WorldWideScience

Sample records for hepatocytes polypeptide assembly

  1. Evaporation-induced assembly of biomimetic polypeptides

    International Nuclear Information System (INIS)

    Keyes, Joseph; Junkin, Michael; Cappello, Joseph; Wu Xiaoyi; Wong, Pak Kin

    2008-01-01

    We report an evaporation assisted plasma lithography (EAPL) process for guided self-assembly of a biomimetic silk-elastinlike protein (SELP). We demonstrate the formation of SELP structures from millimeter to submicrometer range on plasma-treatment surface templates during an evaporation-induced self-assembly process. The self-assembly processes at different humidities and droplet volumes were investigated. The process occurs efficiently in a window of optimized operating conditions found to be at 70% relative humidity and 8 μl volume of SELP solution. The EAPL approach provides a useful technique for the realization of functional devices and systems using these biomimetic materials

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  3. Aqueous cholesteric liquid crystals using uncharged rodlike polypeptides. Polypeptide vesicles by conformation-specific assembly. Ordered chiral macroporous hybrid silica-polypeptide composites

    Science.gov (United States)

    Bellomo, Enrico Giuseppe

    2005-07-01

    Aqueous cholesteric liquid crystals using uncharged rodlike polypeptides . The aqueous, lyotropic liquid-crystalline phase behavior of an alpha helical polypeptide, has been studied using optical microscopy and X-ray scattering. Solutions of optically pure polypeptide were found to form cholesteric liquid crystals at volume fractions that decreased with increasing average chain length. At very high volume fractions, the formation of a hexagonal mesophase was observed. The pitch of the cholesteric phase could be varied by a mixture of enantiomeric samples, where the pitch increased as the mixture approached equimolar. The cholesteric phases could be untwisted, using either magnetic field or shear flow, into nematic phases, which relaxed into cholesterics upon removal of field or shear. We have found that the phase diagram of this polypeptide in aqueous solution parallels that of poly(gamma-benzyl glutamate) in organic solvents, thus providing a useful system for liquid-crystal applications requiring water as solvent. Polypeptide vesicles by conformation-specific assembly. We have found that block copolymers composed of polypeptide segments provide significant advantages in controlling both the function and supramolecular structure of bioinspired self-assemblies. Incorporation of the stable chain conformations found in proteins into block copolymers was found to provide an additional element of control, beyond amphiphilicity and composition that defines self-assembled architecture. The abundance of functionality present in amino acids, and the ease by which they can be incorporated into these materials, also provides a powerful mechanism to impart block copolypeptides with function. This combination of structure and function work synergistically to enable significant advantages in the preparation of therapeutic agents as well as provide insight into design of self-assemblies beginning to approach the complexity of natural structures such as virus capsids. Ordered

  4. 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.

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

    KAUST Repository

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2017-09-01

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

  8. 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.

  9. Reversible end-to-end assembly of gold nanorods using a disulfide-modified polypeptide

    International Nuclear Information System (INIS)

    Walker, David A; Gupta, Vinay K

    2008-01-01

    Directing the self-assembly of colloidal particles into nanostructures is of great interest in nanotechnology. Here, reversible end-to-end assembly of gold nanorods (GNR) is induced by pH-dependent changes in the secondary conformation of a disulfide-modified poly(L-glutamic acid) (SSPLGA). The disulfide anchoring group drives chemisorption of the polyacid onto the end of the gold nanorods in an ethanolic solution. A layer of poly(vinyl pyrrolidone) is adsorbed on the positively charged, surfactant-stabilized GNR to screen the surfactant bilayer charge and provide stability for dispersion of the GNR in ethanol. For comparison, irreversible end-to-end assembly using a bidentate ligand, namely 1,6-hexanedithiol, is also performed. Characterization of the modified GNR and its end-to-end linking behavior using SSPLGA and hexanedithiol is performed using dynamic light scattering (DLS), UV-vis absorption spectroscopy and transmission electron microscopy (TEM). Experimental results show that, in a colloidal solution of GNR-SSPLGA at a pH∼3.5, where the PLGA is in an α-helical conformation, the modified GNR self-assemble into one-dimensional nanostructures. The linking behavior can be reversed by increasing the pH (>8.5) to drive the conformation of the polypeptide to a random coil and this reversal with pH occurs rapidly within minutes. Cycling the pH multiple times between low and high pH values can be used to drive the formation of the nanostructures of the GNR and disperse them in solution.

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

    International Nuclear Information System (INIS)

    She Zhending; Feng Qingling; Liu Weiqiang

    2009-01-01

    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 β-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.

  11. 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.

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

    Science.gov (United States)

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

    2018-05-01

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

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

    Science.gov (United States)

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

    2009-10-12

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

  14. Liver tissue engineering based on aggregate assembly: efficient formation of endothelialized rat hepatocyte aggregates and their immobilization with biodegradable fibres

    International Nuclear Information System (INIS)

    Pang, Y; Shinohara, M; Komori, K; Sakai, Y; Montagne, K

    2012-01-01

    To realize long-term in vitro culture of hepatocytes at a high density while maintaining a high hepatic function for aggregate-based liver tissue engineering, we report here a novel culture method whereby endothelialized rat hepatocyte aggregates were formed using a PDMS microwell device and cultured in a perfusion bioreactor by introducing spacers between aggregates to improve oxygen and nutrient supply. Primary rat hepatocyte aggregates around 100 µm in diameter coated with human umbilical vein endothelial cells were spontaneously and quickly formed after 12 h of incubation, thanks to the continuous supply of oxygen by diffusion through the PDMS honeycomb microwell device. Then, the recovered endothelialized rat hepatocyte aggregates were mixed with biodegradable poly-l-lactic acid fibres in suspension and packed into a PDMS-based bioreactor. Perfusion culture of 7 days was successfully achieved with more than 73.8% cells retained in the bioreactor. As expected, the fibres acted as spacers between aggregates, which was evidenced from the enhanced albumin production and more spherical morphology compared with fibre-free packing. In summary, this study shows the advantages of using PDMS-based microwells to form heterotypic aggregates and also demonstrates the feasibility of spacing tissue elements for improving oxygen and nutrient supply to tissue engineering based on modular assembly. (paper)

  15. Sodium sulfite promotes the assembly and secretion of very low-density lipoprotein in HL-7702 hepatocytes

    Directory of Open Access Journals (Sweden)

    Jianying Bai

    Full Text Available This study investigated the effects of Na2SO3 on the fat metabolism in human normal diploid HL-7702 (referred as L-02 hepatocytes. After 24 h and 48 h, treatment with different concentrations of Na2SO3, the intra and extra-hepatocellular triglyceride (TG contents of L-02 were determined using chemical-enzymatic method. The contents of very low-density lipoprotein (VLDL and apolipoprotein B100 (apoB100 in the culture supernatants were determined using enzyme-linked immunosorbent assay (ELISA. Western blot was applied to detect the expressions of fatty acid oxidation and fat synthesis related proteins, VLDL assembly and secretion in L-02 cells. Results: Na2SO3 treatment (10 mM, 24 h/48 h significantly increased the intra TG level in the hepatocytes. Different concentrations of Na2SO3 increased the extra-hepatocellular TG content. After 24 h exposure, the extracellular VLDL levels and secretions of apoB100 in 0.1–10 mM Na2SO3 groups were significantly higher than that of the negative control (P < 0.05. Meanwhile, the expression of CPT1 and SREBP1 protein were significantly reduced by Na2SO3. MTP and TGH protein expressions were significantly elevated in each Na2SO3 treatment group. The expression level of LDLR in hepatocytes was reduced by Na2SO3. Conclusion: Na2SO3 exposure may promote the hepatocellular VLDL assembly and secretion, through increasing of MTP and TGH expressions and inhibiting the uptake of extracelluar VLDL. Keywords: Sodium sulfite, Hepatocytes, VLDL, Fatty acid oxidation, Fat synthesis, VLDL uptake

  16. Effects of insulin-like growth factor-1 on the assembly and secretion of very low-density lipoproteins in cow hepatocytes in vitro.

    Science.gov (United States)

    Li, Xinwei; Guan, Yuan; Li, Ying; Wu, Dianjun; Liu, Lei; Deng, Qinghua; Li, Xiaobing; Wang, Zhe; Liu, Guowen

    2016-01-15

    Fatty liver is a major metabolic disorder of dairy cows. One important reason is that hepatic very low-density lipoproteins (VLDL) assembly was significant decreased in dairy cows with fatty liver. In addition, the impairment of insulin-like growth factor (IGF)-1 synthesis was involved in the development of fatty liver. Therefore, the objective of this study was to investigate the effects of IGF-1 on the VLDL assembly in cow hepatocytes. In this study, cow hepatocytes were cultured and then transfected with Ad-GFP-IGF-1 (inhibited the IGF-1 expression) and Ad-GFP (negative control), and treated with different concentrations of IGF-1, respectively. The results showed that IGF-1 increased the mRNA abundance of apolipoprotein B100 (ApoB100), apolipoprotein E (ApoE), microsomal triglyceride transfer protein (MTTP), and low-density lipoprotein receptor (LDLR) and then increased the VLDL assembly in cow hepatocytes. Nevertheless, impairment of IGF-1 expression by Ad-GFP-IGF-1 could inhibit above genes expression and VLDL assembly in hepatocytes. Taken together, these results indicate that IGF-1 increases the VLDL assembly and impairment of IGF-1 expression decreases the VLDL assembly in cow hepatocytes. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. 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.

  18. Sequence-Dependent Self-Assembly and Structural Diversity of Islet Amyloid Polypeptide-Derived β-Sheet Fibrils

    International Nuclear Information System (INIS)

    Wang, Shih-Ting; Lin, Yiyang; Spencer, Ryan K.; Thomas, Michael R.; Nguyen, Andy I.

    2017-01-01

    Determining the structural origins of amyloid fibrillation is essential for understanding both the pathology of amyloidosis and the rational design of inhibitors to prevent or reverse amyloid formation. In this work, the decisive roles of peptide structures on amyloid self-assembly and morphological diversity were investigated by the design of eight amyloidogenic peptides derived from islet amyloid polypeptide. Among the segments, two distinct morphologies were highlighted in the form of twisted and planar (untwisted) ribbons with varied diameters, thicknesses, and lengths. In particular, transformation of amyloid fibrils from twisted ribbons into untwisted structures was triggered by substitution of the C-terminal serine with threonine, where the side chain methyl group was responsible for the distinct morphological change. This effect was confirmed following serine substitution with alanine and valine and was ascribed to the restriction of intersheet torsional strain through the increased hydrophobic interactions and hydrogen bonding. We also studied the variation of fibril morphology (i.e., association and helicity) and peptide aggregation propensity by increasing the hydrophobicity of the peptide side group, capping the N-terminus, and extending sequence length. Lastly, we anticipate that our insights into sequence-dependent fibrillation and morphological diversity will shed light on the structural interpretation of amyloidogenesis and development of structure-specific imaging agents and aggregation inhibitors.

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

    Science.gov (United States)

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

    2002-02-28

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

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

    Science.gov (United States)

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

    2016-01-01

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

  1. 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

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

    Science.gov (United States)

    Lim, Dong Woo

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

  3. 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.

  4. Effects of β-hydroxybutyricacid on the synthesis and assembly of very low-density lipoprotein in bovine hepatocytes in vitro.

    Science.gov (United States)

    Deng, Q; Ma, D; Shi, Z; Huang, W; Du, X; Gao, W; Zhu, X; Lei, L; Zhang, M; Sun, G; Yuan, X; Li, X; Wang, Z; Liu, G; Li, X

    2016-04-01

    β-Hydroxybutyricacid (BHBA) is an important metabolite that involved in the development of ketosis and fatty liver in dairy cows. Dairy cows with fatty liver displayed high blood concentration of BHBA and very low-density lipoprotein (VLDL) assembly. The effects of BHBA on VLDL synthesis and assembly in hepatocytes of cows were unclear. In this study, bovine hepatocytes were cultured and treated with different concentrations of BHBA. We found that BHBA treatment upregulated the mRNA and protein levels of apolipoprotein B100 (ApoB 100), apolipoprotein E (ApoE) and microsomal triglyceride transfer protein (MTTP) and showed in a firstly increased and then decreased trend. Meanwhile, the mRNA and protein levels of LDLR showed in a reverse trend. Consequently, VLDL content was significantly increased in medium-dose BHBA treatment group, while decreased in high-dose group. These results indicate that the effects of BHBA on the VLDL synthesis showed in a dose-dependent manner that low levels of BHBA increase VLDL synthesis and high levels of BHBA decrease VLDL synthesis. Journal of Animal Physiology and Animal Nutrition © 2015 Blackwell Verlag GmbH.

  5. Yeast polypeptide exit tunnel ribosomal proteins L17, L35 and L37 are necessary to recruit late-assembling factors required for 27SB pre-rRNA processing.

    Science.gov (United States)

    Gamalinda, Michael; Jakovljevic, Jelena; Babiano, Reyes; Talkish, Jason; de la Cruz, Jesús; Woolford, John L

    2013-02-01

    Ribosome synthesis involves the coordinated folding and processing of pre-rRNAs with assembly of ribosomal proteins. In eukaryotes, these events are facilitated by trans-acting factors that propel ribosome maturation from the nucleolus to the cytoplasm. However, there is a gap in understanding how ribosomal proteins configure pre-ribosomes in vivo to enable processing to occur. Here, we have examined the role of adjacent yeast r-proteins L17, L35 and L37 in folding and processing of pre-rRNAs, and binding of other proteins within assembling ribosomes. These three essential ribosomal proteins, which surround the polypeptide exit tunnel, are required for 60S subunit formation as a consequence of their role in removal of the ITS2 spacer from 27SB pre-rRNA. L17-, L35- and L37-depleted cells exhibit turnover of aberrant pre-60S assembly intermediates. Although the structure of ITS2 does not appear to be grossly affected in their absence, these three ribosomal proteins are necessary for efficient recruitment of factors required for 27SB pre-rRNA processing, namely, Nsa2 and Nog2, which associate with pre-60S ribosomal particles containing 27SB pre-rRNAs. Altogether, these data support that L17, L35 and L37 are specifically required for a recruiting step immediately preceding removal of ITS2.

  6. Endothelial-monocyte activating polypeptide II alters fibronectin based endothelial cell adhesion and matrix assembly via alpha5 beta1 integrin

    International Nuclear Information System (INIS)

    Schwarz, Margaret A.; Zheng, Hiahua; Liu, Jie; Corbett, Siobhan; Schwarz, Roderich E.

    2005-01-01

    Mature Endothelial-Monocyte Activating Polypeptide (mEMAP) II functions as a potent antiangiogenic peptide. Although the anti-tumor effect of mEMAP II has been described, little is known regarding its mechanism of action. Observations that mEMAP II induced apoptosis only in a subset of migrating and proliferating endothelial cells (EC) suggests a targeted effect on cells engaged in angiogenic activities which are known to rely upon cell adhesion and migration. Indeed, we demonstrate that mEMAP II inhibited fibronectin (FN) dependent microvascular EC (MEC) adhesion and spreading and we show that this depends upon the alpha5 beta1 integrin. Immunofluorescence analysis demonstrated that mEMAP II-dependent blockade of FN-alpha5 beta1 interactions was associated with disassembly of both actin stress fiber networks and FN matrix. These findings suggest that mEMAP II blocks MEC adhesion and spreading on fibronectin, via a direct interaction with the integrin alpha5 beta1, thus implicating that alpha5 integrin may be a mediator of mEMAP II's antiangiogenic function

  7. Chirality-selected phase behaviour in ionic polypeptide complexes

    Science.gov (United States)

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

    2015-01-01

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

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

    Lifescience Database Archive (English)

    Full Text Available His.Liv.10.AllAg.Hepatocytes hg19 Histone Liver Hepatocytes SRX1013892,SRX1013890,S...RX1013888,SRX1013889,SRX1013891 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Liv.10.AllAg.Hepatocytes.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available NoD.Liv.50.AllAg.Hepatocytes mm9 No description Liver Hepatocytes ERX113003,ERX1129...,ERX113010,ERX008753,ERX113014,ERX008723 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Liv.50.AllAg.Hepatocytes.bed ...

  10. File list: NoD.Liv.50.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Liv.50.AllAg.Hepatocytes hg19 No description Liver Hepatocytes SRX815538,SRX477...15539,ERX008754,ERX008736 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Liv.50.AllAg.Hepatocytes.bed ...

  11. File list: Oth.Liv.05.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Liv.05.AllAg.Hepatocytes hg19 TFs and others Liver Hepatocytes SRX530184,SRX530...186 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Liv.05.AllAg.Hepatocytes.bed ...

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

    Lifescience Database Archive (English)

    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 ...

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

    Lifescience Database Archive (English)

    Full Text Available ALL.Liv.20.AllAg.Hepatocytes mm9 All antigens Liver Hepatocytes ERX113003,SRX103898...010,ERX008753 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Liv.20.AllAg.Hepatocytes.bed ...

  14. File list: NoD.Liv.20.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Liv.20.AllAg.Hepatocytes mm9 No description Liver Hepatocytes ERX113003,SRX1038...,ERX113014,ERX008746,ERX113010,ERX008753 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Liv.20.AllAg.Hepatocytes.bed ...

  15. File list: NoD.Liv.10.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Liv.10.AllAg.Hepatocytes hg19 No description Liver Hepatocytes SRX815538,SRX815...08749,ERX025732,ERX008736 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Liv.10.AllAg.Hepatocytes.bed ...

  16. File list: ALL.Liv.05.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Liv.05.AllAg.Hepatocytes mm9 All antigens Liver Hepatocytes ERX008721,ERX112964...746,ERX113010 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Liv.05.AllAg.Hepatocytes.bed ...

  17. File list: NoD.Liv.20.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Liv.20.AllAg.Hepatocytes hg19 No description Liver Hepatocytes SRX815538,SRX477...08749,ERX025732,ERX008736 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Liv.20.AllAg.Hepatocytes.bed ...

  18. File list: NoD.Liv.10.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Liv.10.AllAg.Hepatocytes mm9 No description Liver Hepatocytes ERX113003,ERX1130...,ERX113014,ERX008744,ERX008746,ERX113010 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Liv.10.AllAg.Hepatocytes.bed ...

  19. 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 ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Liv.20.AllAg.Hepatocytes mm9 RNA polymerase Liver Hepatocytes ERX204069,ERX2040...60,ERX204064 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Liv.20.AllAg.Hepatocytes.bed ...

  1. File list: Oth.Liv.50.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Liv.50.AllAg.Hepatocytes mm9 TFs and others Liver Hepatocytes SRX019007,SRX1169...04059,ERX204068,ERX204062,ERX204061,ERX204067,ERX204065,SRX019006,ERX204058 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Liv.50.AllAg.Hepatocytes.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Oth.Liv.10.AllAg.Hepatocytes hg19 TFs and others Liver Hepatocytes SRX530184,SRX530...186 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Liv.10.AllAg.Hepatocytes.bed ...

  3. File list: InP.Liv.05.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Liv.05.AllAg.Hepatocytes mm9 Input control Liver Hepatocytes SRX019015,SRX55553...3,SRX1334843 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Liv.05.AllAg.Hepatocytes.bed ...

  4. File list: ALL.Liv.10.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Liv.10.AllAg.Hepatocytes mm9 All antigens Liver Hepatocytes ERX113003,ERX113015...746,ERX113010 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Liv.10.AllAg.Hepatocytes.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Liv.05.AllAg.Hepatocytes mm9 RNA polymerase Liver Hepatocytes ERX204060,ERX2040...69,ERX204064 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Liv.05.AllAg.Hepatocytes.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Oth.Liv.20.AllAg.Hepatocytes mm9 TFs and others Liver Hepatocytes ERX204070,SRX0190...04059,SRX116909,ERX204065,ERX204063,SRX116906,SRX555532,SRX019006,ERX204058 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Liv.20.AllAg.Hepatocytes.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available ALL.Liv.20.AllAg.Hepatocytes hg19 All antigens Liver Hepatocytes SRX815538,SRX47792...RX1013893,SRX1013888,SRX1013891,SRX1013889 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Liv.20.AllAg.Hepatocytes.bed ...

  8. 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 ...

  9. File list: ALL.Liv.05.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Liv.05.AllAg.Hepatocytes hg19 All antigens Liver Hepatocytes ERX008749,SRX81553...RX1013893,SRX1013888,SRX1013889,SRX1013891 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Liv.05.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 mm9 Input control Liver Hepatocytes SRX019015,SRX13348...43,SRX555533 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Liv.20.AllAg.Hepatocytes.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available NoD.Liv.05.AllAg.Hepatocytes hg19 No description Liver Hepatocytes ERX008749,SRX815...08760,ERX008728,ERX008736 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Liv.05.AllAg.Hepatocytes.bed ...

  12. 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 ...

  13. File list: Oth.Liv.50.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Liv.50.AllAg.Hepatocytes hg19 TFs and others Liver Hepatocytes SRX530184,SRX530...186 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Liv.50.AllAg.Hepatocytes.bed ...

  14. 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 ...

  15. File list: ALL.Liv.10.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Liv.10.AllAg.Hepatocytes hg19 All antigens Liver Hepatocytes SRX815538,SRX81553...RX1013893,SRX1013888,SRX1013889,SRX1013891 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Liv.10.AllAg.Hepatocytes.bed ...

  16. File list: ALL.Liv.50.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Liv.50.AllAg.Hepatocytes mm9 All antigens Liver Hepatocytes ERX113003,ERX112977...014,ERX008723 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Liv.50.AllAg.Hepatocytes.bed ...

  17. File list: ALL.Liv.50.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Liv.50.AllAg.Hepatocytes hg19 All antigens Liver Hepatocytes SRX815538,SRX47792...,SRX1013891,SRX1013889,ERX008736 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Liv.50.AllAg.Hepatocytes.bed ...

  18. File list: Pol.Liv.10.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Liv.10.AllAg.Hepatocytes mm9 RNA polymerase Liver Hepatocytes ERX204060,ERX2040...69,ERX204064 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Liv.10.AllAg.Hepatocytes.bed ...

  19. File list: Oth.Liv.05.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Liv.05.AllAg.Hepatocytes mm9 TFs and others Liver Hepatocytes SRX019007,SRX1169...19009,SRX116906,ERX204061,ERX204058,SRX019006,SRX555532,ERX204067,ERX204065 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Liv.05.AllAg.Hepatocytes.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Oth.Liv.20.AllAg.Hepatocytes hg19 TFs and others Liver Hepatocytes SRX530184,SRX530...186 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Liv.20.AllAg.Hepatocytes.bed ...

  1. Polypeptide based hydrogels

    OpenAIRE

    Hanay, Saltuk

    2018-01-01

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

  2. Tunable drug loading and release from polypeptide multilayer nanofilms

    Science.gov (United States)

    Jiang, Bingbing; Li, Bingyun

    2009-01-01

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

  3. Mosaic HIV envelope immunogenic polypeptides

    Science.gov (United States)

    Korber, Bette T. M.; Gnanakaran, S.; Perkins, Simon; Sodroski, Joseph; Haynes, Barton

    2018-01-02

    Disclosed herein are mosaic HIV envelope (Env) polypeptides that can elicit an immune response to HIV (such as cytotoxic T cell (CTL), helper T cell, and/or humoral responses). Also disclosed are sets of the disclosed mosaic Env polypeptides, which include two or more (for example, three) of the polypeptides. Also disclosed herein are methods for treating or inhibiting HIV in a subject including administering one or more of the disclosed immunogenic polypeptides or compositions to a subject infected with HIV or at risk of HIV infection. In some embodiments, the methods include inducing an immune response to HIV in a subject comprising administering to the subject at least one (such as two, three, or more) of the immunogenic polypeptides or at least one (such as two, three, or more) nucleic acids encoding at least one of the immunogenic polypeptides disclosed herein.

  4. Radiolysis of polypeptide

    International Nuclear Information System (INIS)

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

    1981-01-01

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

  5. Polypeptides Based Molecular Electronics

    National Research Council Canada - National Science Library

    Lam, Yeng M; Mhaisalkar, Subodh; Li, Lain-Jong; Dravid, Vinayak P; Shekhawat, Gajendra S; Suri, Raman

    2008-01-01

    ... the formation of molecular devices such as transistors, diodes, and sensors. We have designed the peptides, arranged them on substrates using self-assembly, Dip-PEN nanolithography, and also e-beam assisted lithography...

  6. 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.

  7. Methods for using polypeptides having cellobiohydrolase activity

    Science.gov (United States)

    Morant, Marc D; Harris, Paul

    2016-08-23

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

  8. Repolarization of hepatocytes in culture.

    Science.gov (United States)

    Talamini, M A; Kappus, B; Hubbard, A

    1997-01-01

    We have evaluated the biochemical, morphological, and functional redevelopment of polarity in freshly isolated hepatocytes cultured using a double layer collagen gel sandwich technique. Western blot analysis showed increased cellular levels of the cell adhesion protein uvomorulin as cultured hepatocytes repolarized. Immunofluorescence studies using antibodies against domain-specific membrane proteins showed polarity as early as 48 hours, although the pattern of the polymeric Immunoglobulin-A receptor (pIgA-R) differed from in vivo liver. Electron microscopy showed developing bile canaliculi at 1 day. However, the functional presence of tight junctions was absent at 1 day, but present at 5 days. We further showed functional polarity to be present at 4 days by documenting the ability of cultured hepatocytes to metabolize and excrete fluorescein diacetate into visible bile canaliculi. We conclude that hepatocytes cultured appropriately develop morphological and functional polarity. Hepatocyte culture is therefore a useful tool for the study of mechanisms responsible for the development of polarized function.

  9. 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.

  10. Cotranslational structure acquisition of nascent polypeptides monitored by NMR spectroscopy.

    Science.gov (United States)

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

    2010-05-18

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

  11. Polypeptides having catalase activity and polynucleotides encoding same

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-05-02

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

  12. Polypeptides having xylanase activity and polynucleotides encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Spodsberg, Nikolaj

    2018-02-06

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

  13. Glucose-dependent Insulinotropic Polypeptide

    DEFF Research Database (Denmark)

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

    2014-01-01

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

  14. 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.

  15. 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.

  16. Coulomb repulsion in short polypeptides.

    Science.gov (United States)

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

    2015-01-08

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

  17. GLYCOSYLATED YGHJ POLYPEPTIDES FROM ENTEROTOXIGENIC ESCHERICHIA COLI (ETEC)

    DEFF Research Database (Denmark)

    2017-01-01

    The present invention relates to glycosylated YghJ polypeptides from or derived from enterotoxigenic Escherichia coli (ETEC) that are immunogenic. In particular, the present invention relates to compositions or vaccines comprising the polypeptides and their application in immunization, vaccination...

  18. Biochemical characterization of domain-specific glycoproteins of the rat hepatocyte plasma membrane

    International Nuclear Information System (INIS)

    Bartles, J.R.; Braiterman, L.T.; Hubbard, A.L.

    1985-01-01

    Seven integral proteins (CE 9, HA 21, HA 116, HA 16, HA 4, HA 201, and HA 301) were isolated from rat hepatocyte plasma membranes by immunoaffinity chromatography on monoclonal antibody-Sepharose. Six of the proteins (all but HA 16) exhibit domain-specific localizations (either bile canalicular or sinusoidal/lateral) about the hepatocyte surface. The authors identified three of these protein antigens as leucine aminopeptidase (HA 201), dipeptidyl peptidase IV (HA 301), and the asialoglycoprotein receptor (HA 116). They also developed 125 I-lectin blotting procedures that, when used in conjunction with chemical and glycosidase treatments, permitted a comparison of the types of oligosaccharides present on the seven proteins. All seven are sialoglycoproteins, based upon the effects of prior neuraminidase and periodate-aniline-cyanoborohydride treatments of blots on labeling by 125 I-wheat germ agglutinin. Depending upon the protein, they estimated the presence of 2-26 N-linked oligosaccharides/polypeptide chain from the Mr reductions accompanying chemical or enzymatic deglycosylation. Three of these mature plasma membrane proteins (HA 21, HA 116, and HA 4) have both high mannose-type and complex-type oligosaccharides on every copy of their polypeptide chains

  19. 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

  20. Chimeric polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    Science.gov (United States)

    Wogulis, Mark; Sweeney, Matthew; Heu, Tia

    2017-06-14

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

  1. Polypeptides having xylanase activity and polynucleotides encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Spodsberg, Nikolaj; Shaghasi, Tarana

    2017-06-20

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

  2. Phase transitions in polypeptides: analysis of energy fluctuations

    DEFF Research Database (Denmark)

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

    2009-01-01

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

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

    Science.gov (United States)

    Harris, Paul; Golightly, Elizabeth

    2012-11-27

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

  4. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    Science.gov (United States)

    Morant, Marc D.; Harris, Paul

    2015-10-13

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

  5. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    Science.gov (United States)

    Maiyuran, Suchindra; Kramer, Randall; Harris, Paul

    2013-10-29

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

  6. Polynucleotides encoding polypeptides having beta-glucosidase activity

    Science.gov (United States)

    Harris, Paul; Golightly, Elizabeth

    2010-03-02

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

  7. 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.

  8. Factor VIIa binding and internalization in hepatocytes

    DEFF Research Database (Denmark)

    Hjortoe, G; Sorensen, B B; Petersen, L C

    2005-01-01

    The liver is believed to be the primary clearance organ for coagulation proteases, including factor VIIa (FVIIa). However, at present, clearance mechanisms for FVIIa in liver are unknown. To obtain information on the FVIIa clearance mechanism, we investigated the binding and internalization...... no effect. HEPG2 cells internalized FVIIa with a rate of 10 fmol 10(-5) cells h(-1). In contrast to HEPG2 cells, FVIIa binding to primary rat hepatocytes was completely independent of TF, and excess unlabeled FVIIa partly reduced the binding of 125I-FVIIa to rat hepatocytes. Further, compared with HEPG2...... cells, three- to fourfold more FVIIa bound to rat primary hepatocytes, and the bound FVIIa was internalized at a faster rate. Similar FVIIa binding and internalization profiles were observed in primary human hepatocytes. Plasma inhibitors had no effect on FVIIa binding and internalization in hepatocytes...

  9. Measles virus polypeptides in purified virions and in infected cells

    International Nuclear Information System (INIS)

    Vainionpaeae, R.; Ziola, B.; Salmi, A.

    1978-01-01

    A wild-type measles virus was radiolabeled during growth in VERO cells and purified by two successive potassium tartrate gradient centrifugations. The virion polypeptide composition was determined by SDS-polyacrylamide gel electrophoresis employing two different buffer systems. Six virus-specific polypeptides were consistently detected. The largest (L) had a molecular weight (MW) of greater than 150,000. The second largest polypeptide, G (MW 79,000), was the only glycoprotein found. The proteins designated polypeptide 2 (MW 66 to 70,000) and nucleocapsid protein or NP (MW 61,000) were phosphorylated. The remaining virus-coded proteins were polypeptide 5 (MW 40,000) and the matrix or M protein (MW 37,000). Measles virions also contained a polypeptide (MW 42,000) thought to be actin due to co-migration with this component of uninfected cells. Analysis of in vitro 3 H-acetic anhydride radiolabeled virions confirmed the presence of these seven polypeptides. Acetic anhydride also labeled a protein designated polypeptide 4 (MW 53,000) which was not consistently radiolabeled in vivo, as well as several other minor proteins believed to be cellular in origin. Synthesis of the six virus-specific structural polypeptides was detected in lysates of infected cells by SDS-polyacrylamide slab gel electrophoresis. Virus specificity of polypeptide 4 could not be confirmed due to the similar MW of several cellular polypeptides. Two non-virion, but virus-specified polypeptides, of MW 38,000 and 18,000 were also detected. Synthesis of the virus structural proteins was in the same proportions as the polypeptides found in virions except for under production of polypeptide G and over production of polypeptide 2. (author)

  10. Nanostructured complexes of polyelectrolytes and charged polypeptides

    Czech Academy of Sciences Publication Activity Database

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

    2010-01-01

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

  11. Generation of human hepatocytes by stem cell technology: definition of the hepatocyte.

    Science.gov (United States)

    Hengstler, Jan G; Brulport, Marc; Schormann, Wiebke; Bauer, Alexander; Hermes, Matthias; Nussler, Andreas K; Fandrich, Fred; Ruhnke, Maren; Ungefroren, Hendrik; Griffin, Louise; Bockamp, Ernesto; Oesch, Franz; von Mach, Marc-Alexander

    2005-06-01

    Since 1999, numerous articles have reported the generation of hepatocytes from different types of extrahepatic stem or precursor cells. This opens exciting new possibilities for pharmacology and toxicology, as well as for cell therapy. Hepatocyte marker expression, including albumin, cytokeratin 18, c-met, alpha-fetoprotein and cytochrome P450 3A4 and -2B6, has been observed after transplantation of different types of human stem cells into the liver of laboratory animals or in vitro after incubation with cytokines. These intriguing observations have prompted scientists to classify stem cell-derived cell populations as hepatocytes. However, this conclusion may be premature. It has been shown that factors of the liver microenvironment can induce expression of a limited number of hepatocyte marker genes in nonhepatic cell types. To conclude on the grounds of a limited number of markers that these cells are true hepatocytes is not indicated. In this case one should carefully evaluate crucial hepatocyte-defining enzymatic properties. The present article: i) reviews studies describing the fate of extrahepatic human stem and precursor cells in livers of laboratory animals, including the possibility of cell fusion; and ii) critically discusses the phenotype of stem cells after application of various differentiation protocols aimed at generating human hepatocytes. In addition, the necessary criteria needed for defining a true hepatocyte are suggested. Establishing the necessary properties for stem cell-derived hepatocytes is timely and reasonable, and thus avoids further misleading semantic confusion. Finally, it is essential to understand that the definition of a bona fide hepatocyte should not be limited to qualitative assays, such as reverse transcriptase polymerase chain reaction and immunohistochemistry, but has to include a quantitative analysis of enzymatic activities, which allows direct comparison with primary hepatocytes. Although the stem cell-derived-hepatocyte

  12. Comparison of the biological features between human fetal hepatocyte and immortalized L-02 hepatocyte in vitro

    International Nuclear Information System (INIS)

    Kong Weiwei; Teng Gaojun

    2004-01-01

    Objective: To evaluate the feasibilities of the potential donors in liver cell transplantation using the human fetal hepatocytes and immortalized L-02 hepatocytes by comparing their biological features. Methods: Human fetal hepatocytes were isolated from aborted fetal livers (gestational ages from 14 w to 24 w) by an improved two-stage perfusion method and cultured in a conditioned medium without any growth factors. α-fetal protein (AFP) and albumin (ALB) were detected by radioimmunoassay (RIA) and cytokeratin-19 (CK-19 ) was identified by cellular immunochemistry study. Immortalized L-02 hepatocytes were cultured in the same condition and the characteristic proteins were detected by the same methods. Results: The viability of human fetal hepatocytes was approximately 95% using the perfusion method, and the maximum survival time of the cultured hepatocytes was 3 weeks. The expression of AFP, ALB, and CK19 was detected at the same time, especially during Day 3 to Day 7 in the culture. By comparison, the proliferation ability of L-02 hepatocyte was greater, although with a lower level of ALB secretion. The expression of AFP and CK19 was not detected. Furthermore, during the long culture, L-02 hepatocytes may undergo a morphologic change and fail to express ALB. Conclusion: Human fetal hepatocyte may be a practical donor for hepatocyte transplantation with its high-level protein expression and potential bi-differentiation ability. In view of the absent expression of ALB and the morphologic change in culture, although with better proliferation, L-02 hepatocyte seems not useful for hepatocyte transplantation

  13. 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.

  14. Selective insulin resistance in hepatocyte senescence

    International Nuclear Information System (INIS)

    Aravinthan, Aloysious; Challis, Benjamin; Shannon, Nicholas; Hoare, Matthew; Heaney, Judith; Alexander, Graeme J.M.

    2015-01-01

    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

  15. 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.

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

    Science.gov (United States)

    Fu, Sihai; Feng, Mei; Xiong, Yan

    2017-11-28

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

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

    Science.gov (United States)

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

    2011-01-01

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

  18. 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.

  19. Measles virus-specified polypeptides in infected cells

    International Nuclear Information System (INIS)

    Vainionpaepae, R.

    1979-01-01

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

  20. Caffeine-water-polypeptide interaction in aqueous solution

    Science.gov (United States)

    Ghabi, Habib; Dhahbi, Mahmoud

    1999-04-01

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

  1. 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.

  2. Tissue polypeptide antigen activity in cerebrospinal fluid

    DEFF Research Database (Denmark)

    Bach, F; Söletormos, Georg; Dombernowsky, P

    1991-01-01

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

  3. 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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  6. 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.

  7. Swelling of rat hepatocytes stimulates glycogen synthesis

    NARCIS (Netherlands)

    Baquet, A.; Hue, L.; Meijer, A. J.; van Woerkom, G. M.; Plomp, P. J.

    1990-01-01

    In hepatocytes from fasted rats, several amino acids are known to stimulate glycogen synthesis via activation of glycogen synthase. The hypothesis that an increase in cell volume resulting from amino acid uptake may be involved in the stimulation of glycogen synthesis is supported by the following

  8. 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.

  9. Target organs for avian pancreatic polypeptide

    International Nuclear Information System (INIS)

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

    1981-01-01

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

  10. Fibrillar dimer formation of islet amyloid polypeptides

    Directory of Open Access Journals (Sweden)

    Chi-cheng Chiu

    2015-09-01

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

  11. 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.

  12. Insulin suppresses the AMPK signaling pathway to regulate lipid metabolism in primary cultured hepatocytes of dairy cows.

    Science.gov (United States)

    Li, Xinwei; Li, Yu; Ding, Hongyan; Dong, Jihong; Zhang, Renhe; Huang, Dan; Lei, Lin; Wang, Zhe; Liu, Guowen; Li, Xiaobing

    2018-05-01

    Dairy cows with type II ketosis display hepatic fat accumulation and hyperinsulinemia, but the underlying mechanism is not completely clear. This study aimed to clarify the regulation of lipid metabolism by insulin in cow hepatocytes. In vitro, cow hepatocytes were treated with 0, 1, 10, or 100 nm insulin in the presence or absence of AICAR (an AMP-activated protein kinase alpha (AMPKα) activator). The results showed that insulin decreased AMPKα phosphorylation. This inactivation of AMPKα increased the gene and protein expression levels of carbohydrate responsive element-binding protein (ChREBP) and sterol regulatory element-binding protein-1c (SREBP-1c), which downregulated the expression of lipogenic genes, thereby decreasing lipid biosynthesis. Furthermore, AMPKα inactivation decreased the gene and protein expression levels of peroxisome proliferator-activated receptor-α (PPARα), which upregulated the expression of lipid oxidation genes, thereby increasing lipid oxidation. In addition, insulin decreased the very low density lipoprotein (VLDL) assembly. Consequently, triglyceride content was significantly increased in insulin treated hepatocytes. Activation of AMPKα induced by AICAR could reverse the effect of insulin on PPARα, SREBP-1c, and ChREBP, thereby decreasing triglyceride content. These results indicate that insulin inhibits the AMPKα signaling pathway to increase lipid synthesis and decrease lipid oxidation and VLDL assembly in cow hepatocytes, thereby inducing TG accumulation. This mechanism could partly explain the causal relationship between hepatic fat accumulation and hyperinsulinemia in dairy cows with type II ketosis.

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

    KAUST Repository

    Pahovnik, David; Hadjichristidis, Nikolaos

    2015-01-01

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

  14. Metabolism of lipoproteins by human fetal hepatocytes

    International Nuclear Information System (INIS)

    Carr, B.R.

    1987-01-01

    The rate of clearance of lipoproteins from plasma appears to play a role in the development of atherogenesis. The liver may account for as much as two thirds of the removal of low-density lipoprotein and one third of the clearance of high-density lipoprotein in certain animal species and humans, mainly by receptor-mediated pathways. The purpose of the present investigation was to determine if human fetal hepatocytes maintained in vitro take up and degrade lipoproteins. We first determined that the maximal binding capacity of iodine 125-iodo-LDL was approximately 300 ng of low-density lipoprotein protein/mg of membrane protein and an apparent dissociation constant of approximately 60 micrograms low-density lipoprotein protein/ml in membranes prepared from human fetal liver. We found that the maximal uptake of [ 125 I]iodo-LDL and [ 125 I]iodo-HDL by fetal hepatocytes occurred after 12 hours of incubation. Low-density lipoprotein uptake preceded the appearance of degradation products by 4 hours, and thereafter the degradation of low-density lipoprotein increased linearly for at least 24 hours. In contrast, high-density lipoprotein was not degraded to any extent by fetal hepatocytes. [ 125 I]Iodo-LDL uptake and degradation were inhibited more than 75% by preincubation with low-density lipoprotein but not significantly by high-density lipoprotein, whereas [ 125 I]iodo-HDL uptake was inhibited 70% by preincubation with high-density lipoprotein but not by low-density lipoprotein. In summary, human fetal hepatocytes take up and degrade low-density lipoprotein by a receptor-mediated process similar to that described for human extrahepatic tissues

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

    Science.gov (United States)

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

    2015-07-01

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

  16. Inhibition of hepatocyte gap junctional intercellular communication by tumor promoters

    International Nuclear Information System (INIS)

    Ruch, R.J.

    1988-01-01

    The mechanisms by which tumor promoters enhance neoplasia are poorly understood. One effect common to most tumor promoters is their ability to inhibit the cell-to-cell exchange of small molecules and ions through gap junctions, i.e., gap junctional intercellular communication (IC). IC maybe necessary for normal growth control and the loss of IC may predispose cells to enhanced growth. In the present studies, the effects of liver tumor promoters and other agents on IC between rodent hepatocytes in primary culture has been studied. IC was detected between hepatocytes: (1) autoradiographically following the passage and incorporation of [5- 3 H]uridine nucleotides from pre-labeled donor hepatocytes to non-labeled, adjacent recipient hepatocytes and (2) by fluorescence microscopy after microinjection of fluorescent Lucifer Yellow CH dye into hepatocytes and visualizing dye spread into adjacent hepatocytes

  17. Hepatocyte heterogeneity in the metabolism of carbohydrates.

    Science.gov (United States)

    Jungermann, K; Thurman, R G

    1992-01-01

    Periportal and perivenous hepatocytes possess different amounts and activities of the rate-generating enzymes of carbohydrate and oxidative energy metabolism and thus different metabolic capacities. This is the basis of the model of metabolic zonation, according to which periportal cells catalyze predominantly the oxidative catabolism of fatty and amino acids as well as glucose release and glycogen formation via gluconeogenesis, and perivenous cells carry out preferentially glucose uptake for glycogen synthesis and glycolysis coupled to liponeogenesis. The input of humoral and nervous signals into the periportal and perivenous zones is different; gradients of oxygen, substrates and products, hormones and mediators and nerve densities exist which are important not only for the short-term regulation of carbohydrate metabolism but also for the long-term regulation of zonal gene expression. The specialization of periportal and perivenous hepatocytes in carbohydrate metabolism has been well characterized. In vivo evidence is provided by the complex metabolic situation termed the 'glucose paradox' and by zonal flux differences calculated on the basis of the distribution of enzymes and metabolites. In vitro evidence is given by the different flux rates determined with classical invasive techniques, e.g. in periportal-like and perivenous-like hepatocytes in cell culture, in periportal- and perivenous-enriched hepatocyte populations and in perfused livers during orthograde and retrograde flow, as well as with noninvasive techniques using miniature oxygen electrodes, e.g. in livers perfused in either direction. Differences of opinion in the interpretation of studies with invasive and noninvasive techniques by the authors are discussed. The declining gradient in oxygen concentrations, the decreasing glucagon/insulin ratio and the different innervation could be important factors in the zonal expression of the genes of carbohydrate-metabolizing enzymes. While it is clear that

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

    International Nuclear Information System (INIS)

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

    2010-01-01

    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 ∼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.

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

    Science.gov (United States)

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

    2016-02-08

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

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

    Science.gov (United States)

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

    2018-03-01

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

  1. 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...

  2. Polypeptide profiles of human oocytes and preimplantation embryos.

    Science.gov (United States)

    Capmany, G; Bolton, V N

    1993-11-01

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

  3. 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.

  4. Basal transcription of APOBEC3G is regulated by USF1 gene in hepatocyte

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Yanli [Department of Infectious Diseases, Zhengzhou University People' s Hospital (Henan Provincial People' s Hospital), Zhengzhou, 450003 (China); Li, Hui [The Central Hospital of Wuhan, Tongji Medical College Huazhong University of Science Technology, Wuhan, 430000 (China); Zhang, Xiaoju [Department of Respiratory Medicine, Zhengzhou University People' s Hospital (Henan Provincial People' s Hospital), Zhengzhou, 450003 (China); Shang, Jia [Department of Infectious Diseases, Zhengzhou University People' s Hospital (Henan Provincial People' s Hospital), Zhengzhou, 450003 (China); Kang, Yi, E-mail: kykangyi@163.com [Department of Infectious Diseases, Zhengzhou University People' s Hospital (Henan Provincial People' s Hospital), Zhengzhou, 450003 (China)

    2016-01-29

    Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G, A3G) exert antiviral defense as an important factor of innate immunity. A variety of cytokines such as IFN-γ,IL2,IL15,IL7 could induce the transcription of A3G. However, the regulation of other nuclear factor on the transcription of A3G have not been reported at the present. To gain new insights into the transcriptional regulation of this restriction factor, we cloned and characterized the promoter region of A3G and investigate the modulation of USF1 gene on the transcription of A3G. We identified a 232 bp region that was sufficient to regulate the activity of full promoter. Transcriptional start sites (TSS) were identified by the luciferase reporter assays of plasmids containing full or shorter fragments of the A3G promoter. The results demonstrated that the core promoter of A3G is located within the region -159/-84 relative to the TSS. Transcriptional activity of A3G core promoter regulated by USF1 was dependent on an E-box (located at position -91/-86 relative to the major TSS) and was abolished after mutation of this DNA element. USF1 gene can take part in basal transcription regulation of the human A3G gene in hepatocyte, and the identified E-box represented a binding site for the USF1. - Highlights: • The core promoter of A3G is located within the region −159/−84 relative to the TSS. • Transcriptional activity of A3G core promoter regulated by USF1 was dependent on an E-box (located at position −91/−86 relative to the major TSS). • USF1 gene can take part in basal transcription regulation of the human A3G gene in hepatocyte.

  5. Basal transcription of APOBEC3G is regulated by USF1 gene in hepatocyte

    International Nuclear Information System (INIS)

    Zeng, Yanli; Li, Hui; Zhang, Xiaoju; Shang, Jia; Kang, Yi

    2016-01-01

    Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G, A3G) exert antiviral defense as an important factor of innate immunity. A variety of cytokines such as IFN-γ,IL2,IL15,IL7 could induce the transcription of A3G. However, the regulation of other nuclear factor on the transcription of A3G have not been reported at the present. To gain new insights into the transcriptional regulation of this restriction factor, we cloned and characterized the promoter region of A3G and investigate the modulation of USF1 gene on the transcription of A3G. We identified a 232 bp region that was sufficient to regulate the activity of full promoter. Transcriptional start sites (TSS) were identified by the luciferase reporter assays of plasmids containing full or shorter fragments of the A3G promoter. The results demonstrated that the core promoter of A3G is located within the region -159/-84 relative to the TSS. Transcriptional activity of A3G core promoter regulated by USF1 was dependent on an E-box (located at position -91/-86 relative to the major TSS) and was abolished after mutation of this DNA element. USF1 gene can take part in basal transcription regulation of the human A3G gene in hepatocyte, and the identified E-box represented a binding site for the USF1. - Highlights: • The core promoter of A3G is located within the region −159/−84 relative to the TSS. • Transcriptional activity of A3G core promoter regulated by USF1 was dependent on an E-box (located at position −91/−86 relative to the major TSS). • USF1 gene can take part in basal transcription regulation of the human A3G gene in hepatocyte.

  6. Hepatocyte nuclear factor 4A improves hepatic differentiation of immortalized adult human hepatocytes and improves liver function and survival.

    Science.gov (United States)

    Hang, Hua-Lian; Liu, Xin-Yu; Wang, Hai-Tian; Xu, Ning; Bian, Jian-Min; Zhang, Jian-Jun; Xia, Lei; Xia, Qiang

    2017-11-15

    Immortalized human hepatocytes (IHH) could provide an unlimited supply of hepatocytes, but insufficient differentiation and phenotypic instability restrict their clinical application. This study aimed to determine the role of hepatocyte nuclear factor 4A (HNF4A) in hepatic differentiation of IHH, and whether encapsulation of IHH overexpressing HNF4A could improve liver function and survival in rats with acute liver failure (ALF). Primary human hepatocytes were transduced with lentivirus-mediated catalytic subunit of human telomerase reverse transcriptase (hTERT) to establish IHH. Cells were analyzed for telomerase activity, proliferative capacity, hepatocyte markers, and tumorigenicity (c-myc) expression. Hepatocyte markers, hepatocellular functions, and morphology were studied in the HNF4A-overexpressing IHH. Hepatocyte markers and karyotype analysis were completed in the primary hepatocytes using shRNA knockdown of HNF4A. Nuclear translocation of β-catenin was assessed. Rat models of ALF were treated with encapsulated IHH or HNF4A-overexpressing IHH. A HNF4A-positive IHH line was established, which was non-tumorigenic and conserved properties of primary hepatocytes. HNF4A overexpression significantly enhanced mRNA levels of genes related to hepatic differentiation in IHH. Urea levels were increased by the overexpression of HNF4A, as measured 24h after ammonium chloride addition, similar to that of primary hepatocytes. Chromosomal abnormalities were observed in primary hepatocytes transfected with HNF4A shRNA. HNF4α overexpression could significantly promote β-catenin activation. Transplantation of HNF4A overexpressing IHH resulted in better liver function and survival of rats with ALF compared with IHH. HNF4A improved hepatic differentiation of IHH. Transplantation of HNF4A-overexpressing IHH could improve the liver function and survival in a rat model of ALF. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. The potential of induced pluripotent stem cell derived hepatocytes.

    Science.gov (United States)

    Hannoun, Zara; Steichen, Clara; Dianat, Noushin; Weber, Anne; Dubart-Kupperschmitt, Anne

    2016-07-01

    Orthotopic liver transplantation remains the only curative treatment for liver disease. However, the number of patients who die while on the waiting list (15%) has increased in recent years as a result of severe organ shortages; furthermore the incidence of liver disease is increasing worldwide. Clinical trials involving hepatocyte transplantation have provided encouraging results. However, transplanted cell function appears to often decline after several months, necessitating liver transplantation. The precise aetiology of the loss of cell function is not clear, but poor engraftment and immune-mediated loss appear to be important factors. Also, primary human hepatocytes (PHH) are not readily available, de-differentiate, and die rapidly in culture. Hepatocytes are available from other sources, such as tumour-derived human hepatocyte cell lines and immortalised human hepatocyte cell lines or porcine hepatocytes. However, all these cells suffer from various limitations such as reduced or differences in functions or risk of zoonotic infections. Due to their significant potential, one possible inexhaustible source of hepatocytes is through the directed differentiation of human induced pluripotent stem cells (hiPSCs). This review will discuss the potential applications and existing limitations of hiPSC-derived hepatocytes in regenerative medicine, drug screening, in vitro disease modelling and bioartificial livers. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  8. Scintigraphic evidence of transplanted hepatocytes in spleen and liver

    International Nuclear Information System (INIS)

    Henne-Bruns, D.; Kremer, B.; Gramminger, K.; Broelsch, C.

    1986-01-01

    In rats suffering from hepatic enzymatic deficiency transplanted hepatocytes could be evidenced scintigraphically in liver, spleen and granulomas. In pigs, however, it is very difficult to demonstrate transplanted hepatocytes by scintiscanning because of the thickness of the tissues and the high background radiation in large animals

  9. Targeted deletion of hepatocyte Ikkβ confers growth advantages

    International Nuclear Information System (INIS)

    Koch, Katherine S.; Maeda, Shin; He, Guobin; Karin, Michael; Leffert, Hyam L.

    2009-01-01

    Mice lacking hepatocyte IKKβ (Ikkβ Δhep ) are defective in TNFα-activation of hepatocellular transcription factor NF-κB, and highly susceptible to hepatotoxicity. Following diethylnitrosamine (DEN) exposure, Ikkβ Δhep mice develop more hepatocellular carcinoma (HCC) than control mice due partly to enhanced DEN-induced hepatocyte death. Here we show that Ikkβ Δhep hepatocytes display growth advantages over normal hepatocytes consisting of precocious PCNA and cyclin D1 expression during liver regeneration (shortened hepatocyte G 0 → G 1 transitions), and enhanced recovery efficiency, cyclin D1 expression and cell proliferation after plating. Ex vivo deletion of Ikkβ also accelerates hepatocyte growth. Ikkβ Δhep hepatocyte proliferative responses show heightened sensitivity to TGFα and TNFα, and heightened expression of fibronectin, collagens I/III, nidogen, β-actin and integrin β1 mRNAs. These findings suggest that altered mitogen signaling and expression of extracellular matrix and its associated components underlie growth advantages. Increased HCC development in Ikkβ Δhep mice may also be caused by growth advantages of surviving Ikkβ-deleted hepatocytes.

  10. Cell swelling and glycogen metabolism in hepatocytes from fasted rats

    NARCIS (Netherlands)

    Gustafson, L. A.; Jumelle-Laclau, M. N.; van Woerkom, G. M.; van Kuilenburg, A. B.; Meijer, A. J.

    1997-01-01

    Cell swelling is known to increase net glycogen production from glucose in hepatocytes from fasted rats by activating glycogen synthase. Since both active glycogen synthase and phosphorylase are present in hepatocytes, suppression of flux through phosphorylase may also contribute to the net increase

  11. Ketose induced respiratory inhibition in isolated hepatocytes.

    Science.gov (United States)

    Martínez, P; Carrascosa, J M; Núñez de Castro, I

    1987-06-01

    The addition of 10 mM fructose or 10 mM tagatose to a suspension of hepatocytes caused respiratory inhibition, whereas no change in oxygen uptake was observed following the addition of glucose. However, incubations in the presence of fructose showed a high, aerobic glycolytic activity. Tagatose is phosphorylated to tagatose 1-phosphate but is not further metabolized by cell free liver extract. Moreover, the addition of fructose to glucagon treated cells also caused the Crabtree-like effect. The concentration of adenine nucleotides and inorganic phosphate (Pi) in the mitochondrial and cytosolic compartments during incubation (time 30 min) was determined by the digitonin fractionation procedure. In the presence of 10 mM fructose or tagatose, the total adenine nucleotide pools decreased by 40%; however, glucose produced no change. The addition of ketoses diminished the asymmetric distribution of extramitochondrial (ATP/ADP)e ratio and intramitochondrial (ATP/ADP)i ratio. At the same time the total mitochondrial Pi fell from 17 mM to 6-7 mM. The mitochondrial membrane potential (-161 mV) in the presence of fructose showed no changes during the 30 min experimental period. An increase in the NADH/NAD+ ratio was observed. These results suggest that in hepatocytes the inhibition of respiration is not necessarily linked with the enhanced aerobic glycolysis, by competition for common substrates.

  12. Insulin internalization in isolated rat hepatocytes

    International Nuclear Information System (INIS)

    Galan, J.; Trankina, M.; Noel, R.; Ward, W.

    1990-01-01

    This project was designed to determine whether neomycin, an aminoglycoside antibiotic, has a significant effect upon the pathways of ligand endocytosis in isolated rat hepatocytes. The pathways studied include receptor-mediated endocytosis and fluid-phase endocytosis. Neomycin causes a dose-dependent acceleration of 125 I-insulin internalization. Since fluid-phase endocytosis can also be a significant factor in 125 I-insulin internalization, lucifer yellow (LY), a marker for fluid-phase endocytosis, was incorporated into an assay similar to the 125 I-insulin internalization procedure. In the presence of 5 mM neomycin, a significant increase in LY uptake was evident at 0.2 and 0.4 mg/ml of LY. At 0.8 mg/ml, a decrease in LY uptake was observed. The increased rate of 125 I-insulin internalization in the presence of neomycin was intriguing. Since one action of neomycin is to inhibit phosphoinositidase C, it suggests that the phosphotidylinositol cycle may be involved in ligand internalization by hepatocytes. At low insulin concentrations, receptor-mediated uptake predominates. Fluid-phase uptake can become an important uptake route as insulin concentrations are increased. Since neomycin stimulates fluid-phase endocytosis, it must also be taken into account when measuring ligand internalization

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

    International Nuclear Information System (INIS)

    Sanada, Takahiro; Tsukiyama-Kohara, Kyoko; Yamamoto, Naoki; Ezzikouri, Sayeh; Benjelloun, Soumaya; Murakami, Shuko; Tanaka, Yasuhito; Tateno, Chise; Kohara, Michinori

    2016-01-01

    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"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"4-10"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"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.

  14. 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.

  15. Polypeptides having beta-glucosidase activity and polynucleotides encoding the same

    Science.gov (United States)

    Brown, Kimberly; Harris, Paul

    2013-12-17

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

  16. Bacteriophage Assembly

    Directory of Open Access Journals (Sweden)

    Anastasia A. Aksyuk

    2011-02-01

    Full Text Available Bacteriophages have been a model system to study assembly processes for over half a century. Formation of infectious phage particles involves specific protein-protein and protein-nucleic acid interactions, as well as large conformational changes of assembly precursors. The sequence and molecular mechanisms of phage assembly have been elucidated by a variety of methods. Differences and similarities of assembly processes in several different groups of bacteriophages are discussed in this review. The general principles of phage assembly are applicable to many macromolecular complexes.

  17. Fuel assemblies

    International Nuclear Information System (INIS)

    Nakatsuka, Masafumi.

    1979-01-01

    Purpose: To prevent scattering of gaseous fission products released from fuel assemblies stored in an fbr type reactor. Constitution; A cap provided with means capable of storing gas is adapted to amount to the assembly handling head, for example, by way of threading in a storage rack of spent fuel assemblies consisting of a bottom plate, a top plate and an assembly support mechanism. By previously eliminating the gas inside of the assembly and the cap in the storage rack, gaseous fission products upon loading, if released from fuel rods during storage, are stored in the cap and do not scatter in the storage rack. (Horiuchi, T.)

  18. 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 and...... in genomic DNA, highly expressed genes and alternative transcripts in EST sequences. We summarize existing comparisons of different assemblers and provide a detailed descriptions and directions for download of assembly programs at: http://genome.ku.dk/resources/assembly/methods.html....

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

    DEFF Research Database (Denmark)

    Ottesen, B; Fahrenkrug, J

    1995-01-01

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

  20. 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. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. In Vitro Assembly of Catalase*

    Science.gov (United States)

    Baureder, Michael; Barane, Elisabeth; Hederstedt, Lars

    2014-01-01

    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. PMID:25148685

  2. Hepatocyte growth factor profile with breast cancer

    Directory of Open Access Journals (Sweden)

    Hoda A EL-Attar

    2011-01-01

    Full Text Available Background: The multifunctional hepatocyte growth factor (HGF is the ligand of c-Met receptor; it plays important role in mammary differentiation. HGF-Met signaling is a critical downstream function of c-Src-Stat3 pathway in mammalian tumorigenesis. Aim: Evaluation of tissue c-Met receptor hepatocyte growth factor receptor (HGFR and serum level of HGF in female breast ductal carcinoma. Materials and Methods: Sixty-eight premenopausal females were divided as 30 control females subdivided into: [Group 1] 15 healthy volunteer females and [Group 2] five with fibrocystic disease and 10 having fibroadenoma of the breast and patients group [Group 3] consisted of 38 female patients with breast ductal carcinoma. Thorough clinical examination, preoperative fine needle aspiration cytology, estimation of fasting serum glucose, urea, creatinine, and uric acid levels, alanine aminotransferase activities, C-reactive protein, HGF level, before surgery and histopathological examination of the breast masses, and immunohistochemical detection of HGFR were done. Results and Conclusions: Significant increase in serum HGF levels were found in patients with breast cancer as compared with controls. Significant increase was also seen in patients with breast cancer with and without lymph node metastasis when each subgroup was compared with controls. Serum level of HGF is an independent prognostic indicator of breast cancer. Fibrocystic disease of the breast showed weak HGFR expression, while in normal tissue, HGFR was scanty; meanwhile, breast invasive ductal carcinoma showed homogenous strong reaction to HGFR. HGF is only one of a number of key factors involved in breast cancer and preoperative high serum HGF levels and malignancy occur usually together.

  3. 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.

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

    Science.gov (United States)

    Glassman, Matthew; Olsen, Bradley

    2014-03-01

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

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

    Science.gov (United States)

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

    2015-08-01

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

  6. Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes.

    Science.gov (United States)

    Baxter, Melissa; Withey, Sarah; Harrison, Sean; Segeritz, Charis-Patricia; Zhang, Fang; Atkinson-Dell, Rebecca; Rowe, Cliff; Gerrard, Dave T; Sison-Young, Rowena; Jenkins, Roz; Henry, Joanne; Berry, Andrew A; Mohamet, Lisa; Best, Marie; Fenwick, Stephen W; Malik, Hassan; Kitteringham, Neil R; Goldring, Chris E; Piper Hanley, Karen; Vallier, Ludovic; Hanley, Neil A

    2015-03-01

    Hepatocyte-like cells (HLCs), differentiated from pluripotent stem cells by the use of soluble factors, can model human liver function and toxicity. However, at present HLC maturity and whether any deficit represents a true fetal state or aberrant differentiation is unclear and compounded by comparison to potentially deteriorated adult hepatocytes. Therefore, we generated HLCs from multiple lineages, using two different protocols, for direct comparison with fresh fetal and adult hepatocytes. Protocols were developed for robust differentiation. Multiple transcript, protein and functional analyses compared HLCs to fresh human fetal and adult hepatocytes. HLCs were comparable to those of other laboratories by multiple parameters. Transcriptional changes during differentiation mimicked human embryogenesis and showed more similarity to pericentral than periportal hepatocytes. Unbiased proteomics demonstrated greater proximity to liver than 30 other human organs or tissues. However, by comparison to fresh material, HLC maturity was proven by transcript, protein and function to be fetal-like and short of the adult phenotype. The expression of 81% phase 1 enzymes in HLCs was significantly upregulated and half were statistically not different from fetal hepatocytes. HLCs secreted albumin and metabolized testosterone (CYP3A) and dextrorphan (CYP2D6) like fetal hepatocytes. In seven bespoke tests, devised by principal components analysis to distinguish fetal from adult hepatocytes, HLCs from two different source laboratories consistently demonstrated fetal characteristics. HLCs from different sources are broadly comparable with unbiased proteomic evidence for faithful differentiation down the liver lineage. This current phenotype mimics human fetal rather than adult hepatocytes. Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  7. Organic anion transporting polypeptide 1B transporters modulate hydroxyurea pharmacokinetics

    OpenAIRE

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

    2013-01-01

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

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

    OpenAIRE

    Deming, TJ

    2017-01-01

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

  9. RNA synthesis in primary cultures of adult rat hepatocytes

    International Nuclear Information System (INIS)

    Fugassa, E.; Gallo, G.; Voci, A.; Cordone, A.

    1983-01-01

    The ability of hepatocyte monolayers to synthesize RNA was investigated by measuring [3H]orotic acid incorporation into RNA and the total nuclear RNA polymerase activity as a function of the time in culture. The results demonstrate that primary cultures of hepatocytes maintained in a chemically defined serum- and hormone-free medium are able to synthesize RNA actively. This ability increases within the first 2 d of culture, despite the concomitant decrease in [3H]orotic acid uptake, and decreases only after 3 d. Factors such as serum, insulin, and dexamethasone, known to improve maintenance of functional hepatocytes, markedly stimulate the uptake of labeled precursor without apparently affecting the rate of RNA synthesis by cultured cells. It is suggested that the culture of adult rat hepatocytes provides a useful experimental model for the studies of hormonal regulation of transcription in liver

  10. Generation of polypeptide-templated gold nanoparticles using ionizing radiation.

    Science.gov (United States)

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

    2013-08-13

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

  11. Metabolism of para-aminophenol by rat hepatocytes.

    Science.gov (United States)

    Yan, Z; Nikelly, J G; Killmer, L; Tarloff, J B

    2000-08-01

    Autoxidation of para-aminophenol (PAP) has been proposed to account for the selective nephrotoxicity of this compound. However, other studies suggest that hepatic metabolites of PAP rather than the parent compound may be responsible for renal damage. These studies were designed to investigate PAP metabolism in isolated hepatocytes. We synthesized several proposed metabolites for analysis by HPLC/mass spectrometry and compared those results with HPLC/mass spectrometric analyses of metabolites found after incubating hepatocytes with PAP. Hepatocytes prepared from male Sprague-Dawley rats were incubated in Krebs-Henseleit buffer at 37 degrees C for 5 h with 2.3 mM PAP under an atmosphere of 5% CO2/95% O2. Aliquots were withdrawn at 0.1 h of incubation and then hourly through 5 h of incubation. Reactions were terminated by the addition of acetonitrile. Hepatocyte viability was unaltered with PAP present in the incubation medium. We found that hepatocytes converted PAP to two major metabolites (PAP-GSH conjugates and PAP-N-acetylcysteine conjugates) and several minor metabolites [PAP-O-glucuronide, acetaminophen (APAP), APAP-O-glucuronide, APAP-GSH conjugates, and 4-hydroxyformanilide]. Preincubating hepatoyctes with 1-aminobenzotriazole, an inhibitor of cytochromes P450, did not alter the pattern of PAP metabolism. In conclusion, we found that PAP was metabolized in hepatocytes predominantly to PAP-GSH conjugates and PAP-N-acetylcysteine conjugates in sufficient quantities to account for the nephrotoxicity of PAP.

  12. Hepatocyte polyploidization and its association with pathophysiological processes.

    Science.gov (United States)

    Wang, Min-Jun; Chen, Fei; Lau, Joseph T Y; Hu, Yi-Ping

    2017-05-18

    A characteristic cellular feature of the mammalian liver is the progressive polyploidization of the hepatocytes, where individual cells acquire more than two sets of chromosomes. Polyploidization results from cytokinesis failure that takes place progressively during the course of postnatal development. The proportion of polyploidy also increases with the aging process or with cellular stress such as surgical resection, toxic stimulation, metabolic overload, or oxidative damage, to involve as much as 90% of the hepatocytes in mice and 40% in humans. Hepatocyte polyploidization is generally considered an indicator of terminal differentiation and cellular senescence, and related to the dysfunction of insulin and p53/p21 signaling pathways. Interestingly, the high prevalence of hepatocyte polyploidization in the aged mouse liver can be reversed when the senescent hepatocytes are serially transplanted into young mouse livers. Here we review the current knowledge on the mechanism of hepatocytes polyploidization during postnatal growth, aging, and liver diseases. The biologic significance of polyploidization in senescent reversal, within the context of new ways to think of liver aging and liver diseases is considered.

  13. Functional assessment of hepatocytes after transplantation into rat spleen

    International Nuclear Information System (INIS)

    Woods, R.J.; Fuller, B.J.; Attenburrow, V.D.; Nutt, L.H.; Hobbs, K.E.

    1982-01-01

    The retention of structural integrity and metabolic function by isolated hepatocytes after ectopic transplantation has been investigated in autografted rats. Rats were partially hepatectomized and isolated hepatocytes prepared from the excised liver lobes were implanted into their spleens. Histological examination of the spleens 7 or more weeks after implantation revealed aggregates of hepatocytes in the red pulp. Two tests of biochemical function were applied to the hepatocytes after transplantation. In the first the hepatobiliary imaging agent technetium-99m N-[N'-(2, 6-dimethylphenyl)carbamoylmethyl]iminodiacetic acid (99mTc HIDA), which was shown to be avidly taken up by isolated hepatocytes in vitro, was infused into the tail veins of autograft and control rats. Radioactivity accumulating in the spleens of autografted rats was markedly greater than that in controls implanted with lethally damaged cells or in nontransplanted rats. In the second the presence of bilirubin metabolites was sought in autograft spleens after intravenous infusion of bilirubin. Both mono- and diglucuronides of bilirubin were recovered from the spleens of autograft rats but no conjugates were recovered from the spleens of unoperated controls. We conclude that after autotransplantation isolated hepatocytes retain their morphology and at least some of their functional activities

  14. 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.

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

    Science.gov (United States)

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

    1989-10-01

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

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

    International Nuclear Information System (INIS)

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

    1982-01-01

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

  17. 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.

  18. 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.

  19. Role of macrophages in the immune response to hepatocytes

    International Nuclear Information System (INIS)

    Bumgardner, G.L.; Chen, S.; Almond, S.P.; Ascher, N.L.; Payne, W.D.; Matas, A.J.

    1990-01-01

    The purpose of this study was to determine the role of host macrophages in the development of allospecific cytolytic T cells (allo-CTLs) in response to purified allogeneic MHC Class I+, Class II- hepatocytes in vivo in hepatocyte sponge matrix allografts (HC-SMA). Depletion of antigen-presenting cells (APCs) from responder splenocytes in mixed lymphocyte hepatocyte culture (MLHC) inhibits the development of allo-CTLs in response to purified hepatocytes. First the ability of sponge macrophages to function as accessory cells in indirect presentation of hepatocyte Class I antigen was tested in MLHC. We found that addition of irradiated sponge cells (a source of sponge macrophages) restored the development of allo-CTLs in MLHC depleted of responder APCs. Therefore, radioresistant sponge macrophages can function as accessory cells in MLHC. We next employed silica as an immunotherapy targeted against host macrophages and assessed the effect on development of allo-CTLs in HC-SMA. We found that local (intrasponge) silica treatment completely inhibited the development of allo-CTLs in HC-SMA. Combined local and systemic silica treatment resulted in inhibition of allocytotoxicity comparable to local silica treatment alone in the doses tested. We conclude that host macrophages which infiltrate HC-SMA can function as accessory cells in vitro in MLHC and that both infiltrating host macrophages and lymphocytes participate in the development of an alloimmune response to purified hepatocytes in vivo. This interaction may involve indirect antigen presentation of hepatocyte Class I antigen by macrophages to host lymphocytes which accumulate in HC-SMA

  20. Effects of edaravone, a radical scavenger, on hepatocyte transplantation.

    Science.gov (United States)

    Hayashi, Chihiro; Ito, Masahiro; Ito, Ryoutaro; Murakumo, Akiko; Yamamoto, Naoki; Hiramatsu, Noriko; Fox, Ira J; Horiguchi, Akihiko

    2014-12-01

    Hepatocyte transplantation (HTx) has yielded significant improvements in liver function and survival in experimentally induced acute liver failure and liver-based metabolic disease. However, transplantation is inefficient, and it is thought that transplanted hepatocytes have a shortened lifespan because of inflammation involving excess nitric oxide (NO). The present study aimed to clarify whether edaravone, a free radical scavenger used to treat ischemic stroke, could reduce ischemic changes in hepatocyte-transplanted livers. Edaravone (3 mg/kg) was administered intravenously 24 h before HTx to Nagase analbuminemic rats (NARs). Hepatocytes were isolated, and 30 × 10(6) cells were injected in a 1.0-ml volume directly into the spleens of NARs. All experimental groups studied received FK506 to control rejection. Animals in Group A received medium-only; Group B received HTx only; and Group C received HTx and edaravone. Forty-eight hours after transplantation, the hepatocytes from animals were isolated and analyzed for staining with propidium iodide- and annexin-V using flow cytometry. Liver sections were also studied by immunostaining for albumin, and TUNEL. Peripheral blood serum albumin levels were measured on post-transplant days 0, 3, 5, 7, 10 and 14 using ELISA. The edaravone-treated animals demonstrated an increased number of engrafted donor hepatocytes in the liver. The edaravone-treated liver sections also contained fewer TUNEL-positive cells and animals that received edaravone had higher serum albumin levels post-transplantation. Hepatocytes were also found to have increased in numbers 2 weeks following treatment with edaravone. Edaravone administration during HTx can suppress apoptosis near the transplanted cells, increasing engraftment. These studies indicate its potential usefulness for future clinical application. © 2014 Japanese Society of Hepato-Biliary-Pancreatic Surgery.

  1. Water and nonelectrolyte permeability of isolated rat hepatocytes

    International Nuclear Information System (INIS)

    Alpini, G.; Garrick, R.A.; Jones, M.J.; Nunes, R.; Tavoloni, N.

    1986-01-01

    We have measured the diffusive permeability coefficients of isolated rat hepatocytes to 3 H 2 O, [ 14 C]urea, [ 14 C]erythritol, [ 14 C]mannitol, [ 3 H]sucrose, and [ 3 H]inulin, employing a technique previously developed for erythrocytes (Redwood et al., J. Gen. Physiol 64:706-729, 1974). Diffusion coefficients for the tracer molecules were measured in packed hepatocytes, supernatant fluid, and intracellular medium (lysed hepatocytes) and were calculated assuming one-dimensional semi-infinite diffusion through a homogeneous medium. By applying the series-parallel pathway model, the following permeability coefficients (10(-5) cm/sec) for the hepatocyte plasma membrane were obtained. 3 H 2 O, 98.6 +/- 18.4; [ 14 C]urea, 18.2 +/- 5.3; [ 14 C]erythritol, 4.8 +/- 1.6; [ 14 C]mannitol, 3.1 +/- 1.4; [ 3 H]sucrose, 0; [ 3 H]inulin, 0. These results indicate that isolated rat hepatocytes are highly permeable to water and polar nonelectrolytes, when compared with other transporting epithelia. This relatively high cellular permeability is consistent with a model in which nonelectrolyte permeation is via an aqueous pathway of equivalent pore diameter of 8-12 A. The finding that [ 14 C]erythritol and [ 14 C]mannitol cross the hepatocyte plasma membrane indicates that these molecules enter the bile canaliculus through the transcellular route. Conversely, the failure of [ 3 H]sucrose and [ 3 H]inulin to permeate the hepatocyte in the isolated condition supports the concept that biliary entry of these large carbohydrates, at least that fraction which cannot be accounted for by a vesicular mechanism, must occur via the transjunctional shunt pathway

  2. 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...

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

    OpenAIRE

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

    1984-01-01

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

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

    International Nuclear Information System (INIS)

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

    2012-01-01

    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 ® 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 + -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 concentrations

  5. 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

  6. Primary structure, gene organization and polypeptide expression of poliovirus RNA

    Energy Technology Data Exchange (ETDEWEB)

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

    1981-06-18

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

  7. 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.

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

    International Nuclear Information System (INIS)

    Garrison, W.M.

    1985-01-01

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

  9. NMR study of the cooperative behavior of thermotropic model polypeptides

    Czech Academy of Sciences Publication Activity Database

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

    2007-01-01

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

  10. 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.

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

    Science.gov (United States)

    Deming, Timothy J

    2017-03-15

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

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

    KAUST Repository

    Zhao, Wei; Gnanou, Yves; Hadjichristidis, Nikolaos

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Dimitrios Skoulas

    2017-06-01

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

  14. Hepatocyte specific expression of human cloned genes

    Energy Technology Data Exchange (ETDEWEB)

    Cortese, R

    1986-01-01

    A large number of proteins are specifically synthesized in the hepatocyte. Only the adult liver expresses the complete repertoire of functions which are required at various stages during development. There is therefore a complex series of regulatory mechanisms responsible for the maintenance of the differentiated state and for the developmental and physiological variations in the pattern of gene expression. Human hepatoma cell lines HepG2 and Hep3B display a pattern of gene expression similar to adult and fetal liver, respectively; in contrast, cultured fibroblasts or HeLa cells do not express most of the liver specific genes. They have used these cell lines for transfection experiments with cloned human liver specific genes. DNA segments coding for alpha1-antitrypsin and retinol binding protein (two proteins synthesized both in fetal and adult liver) are expressed in the hepatoma cell lines HepG2 and Hep3B, but not in HeLa cells or fibroblasts. A DNA segment coding for haptoglobin (a protein synthesized only after birth) is only expressed in the hepatoma cell line HepG2 but not in Hep3B nor in non hepatic cell lines. The information for tissue specific expression is located in the 5' flanking region of all three genes. In vivo competition experiments show that these DNA segments bind to a common, apparently limiting, transacting factor. Conventional techniques (Bal deletions, site directed mutagenesis, etc.) have been used to precisely identify the DNA sequences responsible for these effects. The emerging picture is complex: they have identified multiple, separate transcriptional signals, essential for maximal promoter activation and tissue specific expression. Some of these signals show a negative effect on transcription in fibroblast cell lines.

  15. 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

  16. 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

  17. Whole-body γ-irradiation decelerates rat hepatocyte polyploidization.

    Science.gov (United States)

    Ikhtiar, Adnan M

    2015-07-01

    To characterize hepatocyte polyploidization induced by intermediate dose of γ-ray. Male Wistar strain rats were whole-body irradiated (WBI) with 2 Gy of γ-ray at the age of 1 month, and 5-6 rats were sacrificed monthly at 0-25 months after irradiation. The nuclear DNA content of individual hepatocytes was measured by flow cytometry, then hepatocytes were classified into various ploidy classes. Survival percentage, after exposure up to the end of the study, did not indicate any differences between the irradiated groups and controls. The degree of polyploidization in hepatocytes of irradiated rats, was significantly lower than that for the control after 1 month of exposure, and it continued to be lower after up to 8 months. Thereafter, the degree of polyploidization in the irradiated group slowly returned to the control level when the irradiated rats reached the age of 10 months. Intermediate dose of ionizing radiation, in contrast to high doses, decelerate hepatocyte polyploidization, which may coincides with the hypothesis of the beneficial effects of low doses of ionizing radiation.

  18. Fuel assembly

    International Nuclear Information System (INIS)

    Abe, Hideaki; Sakai, Takao; Ishida, Tomio; Yokota, Norikatsu.

    1992-01-01

    The lower ends of a plurality of plate-like shape memory alloys are secured at the periphery of the upper inside of the handling head of a fuel assembly. As the shape memory alloy, a Cu-Zn alloy, a Ti-Pd alloy or a Fe-Ni alloy is used. When high temperature coolants flow out to the handling head, the shape memory alloy deforms by warping to the outer side more greatly toward the upper portion thereof with the temperature increase of the coolants. As the result, the shape of the flow channel of the coolants is changed so as to enlarge at the exit of the upper end of the fuel assembly. Then, the pressure loss of the coolants in the fuel assembly is decreased by the enlargement. Accordingly, the flow rate of the coolants in the fuel assembly is increased to lower the temperature of the coolants. Further, high temperature coolants and low temperature coolants are mixed sufficiently just above the fuel assembly. This can suppress the temperature fluctuation of the mixed coolants in the upper portion of the reactor core, thereby enabling to decrease a fatigue and failures of the structural components in the upper portion of the reactor core. (I.N.)

  19. 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.

  20. Engineered aggregation inhibitor fusion for production of highly amyloidogenic human islet amyloid polypeptide.

    Science.gov (United States)

    Mirecka, Ewa Agnieszka; Gremer, Lothar; Schiefer, Stephanie; Oesterhelt, Filipp; Stoldt, Matthias; Willbold, Dieter; Hoyer, Wolfgang

    2014-12-10

    Human islet amyloid polypeptide (IAPP) is the major component of pancreatic amyloid deposits in type 2 diabetes. The structural conversion of IAPP from a monomeric state into amyloid assemblies is the subject of intense research. Recombinant production of IAPP is, however, difficult due to its extreme aggregation propensity. Here we describe a novel strategy for expression of IAPP in Escherichia coli, based on an engineered protein tag, which sequesters IAPP monomers and prevents IAPP aggregation. The IAPP-binding protein HI18 was selected by phage display from a β-wrapin library. Fusion of HI18 to IAPP enabled the soluble expression of the construct. IAPP was cleaved from the fusion construct and purified to homogeneity with a yield of 3mg of isotopically labeled peptide per liter of culture. In the monomeric state, IAPP was largely disordered as evidenced by far-UV CD and liquid-state NMR spectroscopy but competent to form amyloid fibrils according to atomic force microscopy. These results demonstrate the ability of the engineered β-wrapin HI18 for shielding the hydrophobic sequence of IAPP during expression and purification. Fusion of aggregation-inhibiting β-wrapins is a suitable approach for the recombinant production of aggregation-prone proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. 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.

  2. cDNA encoding a polypeptide including a hevein sequence

    Energy Technology Data Exchange (ETDEWEB)

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

    2000-07-04

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

  3. Light Scattering Characterization of Elastin-Like Polypeptide Trimer Micelles

    Science.gov (United States)

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

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

  4. cDNA encoding a polypeptide including a hevein sequence

    Energy Technology Data Exchange (ETDEWEB)

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

    1999-05-04

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

  5. cDNA encoding a polypeptide including a hevein sequence

    Energy Technology Data Exchange (ETDEWEB)

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

    1999-05-04

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

  6. cDNA encoding a polypeptide including a hevein sequence

    Energy Technology Data Exchange (ETDEWEB)

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

    1995-03-21

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

  7. A refined characterisation of the NeoHepatocyte phenotype necessitates a reappraisal of the transdifferentiation hypothesis.

    Science.gov (United States)

    Riquelme, Paloma; Wundt, Judith; Hutchinson, James A; Brulport, Marc; Jun, Yu; Sotnikova, Anna; Girreser, Ulrich; Braun, Felix; Gövert, Felix; Soria, Bernat; Nüssler, Andreas; Clement, Bernd; Hengstler, Jan G; Fändrich, Fred

    2009-03-01

    Under certain culture conditions human peripheral blood monocytes may be induced to express phenotypic markers of non-haematopoietic lineages, including hepatocyte-defining traits. One such example, the NeoHepatocyte, was previously shown to express a broad panel of hepatocyte-like marker antigens and metabolic activities, both in vitro and following engraftment in the liver of immunodeficient mice. In this report, a refined description of NeoHepatocytes, with regard to their expression of xenobiotic-metabolising enzymes, morphology, hepatocyte marker expression and cell surface phenotype, is presented in comparison with human macrophages in defined states of activation. Contrary to prior assertions, it would seem more likely that NeoHepatocytes express particular hepatocyte-defining genes during a normal programme of macrophage differentiation rather than undergoing a process of transdifferentiation to become hepatocyte-like cells.

  8. Determination of metabolic stability using cryopreserved hepatocytes from rainbow trout (Oncorhynchus mykiss)

    Science.gov (United States)

    Standard protocols for isolating, cryopreserving, and thawing rainbow trout hepatocytes are described, along with procedures for using fresh or cryopreserved hepatocytes to assess chemical metabolic stability in fish by means of a substrate depletion approach. Variations on thes...

  9. 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.

  10. Fuel assembly

    International Nuclear Information System (INIS)

    Nakatsuka, Masafumi; Matsuzuka, Ryuji.

    1976-01-01

    Object: To provide a fuel assembly which can decrease pressure loss of coolant to uniform temperature. Structure: A sectional area of a flow passage in the vicinity of an inner peripheral surface of a wrapper tube is limited over the entire length to prevent the temperature of a fuel element in the outermost peripheral portion from being excessively decreased to thereby flatten temperature distribution. To this end, a plurality of pincture-frame-like sheet metals constituting a spacer for supporting a fuel assembly, which has a plurality of fuel elements planted lengthwise and in given spaced relation within the wrapper tube, is disposed in longitudinal grooves and in stacked fashion to form a substantially honeycomb-like space in cross section. The fuel elements are inserted and supported in the space to form a fuel assembly. (Kamimura, M.)

  11. Fuel assemblies

    International Nuclear Information System (INIS)

    Nagano, Mamoru; Yoshioka, Ritsuo

    1983-01-01

    Purpose: To effectively utilize nuclear fuels by increasing the reactivity of a fuel assembly and reduce the concentration at the central region thereof upon completion of the burning. Constitution: A fuel assembly is bisected into a central region and a peripheral region by disposing an inner channel box within a channel box. The flow rate of coolants passing through the central region is made greater than that in the peripheral region. The concentration of uranium 235 of the fuel rods in the central region is made higher. In such a structure, since the moderating effect in the central region is improved, the reactivity of the fuel assembly is increased and the uranium concentration in the central region upon completion of the burning can be reduced, fuel economy and effective utilization of uranium can be attained. (Kamimura, M.)

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

    Science.gov (United States)

    Sanada, Takahiro; Tsukiyama-Kohara, Kyoko; Yamamoto, Naoki; Ezzikouri, Sayeh; Benjelloun, Soumaya; Murakami, Shuko; Tanaka, Yasuhito; Tateno, Chise; Kohara, Michinori

    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(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(4)-10(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(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. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Interspecies differences in metabolism of arsenic by cultured primary hepatocytes

    International Nuclear Information System (INIS)

    Drobna, Zuzana; Walton, Felecia S.; Harmon, Anne W.; Thomas, David J.; Styblo, Miroslav

    2010-01-01

    Biomethylation is the major pathway for the metabolism of inorganic arsenic (iAs) in many mammalian species, including the human. However, significant interspecies differences have been reported in the rate of in vivo metabolism of iAs and in yields of iAs metabolites found in urine. Liver is considered the primary site for the methylation of iAs and arsenic (+3 oxidation state) methyltransferase (As3mt) is the key enzyme in this pathway. Thus, the As3mt-catalyzed methylation of iAs in the liver determines in part the rate and the pattern of iAs metabolism in various species. We examined kinetics and concentration-response patterns for iAs methylation by cultured primary hepatocytes derived from human, rat, mice, dog, rabbit, and rhesus monkey. Hepatocytes were exposed to [ 73 As]arsenite (iAs III ; 0.3, 0.9, 3.0, 9.0 or 30 nmol As/mg protein) for 24 h and radiolabeled metabolites were analyzed in cells and culture media. Hepatocytes from all six species methylated iAs III to methylarsenic (MAs) and dimethylarsenic (DMAs). Notably, dog, rat and monkey hepatocytes were considerably more efficient methylators of iAs III than mouse, rabbit or human hepatocytes. The low efficiency of mouse, rabbit and human hepatocytes to methylate iAs III was associated with inhibition of DMAs production by moderate concentrations of iAs III and with retention of iAs and MAs in cells. No significant correlations were found between the rate of iAs methylation and the thioredoxin reductase activity or glutathione concentration, two factors that modulate the activity of recombinant As3mt. No associations between the rates of iAs methylation and As3mt protein structures were found for the six species examined. Immunoblot analyses indicate that the superior arsenic methylation capacities of dog, rat and monkey hepatocytes examined in this study may be associated with a higher As3mt expression. However, factors other than As3mt expression may also contribute to the interspecies differences

  14. Microencapsulation of Hepatocytes and Mesenchymal Stem Cells for Therapeutic Applications.

    Science.gov (United States)

    Meier, Raphael P H; Montanari, Elisa; Morel, Philippe; Pimenta, Joël; Schuurman, Henk-Jan; Wandrey, Christine; Gerber-Lemaire, Sandrine; Mahou, Redouan; Bühler, Leo H

    2017-01-01

    Encapsulated hepatocyte transplantation and encapsulated mesenchymal stem cell transplantation are newly developed potential treatments for acute and chronic liver diseases, respectively. Cells are microencapsulated in biocompatible semipermeable alginate-based hydrogels. Microspheres protect cells against antibodies and immune cells, while allowing nutrients, small/medium size proteins and drugs to diffuse inside and outside the polymer matrix. Microencapsulated cells are assessed in vitro and designed for experimental transplantation and for future clinical applications.Here, we describe the protocol for microencapsulation of hepatocytes and mesenchymal stem cells within hybrid poly(ethylene glycol)-alginate hydrogels.

  15. 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

  16. Inducibility of carbamoylphosphate synthetase (ammonia) in cultures of embryonic hepatocytes: ontogenesis of the responsiveness to hormones

    NARCIS (Netherlands)

    Lamers, W. H.; Zonneveld, D.; Charles, R.

    1984-01-01

    Glucocorticosteroids and cyclic AMP induce carbamoylphosphate synthetase (ammonia) (CPS) in rat hepatocytes. Using an enzyme immunoassay applied to hepatocyte cultures fixed in situ, it has been demonstrated that the capacity of hepatocytes to synthesize CPS in the presence of both hormones is

  17. Billion-scale production of hepatocyte-like cells from human induced pluripotent stem cells.

    Science.gov (United States)

    Yamashita, Tomoki; Takayama, Kazuo; Sakurai, Fuminori; Mizuguchi, Hiroyuki

    2018-02-19

    Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells are expected to be utilized in drug screening and regenerative medicine. However, hepatocyte-like cells have not been fully used in such applications because it is difficult to produce such cells on a large scale. In this study, we tried to establish a method to mass produce hepatocyte-like cells using a three-dimensional (3D) cell culture bioreactor called the Rotary Cell Culture System (RCCS). RCCS enabled us to obtain homogenous hepatocyte-like cells on a billion scale (>10 9  cells). The gene expression levels of some hepatocyte markers (alpha-1 antitrypsin, cytochrome (CYP) 1A2, CYP2D6, and hepatocyte nuclear factor 4alpha) were higher in 3D-cultured hepatocyte-like cells than in 2D-cultured hepatocyte-like cells. This result suggests that RCCS could provide more suitable conditions for hepatocyte maturation than the conventional 2D cell culture conditions. In addition, more than 90% of hepatocyte-like cells were positive for albumin and could uptake low-density lipoprotein in the culture medium. We succeeded in the large-scale production of homogenous and functional hepatocyte-like cells from human iPS cells. This technology will be useful in drug screening and regenerative medicine, which require enormous numbers of hepatocyte-like cells. Copyright © 2018 Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    1980-01-01

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

  19. Valve assembly

    International Nuclear Information System (INIS)

    Sandling, M.

    1981-01-01

    An improved valve assembly, used for controlling the flow of radioactive slurry, is described. Radioactive contamination of the air during removal or replacement of the valve is prevented by sucking air from the atmosphere through a portion of the structure above the valve housing. (U.K.)

  20. Fuel assembly

    International Nuclear Information System (INIS)

    Gjertsen, R.K.; Bassler, E.A.; Huckestein, E.A.; Salton, R.B.; Tower, S.N.

    1988-01-01

    A fuel assembly adapted for use with a pressurized water nuclear reactor having capabilities for fluid moderator spectral shift control is described comprising: parallel arranged elongated nuclear fuel elements; means for providing for axial support of the fuel elements and for arranging the fuel elements in a spaced array; thimbles interspersed among the fuel elements adapted for insertion of a rod control cluster therewithin; means for structurally joining the fuel elements and the guide thimbles; fluid moderator control means for providing a volume of low neutron absorbing fluid within the fuel assembly and for removing a substantially equivalent volume of reactor coolant water therefrom, a first flow manifold at one end of the fuel assembly sealingly connected to a first end of the moderator control tubes whereby the first ends are commonly flow connected; and a second flow manifold, having an inlet passage and an outlet passage therein, sealingly connected to a second end of the moderator control tubes at a second end of the fuel assembly

  1. 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.

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

  3. NMR and rotational angles in solution conformation of polypeptides

    Science.gov (United States)

    Bystrov, V. F.

    1985-01-01

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

  4. A radioimmunoassay of gastric inhibitory polypeptide in human plasma

    International Nuclear Information System (INIS)

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

    1980-01-01

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

  5. Gastric inhibitory polypeptide does not inhibit gastric emptying in humans

    DEFF Research Database (Denmark)

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

    2004-01-01

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

  6. 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....

  7. Radiation-induced PKC signaling system in cultured rat hepatocytes

    International Nuclear Information System (INIS)

    Nakajima, Tetsuo; Yukawa, Osami

    1998-01-01

    Radiation effects on living organisms are mainly caused through reactive oxygen species (ROS) on living cells. It is known that ROS damages various membranes and the bio membranes play an important role in cellular signal transduction pathways. The effects of radiation on cellular signal transduction pathways in cultured rat hepatocytes have been studied

  8. 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 parti...

  9. 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.

  10. 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

  11. Hepatocytes in the development of liver support systems

    NARCIS (Netherlands)

    I.H.M. Borel Rinkes (Inne)

    1993-01-01

    textabstractThis thesis focuses on the development of alternative strategies in the treatment of patients with acute fulminant hepatic failure and inborn errors of metabolism, using hepatocytes as the basis of liver support. When compared with transplantation of the liver as an organ, the

  12. Hepatocyte heterogeneity in the metabolism of amino acids and ammonia

    NARCIS (Netherlands)

    Häussinger, D.; Lamers, W. H.; Moorman, A. F.

    1992-01-01

    With respect to hepatocyte heterogeneity in ammonia and amino acid metabolism, two different patterns of sublobular gene expression are distinguished: 'gradient-type' and 'strict- or compartment-type' zonation. An example for strict-type zonation is the reciprocal distribution of carbamoylphosphate

  13. Biosynthesis of human sialophorins and analysis of the polypeptide core

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  14. Induction of hepatocyte polyploidization in rats of different age by ionizing radiation of different LET

    International Nuclear Information System (INIS)

    Gil'yano, N.Ya.; Malinovskij, O.V.; Khair, M.B.

    1992-01-01

    A decrease in the effectiveness of neutron-irradiation with respect to fusion of nonproliferating hepatocytes of animals with age was shown by the method of flow cytometry. There was an inverse relationship between the effectiveness of induction of non-proliferating hepatocytes fusion and neutron energy. The process of hepatocyte fusion induced by neutrons was inhibited by uranyl acetate. No age-dependent changes were noted in the induction of polyploidization of proliferating hepatocytes by sparsely ionizing radiation. A hypothesis is proposed concerning a membrane nature of the target responsible for hepatocyte polyploidization induced by densely ionizing radiation. (authors). 8 refs., 4 figs., 5 tabs

  15. Induction of hepatocyte polyploidization in rats of different age by ionizing radiation of different LET

    International Nuclear Information System (INIS)

    Gil'yano, N.Ya.; Malinovskij, O.V.; Khair, M.B.

    1990-01-01

    A decrease in the effectiveness of neutron-irradiation with respect to fusion of nonproliferating hepatocytes of animals with age was shown by the method of flow cytometry. There was an inverse relationship between the effectiveness of induction of non-proliferating hepatocytes fusion and neutron energy. The process of hepatocyte fusion induced by neutrons was inhibited by uranyl acetate. No age-dependent changes were noted in the induction of polyploidization of proliferating hepatocytes by sparsely ionizing radiation. A hypothesis is proposed concerning a membrane nature of the target responsible for hepatocyte polyploidization induced by densely ionizing radiation

  16. Fuel assembly

    International Nuclear Information System (INIS)

    Yokota, Tokunobu.

    1990-01-01

    A fuel assembly used in a FBR type nuclear reactor comprises a plurality of fuel rods and a moderator guide member (water rod). A moderator exit opening/closing mechanism is formed at the upper portion of the moderator guide member for opening and closing a moderator exit. In the initial fuel charging operation cycle to the reactor, the moderator exit is closed by the moderator exit opening/closing mechanism. Then, voids are accumulated at the inner upper portion of the moderator guide member to harden spectrum and a great amount of plutonium is generated and accumulated in the fuel assembly. Further, in the fuel re-charging operation cycle, the moderator guide member is used having the moderator exit opened. In this case, voids are discharged from the moderator guide member to decrease the ratio, and the plutonium accumulated in the initial charging operation cycle is burnt. In this way, the fuel economy can be improved. (I.N.)

  17. Fuel assemblies

    International Nuclear Information System (INIS)

    Echigoya, Hironori; Nomata, Terumitsu.

    1983-01-01

    Purpose: To render the axial distribution relatively flat. Constitution: First nuclear element comprises a fuel can made of zircalloy i.e., the metal with less neutron absorption, which is filled with a plurality of UO 2 pellets and sealed by using a lower end plug, a plenum spring and an upper end plug by means of welding. Second fuel element is formed by substituting a part of the UO 2 pellets with a water tube which is sealed with water and has a space for allowing the heat expansion. The nuclear fuel assembly is constituted by using the first and second fuel elements together. In such a structure, since water reflects neutrons and decrease their leakage to increase the temperature, reactivity is added at the upper portion of the fuel assembly to thereby flatten the axial power distribution. Accordingly, stable operation is possible only by means of deep control rods while requiring no shallow control rods. (Sekiya, K.)

  18. Fuel assembly

    International Nuclear Information System (INIS)

    Kawai, Mitsuo.

    1988-01-01

    Purpose: To reduce the corrosion rate and suppress the increase of radioactive corrosion products in reactor water of nuclear fuel assemblies for use in BWR type reactors having spacer springs made of nickel based deposition reinforced type alloys. Constitution: Spacer rings made of nickel based deposition reinforced type alloy are incorporated and used as fuel assemblies after applying treatment of dipping and maintaining at high temperature water followed by heating in steams. Since this can remove the nickel leaching into reactor water at the initial stage, Co-58 as the radioactive corrosion products in the reactor water can be reduced, and the operation at in-service inspection or repairement can be facilitated to improve the working efficiency of the nuclear power plant. The dipping time is desirably more than 10 hours and more desirably more than 30 hours. (Horiuchi, T. )

  19. Fuel assembly

    International Nuclear Information System (INIS)

    Watanabe, Shoichi; Hirano, Yasushi.

    1998-01-01

    A one-half or more of entire fuel rods in a fuel assembly comprises MOX fuel rods containing less than 1wt% of burnable poisons, and at least a portion of the burnable poisons comprises gadolinium. Then, surplus reactivity at an initial stage of operation cycle is controlled to eliminate burnable poisons remained unburnt at a final stage, as well as increase thermal reactivity. In addition, the content of fission plutonium is determined to greater than the content of uranium 235, and fuel rods at corner portions are made not to incorporate burnable poisons. Fuel rods not containing burnable poisons are disposed at positions in adjacent with fuel rods facing to a water rod at one or two directions. Local power at radial center of the fuel assembly is increased to flatten the distortion of radial power distribution. (N.H.)

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

    International Nuclear Information System (INIS)

    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

    2015-01-01

    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.

  1. 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.

  2. Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis

    International Nuclear Information System (INIS)

    Ilowski, Maren; Kleespies, Axel; Toni, Enrico N. de; Donabauer, Barbara; Jauch, Karl-Walter; Hengstler, Jan G.; Thasler, Wolfgang E.

    2011-01-01

    Research highlights: → ALR decreases cytochrome c release from mitochondria. → ALR protects hepatocytes against apoptosis induction by ethanol, TRAIL, anti-Apo, TGF-β and actinomycin D. → ALR exerts a liver-specific anti-apoptotic effect. → A possible medical usage of ALR regarding protection of liver cells during apoptosis inducing therapies. -- Abstract: Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-β, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining. ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-β and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines. Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings.

  3. Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Ilowski, Maren [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Kleespies, Axel [Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Toni, Enrico N. de [Department of Medicine II, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Donabauer, Barbara [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Jauch, Karl-Walter [Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Hengstler, Jan G. [Leibniz Research Centre for Working Environment and Human Factors, Technical University, Dortmund (Germany); Thasler, Wolfgang E., E-mail: wolfgang.thasler@med.uni-muenchen.de [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany)

    2011-01-07

    Research highlights: {yields} ALR decreases cytochrome c release from mitochondria. {yields} ALR protects hepatocytes against apoptosis induction by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. {yields} ALR exerts a liver-specific anti-apoptotic effect. {yields} A possible medical usage of ALR regarding protection of liver cells during apoptosis inducing therapies. -- Abstract: Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-{beta}, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining. ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines. Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings.

  4. 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...

  5. Fuel assembly

    International Nuclear Information System (INIS)

    Ueda, Sei; Ando, Ryohei; Mitsutake, Toru.

    1995-01-01

    The present invention concerns a fuel assembly suitable to a BWR-type reactor and improved especially with the nuclear characteristic, heat performance, hydraulic performance, dismantling or assembling performance and economical property. A part of poison rods are formed as a large-diameter/multi-region poison rods having a larger diameter than a fuel rod. A large number of fuel rods are disposed surrounding a large diameter water rod and a group of the large-diameter/multi-region poison rods in adjacent with the water rod. The large-diameter water rod has a burnable poison at the tube wall portion. At least a portion of the large-diameter poison rods has a coolant circulation portion allowing coolants to circulate therethrough. Since the large-diameter poison rods are disposed at a position of high neutron fluxes, a large neutron multiplication factor suppression effect can be provided, thereby enabling to reduce the number of burnable poison rods relative to fuels. As a result, power peaking in the fuel assembly is moderated and a greater amount of plutonium can be loaded. In addition the flow of cooling water which tends to gather around the large diameter water rod can be controlled to improve cooling performance of fuels. (N.H.)

  6. The organic anion transport polypeptide 1d1 (Oatp1d1) mediates hepatocellular uptake of phalloidin and microcystin into skate liver.

    Science.gov (United States)

    Meier-Abt, F; Hammann-Hänni, A; Stieger, B; Ballatori, N; Boyer, J L

    2007-02-01

    Organic anion transporting polypeptides (rodent Oatp; human OATP) mediate cellular uptake of numerous organic compounds including xenobiotic toxins into mammalian hepatocytes. In the little skate Leucoraja erinacea a liver-specific Oatp (Oatp1d1, also called sOatp) has been identified and suggested to represent an evolutionarily ancient precursor of the mammalian liver OATP1B1 (human), Oatp1b2 (rat), and OATP1B3 (human). The present study tested whether Oatp1d1 shares functional transport activity of the xenobiotic oligopeptide toxins phalloidin and microcystin with the mammalian liver Oatps/OATPs. The phalloidin analogue [(3)H]-demethylphalloin was taken up into skate hepatocytes with high affinity (Km approximately 0.4 microM), and uptake could be inhibited by phalloidin and a variety of typical Oatp/OATP substrates such as bromosulfophthalein, bile salts, estrone-3-sulfate, cyclosporine A and high concentrations of microcystin-LR (Ki approximately 150 microM). When expressed in Xenopus laevis oocytes Oatp1d1 increased uptake of demethylphalloin (Km approximately 2.2 microM) and microcystin-LR (Km approximately 27 microM) 2- to 3-fold over water-injected oocytes, whereas the alternative skate liver organic anion transporter, the dimeric Ostalpha/beta, exhibited no phalloidin and only minor microcystin-LR transport. Also, the closest mammalian Oatp1d1 orthologue, the human brain and testis OATP1C1, did not show any phalloidin transport activity. These results demonstrate that the evolutionarily ancient Oatp1d1 is able to mediate uptake of cyclic oligopeptide toxins into skate liver. The findings support the notion that Oatp1d1 is a precursor of the liver-specific mammalian Oatps/OATPs and that its transport properties are closely associated with certain forms of toxic liver injury such as for example protein phosphatase inhibition by the water-borne toxin microcystin.

  7. The organic anion transport polypeptide 1d1 (Oatp1d1) mediates hepatocellular uptake of phalloidin and microcystin into skate liver

    International Nuclear Information System (INIS)

    Meier-Abt, F.; Hammann-Haenni, A.; Stieger, B.; Ballatori, N.; Boyer, J.L.

    2007-01-01

    Organic anion transporting polypeptides (rodent Oatp; human OATP) mediate cellular uptake of numerous organic compounds including xenobiotic toxins into mammalian hepatocytes. In the little skate Leucoraja erinacea a liver-specific Oatp (Oatp1d1, also called sOatp) has been identified and suggested to represent an evolutionarily ancient precursor of the mammalian liver OATP1B1 (human), Oatp1b2 (rat), and OATP1B3 (human). The present study tested whether Oatp1d1 shares functional transport activity of the xenobiotic oligopeptide toxins phalloidin and microcystin with the mammalian liver Oatps/OATPs. The phalloidin analogue [ 3 H]-demethylphalloin was taken up into skate hepatocytes with high affinity (Km ∼ 0.4 μM), and uptake could be inhibited by phalloidin and a variety of typical Oatp/OATP substrates such as bromosulfophthalein, bile salts, estrone-3-sulfate, cyclosporine A and high concentrations of microcystin-LR (Ki ∼ 150 μM). When expressed in Xenopus laevis oocytes Oatp1d1 increased uptake of demethylphalloin (Km ∼ 2.2 μM) and microcystin-LR (Km ∼ 27 μM) 2- to 3-fold over water-injected oocytes, whereas the alternative skate liver organic anion transporter, the dimeric Ostα/β, exhibited no phalloidin and only minor microcystin-LR transport. Also, the closest mammalian Oatp1d1 orthologue, the human brain and testis OATP1C1, did not show any phalloidin transport activity. These results demonstrate that the evolutionarily ancient Oatp1d1 is able to mediate uptake of cyclic oligopeptide toxins into skate liver. The findings support the notion that Oatp1d1 is a precursor of the liver-specific mammalian Oatps/OATPs and that its transport properties are closely associated with certain forms of toxic liver injury such as for example protein phosphatase inhibition by the water-borne toxin microcystin

  8. Vectorial transport of bile salts across MDCK cells expressing both rat Na+-taurocholate cotransporting polypeptide and rat bile salt export pump.

    Science.gov (United States)

    Mita, Sachiko; Suzuki, Hiroshi; Akita, Hidetaka; Stieger, Bruno; Meier, Peter J; Hofmann, Alan F; Sugiyama, Yuichi

    2005-01-01

    Bile salts are predominantly taken up by hepatocytes via the basolateral Na(+)-taurocholate cotransporting polypeptide (NTCP/SLC10A1) and secreted into the bile by the bile salt export pump (BSEP/ABCB11). In the present study, we transfected rat Ntcp and rat Bsep into polarized Madin-Darby canine kidney cells and characterized the transport properties of these cells for eight bile salts. Immunohistochemical staining demonstrated that Ntcp was expressed at the basolateral domains, whereas Bsep was expressed at the apical domains. Basal-to-apical transport of taurocholate across the monolayer expressing only Ntcp and that coexpressing Ntcp/Bsep was observed, whereas the flux across the monolayer of control and Bsep-expressing cells was symmetrical. Basal-to-apical transport of taurocholate across Ntcp/Bsep-coexpressing monolayers was significantly higher than that across monolayers expressing only Ntcp. Kinetic analysis of this vectorial transport of taurocholate gave an apparent K(m) value of 13.9 +/- 4.7 microM for cells expressing Ntcp alone, which is comparable with 22.2 +/- 4.5 microM for cells expressing both Ntcp and Bsep and V(max) values of 15.8 +/- 4.2 and 60.8 +/- 9.0 pmol.min(-1).mg protein(-1) for Ntcp alone and Ntcp and Bsep-coexpressing cells, respectively. Transcellular transport of cholate, glycocholate, taurochenodeoxycholate, chenodeoxycholate, glycochenodeoxycholate, tauroursodeoxycholate, ursodeoxycholate, and glycoursodeoxycholate, but not that of lithocholate was also observed across the double transfectant. This double-expressing system can be used as a model to clarify vectorial transport of bile salts across hepatocytes under physiological conditions.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2006-07-28

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

  10. 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.

  11. Gender differences in methionine accumulation and metabolism in freshly isolated mouse hepatocytes: Potential roles in toxicity

    International Nuclear Information System (INIS)

    Dever, Joseph T.; Elfarra, Adnan A.

    2009-01-01

    L-Methionine (Met) is hepatotoxic at high concentrations. Because Met toxicity in freshly isolated mouse hepatocytes is gender-dependent, the goal of this study was to assess the roles of Met accumulation and metabolism in the increased sensitivity of male hepatocytes to Met toxicity compared with female hepatocytes. Male hepatocytes incubated with Met (30 mM) at 37 o C exhibited higher levels of intracellular Met at 0.5, 1.0, and 1.5 h, respectively, compared to female hepatocytes. Conversely, female hepatocytes had higher levels of S-adenosyl-L-methionine compared to male hepatocytes. Female hepatocytes also exhibited higher L-methionine-L-sulfoxide levels relative to control hepatocytes, whereas the increases in L-methionine-D-sulfoxide (Met-D-O) levels were similar in hepatocytes of both genders. Addition of aminooxyacetic acid (AOAA), an inhibitor of Met transamination, significantly increased Met levels at 1.5 h and increased Met-D-O levels at 1.0 and 1.5 h only in Met-exposed male hepatocytes. No gender differences in cytosolic Met transamination activity by glutamine transaminase K were detected. However, female mouse liver cytosol exhibited higher methionine-DL-sulfoxide (MetO) reductase activity than male mouse liver cytosol at low (0.25 and 0.5 mM) MetO concentrations. Collectively, these results suggest that increased cellular Met accumulation, decreased Met transmethylation, and increased Met and MetO transamination in male mouse hepatocytes may be contributing to the higher sensitivity of the male mouse hepatocytes to Met toxicity in comparison with female mouse hepatocytes.

  12. 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

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

    Science.gov (United States)

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

    2012-12-01

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

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

    Science.gov (United States)

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

    1986-12-30

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

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

    Science.gov (United States)

    Miao, Hua

    2015-01-01

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

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

    DEFF Research Database (Denmark)

    2016-01-01

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

  17. Fuel assembly

    International Nuclear Information System (INIS)

    Fujibayashi, Toru.

    1970-01-01

    Herein disclosed is a fuel assembly in which a fuel rod bundle is easily detachable by rotating a fuel rod fastener rotatably mounted to the upper surface of an upper tie-plate supporting a fuel bundle therebelow. A locking portion at the leading end of each fuel rod protrudes through the upper tie-plate and is engaged with or separated from the tie-plate by the rotation of the fastener. The removal of a desired fuel rod can therefore be remotely accomplished without the necessity of handling pawls, locking washers and nuts. (Owens, K.J.)

  18. 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...... the investigative tools offered by assemblage theory, actor-network theory and non-representational theory. Clear theoretical explanation and methodological innovation, alongside empirical applications of these emerging frameworks will offer readers new and refreshing perspectives on consumer culture and market...... societies. This is an essential reading for both seasoned scholars and advanced students of markets, economies and social forms of consumption....

  19. Cancer Nano technology Using Elastin-Like Polypeptides

    International Nuclear Information System (INIS)

    Siti Najila Mohd Janib

    2014-01-01

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

  20. Multitriggered Tumor-Responsive Drug Delivery Vehicles Based on Protein and Polypeptide Coassembly for Enhanced Photodynamic Tumor Ablation.

    Science.gov (United States)

    Zhang, Ning; Zhao, Fenfang; Zou, Qianli; Li, Yongxin; Ma, Guanghui; Yan, Xuehai

    2016-11-01

    Tumor-responsive nanocarriers are highly valuable and demanded for smart drug delivery particularly in the field of photodynamic therapy (PDT), where a quick release of photosensitizers in tumors is preferred. Herein, it is demonstrated that protein-based nanospheres, prepared by the electrostatic assembly of proteins and polypeptides with intermolecular disulfide cross-linking and surface polyethylene glycol coupling, can be used as versatile tumor-responsive drug delivery vehicles for effective PDT. These nanospheres are capable of encapsulation of various photosensitizers including Chlorin e6 (Ce6), protoporphyrin IX, and verteporfin. The Chlorin e6-encapsulated nanospheres (Ce6-Ns) are responsive to changes in pH, redox potential, and proteinase concentration, resulting in multitriggered rapid release of Ce6 in an environment mimicking tumor tissues. In vivo fluorescence imaging results indicate that Ce6-Ns selectively accumulate near tumors and the quick release of Ce6 from Ce6-Ns can be triggered by tumors. In tumors the fluorescence of released Ce6 from Ce6-Ns is observed at 0.5 h postinjection, while in normal tissues the fluorescence appeared at 12 h postinjection. Tumor ablation is demonstrated by in vivo PDT using Ce6-Ns and the biocompatibility of Ce6-Ns is evident from the histopathology imaging, confirming the enhanced in vivo PDT efficacy and the biocompatibility of the assembled drug delivery vehicles. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Fuel assembly

    International Nuclear Information System (INIS)

    Kurihara, Kunitoshi; Azekura, Kazuo.

    1992-01-01

    In a reactor core of a heavy water moderated light water cooled pressure tube type reactor, no sufficient effects have been obtained for the transfer width to a negative side of void reactivity change in a region of a great void coefficient. Then, a moderation region divided into upper and lower two regions is disposed at the central portion of a fuel assembly. Coolants flown into the lower region can be discharged to the cooling region from an opening disposed at the upper end portion of the lower region. Light water flows from the lower region of the moderator region to the cooling region of the reactor core upper portion, to lower the void coefficient. As a result, the reactivity performance at low void coefficient, i.e., a void reaction rate is transferred to the negative side. Thus, this flattens the power distribution in the fuel assembly, increases the thermal margin and enables rapid operaiton and control of the reactor core, as well as contributes to the increase of fuel burnup ratio and reduction of the fuel cycle cost. (N.H.)

  2. Fuel assembly

    International Nuclear Information System (INIS)

    Chaki, Masao; Nishida, Koji; Karasawa, Hidetoshi; Kanazawa, Toru; Orii, Akihito; Nagayoshi, Takuji; Kashiwai, Shin-ichi; Masuhara, Yasuhiro

    1998-01-01

    The present invention concerns a fuel assembly, for a BWR type nuclear reactor, comprising fuel rods in 9 x 9 matrix. The inner width of the channel box is about 132mm and the length of the fuel rods which are not short fuel rods is about 4m. Two water rods having a circular cross section are arranged on a diagonal line in a portion of 3 x 3 matrix at the center of the fuel assembly, and two fuel rods are disposed at vacant spaces, and the number of fuel rods is 74. Eight fuel rods are determined as short fuel rods among 74 fuel rods. Assuming the fuel inventory in the short fuel rod as X(kg), and the fuel inventory in the fuel rods other than the short fuel rods as Y(kg), X and Y satisfy the relation: X + Y ≥ 173m, Y ≤ - 9.7X + 292, Y ≤ - 0.3X + 203 and X > 0. Then, even when the short fuel rods are used, the fuel inventory is increased and fuel economy can be improved. (I.N.)

  3. Fuel assembly

    International Nuclear Information System (INIS)

    Fushimi, Atsushi; Shimada, Hidemitsu; Aoyama, Motoo; Nakajima, Junjiro

    1998-01-01

    In a fuel assembly for an n x n lattice-like BWR type reactor, n is determined to 9 or greater, and the enrichment degree of plutonium is determined to 4.4% by weight or less. Alternatively, n is determined to 10 or greater, and the enrichment degree of plutonium is determined to 5.2% by weight or less. An average take-out burnup degree is determined to 39GWd/t or less, and the matrix is determined to 9 x 9 or more, or the average take-out burnup degree is determined to 51GWd/t, and the matrix is determined to 10 x 10 or more and the increase of the margin of the maximum power density obtained thereby is utilized for the compensation of the increase of distortion of power distribution due to decrease of the kinds of plutonium enrichment degree, thereby enabling to reduce the kind of the enrichment degree of MOX fuel rods to one. As a result, the manufacturing step for fuel pellets can be simplified to reduce the manufacturing cost for MOX fuel assemblies. (N.H.)

  4. 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...

  5. 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...

  6. 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...

  7. 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...

  8. Fuel assembly

    International Nuclear Information System (INIS)

    Nomata, Terumitsu.

    1993-01-01

    Among fuel pellets to be loaded to fuel cans of a fuel assembly, fuel pellets having a small thermal power are charged in a region from the end of each of spacers up to about 50mm on the upstream of coolants that flow vertically at the periphery of fuel rods. Coolants at the periphery of fuel rods are heated by the heat generation, to result in voids. However, since cooling effect on the upstream of the spacers is low due to influences of the spacers. Further, since the fuel pellets disposed in the upstream region have small thermal power, a void coefficient is not increased. Even if a thermal power exceeding cooling performance should be generated, there is no worry of causing burnout in the upstream region. Even if burnout should be caused, safety margin and reliability relative to burnout are improved, to increase an allowable thermal power, thereby enabling to improve integrity and reliability of fuel rods and fuel assemblies. (N.H.)

  9. 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.

  10. Metabolism of six CYP probe substrates in fetal hepatocytes

    Directory of Open Access Journals (Sweden)

    Abdul Naveed Shaik

    2016-06-01

    Full Text Available Cytochrome P-450 (CYP are the most common drug metabolizing enzymes and are abundantly expressed in liver apart from kidney, lungs, intestine, brain etc. Their expression levels change with physiological conditions and disease states. The expression of these CYPs is less in human foetus and neonates compared to adults, which results in lower clearance of xenobiotics in infants and neonates compared to adults. Hepatocytes are the cells which are largely used to study these CYPs. We have isolated hepatocytes from aborted foetus to study the metabolism of six probe substrates: phenacetin, diclofenac, S-mephenytoin, dextromethorphan, nifedipine and testosterone. The results obtained show the expression of various CYPs (CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4 in human foetus and their involvement in metabolism of CYP probe substrates.

  11. [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.

  12. 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.

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

    Science.gov (United States)

    Seifert, P; Spitznas, M

    1999-06-01

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

  14. 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.

  15. Bovine pancreatic polypeptide as an antagonist of muscarinic cholinergic receptors

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  16. Intestinal mucosa is a target tissue for pancreatic polypeptide

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  17. Ultrastructure of hepatocyte nuclei in irradiated, adrenalectomized rats

    Energy Technology Data Exchange (ETDEWEB)

    Orkisz, S.; Bartel, H.; Kmiec, B. (Military Medical Academy, Lodz (Poland))

    1984-01-01

    A cytochemical study of hepatocyte nuclei of adrenalectomized and irradiated rats was performed. After irradiation alone, the behaviour of the ribonucleoprotein components was studied according to Bernhard. The findings suggest that a delay occurs in the synthesis of preribosomal RNA in the nucleoli and in the transport of messenger RNA to the cytoplasm. The indirect effect of ionizing radiation on nuclear RNA synthesis is assumed to occur through the influence of cortical steroid hormones on the transcription process.

  18. 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.

  19. Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation

    International Nuclear Information System (INIS)

    Yeom, Chul-gon; Kim, Dong-il; Park, Min-jung; Choi, Joo-hee; Jeong, Jieun; Wi, Anjin; Park, Whoashig; Han, Ho-jae; Park, Soo-hyun

    2015-01-01

    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

  20. [Crabtree effect caused by ketoses in isolated rat hepatocytes].

    Science.gov (United States)

    Martínez, P; Carrascosa, J M; Núñez de Castro, I

    1982-01-01

    Oxygen uptake and glycolytic activity were studied in hepatocytes isolated from fed rats. The addition of fructose or tagatose resulted in a 38% and 31% inhibition of cellular respiration respectively. The addition of 10 mM D-glyceraldehyde caused a slight Crabtree effect. Glucose, L-sorbose, or glycerol failed to modify oxygen consumption. Only incubation in the presence of fructose showed a high aerobic glycolysis measured by lactate production.

  1. Hepatocyte polyploidization and its association with pathophysiological processes

    OpenAIRE

    Wang, Min-Jun; Chen, Fei; Lau, Joseph T Y; Hu, Yi-Ping

    2017-01-01

    A characteristic cellular feature of the mammalian liver is the progressive polyploidization of the hepatocytes, where individual cells acquire more than two sets of chromosomes. Polyploidization results from cytokinesis failure that takes place progressively during the course of postnatal development. The proportion of polyploidy also increases with the aging process or with cellular stress such as surgical resection, toxic stimulation, metabolic overload, or oxidative damage, to involve as ...

  2. Functional activity of the rats’ hepatocytes under cancerogenesis

    Directory of Open Access Journals (Sweden)

    V. V. Ivchuk

    2007-10-01

    Full Text Available Enzymatic activity in rat’s hepatocytes under carcinoma Geuren T8 development as well as after introduction of rhenium compounds and cis-platin were studied. It has been determined that the decrease of enzymatic activity contrary to the control animals has been observed under simultaneous injection of cis-platin and cluster rhenium compounds in a liposome form. That confirms possible hepatoprotective properties of the rhenium compounds.

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

    Science.gov (United States)

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

    2015-09-17

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

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

    Science.gov (United States)

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

    2016-09-12

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

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

    DEFF Research Database (Denmark)

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

    2005-01-01

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

  6. Cholangiocarcinoma in Cirrhosis: Value of Hepatocyte Specific Magnetic Resonance Imaging.

    Science.gov (United States)

    Piscaglia, Fabio; Iavarone, Massimo; Galassi, Marzia; Vavassori, Sara; Renzulli, Matteo; Forzenigo, Laura Virginia; Granito, Alessandro; Salvatore, Veronica; Sangiovanni, Angelo; Golfieri, Rita; Colombo, Massimo; Bolondi, Luigi

    2015-10-01

    The diagnosis of intrahepatic cholangiocellular carcinoma (ICC) remains elusive at imaging, which is a critical issue in cirrhotic patients in whom a diagnosis of hepatocellular carcinoma (HCC) can be established only by imaging. The aim of the study was to evaluate the potential of MRI in the diagnosis of ICC in cirrhosis using 'hepatocyte-specific' Gadolinium (Gd)-based contrast agents. Sixteen histologically proven and retrospectively identified ICCs on cirrhosis were investigated with hepatocyte-specific magnetic resonance contrast agents (6 in Bologna with Gd-EOB-DTPA and 10 in Milan with Gd-BOPTA). The control group consisted of 41 consecutively and prospectively collected nodules (31 HCCs) imaged with Gd-EOB-DTPA. Fifteen ICC nodules (94%) displayed hypointensity in the hepatobiliary phase, suggesting malignancy. Thirteen cholangiocarcinomas (81%) showed hyperenhancement in the venous phase. Only 2 cholangiocarcinoma nodules showed hypoenhancement in the venous phase, corresponding to washout, in both cases preceded by rim enhancement in arterial phase. All the hepatocarcinomas showed hypointensity in hepatobiliary phase, but was always preceded by hypointensity in the venous phase; arterial rim enhancement was never observed in any hepatocarcinoma or regenerative nodule. MRI with hepatocyte-specific Gd-based contrast agents showed a pattern of malignancy in almost all the ICCs, concurrently avoiding misdiagnosis with hepatocarcinoma. These findings suggest a greater diagnostic capacity for this technique compared with the results of MRI with conventional contrast agents reported in the literature in this setting. © 2015 S. Karger AG, Basel.

  7. 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.

  8. 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. © 2016 Society for Endocrinology.

  9. 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.

  10. 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

  11. Fuel assembly

    International Nuclear Information System (INIS)

    Ueda, Makoto; Ogiya, Shunsuke.

    1989-01-01

    For improving the economy of a BWR type reactor by making the operation cycle longer, the fuel enrichment degree has to be increased further. However, this makes the subcriticality shallower in the upper portion of the reactor core, to bring about a possibility that the reactor shutdown becomes impossible. In the present invention, a portion of fuel rod is constituted as partial length fuel rods (P-fuel rods) in which the entire stack length in the effective portion is made shorter by reducing the concentration of fissionable materials in the axial portion. A plurality of moderator rods are disposed at least on one diagonal line of a fuel assembly and P-fuel rods are arranged at a position put between the moderator rods. This makes it possible to reactor shutdown and makes the axial power distribution satisfactory even if the fuel enrichment degree is increased. (T.M.)

  12. Fuel assembly

    International Nuclear Information System (INIS)

    Bando, Masaru.

    1993-01-01

    As neutron irradiation progresses on a fuel assembly of an FBR type reactor, a strong force is exerted to cause ruptures if the arrangement of fuel elements is not displaced, whereas the fuel elements may be brought into direct contact with each other not by way of spacers to cause burning damages if the arrangement is displaced. In the present invention, the circumference of fuel elements arranged in a normal triangle lattice is surrounded by a wrapper tube having a hexagonal cross section, wire spacers are wound therearound, and deformable spacers are distributed to optional positions for fuel elements in the wrapper tube. Interaction between the fuel elements caused by irradiation is effectively absorbed, thereby enabling to delay the occurrence of the rupture and burning damages of the elements. (N.H.)

  13. Fuel assembly

    International Nuclear Information System (INIS)

    Ueda, Makoto.

    1991-01-01

    In a fuel assembly in which spectral shift type moderator guide members are arranged, the moderator guide member has a flow channel resistance member, that provides flow resistance against the moderators, in the upstream of a moderator flowing channel, by which the ratio of removing coolants is set greater at the upstream than downstream. With such a constitution, the void distribution increasing upward in the channel box except for the portion of the moderator guide member is moderated by the increase of the area of the void region that expands downward in the guide member. Accordingly, the axial power distribution is flattened throughout the operation cycle and excess distortion is eliminated to improve the fuel integrity. (T.M.)

  14. Fuel assembly

    International Nuclear Information System (INIS)

    Wataumi, Kazutoshi; Tajiri, Hiroshi.

    1992-01-01

    In a fuel assembly of a BWR type reactor, a pellet to be loaded comprises an external layer of fissile materials containing burnable poisons and an internal layer of fissile materials not containing burnable poison. For example, there is provided a dual type pellet comprising an external layer made of UO 2 incorporated with Gd 2 O 3 at a predetermined concentration as the burnable poisons and an internal layer made of UO 2 not containing Gd 2 O 3 . The amount of the burnable poisons required for predetermined places is controlled by the thickness of the ring of the external layer. This can dissipate an unnecessary poisoning effect at the final stage of the combustion cycle. Further, since only one or a few kinds of powder mixture of the burnable poisons and the fissile materials is necessary, production and product control can be facilitated. (I.N.)

  15. Fuel assembly

    International Nuclear Information System (INIS)

    Ishibashi, Yoko; Aoyama, Motoo; Oyama, Jun-ichi.

    1995-01-01

    Burnable poison-incorporating fuel rods of a first group are disposed in a region in adjacent with a water rod having a large diameter (neutron moderator rod) disposed to the central portion of a fuel assembly. Burnable poison-incorporating fuel rods of a second group are disposed to a region other than peripheral zone in adjacent with a channel box and corners positioned at an inner zone, in adjacent with the channel box. The average concentration of burnable poisons of the burnable poison-incorporating fuel rods of the first group is made greater than that of the second group. With such a constitution, when the burnable poisons of the first group are burnt out, the burnable poisons of the second group are also burnt out at the same time. Accordingly, an amount of burnable poisons left unburnt at the final stage of the operation cycle is reduced, to improve the reactivity. This can improve the economical property. (I.N.)

  16. Fuel assemblies

    International Nuclear Information System (INIS)

    Yoshioka, Ritsuo.

    1983-01-01

    Purpose: To improve the operation performance of a BWR type reactor by improving the distribution of the uranium enrichment and the incorporation amount of burnable poisons in fuel assemblies. Constitution: The average enrichment of uranium 235 is increased in the upper portion as compared with that in the lower portion, while the incorporation amount of burnable poisons is increased in an upper portion as compared with that in the lower portion. The difference in the incorporation amount of the burnable poisons between the upper and lower portions is attained by charging two kinds of fuel rods; the ones incorporated with the burnable poisons over the entire length and the others incorporated with the burnable poisons only in the upper portions. (Seki, T.)

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

    Science.gov (United States)

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

    2010-07-01

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

  18. Differential Impacts of Soybean and Fish Oils on Hepatocyte Lipid Droplet Accumulation and Endoplasmic Reticulum Stress in Primary Rabbit Hepatocytes

    OpenAIRE

    Zhu, Xueping; Xiao, Zhihui; Xu, Yumin; Zhao, Xingli; Cheng, Ping; Cui, Ningxun; Cui, Mingling; Li, Jie; Zhu, Xiaoli

    2016-01-01

    Parenteral nutrition-associated liver disease (PNALD) is a severe ailment associated with long-term parenteral nutrition. Soybean oil-based lipid emulsions (SOLE) are thought to promote PNALD development, whereas fish oil-based lipid emulsions (FOLE) are thought to protect against PNALD. This study aimed to investigate the effects of SOLE and FOLE on primary rabbit hepatocytes. The results reveal that SOLE caused significant endoplasmic reticulum (ER) and mitochondrial damage, ultimately resu...

  19. Differential Impacts of Soybean and Fish Oils on Hepatocyte Lipid Droplet Accumulation and Endoplasmic Reticulum Stress in Primary Rabbit Hepatocytes

    Directory of Open Access Journals (Sweden)

    Xueping Zhu

    2016-01-01

    Full Text Available Parenteral nutrition-associated liver disease (PNALD is a severe ailment associated with long-term parenteral nutrition. Soybean oil-based lipid emulsions (SOLE are thought to promote PNALD development, whereas fish oil-based lipid emulsions (FOLE are thought to protect against PNALD. This study aimed to investigate the effects of SOLE and FOLE on primary rabbit hepatocytes. The results reveal that SOLE caused significant endoplasmic reticulum (ER and mitochondrial damage, ultimately resulting in lipid droplets accumulation and ER stress. While these deleterious events induce hepatocyte injury, FOLE at high doses cause only minor ER and mitochondrial damage, which has no effect on hepatic function. SOLE also significantly upregulated glucose-regulated protein 94 mRNA and protein expression. These data indicate that SOLE, but not FOLE, damage the ER and mitochondria, resulting in lipid droplets accumulation and ER stress and, finally, hepatocyte injury. This likely contributes to the differential impacts of SOLE and FOLE on PNALD development and progression.

  20. Hepatocyte and keratinocyte growth factors and their receptors in human lung emphysema

    Directory of Open Access Journals (Sweden)

    Marchal Joëlle

    2005-10-01

    Full Text Available Abstract Background Hepatocyte and keratinocyte growth factors are key growth factors in the process of alveolar repair. We hypothesized that excessive alveolar destruction observed in lung emphysema involves impaired expression of hepatocyte and keratinocyte growth factors or their respective receptors, c-met and keratinocyte growth factor receptor. The aim of our study was to compare the expression of hepatocyte and keratinocyte growth factors and their receptors in lung samples from 3 groups of patients: emphysema; smokers without emphysema and non-smokers without emphysema. Methods Hepatocyte and keratinocyte growth factor proteins were analysed by immunoassay and western blot; mRNA expression was measured by real time quantitative polymerase chain reaction. Results Hepatocyte and keratinocyte growth factors, c-met and keratinocyte growth factor receptor mRNA levels were similar in emphysema and non-emphysema patients. Hepatocyte growth factor mRNA correlated negatively with FEV1 and the FEV1/FVC ratio both in emphysema patients and in smokers with or without emphysema. Hepatocyte and keratinocyte growth factor protein concentrations were similar in all patients' groups. Conclusion The expression of hepatocyte and keratinocyte growth factors and their receptors is preserved in patients with lung emphysema as compared to patients without emphysema. Hepatocyte growth factor mRNA correlates with the severity of airflow obstruction in smokers.

  1. DNA synthesis in periportal and perivenous hepatocytes of intact and hepatectomized young mice.

    Science.gov (United States)

    Fernández-Blanco, A; Inda, A M; Errecalde, A L

    2015-01-01

    DNA synthesis of hepatocytes in two areas of Intact and Hepatectomized young mice liver along a circadian period was studied. DNA synthesis was significantly different at all analyzed time points in Intact and Hepatectomized animals. Differences between periportal and perivenous hepatocytes were found in hepatectomized animals at 04/42 and 08/46 hr of day/hour post-hepatectomy. DNAs peak in periportal hepatocytes regenerating liver occurs 4 hr earlier than in perivenous hepatocytes, probably reflecting their shorter G1 phase. Besides, daily mean values of regenerating livers were higher than those observed in Intact animals, as a consequence of surgical removal.

  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. Lack of direct mitogenic activity of dichloroacetate and trichloroacetate in cultured rat hepatocytes

    International Nuclear Information System (INIS)

    Walgren, Jennie L.; Kurtz, David T.; McMillan, JoEllyn M.

    2005-01-01

    Dichloroacetate (DCA) and trichloroacetate (TCA) are hepatocarcinogenic metabolites of the common groundwater contaminant, 1,1,2-trichloroethylene. DCA and TCA have been shown to induce hepatocyte proliferation in vivo, but it is not known if this response is the result of direct mitogenic activity or whether cell replication occurs indirectly in response to tissue injury or inflammation. In this study we used primary cultures of rat hepatocytes, a species susceptible to DCA- but not TCA-induced hepatocarcinogenesis, to determine whether DCA and TCA are direct hepatocyte mitogens. Rat hepatocytes, cultured in growth factor-free medium, were treated with 0.01-1.0 mM DCA or TCA for 10-40 h; cell replication was then assessed by measuring incorporation of 3 H-thymidine into DNA and by cell counts. DCA or TCA treatment did not alter 3 H-thymidine incorporation in the cultured hepatocytes. Although an increase in cell number was not observed, DCA treatment significantly abrogated the normal background cell loss, suggesting an ability to inhibit apoptotic cell death in primary hepatocyte cultures. Furthermore, treatment with DCA synergistically enhanced the mitogenic response to epidermal growth factor. The data indicate that DCA and TCA are not direct mitogens in hepatocyte cultures, which is of interest in view of their ability to stimulate hepatocyte replication in vivo. Nevertheless, the synergistic enhancement of epidermal growth factor-induced hepatocyte replication by DCA is of particular interest and warrants further study

  4. Gel entrapment culture of rat hepatocytes for investigation of tetracycline-induced toxicity

    International Nuclear Information System (INIS)

    Shen Chong; Meng Qin; Schmelzer, Eva; Bader, Augustinus

    2009-01-01

    This paper aimed to explore three-dimensionally cultured hepatocytes for testing drug-induced nonalcoholic steatohepatitis. Gel entrapped rat hepatocytes were applied for investigation of the tetracycline-induced steatohepatitis, while hepatocyte monolayer was set as a control. The toxic responses of hepatocytes were systematically evaluated by measuring cell viability, liver-specific function, lipid accumulation, oxidative stress, adenosine triphosphate content and mitochondrial membrane potential. The results suggested that gel entrapped hepatocytes showed cell death after 96 h of tetracycline treatment at 25 μM which is equivalent to toxic serum concentration in rats, while hepatocyte monolayer showed cell death at a high dose of 200 μM. The concentration-dependent accumulation of lipid as well as mitochondrial damage were regarded as two early events for tetracycline hepatotoxicity in gel entrapment culture due to their detectability ahead of subsequent increase of oxidative stress and a final cell death. Furthermore, the potent protection of fenofibrate and fructose-1,6-diphosphate were evidenced in only gel entrapment culture with higher expressions on the genes related to β-oxidation than hepatocyte monolayer, suggesting the mediation of lipid metabolism and mitochondrial damage in tetracycline toxicity. Overall, gel entrapped hepatocytes in three-dimension reflected more of the tetracycline toxicity in vivo than hepatocyte monolayer and thus was suggested as a more relevant system for evaluating steatogenic drugs.

  5. Organic anion transporting polypeptide 1B transporters modulate hydroxyurea pharmacokinetics.

    Science.gov (United States)

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

    2013-12-15

    Hydroxyurea is currently the only FDA-approved drug that ameliorates the pathophysiology of sickle cell anemia. Unfortunately, substantial interpatient variability in the pharmacokinetics (PK) of hydroxyurea may result in variation of the drug's efficacy. However, little is known about mechanisms that modulate hydroxyurea PK. Recent in vitro studies identifying hydroxyurea as a substrate for organic anion transporting polypeptide (OATP1B) transporters prompted the current investigation assessing the role of OATP1B transporters in modulating hydroxyurea PK. Using wild-type and Oatp1b knockout (Oatp1b(-/-)) mice, hydroxyurea PK was analyzed in vivo by measuring [(14)C]hydroxyurea distribution in plasma, kidney, liver, urine, or the exhaled (14)CO2 metabolite. Plasma levels were significantly reduced by 20% in Oatp1b(-/-) mice compared with wild-type (area under the curve of 38.64 or 48.45 μg·h(-1)·ml(-1), respectively) after oral administration, whereas no difference was observed between groups following intravenous administration. Accumulation in the kidney was significantly decreased by twofold in Oatp1b(-/-) mice (356.9 vs. 748.1 pmol/g), which correlated with a significant decrease in urinary excretion. Hydroxyurea accumulation in the liver was also decreased (136.6 vs. 107.3 pmol/g in wild-type or Oatp1b(-/-) mice, respectively) correlating with a decrease in exhaled (14)CO2. These findings illustrate that deficiency of Oatp1b transporters alters the absorption, distribution, and elimination of hydroxyurea thus providing the first in vivo evidence that cell membrane transporters may play a significant role in modulating hydroxyurea PK. Future studies to investigate other transporters and their role in hydroxyurea disposition are warranted for understanding the sources of variation in hydroxyurea's PK.

  6. Quantifying polypeptide conformational space: sensitivity to conformation and ensemble definition.

    Science.gov (United States)

    Sullivan, David C; Lim, Carmay

    2006-08-24

    Quantifying the density of conformations over phase space (the conformational distribution) is needed to model important macromolecular processes such as protein folding. In this work, we quantify the conformational distribution for a simple polypeptide (N-mer polyalanine) using the cumulative distribution function (CDF), which gives the probability that two randomly selected conformations are separated by less than a "conformational" distance and whose inverse gives conformation counts as a function of conformational radius. An important finding is that the conformation counts obtained by the CDF inverse depend critically on the assignment of a conformation's distance span and the ensemble (e.g., unfolded state model): varying ensemble and conformation definition (1 --> 2 A) varies the CDF-based conformation counts for Ala(50) from 10(11) to 10(69). In particular, relatively short molecular dynamics (MD) relaxation of Ala(50)'s random-walk ensemble reduces the number of conformers from 10(55) to 10(14) (using a 1 A root-mean-square-deviation radius conformation definition) pointing to potential disconnections in comparing the results from simplified models of unfolded proteins with those from all-atom MD simulations. Explicit waters are found to roughen the landscape considerably. Under some common conformation definitions, the results herein provide (i) an upper limit to the number of accessible conformations that compose unfolded states of proteins, (ii) the optimal clustering radius/conformation radius for counting conformations for a given energy and solvent model, (iii) a means of comparing various studies, and (iv) an assessment of the applicability of random search in protein folding.

  7. Magnetic cell labeling of primary and stem cell-derived pig hepatocytes for MRI-based cell tracking of hepatocyte transplantation.

    Directory of Open Access Journals (Sweden)

    Dwayne R Roach

    Full Text Available Pig hepatocytes are an important investigational tool for optimizing hepatocyte transplantation schemes in both allogeneic and xenogeneic transplant scenarios. MRI can be used to serially monitor the transplanted cells, but only if the hepatocytes can be labeled with a magnetic particle. In this work, we describe culture conditions for magnetic cell labeling of cells from two different pig hepatocyte cell sources; primary pig hepatocytes (ppHEP and stem cell-derived hepatocytes (PICM-19FF. The magnetic particle is a micron-sized iron oxide particle (MPIO that has been extensively studied for magnetic cell labeling for MRI-based cell tracking. ppHEP could endocytose MPIO with labeling percentages as high as 70%, achieving iron content as high as ~55 pg/cell, with >75% viability. PICM-19FF had labeling >97%, achieving iron content ~38 pg/cell, with viability >99%. Extensive morphological and functional assays indicated that magnetic cell labeling was benign to the cells. The results encourage the use of MRI-based cell tracking for the development and clinical use of hepatocyte transplantation methodologies. Further, these results generally highlight the importance of functional cell assays in the evaluation of contrast agent biocompatibility.

  8. Fuel assembly

    International Nuclear Information System (INIS)

    Hirukawa, Koji; Sakurada, Koichi.

    1992-01-01

    In a fuel assembly for a BWR type reactor, water rods or water crosses are disposed between fuel rods, and a value with a spring is disposed at the top of the coolant flow channel thereof, which opens a discharge port when pressure is increased to greater than a predetermined value. Further, a control element for the amount of coolant flow rate is inserted retractable to a control element guide tube formed at the lower portion of the water rod or the water cross. When the amount of control elements inserted to the control element guide tube is small and the inflown coolant flow rate is great, the void coefficient at the inside of the water rod is less than 5%. On the other hand, when the control elements are inserted, the flow resistance is increased, so that the void coefficient in the water rod is greater than 80%. When the pressure in the water rod is increased, the valve with the spring is raised to escape water or steams. Then, since the variation range of the change of the void coefficient can be controlled reliably by the amount of the control elements inserted, and nuclear fuel materials can be utilized effectively. (N.H.)

  9. Fuel assembly

    International Nuclear Information System (INIS)

    Hiraiwa, Koji; Ueda, Makoto

    1989-01-01

    In a fuel assembly used for a light water cooled reactor such as a BWR type reactor, a water rod is divided axially into an upper outer tube and a lower outer tube by means of a plug disposed from the lower end of a water rod to a position 1/4 - 1/2 of the entire length for the water rod. Inlet apertures and exit apertures for moderators are respectively perforated for the divided outer tube and upper and lower portions. Further, an upper inner tube with less neutron irradiation growing amount than the outer tube is perforated on the plug in the outer tube, while a lower inner tube with greater neutron irradiation growing amount than the outer tube is suspended from the lower surface of the plug in the outer tube. Then, the opening area for the exit apertures disposed to the upper outer tube and the lower outer tube is controlled depending on the difference of the neutron irradiation growing amount between the upper inner tube and the upper outer tube, and the difference of the neutron irradiation growing amount between the lower inner tube and the lower outer tube. This enables effective spectral shift operation and improve the fuel economy. (T.M.)

  10. Fuel assembly

    International Nuclear Information System (INIS)

    Yamazaki, Hajime.

    1995-01-01

    In a fuel assembly having fuel rods of different length, fuel pellets of mixed oxides of uranium and plutonium are loaded to a short fuel rod. The volume ratio of a pellet-loaded portion to a plenum portion of the short fuel rod is made greater than the volume ratio of a fuel rod to which uranium fuel pellets are loaded. In addition, the volume of the plenum portion of the short fuel rod is set greater depending on the plutonium content in the loaded fuel pellets. MOX fuel pellets are loaded on the short fuel rods having a greater degree of freedom relevant to the setting for the volume of the plenum portion compared with that of a long rod fuel, and the volume of the plenum portion is ensured greater depending on the plutonium content. Even if a large amount of FP gas and He gas are discharged from the MOX fuels compared with that from the uranium fuels, the internal pressure of the MOX fuel rod during operation is maintained substantially identical with that of the uranium fuel rod, so that a risk of generating excess stresses applied to the fuel cladding tubes and rupture of fuels are greatly reduced. (N.H.)

  11. Fuel assembly

    International Nuclear Information System (INIS)

    Nakajima, Akiyoshi; Bessho, Yasunori; Aoyama, Motoo; Koyama, Jun-ichi; Hirakawa, Hiromasa; Yamashita, Jun-ichi; Hayashi, Tatsuo

    1998-01-01

    In a fuel assembly of a BWR type reactor in which a water rod of a large diameter is disposed at the central portion, the cross sectional area perpendicular to the axial direction comprises a region a of a fuel rod group facing to a wide gap water region to which a control rod is inserted, a region b of a fuel rod group disposed on the side of the wide gap water region other than the region a, a region d of a fuel rod group facing to a narrow gap water region and a region c of a fuel rod group disposed on the side of the narrow gap water region other than the region d. When comparing an amount of fission products contained in the four regions relative to that in the entire regions and average enrichment degrees of fuel rods for the four regions, the relative amount and the average enrichment degree of the fuel rod group of the region a is minimized, and the relative amount and the average enrichment degree of the fuel rod group in the region b is maximized. Then, reactor shut down margin during cold operation can be improved while flattening the power in the cross section perpendicular to the axial direction. (N.H.)

  12. Biomimetic self-assembly of a functional asymmetrical electronic device.

    Science.gov (United States)

    Boncheva, Mila; Gracias, David H; Jacobs, Heiko O; Whitesides, George M

    2002-04-16

    This paper introduces a biomimetic strategy for the fabrication of asymmetrical, three-dimensional electronic devices modeled on the folding of a chain of polypeptide structural motifs into a globular protein. Millimeter-size polyhedra-patterned with logic devices, wires, and solder dots-were connected in a linear string by using flexible wire. On self-assembly, the string folded spontaneously into two domains: one functioned as a ring oscillator, and the other one as a shift register. This example demonstrates that biomimetic principles of design and self-organization can be applied to generate multifunctional electronic systems of complex, three-dimensional architecture.

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

    International Nuclear Information System (INIS)

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

    1990-01-01

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

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

    Science.gov (United States)

    Kozlov, Sergey; Grishin, Eugene

    2011-01-31

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

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

    Directory of Open Access Journals (Sweden)

    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.

  16. Manganese Transport and Toxicity in Polarized WIF-B Hepatocytes.

    Science.gov (United States)

    Thompson, Khristy J; Hein, Jennifer; Baez, Andrew; Sosa, Jose Carlo; Wessling-Resnick, Marianne

    2018-05-24

    Mn toxicity arises from nutritional problems, community and occupational exposures, and genetic risks. Mn blood levels are controlled by hepatobiliary clearance. The goals of this study were to determine the cellular distribution of Mn transporters in polarized hepatocytes, to establish an in vitro assay for hepatocyte Mn efflux, and to examine possible roles the Mn transporters would play in metal import and export. For these experiments, hepatocytoma WIF-B cells were grown for 12-14 days to achieve maximal polarity. Immunoblots showed that Mn transporters ZIP8, ZnT10, ferroportin (Fpn), and ZIP14 were present. Indirect immunofluorescence microscopy localized Fpn and ZIP14 to WIF-B cell basolateral domains while ZnT10 and ZIP8 associated with intracellular vesicular compartments. ZIP8-positive structures were distributed uniformly throughout the cytoplasm, but ZnT10-positive vesicles were adjacent to apical bile compartments. WIF-B cells were sensitive to Mn toxicity, showing decreased viability after 16 h exposure to > 250 M MnCl2. However, the hepatocytes were resistant to 4 h exposures of up to 500 M MnCl2 despite 50-fold increased Mn content. Washout experiments showed time-dependent efflux with 80% Mn released after a 4 h chase period. Hepcidin reduced levels of Fpn in WIF-B cells, clearing Fpn from the cell surface, but Mn efflux was unaffected. The secretory inhibitor brefeldin A did block release of Mn from WIF-B cells, suggesting vesicle fusion may be involved in export. These results point to a possible role of ZnT10 to import Mn into vesicles that subsequently fuse with the apical membrane and empty their contents into bile.

  17. 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.

  18. 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.

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

    Science.gov (United States)

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

    2016-10-01

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

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

    Science.gov (United States)

    Munegumi, Toratane; Tanikawa, Naoya

    2017-09-01

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

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

    Science.gov (United States)

    Munegumi, Toratane; Tanikawa, Naoya

    2017-09-01

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

  2. Avian leukosis virus is a versatile eukaryotic platform for polypeptide display

    International Nuclear Information System (INIS)

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

    2003-01-01

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

  3. 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

  4. 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.

  5. High throughput micro-well generation of hepatocyte micro-aggregates for tissue engineering.

    Directory of Open Access Journals (Sweden)

    Elien Gevaert

    Full Text Available The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the high throughput generation of hepatocyte aggregates, uniform in size. In our study we observed that aggregation of hepatocytes had a beneficial effect on the expression of certain hepatocyte specific markers. Moreover we observed that the beneficial effect was dependent on the aggregate dimensions, indicating that aggregate parameters should be carefully considered. In a second part of the study, the selected aggregates were immobilized by encapsulation in methacrylamide-modified gelatin. Phenotype evaluations revealed that a stable hepatocyte phenotype could be maintained during 21 days when encapsulated in the hydrogel. In conclusion we have demonstrated the beneficial use of micro-well chips for hepatocyte aggregation and the size-dependent effects on hepatocyte phenotype. We also pointed out that methacrylamide-modified gelatin is suitable for the encapsulation of these aggregates.

  6. High Throughput Micro-Well Generation of Hepatocyte Micro-Aggregates for Tissue Engineering

    NARCIS (Netherlands)

    Gevaert, Elien; Dollé, Laurent; Billiet, Thomas; Dubruel, Peter; van Grunsven, Leo; van Apeldoorn, Aart A.; Cornelissen, Ria

    2014-01-01

    The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the

  7. High efficient differentiation of functional hepatocytes from porcine induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Ying Ao

    Full Text Available Hepatocyte transplantation is considered to be a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs provide an unlimited source for the generation of functional hepatocytes. In this study, we generated iPSCs from porcine ear fibroblasts (PEFs by overexpressing Sox2, Klf4, Oct4, and c-Myc (SKOM, and developed a novel strategy for the efficient differentiation of hepatocyte-like cells from porcine iPSCs by following the processes of early liver development. The differentiated cells displayed the phenotypes of hepatocytes, exhibited classic hepatocyte-associated bio-functions, such as LDL uptake, glycogen storage and urea secretion, as well as possessed the metabolic activities of cytochrome P-450 (CYP 3A and 2C. Furthermore, we compared the hepatocyte differentiation efficacy of our protocol with another published method, and the results demonstrated that our differentiation strategy could significantly improve the generation of morphological and functional hepatocyte-like cells from porcine iPSCs. In conclusion, this study establishes an efficient method for in vitro generation of functional hepatocytes from porcine iPSCs, which could represent a promising cell source for preclinical testing of cell-based therapeutics for liver failure and for pharmacological applications.

  8. Integrin-linked kinase is involved in matrix-induced hepatocyte differentiation

    International Nuclear Information System (INIS)

    Gkretsi, Vasiliki; Bowen, William C.; Yang, Yu; Wu, Chuanyue; Michalopoulos, George K.

    2007-01-01

    Hepatocytes have restricted proliferative capacity in culture and when cultured without matrix, lose the hepatocyte-specific gene expression and characteristic cellular micro-architecture. Overlay of matrix-preparations on de-differentiated hepatocytes restores differentiation. Integrin-linked kinase (ILK) is a cell-matrix-adhesion protein crucial in fundamental processes such as differentiation and survival. In this study, we investigated the role of ILK, and its binding partners PINCH, α-parvin, and Mig-2 in matrix-induced hepatocyte differentiation. We report here that ILK is present in the liver and localizes at cell-matrix adhesions of cultured hepatocytes. We also show that ILK, PINCH, α-parvin, and Mig-2 expression level is dramatically reduced in the re-differentiated hepatocytes. Interestingly, hepatocytes lacking ILK undergo matrix-induced differentiation but their differentiation is incomplete, as judged by monitoring cell morphology and production of albumin. Our results show that ILK and cell-matrix adhesion proteins play an important role in the process of matrix-induced hepatocyte differentiation

  9. High throughput micro-well generation of hepatocyte micro-aggregates for tissue engineering.

    Science.gov (United States)

    Gevaert, Elien; Dollé, Laurent; Billiet, Thomas; Dubruel, Peter; van Grunsven, Leo; van Apeldoorn, Aart; Cornelissen, Ria

    2014-01-01

    The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the high throughput generation of hepatocyte aggregates, uniform in size. In our study we observed that aggregation of hepatocytes had a beneficial effect on the expression of certain hepatocyte specific markers. Moreover we observed that the beneficial effect was dependent on the aggregate dimensions, indicating that aggregate parameters should be carefully considered. In a second part of the study, the selected aggregates were immobilized by encapsulation in methacrylamide-modified gelatin. Phenotype evaluations revealed that a stable hepatocyte phenotype could be maintained during 21 days when encapsulated in the hydrogel. In conclusion we have demonstrated the beneficial use of micro-well chips for hepatocyte aggregation and the size-dependent effects on hepatocyte phenotype. We also pointed out that methacrylamide-modified gelatin is suitable for the encapsulation of these aggregates.

  10. Computational Topology Based Quantification Of Hepatocytes Nuclei In Lipopolysaccharide-Induced Liver Injury In Mice

    Directory of Open Access Journals (Sweden)

    Rodrigo Rojas Moraleda

    2016-06-01

    The computational topology approach proposed successfully detected hepatocyte cells under several natural variations. We evaluated on a per-pixel basis how the segmentation performs on: i all nuclei in the images, ii big round nuclei considered belonging to hepatocytes cells (accuracy 87.2%, recall 80.3%, and iii nuclei regarded to non-parenchymal cells.  

  11. Extracellular matrix components influence DNA synthesis of rat hepatocytes in primary culture

    International Nuclear Information System (INIS)

    Sawada, N.; Tomomura, A.; Sattler, C.A.; Sattler, G.L.; Kleinman, H.K.; Pitot, H.C.

    1986-01-01

    The effects of several extracellular matrix components (EMCs) - fibronectin (Fn), laminin (Ln), type I (C-I) and type IV (C-IV) collagen - on DNA synthesis in rat hepatocytes in primary culture were examined by both quantitative scintillation spectrometry and autoradiography of [ 3 H]thymidine incorporation. Hepatocytes cultured on Fn showed the most active DNA synthesis initiated by epidermal growth factor (EGF) with decreasing levels of [ 3 H]thymidine uptake exhibited in the cell cultured on C-IV, C-I, and Ln, respectively. The decreasing level of DNA synthesis in hepatocytes cultured on Fn, C-IV, C-I, and Ln respectively was not influenced by cell density. The number of EGF receptors of hepatocytes was also not influenced by EMCs. These data suggest that EMCs modify hepatocyte DNA synthesis by means of post-EGF-receptor mechanisms which are regulated by both growth factors and cell density

  12. The role of hepatocyte nuclear factor 4 alpha in development and progression of liver diseases

    Directory of Open Access Journals (Sweden)

    YANG Jinlian

    2016-02-01

    Full Text Available Hepatocyte nuclear factor 4 alpha (HNF4α, a member of the nuclear receptor superfamily, has a high expression level in mature hepatocytes. HNF4α can regulate hepatocyte-specific gene expression at a transcriptional level, promote hepatocyte development and differentiation, participate in establishment and maintenance of hepatocyte polarity, and enhance the synthetic, metabolic, and detoxifying functions of the liver. Through inhibiting the activation of hepatic stellate cells, reversing epithelial-mesenchymal transition, and inhibiting the proliferation, invasion, and metastasis of hepatoma cells, HNF4α may be involved in the development and progression of various liver diseases including liver fibrosis, liver cirrhosis, and hepatocellular carcinoma. This paper elaborates on the biological functions of HNF4α, and summarizes and analyzes the research advances in the mechanisms of action of HNF4α in the pathological process of liver diseases, in order to provide references for further investigation of the potential targeted therapies for liver diseases.

  13. Galactosylated DNA lipid nanocapsules for efficient hepatocyte targeting.

    Science.gov (United States)

    Morille, M; Passirani, C; Letrou-Bonneval, E; Benoit, J-P; Pitard, B

    2009-09-11

    The main objective of gene therapy via a systemic pathway is the development of a stable and non-toxic gene vector that can encapsulate and deliver foreign genetic materials into specific cell types with the transfection efficiency of viral vectors. With this objective, DNA complexed with cationic lipids of DOTAP/DOPE was encapsulated into lipid nanocapsules (LNCs) forming nanocarriers (DNA LNCs) with a size suitable for systemic injection (109+/-6 nm). With the goal of increasing systemic delivery, LNCs were stabilised with long chains of poly(ethylene glycol) (PEG), either from a PEG lipid derivative (DSPE-mPEG(2000)) or from an amphiphilic block copolymer (F108). In order to overcome internalisation difficulties encountered with PEG shield, a specific ligand (galactose) was covalently added at the distal end of the PEG chains, in order to provide active targeting of the asialoglycoprotein-receptor present on hepatocytes. This study showed that DNA LNCs were as efficient as positively charged DOTAP/DOPE lipoplexes for transfection. In primary hepatocytes, when non-galactosylated, the two polymers significantly decreased the transfection, probably by creating a barrier around the DNA LNCs. Interestingly, galactosylated F108 coated DNA LNCs led to a 18-fold increase in luciferase expression compared to non-galactosylated ones.

  14. Study of Valproic Acid-Enhanced Hepatocyte Steatosis

    Science.gov (United States)

    Chang, Renin; Chou, Mei-Chia; Hung, Li-Ying; Wang, Mu-En; Hsu, Meng-Chieh; Chiu, Chih-Hsien

    2016-01-01

    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. PMID:27034954

  15. Study of Valproic Acid-Enhanced Hepatocyte Steatosis

    Directory of Open Access Journals (Sweden)

    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.

  16. Transport of heparan sulfate into the nuclei of hepatocytes

    International Nuclear Information System (INIS)

    Ishihara, M.; Fedarko, N.S.; Conrad, H.E.

    1986-01-01

    A rat hepatocyte cell line which accumulates free heparan sulfate (HS) chains enriched in GlcA-2-SO 4 residues in the nucleus was labeled with 35 SO 4 2- and the rate of appearance of [ 35 SO 4 ]HS in the nucleus was measured. [ 35 SO 4 ]HS began to accumulate in the nucleus 2 h after the addition of 35 SO 42- and reached a steady state level after 20 h. HS was lost from the nuclei of prelabeled cells with a t/sub 1/2/ of 8 h. Chloroquine did not inhibit the transport of HS into the nucleus, but increased the t/sub 1/2/ for the exit of HS from the nucleus to 20 h. At both 37 0 C and 16 0 C exogenous [ 35 SO 4 ]proteoHS was taken up by the cells and converted to free chains and about 10% of the internalized [ 35 SO 4 ]HS was transported into the nucleus. The [ 35 SO 4 ]HS isolated from the nucleus was enriched in GlcA-2-SO 4 residues, whereas the [ 35 SO 4 ]HS remaining in the rest of the intra-cellular pool showed a corresponding depletion in GlcA-2-SO 4 residues. The results show that nuclear HS is derived from the pool of a secreted proteoHS and that metabolism of exogenous HS by hepatocytes does not involve lysosomal processing of the internalized HS

  17. Biotransformation of hydralazine (HDZ) in monolayer cultures of rabbit hepatocytes

    International Nuclear Information System (INIS)

    McQueen, C.A.; Rosado, R.R.

    1990-01-01

    Adverse reactions to HDZ have been associated with the acetylator polymorphism; slow acetylators are more likely to develop HDZ-induced lupus erythematosus. In studying the role of this polymorphism in susceptibility to HDZ toxicity, the biotransformation of HDZ was investigated in rabbit hepatocytes. New Zealand white rabbits, like humans, are classified as rapid or slow acetylators. Heptocytes were isolated from rapid acetylator rabbits by collagenase perfusion. Monolayer cultures were initiated and exposed to 14 C-HDZ. Since HDZ is unstable at neutral pH, parallel incubations were done in the absence of cells. Metabolites in the media were determined by reverse phase HPLC. Phthalazine (P), phthalazinone (PZ), triazoloph-thalazine (TP), methyl TP (MTP) and 3-hydroxy MTP were identified. In the absence of cells, more TP was formed than MTP, probably resulting from reaction of HDZ with components in the medium. In the presence of cells, there was a three-fold increase in MTP, while the amount of TP was relatively constant. Only trace amounts of P, PZ 3-hydroxy MTP were detected. These data indicate that monolayer cultures of rapid acetylator rabbit hepatocytes were capable of metabolizing HDZ with acetylation playing a major role. These studies are being extended to cells from slow acetylator rabbits

  18. Rapid and sensitive measure of gluconeogenesis in isolated bovine hepatocytes

    International Nuclear Information System (INIS)

    Azain, M.J.; Kasser, T.R.; Atwell, C.A.; Baile, C.A.

    1986-01-01

    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 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 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 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

  19. Stimulation of glucose phosphorylation by fructose in isolated rat hepatocytes.

    Science.gov (United States)

    Van Schaftingen, E; Vandercammen, A

    1989-01-15

    The phosphorylation of glucose was measured by the formation of [3H]H2O from [2-3H]glucose in suspensions of freshly isolated rat hepatocytes. Fructose (0.2 mM) stimulated 2-4-fold the rate of phosphorylation of 5 mM glucose although not of 40 mM glucose, thus increasing the apparent affinity of the glucose phosphorylating system. A half-maximal stimulatory effect was observed at about 50 microM fructose. Stimulation was maximal 5 min after addition of the ketose and was stable for at least 40 min, during which period 60% of the fructose was consumed. The effect of fructose was reversible upon removal of the ketose. Sorbitol and tagatose were as potent as fructose in stimulating the phosphorylation of 5 mM glucose. D-Glyceraldehyde also had a stimulatory effect but at tenfold higher concentrations. In contrast, dihydroxyacetone had no significant effect and glycerol inhibited the detritiation of glucose. Oleate did not affect the phosphorylation of glucose, even in the presence of fructose, although it stimulated the formation of ketone bodies severalfold, indicating that it was converted to its acyl-CoA derivative. These results allow the conclusion that fructose stimulates glucokinase in the intact hepatocyte. They also suggest that this effect is mediated through the formation of fructose 1-phosphate, which presumably interacts with a competitive inhibitor of glucokinase other than long-chain acyl-CoAs.

  20. Hepatocytes polyploidization and cell cycle control in liver physiopathology.

    Science.gov (United States)

    Gentric, Géraldine; Desdouets, Chantal; Celton-Morizur, Séverine

    2012-01-01

    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.

  1. Hepatocytes Polyploidization and Cell Cycle Control in Liver Physiopathology

    Directory of Open Access Journals (Sweden)

    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.

  2. 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.

  3. Analysis of urine composition in type Ⅱ diabetic mice after intervention therapy using holothurian polypeptides

    Science.gov (United States)

    Li, Yanyan; Xu, Jiajie; Su, Xiurong

    2017-07-01

    Hydrolysates and peptide fractions (PF) obtained from sea cucumber with commercial enzyme were studied on the hpyerglycemic and renal protective effects on db/db rats using urine metabolomics. Compared with the control group the polypeptides from the two species could significantly reduce the urine glucose and urea. We also tried to address the compositions of highly expressed urinary proteins using a proteomics approach. They were serum albumins, AMBP proteins, negative trypsin, elastase and urinary protein, GAPDH, a receptor of urokinase-type plasminogen activator (uPAR), and Ig kappa chain C region. We used the electronic nose to quickly detect changes in the volatile substances in mice urine after holothurian polypeptides fed, and the results show it can identify the difference between treatment groups with the control group without overlapping. The protein express mechanism of holothurian polypeptides treating diabetes was discussed, and we suggested these two peptides with the hypoglycemic and renal protective activity might be utilized as nutraceuticals.

  4. 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.

  5. Effect of oxygen on morphogenesis and polypeptide expression by Mucor racemosus

    International Nuclear Information System (INIS)

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

    1985-01-01

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

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

    Science.gov (United States)

    Capmany, G; Bolton, V N

    1999-09-01

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

  7. 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.

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

    Science.gov (United States)

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

    1985-01-16

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

  9. TGFbeta Induces Binucleation/Polyploidization in Hepatocytes through a Src-Dependent Cytokinesis Failure.

    Directory of Open Access Journals (Sweden)

    Marco De Santis Puzzonia

    Full Text Available In all mammals, the adult liver shows binucleated as well as mononucleated polyploid hepatocytes. The hepatic polyploidization starts after birth with an extensive hepatocyte binucleation and generates hepatocytes of several ploidy classes. While the functional significance of hepatocyte polyploidy is becoming clearer, how it is triggered and maintained needs to be clarified. Aim of this study was to identify a major inducer of hepatocyte binucleation/polyploidization and the cellular and molecular mechanisms involved. We found that, among several cytokines analyzed, known to be involved in early liver development and/or mass control, TGFbeta1 was capable to induce, together with the expected morphological changes, binucleation in hepatocytes in culture. Most importantly, the pharmacological inhibition of TGFbeta signaling in healthy mice during weaning, when the physiological binucleation occurs, induced a significant decrease of hepatocyte binucleation rate, without affecting cell proliferation and hepatic index. The TGFbeta-induced hepatocyte binucleation resulted from a cytokinesis failure, as assessed by video microscopy, and is associated with a delocalization of the cytokinesis regulator RhoA-GTPase from the mid-body of dividing cells. The use of specific chemical inhibitors demonstrated that the observed events are Src-dependent. Finally, the restoration of a fully epithelial phenotype by TGFbeta withdrawal gave rise to a cell progeny capable to maintain the polyploid state. In conclusion, we identified TGFbeta as a major inducer of hepatocyte binucleation both in vitro and in vivo, thus ascribing a novel role to this pleiotropic cytokine. The production of binucleated/tetraploid hepatocytes is due to a cytokinesis failure controlled by the molecular axis TGFbeta/Src/RhoA.

  10. TGFbeta Induces Binucleation/Polyploidization in Hepatocytes through a Src-Dependent Cytokinesis Failure.

    Science.gov (United States)

    De Santis Puzzonia, Marco; Cozzolino, Angela Maria; Grassi, Germana; Bisceglia, Francesca; Strippoli, Raffaele; Guarguaglini, Giulia; Citarella, Franca; Sacchetti, Benedetto; Tripodi, Marco; Marchetti, Alessandra; Amicone, Laura

    2016-01-01

    In all mammals, the adult liver shows binucleated as well as mononucleated polyploid hepatocytes. The hepatic polyploidization starts after birth with an extensive hepatocyte binucleation and generates hepatocytes of several ploidy classes. While the functional significance of hepatocyte polyploidy is becoming clearer, how it is triggered and maintained needs to be clarified. Aim of this study was to identify a major inducer of hepatocyte binucleation/polyploidization and the cellular and molecular mechanisms involved. We found that, among several cytokines analyzed, known to be involved in early liver development and/or mass control, TGFbeta1 was capable to induce, together with the expected morphological changes, binucleation in hepatocytes in culture. Most importantly, the pharmacological inhibition of TGFbeta signaling in healthy mice during weaning, when the physiological binucleation occurs, induced a significant decrease of hepatocyte binucleation rate, without affecting cell proliferation and hepatic index. The TGFbeta-induced hepatocyte binucleation resulted from a cytokinesis failure, as assessed by video microscopy, and is associated with a delocalization of the cytokinesis regulator RhoA-GTPase from the mid-body of dividing cells. The use of specific chemical inhibitors demonstrated that the observed events are Src-dependent. Finally, the restoration of a fully epithelial phenotype by TGFbeta withdrawal gave rise to a cell progeny capable to maintain the polyploid state. In conclusion, we identified TGFbeta as a major inducer of hepatocyte binucleation both in vitro and in vivo, thus ascribing a novel role to this pleiotropic cytokine. The production of binucleated/tetraploid hepatocytes is due to a cytokinesis failure controlled by the molecular axis TGFbeta/Src/RhoA.

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

    Science.gov (United States)

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

    2007-02-01

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

  12. 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.

  13. 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

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

  15. 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.

  16. Controlled cell morphology and liver-specific function of engineered primary hepatocytes by fibroblast layer cell densities.

    Science.gov (United States)

    Sakai, Yusuke; Koike, Makiko; Kawahara, Daisuke; Hasegawa, Hideko; Murai, Tomomi; Yamanouchi, Kosho; Soyama, Akihiko; Hidaka, Masaaki; Takatsuki, Mitsuhisa; Fujita, Fumihiko; Kuroki, Tamotsu; Eguchi, Susumu

    2018-03-05

    Engineered primary hepatocytes, including co-cultured hepatocyte sheets, are an attractive to basic scientific and clinical researchers because they maintain liver-specific functions, have reconstructed cell polarity, and have high transplantation efficiency. However, co-culture conditions regarding engineered primary hepatocytes were suboptimal in promoting these advantages. Here we report that the hepatocyte morphology and liver-specific function levels are controlled by the normal human diploid fibroblast (TIG-118 cell) layer cell density. Primary rat hepatocytes were plated onto TIG-118 cells, previously plated 3 days before at 1.04, 5.21, and 26.1×10 3  cells/cm 2 . Hepatocytes plated onto lower TIG-118 cell densities expanded better during the early culture period. The hepatocytes gathered as colonies and only exhibited small adhesion areas because of the pushing force from proliferating TIG-118 cells. The smaller areas of each hepatocyte result in the development of bile canaliculi. The highest density of TIG-118 cells downregulated albumin synthesis activity of hepatocytes. The hepatocytes may have undergone apoptosis associated with high TGF-β1 concentration and necrosis due to a lack of oxygen. These occurrences were supported by apoptotic chromatin condensation and high expression of both proteins HIF-1a and HIF-1b. Three types of engineered hepatocyte/fibroblast sheets comprising different TIG-118 cell densities were harvested after 4 days of hepatocyte culture and showed a complete cell sheet format without any holes. Hepatocyte morphology and liver-specific function levels are controlled by TIG-118 cell density, which helps to design better engineered hepatocytes for future applications such as in vitro cell-based assays and transplantable hepatocyte tissues. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  18. Tuning the conformation of synthetic co-polypeptides of serine and glutamic acid through control over polymer composition

    NARCIS (Netherlands)

    Canning, A.; Pasquazi, A.; Fijten, M.; Rajput, S.; Buttery, L.; Aylott, J.W.; Zelzer, M.

    2016-01-01

    Ring opening polymerization (ROP) of N-carboxy anhydride (NCA) amino acids presents a rapid way to synthesize high molecular weight polypeptides with different amino acid compositions. The compositional and functional versatility of polypeptides make these materials an attractive choice for

  19. 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

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

    Science.gov (United States)

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

    2016-01-01

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

  1. Transplant of Hepatocytes, Undifferentiated Mesenchymal Stem Cells, and In Vitro Hepatocyte-Differentiated Mesenchymal Stem Cells in a Chronic Liver Failure Experimental Model: A Comparative Study.

    Science.gov (United States)

    El Baz, Hanan; Demerdash, Zeinab; Kamel, Manal; Atta, Shimaa; Salah, Faten; Hassan, Salwa; Hammam, Olfat; Khalil, Heba; Meshaal, Safa; Raafat, Inas

    2018-02-01

    Liver transplant is the cornerstone line of treatment for chronic liver diseases; however, the long list of complications and obstacles stand against this operation. Searching for new modalities for treatment of chronic liver illness is a must. In the present research, we aimed to compare the effects of transplant of undifferentiated human mesenchymal stem cells, in vitro differentiated mesenchymal stem cells, and adult hepatocytes in an experimental model of chronic liver failure. Undifferentiated human cord blood mesenchymal stem cells were isolated, pro-pagated, and characterized by morphology, gene expression analysis, and flow cytometry of surface markers and in vitro differentiated into hepatocyte-like cells. Rat hepatocytes were isolated by double perfusion technique. An animal model of chronic liver failure was developed, and undifferentiated human cord blood mesenchymal stem cells, in vitro hepato-genically differentiated mesenchymal stem cells, or freshly isolated rat hepatocytes were transplanted into a CCL4 cirrhotic experimental model. Animals were killed 3 months after transplant, and liver functions and histopathology were assessed. Compared with the cirrhotic control group, the 3 cell-treated groups showed improved alanine aminotransferase, aspartate aminotransferase, albumin, and bilirubin levels, with best results shown in the hepatocyte-treated group. Histopathologic examination of the treated groups showed improved fibrosis, with best results obtained in the undifferentiated mesenchymal stem cell-treated group. Both adult hepatocytes and cord blood mesenchymal stem cells proved to be promising candidates for cell-based therapy in liver regeneration on an experimental level. Improved liver function was evident in the hepatocyte-treated group, and fibrosis control was more evident in the undifferentiated mesenchymal stem cell-treated group.

  2. 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.

  3. Sodium-independent, bicuculline-sensitive [3H]GABA binding to isolated rat hepatocytes

    International Nuclear Information System (INIS)

    Minuk, G.Y.; Bear, C.E.; Sarjeant, E.J.

    1987-01-01

    To determine whether hepatocytes possess specific receptor sites for gamma-aminobutyric acid (GABA), a potent amino acid neurotransmitter, [ 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 [ 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

  4. Dendritic cells and hepatocytes use distinct pathways to process protective antigen from plasmodium in vivo.

    Directory of Open Access Journals (Sweden)

    Ian A Cockburn

    2011-03-01

    Full Text Available Malaria-protective CD8+ T cells specific for the circumsporozoite (CS protein are primed by dendritic cells (DCs after sporozoite injection by infected mosquitoes. The primed cells then eliminate parasite liver stages after recognizing the CS epitopes presented by hepatocytes. To define the in vivo processing of CS by DCs and hepatocytes, we generated parasites carrying a mutant CS protein containing the H-2K(b epitope SIINFEKL, and evaluated the T cell response using transgenic and mutant mice. We determined that in both DCs and hepatocytes CS epitopes must reach the cytosol and use the TAP transporters to access the ER. Furthermore, we used endosomal mutant (3d and cytochrome c treated mice to address the role of cross-presentation in the priming and effector phases of the T cell response. We determined that in DCs, CS is cross-presented via endosomes while, conversely, in hepatocytes protein must be secreted directly into the cytosol. This suggests that the main targets of protective CD8+ T cells are parasite proteins exported to the hepatocyte cytosol. Surprisingly, however, secretion of the CS protein into hepatocytes was not dependent upon parasite-export (Pexel/VTS motifs in this protein. Together, these results indicate that the presentation of epitopes to CD8+ T cells follows distinct pathways in DCs when the immune response is induced and in hepatocytes during the effector phase.

  5. Human hepatocyte depletion in the presence of HIV-1 infection in dual reconstituted humanized mice

    Science.gov (United States)

    Wang, Weimin; Cheng, Yan; Makarov, Edward; Ganesan, Murali; Gebhart, Catherine L.; Gorantla, Santhi; Osna, Natalia

    2018-01-01

    ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infection impairs liver function, and liver diseases have become a leading cause of morbidity in infected patients. The immunopathology of liver damage caused by HIV-1 remains unclear. We used chimeric mice dually reconstituted with a human immune system and hepatocytes to address the relevance of the model to pathobiology questions related to human hepatocyte survival in the presence of systemic infection. TK-NOG males were transplanted with mismatched human hematopoietic stem/progenitor cells and hepatocytes, human albumin concentration and the presence of human immune cells in blood were monitored for hepatocytes and immune reconstitution, and mice were infected with HIV-1. HIV-1-infected animals showed a decline in human albumin concentration with a significant reduction in percentage of human hepatocytes compared to uninfected mice. The decrease in human albumin levels correlated with a decline in CD4+ cells in the liver and with an increase in HIV-1 viral load. HIV-1 infection elicited proinflammatory response in the immunological milieu of the liver in HIV-infected mice compared to uninfected animals, as determined by upregulation of IL23, CXCL10 and multiple toll-like receptor expression. The inflammatory reaction associated with HIV-1 infection in vivo could contribute to the depletion and dysfunction of hepatocytes. The dual reconstituted TK-NOG mouse model is a feasible platform to investigate hepatocyte-related HIV-1 immunopathogenesis. This article has an associated First Person interview with the first author of the paper. PMID:29361613

  6. Human hepatocyte depletion in the presence of HIV-1 infection in dual reconstituted humanized mice

    Directory of Open Access Journals (Sweden)

    Raghubendra Singh Dagur

    2018-02-01

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 infection impairs liver function, and liver diseases have become a leading cause of morbidity in infected patients. The immunopathology of liver damage caused by HIV-1 remains unclear. We used chimeric mice dually reconstituted with a human immune system and hepatocytes to address the relevance of the model to pathobiology questions related to human hepatocyte survival in the presence of systemic infection. TK-NOG males were transplanted with mismatched human hematopoietic stem/progenitor cells and hepatocytes, human albumin concentration and the presence of human immune cells in blood were monitored for hepatocytes and immune reconstitution, and mice were infected with HIV-1. HIV-1-infected animals showed a decline in human albumin concentration with a significant reduction in percentage of human hepatocytes compared to uninfected mice. The decrease in human albumin levels correlated with a decline in CD4+ cells in the liver and with an increase in HIV-1 viral load. HIV-1 infection elicited proinflammatory response in the immunological milieu of the liver in HIV-infected mice compared to uninfected animals, as determined by upregulation of IL23, CXCL10 and multiple toll-like receptor expression. The inflammatory reaction associated with HIV-1 infection in vivo could contribute to the depletion and dysfunction of hepatocytes. The dual reconstituted TK-NOG mouse model is a feasible platform to investigate hepatocyte-related HIV-1 immunopathogenesis. This article has an associated First Person interview with the first author of the paper.

  7. Cell therapy from bench to bedside: Hepatocytes from fibroblasts - the truth and myth of transdifferentiation.

    Science.gov (United States)

    Sanal, Madhusudana Girija

    2015-06-07

    Hepatocyte transplantation is an alternative to liver transplantation in certain disorders such as inherited liver diseases and liver failure. It is a relatively less complicated surgical procedure, and has the advantage that it can be repeated several times if unsuccessful. Another advantage is that hepatocytes can be isolated from partly damaged livers which are not suitable for liver transplantation. Despite these advantages hepatocyte transplantation is less popular. Important issues are poor engraftment of the transplanted cells and the scarcity of donor hepatocytes. Generation of "hepatocyte like cells"/iHeps from embryonic stem cells (ES) and induced pluripotent stem cells (iPSCs) by directed differentiation is an emerging solution to the latter issue. Direct conversation or trans-differentiation of fibroblasts to "hepatocyte like cells" is another way which is, being explored. However this method has several inherent and technical disadvantages compared to the directed differentiation from ES or iPSC. There are several methods claiming to be "highly efficient" for generating "highly functional" "hepatocyte like cells". Currently different groups are working independently and coming up with differentiation protocols and each group claiming an advantage for their protocol. Directed differentiation protocols need to be designed, compared, analyzed and tweaked systematically and logically than empirically. There is a need for a well-coordinated global initiative comparable to the Human Genome Project to achieve this goal in the near future.

  8. Comparison of para-aminophenol cytotoxicity in rat renal epithelial cells and hepatocytes.

    Science.gov (United States)

    Li, Ying; Bentzley, Catherine M; Tarloff, Joan B

    2005-04-01

    Several chemicals, including para-aminophenol (PAP), produce kidney damage in the absence of hepatic damage. Selective nephrotoxicity may be related to the ability of the kidney to reabsorb filtered water, thereby raising the intraluminal concentration of toxicants and exposing tubular epithelial cells to higher concentrations than would be present in other tissues. The present experiments tested the hypothesis that hepatocytes and renal epithelial cells exposed to equivalent concentrations of PAP would be equally susceptible to toxicity. Hepatocytes and renal epithelial cells were prepared by collagenase digestion of tissues obtained from female Sprague-Dawley rats. Toxicity was monitored using trypan blue exclusion, oxygen consumption and ATP content. We measured the rate of PAP clearance and formation of PAP-glutathione conjugate by HPLC. We found that renal epithelial cells accumulated trypan blue and showed declines in oxygen consumption and ATP content at significantly lower concentrations of PAP and at earlier time points than hepatocytes. The half-life of PAP in hepatocyte incubations was significantly shorter (0.71+/-0.07 h) than in renal epithelial cell incubations (1.33+/-0.23 h), suggesting that renal epithelial cells were exposed to PAP for longer time periods than hepatocytes. Renal epithelial cells formed significantly less glutathione conjugates of PAP (PAP-SG) than did hepatocytes, consistent with less efficient detoxification of reactive PAP intermediates by renal epithelial cells. Finally, hepatocytes contained significant more reduced glutathione (NPSH) than did renal epithelial cells, possibly explaining the enhanced formation of PAP-SG by this cell population. In conclusion, our data indicates that renal epithelial cells are intrinsically more susceptible to PAP cytotoxicity than are hepatocytes. This enhanced cytotoxicity may be due to longer exposure to PAP and/or reduced detoxification of reactive intermediates due to lower concentrations

  9. 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

  10. Peroxisomal abnormalities in the immortalized human hepatocyte (IHH) cell line.

    Science.gov (United States)

    Klouwer, Femke C C; Koster, Janet; Ferdinandusse, Sacha; Waterham, Hans R

    2017-04-01

    The immortalized human hepatocyte (IHH) cell line is increasingly used for studies related to liver metabolism, including hepatic glucose, lipid, lipoprotein and triglyceride metabolism, and the effect of therapeutic interventions. To determine whether the IHH cell line is a good model to investigate hepatic peroxisomal metabolism, we measured several peroxisomal parameters in IHH cells and, for comparison, HepG2 cells and primary skin fibroblasts. This revealed a marked plasmalogen deficiency and a deficient fatty acid α-oxidation in the IHH cells, due to a defect of PEX7, a cytosolic receptor protein required for peroxisomal import of a subset of peroxisomal proteins. These abnormalities have consequences for the lipid homeostasis of these cells and thus should be taken into account for the interpretation of data previously generated by using this cell line and when considering using this cell line for future research.

  11. Hepatocyte growth factor inhibitor-2 prevents shedding of matritpase

    DEFF Research Database (Denmark)

    Larsen, Brian R; Steffensen, Simon D; Nielsen, Nis V L

    2013-01-01

    Hepatocyte growth factor activator inhibitor-2 (HAI-2) is an inhibitor of many proteases in vitro, including the membrane-bound serine protease, matriptase. Studies of knock-out mice have shown that HAI-2 is essential for placental development only in mice expressing matriptase, suggesting that HAI......-2 is important for regulation of matriptase. Previous studies have shown that recombinant expression of matriptase was unsuccessful unless co-expressed with another HAI, HAI-1. In the present study we show that when human matriptase is recombinantly expressed alone in the canine cell line MDCK......, then human matriptase mRNA can be detected and the human matriptase ectodomain is shed to the media, suggesting that matriptase expressed alone is rapidly transported through the secretory pathway and shed. Whereas matriptase expressed together with HAI-1 or HAI-2 accumulates on the plasma membrane where...

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

    NARCIS (Netherlands)

    Habraken, G.J.M.

    2011-01-01

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

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

    NARCIS (Netherlands)

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

    1985-01-01

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

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

    DEFF Research Database (Denmark)

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

    2003-01-01

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

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

    International Nuclear Information System (INIS)

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

    1990-01-01

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

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

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

    Science.gov (United States)

    Dunlap, Dacey; Patrick, Patricia

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Guangxin Yuan

    2018-01-01

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

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

    NARCIS (Netherlands)

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

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

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

    Science.gov (United States)

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

    2003-02-01

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

  1. 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

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

    Science.gov (United States)

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

    2013-05-01

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

  3. Permanent Electric Dipole-Dipole Interactions in Lyotropic Polypeptide Liquid Crystals

    OpenAIRE

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

    1981-01-01

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

  4. Interaction of propionate and carnitine metabolism in isolated rat hepatocytes

    International Nuclear Information System (INIS)

    Brass, E.P.; Beyerinck, R.A.

    1987-01-01

    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. 14 C-P was converted to CO 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 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 μM 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 μM or 150 μM. 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

  5. Glycogen synthase activation by sugars in isolated hepatocytes.

    Science.gov (United States)

    Ciudad, C J; Carabaza, A; Bosch, F; Gòmez I Foix, A M; Guinovart, J J

    1988-07-01

    We have investigated the activation by sugars of glycogen synthase in relation to (i) phosphorylase a activity and (ii) changes in the intracellular concentration of glucose 6-phosphate and adenine nucleotides. All the sugars tested in this work present the common denominator of activating glycogen synthase. On the other hand, phosphorylase a activity is decreased by mannose and glucose, unchanged by galactose and xylitol, and increased by tagatose, glyceraldehyde, and fructose. Dihydroxyacetone exerts a biphasic effect on phosphorylase. These findings provide additional evidence proving that glycogen synthase can be activated regardless of the levels of phosphorylase a, clearly establishing that a nonsequential mechanism for the activation of glycogen synthase occurs in liver cells. The glycogen synthase activation state is related to the concentrations of glucose 6-phosphate and adenine nucleotides. In this respect, tagatose, glyceraldehyde, and fructose deplete ATP and increase AMP contents, whereas glucose, mannose, galactose, xylitol, and dihydroxyacetone do not alter the concentration of these nucleotides. In addition, all these sugars, except glyceraldehyde, increase the intracellular content of glucose 6-phosphate. The activation of glycogen synthase by sugars is reflected in decreases on both kinetic constants of the enzyme, M0.5 (for glucose 6-phosphate) and S0.5 (for UDP-glucose). We propose that hepatocyte glycogen synthase is activated by monosaccharides by a mechanism triggered by changes in glucose 6-phosphate and adenine nucleotide concentrations which have been described to modify glycogen synthase phosphatase activity. This mechanism represents a metabolite control of the sugar-induced activation of hepatocyte glycogen synthase.

  6. 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

  7. 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

  8. Host apolipoprotein B messenger RNA-editing enzyme catalytic polypeptide-like 3G is an innate defensive factor and drug target against hepatitis C virus.

    Science.gov (United States)

    Peng, Zong-Gen; Zhao, Zhi-Yun; Li, Yan-Ping; Wang, Yu-Ping; Hao, Lan-Hu; Fan, Bo; Li, Yu-Huan; Wang, Yue-Ming; Shan, Yong-Qiang; Han, Yan-Xing; Zhu, Yan-Ping; Li, Jian-Rui; You, Xue-Fu; Li, Zhuo-Rong; Jiang, Jian-Dong

    2011-04-01

    Host cellular factor apolipoprotein B messenger RNA (mRNA)-editing enzyme catalytic polypeptide-like 3G (hA3G) is a cytidine deaminase that inhibits a group of viruses including human immunodeficiency virus-1 (HIV-1). In the continuation of our research on hA3G, we found that hA3G stabilizing compounds significantly inhibited hepatitis C virus (HCV) replication. Therefore, this study investigated the role of hA3G in HCV replication. Introduction of external hA3G into HCV-infected Huh7.5 human hepatocytes inhibited HCV replication; knockdown of endogenous hA3G enhanced HCV replication. Exogenous HIV-1 virion infectivity factor (Vif) decreased intracellular hA3G and therefore enhanced HCV proliferation, suggesting that the presence of Vif might be an explanation for the HIV-1/HCV coinfection often observed in HIV-1(+) individuals. Treatment of the HCV-infected Huh7.5 cells with RN-5 or IMB-26, two known hA3G stabilizing compounds, increased intracellular hA3G and accordingly inhibited HCV replication. The compounds inhibit HCV through increasing the level of hA3G incorporated into HCV particles, but not through inhibiting HCV enzymes. However, G/A hypermutation in the HCV genome were not detected, suggesting a new antiviral mechanism of hA3G in HCV, different from that in HIV-1. Stabilization of hA3G by RN-5 was safe in vivo. hA3G appears to be a cellular restrict factor against HCV and could be a potential target for drug discovery. 2011 American Association for the Study of Liver Diseases.

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

    Science.gov (United States)

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

    2010-02-01

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

  10. Fuel assembly guide tube

    International Nuclear Information System (INIS)

    Jabsen, F.S.

    1979-01-01

    This invention is directed toward a nuclear fuel assembly guide tube arrangement which restrains spacer grid movement due to coolant flow and which offers secondary means for supporting a fuel assembly during handling and transfer operations

  11. 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

  12. Nuclear reactor fuel assembly

    International Nuclear Information System (INIS)

    Sasaki, Y.; Tashima, J.

    1975-01-01

    A description is given of nuclear reactor fuel assemblies arranged in the form of a lattice wherein there is attached to the interface of one of two adjacent fuel assemblies a plate spring having a concave portion curved toward said interface and to the interface of the other fuel assembly a plate spring having a convex portion curved away from said interface

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

    Science.gov (United States)

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

    2015-10-01

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

  14. Assembly and Function of a Spore Coat-Associated Transglutaminase of Bacillus subtilis

    Science.gov (United States)

    Zilhão, Rita; Isticato, Rachele; Martins, Lígia O.; Steil, Leif; Völker, Uwe; Ricca, Ezio; Moran, Charles P.; Henriques, Adriano O.

    2005-01-01

    The assembly of a multiprotein coat around the Bacillus subtilis spore confers resistance to lytic enzymes and noxious chemicals and ensures normal germination. Part of the coat is cross-linked and resistant to solubilization. The coat contains ɛ-(γ-glutamyl)lysyl cross-links, and the expression of the gene (tgl) for a spore-associated transglutaminase was shown before to be required for the cross-linking of coat protein GerQ. Here, we have investigated the assembly and function of Tgl. We found that Tgl associates, albeit at somewhat reduced levels, with the coats of mutants that are unable to assemble the outer coat (cotE), that are missing the inner coat and with a greatly altered outer coat (gerE), or that are lacking discernible inner and outer coat structures (cotE gerE double mutant). This suggests that Tgl is present at various levels within the coat lattice. The assembly of Tgl occurs independently of its own activity, as a single amino acid substitution of a cysteine to an alanine (C116A) at the active site of Tgl does not affect its accumulation or assembly. However, like a tgl insertional mutation, the tglC116A allele causes increased extractability of polypeptides of about 40, 28, and 16 kDa in addition to GerQ (20 kDa) and affects the structural integrity of the coat. We show that most Tgl is assembled onto the spore surface soon after its synthesis in the mother cell under σK control but that the complete insolubilization of at least two of the Tgl-controlled polypeptides occurs several hours later. We also show that a multicopy allele of tgl causes increased assembly of Tgl and affects the assembly, structure, and functional properties of the coat. PMID:16267299

  15. 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.

  16. Glucose-induced lipid deposition in goose primary hepatocytes is dependent on the PI3K-Akt-mTOR signaling pathway

    Directory of Open Access Journals (Sweden)

    Han Chunchun

    2016-01-01

    Full Text Available Previously we showed that fatty liver formation in overfed geese was accompanied by PI3K-Akt-mTOR pathway activation and changes in plasma glucose concentrations. Here, we show that glucose acts in goose hepatocellular lipid metabolism through the PI3K-Akt-mTOR signaling pathway. We observed that glucose increased lipogenesis, decreased fatty acid oxidation and increased very low density lipoprotein triglyceride (VLDL-TG assembly and secretion. Co-treatment with glucose and inhibitors of the PI3K-Akt-mTOR pathway (LY294002, rapamycin, NVP-BEZ235 decreased the levels of factors involved in lipogenesis and increased the levels of factors involved in fatty acid oxidation and VLDL-TG assembly and secretion. These findings show that inhibition of the PI3K-Akt-mTOR pathway decreased glucose-induced lipogenesis, inhibited the downregulation of fatty acid oxidation by glucose and increased the upregulation of VLDL-TG assembly and secretion by glucose. The results presented herein provide further support for the role of the PI3K-Akt-mTOR pathway in lipid metabolism as we showed that in goose primary hepatocytes, glucose acts through the PI3K-Akt-mTOR-dependent pathway to stimulate lipid deposition by increasing lipogenesis and decreasing fatty acid oxidation and VLDL-TG assembly and secretion.

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

    Science.gov (United States)

    Han, P; Lucero, M T

    2005-01-01

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

  18. Impaired mitochondrial functions contribute to 3-bromopyruvate toxicity in primary rat and mouse hepatocytes

    Czech Academy of Sciences Publication Activity Database

    Sobotka, O.; Endlicher, R.; Drahota, Zdeněk; Kučera, O.; Rychtrmoc, D.; Raad, M.; Hakeem, K.; Červinková, Z.

    2016-01-01

    Roč. 48, č. 4 (2016), s. 363-373 ISSN 0145-479X Institutional support: RVO:67985823 Keywords : 3-bromopyruvate * toxicity * liver * hepatocyte * mitochondria Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.576, year: 2016

  19. SELENIUM MODIFIES THE METABOLISM AND TOXICITY OF ARSENIC IN PRIMARY RAT HEPATOCYTES

    Science.gov (United States)

    ABSTRACTSelenium Modifies the Metabolism and Toxicity of Arsenic in Primary Rat Hepatocytes. Miroslav Styblo, David J. Thomas (2000) Toxicol. Appl. Pharmacol. Arsenic and selenium are metalloids with similar chemical properties and metabolic fates. Inorganic arsenic (iAs...

  20. Acidosis-induced downregulation of hepatocyte mitochondrial aquaporin-8 and ureagenesis from ammonia.

    Science.gov (United States)

    Molinas, Sara M; Soria, Leandro R; Marrone, Julieta; Danielli, Mauro; Trumper, Laura; Marinelli, Raúl A

    2015-08-01

    It has been proposed that, during metabolic acidosis, the liver downregulates mitochondrial ammonia detoxification via ureagenesis, a bicarbonate-consuming process. Since we previously demonstrated that hepatocyte mitochondrial aquaporin-8 channels (mtAQP8) facilitate the uptake of ammonia and its metabolism into urea, we studied whether mtAQP8 is involved in the liver adaptive response to acidosis. Primary cultured rat hepatocytes were adapted to acidosis by exposing them to culture medium at pH 7.0 for 40 h. Control cells were exposed to pH 7.4. Hepatocytes exposed to acid medium showed a decrease in mtAQP8 protein expression (-30%, p ammonia was assessed by incubating the cells with (15)N-labeled ammonia and measuring (15)N-labeled urea synthesis by nuclear magnetic resonance. Reduced ureagenesis was found in acidified hepatocytes (-31%, p ammonia in response to acidosis.

  1. Hepatocyte produced matrix metalloproteinases are regulated by CD147 in liver fibrogenesis.

    Science.gov (United States)

    Calabro, Sarah R; Maczurek, Annette E; Morgan, Alison J; Tu, Thomas; Wen, Victoria W; Yee, Christine; Mridha, Auvro; Lee, Maggie; d'Avigdor, William; Locarnini, Stephen A; McCaughan, Geoffrey W; Warner, Fiona J; McLennan, Susan V; Shackel, Nicholas A

    2014-01-01

    The classical paradigm of liver injury asserts that hepatic stellate cells (HSC) produce, remodel and turnover the abnormal extracellular matrix (ECM) of fibrosis via matrix metalloproteinases (MMPs). In extrahepatic tissues MMP production is regulated by a number of mechanisms including expression of the glycoprotein CD147. Previously, we have shown that CD147 is expressed on hepatocytes but not within the fibrotic septa in cirrhosis [1]. Therefore, we investigated if hepatocytes produce MMPs, regulated by CD147, which are capable of remodelling fibrotic ECM independent of the HSC. Non-diseased, fibrotic and cirrhotic livers were examined for MMP activity and markers of fibrosis in humans and mice. CD147 expression and MMP activity were co-localised by in-situ zymography. The role of CD147 was studied in-vitro with siRNA to CD147 in hepatocytes and in-vivo in mice with CCl4 induced liver injury using ãCD147 antibody intervention. In liver fibrosis in both human and mouse tissue MMP expression and activity (MMP-2, -9, -13 and -14) increased with progressive injury and localised to hepatocytes. Additionally, as expected, MMPs were abundantly expressed by activated HSC. Further, with progressive fibrosis there was expression of CD147, which localised to hepatocytes but not to HSC. Functionally significant in-vitro regulation of hepatocyte MMP production by CD147 was demonstrated using siRNA to CD147 that decreased hepatocyte MMP-2 and -9 expression/activity. Further, in-vivo α-CD147 antibody intervention decreased liver MMP-2, -9, -13, -14, TGF-β and α-SMA expression in CCl4 treated mice compared to controls. We have shown that hepatocytes produce active MMPs and that the glycoprotein CD147 regulates hepatocyte MMP expression. Targeting CD147 regulates hepatocyte MMP production both in-vitro and in-vivo, with the net result being reduced fibrotic matrix turnover in-vivo. Therefore, CD147 regulation of hepatocyte MMP is a novel pathway that could be targeted by

  2. Hepatocyte produced matrix metalloproteinases are regulated by CD147 in liver fibrogenesis.

    Directory of Open Access Journals (Sweden)

    Sarah R Calabro

    Full Text Available The classical paradigm of liver injury asserts that hepatic stellate cells (HSC produce, remodel and turnover the abnormal extracellular matrix (ECM of fibrosis via matrix metalloproteinases (MMPs. In extrahepatic tissues MMP production is regulated by a number of mechanisms including expression of the glycoprotein CD147. Previously, we have shown that CD147 is expressed on hepatocytes but not within the fibrotic septa in cirrhosis [1]. Therefore, we investigated if hepatocytes produce MMPs, regulated by CD147, which are capable of remodelling fibrotic ECM independent of the HSC.Non-diseased, fibrotic and cirrhotic livers were examined for MMP activity and markers of fibrosis in humans and mice. CD147 expression and MMP activity were co-localised by in-situ zymography. The role of CD147 was studied in-vitro with siRNA to CD147 in hepatocytes and in-vivo in mice with CCl4 induced liver injury using ãCD147 antibody intervention.In liver fibrosis in both human and mouse tissue MMP expression and activity (MMP-2, -9, -13 and -14 increased with progressive injury and localised to hepatocytes. Additionally, as expected, MMPs were abundantly expressed by activated HSC. Further, with progressive fibrosis there was expression of CD147, which localised to hepatocytes but not to HSC. Functionally significant in-vitro regulation of hepatocyte MMP production by CD147 was demonstrated using siRNA to CD147 that decreased hepatocyte MMP-2 and -9 expression/activity. Further, in-vivo α-CD147 antibody intervention decreased liver MMP-2, -9, -13, -14, TGF-β and α-SMA expression in CCl4 treated mice compared to controls.We have shown that hepatocytes produce active MMPs and that the glycoprotein CD147 regulates hepatocyte MMP expression. Targeting CD147 regulates hepatocyte MMP production both in-vitro and in-vivo, with the net result being reduced fibrotic matrix turnover in-vivo. Therefore, CD147 regulation of hepatocyte MMP is a novel pathway that could be

  3. A fat option for the pig: Hepatocytic differentiated mesenchymal stem cells for translational research

    Energy Technology Data Exchange (ETDEWEB)

    Brückner, Sandra, E-mail: sandra.brueckner@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); Tautenhahn, Hans-Michael, E-mail: hans-michael.tautenhahn@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); TRM, Translational Centre for Regenerative Medicine, Philipp-Rosenthal-Str. 55, Leipzig D-04103 (Germany); Winkler, Sandra, E-mail: sandra.pelz@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); Stock, Peggy, E-mail: peggy.stock@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); Dollinger, Matthias, E-mail: matthias.dollinger@uniklinik-ulm.de [University Hospital Ulm, First Department of Medicine, Albert-Einstein-Allee 23, Ulm D-89081 (Germany); Christ, Bruno, E-mail: bruno.christ@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); TRM, Translational Centre for Regenerative Medicine, Philipp-Rosenthal-Str. 55, Leipzig D-04103 (Germany)

    2014-02-15

    Study background: Extended liver resection is the only curative treatment option of liver cancer. Yet, the residual liver may not accomplish the high metabolic and regenerative capacity needed, which frequently leads to acute liver failure. Because of their anti-inflammatory and -apoptotic as well as pro-proliferative features, mesenchymal stem cells differentiated into hepatocyte-like cells might provide functional and regenerative compensation. Clinical translation of basic research requires pre-clinical approval in large animals. Therefore, we characterized porcine mesenchymal stem cells (MSC) from adipose tissue and bone marrow and their hepatocyte differentiation potential for future assessment of functional liver support after surgical intervention in the pig model. Methods: Mesenchymal surface antigens and multi-lineage differentiation potential of porcine MSC isolated by collagenase digestion either from bone marrow or adipose tissue (subcutaneous/visceral) were assessed by flow cytometry. Morphology and functional properties (urea-, glycogen synthesis and cytochrome P450 activity) were determined during culture under differentiation conditions and compared with primary porcine hepatocytes. Results: MSC from porcine adipose tissue and from bone marrow express the typical mesenchymal markers CD44, CD29, CD90 and CD105 but not haematopoietic markers. MSC from both sources displayed differentiation into the osteogenic as well as adipogenic lineage. After hepatocyte differentiation, expression of CD105 decreased significantly and cells adopted the typical polygonal morphology of hepatocytes. Glycogen storage was comparable in adipose tissue- and bone marrow-derived cells. Urea synthesis was about 35% lower in visceral than in subcutaneous adipose tissue-derived MSC. Cytochrome P450 activity increased significantly during differentiation and was twice as high in hepatocyte-like cells generated from bone marrow as from adipose tissue. Conclusion: The hepatocyte

  4. A fat option for the pig: Hepatocytic differentiated mesenchymal stem cells for translational research

    International Nuclear Information System (INIS)

    Brückner, Sandra; Tautenhahn, Hans-Michael; Winkler, Sandra; Stock, Peggy; Dollinger, Matthias; Christ, Bruno

    2014-01-01

    Study background: Extended liver resection is the only curative treatment option of liver cancer. Yet, the residual liver may not accomplish the high metabolic and regenerative capacity needed, which frequently leads to acute liver failure. Because of their anti-inflammatory and -apoptotic as well as pro-proliferative features, mesenchymal stem cells differentiated into hepatocyte-like cells might provide functional and regenerative compensation. Clinical translation of basic research requires pre-clinical approval in large animals. Therefore, we characterized porcine mesenchymal stem cells (MSC) from adipose tissue and bone marrow and their hepatocyte differentiation potential for future assessment of functional liver support after surgical intervention in the pig model. Methods: Mesenchymal surface antigens and multi-lineage differentiation potential of porcine MSC isolated by collagenase digestion either from bone marrow or adipose tissue (subcutaneous/visceral) were assessed by flow cytometry. Morphology and functional properties (urea-, glycogen synthesis and cytochrome P450 activity) were determined during culture under differentiation conditions and compared with primary porcine hepatocytes. Results: MSC from porcine adipose tissue and from bone marrow express the typical mesenchymal markers CD44, CD29, CD90 and CD105 but not haematopoietic markers. MSC from both sources displayed differentiation into the osteogenic as well as adipogenic lineage. After hepatocyte differentiation, expression of CD105 decreased significantly and cells adopted the typical polygonal morphology of hepatocytes. Glycogen storage was comparable in adipose tissue- and bone marrow-derived cells. Urea synthesis was about 35% lower in visceral than in subcutaneous adipose tissue-derived MSC. Cytochrome P450 activity increased significantly during differentiation and was twice as high in hepatocyte-like cells generated from bone marrow as from adipose tissue. Conclusion: The hepatocyte

  5. 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

  6. Nuclear fuel string assembly

    International Nuclear Information System (INIS)

    Ip, A.K.; Koyanagi, K.; Tarasuk, W.R.

    1976-01-01

    A method of fabricating rodded fuels suitable for use in pressure tube type reactors and in pressure vessel type reactors is described. Fuel rods are secured as an inner and an outer sub-assembly, each rod attached between mounting rings secured to the rod ends. The two sub-assemblies are telescoped together and positioned by spaced thimbles located between them to provide precise positioning while permittng differential axial movement between the sub-assemblies. Such sub-assemblies are particularly suited for mounting as bundle strings. The method provides particular advantages in the assembly of annular-section fuel pins, which includes booster fuel containing enriched fuel material. (LL)

  7. Nuclear reactor fuel assembly

    International Nuclear Information System (INIS)

    Marmonier, Pierre; Mesnage, Bernard; Nervi, J.C.

    1975-01-01

    This invention refers to fuel assemblies for a liquid metal cooled fast neutron reactor. Each assembly is composed of a hollow vertical casing, of regular polygonal section, containing a bundle of clad pins filled with a fissile or fertile substance. The casing is open at its upper end and has a cylindrical foot at its lower end for positioning the assembly in a housing provided in the horizontal diagrid, on which the core assembly rests. A set of flat bars located on the external surface of the casing enables it to be correctly orientated in its housing among the other core assemblies [fr

  8. Hypoxia-inducible factor-dependent production of profibrotic mediators by hypoxic hepatocytes.

    Science.gov (United States)

    Copple, Bryan L; Bustamante, Juan J; Welch, Timothy P; Kim, Nam Deuk; Moon, Jeon-Ok

    2009-08-01

    During the development of liver fibrosis, mediators are produced that stimulate cells in the liver to differentiate into myofibroblasts and to produce collagen. Recent studies demonstrated that the transcription factor, hypoxia-inducible factor-1alpha (HIF-1alpha), is critical for upregulation of profibrotic mediators, such as platelet-derived growth factor-A (PDGF-A), PDGF-B and plasminogen activator inhibitor-1 (PAI-1) in the liver, during the development of fibrosis. What remains unknown is the cell type-specific regulation of these genes by HIF-1alpha in liver cell types. Accordingly, the hypothesis was tested that HIF-1alpha is activated in hypoxic hepatocytes and regulates the production of profibrotic mediators by these cells. In this study, hepatocytes were isolated from the livers of control and HIF-1alpha- or HIF-1beta-deficient mice and exposed to hypoxia. Exposure of primary mouse hepatocytes to 1% oxygen stimulated nuclear accumulation of HIF-1alpha and upregulated PAI-1, vascular endothelial cell growth factor and the vasoactive peptides adrenomedullin-1 (ADM-1) and ADM-2. In contrast, the levels of PDGF-A and PDGF-B mRNAs were unaffected in these cells by hypoxia. Exposure of HIF-1alpha-deficient hepatocytes to 1% oxygen only partially prevented upregulation of these genes, suggesting that other hypoxia-regulated transcription factors, such as HIF-2alpha, may also regulate these genes. In support of this, HIF-2alpha was activated in hypoxic hepatocytes, and exposure of HIF-1beta-deficient hepatocytes to 1% oxygen completely prevented upregulation of PAI-1, vascular endothelial cell growth factor and ADM-1, suggesting that HIF-2alpha may also contribute to upregulation of these genes in hypoxic hepatocytes. Collectively, our results suggest that HIFs may be important regulators of profibrotic and vasoactive mediators by hypoxic hepatocytes.

  9. Bile acid-induced necrosis in primary human hepatocytes and in patients with obstructive cholestasis

    Energy Technology Data Exchange (ETDEWEB)

    Woolbright, Benjamin L.; Dorko, Kenneth [Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Antoine, Daniel J.; Clarke, Joanna I. [MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool (United Kingdom); Gholami, Parviz [Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS (United States); Li, Feng [Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Kumer, Sean C.; Schmitt, Timothy M.; Forster, Jameson [Department of Surgery, University of Kansas Medical Center, Kansas City, KS (United States); Fan, Fang [Department of Pathology, University of Kansas Medical Center, Kansas City, KS (United States); Jenkins, Rosalind E.; Park, B. Kevin [MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool (United Kingdom); Hagenbuch, Bruno [Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Olyaee, Mojtaba [Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS (United States); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States)

    2015-03-15

    Accumulation of bile acids is a major mediator of cholestatic liver injury. Recent studies indicate bile acid composition between humans and rodents is dramatically different, as humans have a higher percent of glycine conjugated bile acids and increased chenodeoxycholate content, which increases the hydrophobicity index of bile acids. This increase may lead to direct toxicity that kills hepatocytes, and promotes inflammation. To address this issue, this study assessed how pathophysiological concentrations of bile acids measured in cholestatic patients affected primary human hepatocytes. Individual bile acid levels were determined in serum and bile by UPLC/QTOFMS in patients with extrahepatic cholestasis with, or without, concurrent increases in serum transaminases. Bile acid levels increased in serum of patients with liver injury, while biliary levels decreased, implicating infarction of the biliary tracts. To assess bile acid-induced toxicity in man, primary human hepatocytes were treated with relevant concentrations, derived from patient data, of the model bile acid glycochenodeoxycholic acid (GCDC). Treatment with GCDC resulted in necrosis with no increase in apoptotic parameters. This was recapitulated by treatment with biliary bile acid concentrations, but not serum concentrations. Marked elevations in serum full-length cytokeratin-18, high mobility group box 1 protein (HMGB1), and acetylated HMGB1 confirmed inflammatory necrosis in injured patients; only modest elevations in caspase-cleaved cytokeratin-18 were observed. These data suggest human hepatocytes are more resistant to human-relevant bile acids than rodent hepatocytes, and die through necrosis when exposed to bile acids. These mechanisms of cholestasis in humans are fundamentally different to mechanisms observed in rodent models. - Highlights: • Cholestatic liver injury is due to cytoplasmic bile acid accumulation in hepatocytes. • Primary human hepatocytes are resistant to BA-induced injury

  10. Acrolein cytotoxicity in hepatocytes involves endoplasmic reticulum stress, mitochondrial dysfunction and oxidative stress

    OpenAIRE

    Mohammad, Mohammad K; Avila, Diana; Zhang, Jingwen; Barve, Shirish; Arteel, Gavin; McClain, Craig; Joshi-Barve, Swati

    2012-01-01

    Acrolein is a common environmental, food and water pollutant and a major component of cigarette smoke. Also, it is produced endogenously via lipid peroxidation and cellular metabolism of certain amino acids and drugs. Acrolein is cytotoxic to many cell types including hepatocytes; however the mechanisms are not fully understood. We examined the molecular mechanisms underlying acrolein hepatotoxicity in primary human hepatocytes and hepatoma cells. Acrolein, at pathophysiological concentration...

  11. Role of CYP2B in Phenobarbital-Induced Hepatocyte Proliferation in Mice.

    Science.gov (United States)

    Li, Lei; Bao, Xiaochen; Zhang, Qing-Yu; Negishi, Masahiko; Ding, Xinxin

    2017-08-01

    Phenobarbital (PB) promotes liver tumorigenesis in rodents, in part through activation of the constitutive androstane receptor (CAR) and the consequent changes in hepatic gene expression and increases in hepatocyte proliferation. A typical effect of CAR activation by PB is a marked induction of Cyp2b10 expression in the liver; the latter has been suspected to be vital for PB-induced hepatocellular proliferation. This hypothesis was tested here by using a Cyp2a(4/5)bgs -null (null) mouse model in which all Cyp2b genes are deleted. Adult male and female wild-type (WT) and null mice were treated intraperitoneally with PB at 50 mg/kg once daily for 5 successive days and tested on day 6. The liver-to-body weight ratio, an indicator of liver hypertrophy, was increased by 47% in male WT mice, but by only 22% in male Cyp2a(4/5)bgs -null mice, by the PB treatment. The fractions of bromodeoxyuridine-positive hepatocyte nuclei, assessed as a measure of the rate of hepatocyte proliferation, were also significantly lower in PB-treated male null mice compared with PB-treated male WT mice. However, whereas few proliferating hepatocytes were detected in saline-treated mice, many proliferating hepatocytes were still detected in PB-treated male null mice. In contrast, female WT mice were much less sensitive than male WT mice to PB-induced hepatocyte proliferation, and PB-treated female WT and PB-treated female null mice did not show significant difference in rates of hepatocyte proliferation. These results indicate that CYP2B induction plays a significant, but partial, role in PB-induced hepatocyte proliferation in male mice. U.S. Government work not protected by U.S. copyright.

  12. Optimization of the isolation and cultivation of Cyprinus carpio primary hepatocytes

    OpenAIRE

    Yanhong, Fan; Chenghua, He; Guofang, Liu; Haibin, Zhang

    2008-01-01

    The aquatic environment is affected by numerous chemical contaminants. There is an increasing need to identify these chemicals and to evaluate their potential toxicity towards aquatic life. In this research we optimized techniques for primary cell culture of Cyprinus carpio hepatocytes as one adjunct model for ecotoxicological evaluation of the potential hazards of xenobiotics in the aquatic environment. In this study, Cyprinus carpio hepatocytes were isolated by mechanical separation, two-st...

  13. Subtoxic Alterations in Hepatocyte-Derived Exosomes: An Early Step in Drug-Induced Liver Injury?

    Science.gov (United States)

    Holman, Natalie S; Mosedale, Merrie; Wolf, Kristina K; LeCluyse, Edward L; Watkins, Paul B

    2016-06-01

    Drug-induced liver injury (DILI) is a significant clinical and economic problem in the United States, yet the mechanisms that underlie DILI remain poorly understood. Recent evidence suggests that signaling molecules released by stressed hepatocytes can trigger immune responses that may be common across DILI mechanisms. Extracellular vesicles released by hepatocytes, principally hepatocyte-derived exosomes (HDEs), may constitute one such signal. To examine HDE alterations as a function of drug-induced stress, this work utilized prototypical hepatotoxicant acetaminophen (APAP) in male Sprague-Dawley (SD) rats, SD rat hepatocytes, and primary human hepatocytes. HDE were isolated using ExoQuick precipitation reagent and analyzed by quantification of the liver-specific RNAs albumin and microRNA-122 (miR-122). In vivo, significant elevations in circulating exosomal albumin mRNA were observed at subtoxic APAP exposures. Significant increases in exosomal albumin mRNA were also observed in primary rat hepatocytes at subtoxic APAP concentrations. In primary human hepatocytes, APAP elicited increases in both exosomal albumin mRNA and exosomal miR-122 without overt cytotoxicity. However, the number of HDE produced in vitro in response to APAP did not increase with exosomal RNA quantity. We conclude that significant drug-induced alterations in the liver-specific RNA content of HDE occur at subtoxic APAP exposures in vivo and in vitro, and that these changes appear to reflect selective packaging rather than changes in exosome number. The current findings demonstrate that translationally relevant HDE alterations occur in the absence of overt hepatocellular toxicity, and support the hypothesis that HDE released by stressed hepatocytes may mediate early immune responses in DILI. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  14. Effects of Cytochrome P 450 Inhibitors on Itraconazole and Fluconazole Induced Cytotoxicity in Hepatocytes

    International Nuclear Information System (INIS)

    Somchit, N.; Ngee, C.S.; Yaakob, A.; Ahmad, Z.; Zakaria, Z.A.

    2009-01-01

    Itraconazole and fluconazole have been reported to induce hepatotoxicity in patients. The present study was designed to investigate the role of cytochrome P450 inhibitors, SKF 525A, and curcumin pretreatment on the cytotoxicity of antifungal drugs fluconazole and itraconazole. For 3 consecutive days, female rats were administered daily SKF 525A or curcumin (5 and 25?mg/kg). Control rats received an equivalent amount of dosed vehicle. The animals were anaesthetised 24 hours after receiving the last dose for liver perfusion. Hepatocytes were then exposed to various concentrations of antifungal drugs. In vitro incubation of hepatocytes with itraconazole revealed significantly lower viability when compared to fluconazole as assessed by lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase activities. The cytotoxicity of itraconazole was enhanced when incubated with hepatocytes pretreated with SKF 525A. SKF 525A had no effects on the cytotoxicity of fluconazole. Curcumin failed to either increase or decrease the cytotoxicity of both antifungal drugs. ATP levels also showed significant decrease in both itraconazole and fluconazole incubated hepatocytes. However, SKF 525A pretreated hepatocytes had significantly lower ATP levels after itraconazole incubations. Collectively, these results confirm the involvement of cytochrome P450 in the cytoprotection in itraconazole induced hepatocyte toxicity. Differences of the effects of SKF 525A on the cytotoxicity induced by itraconazole and fluconazole may be due to the differences on the metabolism of each antifungal drug in vivo.

  15. YAP Inhibition Restores Hepatocyte Differentiation in Advanced HCC, Leading to Tumor Regression

    Directory of Open Access Journals (Sweden)

    Julien Fitamant

    2015-03-01

    Full Text Available Defective Hippo/YAP signaling in the liver results in tissue overgrowth and development of hepatocellular carcinoma (HCC. Here, we uncover mechanisms of YAP-mediated hepatocyte reprogramming and HCC pathogenesis. YAP functions as a rheostat in maintaining metabolic specialization, differentiation, and quiescence within the hepatocyte compartment. Increased or decreased YAP activity reprograms subsets of hepatocytes to different fates associated with deregulation of the HNF4A, CTNNB1, and E2F transcriptional programs that control hepatocyte quiescence and differentiation. Importantly, treatment with small interfering RNA-lipid nanoparticles (siRNA-LNPs targeting YAP restores hepatocyte differentiation and causes pronounced tumor regression in a genetically engineered mouse HCC model. Furthermore, YAP targets are enriched in an aggressive human HCC subtype characterized by a proliferative signature and absence of CTNNB1 mutations. Thus, our work reveals Hippo signaling as a key regulator of the positional identity of hepatocytes, supports targeting of YAP using siRNA-LNPs as a paradigm of differentiation-based therapy, and identifies an HCC subtype that is potentially responsive to this approach.

  16. [The polyploidization characteristics of the hepatocytes of the mouse-like hamster Calomyscus mystax].

    Science.gov (United States)

    Anatskaia, O V; Malikov, V G; Meĭer, M N; Kudriavtsev, B N

    1995-01-01

    A cytophotometric measurement of DNA content in hepatocytes of maturing mouse-like hamsters was made. Cells belonging to ordinary mammalian ploidy classes 2c, 2c x 2, 4c, and 4c x 2 made about 90% of the hepatocyte population. The share of binucleated cells wa high (about 80%), the majority of these cells being 2c X 2 hepatocytes. Binucleated cells with tetraploid and diploid nuclei occur in almost every animal. An average hepatocyte ploidy level in mouse-like hamster is 4.6c. The main peculiarity of parenchymal liver cell populations is that up 5% of hepatocytes contain 3--11 nuclei of different ploidy classes. Multinucleated cells increase in number from 1.5% to 4% within the period from one year (the age of maturation) to two years. Later on their percentage does not change. It is found that in binucleated and multinucleated hepatocytes DNA synthesis can proceed asynchronously. Asynchrony in DNA synthesis elevates as the number of nuclei increases. Among the 2c x 2 and 2c x 3 cells an uneven distribution of 3H-thymidine label can occur, respectively, in 5 and in 50% cases, whereas all the cells with more than 3 nuclei display an uneven an uneven 3H-thymidin label distribution. The formation of multinucleated cells is supposed to be associated with asynchrony in DNA-synthesis in binucleated cells and with the restitution of mitosis.

  17. Hepatocyte transplantation and advancements in alternative cell sources for liver-based regenerative medicine.

    Science.gov (United States)

    Lee, Charlotte A; Sinha, Siddharth; Fitzpatrick, Emer; Dhawan, Anil

    2018-06-01

    Human hepatocyte transplantation has been actively perused as an alternative to liver replacement for acute liver failure and liver-based metabolic defects. Current challenges in this field include a limited cell source, reduced cell viability following cryopreservation and poor engraftment of cells into the recipient liver with consequent limited life span. As a result, alternative stem cell sources such as pluripotent stem cells, fibroblasts, hepatic progenitor cells, amniotic epithelial cells and mesenchymal stem/stromal cells (MSCs) can be used to generate induced hepatocyte like cells (HLC) with each technique exhibiting advantages and disadvantages. HLCs may have comparable function to primary human hepatocytes and could offer patient-specific treatment. However, long-term functionality of transplanted HLCs and the potential oncogenic risks of using stem cells have yet to be established. The immunomodulatory effects of MSCs are promising, and multiple clinical trials are investigating their effect in cirrhosis and acute liver failure. Here, we review the current status of hepatocyte transplantation, alternative cell sources to primary human hepatocytes and their potential in liver regeneration. We also describe recent clinical trials using hepatocytes derived from stem cells and their role in improving the phenotype of several liver diseases.

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

    Science.gov (United States)

    Beck, P; Nicholas, H

    1975-03-01

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

  19. Progress Toward the Clinical Translation of Bioinspired Peptide and Protein Assemblies.

    Science.gov (United States)

    Hainline, Kelly M; Fries, Chelsea N; Collier, Joel H

    2018-03-01

    Supramolecular materials composed of proteins and peptides have been receiving considerable attention toward a range of diseases and conditions from vaccines to drug delivery. Owing to the relative newness of this class of materials, the bulk of work to date has been preclinical. However, examples of approved treatments particularly in vaccines, dentistry, and hemostasis demonstrate the translational potential of supramolecular polypeptides. Critical milestones in the clinical development of this class of materials and currently approved supramolecular polypeptide therapies are described in this study. Additional examples of not-yet-approved materials that are steadily advancing toward clinical use are also featured. Spherical assemblies such as virus-like particles, designed protein nanoparticles, and spherical peptide amphiphiles are highlighted, followed by fiber-forming systems such as fibrillizing peptides, fiber-forming peptide-amphiphiles, and filamentous bacteriophages. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Science.gov (United States)

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

    2014-06-01

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

  1. Chronic Ethanol Consumption in Mice Alters Hepatocyte Lipid Droplet Properties

    Science.gov (United States)

    Orlicky, David J.; Roede, James R.; Bales, Elise; Greenwood, Carrie; Greenberg, Andrew; Petersen, Dennis; McManaman, James L.

    2014-01-01

    Background Hepatosteatosis is a common pathological feature of impaired hepatic metabolism following chronic alcohol consumption. Although often benign and reversible, it is widely believed that steatosis is a risk factor for development of advanced liver pathologies, including steatohepatitis and fibrosis. The hepatocyte alterations accompanying the initiation of steatosis are not yet clearly defined. Methods Induction of hepatosteatosis by chronic ethanol consumption was investigated using the Lieber-DeCarli (LD) high fat diet model. Effects were assessed by immunohistochemistry and blood and tissue enzymatic assays. Cell culture models were employed for mechanistic studies. Results Pair feeding mice ethanol (LD-Et) or isocaloric control (LD-Co) diets for 6 weeks progressively increased hepatocyte triglyceride accumulation in morphological, biochemical, and zonally distinct cytoplasmic lipid droplets (CLD). The LD-Et diet induced zone 2-specific triglyceride accumulation in large CLD coated with perilipin, adipophilin (ADPH), and TIP47. In LD-Co- fed mice, CLD were significantly smaller than those in LD-Et-fed mice and lacked perilipin. A direct role of perilipin in formation of large CLD was further suggested by cell culture studies showing that perilipin-coated CLD were significantly larger than those coated with ADPH or TIP47. LD-Co- and LD-Et-fed animals also differed in hepatic metabolic stress responses. In LD-Et but not LD-Co-fed mice, inductions were observed in the following: microsomal ethanol-oxidizing system [cytochrome P-4502E1 (CYP2E1)], hypoxia response pathway (hypoxia-inducible factor 1 alpha, HIF1α), endoplasmic reticulum stress pathway (calreticulin), and synthesis of lipid peroxidation products [4-hydroxynonenal (4-HNE)]. CYP2E1 and HIF1 α immunostaining localized to zone 3 and did not correlate with accumulation of large CLD. In contrast, calreticulin and 4-HNE immunostaining closely correlated with large CLD accumulation. Importantly, 4

  2. DDX4 (DEAD box polypeptide 4) colocalizes with cancer stem cell marker CD133 in ovarian cancers

    International Nuclear Information System (INIS)

    Kim, Ki Hyung; Kang, Yun-Jeong; Jo, Jin-Ok; Ock, Mee Sun; Moon, Soo Hyun; Suh, Dong Soo; Yoon, Man Soo; Park, Eun-Sil; Jeong, Namkung; Eo, Wan-Kyu; Kim, Heung Yeol; Cha, Hee-Jae

    2014-01-01

    Highlights: • Germ cell marker DDX4 was significantly increased in ovarian cancer. • Ovarian cancer stem cell marker CD133 was significantly increased in ovarian cancer. • DDX4 and CD133 were mostly colocalized in various types of ovarian cancer tissues. • CD133 positive ovarian cancer cells also express DDX4 whereas CD133-negative cells did not possess DDX4. • Germ cell marker DDX4 has the potential of ovarian cancer stem cell marker. - Abstract: DDX4 (DEAD box polypeptide 4), characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD), is an RNA helicase which is implicated in various cellular processes involving the alteration of RNA secondary structure, such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. DDX4 is known to be a germ cell-specific protein and is used as a sorting marker of germline stem cells for the production of oocytes. A recent report about DDX4 in ovarian cancer showed that DDX4 is overexpressed in epithelial ovarian cancer and disrupts a DNA damage-induced G2 checkpoint. We investigated the relationship between DDX4 and ovarian cancer stem cells by analyzing the expression patterns of DDX4 and the cancer stem cell marker CD133 in ovarian cancers via tissue microarray. Both DDX4 and CD133 were significantly increased in ovarian cancer compared to benign tumors, and showed similar patterns of expression. In addition, DDX4 and CD133 were mostly colocalized in various types of ovarian cancer tissues. Furthermore, almost all CD133 positive ovarian cancer cells also express DDX4 whereas CD133-negative cells did not possess DDX4, suggesting a strong possibility that DDX4 plays an important role in cancer stem cells, and/or can be used as an ovarian cancer stem cell marker

  3. Amyloid Structure and Assembly: Insights from Scanning Transmission Electron Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Goldsbury, C.; Wall, J.; Baxa, U.; Simon, M. N.; Steven, A. C.; Engel, A.; Aebi, U.; Muller, S. A.

    2011-01-01

    Amyloid fibrils are filamentous protein aggregates implicated in several common diseases such as Alzheimer's disease and type II diabetes. Similar structures are also the molecular principle of the infectious spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, scrapie in sheep, and of the so-called yeast prions, inherited non-chromosomal elements found in yeast and fungi. Scanning transmission electron microscopy (STEM) is often used to delineate the assembly mechanism and structural properties of amyloid aggregates. In this review we consider specifically contributions and limitations of STEM for the investigation of amyloid assembly pathways, fibril polymorphisms and structural models of amyloid fibrils. This type of microscopy provides the only method to directly measure the mass-per-length (MPL) of individual filaments. Made on both in vitro assembled and ex vivo samples, STEM mass measurements have illuminated the hierarchical relationships between amyloid fibrils and revealed that polymorphic fibrils and various globular oligomers can assemble simultaneously from a single polypeptide. The MPLs also impose strong constraints on possible packing schemes, assisting in molecular model building when combined with high-resolution methods like solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR).

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

    Science.gov (United States)

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

    2014-03-01

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

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

    Science.gov (United States)

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

    2016-10-28

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

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

    Science.gov (United States)

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

    2013-07-01

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

  7. Polypeptide composition of fraction 1 protein of the somatic hybrid between Petunia parodii and Petunia parviflora.

    Science.gov (United States)

    Kumar, A; Wilson, D; Cocking, E C

    1981-04-01

    The analysis of the subunit polypeptide composition of Fraction 1 protein provides information on the expression of both chloroplast and nuclear genomes. Fraction 1 protein, isolated from leaves of the somatic hybrid plants derived form the fusion of protoplasts of Petunia parodii and P. parviflora, was analyzed for its subunit polypeptide composition by isoelectric focusing in 8 M urea. The fraction 1 protein enzyme oligomer in the somatic hybrid plants contained small subunits resulting from the expression of both parental nuclear genomes, but probably only one of the parental large subunits, namely that of P. parodii. The relevance of such somatic hybrid material for the study of nucleocytoplasmic interrelationship is discussed, as well as the use of these fraction 1 protein isoelectric focusing patterns for the analysis of taxonomic relationships in Petunia.

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

    Science.gov (United States)

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

    2018-06-12

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

  9. Requirements of glycerol and fatty acid for triglyceride synthesis and ketogenesis by hepatocytes from normal and triiodothyronine-treated rats

    International Nuclear Information System (INIS)

    Olubadewo, J.O.; Heimberg, M.

    1985-01-01

    Hepatocytes from T3-treated rats synthesized less triglyceride and more ketone bodies from [1- 14 C]oleate at all concentrations from 0-2 mM, than did hepatocytes from euthyroid animals; addition of 1.0 mM glycerol increased triglyceride synthesis and reduced ketogenesis in hepatocytes from T3-treated rats to the rates observed in euthyroid hepatocytes in the absence of added glycerol. Glycerol did not alter triglyceride synthesis, but reduced ketogenesis genesis by euthyroid hepatocytes. It is probable from these and other data that, in the hyperthyroid rat, glycero-3-P, and not fatty acid, is rate limiting for synthesis of triglyceride, and, secondarily for reducing rates of ketogenesis in the hepatocyte

  10. Spinoculation Enhances HBV Infection in NTCP-Reconstituted Hepatocytes

    Science.gov (United States)

    Yan, Ran; Zhang, Yongmei; Cai, Dawei; Liu, Yuanjie; Cuconati, Andrea; Guo, Haitao

    2015-01-01

    Hepatitis B virus (HBV) infection and its sequelae remain a major public health burden, but both HBV basic research and the development of antiviral therapeutics have been hindered by the lack of an efficient in vitro infection system. Recently, sodium taurocholate cotransporting polypeptide (NTCP) has been identified as the HBV receptor. We herein report that we established a NTCP-complemented HepG2 cell line (HepG2-NTCP12) that supports HBV infection, albeit at a low infectivity level following the reported infection procedures. In our attempts to optimize the infection conditions, we found that the centrifugation of HepG2-NTCP12 cells during HBV inoculation (termed “spinoculation”) significantly enhanced the virus infectivity. Moreover, the infection level gradually increased with accelerated speed of spinoculation up to 1,000g tested. However, the enhancement of HBV infection was not significantly dependent upon the duration of centrifugation. Furthermore, covalently closed circular (ccc) DNA was detected in infected cells under optimized infection condition by conventional Southern blot, suggesting a successful establishment of HBV infection after spinoculation. Finally, the parental HepG2 cells remained uninfected under HBV spinoculation, and HBV entry inhibitors targeting NTCP blocked HBV infection when cells were spinoculated, suggesting the authentic virus entry mechanism is unaltered under centrifugal inoculation. Our data suggest that spinoculation could serve as a standard protocol for enhancing the efficiency of HBV infection in vitro. PMID:26070202

  11. Somatomedin-C stimulates glycogen synthesis in fetal rat hepatocytes

    International Nuclear Information System (INIS)

    Freemark, M.; D'Ercole, A.J.; Handwerger, S.

    1985-01-01

    The effects of somatomedin-C/insulin-like growth factor I (Sm-C) on glycogen metabolism in cultured hepatocytes from 20-day-old rat fetuses have been examined and compared with the effects of insulin. Sm-C (25-375 ng/ml; 3.25-50 nM) stimulated dose-dependent increases in [ 14 C]glucose incorporation into glycogen (14.4-72.9% and total cell glycogen content (10.6-34.3%. Maximal stimulation of glycogen synthesis by Sm-C occurred at 2-4 h of incubation. Insulin (10 nM to 10 microM) also stimulated [ 14 C]glucose incorporation but its potency was only 1/20th that of Sm-C. The time course of stimulation of glucose incorporation by insulin was identical to that of Sm-C, the dose-response curves of the two hormones were parallel, and the maximal effects of insulin were not enhanced by simultaneous exposure of cells to Sm-C. These findings suggest that Sm-C and insulin stimulate glycogenesis in fetal liver through similar or identical mechanisms. Since the potency of Sm-C was 20 times greater than that of insulin, the glycogenic action of insulin in fetal liver may be mediated through binding to a hepatic receptor which also binds Sm-C. In addition to having mitogenic effects on fetal tissues, Sm-C may have direct anabolic effects on fetal carbohydrate metabolism

  12. Protein phosphorylation in isolated hepatocytes of septic and endotoxemic rats

    International Nuclear Information System (INIS)

    Deaciuc, I.V.; Spitzer, J.A.

    1989-01-01

    The purpose of this study was to investigate possible alterations induced by sepsis and endotoxicosis in the late phase of Ca2+-dependent signaling in rat liver. Hepatocytes isolated from septic or chronically endotoxin (ET)-treated rats were labeled with [32P]H3PO4 and stimulated with various agents. Proteins were resolved by one-dimensional polyacrylamide gel electrophoresis and autoradiographed. Vasopressin (VP)- and phenylephrine (PE)-induced responses were attenuated in both septic and ET-treated rats for cytosolic and membrane proteins compared with their respective controls. Glucagon and 12-O-myristate phorbol-13-acetate (TPA) affected only the phosphorylation of membrane proteins. Glucagon-induced changes in the phosphorylation of membrane proteins were affected by both sepsis and endotoxicosis, whereas TPA-stimulated phosphorylation was lowered only in endotoxicosis. Response to the Ca2+ ionophore A23187 was depressed in septic rats for cytosolic proteins. The phosphorylation of two cytosolic proteins, i.e., 93 and 61 kDa (previously identified as glycogen phosphorylase and pyruvate kinase, respectively), in response to VP, PE, and A23187 was severely impaired by endotoxicosis and sepsis. TPA did not affect the phosphorylation state of these two proteins. The results show that sepsis and endotoxicosis produce perturbations of the phosphorylation step in Ca2+ transmembrane signaling. Such changes can explain alterations of glycogenolysis and gluconeogenesis associated with sepsis and endotoxicosis

  13. Stabilization of glucocorticoid receptors in isolated rat hepatocytes by radioprotectants

    International Nuclear Information System (INIS)

    Karle, J.M.; Ridder, W.E.; Wright, N.; Olmeda, R.; Nielsen, C.J.

    1986-01-01

    Previous work has shown that glucocorticoid receptors in rat liver homogenate can be stabilized by the addition of MoO 4 plus the sulfhydryl-containing compounds dithiothreitol and WR 1065. The latter is the dephosphorylated, principal metabolite of the radioprotectant WR 2721 (or S-2-(3-aminopropylamino)ethanesphosphorothioic acid). The current work results from applying this knowledge to intact rat hepatocytes. Cells were isolated by collagenase perfusion and incubated in supplemented minimum essential medium at 37 0 C with various concentrations of WR 2721, WR 1065, or vehicle. Samples of these cell suspensions were analyzed at various times for steroid binding capacity by incubating homogenates (27,000 x g supernates) with 50 nM 3 H-triamcinolone acetonide in the presence or absence of excess unlabelled dexamethasone. Concentrations of 10 mM WR 2721 provided marked preservation of the binding capacity (>85% of the initial value at 5 hours) compared to control at 60% of the binding capacity. WR 1065 at 10 mM provided no such protection. This is consistent with the observation that WR 1065 does not pass cell membranes. The authors propose that supplying reducing equivalents to intracellular components such as the glucocorticoid receptor may be one mechanism of the radioprotection afforded by WR 2721

  14. Relationship of histochemically detectable altered hepatocyte foci to hepatic tumorigenesis

    Energy Technology Data Exchange (ETDEWEB)

    Peraino, C.; Staffeldt, E.F.; Carnes, B.A.; Ludeman, V.A.; Blomquist, J.A.; Vesselinovitch, S.D.

    1984-01-01

    A new experimental system was used to examine the stages of chemically induced hepatic neoplasia in the rat. The treatment protocol involved the intraperitoneal injection of a single non-necrogenic dose of carcinogen (N-nitrosodiethylamine (NDEA) or benzo(a)pyrene (BP)) into male and female rats within one day after birth, followed by dietary exposure to promoter (0.05% phenobarbital) from weaning. Rats were killed at intervals, and their livers were examined for tumors and for histochemically detectable foci of altered hepatocytes. The data showed that (1) the new treatment protocol was highly efficient in foci and tumor production; (2) growth rates and incidence levels of foci were directly related to hepatocarcinogenic effectiveness (NDEA > BP), whereas both carcinogens had similar effects on foci phenotypic properties; (3) after their formation, foci at a given level of phenotypic complexity did not become progressively more complex; (4) incidence levels of foci were sex-dependent (females > males), but growth rates of foci were the same for both sexes; (5) growth rates and growth capacities (ranges of possible growth rates) of foci were directly related to phenotypic complexity levels of foci; (6) frequencies and phenotypic complexities of foci were inversely related; the reverse was true for tumors, although 10% of the tumors were relatively simple (three markers or fewer); (7) marker frequency distribution patterns were completely different in foci and in tumors.

  15. Anabolic regulation of gluconeogenesis by insulin in isolated rat hepatocytes

    International Nuclear Information System (INIS)

    Mohan, C.; Bessman, S.P.

    1985-01-01

    The role of substrate availability in the regulation of gluconeogenesis in isolated rat hepatocytes was studied using [U- 14 C]alanine as a tracer in the presence of different concentrations of L-alanine in the incubation medium. At low alanine concentrations (0.5 mM) insulin decreased the 14 C incorporation into the glucose pool and increased the incorporation of tracer carbons into the protein and lipid pools and into CO 2 . The net radioactivity lost from the glucose pool was only a small percentage of the total increase in the activity of the protein, lipid, CO 2 , or glycogen pools, supporting the notion that the effect of insulin in diminishing gluconeogenesis is secondary to its effects on pathways using pyruvate. At higher concentrations of alanine (2.5, 5.0, and 10.0 mM) in the incubation medium insulin increased the movement of alanine carbons into protein and glucose. These results were further confirmed by using [U- 14 C]lactate. The increases in observed specific activity of glucose following insulin administration would not be possible if insulin acted by affecting the activity of any enzyme directly involved in the formation or utilization of pyruvate, most of which have been proposed as sites of insulin action. Data presented show that insulin inhibits gluconeogenesis by affecting a change in substrate availability

  16. Nuclear lactate dehydrogenase modulates histone modification in human hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Castonguay, Zachary; Auger, Christopher; Thomas, Sean C.; Chahma, M’hamed; Appanna, Vasu D., E-mail: vappanna@laurentian.ca

    2014-11-07

    Highlights: • Nuclear LDH is up-regulated under oxidative stress. • SIRT1 is co-immunoprecipitated bound to nuclear LDH. • Nuclear LDH is involved in histone deacetylation and epigenetics. - Abstract: It is becoming increasingly apparent that the nucleus harbors metabolic enzymes that affect genetic transforming events. Here, we describe a nuclear isoform of lactate dehydrogenase (nLDH) and its ability to orchestrate histone deacetylation by controlling the availability of nicotinamide adenine dinucleotide (NAD{sup +}), a key ingredient of the sirtuin-1 (SIRT1) deacetylase system. There was an increase in the expression of nLDH concomitant with the presence of hydrogen peroxide (H{sub 2}O{sub 2}) in the culture medium. Under oxidative stress, the NAD{sup +} generated by nLDH resulted in the enhanced deacetylation of histones compared to the control hepatocytes despite no discernable change in the levels of SIRT1. There appeared to be an intimate association between nLDH and SIRT1 as these two enzymes co-immunoprecipitated. The ability of nLDH to regulate epigenetic modifications by manipulating NAD{sup +} reveals an intricate link between metabolism and the processing of genetic information.

  17. Translation of mitochondrial proteins in digitonin-treated rat hepatocytes

    International Nuclear Information System (INIS)

    Kuzela, S.; Wielburski, A.; Nelson, B.D.

    1981-01-01

    Although it is now clear that up to 13 peptides may be encoded in mammalian mitochondrial DNA there is little agreement concerning the numbers of stable translation products detectable in these mitochondria. Part of this uncertainty is due to the low rates of labeling of mammalian mitochondrial translations products resulting from the relatively slow growth rates of mammalian cells. Indeed, it is often necessary to isolate mammalian mitochondria in order to analyze their translation products, and the isolation procedures could conceivably lead to artifacts from proteolysis or from the early release of nascent peptides. To circumvent this problem, it would be desirable to have available a mammalian system which combines the advantage of high rates of labeling of mitochondrial proteins with rapid preparation times. The authors report the novel use of digitonin-treated rat hepatocytes, which provide such a system. This preparation, which is complete in <10 min, does not carry out cytosolic protein synthesis, but labels mitochondrial translation products at rates much higher than intact cells or isolated, in vitro labeled mitochondria. (Auth.)

  18. Hepatocyte cotransport of taurocholate and bilirubin glucuronides: Role of microtubules

    International Nuclear Information System (INIS)

    Crawford, J.M.; Gollan, J.L.

    1988-01-01

    Modulation of bile pigment excretion by bile salts has been attributed to modification of canalicular membrane transport or a physical interaction in bile. Based on the observation that a microtubule-dependent pathway is involved in the hepatocellular transport of bile salts, the authors investigated the possibility that bilirubin glucuronides are associated with bile salts during intracellular transport. Experiments were conducted in intact rats (basal) or after overnight biliary diversion and intravenous reinfusion of taurocholate (depleted/reinfused). All rats were pretreated with intravenous low-dose colchicine or its inactive isomer lumicolchicine. Biliary excretion of radiolabeled bilirubin glucuronides derived from tracer [ 14 C]bilirubin-[ 3 H]bilirubin monoglucuronide (coinjected iv) was unchanged in basal rats but was consistently delayed in depleted/reinfused rats. This was accompanied by a significant shift toward bilirubin diglucuronide formation from both substrates. In basal Gunn rats, with deficient bilirubin glucuronidation, biliary excretion of intravenous [ 14 C]bilirubin monoglucuronide-[ 3 H]bilirubin diglucuronide was unaffected by colchicine but was retarded in depleted/reinfused Gunn rats. Colchicine had no effect on the rate of bilirubin glucuronidation in vitro in rat liver microsomes. They conclude that a portion of the bilirubin glucuronides generated endogenously in hepatocytes or taken up directly from plasma may be cotransported with bile salts to the bile canalicular membrane via a microtubule-dependent mechanism

  19. [Cadmium citotoxicity in mice hepatocytes and implications on tropical environments].

    Science.gov (United States)

    Marcano, Letty; Faría, Clarisa de R; Carruyo, Ingrid; Montiel, Xiomara

    2006-06-01

    We analyzed phenotypic, structural and ultrastructural alterations induced by Cd+2 in hepatocytes extracted from Swiss Albino mice. Cadmium was given orally in watery solution of CdCl2 during 100 days at concentrations of 50 ppm, 100 ppm and 150 ppm. In controls, distilled water alone was used. The samples were processed with the paraffin inclusion and hematoxilin-eosin coloration techniques for light microscopy. For transmission electron microscopy we used the conventional technique. We found phenotypic (size and weight differences) and physiologic changes (muscular weakness, unrest); at the structural level we noticed loss of trabecular disposition and of lobulillar architecture, lymphocyte agglomeration, vacuolization, dilatation of sinusoid and central vein, among others. The ultrastructural study evidenced alterations coincident with those seen with light microscopy, which were accentuated with the increase of metal concentration: nucleolus with a high number of fibrillar centers (50 ppm); voluminous lipidic drops in the cytoplasm, loose endoplasmic rough reticulum, citoplasmatic vacuolization, altered lisosomes and peroxisomes (100 ppm); contracted nuclei with condensed cromatine, dilatation of intracellular space and mitochondria, and loss of fibrillar areas (150 ppm). Cadmium produces a toxic effect in the hepatic cells; the effect is more severe at higher concentration, leading to cellular necrosis.

  20. Hepatocyte growth factor in renal failure: promise and reality.

    Science.gov (United States)

    Vargas, G A; Hoeflich, A; Jehle, P M

    2000-04-01

    Can science discover some secrets of Greek mythology? In the case of Prometheus, we can now suppose that his amazing hepatic regeneration was caused by a peptide growth factor called hepatocyte growth factor (HGF). Increasing evidence indicates that HGF acts as a multifunctional cytokine on different cell types. This review addresses the molecular mechanisms that are responsible for the pleiotropic effects of HGF. HGF binds with high affinity to its specific tyrosine kinase receptor c-met, thereby stimulating not only cell proliferation and differentiation, but also cell migration and tumorigenesis. The three fundamental principles of medicine-prevention, diagnosis, and therapy-may be benefited by the rational use of HGF. In renal tubular cells, HGF induces mitogenic and morphogenetic responses. In animal models of toxic or ischemic acute renal failure, HGF acts in a renotropic and nephroprotective manner. HGF expression is rapidly up-regulated in the remnant kidney of nephrectomized rats, inducing compensatory growth. In a mouse model of chronic renal disease, HGF inhibits the progression of tubulointerstitial fibrosis and kidney dysfunction. Increased HGF mRNA transcripts were detected in mesenchymal and tubular epithelial cells of rejecting kidney. In transplanted patients, elevated HGF levels may indicate renal rejection. When HGF is considered as a therapeutic agent in human medicine, for example, to stimulate kidney regeneration after acute injury, strategies need to be developed to stimulate cell regeneration and differentiation without an induction of tumorigenesis.

  1. Acylation and metabolism of (n-6) fatty acids in hepatocytes

    International Nuclear Information System (INIS)

    Voss, A.C.; Sprecher, H.

    1986-01-01

    Isolated hepatocytes (5 x 10 6 in 2ml) from chow fed rats were incubated from 20 to 60 min. with increasing concentrations of [1- 14 C] labeled 18:2 (n-6), 18:3 (n-6) or 20:3 (n-6) to define optimum conditions for measuring acylation and metabolism to other (n-6) acids with subsequent incorporation into lipids. The triglycerides (TG) and phospholipids (PL) contained 157 and 80 nmols of 18:2 (n-6) and 6.0 and 6.1 nmols of other (n-6) acids, respectively, when cells were incubated with 0.3mM [1- 14 C] 18:2 (n-6) for 40 min. When cells were incubated with 0.3mM [1- 14 C] 18:2 (n-6) plus 0.15 to 0.45mM 18:3 (n-6) or 20:3 (n-6), the metabolism of 18:2 (n-6) to other (n-6) acids was inhibited but not totally abolished. These results may suggest that (n-6) acid made from linoleate do not totally equilibrate with exogenous 18:3 (n-6) or 20:3

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

    Science.gov (United States)

    Yang, Zhenbiao; Karr, Stephen

    2014-02-11

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

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

    OpenAIRE

    Arnelo, Urban

    1997-01-01

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

  4. Sustained Release of Antibiotics from Injectable and Thermally Responsive Polypeptide Depots

    OpenAIRE

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

    2009-01-01

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

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

    International Nuclear Information System (INIS)

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

    1977-01-01

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

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

    OpenAIRE

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

    1981-01-01

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

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

    International Nuclear Information System (INIS)

    Perly, Bruno

    1980-01-01

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

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

    Science.gov (United States)

    Gharakhanian, Eric G; Deming, Timothy J

    2016-07-07

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

  9. Assembly tool design

    International Nuclear Information System (INIS)

    Kanamori, Naokazu; Nakahira, Masataka; Ohkawa, Yoshinao; Tada, Eisuke; Seki, Masahiro

    1996-06-01

    The reactor core of the International Thermonuclear Experimental Reactor (ITER) is assembled with a number of large and asymmetric components within a tight tolerance in order to assure the structural integrity for various loads and to provide the tritium confinement. In addition, the assembly procedure should be compatible with remote operation since the core structures will be activated by 14-MeV neutrons once it starts operation and thus personal access will be prohibited. Accordingly, the assembly procedure and tool design are quite essential and should be designed from the beginning to facilitate remote operation. According to the ITER Design Task Agreement, the Japan Atomic Energy Research Institute (JAERI) has performed design study to develop the assembly procedures and associated tool design for the ITER tokamak assembly. This report describes outlines of the assembly tools and the remaining issues obtained in this design study. (author)

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

    Directory of Open Access Journals (Sweden)

    Carmen M. González-Henríquez

    2017-10-01

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

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

    Science.gov (United States)

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

    2018-06-11

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

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

    Science.gov (United States)

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

    2018-04-30

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

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

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

    Science.gov (United States)

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

    2016-12-01

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

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

    Science.gov (United States)

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

    2007-01-01

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

  16. Effect of hepatocyte growth factor on radiation response of HeLa, V79, CHO and primary cultured parenchymal hepatocyte in vitro

    International Nuclear Information System (INIS)

    Yamazaki, Hideya; Inoue, Takehiro; Nose, Takayuki; Murayama, Shigeyuki; Teshima, Teruki; Ozeki, Syuji; Koizumi, Masahiko; Inoue, Toshihiko.

    1996-01-01

    Hepatocyte growth factor (HGF) is a multipotent cytokine enhancing regeneration of injured organs as liver, kidney and lung after injury. HGF enhances proliferation of various type of cells, inhibits proliferation of carcinoma cells, enhances motility of epithelial cells. We examined three cell lines (CHO, HeLa, V79) and primary cultured normal rat parenchymal hepatocytes to determine the effect of HGF on radiation response. HGF diminished survival of CHO and V79 cells determined by colony formation assay, whereas no significant change of survival was found in HeLa cells. No synergistic changes of survival were found when these three cell lines were irradiated with the addition of HGF. Thus, HGF did not enhance the radiation effect. We also analyzed the impact of irradiation with HGF on primary cultured normal rat parenchymal hepatocytes. At first, the release of glutamic-oxaloacetic amino-transaminase (GOT) in the supernatant was estimated. Irradiation (40 Gy) with or without HGF did not change GOT release in acute phase by 4 days after irradiation compared with the unirradiated control. Second, the DNA synthesis of rat parenchymal hepatocytes was analyzed using radioactive iodine-labeled deoxyuridine incorporation. HGF counteracted the suppression of DNA synthesis induced by irradiation. Thus, HGF may act as a mitogen even for irradiation-damaged normal cells. (author)

  17. Fuel assembly storage pool

    International Nuclear Information System (INIS)

    Hiranuma, Hiroshi.

    1976-01-01

    Object: To remove limitation of the number of storage of fuel assemblies to increase the number of storage thereof so as to relatively reduce the water depth required for shielding radioactive rays. Structure: Fuel assembly storage rack containers for receiving a plurality of spent fuel assembly racks are stacked in multi-layer fashion within a storage pool filled with water for shielding radioactive rays and removing heat. (Furukawa, Y.)

  18. Hepatocyte polarization is essential for the productive entry of the hepatitis B virus.

    Science.gov (United States)

    Schulze, Andreas; Mills, Kerry; Weiss, Thomas S; Urban, Stephan

    2012-02-01

    Human hepatitis B virus (HBV) is characterized by a high species specificity and a distinct liver tropism. Within the liver, HBV replication occurs in differentiated and polarized hepatocytes. Accordingly, the in vitro HBV infection of primary human hepatocytes (PHHs) and the human hepatoma cell line, HepaRG, is restricted to differentiated, hepatocyte-like cells. Though preparations of PHH contain up to 100% hepatic cells, cultures of differentiated HepaRG cells are a mixture of hepatocyte-like and biliary-like epithelial cells. We used PHH and HepaRG cells and compared the influence of virus inoculation dose, cell differentiation, and polarization on productive HBV infection. At multiplicities of genome equivalents (mge) >8,000, almost 100% of PHHs could be infected. In contrast, only a subset of HepaRG cells stained positive for HBcAg at comparable or even higher mge. Infection predominantly occurred at the edges of islands of hepatocyte-like HepaRG cells. This indicates a limited accessibility of the HBV receptor, possibly as a result of its polar sorting. Multidrug resistance protein 2 (MRP2), a marker selectively transported to the apical (i.e., canalicular) cell membrane, revealed two polarization phenotypes of HepaRG cells. HBV infection within the islands of hepatocyte-like HepaRG cells preferentially occurred in cells that resemble PHH, exhibiting canalicular structures. However, disruption of cell-cell junctions allowed the additional infection of cells that do not display a PHH-like polarization. HBV enters hepatocytes via the basolateral membrane. This model, at least partially, explains the difference of PHH and HepaRG cells in infection efficacy, provides insights into natural HBV infection, and establishes a basis for optimization of the HepaRG infection system. Copyright © 2011 American Association for the Study of Liver Diseases.

  19. β-Adrenergic induction of lipolysis in hepatocytes is inhibited by ethanol exposure.

    Science.gov (United States)

    Schott, Micah B; Rasineni, Karuna; Weller, Shaun G; Schulze, Ryan J; Sletten, Arthur C; Casey, Carol A; McNiven, Mark A

    2017-07-14

    In liver steatosis ( i.e. fatty liver), hepatocytes accumulate many large neutral lipid storage organelles known as lipid droplets (LDs). LDs are important in the maintenance of energy homeostasis, but the signaling mechanisms that stimulate LD metabolism in hepatocytes are poorly defined. In adipocytes, catecholamines target the β-adrenergic (β-AR)/cAMP pathway to activate cytosolic lipases and induce their recruitment to the LD surface. Therefore, the goal of this study was to determine whether hepatocytes, like adipocytes, also undergo cAMP-mediated lipolysis in response to β-AR stimulation. Using primary rat hepatocytes and human hepatoma cells, we found that treatment with the β-AR agent isoproterenol caused substantial LD loss via activation of cytosolic lipases adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). β-Adrenergic stimulation rapidly activated PKA, which led to the phosphorylation of ATGL and HSL and their recruitment to the LD surface. To test whether this β-AR-dependent lipolysis pathway was altered in a model of alcoholic fatty liver, primary hepatocytes from rats fed a 6-week EtOH-containing Lieber-DeCarli diet were treated with cAMP agonists. Compared with controls, EtOH-exposed hepatocytes showed a drastic inhibition in β-AR/cAMP-induced LD breakdown and the phosphorylation of PKA substrates, including HSL. This observation was supported in VA-13 cells, an EtOH-metabolizing human hepatoma cell line, which displayed marked defects in both PKA activation and isoproterenol-induced ATGL translocation to the LD periphery. In summary, these findings suggest that β-AR stimulation mobilizes cytosolic lipases for LD breakdown in hepatocytes, and perturbation of this pathway could be a major consequence of chronic EtOH insult leading to fatty liver.

  20. Lipopolysaccharide stimulates p62-dependent autophagy-like aggregate clearance in hepatocytes.

    Science.gov (United States)

    Chen, Christine; Deng, Meihong; Sun, Qian; Loughran, Patricia; Billiar, Timothy R; Scott, Melanie J

    2014-01-01

    Impairment of autophagy has been associated with liver injury. TLR4-stimulation by LPS upregulates autophagy in hepatocytes, although the signaling pathways involved remain elusive. The objective of this study was to determine the signaling pathway leading to LPS-stimulated autophagy in hepatocytes. Cell lysates from livers of wild type (WT; C57BL/6) mice given LPS (5 mg/kg-IP) and hepatocytes from WT, TLR4ko, and MyD88ko mice treated with LPS (100 ng/mL) up to 24 h were collected. LC3II, p62/SQSTM1, Nrf2, and beclin1 levels were determined by immunoblot, immunofluorescence, and qPCR. Autophagy-like activation was measured by GFP-LC3-puncta formation and LC3II-expression. Beclin1, Nrf2, p62, MyD88, and TIRAP were knocked-down using siRNA. LC3II-expression increased in both liver and hepatocytes after LPS and was dependent on TLR4. Beclin1 expression did not increase after LPS in hepatocytes and beclin1-knockdown did not affect LC3II levels. In hepatocytes given LPS, expression of p62 increased and p62 colocalized with LC3. p62-knockdown prevented LC3II puncta formation. LPS-induced LC3II/p62-puncta also required MyD88/TIRAP signaling and localization of both Nrf2 and NF κ B transcription factors to the nucleus to upregulate p62-expression. Therefore, TLR4-activation by LPS in hepatocytes induces a p62-mediated, not beclin1-mediated, autophagy-like clearance pathway that is hepatoprotective by clearing aggregate-prone or misfolded proteins from the cytosol and preserving energy homeostasis under stress.

  1. DISTINCT FUNCTIONS OF JNK AND C-JUN IN OXIDANT-INDUCED HEPATOCYTE DEATH

    Science.gov (United States)

    Amir, Muhammad; Liu, Kun; Zhao, Enpeng; Czaja, Mark J.

    2013-01-01

    Overactivation of c-Jun N-terminal kinase (JNK)/c-Jun signaling is a central mechanism of hepatocyte injury and death including that from oxidative stress. However, the functions of JNK and c-Jun are still unclear, and this pathway also inhibits hepatocyte death. Previous studies of menadione-induced oxidant stress demonstrated that toxicity resulted from sustained JNK/c-Jun activation as death was blocked by the c-Jun dominant negative TAM67. To further delineate the function of JNK/c-Jun signaling in hepatocyte injury from oxidant stress, the effects of direct JNK inhibition on menadione-induced death were examined. In contrast to the inhibitory effect of TAM67, pharmacological JNK inhibition by SP600125 sensitized the rat hepatocyte cell line RALA255-10G to death from menadione. SP600125 similarly sensitized mouse primary hepatocytes to menadione toxicity. Death from SP600125/menadione was c-Jun dependent as it was blocked by TAM67, but independent of c-Jun phosphorylation. Death occurred by apoptosis and necrosis and activation of the mitochondrial death pathway. Short hairpin RNA knockdowns of total JNK or JNK2 sensitized to death from menadione, whereas a jnk1 knockdown was protective. Jnk2 null mouse primary hepatocytes were also sensitized to menadione death. JNK inhibition magnified decreases in cellular ATP content and β-oxidation induced by menadione. This effect mediated cell death as chemical inhibition of β-oxidation also sensitized cells to death from menadione, and supplementation with the β-oxidation substrate oleate blocked death. Components of the JNK/c-Jun signaling pathway have opposing functions in hepatocyte oxidant stress with JNK2 mediating resistance to cell death and c-Jun promoting death. PMID:22644775

  2. Functional properties of hepatocytes in vitro are correlated with cell polarity maintenance.

    Science.gov (United States)

    Zeigerer, Anja; Wuttke, Anne; Marsico, Giovanni; Seifert, Sarah; Kalaidzidis, Yannis; Zerial, Marino

    2017-01-01

    Exploring the cell biology of hepatocytes in vitro could be a powerful strategy to dissect the molecular mechanisms underlying the structure and function of the liver in vivo. However, this approach relies on appropriate in vitro cell culture systems that can recapitulate the cell biological and metabolic features of the hepatocytes in the liver whilst being accessible to experimental manipulations. Here, we adapted protocols for high-resolution fluorescence microscopy and quantitative image analysis to compare two primary hepatocyte culture systems, monolayer and collagen sandwich, with respect to the distribution of two distinct populations of early endosomes (APPL1 and EEA1-positive), endocytic capacity, metabolic and signaling activities. In addition to the re-acquisition of hepatocellular polarity, primary hepatocytes grown in collagen sandwich but not in monolayer culture recapitulated the apico-basal distribution of EEA1 endosomes observed in liver tissue. We found that such distribution correlated with the organization of the actin cytoskeleton in vitro and, surprisingly, was dependent on the nutritional state in vivo. Hepatocytes in collagen sandwich also exhibited faster kinetics of low-density lipoprotein (LDL) and epidermal growth factor (EGF) internalization, showed improved insulin sensitivity and preserved their ability for glucose production, compared to hepatocytes in monolayer cultures. Although no in vitro culture system can reproduce the exquisite structural features of liver tissue, our data nevertheless highlight the ability of the collagen sandwich system to recapitulate key structural and functional properties of the hepatocytes in the liver and, therefore, support the usage of this system to study aspects of hepatocellular biology in vitro. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Oxidative stress is involved in Dasatinib-induced apoptosis in rat primary hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Xue, Tao; Luo, Peihua; Zhu, Hong; Zhao, Yuqin [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Wu, Honghai; Gai, Renhua; Wu, Youping [Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou 310058 (China); Yang, Bo [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Yang, Xiaochun, E-mail: yangxiaochun@zju.edu.cn [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou 310058 (China); He, Qiaojun, E-mail: qiaojunhe@zju.edu.cn [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou 310058 (China)

    2012-06-15

    Dasatinib, a multitargeted inhibitor of BCR–ABL and SRC kinases, exhibits antitumor activity and extends the survival of patients with chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL). However, some patients suffer from hepatotoxicity, which occurs through an unknown mechanism. In the present study, we found that Dasatinib could induce hepatotoxicity both in vitro and in vivo. Dasatinib reduced the cell viability of rat primary hepatocytes, induced the release of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in vitro, and triggered the ballooning degeneration of hepatocytes in Sprague–Dawley rats in vivo. Apoptotic markers (chromatin condensation, cleaved caspase-3 and cleaved PARP) were detected to indicate that the injury induced by Dasatinib in hepatocytes in vitro was mediated by apoptosis. This result was further validated in vivo using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Here we found that Dasatinib dramatically increased the level of reactive oxygen species (ROS) in hepatocytes, reduced the intracellular glutathione (GSH) content, attenuated the activity of superoxide dismutase (SOD), generated malondialdehyde (MDA), a product of lipid peroxidation, decreased the mitochondrial membrane potential, and activated nuclear factor erythroid 2-related factor 2 (Nrf2) and mitogen-activated protein kinases (MAPK) related to oxidative stress and survival. These results confirm that oxidative stress plays a pivotal role in Dasatinib-mediated hepatotoxicity. N-acetylcysteine (NAC), a typical antioxidant, can scavenge free radicals, attenuate oxidative stress, and protect hepatocytes against Dasatinib-induced injury. Thus, relieving oxidative stress is a viable strategy for reducing Dasatinib-induced hepatotoxicity. -- Highlights: ►Dasatinib shows potential hepatotoxicity both in vitro and in vivo. ►Apoptosis plays a vital role in Dasatinib

  4. Oncostatin M induces upregulation of claudin-2 in rodent hepatocytes coinciding with changes in morphology and function of tight junctions

    International Nuclear Information System (INIS)

    Imamura, Masafumi; Kojima, Takashi; Lan, Mengdong; Son, Seiichi; Murata, Masaki; Osanai, Makoto; Chiba, Hideki; Hirata, Koichi; Sawada, Norimasa

    2007-01-01

    In rodent livers, integral tight junction (TJ) proteins claudin-1, -2, -3, -5 and -14 are detected and play crucial roles in the barrier to keep bile in bile canaculi away from the blood circulation. Claudin-2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, whereas claudin-1 and -3 are expressed in the whole liver lobule. Although claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells, the physiological functions and regulation of claudin-2 in hepatocytes remain unclear. Oncostatin M (OSM) is a multifunctional cytokine implicated in the differentiation of hepatocytes that induces formation of E-cadherin-based adherens junctions in fetal hepatocytes. In this study, we examined whether OSM could induce expression and function of claudin-2 in rodent hepatocytes, immortalized mouse and primary cultured proliferative rat hepatocytes. In the immortalized mouse and primary cultured proliferative rat hepatocytes, treatment with OSM markedly increased mRNA and protein of claudin-2 together with formation of developed networks of TJ strands. The increase of claudin-2 enhanced the paracellular barrier function which depended on molecular size. The increase of claudin-2 expression induced by OSM in rodent hepatocytes was regulated through distinct signaling pathways including PKC. These results suggest that expression of claudin-2 in rodent hepatocytes may play a specific role as controlling the size of paracellular permeability in the barrier to keep bile in bile canaculi

  5. Target Assembly Facility

    Data.gov (United States)

    Federal Laboratory Consortium — The Target Assembly Facility integrates new armor concepts into actual armored vehicles. Featuring the capability ofmachining and cutting radioactive materials, it...

  6. Composite turbine bucket assembly

    Science.gov (United States)

    Liotta, Gary Charles; Garcia-Crespo, Andres

    2014-05-20

    A composite turbine blade assembly includes a ceramic blade including an airfoil portion, a shank portion and an attachment portion; and a transition assembly adapted to attach the ceramic blade to a turbine disk or rotor, the transition assembly including first and second transition components clamped together, trapping said ceramic airfoil therebetween. Interior surfaces of the first and second transition portions are formed to mate with the shank portion and the attachment portion of the ceramic blade, and exterior surfaces of said first and second transition components are formed to include an attachment feature enabling the transition assembly to be attached to the turbine rotor or disk.

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

    Science.gov (United States)

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

    2016-02-18

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

  8. Renal medullary AA amyloidosis, hepatocyte dissociation and multinucleated hepatocytes in a 14-year-old free-ranging lioness (Panthera leo

    Directory of Open Access Journals (Sweden)

    J.H. Williams

    2005-06-01

    Full Text Available A 14-year-old lioness, originating from Etosha in Namibia, and a member of a pride in Pilanesberg National Park since translocation in 1994, was euthanased due to fight-related vertebral fracture and spinal injury, incurred approximately 6-8 weeks previously. Blood specimens collected at the time of death showed mild anaemia and a leukogram reflecting stress and chronic infection. Necropsy conducted within 2 hours of death was on a dehydrated, emaciated animal with hindquarter wasting and chronic traumatic friction injuries from dragging her hindlegs. There was cellulitis in the region of bite-wounds adjacent to the thoraco-lumbar vertebral fracture, at which site there was spinal cord compression, and there was marked intestinal helminthiasis. The outer renal medullae appeared pale and waxy and the liver was macroscopically unremarkable. Histopathology and electron microscopy of the kidneys revealed multifocal to coalescing deposits of proximal medullary interstitial amyloid, which fluoresced strongly with thioflavine T, and was sensitive to potassium permanganate treatment prior to Congo Red staining, thus indicating inflammatory (AA origin. There was diffuse hepatocyte dissociation, as well as numerous binucleated and scattered multinucleated (up to 8 nuclei/cell hepatocytes, with swollen hepatocyte mitochondria, in liver examined light microscopically. Ultrastructurally, the mono-, bi- and multinucleated hepatocytes contained multifocal irregular membrane-bound accumulations of finely-granular, amorphous material both intra-cytoplasmically and intra-nuclearly, as well as evidence of irreversible mitochondrial injury. The incidence and relevance in cats and other species of amyloidosis, particularly with renal medullary distribution, as well as of hepatocyte dissociation and multinucleation, as reported in selected literature, is briefly overviewed and their occurrence in this lioness is discussed.

  9. MiR-375 inhibits the hepatocyte growth factor-elicited migration of mesenchymal stem cells by downregulating Akt signaling.

    Science.gov (United States)

    He, Lihong; Wang, Xianyao; Kang, Naixin; Xu, Jianwei; Dai, Nan; Xu, Xiaojing; Zhang, Huanxiang

    2018-04-01

    The migration of mesenchymal stem cells (MSCs) is critical for their use in cell-based therapies. Accumulating evidence suggests that microRNAs are important regulators of MSC migration. Here, we report that the expression of miR-375 was downregulated in MSCs treated with hepatocyte growth factor (HGF), which strongly stimulates the migration of these cells. Overexpression of miR-375 decreased the transfilter migration and the migration velocity of MSCs triggered by HGF. In our efforts to determine the mechanism by which miR-375 affects MSC migration, we found that miR-375 significantly inhibited the activation of Akt by downregulating its phosphorylation at T308 and S473, but had no effect on the activity of mitogen-activated protein kinases. Further, we showed that 3'phosphoinositide-dependent protein kinase-1 (PDK1), an upstream kinase necessary for full activation of Akt, was negatively regulated by miR-375 at the protein level. Moreover, miR-375 suppressed the phosphorylation of focal adhesion kinase (FAK) and paxillin, two important regulators of focal adhesion (FA) assembly and turnover, and decreased the number of FAs at cell periphery. Taken together, our results demonstrate that miR-375 inhibits HGF-elicited migration of MSCs through downregulating the expression of PDK1 and suppressing the activation of Akt, as well as influencing the tyrosine phosphorylation of FAK and paxillin and FA periphery distribution.

  10. Live cell imaging of cytosolic NADH/NAD+ ratio in hepatocytes and liver slices.

    Science.gov (United States)

    Masia, Ricard; McCarty, William J; Lahmann, Carolina; Luther, Jay; Chung, Raymond T; Yarmush, Martin L; Yellen, Gary

    2018-01-01

    Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD + ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD + ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD + ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD + ratio, and this increase is mitigated by the presence of NAD + -generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD + ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes. NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD + ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real

  11. Activated human neutrophils release hepatocyte growth factor/scatter factor.

    LENUS (Irish Health Repository)

    McCourt, M

    2012-02-03

    BACKGROUND: Hepatocyte growth factor or scatter factor (HGF\\/SF) is a pleiotropic cytokine that has potent angiogenic properties. We have previously demonstrated that neutrophils (PMN) are directly angiogenic by releasing vascular endothelial growth factor (VEGF). We hypothesized that the acute inflammatory response can stimulate PMN to release HGF. AIMS: To examine the effects of inflammatory mediators on PMN HGF release and the effect of recombinant human HGF (rhHGF) on PMN adhesion receptor expression and PMN VEGF release. METHODS: In the first experiment, PMN were isolated from healthy volunteers and stimulated with tumour necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), interleukin-8 (IL-8), and formyl methionyl-leucyl-phenylalanine (fMLP). Culture supernatants were assayed for HGF using ELISA. In the second experiment, PMN were lysed to measure total HGF release and HGF expression in the PMN was detected by Western immunoblotting. Finally, PMN were stimulated with rhHGF. PMN CD 11a, CD 11b, and CD 18 receptor expression and VEGF release was measured using flow cytometry and ELISA respectively. RESULTS: TNF-alpha, LPS and fMLP stimulation resulted in significantly increased release of PMN HGF (755+\\/-216, 484+\\/-221 and 565+\\/-278 pg\\/ml, respectively) compared to controls (118+\\/-42 pg\\/ml). IL-8 had no effect. Total HGF release following cell lysis and Western blot suggests that HGF is released from intracellular stores. Recombinant human HGF did not alter PMN adhesion receptor expression and had no effect on PMN VEGF release. CONCLUSIONS: This study demonstrates that pro-inflammatory mediators can stimulate HGF release from a PMN intracellular store and that activated PMN in addition to secreting VEGF have further angiogenic potential by releasing HGF.

  12. Differentiation of hepatocytes from induced pluripotent stem cells derived from human hair follicle mesenchymal stem cells.

    Science.gov (United States)

    Shi, Xu; Lv, Shuang; He, Xia; Liu, Xiaomei; Sun, Meiyu; Li, Meiying; Chi, Guangfan; Li, Yulin

    2016-10-01

    Due to the limitations of organ donors and immune rejection in severe liver diseases, stem cell-based therapy presents a promising application for tissue repair and regeneration. As a novel cell source, mesenchymal stem cells separated from human hair follicles (HF-MSCs) are convenient to obtain and have no age limit. To date, the differentiation of HF-MSCs into hepatocytes has not been reported. In this study, we explored whether HF-MSCs and HF-MSC-derived-induced pluripotent stem cells (HF-iPS) could differentiate into hepatocytes in vitro. Flow cytometry, Oil Red O stain and Alizarin Red stain were used to identify the characteristics of HF-MSCs. The expression of liver-specific gene was detected by immunofluorescence and Quantitative Polymerase Chain Reaction. Periodic Acid-Schiff stain, Indocyanine Green stain and Low-Density Lipoprotein stain were performed to evaluate the functions of induced hepatocyte-like cells (HLCs). HF-MSCs were unable to differentiate into HLCs using previously reported procedures for MSCs from other tissues. However, HF-iPS efficiently induced the generation of HLCs that expressed hepatocyte markers and drug metabolism-related genes. HF-iPS can be used as novel and alternative cellular tools for inducing hepatocytes in vitro, simultaneously benefiting from utilizing HF-MSCs as a noninvasive and convenient cell source for reprogramming.

  13. Generation of human pluripotent stem cell-derived hepatocyte-like cells for drug toxicity screening.

    Science.gov (United States)

    Takayama, Kazuo; Mizuguchi, Hiroyuki

    2017-02-01

    Because drug-induced liver injury is one of the main reasons for drug development failures, it is important to perform drug toxicity screening in the early phase of pharmaceutical development. Currently, primary human hepatocytes are most widely used for the prediction of drug-induced liver injury. However, the sources of primary human hepatocytes are limited, making it difficult to supply the abundant quantities required for large-scale drug toxicity screening. Therefore, there is an urgent need for a novel unlimited, efficient, inexpensive, and predictive model which can be applied for large-scale drug toxicity screening. Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are able to replicate indefinitely and differentiate into most of the body's cell types, including hepatocytes. It is expected that hepatocyte-like cells generated from human ES/iPS cells (human ES/iPS-HLCs) will be a useful tool for drug toxicity screening. To apply human ES/iPS-HLCs to various applications including drug toxicity screening, homogenous and functional HLCs must be differentiated from human ES/iPS cells. In this review, we will introduce the current status of hepatocyte differentiation technology from human ES/iPS cells and a novel method to predict drug-induced liver injury using human ES/iPS-HLCs. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

  14. Prolongation of liver-specific function for primary hepatocytes maintenance in 3D printed architectures.

    Science.gov (United States)

    Kim, Yohan; Kang, Kyojin; Yoon, Sangtae; Kim, Ji Sook; Park, Su A; Kim, Wan Doo; Lee, Seung Bum; Ryu, Ki-Young; Jeong, Jaemin; Choi, Dongho

    2018-01-23

    Isolated primary hepatocytes from the liver are very similar to in vivo native liver hepatocytes, but they have the disadvantage of a limited lifespan in 2D culture. Although a sandwich culture and 3D organoids with mesenchymal stem cells (MSCs) as an attractive assistant cell source to extend lifespan can be used, it cannot fully reproduce the in vivo architecture. Moreover, long-term 3D culture leads to cell death because of hypoxic stress. Therefore, to overcome the drawback of 2D and 3D organoids, we try to use a 3D printing technique using alginate hydrogels with primary hepatocytes and MSCs. The viability of isolated hepatocytes was more than 90%, and the cells remained alive for 7 days without morphological changes in the 3D hepatic architecture with MSCs. Compared to a 2D system, the expression level of functional hepatic genes and proteins was higher for up to 7 days in the 3D hepatic architecture. These results suggest that both the 3D bio-printing technique and paracrine molecules secreted by MSCs supported long-term culture of hepatocytes without morphological changes. Thus, this technique allows for widespread expansion of cells while forming multicellular aggregates, may be applied to drug screening and could be an efficient method for developing an artificial liver.

  15. Glutamic Acid as Enhancer of Protein Synthesis Kinetics in Hepatocytes from Old Rats.

    Science.gov (United States)

    Brodsky, V Y; Malchenko, L A; Butorina, N N; Lazarev Konchenko, D S; Zvezdina, N D; Dubovaya, T K

    2017-08-01

    Dense cultures of hepatocytes from old rats (~2 years old, body weight 530-610 g) are different from similar cultures of hepatocytes from young rats by the low amplitude of protein synthesis rhythm. Addition of glutamic acid (0.2, 0.4, or 0.6 mg/ml) into the culture medium with hepatocytes of old rats resulted in increase in the oscillation amplitudes of the protein synthesis rhythm to the level of young rats. A similar action of glutamic acid on the protein synthesis kinetics was observed in vivo after feeding old rats with glutamic acid. Inhibition of metabotropic receptors of glutamic acid with α-methyl-4-carboxyphenylglycine (0.01 mg/ml) abolished the effect of glutamic acid. The amplitude of oscillation of the protein synthesis rhythm in a cell population characterizes synchronization of individual oscillations caused by direct cell-cell communications. Hence, glutamic acid, acting as a receptor-dependent transmitter, enhanced direct cell-cell communications of hepatocytes that were decreased with aging. As differentiated from other known membrane signaling factors (gangliosides, norepinephrine, serotonin, dopamine), glutamic acid can penetrate into the brain and thus influence the communications and protein synthesis kinetics that are disturbed with aging not only in hepatocytes, but also in neurons.

  16. Isolation of primary human hepatocytes from normal and diseased liver tissue: a one hundred liver experience.

    Directory of Open Access Journals (Sweden)

    Ricky H Bhogal

    2011-03-01

    Full Text Available Successful and consistent isolation of primary human hepatocytes remains a challenge for both cell-based therapeutics/transplantation and laboratory research. Several centres around the world have extensive experience in the isolation of human hepatocytes from non-diseased livers obtained from donor liver surplus to surgical requirement or at hepatic resection for tumours. These livers are an important but limited source of cells for therapy or research. The capacity to isolate cells from diseased liver tissue removed at transplantation would substantially increase availability of cells for research. However no studies comparing the outcome of human hepatocytes isolation from diseased and non-diseased livers presently exist. Here we report our experience isolating human hepatocytes from organ donors, non-diseased resected liver and cirrhotic tissue. We report the cell yields and functional qualities of cells isolated from the different types of liver and demonstrate that a single rigorous protocol allows the routine harvest of good quality primary hepatocytes from the most commonly accessible human liver tissue samples.

  17. Acquisition of lipid metabolic capability in hepatocyte-like cells directly induced from mouse fibroblasts

    Directory of Open Access Journals (Sweden)

    Shizuka eMiura

    2014-08-01

    Full Text Available Recently, the numbers of patients with non-alcoholic fatty liver disease (NAFLD and non-alcoholic steatohepatitis (NASH have increased worldwide. NAFLD and NASH are known as risk factors for liver cirrhosis and hepatocellular carcinoma. Because many factors can promote the progression of NAFLD and NASH, the treatment of these patients involves various strategies. Thus, it is desired that drugs for patients with NAFLD and NASH should be developed more easily and rapidly using cultures of primary hepatocytes. However, it is difficult to use hepatocytes as a tool for drug screening, because these cells cannot be functionally maintained in culture. Thus, in this study, we sought to examine whether induced hepatocyte-like (iHep cells, which were directly induced from mouse dermal fibroblasts by infection with a retrovirus expressing Hnf4α and Foxa3, possess the potential for lipid metabolism, similar to hepatocytes. Our data showed that iHep cells were capable of synthesizing lipids from a cis-unsaturated fatty acid, a trans-unsaturated fatty acid, and a saturated fatty acid, accumulating the synthesized lipids in cellular vesicles, and secreting the lipids into the culture medium. Moreover, the lipid synthesis in iHep cells was significantly inhibited in cultures with lipid metabolism improvers. These results demonstrate that iHep cells could be useful not only for screening of drugs for patients with NAFLD and NASH, but also for elucidation of the mechanisms underlying hereditary lipid metabolism disorders, as an alternative to hepatocytes.

  18. Evaluation and optimization of hepatocyte culture media factors by design of experiments (DoE) methodology.

    Science.gov (United States)

    Dong, Jia; Mandenius, Carl-Fredrik; Lübberstedt, Marc; Urbaniak, Thomas; Nüssler, Andreas K N; Knobeloch, Daniel; Gerlach, Jörg C; Zeilinger, Katrin

    2008-07-01

    Optimization of cell culture media based on statistical experimental design methodology is a widely used approach for improving cultivation conditions. We applied this methodology to refine the composition of an established culture medium for growth of a human hepatoma cell line, C3A. A selection of growth factors and nutrient supplements were systematically screened according to standard design of experiments (DoE) procedures. The results of the screening indicated that the medium additives hepatocyte growth factor, oncostatin M, and fibroblast growth factor 4 significantly influenced the metabolic activities of the C3A cell line. Surface response methodology revealed that the optimum levels for these factors were 30 ng/ml for hepatocyte growth factor and 35 ng/ml for oncostatin M. Additional experiments on primary human hepatocyte cultures showed high variance in metabolic activities between cells from different individuals, making determination of optimal levels of factors more difficult. Still, it was possible to conclude that hepatocyte growth factor, epidermal growth factor, and oncostatin M had decisive effects on the metabolic functions of primary human hepatocytes.

  19. Deletion of hepatocyte nuclear factor-1-beta in an infant with prune belly syndrome.

    Science.gov (United States)

    Haeri, Sina; Devers, Patricia L; Kaiser-Rogers, Kathleen A; Moylan, Vincent J; Torchia, Beth S; Horton, Amanda L; Wolfe, Honor M; Aylsworth, Arthur S

    2010-08-01

    Prune belly syndrome is a rare congenital disorder characterized by deficiency of abdominal wall muscles, cryptorchidism, and urinary tract anomalies. We have had the opportunity to study a baby with prune belly syndrome associated with an apparently de novo 1.3-megabase interstitial 17q12 microdeletion that includes the hepatocyte nuclear factor-1-beta gene at 17q12. One previous patient, an adult, has been reported with prune belly syndrome and a hepatocyte nuclear factor-1-beta microdeletion. Hepatocyte nuclear factor-1-beta is a widely expressed transcription factor that regulates tissue-specific gene expression and is expressed in numerous tissues including mesonephric duct derivatives, the renal tubule of the metanephros, and the developing prostate of the mouse. Mutations in hepatocyte nuclear factor-1-beta cause the "renal cysts and diabetes syndrome," isolated renal cystic dysplasia, and a variety of other malformations. Based on its expression pattern and the observation of two affected cases, we propose that haploinsufficiency of hepatocyte nuclear factor-1-beta may be causally related to the production of the prune belly syndrome phenotype through a mechanism of prostatic and ureteral hypoplasia that results in severe obstructive uropathy with urinary tract and abdominal distension. Copyright Thieme Medical Publishers.

  20. Optimization of the isolation and cultivation of Cyprinus carpio primary hepatocytes.

    Science.gov (United States)

    Yanhong, Fan; Chenghua, He; Guofang, Liu; Haibin, Zhang

    2008-10-01

    The aquatic environment is affected by numerous chemical contaminants. There is an increasing need to identify these chemicals and to evaluate their potential toxicity towards aquatic life. In this research we optimized techniques for primary cell culture of Cyprinus carpio hepatocytes as one adjunct model for ecotoxicological evaluation of the potential hazards of xenobiotics in the aquatic environment. In this study, Cyprinus carpio hepatocytes were isolated by mechanical separation, two-step collagenase perfusion, and pancreatin digestion. The hepatocytes or parenchymal cells could be separated from cell debris and from non-parenchymal cells by low-speed centrifugation (Percoll gradient centrifugation). The harvested hepatocytes were suspended in DMEM, M199 (cultured in 5% CO(2)), or L-15 (cultured without 5% CO(2)) medium then cultured at 17, 27, or 37 degrees C. Cell yield was counted by use of a hemocytometer, and the viability of the cells was assessed by use of the Trypan blue exclusion test. Results from these studies showed that the best method of isolation was pancreatin digestion (the cell yield was 2.7 x 10(8) per g (liver weight) and the viability was 98.4%) and the best medium was M199 (cultured in 5% CO(2)) or L-15 (cultured without 5% CO(2)). The optimum culture temperature was 27 degrees C. The primary hepatocytes culture of Cyprimus carpio grew well and satisfied requirements for most toxicological experiments in this condition.

  1. Generation, characterization and potential therapeutic applications of mature and functional hepatocytes from stem cells.

    Science.gov (United States)

    Zhang, Zhenzhen; Liu, Jianfang; Liu, Yang; Li, Zheng; Gao, Wei-Qiang; He, Zuping

    2013-02-01

    Liver cancer is the sixth most common tumor in the world and the majority of patients with this disease usually die within 1 year. The effective treatment for end-stage liver disease (also known as liver failure), including liver cancer or cirrhosis, is liver transplantation. However, there is a severe shortage of liver donors worldwide, which is the major handicap for the treatment of patients with liver failure. Scarcity of liver donors underscores the urgent need of using stem cell therapy to the end-stage liver disease. Notably, hepatocytes have recently been generated from hepatic and extra-hepatic stem cells. We have obtained mature and functional hepatocytes from rat hepatic stem cells. Here, we review the advancements on hepatic differentiation from various stem cells, including hepatic stem cells, embryonic stem cells, the induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, and probably spermatogonial stem cells. The advantages, disadvantages, and concerns on differentiation of these stem cells into hepatic cells are highlighted. We further address the methodologies, phenotypes, and functional characterization on the differentiation of numerous stem cells into hepatic cells. Differentiation of stem cells into mature and functional hepatocytes, especially from an extra-hepatic stem cell source, would circumvent the scarcity of liver donors and human hepatocytes, and most importantly it would offer an ideal and promising source of hepatocytes for cell therapy and tissue engineering in treating liver disease. Copyright © 2012 Wiley Periodicals, Inc.

  2. Nuclear fuel assembly

    International Nuclear Information System (INIS)

    Delafosse, Jacques.

    1977-01-01

    This invention relates to a nuclear fuel assembly for a light or heavy water reactor, or for a fast reactor of the kind with a bundle of cladded pins, maintained parallel to each other in a regular network by an assembly of separate supporting grids, fitted with elastic bearing surfaces on these pins [fr

  3. Assembling Transgender Moments

    Science.gov (United States)

    Greteman, Adam J.

    2017-01-01

    In this article, the author seeks to assemble moments--scholarly, popular, and aesthetic--in order to explore the possibilities that emerge as moments collect in education's encounters with the needs, struggles, and possibilities of transgender lives and practices. Assembling moments, the author argues, illustrates the value of "moments"…

  4. Vertical pump assembly

    International Nuclear Information System (INIS)

    Dohnal, M.; Rosel, J.; Skarka, V.

    1988-01-01

    The mounting is described of the drive assembly of a vertical pump for nuclear power plants in areas with seismic risk. The assembly is attached to the building floor using flexible and damping elements. The design allows producing seismically resistant pumps without major design changes in the existing types of vertical pumps. (E.S.). 1 fig

  5. Roles of Acyl-CoA:Diacylglycerol Acyltransferases 1 and 2 in Triacylglycerol Synthesis and Secretion in Primary Hepatocytes.

    Science.gov (United States)

    Li, Chen; Li, Lena; Lian, Jihong; Watts, Russell; Nelson, Randal; Goodwin, Bryan; Lehner, Richard

    2015-05-01

    Very low-density lipoprotein assembly and secretion are regulated by the availability of triacylglycerol. Although compelling evidence indicates that the majority of triacylglycerol in very low-density lipoprotein is derived from re-esterification of lipolytic products released by endoplasmic reticulum-associated lipases, little is known about roles of acyl-CoA:diacylglycerol acyltransferases (DGATs) in this process. We aimed to investigate the contribution of DGAT1 and DGAT2 in lipid metabolism and lipoprotein secretion in primary mouse and human hepatocytes. We used highly selective small-molecule inhibitors of DGAT1 and DGAT2, and we tracked storage and secretion of lipids synthesized de novo from [(3)H]acetic acid and from exogenously supplied [(3)H]oleic acid. Inactivation of individual DGAT activity did not affect incorporation of either radiolabeled precursor into intracellular triacylglycerol, whereas combined inactivation of both DGATs severely attenuated triacylglycerol synthesis. However, inhibition of DGAT2 augmented fatty acid oxidation, whereas inhibition of DGAT1 increased triacylglycerol secretion, suggesting preferential channeling of separate DGAT-derived triacylglycerol pools to distinct metabolic pathways. Inactivation of DGAT2 impaired cytosolic lipid droplet expansion, whereas DGAT1 inactivation promoted large lipid droplet formation. Moreover, inactivation of DGAT2 attenuated expression of lipogenic genes. Finally, triacylglycerol secretion was significantly reduced on DGAT2 inhibition without altering extracellular apolipoprotein B levels. Our data suggest that DGAT1 and DGAT2 can compensate for each other to synthesize triacylglycerol, but triacylglycerol synthesized by DGAT1 is preferentially channeled to oxidation, whereas DGAT2 synthesizes triacylglycerol destined for very low-density lipoprotein assembly. © 2015 American Heart Association, Inc.

  6. Fuel Assembly Damping Summary

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Kanghee; Kang, Heungseok; Oh, Dongseok; Yoon, Kyungho; Kim, Hyungkyu; Kim, Jaeyong [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2013-10-15

    This paper summary the fuel assembly damping data in air/in still water/under flow, released from foreign fuel vendors, compared our data with the published data. Some technical issues in fuel assembly damping measurement testing are also briefly discussed. Understanding of each fuel assembly damping mechanisms according to the surrounding medium and flow velocity can support the fuel design improvement in fuel assembly dynamics and structural integrity aspect. Because the upgraded requirements of the newly-developed advanced reactor system will demands to minimize fuel design margin in integrity evaluation, reduction in conservatism of fuel assembly damping can contribute to alleviate the fuel design margin for sure. Damping is an energy dissipation mechanism in a vibrating mechanical structure and prevents a resonant structure from having infinite vibration amplitudes. The sources of fuel assembly damping are various from support friction to flow contribution, and it can be increased by the viscosity or drag of surrounding fluid medium or the average velocity of water flowing. Fuel licensing requires fuel design evaluation in transient or accidental condition. Dynamic response analysis of fuel assembly is to show fuel integrity and requires information on assembly-wise damping in dry condition and under wet or water flowing condition. However, damping measurement test for the full-scale fuel assembly prototype is not easy to carry out because of the scale (fuel prototype, test facility), unsteadiness of test data (scattering, random sampling and processing), instrumentation under water flowing (water-proof response measurement), and noise. LWR fuel technology division in KAERI is preparing the infra structure for damping measurement test of full-scale fuel assembly, to support fuel industries and related research activities. Here is a preliminary summary of fuel assembly damping, published in the literature. Some technical issues in fuel assembly damping

  7. Self-assembled nanostructures

    CERN Document Server

    Zhang, Jin Z; Liu, Jun; Chen, Shaowei; Liu, Gang-yu

    2003-01-01

    Nanostructures refer to materials that have relevant dimensions on the nanometer length scales and reside in the mesoscopic regime between isolated atoms and molecules in bulk matter. These materials have unique physical properties that are distinctly different from bulk materials. Self-Assembled Nanostructures provides systematic coverage of basic nanomaterials science including materials assembly and synthesis, characterization, and application. Suitable for both beginners and experts, it balances the chemistry aspects of nanomaterials with physical principles. It also highlights nanomaterial-based architectures including assembled or self-assembled systems. Filled with in-depth discussion of important applications of nano-architectures as well as potential applications ranging from physical to chemical and biological systems, Self-Assembled Nanostructures is the essential reference or text for scientists involved with nanostructures.

  8. Low-protein diet induces, whereas high-protein diet reduces hepatic FGF21 production in mice, but glucose and not amino acids up-regulate FGF21 in cultured hepatocytes.

    Science.gov (United States)

    Chalvon-Demersay, Tristan; Even, Patrick C; Tomé, Daniel; Chaumontet, Catherine; Piedcoq, Julien; Gaudichon, Claire; Azzout-Marniche, Dalila

    2016-10-01

    Fibroblast growth factor 21 (FGF21) is a polypeptide secreted by the liver and involved in several metabolic processes such as thermogenesis and lipid oxidation. The nutritional mechanisms controlling FGF21 production are poorly understood. This study aimed to investigate how dietary carbohydrates and proteins impact FGF21 production and how in turn, FGF21 is involved in the metabolic adaptation to changes in the carbohydrate and protein contents of the diet. For that purpose, we fed 25 male C57BL/6 mice diets composed of different protein and carbohydrate contents (normal-protein and carbohydrate diet (N=9, NPNC), low-protein high-carbohydrate diet (N=8, LPHC), high-protein low-carbohydrate diet (N=8, HPLC) for 3 weeks. We measured liver Fgf21 gene expression, synthesis and secretion as well as different parameters related to energy and glucose metabolism. We also investigated the direct role of amino acids and glucose in the control of Fgf21 gene expression in hepatocyte primary cultures (n=6). In vivo, FGF21 responds acutely to LPHC intake whereas under an HPLC diet, plasma FGF21 circulating levels are low in the fasted and refed states. In hepatocytes, Fgf21 expression was controlled by glucose but not amino acids. Both diets increased the thermic effect of feeding (TEF) and ketogenesis was increased in fasted HPLC mice. The results presented suggest that dietary glucose, rather than amino acids, directly controls FGF21 secretion, and that FGF21 may be involved in the increased TEF response to LPHC. The effects of the HPLC diet on ketogenesis and TEF are probably controlled by other metabolic pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Whole-body X-irradiation of mice accelerates polyploidization of hepatocytes

    International Nuclear Information System (INIS)

    Shima, A.; Egami, N.

    1985-01-01

    Male C57BL/6 mice were whole-body irradiated with 4.75 gy of X-rays at the age of 2 months and killed at 2, 6, 12 and 19 months after irradiation. The percentage survival began to decline earlier and faster in the irradiated group than the controls up to 19 months after exposure when the study was terminated. The nuclear DNA content of individual hepatocytes was measured by a Feulgen-DNA microfluorometric method, and hepatocytes were classified into various ploidy classes. In the irradiated mice, the degree of polyploidization was significantly higher than the controls by 2 months after exposure and steadily increased up to 6 months after exposure. Thereafter, however, a slow return to the control level was found up to 19 months after irradiation. These results appear to support a hypothesis that radiation accelerates the ageing process as judged from hepatocyte polyploidization. (author)

  10. Preventing hepatocyte oxidative stress cytotoxicity with Mangifera indica L. extract (Vimang).

    Science.gov (United States)

    Remirez, Diadelis; Tafazoli, Shahrzad; Delgado, Rene; Harandi, Asghar A; O'Brien, Peter J

    2005-01-01

    Vimang is an aqueous extract of Mangifera indica used in Cuba to improve the quality of life in patients suffering from inflammatory diseases. In the present study we evaluated the effects of Vimang at preventing reactive oxygen species (ROS) formation and lipid peroxidation in intact isolated rat hepatocytes. Vimang at 20, 50 and 100 microg/ml inhibited hepatocyte ROS formation induced by glucose-glucose oxidase. Hepatocyte cytotoxicity and lipid peroxidation induced by cumene hydroperoxide was also inhibited by Vimang in a dose and time dependent manner at the same concentration. Vimang also inhibited superoxide radical formation by xanthine oxidase and hypoxanthine. The superoxide radical scavenging and antioxidant activity of the Vimang extract was likely related to its gallates, catechins and mangiferin content. To our knowledge, this is the first report of cytoprotective antioxidant effects of Vimang in cellular oxidative stress models.

  11. Choline or methionine reverses impaired secretion of VLDL by hepatocytes from choline-deficient rats

    International Nuclear Information System (INIS)

    Yao, Z.; Vance, D.E.

    1987-01-01

    Male rats fed a choline-deficient (CD) diet for three days accumulated triacylglycerol (TG) in the liver. Hepatocytes from these rats were cultured and maintained in a medium + choline. The rate of secretion of TG was reduced by 50% in the CD cells. Correspondingly, [ 3 H]oleate and [ 3 H]glycerol were incorporated at a 2-fold higher rate into TG secreted by choline-supplemented cells compared to CD cells. Isolation of lipoprotein fractions by ultracentrifugation showed that the reduced secretion of TG by CD hepatocytes was mainly due to an impaired secretion of very low density lipoprotein (VLDL). Incorporation of [ 3 H]leucine into secreted apoB/sub H/, apoB/sub L/ and apoE was markedly reduced in CD cells compared to choline-supplemented cells. Secretion of high density lipoprotein was not reduced in the CD hepatocytes. Normal secretion of VLDL was resumed upon addition of methionine to the CD cells

  12. Lipopolysaccharide impairs hepatocyte ureagenesis from ammonia: involvement of mitochondrial aquaporin-8.

    Science.gov (United States)

    Soria, Leandro R; Marrone, Julieta; Molinas, Sara M; Lehmann, Guillermo L; Calamita, Giuseppe; Marinelli, Raúl A

    2014-05-02

    We recently reported that hepatocyte mitochondrial aquaporin-8 (mtAQP8) channels facilitate the uptake of ammonia and its metabolism into urea. Here we studied the effect of bacterial lipopolysaccharides (LPS) on ammonia-derived ureagenesis. In LPS-treated rats, hepatic mtAQP8 protein expression and diffusional ammonia permeability (measured utilizing ammonia analogues) of liver inner mitochondrial membranes were downregulated. NMR studies using 15N-labeled ammonia indicated that basal and glucagon-induced ureagenesis from ammonia were significantly reduced in hepatocytes from LPS-treated rats. Our data suggest that hepatocyte mtAQP8-mediated ammonia removal via ureagenesis is impaired by LPS, a mechanism potentially relevant to the molecular pathogenesis of defective hepatic ammonia detoxification in sepsis. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  13. Xenobiotic-Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes Modulated by Toxcast Chemicals

    Science.gov (United States)

    Primary human hepatocyte cultures are useful in vitro model systems of human liver because when cultured under appropriate conditions the hepatocytes retain liver-like functionality such as metabolism, transport, and cell signaling. This model system was used to characterize the ...

  14. Diclofenac inhibits tumor necrosis factor-a-induced nuclear factor-kB activation causing synergic hepatocyte apoptosis

    NARCIS (Netherlands)

    Frederiksson, L; Herpers, B; Benedetti, G; Matadin, Q; Puigvert, J.C.; de Bont, H; Dragovic, S.; Vermeulen, N.P.E.; Commandeur, J.N.M.; Danen, E; de Graauw, M; van de Water, B.

    2011-01-01

    Drug-induced liver injury (DILI) is an important clinical problem. It involves crosstalk between drug toxicity and the immune system, but the exact mechanism at the cellular hepatocyte level is not well understood. Here we studied the mechanism of crosstalk in hepatocyte apoptosis caused by

  15. Induced Mitogenic Activity in AML-12 Mouse Hepatocytes Exposed to Low-dose Ultra-Wideband Electromagnetic Radiation

    Directory of Open Access Journals (Sweden)

    P. B. Tchounwou

    2005-04-01

    Full Text Available Ultra–wideband (UWB technology has increased with the use of various civilian and military applications. In the present study, we hypothesized that low-dose UWB electromagnetic radiation (UWBR could elicit a mitogenic effect in AML-12 mouse hepatocytes, in vitro. To test this hypothesis, we exposed AML-12 mouse hepatocytes, to UWBR in a specially constructed gigahertz transverse electromagnetic mode (GTEM cell. Cells were exposed to UWBR for 2 h at a temperature of 23°C, a pulse width of 10 ns, a repetition rate of 1 kHz, and field strength of 5-20 kV/m. UWB pulses were triggered by an external pulse generator for UWBR exposure but were not triggered for the sham exposure. We performed an MTT Assay to assess cell viability for UWBR-treated and sham-exposed hepatocytes. Data from viability studies indicated a time-related increase in hepatocytes at time intervals from 8-24 h post exposure. UWBR exerted a statistically significant (p < 0.05 dose-dependent response in cell viability in both serum-treated and serum free medium (SFM -treated hepatocytes. Western blot analysis of hepatocyte lysates demonstrated that cyclin A protein was induced in hepatocytes, suggesting that increased MTT activity after UWBR exposure was due to cell proliferation. This study indicates that UWBR has a mitogenic effect on AML-12 mouse hepatocytes and implicates a possible role for UWBR in hepatocarcinoma.

  16. IL-6 modulates hepatocyte proliferation via induction of HGF/p21cip1: Regulation by SOCS3

    International Nuclear Information System (INIS)

    Sun Rui; Jaruga, Barbara; Kulkarni, Shailin; Sun Haoyu; Gao Bin

    2005-01-01

    The precise role of IL-6 in liver regeneration and hepatocyte proliferation is controversial and the role of SOCS3 in liver regeneration remains unknown. Here we show that in vitro treatment with IL-6 inhibited primary mouse hepatocyte proliferation. IL-6 induced p21 cip1 protein expression in primary mouse hepatocytes. Disruption of the p21 cip1 gene abolished the inhibitory effect of IL-6 on cell proliferation. Co-culture with nonparenchymal liver cells diminished IL-6 inhibition of hepatocyte proliferation, which was likely due to IL-6 stimulation of nonparenchymal cells to produce HGF. Finally, IL-6 induced higher levels of p21 cip1 protein expression and a slightly stronger inhibition of cell proliferation in SOCS3 +/- mouse hepatocytes compared to wild-type hepatocytes, while liver regeneration was enhanced and prolonged in SOCS3 +/- mice. Our findings suggest that IL-6 directly inhibits hepatocyte proliferation via a p21 cip1 -dependent mechanism and indirectly enhances hepatocyte proliferation via stimulating nonparenchymal cells to produce HGF. SOCS3 negatively regulates liver regeneration

  17. Palm kernel cake extract exerts hepatoprotective activity in heat-induced oxidative stress in chicken hepatocytes.

    Science.gov (United States)

    Oskoueian, Ehsan; Abdullah, Norhani; Idrus, Zulkifli; Ebrahimi, Mahdi; Goh, Yong Meng; Shakeri, Majid; Oskoueian, Armin

    2014-10-02

    Palm kernel cake (PKC), the most abundant by-product of oil palm industry is believed to contain bioactive compounds with hepatoprotective potential. These compounds may serve as hepatoprotective agents which could help the poultry industry to alleviate adverse effects of heat stress on liver function in chickens. This study was performed to evaluate the hepatoprotective potential of PKC extract in heat-induced oxidative stress in chicken hepatocytes. The nature of the active metabolites and elucidation of the possible mechanism involved were also investigated. The PKC extract possessed free radical scavenging activity with values significantly (p < 0.05) lower than silymarin as the reference antioxidant. Heat-induced oxidative stress in chicken hepatocyte impaired the total protein, lipid peroxidation and antioxidant enzymes activity significantly (p < 0.05). Treatment of heat-induced hepatocytes with PKC extract (125 μg/ml) and silymarin as positive control increased these values significantly (p < 0.05). The real time PCR and western blot analyses revealed the significant (p < 0.05) up-regulation of oxidative stress biomarkers including TNF-like, IFN-γ and IL-1β genes; NF-κB, COX-2, iNOS and Hsp70 proteins expression upon heat stress in chicken hepatocytes. The PKC extract and silymarin were able to alleviate the expression of all of these biomarkers in heat-induced chicken hepatocytes. The gas chromatography-mass spectrometry analysis of PKC extract showed the presence of fatty acids, phenolic compounds, sugar derivatives and other organic compounds such as furfural which could be responsible for the observed hepatoprotective activity. Palm kernel cake extract could be a potential agent to protect hepatocytes function under heat induced oxidative stress.

  18. Assessment of mitochondrial dysfunction-related, drug-induced hepatotoxicity in primary rat hepatocytes

    International Nuclear Information System (INIS)

    Liu, Cong; Sekine, Shuichi; Ito, Kousei

    2016-01-01

    Evidence that mitochondrial dysfunction plays a central role in drug-induced liver injury is rapidly accumulating. In contrast to physiological conditions, in which almost all adenosine triphosphate (ATP) in hepatocytes is generated in mitochondria via aerobic respiration, the high glucose content and limited oxygen supply of conventional culture systems force primary hepatocytes to generate most ATP via cytosolic glycolysis. Thus, such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, and are not suitable to identify drugs with mitochondrial liabilities. In this study, primary rat hepatocytes were cultured in galactose-based medium, instead of the conventional glucose-based medium, and in hyperoxia to improve the reliance of energy generation on aerobic respiration. Activation of mitochondria was verified by diminished cellular lactate release and increased oxygen consumption. These conditions improved sensitivity to the mitochondrial complex I inhibitor rotenone. Since oxidative stress is also a general cause of mitochondrial impairment, cells were exposed to test compounds in the presence of transferrin to increase the generation of reactive oxygen species via increased uptake of iron. Finally, 14 compounds with reported mitochondrial liabilities were tested to validate this new drug-induced mitochondrial toxicity assay. Overall, the culture of primary rat hepatocytes in galactose, hyperoxia and transferrin is a useful model for the identification of mitochondrial dysfunction-related drug-induced hepatotoxicity. - Highlights: • Drug-induced mitochondrial toxicity was evaluated using primary rat hepatocytes. • Galactose and hyperoxia could activate OXPHOS in primary rat hepatocytes. • Cells with enhanced OXPHOS exhibit improved sensitivity to mitochondrial toxins. • Transferrin potentiate mitochondrial toxicity via increased ROS production.

  19. Resveratrol Differentially Regulates NAMPT and SIRT1 in Hepatocarcinoma Cells and Primary Human Hepatocytes

    Science.gov (United States)

    Schuster, Susanne; Penke, Melanie; Gorski, Theresa; Petzold-Quinque, Stefanie; Damm, Georg; Gebhardt, Rolf; Kiess, Wieland; Garten, Antje

    2014-01-01

    Resveratrol is reported to possess chemotherapeutic properties in several cancers. In this study, we wanted to investigate the molecular mechanisms of resveratrol-induced cell cycle arrest and apoptosis as well as the impact of resveratrol on NAMPT and SIRT1 protein function and asked whether there are differences in hepatocarcinoma cells (HepG2, Hep3B cells) and non-cancerous primary human hepatocytes. We found a lower basal NAMPT mRNA and protein expression in hepatocarcinoma cells compared to primary hepatocytes. In contrast, SIRT1 was significantly higher expressed in hepatocarcinoma cells than in primary hepatocytes. Resveratrol induced cell cycle arrest in the S- and G2/M- phase and apoptosis was mediated by activation of p53 and caspase-3 in HepG2 cells. In contrast to primary hepatocytes, resveratrol treated HepG2 cells showed a reduction of NAMPT enzymatic activity and increased p53 acetylation (K382). Resveratrol induced NAMPT release from HepG2 cells which was associated with increased NAMPT mRNA expression. This effect was absent in primary hepatocytes where resveratrol was shown to function as NAMPT and SIRT1 activator. SIRT1 inhibition by EX527 resembled resveratrol effects on HepG2 cells. Furthermore, a SIRT1 overexpression significantly decreased both p53 hyperacetylation and resveratrol-induced NAMPT release as well as S-phase arrest in HepG2 cells. We could show that NAMPT and SIRT1 are differentially regulated by resveratrol in hepatocarcinoma cells and primary hepatocytes and that resveratrol did not act as a SIRT1 activator in hepatocarcinoma cells. PMID:24603648

  20. EXPERIMENTAL SUBSTANTIATION OF PERMEABILIZED HEPATOCYTES MODEL FOR INVESTIGATION OF MITOCHONDRIA IN SITU RESPIRATION.

    Science.gov (United States)

    Merlavsky, V M; Manko, B O; Ikkert, O V; Manko, V V

    2015-01-01

    To verify experimentally the model of permeabilized hepatocytes, the degree of cell permeability was assessed using trypan blue and polarographycally determined cell respiration rate upon succinate (0.35 mM) and a-ketoglutarate (1 mM) oxidation. Oxidative phosphorylation was stimulated by ADP (750 μM). Hepatocyte permeabilization depends on digitonin concentraion in medium and on the number of cells in suspension. Thus, the permeabilization of 0.9-1.7 million cells/ml was completed by 25 μg/ml of digitonin, permeabilization of 2.0-3.0 million cells/ml--by 50 μg/ml of digitonin and permeabilization of 4.0-5.6 million cells/ml--by 100 μg/ml. Thus, the higher is the suspension density, the higher digitonin concentration is required. Treatment of hepatocytes with digitonin resulted in a decrease of endogenous respiration rate to a minimum upon 20-22 μg of digitonin per 1 million cells. Supplementation of permeabilized hepatocytes with α-ketoglutarate maintained stable respiration rate, on the level higher than endogenous respiration at the corresponding digitonin concentration, unlike the intact cells. Respiration rate of permeabilized hepatocytes at the simultaneous addition of α-ketoglutarate and ADP increased to the level of intact cell respiration, irrespective of digitonin concentration. Addition of solely succinate and especially succinate plus ADP markedly intensified the respiration of permeabilized hepatocytes to the level higher than that of intact cells. The dependence of succinate-stimulated respiration on digitonin concentration reached maximum at 20-22 αg of digitonin per 1 million cells. Optimal ratio of digitonin amount and the cell number in suspension is expected to be different in various tissues.

  1. Connexin 32 and connexin 43 are involved in lineage restriction of hepatic progenitor cells to hepatocytes

    Directory of Open Access Journals (Sweden)

    Haiyun Pei

    2017-11-01

    Full Text Available Abstract Background Bi-potential hepatic progenitor cells can give rise to both hepatocytes and cholangiocytes, which is the last phase and critical juncture in terms of sequentially hepatic lineage restriction from any kind of stem cells. If their differentiation can be controlled, it might access to functional hepatocytes to develop pharmaceutical and biotechnology industries as well as cell therapies for end-stage liver diseases. Methods In this study, we investigated the influence of Cx32 and Cx43 on hepatocyte differentiation of WB-F344 cells by in vitro gain and loss of function analyses. An inhibitor of Cx32 was also used to make further clarification. To reveal p38 MAPK pathway is closely related to Cxs, rats with 70% partial hepatectomy were injected intraperitoneally with a p38 inhibitor, SB203580. Besides, the effects of p38 MAPK pathway on differentiation of hepatoblasts isolated from fetal rat livers were evaluated by addition of SB203580 in culture medium. Results In vitro gain and loss of function analyses showed overexpression of Connexin 32 and knockdown of Connexin 43 promoted hepatocytes differentiation from hepatic progenitor cells. In addition, in vitro and ex vivo research revealed inhibition of p38 mitogen-activated protein kinase pathway can improve hepatocytes differentiation correlating with upregulation of Connexin 32 expression and downregulation of Connexin 43 expression. Conclusions Here we demonstrate that Connexins play crucial roles in facilitating differentiation of hepatic progenitors. Our work further implicates that regulators of Connexins and their related pathways might provide new insights to improve lineage restriction of stem cells to mature hepatocytes.

  2. Regulation of lipid synthesis in hepatocytes from lean and obese Zucker rats

    International Nuclear Information System (INIS)

    Triscari, J.; Greenwood, M.R.; Sullivan, A.C.

    1981-01-01

    Fatty acid synthesis and CO 2 production were evaluated in hepatocytes from lean and obese Zucker rats in the presence of 3 H 2 O, and several carbon precursors. The incorporation of 3 H 2 O into fatty acids was greater in obese compared to lean rats in both the isolated hepatocyte and in vivo. The rates of incorporation of 3 H 2 O into fatty acids and cholesterol in hepatocytes of both lean and obese rats were linear for 2 hr, in the absence or presence of 16.7 mM glucose. Rates of fatty acid synthesis were higher in the presence of 16.7 mM glucose compared to the absence of glucose in both lean and obese while rates of cholesterol synthesis were similar. The incorporation of 3H2O into fatty acids, but not into cholesterol, was correlated with increasing glucose concentration and was 2 to three-fold higher in hepatocytes of obese compared to lean rats in the presence of several carbon precursors. Differences in CO 2 production between lean and obese rats suggested increased pentose phosphate shunt activity, decreased pyruvate dehydrogenase activity, and lower tricarboxylic acid cycle activity in obese rats. Fatty acid synthesis and CO 2 production from 3 H 2 O and [U- 14 C]glucose in hepatocytes of lean and obese rats was similarly elevated by insulin and depressed by glucagon at several concentrations, suggesting that hepatocytes of obese animals respond to these hormones. These data indicate that rates of hepatic fatty acid synthesis although higher in obese rats respond to modulation in a fashion which is similar to the response in lean rats. The present studies suggest that the oxidation of several carbon precursors in the tricarboxylic acid cycle is diminished in obese compared to lean rats, but pentose phosphate shunt activity is greater in the obese Zucker rats

  3. 3D-printed gelatin scaffolds of differing pore geometry modulate hepatocyte function and gene expression.

    Science.gov (United States)

    Lewis, Phillip L; Green, Richard M; Shah, Ramille N

    2018-03-15

    Three dimensional (3D) printing is highly amenable to the fabrication of tissue-engineered organs of a repetitive microstructure such as the liver. The creation of uniform and geometrically repetitive tissue scaffolds can also allow for the control over cellular aggregation and nutrient diffusion. However, the effect of differing geometries, while controlling for pore size, has yet to be investigated in the context of hepatocyte function. In this study, we show the ability to precisely control pore geometry of 3D-printed gelatin scaffolds. An undifferentiated hepatocyte cell line (HUH7) demonstrated high viability and proliferation when seeded on 3D-printed scaffolds of two different geometries. However, hepatocyte specific functions (albumin secretion, CYP activity, and bile transport) increases in more interconnected 3D-printed gelatin cultures compared to a less interconnected geometry and to 2D controls. Additionally, we also illustrate the disparity between gene expression and protein function in simple 2D culture modes, and that recreation of a physiologically mimetic 3D environment is necessary to induce both expression and function of cultured hepatocytes. Three dimensional (3D) printing provides tissue engineers the ability spatially pattern cells and materials in precise geometries, however the biological effects of scaffold geometry on soft tissues such as the liver have not been rigorously investigated. In this manuscript, we describe a method to 3D print gelatin into well-defined repetitive geometries that show clear differences in biological effects on seeded hepatocytes. We show that a relatively simple and widely used biomaterial, such as gelatin, can significantly modulate biological processes when fabricated into specific 3D geometries. Furthermore, this study expands upon past research into hepatocyte aggregation by demonstrating how it can be manipulated to enhance protein function, and how function and expression may not precisely correlate in

  4. Cell proliferation studies in rodent hepatocytes during 1,4-dichlorobenzene administration

    International Nuclear Information System (INIS)

    Eldridge, S.R.; Tilbury, L.F.; Randall, H.; Goldsworthy, T.L.; Butterworth, B.E.

    1990-01-01

    In the NTP bioassay, 1,4-dichlorobenzene (DCB) induced hepatocellular carcinomas in mice, but not in rats. Because DCB is not DNA reactive, a cell proliferation study under conditions of the bioassay was undertaken to determine whether increased cell proliferation might play a role in DCB-induced hepatocarcinogenicity. DCB was administered in corn oil by gavage at the highest bioassay dose to male B6C3F1 mice (600 mg/kg) and male F344 rats (300 mg/kg) for five consecutive days. Cell proliferation was detected by labeling hepatocytes with either 5-bromo-2'-deoxyuridine (BRDU) or 3 H-thymidine delivered during the entire treatment period by subcutaneously implanted osmotic pumps. An increase in liver weight as a percentage of body weight was observed in treated mice (6.7±0.6 vs. 5.9±0.2) and rats (4.7±0.1 vs. 4.0±0.2) compared to controls. No significant elevations in plasma enzymes were found in either treated species, indicating a lack of overt hepatotoxicity. Histopathological evaluation revealed no evidence of hepatotoxicity in either species. The percentage of hepatocytes in S-phase was increased approximately 10-fold in both treated mice and rats compared to the respective control animals. Mice exhibited a centrilobular pattern of labeled hepatocytes, whereas rat hepatocytes were labeled hepatocytes, whereas rat hepatocytes were labeled throughout the lobules. These data demonstrate the hepatic mitogenic activity of DCB in mice and rats. However, this response dose not correlate with DCB-induced hepatocarcinogenicity. Further studies are required to examine the extent, duration and nature of the proliferative response in order to understand the species-specific effects of DCB

  5. Implications of the simultaneous occurrence of glycolysis and gluconeogenesis in hepatocytes from normal and hyperthyroid rats

    International Nuclear Information System (INIS)

    Phillips, J.W.; Berry, M.N.

    2001-01-01

    The mammalian liver has the capability for both glycolysis and gluconeogenesis. In the fasting state, metabolites such as lactate and glycerol, generated in the peripheral tissues, are taken up by the liver and converted to glucose. However, hepatocytes from fasted animals are also capable of substantial rates of glycolysis. It is generally assumed that glycolysis and gluconeogenesis do not occur simultaneously in the same cell, but rather the metabolic conditions that facilitate flux through one pathway impair flow in the opposite direction. The actual direction of flow at any given moment is thought to be determined by regulatory mechanisms that control flux through the enzymatic steps specific to glycolysis and gluconeogenesis. The rates of glycolysis from [6- 3 H]glucose and gluconeogenesis from [U- 14 C]glycerol were determined in isolated hepatocytes from fasted normal and hyperthyroid rats. We observed that glycolysis from glucose and glucose synthesis from glycerol occurred simultaneously at substantial rates in hepatocytes from normal rats and that gluconeogenesis, but not glycolysis, was increased twofold in hepatocytes from thyroid treated rats. In the hyperthyroid state, the rate of glycolysis from glucose was approximately equal to the rate of glucose formation from glycerol. Hence, metabolism and ATP turnover were stimulated without substantially altering steady-state concentrations of glucose. The concomitant operation of hepatic glycolysis and gluconeogenesis may be a mechanism that accounts in part for the calorigenic effect of thyroid hormone. Since hepatocyles are generally impermeable to phosphorylated metabolites, our observations suggest that glycolysis, and phosphorylation of glycerol take place in the same cells, and that the occurrence of simultaneous glycolysis and gluconeogenesis is an indication of channelling within the hepatocyte cytoplasm of individual hepatocytes

  6. Hepatocyte transplants improve liver function and encephalopathy in portacaval shunted rats.

    Science.gov (United States)

    Fogel, Wieslawa Agnieszka; Stasiak, Anna; Maksymowicz, Michał; Kobos, Jozef; Unzeta, Mercedes; Mussur, Miroslaw

    2014-07-01

    Rats with portacaval shunt (PCS) are useful experimental models of human hepatic encephalopathy in chronic liver dysfunction. We have previously shown that PCS modifies amine neurotransmitter systems in the CNS and increases voluntary alcohol intake by rats. Hepatocyte transplantation, used in acute liver failure, has recently also been applied to chronic liver diseases, which prompted us to investigate whether the altered brain amine system and the drinking behavior in long-term shunted rats could be normalized by hepatocyte transplants. Hepatocytes, isolated from syngeneic donors by collagenase digestion, were injected (3 × 10(6) cells/rat) into the pancreatic tail region, 6 months after PCS. Hepatic function was evaluated by measuring urine urea and plasma L-histidine concentrations. A free choice test with two bottles (tap water and 10% ethyl alcohol) was performed for 3 days to assess the rats' preference for alcohol. The rats were euthanized 2 months posttransplantation. Brain histamine and 5-hydroxyindoleacetic acid (5-HIAA) levels were measured by radioenzymatic assay and by HPLC-EC, respectively, N-tele-methylhistamine by GC/MS while MAOA and MAOB activities by isotopic procedures. Portacaval shunt rats with hepatocyte transplants gave more urea than before transplantation, with lower plasma L-His levels and higher body weight versus the PCS counterparts. Also, those rats consumed less alcohol. The CNS amines and 5-HIAA concentrations, as well as MAO-B activity, being abnormally high in untreated PCS rats, significantly reduced after PCS hepatocyte treatment. The results support the therapeutic values of hepatocyte transplants in chronic liver diseases and the temporary character of PCS-exerted CNS dysfunctions. © 2014 John Wiley & Sons Ltd.

  7. Cytotoxic mechanisms of hydrosulfide anion and cyanide anion in primary rat hepatocyte cultures

    International Nuclear Information System (INIS)

    Thompson, Rodney W.; Valentine, Holly L.; Valentine, William M.

    2003-01-01

    Hydrogen sulfide and hydrogen cyanide are known to compromise mitochondrial respiration through inhibition of cytochrome c oxidase and this is generally considered to be their primary mechanism of toxicity. Experimental studies and the efficiency of current treatment protocols suggest that H 2 S may exert adverse physiological effects through additional mechanisms. To evaluate the role of alternative mechanisms in H 2 S toxicity, the relative contributions of electron transport inhibition, uncoupling of mitochondrial respiration, and opening of the mitochondrial permeability transition pore (MPTP) to hydrosulfide and cyanide anion cytotoxicity in primary hepatocyte cultures were examined. Supplementation of hepatocytes with the glycolytic substrate, fructose, rescued hepatocytes from cyanide anion induced toxicity, whereas fructose supplementation increased hydrosulfide anion toxicity suggesting that hydrosulfide anion may compromise glycolysis in hepatocytes. Although inhibitors of the MPTP opening were protective for hydrosulfide anion, they had no effect on cyanide anion toxicity, consistent with an involvement of the permeability transition pore in hydrosulfide anion toxicity but not cyanide anion toxicity. Exposure of isolated rat liver mitochondria to hydrosulfide did not result in large amplitude swelling suggesting that if H 2 S induces the permeability transition it does so indirectly through a mechanism requiring other cellular components. Hydrosulfide anion did not appear to be an uncoupler of mitochondrial respiration in hepatocytes based upon the inability of oligomycin and fructose to protect hepatocytes from hydrosulfide anion toxicity. These findings support mechanisms additional to inhibition of cytochrome c oxidase in hydrogen sulfide toxicity. Further investigations are required to assess the role of the permeability transition in H 2 S toxicity, determine whether similar affects occur in other cell types or in vivo and evaluate whether this may

  8. Titanium Dioxide Nanoparticles Trigger Loss of Function and Perturbation of Mitochondrial Dynamics in Primary Hepatocytes.

    Directory of Open Access Journals (Sweden)

    Vaishaali Natarajan

    Full Text Available Titanium dioxide (TiO2 nanoparticles are one of the most highly manufactured and employed nanomaterials in the world with applications in copious industrial and consumer products. The liver is a major accumulation site for many nanoparticles, including TiO2, directly through intentional exposure or indirectly through unintentional ingestion via water, food or animals and increased environmental contamination. Growing concerns over the current usage of TiO2 coupled with the lack of mechanistic understanding of its potential health risk is the motivation for this study. Here we determined the toxic effect of three different TiO2 nanoparticles (commercially available rutile, anatase and P25 on primary rat hepatocytes. Specifically, we evaluated events related to hepatocyte functions and mitochondrial dynamics: (1 urea and albumin synthesis using colorimetric and ELISA assays, respectively; (2 redox signaling mechanisms by measuring reactive oxygen species (ROS production, manganese superoxide dismutase (MnSOD activity and mitochondrial membrane potential (MMP; (3 OPA1 and Mfn-1 expression that mediates the mitochondrial dynamics by PCR; and (4 mitochondrial morphology by MitoTracker Green FM staining. All three TiO2 nanoparticles induced a significant loss (p < 0.05 in hepatocyte functions even at concentrations as low as 50 ppm with commercially used P25 causing maximum damage. TiO2 nanoparticles induced a strong oxidative stress in primary hepatocytes. TiO2 nanoparticles exposure also resulted in morphological changes in mitochondria and substantial loss in the fusion process, thus impairing the mitochondrial dynamics. Although this study demonstrated that TiO2 nanoparticles exposure resulted in substantial damage to primary hepatocytes, more in vitro and in vivo studies are required to determine the complete toxicological mechanism in primary hepatocytes and subsequently liver function.

  9. Assessment of mitochondrial dysfunction-related, drug-induced hepatotoxicity in primary rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Cong; Sekine, Shuichi, E-mail: ssekine@faculty.chiba-u.jp; Ito, Kousei

    2016-07-01

    Evidence that mitochondrial dysfunction plays a central role in drug-induced liver injury is rapidly accumulating. In contrast to physiological conditions, in which almost all adenosine triphosphate (ATP) in hepatocytes is generated in mitochondria via aerobic respiration, the high glucose content and limited oxygen supply of conventional culture systems force primary hepatocytes to generate most ATP via cytosolic glycolysis. Thus, such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, and are not suitable to identify drugs with mitochondrial liabilities. In this study, primary rat hepatocytes were cultured in galactose-based medium, instead of the conventional glucose-based medium, and in hyperoxia to improve the reliance of energy generation on aerobic respiration. Activation of mitochondria was verified by diminished cellular lactate release and increased oxygen consumption. These conditions improved sensitivity to the mitochondrial complex I inhibitor rotenone. Since oxidative stress is also a general cause of mitochondrial impairment, cells were exposed to test compounds in the presence of transferrin to increase the generation of reactive oxygen species via increased uptake of iron. Finally, 14 compounds with reported mitochondrial liabilities were tested to validate this new drug-induced mitochondrial toxicity assay. Overall, the culture of primary rat hepatocytes in galactose, hyperoxia and transferrin is a useful model for the identification of mitochondrial dysfunction-related drug-induced hepatotoxicity. - Highlights: • Drug-induced mitochondrial toxicity was evaluated using primary rat hepatocytes. • Galactose and hyperoxia could activate OXPHOS in primary rat hepatocytes. • Cells with enhanced OXPHOS exhibit improved sensitivity to mitochondrial toxins. • Transferrin potentiate mitochondrial toxicity via increased ROS production.

  10. Resveratrol differentially regulates NAMPT and SIRT1 in Hepatocarcinoma cells and primary human hepatocytes.

    Directory of Open Access Journals (Sweden)

    Susanne Schuster

    Full Text Available Resveratrol is reported to possess chemotherapeutic properties in several cancers. In this study, we wanted to investigate the molecular mechanisms of resveratrol-induced cell cycle arrest and apoptosis as well as the impact of resveratrol on NAMPT and SIRT1 protein function and asked whether there are differences in hepatocarcinoma cells (HepG2, Hep3B cells and non-cancerous primary human hepatocytes. We found a lower basal NAMPT mRNA and protein expression in hepatocarcinoma cells compared to primary hepatocytes. In contrast, SIRT1 was significantly higher expressed in hepatocarcinoma cells than in primary hepatocytes. Resveratrol induced cell cycle arrest in the S- and G2/M- phase and apoptosis was mediated by activation of p53 and caspase-3 in HepG2 cells. In contrast to primary hepatocytes, resveratrol treated HepG2 cells showed a reduction of NAMPT enzymatic activity and increased p53 acetylation (K382. Resveratrol induced NAMPT release from HepG2 cells which was associated with increased NAMPT mRNA expression. This effect was absent in primary hepatocytes where resveratrol was shown to function as NAMPT and SIRT1 activator. SIRT1 inhibition by EX527 resembled resveratrol effects on HepG2 cells. Furthermore, a SIRT1 overexpression significantly decreased both p53 hyperacetylation and resveratrol-induced NAMPT release as well as S-phase arrest in HepG2 cells. We could show that NAMPT and SIRT1 are differentially regulated by resveratrol in hepatocarcinoma cells and primary hepatocytes and that resveratrol did not act as a SIRT1 activator in hepatocarcinoma cells.

  11. Possibility of Undifferentiated Human Thigh Adipose Stem Cells Differentiating into Functional Hepatocytes

    Directory of Open Access Journals (Sweden)

    Jong Hoon Lee

    2012-11-01

    Full Text Available BackgroundThis study aimed to investigate the possibility of isolating mesenchymal stem cells (MSCs from human thigh adipose tissue and the ability of human thigh adipose stem cells (HTASCs to differentiate into hepatocytes.MethodsThe adipose-derived stem cells (ADSCs were isolated from thigh adipose tissue. Growth factors, cytokines, and hormones were added to the collagen coated dishes to induce the undifferentiated HTASCs to differentiate into hepatocyte-like cells. To confirm the experimental results, the expression of hepatocyte-specific markers on undifferentiated and differentiated HTASCs was analyzed using reverse transcription polymerase chain reaction and immunocytochemical staining. Differentiation efficiency was evaluated using functional tests such as periodic acid schiff (PAS staining and detection of the albumin secretion level using enzyme-linked immunosorbent assay (ELISA.ResultsThe majority of the undifferentiated HTASCs were changed into a more polygonal shape showing tight interactions between the cells. The differentiated HTASCs up-regulated mRNA of hepatocyte markers. Immunocytochemical analysis showed that they were intensely stained with anti-albumin antibody compared with undifferentiated HTASCs. PAS staining showed that HTASCs submitted to the hepatocyte differentiation protocol were able to more specifically store glycogen than undifferentiated HTASCs, displaying a purple color in the cytoplasm of the differentiated HTASCs. ELISA analyses showed that differentiated HTASCs could secrete albumin, which is one of the hepatocyte markers.ConclusionsMSCs were islolated from human thigh adipose tissue differentiate to heapatocytes. The source of ADSCs is not only abundant abdominal adipose tissue, but also thigh adipose tissue for cell therapy in liver regeneration and tissue regeneration.

  12. Serum Hepatocyte Growth Factor as A Non-Invasive Marker For Evaluation of Hepatocellular Carcinoma

    International Nuclear Information System (INIS)

    Abdelgawad, M.R.; Wahba, M.A.

    2012-01-01

    The change and the prognostic value of serum hepatocyte growth factor and AFP level in patients with cirrhosis and/or primary liver cancer (HCC) were investigated. The level of serum hepatocyte growth factor was determined by using enzyme-linked immunosorbent assay, and AFP was determined by using radioimmunoassay in 29 patients with cirrhosis. Twenty five patients with primary liver cancer (13 patients without nodular cirrhosis and 12 patients with nodular cirrhosis) were categorized according to tumour size (≤ or >5 cm) and the level of AFP (≤ or > 200 ng/dl). The correlation between serum AFP and hepatocyte growth factor were significantly increased (P 0.05). Serum AFP can significantly discriminate between all studied groups (P 0.001) except for the comparison between control and cirrhosis (P>0.05), and also between HCC and HCC without nodular cirrhosis and HCC with cirrhosis (P>0.05). Serum HGF and AFP levels were positively affected by tumour size and nodular cirrhosis (P<0.001). Also, serum HGF level was highly affected by the levels of serum AFP in HCC patients. Non-significant correlation was observed between serum hepatocyte growth factor and AFP in control, cirrhosis, cirrhosis and HCC patients with AFP ? 200 ng/dl. It could be concluded that the over expressions of the hepatocyte growth factor and AFP may indicate an adverse prognosis for patients with cirrhosis and/or liver cancer. The sustained high level of serum hepatocyte growth factor in cirrhosis and/or HCC could be considered a factor related to early tumour diagnosis, so, serum HGF level may be used as a non-invasive marker in diagnosis and prognosis of liver malignancy. However, further studies are highly recommended to evaluate the role of HGF or its constituents in diagnosis and/or therapy in the future in a larger cohort of patients with different stages of liver malignancy

  13. TPX assembly plan

    International Nuclear Information System (INIS)

    Knutson, D.

    1993-01-01

    The TPX machine will be assembled in the TFTR Test Cell at the Plasma Physics Laboratory, utilizing the existing TFTR machine foundation. Preparation of the area for assembly will begin after completion of the decontamination and decommissioning phase on TFTR and certification that the radiation levels remaining, if any, are consistent with the types of operations planned. Assembly operations begin with the arrival of the first components, and conclude, approximately 24 months later, with the successful completion of the integrated systems tests and the achievement of a first plasma

  14. Nuclear reactor control assembly

    International Nuclear Information System (INIS)

    Negron, S.B.

    1991-01-01

    This patent describes an assembly for providing global power control in a nuclear reactor having the core split into two halves. It comprises a disk assembly formed from at least two disks each machined with an identical surface hole pattern such that rotation of one disk relative to the other causes the hole pattern to open or close, the disk assembly being positioned substantially at the longitudinal center of and coaxial with the core halves; and means for rotating at least one of the disks relative to the other

  15. Membrane fractions active in poliovirus RNA replication contain VPg precursor polypeptides

    International Nuclear Information System (INIS)

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

    1983-01-01

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

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

    Science.gov (United States)

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

    2016-11-16

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

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

    International Nuclear Information System (INIS)

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

    2005-07-01

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

  18. Isolation of Kupffer Cells and Hepatocytes from a Single Mouse Liver

    DEFF Research Database (Denmark)

    Aparicio-Vergara, Marcela; Tencerova, Michaela; Morgantini, Cecilia

    2017-01-01

    Liver perfusion is a common technique used to isolate parenchymal and non-parenchymal liver cells for in vitro experiments. This method allows hepatic cells to be separated based on their size and weight, by centrifugation using a density gradient. To date, other methods allow the isolation of only...... one viable hepatic cellular fraction from a single mouse; either parenchymal (hepatocytes) or non-parenchymal cells (i.e., Kupffer cells or hepatic stellate cells). Here, we describe a method to isolate both hepatocytes and Kupffer cells from a single mouse liver, thereby providing the unique...... advantage of studying different liver cell types that have been isolated from the same organism....

  19. Bidirectional transport of iminodiacetic organic anion analogues between plasma and hepatocyte

    International Nuclear Information System (INIS)

    Peters, A.M.; Myers, M.J.; Mohammadtaghi, S.; Mubashar, M.; Mathie, R.T.

    1998-01-01

    The kinetics of organic anions are well described and back-diffusion from hepatocyte to plasma is accepted. Although iminodiacetic (IDA) analogues, as organic anions, should also show bidirectional transport between hepatocyte and plasma, this has not been directly demonstrated heretofore. The aim of this study was to directly demonstrate back-diffusion and to quantify it in terms of its fractional rate constant. Kinetics of diethyl IDA were studied in three anaesthetised dogs in which femoral arterial and hepatic venous samples were obtained after injection of tracer into (a) a peripheral vein or (b) hepatic artery or portal vein. Arterial time-concentration curves were also compared between peripheral venous and either hepatic arterial or portal venous injections. Time-activity curves were recorded from regions of interest over the cardiac blood pool and peripheral hepatic parenchyma in 30 patients undergoing routine IDA hepatobiliary imaging with diethyl IDA or mebrofenin and fractional rate constants of clearance of IDA from the hepatocyte compared between compartmental and deconvolution analyses. After peripheral injection in dogs, there was an early arteriovenous concentration gradient across the liver indicating an hepatocyte extraction fraction in the three animals of 0.9, 0.8 and 0.6. The net extraction fraction decreased exponentially over 40 min. Time-concentration curves from hepatic vein and femoral artery were virtually superimposed following intrahepatic injections. Peripheral arterial curves, however, had different shapes according to whether injections were intrahepatic or peripheral, and were consistent with significant back-diffusion. In clinical studies, the blood disappearance curves were fitted as the sum of two exponentials and the liver curves as the difference of two exponentials (with rate constants denoted α 1 h and α 2 h ). Based on compartmental analysis of the blood curves, the sum of the fractional rate constants of tracer movement

  20. Evaluation of perfluoroalkyl acid activity using primary mouse and human hepatocytes

    International Nuclear Information System (INIS)

    Rosen, Mitchell B.; Das, Kaberi P.; Wood, Carmen R.; Wolf, Cynthia J.; Abbott, Barbara D.; Lau, Christopher

    2013-01-01

    While perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) have been studied at length, less is known about the biological activity of other perfluoroalkyl acids (PFAAs) detected in the environment. Using a transient transfection assay developed in COS-1 cells, our group has previously evaluated a variety of PFAAs for activity associated with activation of peroxisome proliferator-activated receptor alpha (PPARα). Here we use primary heptatocytes to further assess the biological activity of a similar group of PFAAs using custom designed Taqman Low Density Arrays. Primary mouse and human hepatoyctes were cultured for 48 h in the presence of varying concentrations of 12 different PFAAs or Wy14,643, a known activator of PPARα. Total RNA was collected and the expression of 48 mouse or human genes evaluated. Gene selection was based on either in-house liver microarray data (mouse) or published data using primary hepatocytes (human). Gene expression in primary mouse hepatocytes was more restricted than expected. Genes typically regulated in whole tissue by PPARα agonists were not altered in mouse cells including Acox1, Me1, Acaa1a, Hmgcs1, and Slc27a1. Cyp2b10, a gene regulated by the constitutive androstane receptor and a transcript normally up-regulated by in vivo exposure to PFAAs, was also unchanged in cultured mouse hepatocytes. Cyp4a14, Ehhadh, Pdk4, Cpt1b, and Fabp1 were regulated as expected in mouse cells. A larger group of genes were differentially expressed in human primary hepatocytes, however, little consistency was observed across compounds with respect to which genes produced a significant dose response making the determination of relative biological activity difficult. This likely reflects weaker activation of PPARα in human versus rodent cells as well as variation among individual cell donors. Unlike mouse cells, CYP2B6 was up-regulated in human hepatocytes by a number of PFAAs as was PPARδ. Rankings were conducted on the limited