partial hepatectomy (PH and also exhibited delayed and reduced E2 induction. Both E2 administration and PH induced the gene expression of estrogen receptor α (ERα. The transcripts of ERα were detected specifically in periportal hepatocytes after E2 administration and PH. Moreover, the E2 concentrations and hepatocyte proliferation rates were highest in the proestrus period of the estrous cycle. Taken together, these findings indicate that E2 accelerated ERα expression in periportal hepatocytes and hepatocyte proliferation in the female mice.Keywords: estrogen, ER, estrous cycle, hepatocyte proliferation, liver regeneration

  7. [Comparison of conformational possibilities of polypeptides representing the terminal segments of different histones].

    Science.gov (United States)

    Ramm, E I; Kamilova, R R; Polozova, O L; Vorob'ev, V I; Burichenko, V K

    1979-01-01

    Regular polypeptides--models of the N-terminal fragments of histones H2A and H4 and the C-terminal half of histone H1 were synthesized. Conformations of these polypeptides were investigated by using the methods of circular dichroism and optical rotatory dispersion. It was shown that all polypeptides studied in aqueous solutions at neutral pH and at low temperature (+2 degrees C) had the conformation of left-handed helix (LHH) or poly-L-proline type. The neutralization of positive charges of side groups at alkaline pH of screening of charged groups at a high ionic strength (up to 1 M NaF) results in increase of the degree of defectness of this conformation. There occur no transition of LHH to such an ordered conformation as alpha-helix or beta-sheet structure or complete disappearance of LHH. The influence of temperature, 80% ethanol and 1% sodium dodecylsulphate on the structure of these polypeptides was also studied. Differences in conformational potencies of two studied groups of polypeptides which are the models of the terminal fragments of various histones were discovered and associated with different biological functions of these histones in chromatin.

  8. Identification of a 32-34-kilodalton polypeptide as a herbicide receptor protein in photosystem II.

    Science.gov (United States)

    Mullet, J E; Arntzen, C J

    1981-04-13

    Photosystem II particles which retained high rates of herbicide-sensitive activity were used to examine the site(s) of action of various herbicides. A polypeptide of 32-34 kdaltons was identified as the triazine-herbicide binding site based upon: (a) parallel loss of atrazine activity and the polypeptide during either trypsin treatment or selective detergent depletion of protein in the Photosystem II complex, and (b) covalent labeling of the polypeptide by a 14C-labeled photoaffinity triazine. In Photosystem II particles depleted of the 32-34-kdalton polypeptide, electron transport was still active and was slightly sensitive to DCMU and largely sensitive to dinoseb (urea and nitrophenol herbicides, respectively). On the basis of this result it is proposed that the general herbicide binding site common to atrazine, DCMU and dinoseb is formed by a minimum of two polypeptides which determine affinity and/or mediate herbicide-induced inhibition of electron transport on the acceptor side of Photosystem II.

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

    Directory of Open Access Journals (Sweden)

    Santos P.R.P.

    1997-01-01

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

  10. Chlorophyll-Protein Complexes from Euglena gracilis and Mutants Deficient in Chlorophyll b: II. Polypeptide Composition.

    Science.gov (United States)

    Cunningham, F X; Schiff, J A

    1986-01-01

    Chlorophyll-protein complexes (CPs) obtained from thylakoids of Euglena gracilis Klebs var bacillaris Cori contain the following polypeptides (listed in parentheses in order of prominence after Coomassie R-250 staining of polyacrylamide gels): CP Ia (66, 18, 22, 22.5, 27.5, 21, 28, 24, 25.5, and 26 kilodaltons [kD]); CP I (66 kD); CPx (41 kD); LHCP(2) (an oligomer of LHCP) (26.5, 28, and 26 kD); CPy (27 and 19 kD); CPa (54 kD); and LHCP (26.5, 28, and 26 kD). Mutants of bacillaris low in chlorophyll b (Gr(1)BSL, G(1)BU, and O(4)BSL; Chl a/b [mol/mol] = 50-100) which lack CP Ia, LHCP(2), and LHCP also lack or are deficient in polypeptides associated with these complexes in wild-type cells. Mutants G(1) and O(4), which also lack CPy, lack the CPy-associated polypeptides found in wild-type and Gr(1). Using an antiserum which was elicited by and reacts strongly and selectively with the SDS-treated major polypeptide (26.5 kD) of the LHCP complexes of wild-type, this polypeptide is undetectable in the mutants (saturation, consistent with the selective loss of major antenna components but not CP I or CPa from the mutants.

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

    Science.gov (United States)

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

    2015-07-07

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

  12. Prolonged Survival of Mice with Acute Liver Failure with Transplantation of Monkey Hepatocytes Cultured with an Antiapoptotic Pentapeptide V5

    OpenAIRE

    田中, 公章

    2007-01-01

    BACKGROUND: Because hepatocyte transplantation has been considered to be an attractive method to treat acute liver failure (ALF), efficient recovery of hepatocytes and maintenance of differentiated hepatocyte functions is of extreme importance. We here report the usefulness of an antiapoptotic pentapeptide V5, composed of Val-Pro-Met-Leu-Lys, in the monkey hepatocyte cultures. METHODS: We evaluated albumin production, metabolizing abilities of ammonia, lidocaine, and diazepam of monkey hepato...

  13. Tauroursodeoxycholate Protects Rat Hepatocytes from Bile Acid-Induced Apoptosis via β1-Integrin- and Protein Kinase A-Dependent Mechanisms

    Directory of Open Access Journals (Sweden)

    Annika Sommerfeld

    2015-05-01

    Full Text Available Background/Aims: Ursodeoxycholic acid, which in vivo is rapidly converted into its taurine conjugate, is frequently used for the treatment of cholestatic liver disease. Apart from its choleretic effects, tauroursodeoxycholate (TUDC can protect hepatocytes from bile acid-induced apoptosis, but the mechanisms underlying its anti-apoptotic effects are poorly understood. Methods: These mechanisms were investigated in perfused rat liver and isolated rat hepatocytes. Results: It was found that TUDC inhibited the glycochenodeoxycholate (GCDC-induced activation of the CD95 death receptor at the level of association between CD95 and the epidermal growth factor receptor. This was due to a rapid TUDC-induced β1-integrin-dependent cyclic AMP (cAMP signal with induction of the dual specificity mitogen-activated protein (MAP kinase phosphatase 1 (MKP-1, which prevented GCDC-induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4 and c-jun-NH2-terminal kinase (JNK activation. Furthermore, TUDC induced a protein kinase A (PKA-mediated serine/threonine phosphorylation of the CD95, which was recently identified as an internalization signal for CD95. Furthermore, TUDC inhibited GCDC-induced CD95 targeting to the plasma membrane in a β1-integrin-and PKA-dependent manner. In line with this, the β1-integrin siRNA knockdown in sodium taurocholate cotransporting polypeptide (Ntcp-transfected HepG2 cells abolished the protective effect of TUDC against GCDC-induced apoptosis. Conclusion: TUDC exerts its anti-apoptotic effect via a β1-integrin-mediated formation of cAMP, which prevents CD95 activation by hydrophobic bile acids at the levels of JNK activation and CD95 serine/threonine phosphorylation.

  14. Study of Valproic Acid-Enhanced Hepatocyte Steatosis

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    Renin Chang

    2016-01-01

    Full Text Available Valproic acid (VPA is one of the most widely used antiepilepsy drugs. However, several side effects, including weight gain and fatty liver, have been reported in patients following VPA treatment. In this study, we explored the molecular mechanisms of VPA-induced hepatic steatosis using FL83B cell line-based in vitro model. Using fluorescent lipid staining technique, we found that VPA enhanced oleic acid- (OLA- induced lipid accumulation in a dose-dependent manner in hepatocytes; this may be due to upregulated lipid uptake, triacylglycerol (TAG synthesis, and lipid droplet formation. Real-time PCR results showed that, following VPA treatment, the expression levels of genes encoding cluster of differentiation 36 (Cd36, low-density lipoprotein receptor-related protein 1 (Lrp1, diacylglycerol acyltransferase 2 (Dgat2, and perilipin 2 (Plin2 were increased, that of carnitine palmitoyltransferase I a (Cpt1a was not affected, and those of acetyl-Co A carboxylase α (Acca and fatty acid synthase (Fasn were decreased. Furthermore, using immunofluorescence staining and flow cytometry analyses, we found that VPA also induced peroxisome proliferator-activated receptor γ (PPARγ nuclear translocation and increased levels of cell-surface CD36. Based on these results, we propose that VPA may enhance OLA-induced hepatocyte steatosis through the upregulation of PPARγ- and CD36-dependent lipid uptake, TAG synthesis, and lipid droplet formation.

  15. Discovering networks of perturbed biological processes in hepatocyte cultures.

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    Christopher D Lasher

    Full Text Available The liver plays a vital role in glucose homeostasis, the synthesis of bile acids and the detoxification of foreign substances. Liver culture systems are widely used to test adverse effects of drugs and environmental toxicants. The two most prevalent liver culture systems are hepatocyte monolayers (HMs and collagen sandwiches (CS. Despite their wide use, comprehensive transcriptional programs and interaction networks in these culture systems have not been systematically investigated. We integrated an existing temporal transcriptional dataset for HM and CS cultures of rat hepatocytes with a functional interaction network of rat genes. We aimed to exploit the functional interactions to identify statistically significant linkages between perturbed biological processes. To this end, we developed a novel approach to compute Contextual Biological Process Linkage Networks (CBPLNs. CBPLNs revealed numerous meaningful connections between different biological processes and gene sets, which we were successful in interpreting within the context of liver metabolism. Multiple phenomena captured by CBPLNs at the process level such as regulation, downstream effects, and feedback loops have well described counterparts at the gene and protein level. CBPLNs reveal high-level linkages between pathways and processes, making the identification of important biological trends more tractable than through interactions between individual genes and molecules alone. Our approach may provide a new route to explore, analyze, and understand cellular responses to internal and external cues within the context of the intricate networks of molecular interactions that control cellular behavior.

  16. Metformin protects rat hepatocytes against bile acid-induced apoptosis.

    Directory of Open Access Journals (Sweden)

    Titia E Woudenberg-Vrenken

    Full Text Available BACKGROUND: Metformin is used in the treatment of Diabetes Mellitus type II and improves liver function in patients with non-alcoholic fatty liver disease (NAFLD. Metformin activates AMP-activated protein kinase (AMPK, the cellular energy sensor that is sensitive to changes in the AMP/ATP-ratio. AMPK is an inhibitor of mammalian target of rapamycin (mTOR. Both AMPK and mTOR are able to modulate cell death. AIM: To evaluate the effects of metformin on hepatocyte cell death. METHODS: Apoptotic cell death was induced in primary rat hepatocytes using either the bile acid glycochenodeoxycholic acid (GCDCA or TNFα in combination with actinomycin D (actD. AMPK, mTOR and phosphoinositide-3 kinase (PI3K/Akt were inhibited using pharmacological inhibitors. Apoptosis and necrosis were quantified by caspase activation, acridine orange staining and Sytox green staining respectively. RESULTS: Metformin dose-dependently reduces GCDCA-induced apoptosis, even when added 2 hours after GCDCA, without increasing necrotic cell death. Metformin does not protect against TNFα/ActD-induced apoptosis. The protective effect of metformin is dependent on an intact PI3-kinase/Akt pathway, but does not require AMPK/mTOR-signaling. Metformin does not inhibit NF-κB activation. CONCLUSION: Metformin protects against bile acid-induced apoptosis and could be considered in the treatment of chronic liver diseases accompanied by inflammation.

  17. Cytotoxicity of ortho-phenylphenol in isolated rat hepatocytes.

    Science.gov (United States)

    Nakagawa, Y; Moldéus, P; Moore, G A

    1992-01-22

    The effects of ortho-phenylphenol (OPP) and its metabolites, phenyl-hydroquinol (PHQ) and phenyl-benzoquinone (PBQ), on isolated rat hepatocytes were investigated. Addition of OPP (0.5-1.0 mM) to cells caused a dose-dependent cell death accompanied by the depletion of intracellular levels of ATP, glutathione (GSH) and protein thiols. GSH loss correlated with the formation of oxidized GSH. In addition, PHQ and especially PBQ (both at 0.5 mM) resulted in acute cell death with rapid depletion of ATP, GSH and protein thiols, and further low doses of PBQ (10-50 microM) elicited serious impairment of mitochondrial functions related to oxidative phosphorylation and Ca fluxes in isolated liver mitochondria. These results indicate that mitochondria are a target for these compounds and that OPP is itself toxic to hepatocytes even when metabolism is inhibited. The loss of cellular GSH and protein thiols accompanied by the impairment of mitochondrial function may be the main mechanisms of cytotoxicity induced by OPP and its metabolites.

  18. Hepatocytes Polyploidization and Cell Cycle Control in Liver Physiopathology

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    Géraldine Gentric

    2012-01-01

    Full Text Available Most cells in mammalian tissues usually contain a diploid complement of chromosomes. However, numerous studies have demonstrated a major role of “diploid-polyploid conversion” during physiopathological processes in several tissues. In the liver parenchyma, progressive polyploidization of hepatocytes takes place during postnatal growth. Indeed, at the suckling-weaning transition, cytokinesis failure events induce the genesis of binucleated tetraploid liver cells. Insulin signalling, through regulation of the PI3K/Akt signalling pathway, is essential in the establishment of liver tetraploidization by controlling cytoskeletal organisation and consequently mitosis progression. Liver cell polyploidy is generally considered to indicate terminal differentiation and senescence, and both lead to a progressive loss of cell pluripotency associated to a markedly decreased replication capacity. Although adult liver is a quiescent organ, it retains a capacity to proliferate and to modulate its ploidy in response to various stimuli or aggression (partial hepatectomy, metabolic overload (i.e., high copper and iron hepatic levels, oxidative stress, toxic insult, and chronic hepatitis etc.. Here we review the mechanisms and functional consequences of hepatocytes polyploidization during normal and pathological liver growth.

  19. Rapid and sensitive measure of gluconeogenesis in isolated bovine hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Azain, M.J.; Kasser, T.R.; Atwell, C.A.; Baile, C.A.

    1986-03-05

    Available methods for determining glucose synthesis from radiolabelled precursors using ion exchange column chromatography limit the number of samples that can be processed. To facilitate this process, a rapid method for determining glucose synthesis from 3-carbon precursors was developed using suspensions of anion and cation exchange resins. Hepatocytes were prepared from calf liver by collagenase perfusion of the caudate lobe. Isolated cells were incubated with /sup 14/C-labelled lactate or propionate in the presence or absence of glucagen and/or palmitate. Glucose synthesis was determined by vortexing an aliquot of cell suspension with a 50% slurry of anion exchange resin (acetate form), followed by cation exchange resin. After centrifugation /sup 14/C-glucose was recovered in the supernatant and measured by scintillation counting. Using this method, more than 95% of unused labelled precursor was bound to the ion exchange resin and essentially 100% of /sup 14/C-glucose tracer was recovered in the supernatant. In hepatocyte suspensions, radioactivity recovered in the supernatants was confirmed to be glucose by pre-incubating aliquots of media with glucose oxidase prior to addition of ion exchange resins. The present system allows determination of hepatic gluconeogenesis, is sensitive to substrate and hormonal manipulations and has the capacity for processing several hundred samples per day.

  20. The mechanism of methyl mercury toxicity in isolated rat hepatocytes.

    Science.gov (United States)

    Ashour, H; Abdel-Rahman, M; Khodair, A

    1993-07-01

    Mercury is the major component of dental amalgam restorative material, which typically has 50% pure elemental mercury. It is also used in some skin creams, and in the manufacturing of plastic, drugs and fungicides. The present study was designed to investigate the toxicity of methyl mercury (MeHg+) on isolated rat hepatocytes using several toxicity parameters. The hepatocytes were isolated by a collagenase perfusion technique and were incubated with different concentrations of MeHg+ (0.1-100 ppm) for 2 h. Through the incubation period the viability was determined by Trypan blue exclusion. Reduced glutathione (GSH) content and its enzymes, glutathione peroxidase (GSH-PX) and glutathione reductase (GSH-RX) were measured. Leakage of enzymes such as aspartate transaminase (AST), and alanine transaminase (ALT) were determined. The cell viability was reduced significantly after 1 h incubation when 0.1 and 1 ppm MeHg+ were applied. The decrease in the cell viability was dose- and time-dependent. A depletion of GSH content was observed with 100 ppm MeHg+ after 30 min of incubation. A significant decrease in GSH-RX was observed with 100 ppm during 15 and 30 min of incubation, while 10 ppm of MeHg+ significantly increased ALT leakage after 60 min. However, there was a significant increase in AST leakage with 100 ppm only. The present investigation indicates that the toxic effect of MeHg+ is most likely cytosolic enzyme related.

  1. Autonomous electrochromic assembly

    Energy Technology Data Exchange (ETDEWEB)

    Berland, Brian Spencer; Lanning, Bruce Roy; Stowell, Jr., Michael Wayne

    2015-03-10

    This disclosure describes system and methods for creating an autonomous electrochromic assembly, and systems and methods for use of the autonomous electrochromic assembly in combination with a window. Embodiments described herein include an electrochromic assembly that has an electrochromic device, an energy storage device, an energy collection device, and an electrochromic controller device. These devices may be combined into a unitary electrochromic insert assembly. The electrochromic assembly may have the capability of generating power sufficient to operate and control an electrochromic device. This control may occur through the application of a voltage to an electrochromic device to change its opacity state. The electrochromic assembly may be used in combination with a window.

  2. Differentiation of human embryonic stem cells along a hepatocyte lineage and its application in liver regeneration

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Hepatocyte transplantation and bioartificial liver(BAL)as alternatives to liver transplantation offer the possibility of effective treatment for many inherited and acquired hepatic disorders.Unfortunately,the limited availability of donated livers and the variability of their derived hepatocytes make it difficult to obtain enough viable human hepatocytes for the hepatocyte-based therapies.Embryonic stem cells (ESCs),which could be isolated directly from the blastocyst inner cell mass,have permanent self-renewal capability and developmental pluripotency and therefore might be an ideal cell source in the treatment of hepatic discords.However,differentiation of hESCS into hepatocytes with significant numbers remains a challenge.This review updates our current understanding of differentiation of ESCs into hepatic lineage cells,their future therapeutic uses and problems in liver regeneration.

  3. In vivo N-acetyl cysteine reduce hepatocyte death by induced acetaminophen

    Science.gov (United States)

    Lin, Chih-Ju; Li, Feng-Chieh; Wang, Sheng-Shun; Lee, Hsuan-Shu; Dong, Chen-Yuan

    2011-07-01

    Acetaminophen (APAP) is the famous drug in global, and taking overdose Acetaminophen will intake hepatic cell injure. Desptie substantial progress in our understanding of the mechanism of hepatocellular injury during the last 40 years, many aspects of the pathophysiology are still unknown or controversial.1 In this study, mice are injected APAP overdose to damage hepatocyte. APAP deplete glutathione and ATP of cell, N-Acetyl Cysteine (NAC) plays an important role to protect hepatocytes be injury. N-Acetyl Cysteine provides mitochondrial to produce glutathione to release drug effect hepatocyte. By 6-carboxyfluorescein diacetate (6-CFDA) metabolism in vivo, glutathione keep depleting to observe the hepatocyte morphology in time. Without NAC, cell necrosis increase to plasma membrane damage to release 6-CFDA, that's rupture. After 6-CFDA injection, fluorescence will be retained in hepatocyte. For cell retain with NAC and without NAC are almost the same. With NAC, the number of cell rupture decreases about 75%.

  4. Depot-dependent effects of adipose tissue explants on co-cultured hepatocytes

    DEFF Research Database (Denmark)

    Du, Zhen-Yu; Ma, Tao; Lock, Erik-Jan;

    2011-01-01

    We have developed an in vitro hepatocyte-adipose tissue explant (ATE) co-culture model enabling examination of the effect of visceral and subcutaneous adipose tissues on primary rat hepatocytes. Initial analyses of inflammatory marker genes were performed in fractionated epididymal or inguinal...... levels of IL-6, TNF-a and PGE(2) in the media from inguinal ATEs co-cultured with primary rat hepatocytes were higher than that in the media from epididymal ATEs co-cultured with hepatocytes, although the significant difference was only seen in PGE(2). Lipolysis, measured as glycerol release, was similar...... in the ATEs isolated from inguinal and epididymal adipose tissues when cultured alone, but the glycerol release was higher in the ATEs isolated from epididymal than from inguinal adipose tissue when co-cultured with hepatocytes. Compared to epididymal ATEs, the ATEs from inguinal adipose tissue elicited...

  5. EFFECT OF THE ORTHOSIPHON STAMINEUS, BENTH ON AMINOPYRINE METABOLISM IN RAT HEPATOCYTES

    Directory of Open Access Journals (Sweden)

    CHIN JIN HAN1

    2007-01-01

    Full Text Available One of the commonly used herbal medicines in Malaysia is Orthosiphon stamineus, Benth (family: Lamiaceae or locally known as Misai Kucing. This experiment was undertaken to evaluate possible interaction of methanol extract of O. stamineus with aminopyrine, a model drug, in different age groups (young, adult and old of Sprague-Dawley (SD female rat hepatocytes. Hepatocytes were prepared by collagenase perfusion technique. Determination of aminopyrine N-demethylase activity was done by measuring formaldehyde formed. From these findings, only normal young female rat hepatocytes in the presence of 0.001 mg/ml of methanol extract of O. stamineus showed significant increase in aminopyrine N-demethylase activity. However, aminopyrine N-demethylase activity was not affected in hepatocytes of normal adult and old female SD rats. In conclusion, exposure of methanol extract of O. stamineus could affect phase I aminopyrine metabolism in normal young female SD rat hepatocytes but this effect was age-dependent.

  6. Measurement of Blood Coagulation Factor Synthesis in Cultures of Human Hepatocytes.

    Science.gov (United States)

    Heinz, Stefan; Braspenning, Joris

    2015-01-01

    An important function of the liver is the synthesis and secretion of blood coagulation factors. Within the liver, hepatocytes are involved in the synthesis of most blood coagulation factors, such as fibrinogen, prothrombin, factor V, VII, IX, X, XI, XII, as well as protein C and S, and antithrombin, whereas liver sinusoidal endothelial cells produce factor VIII and von Willebrand factor. Here, we describe methods for the detection and quantification of most blood coagulation factors in hepatocytes in vitro. Hepatocyte cultures indeed provide a valuable tool to study blood coagulation factors. In addition, the generation and expansion of hepatocytes or hepatocyte-like cells may be used in future for cell-based therapies of liver diseases, including blood coagulation factor deficiencies.

  7. Lineage tracing reveals conversion of liver sinusoidal endothelial cells into hepatocytes.

    Science.gov (United States)

    Tan, Zhaoli; Chen, Keyan; Shao, Yong; Gao, Lihua; Wang, Yan; Xu, Jianming; Jin, Yang; Hu, Xianwen; Wang, Youliang

    2016-09-01

    Although liver sinusoidal endothelial cells (LSECs) have long been known to contribute to liver regeneration following injury, the exact role of these cells in liver regeneration remains poorly understood. In this work, we performed lineage tracing of LSECs in mice carrying Tie2-Cre or VE-cadherin-Cre constructs to facilitate fate-mapping of LSECs in liver regeneration. Some YFP-positive LSECs were observed to convert into hepatocytes following a two-thirds partial hepatectomy (PH). Furthermore, human umbilical vein endothelial cells (HUVECs) could be triggered to convert into cells that closely resembled hepatocytes when cultured with serum from mice that underwent an extended PH. These findings suggest that mature non-hepatocyte LSECs play an essential role in mammalian liver regeneration by converting to hepatocytes. The conversion of LSECs to hepatocyte-like (iHep) cells may provide a new approach to tissue engineering.

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

    Science.gov (United States)

    Munegumi, Toratane; Tanikawa, Naoya

    2017-07-01

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

  9. Fipronil induces CYP isoforms and cytotoxicity in human hepatocytes.

    Science.gov (United States)

    Das, Parikshit C; Cao, Yan; Cherrington, Nathan; Hodgson, Ernest; Rose, Randy L

    2006-12-15

    Recent studies have demonstrated the potential of pesticides to either inhibit or induce xenobiotic metabolizing enzymes in humans. Exposure of human hepatocytes to doses of fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl) sulfinyl]-1H-pyrazole-3-carbonitrile) ranging from 0.1 to 25 microM resulted in a dose dependent increase in CYP1A1 mRNA expression (3.5 to approximately 55-fold) as measured by the branched DNA assay. In a similar manner, CYP3A4 mRNA expression was also induced (10-30-fold), although at the higher doses induction returned to near control levels. CYP2B6 and 3A5 were also induced by fipronil, although at lower levels (2-3-fold). Confirmation of bDNA results were sought through western blotting and/or enzyme activity assays. Western blots using CYP3A4 antibody demonstrated a dose responsive increase from 0.5 to 1 microM followed by decreasing responses at higher concentrations. Similar increases and decreases were observed in CYP3A4-specific activity levels as measured using 6beta-hydroxytestosterone formation following incubation with testosterone. Likewise, activity levels for a CYP1A1-specific substrate, luciferin CEE, demonstrated that CYP1A1 enzyme activities were maximally induced by 1 microM fipronil followed by dramatically declining activity measurements at 10 and 25 microM. Cytotoxic effects of fipronil and fipronil sulfone were examined using the adenylate kinase and the trypan blue exclusion assays in HepG2 cells and human hepatocytes. The results indicate both that HepG2 cells and primary human hepatocytes are sensitive to the cytotoxic effects of fipronil. The maximum induction of adenylate kinase was ca. 3-fold greater than the respective controls in HepG2 and 6-10-fold in the case of primary hepatocytes. A significant time- and dose-dependent induction of adenylate kinase activity in HepG2 cells was noted from 0.1 to 12.5 microM fipronil followed by decreasing activities at 25 and 50 microM. For

  10. Macrophage secretory products induce an inflammatory phenotype in hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Michelle Melino; Gethin P Thomas; Andrew D Clouston; Julie R Jonsson; Elizabeth E Powell; Victoria L Gadd; Gene V Walker; Richard Skoien; Helen D Barrie; Dinesh Jothimani; Leigh Horsfall; Alun Jones; Matthew J Sweet

    2012-01-01

    AIM:To investigate the influence of macrophages on hepatocyte phenotype and function.METHODS:Macrophages were differentiated from THP-1 monocytes via phorbol myristate acetate stimulation and the effects of monocyte or macrophageconditioned medium on HepG2 mRNA and protein expression determined.The in vivo relevance of these findings was confirmed using liver biopsies from 147patients with hepatitis C virus (HCV) infection.RESULTS:Conditioned media from macrophages,but not monocytes,induced a transient morphological change in hepatocytes associated with upregulation of vimentin (7.8 ± 2.5-fold,P =0.045) and transforming growth factor (TGF)-β1 (2.6 ± 0.2-fold,P < 0.001) and downregulation of epithelial cadherin (1.7 ± 0.02-fold,P =0.017) mRNA expression.Microarray analysis revealed significant upregulation of lipocalin-2 (17-fold,P < 0.001) and pathways associated with inflammation,and substantial downregulation of pathways related to hepatocyte function.In patients with chronic HCV,realtime polymerase chain reaction and immunohistochemistry confirmed an increase in lipocalin-2 mRNA (F0 1.0± 0.3,F1 2.2 ± 0.2,F2 3.0 ± 9.3,F3/4 4.0 ± 0.8,P =0.003) and protein expression (F1 1.0 ± 0.5,F2 1.3 ±0.4,F3/4 3.6 ± 0.4,P =0.014) with increasing liver injury.High performance liquid chromatography-tandem mass spectrometry analysis identified elevated levels of matrix metalloproteinase (MMP)-9 in macrophageconditioned medium,and a chemical inhibitor of MMP-9attenuated the change in morphology and mRNA expression of TGF-β1 (2.9 ± 0.2 vs 1.04 ± 0.1,P < 0.001)in macrophage-conditioned media treated HepG2 cells.In patients with chronic HCV infection,hepatic mRNA expression of CD163 (F0 1.0 ± 0.2,F1/2 2.8 ± 0.3,F3/4 5.3 ± 1.0,P =0.001) and MMP-9 (F0 1.0 ± 0.4,F1/2 2.8 ± 0.3,F3/4 4.1 ± 0.8,P =0.011) was significantly associated with increasing stage of fibrosis.CONCLUSION:Secreted macrophage products alter the phenotype and function of hepatocytes

  11. Influence of 48 hours of cold storage in University of Wisconsin organ preservation solution on metabolic capacity of rat hepatocytes

    NARCIS (Netherlands)

    Olinga, Peter; Slooff, M.JH; Meijer, D.K F; Groothuis, Geny; Merema, M.T.

    1997-01-01

    Background/Aims: Suspensions of isolated hepatocytes are a valuable tool to study liver functions, For optimal use of the isolated hepatocytes, methods are needed to preserve the hepatocytes while maintaining their viability, metabolic and transport functions, Until now little has been known about t

  12. Endotoxin-stimulated Rat Hepatic Stellate Cells Induce Autophagy in Hepatocytes as a Survival Mechanism.

    Science.gov (United States)

    Dangi, Anil; Huang, Chao; Tandon, Ashish; Stolz, Donna; Wu, Tong; Gandhi, Chandrashekhar R

    2016-01-01

    Bacterial lipopolysaccharide (LPS)-stimulated hepatic stellate cells (HSCs) produce many cytokines including IFNβ, TNFα, and IL6, strongly inhibit DNA synthesis, but induce apoptosis of a small number of hepatocytes. In vivo administration of LPS (up to 10 mg/mL) causes modest inflammation and weight loss in rats but not mortality. We determined whether LPS-stimulated HSCs instigate mechanisms of hepatocyte survival. Rats received 10 mg/kg LPS (i.p.) and determinations were made at 6 h. In vitro, HSCs were treated with 100 ng/mL LPS till 24 h. The medium was transferred to hepatocytes, and determinations were made at 0-12 h. Controls were HSC-conditioned medium or medium-containing LPS. LPS treatment of rats caused autophagy in hepatocytes, a physiological process for clearance of undesirable material including injured or damaged organelles. This was accompanied by activation of c-Jun NH2 terminal kinase (JNK) and apoptosis of ~4-5% of hepatocytes. In vitro, LPS-conditioned HSC medium (LPS/HSC) induced autophagy in hepatocytes but apoptosis of only ~10% of hepatocytes. While LPS/HSC stimulated activation of JNK (associated with cell death), it also activated NFkB and ERK1/2 (associated with cell survival). LPS-stimulated HSCs produced IFNβ, and LPS/HSC-induced autophagy in hepatocytes and their apoptosis were significantly inhibited by anti-IFNβ antibody. Blockade of autophagy, on the other hand, strongly augmented hepatocyte apoptosis. While LPS-stimulated HSCs cause apoptosis of a subpopulation of hepatocytes by producing IFNβ, they also induce cell survival mechanisms, which may be of critical importance in resistance to liver injury during endotoxemia.

  13. [POLYPEPTIDES INFLUENCE ON TISSUE CELL CULTURES REGENERATION OF VARIOUS AGE RATS].

    Science.gov (United States)

    Ryzhak, A P; Chalisova, N I; Lin'kova, N S; Khalimov, R I; Ryzhak, G A; Zhekalov, A N

    2015-01-01

    A comparative study of polypeptides extracted from the tissues of calves: Cortexin (from brain cortex), Epinorm (from pineal gland), Ventvil (from liver), Prostatilen (from prostate), Thymalin (from thymus), Chelohart (from heart), Chondrolux (from cartilage) on the relevant organotypic tissue cultures of young and old rats, in concentration 0,01-100 ng/ml was performed. Polypeptides specifically stimulated "young" and "old" cell cultures growth in concentration 20-50 ng/ml. This effect correlates with increasing of PCNA and decreasing of p53 expression in brain cortex, pineal gland, liver, prostate, heart, cartilage. Moreover, Thymalin activated CD5, CD20 expression--markers of B-cells differentiation. These data show that polypeptides isolated from different tissues have selective molecular activity on the regeneration of suitable tissues in aging.

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

    Science.gov (United States)

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

    1997-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Yong-Liang Zhuang

    2010-01-01

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

  16. Bacillus anthracis SlaQ Promotes S-Layer Protein Assembly

    OpenAIRE

    Nguyen-Mau, Sao-Mai; Oh, So-Young; Schneewind, Daphne I.; Missiakas, Dominique; Schneewind, Olaf

    2015-01-01

    Bacillus anthracis vegetative forms assemble an S-layer comprised of two S-layer proteins, Sap and EA1. A hallmark of S-layer proteins are their C-terminal crystallization domains, which assemble into a crystalline lattice once these polypeptides are deposited on the bacterial surface via association between their N-terminal S-layer homology domains and the secondary cell wall polysaccharide. Here we show that slaQ, encoding a small cytoplasmic protein conserved among pathogenic bacilli elabo...

  17. Photoinduced Electron Transfer in Ordered Macromolecular Assemblies. Final report for May 1, 1988 - June 30, 2002

    Energy Technology Data Exchange (ETDEWEB)

    Jones, G.

    2005-02-11

    The final report describes studies over a 13 year period having to do with photoinduced electron transfer for active chromophores and redox agents, including assembly of the components in water soluble polymers or polypeptides. The findings include observation of long range charge separation and electron transport using laser phototransient spectroscopy. The systems targeted in these studies include peptide assemblies for which helical conformations and aggregation are documented. Oligomeric peptides modified with non-native redox active groups were also selected for investigation. Highly charged polymers or peptides were investigated as host agents that resemble proteins. The overall goal of these investigations focused on the design and characterization of systems capable of artificial photosynthesis.

  18. Effects of cholesterol on pore formation in lipid bilayers induced by human islet amyloid polypeptide fragments: A coarse-grained molecular dynamics study

    Science.gov (United States)

    Xu, Weixin; Wei, Guanghong; Su, Haibin; Nordenskiöld, Lars; Mu, Yuguang

    2011-11-01

    Disruption of the cellular membrane by the amyloidogenic peptide, islet amyloid polypeptide (IAPP), has been considered as one of the mechanisms of β-cell death during type 2 diabetes. The N-terminal region (residues 1-19) of the human version of IAPP is suggested to be primarily responsible for the membrane-disrupting effect of the full-length hIAPP peptide. However, the detailed assembly mode of hIAPP1-19 with membrane remains unclear. To gain insight into the interactions of hIAPP1-19 oligomer with the model membrane, we have employed coarse-grained molecular dynamics self-assembly simulations to study the aggregation of hIAPP1-19 fragments in the binary lipid made of zwitterionic dipalmitoylphosphatidylcholine (DPPC) and anionic dipalmitoylphosphatidylserine (DPPS) in the presence and absence of different levels of cholesterol content. The membrane-destabilizing effect of hIAPP1-19 is found to be modulated by the presence of cholesterol. In the absence of cholesterol, hIAPP1-19 aggregates prefer to locate inside the bilayer, forming pore-like assemblies. While in the presence of cholesterol molecules, the lipid bilayer becomes more ordered and stiff, and the hIAPP1-19 aggregates are dominantly positioned at the bilayer-water interface. The action of cholesterol may suggest a possible way to maintain the membrane integrity by small molecule interference.

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

    Science.gov (United States)

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

    2017-05-01

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

  20. Medial septal and median raphe innervation of vasoactive intestinal polypeptide-containing interneurons in the hippocampus.

    Science.gov (United States)

    Papp, E C; Hajos, N; Acsády, L; Freund, T F

    1999-05-01

    Vasoactive intestinal polypeptide-immunoreactive interneurons are known to form three anatomically and neurochemically well-characterized neuron populations in the hippocampus. Two of these establish synaptic contacts selectively with other GABAergic cells (interneuron-selective cells), whereas the third type innervates pyramidal cell bodies and proximal dendrites like a conventional basket cell. Our aim was to examine which of the vasoactive intestinal polypeptide-containing interneuron populations are among the targets of GABAergic septohippocampal and serotonergic raphe-hippocampal pathways. Anterograde tracing with Phaseolus vulgaris leucoagglutinin combined with double immunocytochemistry for vasoactive intestinal polypeptide was used at the light and electron microscopic levels. Our results show that both interneuron-selective cells and vasoactive intestinal polypeptide-containing basket cells receive synaptic input from the medial septum and median raphe nucleus. The GABAergic component of the septohippocampal pathway establishes multiple contacts on both cell types. In the case of the raphe-hippocampal projection, single or double contacts were more frequent on vasoactive intestinal polypeptide-positive interneuron selective cells (76%), whereas multiple contacts predominated on basket cells (83%). The extrinsic GABAergic innervation of interneuron-selective cells in the hippocampus indicates a complex interaction among GABAergic systems, which might ensure the timing and rhythmic synchronization of inhibitory processes in the hippocampus. On the other hand, our results suggest that the serotonergic effect on perisomatic inhibition is exerted via vasoactive intestinal polypeptide-containing basket cells that are functionally distinct from their parvalbumin-positive relatives, which appear to escape control of serotonergic as well as local interneuron-selective cells.

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

    Science.gov (United States)

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

    2017-09-01

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

  2. Bioengineering Multifunctional Quantum Dot-Polypeptide Assemblies and Immunoconjugates for the Ablation of Advanced Prostate Cancer Disease

    Science.gov (United States)

    2008-02-01

    urgency for the discovery of site-specific therapeutics for residual disease and metastasis in advanced prostate cancer. PROSTATE-SPECIFIC MEMBRANE...Chromek, M., et al., The antimicrobial peptide cathelicidin protects the urinary tract against invasive bacterial infection. Nat. Med., 2006. 12(6): p. 636...cathelicidin protects the urinary tract against invasive bacterial infection. Nat Med 2006;12:636–41. 22. Berezin V, Bock E. NCAM mimetic peptides: Phar

  3. Bioengineering Multifunctional Quantum Dot-Polypeptide Assemblies and Immunoconjugates for the Ablation of Advanced Prostate Cancer Disease

    Science.gov (United States)

    2009-02-01

    5d. PROJECT NUMBER Srivatsan Kidambi, Ph.D. 5e. TASK NUMBER E-Mail: srivatsan.kidambi@gmail.com 5f. WORK UNIT NUMBER 7. PERFORMING...2009 Wiley-VCH Verlag Gm[39] J. Silver, W. Ou, Nano Lett. 2005, 5, 1445–1449. [40] O. Harush-Frenkel, E. Rozentur, S. Benita, Y. Altschuler, Bioma

  4. Structure and assembly of calf hoof keratin filaments.

    Science.gov (United States)

    Sayers, Z; Michon, A M; Sicre, P; Koch, M H

    1990-05-01

    Keratin filament polypeptides were purified from calf hoof stratum corneum with the aim of studying the in vitro assembly process and determining structural parameters of reconstituted filaments. Anion exchange chromatography was used to obtain the most complete fractionation and identification of the acidic and basic components in the purified polypeptide mixture to date. The reassembly products of the fractionated components were investigated by electron microscopy. Fully reconstituted filaments yield homogeneous solutions, and values of 9.8 nm for the filament diameter and 25 kDa/nm for the mass per unit length (M/L) were obtained by X-ray solution scattering. The structures formed in solution at various stages of filament assembly were not sufficiently homogeneous to be studied by this technique. X-ray diffraction patterns from native stratum corneum display strong maxima at 3.6 and 5.4 nm. Contrary to previous reports, these maxima do not appear to be due to lipids since they are also observed with delipidated rehydrated specimens. A series of weak maxima is also detected in the patterns of dry tissue. The absence of these features in the patterns of reconstituted filaments suggests that, in contrast to some electron microscopic observations, there are no prominent regularities in the structure of calf hoof keratin filaments.

  5. Polymer Directed Protein Assemblies

    NARCIS (Netherlands)

    van Rijn, Patrick

    2013-01-01

    Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e. g., virus particles. Viruses are a multi-protein assembly of which the morphology is

  6. Polymer Directed Protein Assemblies

    NARCIS (Netherlands)

    van Rijn, Patrick

    Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e. g., virus particles. Viruses are a multi-protein assembly of which the morphology is

  7. Self-assembled bionanostructures: proteins following the lead of DNA nanostructures.

    Science.gov (United States)

    Gradišar, Helena; Jerala, Roman

    2014-02-03

    Natural polymers are able to self-assemble into versatile nanostructures based on the information encoded into their primary structure. The structural richness of biopolymer-based nanostructures depends on the information content of building blocks and the available biological machinery to assemble and decode polymers with a defined sequence. Natural polypeptides comprise 20 amino acids with very different properties in comparison to only 4 structurally similar nucleotides, building elements of nucleic acids. Nevertheless the ease of synthesizing polynucleotides with selected sequence and the ability to encode the nanostructural assembly based on the two specific nucleotide pairs underlay the development of techniques to self-assemble almost any selected three-dimensional nanostructure from polynucleotides. Despite more complex design rules, peptides were successfully used to assemble symmetric nanostructures, such as fibrils and spheres. While earlier designed protein-based nanostructures used linked natural oligomerizing domains, recent design of new oligomerizing interaction surfaces and introduction of the platform for topologically designed protein fold may enable polypeptide-based design to follow the track of DNA nanostructures. The advantages of protein-based nanostructures, such as the functional versatility and cost effective and sustainable production methods provide strong incentive for further development in this direction.

  8. Sensor mount assemblies and sensor assemblies

    Science.gov (United States)

    Miller, David H [Redondo Beach, CA

    2012-04-10

    Sensor mount assemblies and sensor assemblies are provided. In an embodiment, by way of example only, a sensor mount assembly includes a busbar, a main body, a backing surface, and a first finger. The busbar has a first end and a second end. The main body is overmolded onto the busbar. The backing surface extends radially outwardly relative to the main body. The first finger extends axially from the backing surface, and the first finger has a first end, a second end, and a tooth. The first end of the first finger is disposed on the backing surface, and the tooth is formed on the second end of the first finger.

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

    Science.gov (United States)

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

    2016-04-20

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

  10. CYP2E1-dependent hepatotoxicity and oxidative damage after ethanol administration in human primary hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Lie-Gang Liu; Hong Yan; Ping Yao; Wen Zhang; Li-Jun Zou; Fang-Fang Song; Ke Li; Xiu-Fa Sun

    2005-01-01

    AIM: To observe the relationship between ethanol-induced oxidative damage in human primary cultured hepatocytes and cytochrome P450 2E1 (CYP2E1) activity, in order to address if inhibition of CYP2E1 could attenuate ethanol-induced cellular damage.METHODS: The dose-dependent (25-100 mmol/L) and time-dependent (0-24 h) exposures of primary human cultured hepatocytes to ethanol were carried out. CYP2E1 activity and protein expression were detected by spectrophotometer and Western blot analysis respectively.Hepatotoxicity was investigated by determination of lactate dehydrogenase (LDH) and aspartate transaminase (AST) level in hepatocyte culture supernatants, as well as the intracellular formation of malondialdehyde (MDA).RESULTS: A dose-and time-dependent response between ethanol exposure and CYP2E1 activity in human hepatocytes was demonstrated. Moreover, there was a time-dependent increase of CYP2E1 protein after 100 mmol/L ethanol exposure. Meanwhile, ethanol exposure of hepatocytes caused a time-dependent increase of ceilular MDA level, LDH, and AST activities in supernatants.Furthermore, the inhibitor of CYP2E1, diallyl sulfide (DAS) could partly attenuate the increases of MDA, LDH, and AST in human hepatocytes.CONCLUSION: A positive relationship between ethanol-induced oxidative aamage in human primary cultured hepatocytes and CYP2E1 activity was exhibited, and the inhibition of CYP2E1 could partly attenuate ethanol-induced oxidative damage.

  11. Asialoglycoprotein Receptor-Mediated Gene Delivery to Hepatocytes Using Galactosylated Polymers.

    Science.gov (United States)

    Thapa, Bindu; Kumar, Piyush; Zeng, Hongbo; Narain, Ravin

    2015-09-14

    Highly efficient, specific, and nontoxic gene delivery vector is required for gene therapy to the liver. Hepatocytes exclusively express asialoglycoprotein receptor (ASGPR), which can recognize and bind to galactose or N-acetylgalactosamine. Galactosylated polymers are therefore explored for targeted gene delivery to the liver. A library of safe and stable galactose-based glycopolymers that can specifically deliver genes to hepatocytes were synthesized having different architectures, compositions, and molecular weights via the reversible addition-fragmentation chain transfer process. The physical and chemical properties of these polymers have a great impact on gene delivery efficacy into hepatocytes, as such block copolymers are found to form more stable complexes with plasmid and have high gene delivery efficiency into ASGPR expressing hepatocytes. Transfection efficiency and uptake of polyplexes with these polymers decreased significantly by preincubation of hepatocytes with free asialofetuin or by adding free asialofetuin together with polyplexes into hepatocytes. The results confirmed that polyplexes with these polymers were taken up specifically by hepatocytes via ASGPR-mediated endocytosis. The results from transfection efficiency and uptake of these polymers in cells without ASGPR, such as SK Hep1 and HeLa cells, further support this mechanism. Since in vitro cytotoxicity assays prove these glycopolymers to be nontoxic, they may be useful for delivery of clinically important genes specifically to the liver.

  12. Nrf2 is involved in maintaining hepatocyte identity during liver regeneration.

    Directory of Open Access Journals (Sweden)

    Yuhong Zou

    Full Text Available Nrf2, a central regulator of the cellular defense against oxidative stress and inflammation, participates in modulating hepatocyte proliferation during liver regeneration. It is not clear, however, whether Nrf2 regulates hepatocyte growth, an important cellular mechanism to regain the lost liver mass after partial hepatectomy (PH. To determine this, various analyses were performed in wild-type and Nrf2-null mice following PH. We found that, at 60 h post-PH, the vast majority of hepatocytes lacking Nrf2 reduced their sizes, activated hepatic progenitor markers (CD133, TWEAK receptor, and trefoil factor family 3, depleted HNF4α protein, and downregulated the expression of a group of genes critical for their functions. Thus, the identity of hepatocytes deficient in Nrf2 was transiently but massively impaired in response to liver mass loss. This event was associated with the coupling of protein depletion of hepatic HNF4α, a master regulator of hepatocyte differentiation, and concomitant inactivation of hepatic Akt1 and p70S6K, critical hepatocyte growth signaling molecules. We conclude that Nrf2 participates in maintaining newly regenerated hepatocytes in a fully differentiated state by ensuring proper regulation of HNF4α, Akt1, and p70S6K during liver regeneration.

  13. Improved isolation of murine hepatocytes for in vitro malaria liver stage studies

    Directory of Open Access Journals (Sweden)

    Penha-Gonçalves Carlos

    2007-12-01

    Full Text Available Abstract Background Primary hepatocyte cultures are a valuable tool for the understanding of cellular and molecular phenomena occurring during malaria liver stage. This paper describes an improved perfusion/dissociation procedure to isolate hepatocytes from mouse liver that is suitable for malaria studies and allows reproducible preparation of primary hepatocytes with consistent cell yields and controlled purity. Results This protocol is a detailed description of a technique to isolate and culture mouse hepatocytes and represents an improvement over previous descriptions of hepatocyte isolation for malaria studies, regarding three technical aspects: (1 dissociation reagents choice; (2 cell separation gradient and (3 cell purity control. Cell dissociation was optimized for a specific collagenase digestion media. The cell dissociation step was improved by using a three-layer discontinuous gradient. A cell purity check was introduced to monitor the expression of CD95 on hepatocytes using flow cytometry methods. Conclusion The procedure described allows reproducible recovery of one to three million hepatocytes per preparation with cell purity of about 90% as determined by FACS analysis. Completion of the protocol is usually achieved in about four hours per preparation and pooling is suggested for multiple preparations of larger number of cells.

  14. Mycelial culture of Phellinus linteus protects primary cultured rat hepatocytes against hepatotoxins.

    Science.gov (United States)

    Kim, S H; Lee, H S; Lee, S; Cho, J; Ze, K; Sung, J; Kim, Y C

    2004-12-01

    Hepatoprotective activity of Phellinus linteus was studied using H(2)O(2)- or galactosamine-injured primary cultures of rat hepatocytes as screening systems. The methanolic extract of the mycelial culture of Phellinus linteus significantly protected against hepatotoxins-induced toxicity in primary cultured rat hepatocytes as seen from the decreased level of glutamic pyruvic transaminase released from the injured hepatocytes. The methanolic extract of the mycelial culture of Phellinus linteus was subsequently fractionated with n-hexane, ethyl acetate, n-butanol and water. Among these fractions, 100 microg/mL of the ethyl acetate fraction was the most active one. The relative protections were 68.9 +/- 5.3% in H(2)O(2)-injured hepatocytes and 46.8 +/- 3.9% in galactosamine-injured hepatocytes, respectively. The ethyl acetate fraction appeared to maintain the glutathione level which was decreased by the treatment of H(2)O(2) or galactosamine and restored the level of RNA synthesis more than two times compared to galactosamine-injured hepatocytes. These results suggest that the ethyl acetate fraction of the mycelial culture of Phellinus linteus protects hepatocytes from H(2)O(2)- or galactosamine-induced injury by maintaining hepatic glutathione level and RNA synthesis as well.

  15. Sodium-independent, bicuculline-sensitive (/sup 3/H)GABA binding to isolated rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Minuk, G.Y.; Bear, C.E.; Sarjeant, E.J.

    1987-05-01

    To determine whether hepatocytes possess specific receptor sites for gamma-aminobutyric acid (GABA), a potent amino acid neurotransmitter, (/sup 3/H)GABA, was added to sodium-free suspensions of Percoll-purified hepatocytes derived from collagenase-perfused rat livers under various experimental conditions and in the presence or absence of specific GABA receptor agonists (muscimol) and antagonists (bicuculline). The effects of GABA, muscimol, and bicuculline on hepatocyte resting membrane potentials were also determined. Specific binding of (/sup 3/H)GABA to hepatocytes was a consistent finding. GABA-hepatocyte interactions were reversible and temperature dependent. Muscimol and bicuculline inhibited binding in a dose-dependent manner (IC50, 30 nM and 50 microM, respectively), whereas strychnine (1.0-100 microM), a nonspecific central nervous system stimulant, had no appreciable effect. Both GABA and muscimol (100 microM) caused significant hyperpolarization of hepatocyte resting membrane potential (delta PD 5.4 +/- 3.1 and 22.2 +/- 16.2 mV, respectively, means +/- SD, P less than 0.0005). Bicuculline (100 microM) inhibited the effect of muscimol (P less than 0.05). The results of this study suggest that specific GABA receptor sites exist on the surface of isolated rat hepatocytes. The presence of such sites raises the possibility that, in addition to adrenergic and cholinergic innervation, hepatic function may be influenced by GABA-ergic neurotransmitter mechanisms.

  16. Discovering multiple transcripts of human hepatocytes using massively parallel signature sequencing (MPSS

    Directory of Open Access Journals (Sweden)

    Li Yi-Xue

    2007-07-01

    Full Text Available Abstract Background The liver is the largest human internal organ – it is composed of multiple cell types and plays a vital role in fulfilling the body's metabolic needs and maintaining homeostasis. Of these cell types the hepatocytes, which account for three-quarters of the liver's volume, perform its main functions. To discover the molecular basis of hepatocyte function, we employed Massively Parallel Signature Sequencing (MPSS to determine the transcriptomic profile of adult human hepatocytes obtained by laser capture microdissection (LCM. Results 10,279 UniGene clusters, representing 7,475 known genes, were detected in human hepatocytes. In addition, 1,819 unique MPSS signatures matching the antisense strand of 1,605 non-redundant UniGene clusters (such as APOC1, APOC2, APOB and APOH were highly expressed in hepatocytes. Conclusion Apart from a large number of protein-coding genes, some of the antisense transcripts expressed in hepatocytes could play important roles in transcriptional interference via a cis-/trans-regulation mechanism. Our result provided a comprehensively transcriptomic atlas of human hepatocytes using MPSS technique, which could be served as an available resource for an in-depth understanding of human liver biology and diseases.

  17. In vitro induction of human embryonic stem cells into hepatocyte-like cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Human embryonic stem cells (hES cells) are pluripotent and provide a unique, unlimited resource for human hepatocytes, which can serve as a novel cell source for cell transplantation and bioartificial liver (BAL). Here, we have developed a procedure by which hES cells can differentiate into hepatocyte-like cells. After being cultured in suspension in bacteriological petri dishes for 7 d, hES cells developed into cystic embryoid bodies (EBs). The EBs were then cultured in conditional medium containing dexamethasone and insulin in collagen type I-coated tissue culture dishes for two weeks. The hES cell-derived hepatocyte-like cells (HLCs) displayed some morphologic characteristics of hepatocytes. Reverse-transcription polymerase chain reaction (RT-PCR) and immunofluorescence cell staining proved that the induced HLCs expressed several hepatocyte specific genes including AFP, ALB, CYP1B1 and cytokeratins CK18 and CK19. Furthermore, the induced cells executed a range of hepatocyte functions, such as ICG uptake/excretion, glycogen deposits, albumin production and ammonium metabolism. Taken together, our results show that HLCs exhibit similar morphologic, phenotypic, and functional characteristics to hepatocytes.

  18. Auxiliary liver organ formation by implantation of spleen-encapsulated hepatocytes.

    Science.gov (United States)

    Nishio, Reiji; Nakayama, Miyuki; Ikekita, Masahiko; Watanabe, Yoshifumi

    2006-09-01

    Hepatocyte transplantation is an attractive alternative to orthotopic liver transplantation. However, its application has been limited because of its short-term success only. Here we report a new approach to hepatocyte transplantation resulting in the generation of an auxiliary liver in vivo. Isolated primary hepatocytes were encapsulated in isolated spleens and then transplanted by attaching the spleens to the livers of recipient animals (mice or rats) using biodegradable adhesive. A vascular network was rapidly established, and protein molecules circulated freely between the transplanted spleen and the liver, to which they adhered. In contrast, the spleen, which did not adhere to the liver or adhered elsewhere (adipose tissue or peritoneum), did not become vascularized but shrank and died. Encapsulation of hepatocytes in an isolated spleen enhanced their survival significantly, and co-encapsulation of Engelbreth- Holm-Swarm gel together with the hepatocytes further enhanced it. The encapsulated hepatocytes expressed liver-specific differentiation genes for more than 3 weeks. Plasma albumin concentrations in Nagase analbuminemic rats began to increase 3 days after transplantation. The transplanted hepatic cells migrated into the liver parenchyma, whereas the spleen was absorbed. Thus, we have developed a novel, simple approach for the rapid and efficient formation of functional auxiliary liver using a modified hepatocyte transplantation method.

  19. Circadian rhythms of Per2::Luc in individual primary mouse hepatocytes and cultures.

    Directory of Open Access Journals (Sweden)

    Casey J Guenthner

    Full Text Available BACKGROUND: Hepatocytes, the parenchymal cells of the liver, express core clock genes, such as Period2 and Cryptochrome2, which are involved in the transcriptional/translational feedback loop of the circadian clock. Whether or not the liver is capable of sustaining rhythms independent of a central pacemaker is controversial. Whether and how circadian information may be shared among cells in the liver in order to sustain oscillations is currently unknown. RESULTS: In this study we isolated primary hepatocytes from transgenic Per2(Luc mice and used bioluminescence as a read-out of the state of the circadian clock. Hepatocytes cultured in a collagen gel sandwich configuration exhibited persistent circadian rhythms for several weeks. The amplitude of the rhythms damped, but medium changes consistently reset the phase and amplitude of the cultures. Cry2(-/- Per2(Luc cells oscillated robustly and expressed a longer period. Co-culturing with wildtype cells did not significantly shorten the period, indicating that coupling among hepatocytes is insufficient to synchronize cells with significantly differing periods. However, spatial patterns revealed by cellular imaging of wildtype cultures provided evidence of weak local coupling among the hepatocytes. CONCLUSIONS: Our results with primary hepatocyte cultures demonstrate that cultured hepatocytes are weakly coupled. While this coupling is not sufficient to sustain global synchrony, it does increase local synchrony, which may stabilize the circadian rhythms of peripheral oscillators, such as the liver, against noise in the entraining signals.

  20. Restoration of cell polarity and bile excretion function of hepatocytes in sandwich-culture

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xian-jie; WANG Ying; SUN Jia-bang; SONG Mao-min; QIAO Xin

    2007-01-01

    Objective:To investigate the nature of the restoration of cell polarity and bile excretion function in Sandwich-cultured hepatocytes.Methods:Freshly isolated hepatocytes from male Sprague-Dawley rats were cultured in a double layer collagen gel Sandwich configuration.Morphological changes were observed under a inverted microscope.The domain specific membrane associated protein DPP Ⅳ was tested by immunofluorescenee,and the bile excretion function was determined by using fluorescein diacetate.Hepatocytes cultured on a single layer of collagen gel were taken as control.Results:Adult rat hepatocytes cultured in a double layer collagen gel sandwich configuration regained its morphological and functional polarity and maintained polygonal morphology for at least 4 weeks.Immunofluorescence studies USing antibodies against DPP Ⅳ showed polarity restoration as early as 48 h.After cultured in the double layer collagen gel Sandwich configuration for 96 h the hepatocytes began to excrete bile;while hepatocytes cultured on a single layer collagen gel had no bile excretion.Conclusion:Hepatocytes cultured in a double layer collagen gel Sandwich configuration are able to regain their morphological and functional polarity givan certain conditions.Hepaotcyte culture is a useful tool for the study of polarity restoration.

  1. Soldering in electronics assembly

    CERN Document Server

    Judd, Mike

    2013-01-01

    Soldering in Electronics Assembly discusses several concerns in soldering of electronic assemblies. The book is comprised of nine chapters that tackle different areas in electronic assembly soldering. Chapter 1 discusses the soldering process itself, while Chapter 2 covers the electronic assemblies. Chapter 3 talks about solders and Chapter 4 deals with flux. The text also tackles the CS and SC soldering process. The cleaning of soldered assemblies, solder quality, and standards and specifications are also discussed. The book will be of great use to professionals who deal with electronic assem

  2. Hepatocyte growth factor reduces CXCL10 expression in keratinocytes.

    Science.gov (United States)

    Hisadome, Mitsuhiro; Ohnishi, Tomokazu; Kakimoto, Kyoko; Kusuyama, Joji; Bandow, Kenjiro; Kanekura, Takuro; Matsuguchi, Tetsuya

    2016-10-01

    Keratinocytes secrete vascular endothelial growth factor (VEGF) and angioregulatory chemokines during cutaneous wound healing. Hepatocyte growth factor (HGF) promotes skin re-epithelialization by increasing VEGF expression in keratinocytes. Here, we investigated the regulatory roles of HGF in the expression of genes encoding angiogenic and angiostatic chemokines in keratinocytes and found that HGF specifically inhibits mRNA expression of the angiostatic chemokine CXCL10 in both mouse primary keratinocytes and in the human keratinocyte cell line HaCaT through the MEK/ERK cascade. Furthermore, HGF inhibited tumor necrosis factor-α-induced CXCL10 expression at both mRNA and protein levels in HaCaT cells. Thus, HGF may orchestrate angiogenesis in wounded skin by modulating both VEGF and CXCL10 expression in keratinocytes. © 2016 Federation of European Biochemical Societies.

  3. Hepatocyte growth factor in lung repair and pulmonary fibrosis

    Institute of Scientific and Technical Information of China (English)

    Ronald Allan M PANGANIBAN; Regina M DAY

    2011-01-01

    Pulmonary remodeling is characterized by the permanent and progressive loss of the normal alveolar architecture, especially the loss of alveolar epithelial and endothelial cells, persistent proliferation of activated fibroblasts, or myoflbroblasts, and alteration of extracellular matrix. Hepatocyte growth factor (HGF) is a pleiotropic factor, which induces cellular motility, survival, proliferation, and morphogenesis, depending upon the cell type. In the adult, HGF has been demonstrated to play a critical role in tissue repair, including in the lung. Administration of HGF protein or ectopic expression of HGF has been demonstrated in animal models of pulmonary fibrosis to induce normal tissue repair and to prevent fibrotic remodeling. HGF-induced inhibition of fibrotic remodeling may occur via multiple direct and indirect mechanisms including the induction of cell survival and proliferation of pulmonary epithelial and endothelial cells, and the reduction of myofibroblast accumulation.

  4. 3D hepatic cultures simultaneously maintain primary hepatocyte and liver sinusoidal endothelial cell phenotypes.

    Directory of Open Access Journals (Sweden)

    Yeonhee Kim

    Full Text Available Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes and non-parenchymal (liver sinusoidal endothelial, LSEC cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs were cultured in a layered three-dimensional (3D configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM, which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1 demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism

  5. Hepatocyte IKK2 protects Mdr2-/- mice from chronic liver failure.

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    Hanno Ehlken

    Full Text Available Mice lacking the Abc4 protein encoded by the multidrug resistance-2 gene (Mdr2(-/- develop chronic periductular inflammation and cholestatic liver disease resulting in the development of hepatocellular carcinoma (HCC. Inhibition of NF-κB by expression of an IκBα super-repressor (IκBαSR transgene in hepatocytes was shown to prevent HCC development in Mdr2(-/- mice, suggesting that NF-κB acts as a tumour promoter in this model of inflammation-associated carcinogenesis. On the other hand, inhibition of NF-κB by hepatocyte specific ablation of IKK2 resulted in increased liver tumour development induced by the chemical carcinogen DEN. To address the role of IKK2-mediated NF-κB activation in hepatocytes in the pathogenesis of liver disease and HCC in Mdr2(-/- mice, we generated Mdr2-deficient animals lacking IKK2 specifically in hepatocytes using the Cre-loxP system. Mdr2(-/- mice lacking IKK2 in hepatocytes developed spontaneously a severe liver disease characterized by cholestasis, major hyperbilirubinemia and severe to end-stage fibrosis, which caused muscle wasting, loss of body weight, lethargy and early spontaneous death. Cell culture experiments showed that primary hepatocytes lacking IKK2 were more sensitive to bile acid induced death, suggesting that hepatocyte-specific IKK2 deficiency sensitized hepatocytes to the toxicity of bile acids under conditions of cholestasis resulting in greatly exacerbated liver damage. Mdr2(-/-IKK2(Hep-KO mice remarkably recapitulate chronic liver failure in humans and might be of special importance for the study of the mechanisms contributing to the pathogenesis of end-stage chronic liver disease or its implications on other organs.IKK2-mediated signaling in hepatocytes protects the liver from damage under conditions of chronic inflammatory cholestasis and prevents the development of severe fibrosis and liver failure.

  6. Development of mouse hepatocyte lines permissive for hepatitis C virus (HCV.

    Directory of Open Access Journals (Sweden)

    Hussein Hassan Aly

    Full Text Available The lack of a suitable small animal model for the analysis of hepatitis C virus (HCV infection has hampered elucidation of the HCV life cycle and the development of both protective and therapeutic strategies against HCV infection. Human and mouse harbor a comparable system for antiviral type I interferon (IFN induction and amplification, which regulates viral infection and replication. Using hepatocytes from knockout (ko mice, we determined the critical step of the IFN-inducing/amplification pathways regulating HCV replication in mouse. The results infer that interferon-beta promoter stimulator (IPS-1 or interferon A receptor (IFNAR were a crucial barrier to HCV replication in mouse hepatocytes. Although both IFNARko and IPS-1ko hepatocytes showed a reduced induction of type I interferons in response to viral infection, only IPS-1-/- cells circumvented cell death from HCV cytopathic effect and significantly improved J6JFH1 replication, suggesting IPS-1 to be a key player regulating HCV replication in mouse hepatocytes. We then established mouse hepatocyte lines lacking IPS-1 or IFNAR through immortalization with SV40T antigen. Expression of human (hCD81 on these hepatocyte lines rendered both lines HCVcc-permissive. We also found that the chimeric J6JFH1 construct, having the structure region from J6 isolate enhanced HCV replication in mouse hepatocytes rather than the full length original JFH1 construct, a new finding that suggests the possible role of the HCV structural region in HCV replication. This is the first report on the entry and replication of HCV infectious particles in mouse hepatocytes. These mouse hepatocyte lines will facilitate establishing a mouse HCV infection model with multifarious applications.

  7. Proliferation of L02 human hepatocytes in tolerized geneticall yimmunocompetent rats

    Institute of Scientific and Technical Information of China (English)

    Hu Lin; Qing Mao; Yu-Ming Wang; Li Jiang

    2008-01-01

    AIM: To investigate whether human hepatocytes could proliferate after transplantation to normal immunocompetent rats treated with 2-acetaminofluorene or Retrorsine and partial hepatectomy.METHODS: L02 hepatocyte-tolerant Sprague-Dawley rats were injected with Retrorsine, 2-acetaminofluorene or normal saline. L02 hepatocytes were then transplanted via the spleen. Human albumin and its mRNA, specific proliferating cell nuclear antigen (PCNA), L02 hepatocyte dynamic distribution, number density and area density of PCNA-positive cells in the liver were determined.RESULTS: All the examined indicators were not significantly different between the rats treated with 2-acetaminofluorene and normal saline, which was not the case with rats treated with Retrorsine. A dynamic distribution of L02 hepatocytes in the rat liver was detected from wk 1 to mo 6 after transplantation in the Retrorsine group and from wk 1 to 10 in the 2-acetaminofluorene group. Human albumin and its mRNA were detected from wk 2 to mo 6 in the Retrorsine group and from wk 1 to 8 in the 2-acetaminofiuorene group.Specific human PCNA was detected in the rat liver from wk 2 to mo 6 in the Retrorsine group and from wk 2 to 6 in the 2-acetaminofluorene group. Human albumin and its mRNA contents as well as the number of PCNA positive cells reached a peak at wk 4.CONCLUSION: L02 human hepatocytes could not proliferate significiantly after transplantation to the normal,immunocompetent rats treated with 2-acetaminofluorene.L02 human hepatocytes can survive for 10 wk after transplantation and express human albumin for 8 wk. L02human hepatocytes can proliferate and express human albumin for 6 mo after transplantation to the rats treated with Retrorsine. The chimeric L02 human hepatocytes,which then underwent transplantation into tolerant rats,were normal in morphogenesis, biochemistry and function.

  8. Response of porcine hepatocytes in primary culture to plasma from severe viral hepatitis patients

    Institute of Scientific and Technical Information of China (English)

    Yong-Bo Cheng; Ying-Jie Wang; Shi-Chang Zhang; Jun Liu; Zhi Chen; Jia-Jia Li

    2005-01-01

    AIM: To observe the effects of plasma from patients with severe viral hepatitis (SVHP) on the growth and metabolism of porcine hepatocytes and the clinical efficiency of bioartificial liver device.METHODS: Hepatocytes were isolated from male porcines by collagenase perfusion. The synthesis of DNA and total protein, leakages of AST and LDH, changes in glutathione (GSH), catalase and morphology of porcine hepatocytes exposed to SVHP were investigated to indicate the effect of plasma from patients with severe hepatitis on the growth, injury, detoxification, and morphology of porcine hepatocytes.RESULTS: The synthesis of DNA and protein was inhibited in the medium containing 100% SVHP compared to the controls. The leakages of LDH and AST increased in porcine hepatocytes following exposure to 100% SVHP for 5 h. The difference between 100% SVHP and 10% newborn calf serum (NCS) was significant in t-test (LDH: t = 24.552, P = 0.001; AST: t = 4.169, P =0.014). After exposure to SVHP for 24 h, alterations in GSH status were significant (F = 2.746, P<0.05) between porcine hepatocytes in 100% SVHP and 10% NCS, but no alteration occurred in the culture medium after 48 h (F = 4.378, P<0.05). A similar profile was observed in catalase activity. Many round vacuoles were observed in porcine hepatocytes cultured in SVHP. The membranes of these cells became indistinct and almost all the cells died on d 5.CONCLUSION: Plasma from patients with severe hepatitis inhibits the growth, injures membrane, disturbs GSH homeostasis and induces morphological changes of porcine hepatocytes. It is suggested that SVHP should be pretreated to reduce the toxin load and improve the performance of porcine hepatocytes in extracorporeal liver-support devices.

  9. Interaction of propionate and carnitine metabolism in isolated rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Brass, E.P.; Beyerinck, R.A.

    1987-05-01

    Propionate (P) and its metabolic products P-CoA and methylmalonyl-CoA can disrupt normal hepatic metabolism. Carnitine (Cn) has been shown to partially restore cellular function in the presence of P. This effect of Cn may result from removal of propionyl groups as propionylcarnitine (P-Cn). The present study examined the kinetics of P-Cn formation in rat hepatocytes, and the consequence of P-Cn formation on P and Cn metabolism. /sup 14/C-P was converted to CO/sub 2/, glucose and P-Cn in the hepatocyte system. Increasing concentrations of Cn up to 10.0 mM increased P-Cn formation from P without affecting CO/sub 2/ or glucose formation. Thus, 10.0 mM Cn increased total P metabolism by 40%. Metabolism of P was associated with a decrease in Cn concentration and an increase in short chain acylcarnitines (SCCn). In the absence of added Cn, 60 min incubation with P decreased Cn from 6.8 to 2.5 ..mu..M with a corresponding increase in SCCn. This effect of P to deplete free Cn was not seen to the same degree with butyrate in place of P. Similar increases in the formation of SCCn in the presence of P at the expense of free Cn were seen when the incubation Cn concentration was increased to 50 ..mu..M or 150 ..mu..M. HPLC methodologies to study specific acylcarnitines demonstrated the accumulation of large amounts of P-Cn in the incubations containing P, accounting for the depletion of free Cn.

  10. Non-chromatographic purification of recombinant elastin-like polypeptides and their fusions with peptides and proteins from Escherichia coli.

    Science.gov (United States)

    MacEwan, Sarah R; Hassouneh, Wafa; Chilkoti, Ashutosh

    2014-06-09

    Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

  11. Evidence against a stem cell origin of new hepatocytes in a common mouse model of chronic liver injury.

    Science.gov (United States)

    Schaub, Johanna R; Malato, Yann; Gormond, Coralie; Willenbring, Holger

    2014-08-21

    Hepatocytes provide most liver functions, but they can also proliferate and regenerate the liver after injury. However, under some liver injury conditions, particularly chronic liver injury where hepatocyte proliferation is impaired, liver stem cells (LSCs) are thought to replenish lost hepatocytes. Conflicting results have been reported about the identity of LSCs and their contribution to liver regeneration. To address this uncertainty, we followed candidate LSC populations by genetic fate tracing in adult mice with chronic liver injury due to a choline-deficient, ethionine-supplemented diet. In contrast to previous studies, we failed to detect hepatocytes derived from biliary epithelial cells or mesenchymal liver cells beyond a negligible frequency. In fact, we failed to detect hepatocytes that were not derived from pre-existing hepatocytes. In conclusion, our findings argue against LSCs, or other nonhepatocyte cell types, providing a backup system for hepatocyte regeneration in this common mouse model of chronic liver injury.

  12. Evidence against a Stem Cell Origin of New Hepatocytes in a Common Mouse Model of Chronic Liver Injury

    Directory of Open Access Journals (Sweden)

    Johanna R. Schaub

    2014-08-01

    Full Text Available Hepatocytes provide most liver functions, but they can also proliferate and regenerate the liver after injury. However, under some liver injury conditions, particularly chronic liver injury where hepatocyte proliferation is impaired, liver stem cells (LSCs are thought to replenish lost hepatocytes. Conflicting results have been reported about the identity of LSCs and their contribution to liver regeneration. To address this uncertainty, we followed candidate LSC populations by genetic fate tracing in adult mice with chronic liver injury due to a choline-deficient, ethionine-supplemented diet. In contrast to previous studies, we failed to detect hepatocytes derived from biliary epithelial cells or mesenchymal liver cells beyond a negligible frequency. In fact, we failed to detect hepatocytes that were not derived from pre-existing hepatocytes. In conclusion, our findings argue against LSCs, or other nonhepatocyte cell types, providing a backup system for hepatocyte regeneration in this common mouse model of chronic liver injury.

  13. Polypeptoids: A model system for exploring sequence and shape effects on block copolymer self-assembly

    Science.gov (United States)

    Segalman, Rachel

    2015-03-01

    While our ability to understand the detailed relationship between block copolymer chemistry and mesoscopic self-assembly has made remarkable progress over the last many years, yet we are still limited to a relatively small number of blocks in terms of structure-property understanding. Thus, there is a need to explore self-assembly phase space with sequence using a model system. Polypeptoids are non-natural, sequence specific polymers that offer the opportunity to probe the effect of sequence on self-assembly with much simpler molecular interactions and more scalable synthesis than traditional polypeptides. In this talk, I will discuss the use of this model system to understand the role of sequence on chain collapse and globule formation in solution, polymer crystallization, and block copolymer self-assembly. I will then discuss potential application as surface active agents for anti-fouling.

  14. Composite fan stator assembly

    Energy Technology Data Exchange (ETDEWEB)

    Donges, G.L.

    1993-07-13

    A composite fan stator assembly is described for a gas turbine engine having at least two fan rotor stages, the composite stator assembly comprising: an annular composite fan case assembly including an access port, the fan case assembly circumferentially disposed around first and second fan rotor stage locations, a composite fan stator stage supported by and extending radially inward of the fan case assembly and axially disposed between the two fan rotor stage locations, the fan stator stage includes at least one removable vane segment accessible for removal through the access port for assembly and reassembly, the composite fan case assembly including a separable composite forward fan case assembly and a separable composite aft fan case assembly spaced axially aft of the forward fan case assembly, the forward fan case assembly being bolted to the aft fan case assembly, wherein the composite fan stator stage is axially and radially trapped and supported by the forward and aft fan case assemblies. A composite stator vane assembly comprising: a composite inner shroud, a composite outer shroud disposed radially outward of the inner shroud, a plurality of vanes disposed between the shrouds, the vanes including a suction side and a pressure side and radially inner and outer roots, the roots extending through platforms of corresponding ones of the inner and outer shrouds, four box-type attachment elements corresponding to curved suction and pressure sides of the inner and outer roots, the box-type attachment elements having two connected legs angled with respect to each other, a first one of the legs extending along, conforming to the curve of, and bonded to a corresponding one of the airfoil root sides, and a second one of the legs extending along and bonded to a composite shroud surface.

  15. In vitro drug metabolism of green tea catechins in human, monkey, dog, rat and mouse hepatocytes.

    Science.gov (United States)

    Chen, Wendy W; Qin, Geng-Yao; Zhang, Ting; Feng, Wan-Yong

    2012-06-01

    The metabolic fate of green tea catechins [(-)-epicatechin ((-)-EC), (-)-epicatechin-3-gallate (ECG) (-)- epigallocatechin (EGC) and (-)-epigallocatechin-3-gallate (EGCG)] in cryopreserved human, monkey, dog, rat and mouse hepatocytes was studied. Methylation, glucuronidation, sulfation and isomerization pathways of (-)-EC in all five species were found. Methylation, glucuronidation, sulfation, hydrolysis, isomerization and glucosidation pathways of ECG were found. Species differences in metabolism of (-)-EC or ECG were observed. Surprisingly, no metabolites of EGC or EGCG were detected, but chemical oxidation and polymerization were observed under these experimental conditions. It appeared that enzymatic reactions and chemical reactions were differentiated by an additional hydroxyl group on the B-ring between (-)-EC/ECG and EGC/EGCG. For (-)-EC, thirty-five metabolites including isomerized (M6. M10 and M25), glucuronidated (M3, M5 and M11), sulfated (M7, M15, M16, M18, M20, M23, M26), methylated (M2, M9, M12, M17, M19, M21, M27, M30, M32), glucuronated/methylated (M4, M8, M13, M14), sulfated/methylated (M22, M24, M28, M29, M31, M33, M34, M35) and diglucuronidate (M1), were detected and characterized. M11, M18, M19 and M23 were major metabolites in human hepatocytes; M11, M26 and M31 were major metabolites in monkey hepatocytes; M10, M20, M22, M26 and M31 were major metabolites in dog hepatocytes; M5, M6 and M10 were major metabolites in rat hepatocytes; and M5, M6 and M13 were major metabolites in mouse hepatocytes. For ECG, twelve metabolites including isomerized (M1), hydrolyzed (M3), glucosidated (M2), glucuronidated (M4 and M6), sulfated (M9, M11 and M12), methylated (M7), sulfated/glucuronidated/methylated (M8 and M10) and diglucuronidated (M5), were detected and characterized. M4, M11 and M12 were major metabolites in human hepatocytes; M11 and M12 were major metabolites in monkey hepatocytes; M3 and M11 were major metabolites in dog hepatocytes; M4, M6 and

  16. Role of Nuclear Constitutive Androstane Receptor in Regulation of Hepatocyte Proliferation and Hepatocarcinogenesis.

    Science.gov (United States)

    Kazantseva, Y A; Pustylnyak, Y A; Pustylnyak, V O

    2016-04-01

    Activation of the constitutive androstane receptor (CAR) in hepatocytes occurs as a body adaptation in response to a number of external influences, and its functional activity is primarily related to induction of enzymes detoxifying xenobiotics. However, special attention was recently given to CAR due to the fact that its key role becomes unveiled in various physiological and pathophysiological processes occurring in the liver: gluconeogenesis, metabolism of fatty acids and bilirubin, hormonal regulation, proliferation of hepatocytes, and hepatocarcinogenesis. Here we review the main pathways and mechanisms that elevate hepatocyte proliferative activity related to CAR and whose disturbance may be a pivotal factor in hepatocarcinogenesis.

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

    NARCIS (Netherlands)

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

    1993-01-01

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

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

    DEFF Research Database (Denmark)

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

    2006-01-01

    carboxylic groups relative to competing COOH losses (45 Da) from neutral carboxylic groups. Loss of CO2 is a strong indication of the presence of a zwitterionic [(+)...(-)...(+)] salt bridge in the gas-phase polypeptide cation. This method provides a tool for studying, for example, the nature of binding...

  19. Endothelial monocyte-activating polypeptide-II and its functions in (patho)physiological processes.

    NARCIS (Netherlands)

    Horssen, R. van; Eggermont, A.M.M.; Hagen, T.L.M. ten

    2006-01-01

    Endothelial monocyte-activating polypeptide-II (EMAP-II) is a pro-inflammatory cytokine with anti-angiogenic properties. Its precursor, proEMAP, is identical to the p43 auxiliary component of the tRNA multisynthetase complex and therefore involved in protein translation. Although most of the activit

  20. The role of the disulfide bond in the interaction of islet amyloid polypeptide with membranes

    NARCIS (Netherlands)

    Khemtemourian, L.P.; Engel, M.F.M.; Kruijtzer, J.A.W.; Hoppener, J.W.M.; Liskamp, R.M.J.; Killian, J.A.

    2010-01-01

    Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils in pancreatic islets of patients with type 2 diabetes mellitus. It has been suggested that the N-terminal part, which contains a conserved intramolecular disulfide bond between residues 2 and 7, interacts with membranes, ultimately

  1. Ectomycorrhizin Synthesis and Polypeptide Changes during the Early Stage of Eucalypt Mycorrhiza Development 1

    Science.gov (United States)

    Hilbert, Jean-Louis; Costa, Guy; Martin, Francis

    1991-01-01

    In functioning eucalypt ectomycorrhizas, biochemical alterations are accompanied by a differential accumulation of polypeptides including the synthesis of symbiosis-related proteins (JL Hilbert, Martin FM [1988] New Phytol 110: 339-346). In the present study, protein biosynthesis in the early stages of ectomycorrhiza formation on Eucalyptus globulus subsp. bicostata Kirkp. was examined using compatible and incompatible isolates of the basidiomycete Pisolithus tinctorius (Coker & Couch). Changes in polypeptide composition were observed within hours following contact of the compatible mycelium with the roots, well before the differentiation of typical symbiotic tissues. At this stage, at least seven symbiosis-related proteins (ectomycorrhizins) accumulated in root tissues. In vivo incorporation of [35S]methionine by ectomycorrhizas followed by electrophoresis of the labeled proteins revealed that most of these differences in polypeptide concentrations, including the ectomycorrhizin accumulation, are the result of differential protein biosynthesis rather than posttranslational modifications of the polypeptides. The initial development of eucalypt ectomycorrhizas, therefore, coincides with the synthesis of symbiosis-related proteins and the data presented here provide essential evidence to ascribe a functional developmental role to these proteins. ImagesFigure 2Figure 3Figure 4Figure 5 PMID:16668539

  2. Proline-rich polypeptides in Alzheimer's disease and neurodegenerative disorders - Therapeutic potential or a mirage?

    NARCIS (Netherlands)

    Gladkevich, A.; Bosker, F.; Korf, J.; Yenkoyan, K.; Vahradyan, H.; Aghajanov, M.

    2007-01-01

    The development of effective and safe drugs for a growing Alzheimer disease population is an increasing need at present. Both experimental and clinical evidence support a beneficial effect of proline-rich polypeptides in a number of neurodegenerative diseases, including Alzheimer disease. Experiment

  3. Expression of polypeptide GalNAc-transferases in stratified epithelia and squamous cell carcinomas

    DEFF Research Database (Denmark)

    Mandel, U; Hassan, H; Therkildsen, M H

    1999-01-01

    Mucin-type O-glycosylation is initiated by a large family of UDP-GalNAc: polypeptide N -acetyl-galactosaminyltransferases (GalNAc-transferases). Individual GalNAc-transferases appear to have different functions and Northern analysis indicates that they are differently expressed in different organ...

  4. Antipeptide antibodies that can distinguish specific subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.)

    Science.gov (United States)

    Cai, X.; Henry, R. L.; Takemoto, L. J.; Guikema, J. A.; Wong, P. P.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    The amino acid sequences of the beta and gamma subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (gamma 2-9, gamma 264-274, and beta 264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-beta 264-274 antibodies reacted specifically with the beta polypeptide and the anti-gamma 264-274 and anti-gamma 2-9 antibodies reacted specifically with the gamma polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.

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

    DEFF Research Database (Denmark)

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

    1993-01-01

    The pancreatic polypeptide (PP) response to a mixed meal was investigated in seven insulin-dependent diabetics without measurable signs of diabetic autonomic neuropathy, and in seven healthy subjects. Since acute changes in metabolic regulation might influence the meal-induced PP response, the in...

  6. /sup 1/H NMR studied of cardiostimulant polypeptides from sea anemones

    Energy Technology Data Exchange (ETDEWEB)

    Gooley, P.R.

    1985-01-01

    Sea anemones contain a series of homologous polypeptides which are potent cardiac stimulants. They exert their effect by binding to the Na/sup +/ channel of excitable membranes and delaying its closure. Although their physiological and pharmacological properties have been extensively studied, little is known about their conformations. The aim of the work described in this thesis is to characterize the solution conformation of several of these polypeptides, viz. ATXI and II from Anemonia sulcata and AP-A from Anthopleura xanthogrammica, by using high resolution /sup 1/H Nuclear Magnetic Resonance spectroscopy at 300 MHz. Resonances are assigned to amino acid types by a variety of techniques, including pH dependence and two-dimensional homonuclear correlated spectroscopy (COSY). The assignments reveal extensive conformational heterogeneity in AP-A and ATXII, but not in ATXI. The heterogeneity, manifested in the splitting of a number of resonances, is explained in terms of cis-trans isomerism of the peptide bond preceding Pro-41. Information on the secondary and tertiary structures of these polypeptides has been obtained from NOE data. The polypeptides undergo a conformational change at low pH reflecting pK/sub a/ values of 1.7/2.5, 3.0 and 1.9/2.3 for AP-A, ATXI and ATXII, respectively.

  7. Monte carlo generation of polypeptide random coil conformations; the persistence vector and the chain vector distribution

    NARCIS (Netherlands)

    Hesselink, F.Th.

    Polypeptide random coil conformations of various chain lenghts (N = 5, 10, 20, 40, 80 peptide units) are generated by a Monte Carlo procedure. The characteristic ratio obtained for the sets of generated conformations is identical with the exact value calculated with the average transformation matrix

  8. Antipeptide antibodies that can distinguish specific subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.)

    Science.gov (United States)

    Cai, X.; Henry, R. L.; Takemoto, L. J.; Guikema, J. A.; Wong, P. P.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    The amino acid sequences of the beta and gamma subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (gamma 2-9, gamma 264-274, and beta 264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-beta 264-274 antibodies reacted specifically with the beta polypeptide and the anti-gamma 264-274 and anti-gamma 2-9 antibodies reacted specifically with the gamma polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.

  9. Revisiting the human polypeptide GalNAc-T1 and T13 paralogs

    DEFF Research Database (Denmark)

    Festari, María Florencia; Trajtenberg, Felipe; Berois, Nora

    2017-01-01

    Polypeptide GalNAc-transferases (GalNAc-Ts) constitute a family of 20 human glycosyltransferases (comprising 9 subfamilies), which initiate mucin-type O-glycosylation. The O-glycoproteome is thought to be differentially regulated via the different substrate specificities and expression patterns o...

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

    NARCIS (Netherlands)

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

    1993-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Holm Liisa

    2011-05-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-02-01

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

  13. Effect of Polypeptide Chromium Chelate on Hepatic Protein Expression in Diabetic Mice%多肽铬螯合物对糖尿病小鼠肝脏蛋白质表达的影响

    Institute of Scientific and Technical Information of China (English)

    郎婧; 刘安军; 张国蓉

    2009-01-01

    目的:研究多肽铬螯合的对糖尿病小鼠肝脏蛋白质表达的影响,探讨其治疗糖尿病的机理.方法:通过腹腔注射四氧嘧啶建立糖尿病小鼠模型.将小鼠分为正常组、模型组和胶铬组,胶铬组以灌胃方式加入多肽铬螯合物;以光镜及HE染色观察三组小鼠肝脏形态及组织学的变化,采用SDS-PAGE实验和非SDS-PAGE检测三组小鼠肝脏蛋白质表达.结果:光镜及组织学观察结果显示多肽铬螫合物可以有效地减轻四氧嘧啶对肝细胞造成的损伤.模型组肝脏25kDa-35kDa之间的某种蛋白表达升高而多肽铬螯舍物可以降低此种蛋白的表达,初步推断这种蛋白质为SOD.结论:多肽铬螯合物能够通过降低四氧嘧啶造成的肝脏中抗氧化有关的蛋白质代偿性升高而祈祷保护肝脏的作用,是一种新型的治疗糖尿病所致的肝损伤的活性物质.%Objective:To study the effect of polypeptide chromium chelate on hepatic protein expression in diabetic mice.Moeh ods:Diabetic mouse model was set up by i.p.injection of alloxan.The mice were divided into normal group(NG),model group(MG) and polypeptide chromium group(PEG).Polypeptide chromium chelate wag added into PCG by i.g.method.The changes of hepatic morphology and histology in the three groups were observed by HE staining under microscope,and hepatic protein expression was examined by SDS-PAGE and non-SDS-PAGE experiments in the three egroups.Results:The results showed that polypeptide chromium chelate could effectively alleviate the hepatocytes lesion induced by alloxan.Expression of a kind of protein between 25kDa and 35kDa in model group increased but polypeptide chromium chelate could decrease its expression.This protein was deduced to bc SOD initially.Conclusions:Polypeptide chromium chelate could decrease the compesatory elevation of antioxidant related protein in the liver induced by alloxan and perform the hepatoprotective function,which is a novel active

  14. Synthesis of gluten-forming polypeptides. 1. Biosynthesis of gliadins and glutenin subunits.

    Science.gov (United States)

    Abonyi, Tibor; Király, István; Tömösközi, Sándor; Baticz, Orsolya; Guóth, Adrienn; Gergely, Szilveszter; Scholz, Eva; Lásztity, Demeter; Lásztity, Radomir

    2007-05-02

    Five winter wheat cultivars--GK Othalom (HMW-GS composition 2*, 7+8, 5+10), Ukrainka (1, 7+8, 5+10), Palotás (2*, 7+9, 5+10), Ködmön (2*, 7+8, 5+10), and Csongrád (2*, 7+9, 2+12)--grown in Hungary and harvested in the year 2005 were studied. The biosynthesis of gluten-forming polypeptides was followed starting at the 12th day after anthesis to the 53rd. Fresh kernel weight, moisture, and dry matter content of fresh kernels and gliadin and glutenin contents were determined. Gliadin components, total amounts of HMW and LMW polypeptides, and individual HMW polypeptides were determined using a RP-HPLC technique. Although considerable quantitative differences were observed concerning the content of total protein, gliadin, glutenin, and individual gluten-forming polypeptides, the character of accumulation of protein components--determined on the basis protein mass/kernel--was the same for the all of the cultivars studied and could be presented by a sigmoid curve. Small quantities of the gliadin and glutenin monomers may be detected in early stages of kernel development, but the bulk of these proteins is synthesized in later stages of development. It is generally suggested by specialists that the formation and accumulation of glutenin polymers starts later than the synthesis of monomers. Experimental data presented in this paper confirm this suggestion and show that in the first phase of protein synthesis the monomers are in "free" form; polymeric glutenin is detected only later. HMW glutenin subunits are synthesized synchronously, and quantitatively the polypeptides coded by chromosomes D and B dominate.

  15. [Clinical analysis of Cervus and Cucumis Polypeptide injection based on real world hospital information system].

    Science.gov (United States)

    Sun, Shuai-Ling; Xie, Yan-Ming; Li, Yuan-Yuan; Zhang, Yin; Yi, Dan-Hui; Zhuang, Yan

    2016-11-01

    To analyze the clinical application of Cervus and Cucumis Polypeptide injection in the real world, in order to define the characteristics of clinical drug use and correlation, and provide reference for risk management and further study for Cervus and Cucumis Polypeptide injection. Descriptive analysis and association rules analysis were performed on 37 721 cases using Cervus and Cucumis Polypeptide injection in 26 hospitals nationwide. Cervus and Cucumis Polypeptide injection were mostly adopted by patients aged between 45 and 64(39.84%); mainly used to treat fracture patients in clinic(17 362 cases, 33.97%); 12 mL(41.81%) was the commonest dosage. And the course of treatment mainly lasted for 1-3 days(28 467 cases, 76.26%), which was basically consistent with the description of package insert. In clinic, traditional Chinese medicines, such as blood activating and stasis removing agents and Bushen Zhuanggu agents, were frequently combined with it(rule support degree of 19.38%). Such western medicine as antibiotics and nutritional drugs were frequently combined with it(rule support 39.9%). The main single combined medicine were vitamin C(13 202 cases, 35%), and Jintiange capsule(7 285 cases, 19.31%). The commonly used combined drug pairs were Hulisan capsule and Jintiange capsule (rule support 4.458%), phenobarbital and ceftazidime azole oxazoline(rule support degree of 10.62%). Cervus and Cucumis Polypeptide injection is mainly adopted by elderly patients in clinic, used to treat fracture patients, and often combined with blood activating and stasis removing agents, Bushen Zhuanggu agents, antibiotics, and nutritional medicine to enhance fracture healing. In clinical application, attention shall be paid to drug safety of elderly patients and types of combined medicines and their interaction, so as to prevent adverse reactions. Copyright© by the Chinese Pharmaceutical Association.

  16. Short-term exercise reduces markers of hepatocyte apoptosis in nonalcoholic fatty liver disease

    DEFF Research Database (Denmark)

    Fealy, Ciaran E; Haus, Jacob M; Solomon, Thomas

    2012-01-01

    Increased hepatocyte apoptosis is a hallmark of nonalcoholic fatty liver disease (NAFLD) and contributes to the profibrogenic state responsible for the progression to nonalcoholic steatohepatitis (NASH). Strategies aimed at reducing apoptosis may result in better outcomes for individuals with NAF...

  17. Asialoglycoprotein receptor and liposome synergistically mediate the gene transfer into primary rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    李崇辉; 温守明; 翟海峰; 孙曼霁

    1999-01-01

    Gene transfer into primary rat hepatocytes was performed by employing cationic liposome as DNA carrier and the specific ligand of hepatic asialoglycoprotein receptor (ASGPR), asialofetuin, as liver-targeting ligand. The resuits showed that asialofetuin, when added to the gene transfer complexes, could significantly increase the hepatocyte transfeetion efficiency, and alleviate the cellular toxicity of Lipofectin. Several synthetic ligands of ASGPR (galactosyl albumin) could also increase the transfection efficiency of hepatocyte like asialofetuin. It was proved that ASGPR and cationic liposome could synergistically mediate the gene transfer into primary rat hepatoeytes. This novel gene delivery system provided a safer, more simple and efficient gene transfer method for primary hepatocytes, and showed prospecting application in hepatic gene therapy.

  18. Preparation and property analysis of a hepatocyte targeting pH-sensitive liposome

    Institute of Scientific and Technical Information of China (English)

    Si-Yuan Wen; Xiao-Hong Wang; Li Lin; Wei Guan; Sheng-Qi Wang

    2004-01-01

    AIM: To develop a hepatocyte targeting pH-sensitive liposome for drug delivery based on active targeting technology mediated by asialoglycoprotein receptors.METHODS: Four types of targeting molecules with galactose residue were synthesized and mixed with pH-sensitive lipids DC-chol/DOPE to prepare liposome with integrated property of hepatocyte specificity and pH sensitivity. Liposome 18-gal was selected with the best transfection activity through cellular uptake experiment. Property analysis was made through experiments of competitive inhibition of receptors,red blood cell hemolysis,in vitro cytotoxicity test by MTS assay and mediation of inhibitory effects of antisense phosphorothioate ODN on gene expression, etc.RESULTS: Liposome L8-gal had the desired properties of hepatocyte specificity, pH sensitivity, low cytotoxicity, and high transfection efficiency.CONCLUSION: Liposome 18-gal can be further developed as a potential hepatocyte- targeting delivery system.

  19. Recombinant Laminins Drive the Differentiation and Self-Organization of hESC-Derived Hepatocytes

    Directory of Open Access Journals (Sweden)

    Kate Cameron

    2015-12-01

    Full Text Available Stem cell-derived somatic cells represent an unlimited resource for basic and translational science. Although promising, there are significant hurdles that must be overcome. Our focus is on the generation of the major cell type of the human liver, the hepatocyte. Current protocols produce variable populations of hepatocytes that are the product of using undefined components in the differentiation process. This serves as a significant barrier to scale-up and application. To tackle this issue, we designed a defined differentiation process using recombinant laminin substrates to provide instruction. We demonstrate efficient hepatocyte specification, cell organization, and significant improvements in cell function and phenotype. This is driven in part by the suppression of unfavorable gene regulatory networks that control cell proliferation and migration, pluripotent stem cell self-renewal, and fibroblast and colon specification. We believe that this represents a significant advance, moving stem cell-based hepatocytes closer toward biomedical application.

  20. Recombinant Laminins Drive the Differentiation and Self-Organization of hESC-Derived Hepatocytes.

    Science.gov (United States)

    Cameron, Kate; Tan, Rosanne; Schmidt-Heck, Wolfgang; Campos, Gisela; Lyall, Marcus J; Wang, Yu; Lucendo-Villarin, Baltasar; Szkolnicka, Dagmara; Bates, Nicola; Kimber, Susan J; Hengstler, Jan G; Godoy, Patricio; Forbes, Stuart J; Hay, David C

    2015-12-08

    Stem cell-derived somatic cells represent an unlimited resource for basic and translational science. Although promising, there are significant hurdles that must be overcome. Our focus is on the generation of the major cell type of the human liver, the hepatocyte. Current protocols produce variable populations of hepatocytes that are the product of using undefined components in the differentiation process. This serves as a significant barrier to scale-up and application. To tackle this issue, we designed a defined differentiation process using recombinant laminin substrates to provide instruction. We demonstrate efficient hepatocyte specification, cell organization, and significant improvements in cell function and phenotype. This is driven in part by the suppression of unfavorable gene regulatory networks that control cell proliferation and migration, pluripotent stem cell self-renewal, and fibroblast and colon specification. We believe that this represents a significant advance, moving stem cell-based hepatocytes closer toward biomedical application.